Mechanisms of Cellular injury

Transcription

Mechanisms of Cellular injury
Mark R. Ackermann
Department of Veterinary Pathology
College of Veterinary Medicine
Iowa State University
Ames Iowa 50011-1250
[email protected]
515 294 3647
Injury resulting in ATP loss
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Hypoxia/ischemia
Burn, cold
Radiation
Chemicals/drugs/toxins
Infectious agents
Immunologic/autoimmune
Inflammation
Nutrition
Genetics
Normal cell volume control
• Na+ is extracellular
• K+ is intracellular
• Membrane permeability
– Selective passage
• Water, carbon dioxide, oxygen, benzene, urea, glycerol
– No passge
• H+, sodium, bicarbonate (HCO3), potassium (K+),
calcium, chloride, magnesium, glucose (some cells),
sucrose
• Many ions and molecules are regulated by:
– Transportors, Channels, and ATPase pumps
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Transportors
• Transportors are often
– Slow, 102 to 104 molecules/second
– Do not use ATP
Na+-glucose
• Unitransportors
– Transport
T
t one ion/molecule
i / l
l
• Coupled transportors
Symport
– Transport two ions/molecules
• Symport (both transported in the same direction)
• Antiport (transported in opposite directions)
Na+
Antiport
glucose
Transportors
• Unitransportors
– Glucose
• Can also be coupled with Na+ in the small intestine and
renal tubules
– Fructose
• Intestine and liver
• Coupled transportors
– GABA, Norepinephrine, glutamate
• Symport with Na+
– Peptides and amino acids
• Symport with Na+/H
• Transportors also for:
– Pyruvate, fatty acyl Co A, and ATP
Channels
•
Channels are often
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Fast 107 to 108 ions/second
Do not use ATP
Connect cytosol to cell exterior through narrow passages (ie. pores)
Selective
• Mainly transport Na+, K+, Ca2+ and Cl-
Transport efficiency is greater than carrier proteins
Transport is always passive
Secondary to membrane depolarization (voltage-gated)
–
Next page
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Channels
Secondary to membrane depolarization (voltage-gated), continued
Examples
Na+/K+ channels for nerve fiber conduction
Nearly 20 disorders known involving defects in skeletal muscle
to nervous system
Long QT syndrome
Loss of K+ channel function or gain of Na+ channel
function
Ca++ channels in sarcoplasmic reticulum of muscle
Defects in Ryanodine receptor
malignant hyperthermia
Chloride (Cl-) channels
Ligand-gated channels in synapses, CFTR channels, and CLC
channels (numerous functions)
Defects in CFTR Cl- channel result in cystic fibrosis
Although CFTR is a ABC pump that utilizes ATP, it is
considered a channel due to the rapid influx of chloride it
can allow
Defects in voltage-gated CLC chloride channels
Myoclonia congenita, Dent’s disease, Bartter’s disease,
osteopetrosis
ATPase pumps
• ATPase pumps are often
– Slow, 10 to 103 ions/molecules/second
– All require ATP (by definition)
• ATP
ATPase
ADP+Pi
– ½ of all ATP is used to maintain transport
gradients
• Types of ATPase pumps
– P, F, V, ABC
ATPase pumps
• P type ATPase pumps
– Na/K, H, Ca transported
– Locations
• Plasma membrane for Na/K
– 3 Na+ out, 2 K+ in
– For cell homeostasis
• Apical plasma membrane of stomach (H+/K+)
• Plasma membrane of all cells (Ca++)
• Sarcoplasmic reticulum (Ca++)
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ATPase pumps
• F type ATPase
– H+ ion only
• Inner mitochondrial membrane
– And chloroplast thylakoid membrane (plants)
ATPase pumps
• V type ATPase
– H ion only
• Especially for pH regulation
– Locations
• Endosome, lysosome, secretory vesicles
• Plasma membrane of osteoclasts, renal epithelial cells,
neutrophils, macrophages
– Activities
• Endosomal killing/degradation, bone resorption,
breakdown of ligand/receptor complexes (thus controlling
regulation of receptor density), release of enzymes from
Mannose-6-phosphate receptors (thus regulating enzyme
activity)
ATPase pumps
• ABC type ATPase pumps
– Pump ions (many types) and lipophilic substances
– Location and substance
• Endoplasmic reticulum
– Peptide transport for MHC/Ag processing
• Plasma membrane
– MDR (P-glycoprotein)
» Transports lipid-like toxins/drugs
• Macrophages
– ABCA1
» Transports cholesterol and phospholipids
– defects contribute to atherosclerosis
• Rod photoreceptors
– ABCA4
» Transports retinoyl derivates
» Defects result in macular degeneration
• Liver
– ABCB11 (BSEP)
» Transports bile salts
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Cellular energy: required for ATP
Glucose/glycogen
Proteins
Pyruvate
Fatty acid
Fatty Acyl Co A
ACETYL CO A
KREBS (produces NADH, FADH2 for Oxidative phosphorylation)
OXIDATIVE PHOSPHORYLATION
+ NADH
+02
=ATP
Mitochondria
ATP for:
ATPase pumps, signaling, etc.
A transporter is required for:
Fatty Acyl Co A, pyruvate and ATP
Cell plasma membrane
Mitochondrial ATP formation
• The outer mitochondrial membrane is
permeable to H+ ions
• The inner mitochondrial membrane is
impermeable
• NADH generated in Kreb’s cycle
– Releases electrons (H
(H+)) that enters oxidative
phophorylation
• H+ ion is moved from the inner mitochondrial
matrix to the area between the inner and
outer mitochondrial membranes
• Oxygen enters, is converted to water
• H+ enters the F1 ATPase
– Converts ADP to ATP as H+ passes back to the
inner mitochondrial area
Mitochondrial formation of ATP
Cytoplasm
Outer mitochondrial
membrane
H+ H+
H+
FO/F1 ATPase
Impermeable to H+
Inner mitochondrial
membrane
H+
NADH+
NAD
½ O2
ADP
Pi
H20
H+
ATP
Mitochondrial matrix
5
Sites of inhibition to ATP synthesis
H+ H+
NADH/CoQ10 (Complex I):
ROTENONE, MPP
H+
FO/F1 ATPase
H+
NADH+
NAD
½ O2
H20
ADP
Pi
H+
Cytochrome oxidase:
CN (Complex IV), AZIDE, HYDROGEN SULFIDE
HERBICIDES, NO, Antimycin & myxothiazol (complex III)
ATP
Loss of membrane
potential:
DNP, SALICYLATE,
VALINOMYCIN,
GRANICIDIN
FO/FA ATPase:
DDT, oligomycin (Fo subunit)
Lack of oxygen:
RESPIRATORY PARALYSIS
HYPOXIA, ISCHEMIA (ergot alkaloids,
Infarct, cocaine toxicity), CO, MET Hb
Impairment of mitochondrial synthesis of
ATP
• Inhibition of NADH production
• Lack of glucose
• Fatty acid oxidation
• Loss of protein
Effect of ischemia on cells
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Decreased oxidative phosphorylation
Decreased ATP synthesis
Decreased ATPase pump activity
Loss of inner mitochondrial membrane potential
Loss of K+ from the cytoplasm
Influx of Na+
Increased intracellular Ca++
Glucose/glycogen breakdown
Lactate formation
– decreased pH
• Decreased transport vesicles
• Decreased synthesis of proteins, lipids, loss of phospholipid
turnover in membranes
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Increased intracellular calcium
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Cytosol levels normally are 10-100nM (extracellular levels are 1-2 mM)
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Inositol triphosphate (IP3) and other factors can induce release
Increased Ca++ levels can pass the external mitochondrial membrane
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Intracellular storage in smooth and rough endoplasmic reticulum
Storage kept in check with Ca++ATPase pumps
If persistent, the calcium can then induce the internal mitochondrial membrane to form
pores
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Pores are formed with adenine nucleotide translocase (ANT), voltage-dependent anion channel
(VDAC), and cyclophilin
Mitochondrial permeability transition, a state of calcium permeability by the inner membrane
Enhances oxidative phosphorylation temporarily and free radical formation
Eventually damages the inner mitochondrial area and calcium aggregates form
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Simultaneously, calcium activates phospholipases and calpain
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Reduces ATPase activity
Enhances calcium-dependent endonuclease activity and DNA degradation
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Phospholipase A2 degrades plasma membranes
Calpain is a cysteine protease
Robbins and Contran Pathologic basis of disease, 7th edition
Cell injury: reversible/irreversible changes
7
Brown fat utilization and thermogenesis
• Cold increases
– PGC1 (a powerful transcriptional coactivator) for PPAR
gamma (peroxisome proliferator activator receptor)
– TR (thyroid hormone receptor)
– RAR (retinoic acid receptor)
– ER (estrogen receptor)
• PGC1
PGC1, TR
TR, RAR and
d ER activate
ti t UCP ((uncoupling
li proteins)
t i ) and
d
genes of mitochondrial respiratory chain for ATP synthesis
(ATPase and cytochrome oxidase C)
• The UCP (uncoupling proteins) are in the inner mitochondrial
membrane along with F0/F1 ATPase.
• The UCP allow H+ passage with loss of gradient resulting in heat
with reduced ATP.
– PGC1 also activates NRF 1and 2 (nuclear respiratory
factors) for mitochondrial biosynthesis and increases
conversion of type I to type II muscle fibers
• Increased mitochondria for more heat
Examples of channelopathies
• Na+ channel
genes:
– SCN1A, SCN2A,
SCN4A, SCN5A,
SCN1B
• K+ channel genes
– KCNQ1, KCNH2,
KCNJ2, KCNH2
• Resulting condition:
– Epilepsy,
epilepsy,
p p y
paramyotonia,
LQTS, epilepsy
– LQTS, LQTS,
LQTS, LQTS
Examples of channelopathies
• Ca2+
– CACNA1, RyR
• CL– CFTR, CLCN1,
CLCN5, CLCN7,
CLCNNK8
– Timothy Syndrome
(multisystem disorder),
malignant
li
th
hyperthermia
th
i
– Cystic fibrosis, Myotonia,
Dent disease,
Osteopetrosis, Bartter
syndrome
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Several defects can alter the
neuromuscular junction
Neuromuscular junction defects, previous
slide
1. Acetylcholine receptor (AchR) mutation:
myasthenia gravis
2. Loss of K+ channel (presynaptic) increases Ach
release and increases contraction
3. Down-regulation of Ca2+ channel prevents Ach
release causing myasthenia gravis
4. Na+ channel gain-of-function increases contraction
5. Loss of CLC results in contraction
6. Loss of K+ results in contraction
7. Mutation in CaV impair Ca2+ release causing
malignant hyperthermia
8. RYR channel defect reduces Ca2+ release causing
malignant hyperthermia.
Stars: antibodies that can cause myasthenia gravis
Inherited loss of channel function: Malignant
hyperthermia
• Dysfunctional ryanodine receptor (RYR 1) activity
– A calcium channel in the sarcoplasmic reticulum
• “A” site of RYR1 is high affinity for Ca2+ and allows
opening, while “I” site is low affinity and mediates
closing
• Mg2+ also binds these sites and promotes closure, but
with defect binds less strongly
• Sensitive to halothane, stress, caffeine
• Muscle contraction
– Myosin ATPase activity, glycogenolysis,
glucolysis, further release of calcium, excessive
heat, lactic acidosis, ATP loss
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Inherited loss of channel function: Cystic
fibrosis
• Inherited defect of Chloride CFTR channel
– CFTR (cystic fibrosis transmembrane
conductance regulator)
• Sweat test is done clinically, but not definitive
• Loss of ions in respiratory secretions
– Mucus layer becomes thick
– Also affects gastrointestinal secretions and pancreatic
secretions
• Increased susceptibility to respiratory infections,
especially Staphylcoccus aureus, Pseudomonas and
Burkholderi aeruginosa which are almost impossible
to eliminate
– Loss of ionic concentrations
– Loss of antimicrobial peptide (defensin activity)
– CFTR a receptor for Pseudomonas
Eur J Ped 167:839, 2008
Normal lung air-surface liquid
• Upper gel (mucus) layer
• Lower sol (periciliary liquid) layer (PCL)
– 7 um in height
– Maintained by Na+ absorption, Cl secretion
• Apical Na+ channel (ENaC)
• CFTR (Cl) channel
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CFTR defect
• Loss of PCL fluid with CFTR defect due
to:
– Excessive Na+ absorption
– Defective Cl secretion
– Loss of cilia activity
• Reduced PCL is counter-balanced by
– Increased epithelial ATP release
– Activates purinoreceptors (P2Y2)
• Inhibit Na+ absorption and increase Cl
secretion
• Unable to sustain PCL fluid levels; however.
CFTR defect
• Reduced PCL and ciliary activity lead to
– S. aureus, H. influenzae, P. and B.
aeruginosa
g
colonization
• Pa quorum sensing, biofilm deposition
– Continued inflammation
– Reduced lung function with thick, mucoid
secretions
– Reduced lifespan (mid-30’s to 50’s)
CF defects
Proesmans et al Eur J Ped 167:839, 2008
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Cystic fibrosis
• More than 1000 mutations
• Class I defect
– Large deletions and premature stop codons
Classic CF
phenotype
• Class II defect
– Abnormally folded CFTR protein, degraded
• DeltaF508 mutation in allele
– 70% off CF patients
ti t h
have thi
this mutation
t ti
• Class III defect
– CFTR protein has defective regulation
• Class IV defect
Mild CF
No pancreatic
dz
– Defective chloride conductance
• Class V defect
– Transcription dysregulation; reduced CFTR levels
Cystic fibrosis models
• Mice with CFTR defect
– Develop pancreatic fibrosis but do not have
increased respiratory diseases
• Sheep
– Molecular defect identified in mass screens, but no
breeding pairs
• Pigs with CFTR defect
– Developed by Welsh, McCray and others at the
University of Iowa
– No pulmonary lesions in newborn pigs
– Studies ongoing to determine if they have
respiratory defects
CLC Chloride Channels
• Nine mammalian genes encode for
channels denoted CLCN1 to CLCN7 and
CLCNKa and CLCNKb
• Present in plasma membrane and
membrane of intracellular organelles
• Function to stabilize membrane potential,
synaptic inhibition, cell fluid volume
regulation, transepithelial transport,
extracellular and vesicular acidification
and endocytotic trafficking
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CLC Chloride Channel Structure
• Double-barreled channels
• Each pore is gated and can be opened
and closed individually; gating is
independent
• Common gate exists to close both pores
simultaneously
• Single gate mutations
• Common gate mutations
Myotonia Congenita
• Impairment of skeletal muscle relaxation
after contraction
• Mutations in CLC1 which can cause either
dominant ((Thomsen’s)) or recessive
(Becker-type) disease
• Recessive form more common and more
severe
• Dominant mutations act on common gate
affecting both pores
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Myotonia Congenita
• Treatment usually not necessary
• Mexiletine (sodium channel blocker)
Myotonia in goats and mice (KO)
Bartter Syndrome III
• Renal tubular disease characterized by loss of NaCl
reabsorption; PU/PD, bouts of severe dehydration,
failure to thrive
• Mutation in CLC-Kb with autosomal recessive
i h it
inheritance
• Deafness not present because both CLC-Ka and
CLC-Kb are present in inner ear
• Mutation in barttin (beta subunit present in both
CLC-Ka and CLC-Kb) leads to deafness
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Bartter Syndrome III
• Treatment based on
clinical presentation,
not genetic mutations
• Na+ and K+
supplements
• ACE inhibitors
• Indomethacin
• Growth hormone
• Response to therapy
variable
Dent’s Disease
• X-linked recessive gene mutation in
CLC5 with proteinuria,
hyperphosphaturia, hypercalciuria and
nephrocalcinosis
• Endocytosis is impaired
• Luminal parathyroid hormone increases
causing decrease of Na-PO4
cotransporter
• Serum concentrations of active vitD3
variable
Dent’s Disease
• Treatment for nephrocalcinosis may slow
progression to end-stage renal failure
• Thiazide diuretics used which enhance Ca2+
reabsorption
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Dent’s Disease
Osteopetrosis
• Reduced or absent acidification of
resorption lacuna leading to failed bone
resorption by osteoclasts which results in
brittle bones
• Autosomal
A t
l recessive
i or dominant
d i
t linked
li k d
mutation in CLCN7
• CNS neurodegeneration resembling
lysosomal storage diseases produced in
knockout mice
• Retinal degeneration
Osteopetrosis
• Treatment success variable
• Bone marrow transplant
• Vitamin D administration (calcitriol) to
enhance bone resorption
• Prednisone given to improve
hematological alterations (neutropenia
and anemia)
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CLC Defects
Numerous diseases are associated with
CLC defects attributes to their wide
range of physiologic functions and
emphasizes their medical importance
Mitochondrial disorders
www.missinglink.ucsf.edu
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Mitochondria
Provide energy from aerobic metabolism
Regulatory role in apoptosis
Produce and detoxify free radicals
Serve as a cellular calcium buffer
Diseases of mitochondria
– not common
• But similar to Duchenne muscular dystrophy and more
common than Wilson’s disease
– probably often misdiagnosed
– exemplify the vital role of mitochondria (other than
just providing energy and regulating apoptosis)
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Classification of Mitochondrial Involvement in
Neurodegenerative Diseases
Class of mitochondrial involvement
1. OXPHOS abnormalities due to
mtDNA mutations
2. OXPHOS abnormalities due to
mutations in nuclear genes
encoding OXPHOS proteins
3. Mutations in nuclear g
genes
encoding mitochondrial nonOXPHOS proteins
4. Mutations in nuclear genes
encoding non-mitochondrial
proteins affecting OXPHOS
function
5. Disorders with evidence for
mitochondrial involvement
which cannot as yet be
assigned
Examples of associated diseases
1. CPEO, MELAS, MERRF, pure
myopathies, LHON
2. Complex I deficiency, Complex II
deficiency
3. Friedreich's ataxia, hereditary
spastic
p
p
paraplegia,
p g , Wilson
disease, X-linked deafnessdystonia, Complex IV
deficiency, dominant optic
atrophy
4. Huntington disease
5. Parkinson disease, amyotrophic
lateral sclerosis, Alzheimer
disease
(Orth and Schapara, 2001)
Mitochondrial Disorders
• Often alterations of oxidative phosphorylation
– Secondary to MtDNA mutation
• MtDNA mutations first identified in 1988 (recent)
• Affect tissues with the highest energy demand
(i.e., brain, heart, liver, skeletal muscles,
kidney and the endocrine and respiratory
systems)
• Childhood onset for some
– Rule outs: Maple syrup urine disease,
endocrinopathies, homocyteinuria, tyrosinemia,
urea hydrate disorders
• Adult onset for others
Some subunits encoded by nDNA (shaded, no labels);
whereas others are encoded by mtDNA which are
labeled.
Ann NY Acad Sci 1142:133-158, 2008
18
Mitochondria
• Complexes I, II, IV, V
– Encoded by both n and mt DNA
• Complex II encoded entirely by n DNA
• Mt DNA encodes 37 genes
– 2 for rRNA
– 22 for tRNA
• mRNAS are translated on mt ribosomes
– 13 for electron transport system (OXPHOS)
mtDNA, continued
• 16,569 nucleotide-pair (np)
• Closed circular molecule
• Mitochondria has an independent
replication, transcription and translation
system.
Mitochondrial disorders
1. Kearns-Sayre syndrome (KSS)
2. Leigh's syndrome (MILS)
3. LHON (Leber’s hereditary optic
neuropathy)
4. Mitochondrial encephalomyopathy,
lactic acidosis and strokelike
episodes (MELAS)
5. Myoclonus epilepsy with ragged
red fibers (MERRF)
6. Mitochondrial neurogastrointestinal
encephalomyopathy (MNGIE)
7. Neuropathy, ataxia and retinitis
pigmentosa (NARP)
8. Pearson syndrome (PS)
9. Progressive external
ophthalmoplegia (PEO)
1. Sporadic inheritance, ataxia
2. Maternal inheritance, seizures
3. Maternal inheritance, dystonia,
optic atrophy
5. Maternal inheritance, seizures,
myoclonus
7. Maternal inheritance, ataxia
8. Sporadic inheritance,
ophthaloplegia
9. Sporadic inheritance,
ophthaloplegia
Muravchick S: Adv Drug Deliv Rev 60:1553, 2008
19
Fig. 1. The human mtDNA map. The mtDNA encompasscs 16,569 nps with numbering starting at
OH and proceeding counterclockwise (Wallace, 1992).
Fig. 2. Known mutations in mtDNA that cause diseases in humans (Dimauro, et., al, 2001).
Point Mutation in mitochondria
• 80% are an A to G
transition mutation at
nucleotide pair 8344 in
human mitochondrial
DNA ((mtDNA)
tDNA)
• Alters the TψC loop of the
tRNALys gene and
creates a CviJI restriction
site (use as a diagnostic
test)
• 10% have the mutation at
8356T>C
www.biology.plosjournals.org/archive/
Fig. 3. Diagram of tRNA-Lys gene.
20
Pathogenesis cont.
• When a cell is filled with defective mitochondria, it
not only becomes deprived of ATP
– Shunting of pyruvate to lactate
• Less pyruvate for electron transport system (OXPHOS)
• This can lead to the build-up of lactic acid and
unused oxygen can be converted into reactive
oxygen species.
i
• The combined effects of energy deprivation and
toxin accumulation in these cells probably give rise
to the main symptoms of mitochondrial
myopathies and encephalomyopathies.
• Question: If most mitochondrial disease affect
OXPHOS, then why are there different
syndromes?
Maternal Pattern
• Unlike nDNA,
mtDNA passes only
from mother to child.
• During conception
conception,
when the sperm
fuses with the egg,
the sperm's
mitochondria and its
mtDNA are
destroyed.
Fig. 5. Diagram of maternal inheritance of mitochondrial
mutations.
(MDA, 2003
Heteroplasmy
• Disease manifestations are dependent on replicative
segregation of mutant and wild type (“normal”)`
mitochondrial DNAs.
• For example, all MERRF patients and their less-affected
maternal relatives have between 2% and 27% wild-type
mtDNAs
tDNA and
d show
h
an age-related
l t d association
i ti b
between
t
genotype and phenotype (Shoffner, 2001).
• This suggests that a small percentage of normal mtDNAs
has a large protective effect on phenotype. This
mutation provides molecular confirmation that some
forms of epilepsy are the result of deficiencies in
mitochondrial energy production.
21
What is MERRF?
• Myoclonic Epilepsy with Ragged-Red
Fibers
• Mitochondrial myopathy
• Rare (<200,000 Americans affected)
Pathogenesis
• Mitochondrial tRNA Lys
gene mutations result in
multiple mitochondrial
respiratory chain
deficiencies. Enzyme
deficiencies which are
deficiencies,
most pronounced in
complex I and complex
IV, are secondary to
defects in mitochondrial
protein synthesis
caused by mutations in
tRNA Lys.
Fig. 4. Diagram of normal mitochondrial function.
(MDA, 2003)
Hallmarks of MERRF
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Seizures
Muscle weakness
Exercise intolerance
H i
Hearing
i
impairment
i
Ataxia
Learning disabilities
Cataracts
Diabetes
Stunted growth
A combination of three
or more of these
symptoms in one
l points
i
person strongly
to mitochondrial
disease, especially
when the symptoms
involve more than one
organ system.
22
Symptons of MERRF
• Myoclonic seizures (sudden, brief,
jerking, spasms that can affect the limbs
or the entire body)
• Progressive ataxia
• Lactic acidosis
• Dysarthria, optic atrophy, short stature,
hearing loss, dementia, and (nystagmus)
• Late childhood to adolescence
Diagnostics
•
•
•
•
•
•
Family history/PE
Serum chemistry
Biochemistry
IHC
Muscle biopsy
Gomori trichrome
stain
• Electron microscopy
Fig. 6. The red color of these fibers is
due to large numbers of abnormal
mitochondria that represent a
compensatory proliferation. In addition
to their red color, the abnormal fibers
are coarse and disorganized.
www.neuropathologyweb.org/chapter13/chapter13...
Fig. 7. There is an increased number of mitochondria in the subsarcolemmal
space. They also contain paracrystalline inclusions.
www.pathology.vcu.edu/.../em/muscle.path.f.html
23
Fig. 8. In addition to increased numbers, the mitochondria often appear large and
abnormal. The mitochondrial inclusions sometimes are described as having a "parking
lot" appearance.
www.pathology.vcu.edu/.../em/muscle.path.f.html
Treatment
• Seizures: anti-epileptics
• Hearing impairment: hearing aide
• Ataxia/muscle weakness: occupational
and physical therapy
• Cardiomyopathy: pacemaker
• Dietary supplements: creatinine
phosphate, carnitine, coQ10
– Other supplements include L-carnitine,
riboflavin, thiamine, nicotinamide, Vit E, C,
K, selenium, biotin, folic acid, calcium,
magnesium, phosphorus
Muravchick S: Adv Drug Deliv Rev 60:1553, 2008
MELAS
• A-G transition mutation
– At position 3243
– Encodes mt tRNA
– 29 point mutations associated
– Encephalopathy (seizures, migranes),
lactic acidosis, strokelike episodes
24
Other species
• Suspected in a springer spaniel
– Vet Rec 163:396, 2008
• Suspected in a horse
– Muscle Nerve 17:305, 1994
Radical formation
• Radicals, reactive oxygen species (ROS)
– Form secondary to:
• Ultraviolet light, cell metabolism, inflammation
– Cell metabolism
• Of all oxygen used by cells
– 2% is converted to ROS by mitochondrial electron transport
system (ETS)
– 1/100 oxygen molecules cause protein damage
– 1/200 oxygen molecules cause DNA damage
– Oxidation: loss of an electron
– Reductant: Donates electrons via addition of a
hydrogen ion or removal of an oxygen molecule.
25
Free radicals
• Free radicals
. :
– An unpaired electron in an outer orbital
– Example: hydroxy radical
• :O:H hydroxy radical (.0H) a radical
: :
An unpaired
p
electron
• :O:H hydroxy ion (-OH) not a radical
• The dot denotes an unpaired electron, but no inference about
charge; the negative sign denotes charge
• Singlet oxygen (O2)
•
•
•
•
.
Superoxide anion (O2 -)
Hydroxy radical (.OH)
.
Carbon tetrachloride (CCL3 )
Peroxyl (ROO.) and alkoxyl radicals (RO.)
Nonradical oxidants that can form radicals
•
•
•
•
•
•
•
•
•
Hydrogen peroxide (H2O2)
Hypochlorous acid (HCLO)
Hydroperxoyfatty acids
Aldehydes
Quinones
Ozone
Singlet oxygen (1O2)
Peroxynitrite (ONOOH)
Disulfides
Non-radicals that can react with thiols
•
•
•
•
•
Conjugated aldehydes (acrolein)
4-hydroxynonenal, malondialdehyde
Quinones
Expoxides
Zinc, Mercury, other metals
26
Terms
• Reactive oxygen species (ROS), oxygenderived species (ODS), oxidants, and
reactive nitrogen species (RNS)
– These are all often used interchangeably.
Free Radical Formation
•
Reactions for Formation
»
Fe++ Fe +++
• Fenton Reaction
H2O2 Æ OH.
• Haber Weiss reaction
H2O2 + O2. Æ OH.
• Electron transport system Æ O2. - = O = O. - (unpaired
electron)
• Cytochrome P450, xanthine oxidase Æ O2. • Ionizing Radiation H20 Æ .OH
• NADPH
oxidase NADPH -2 e’s + 2O2Æ NADP+ + H+ + 2 O2
-
• Lysyl oxidase-crosslinks collagen
• NOS nitric oxide synthtase 1, 2, 3 (neuronal, endothelial,
macrophage) Æ (peroxynitrite) OONO.
• Radical + Nonradical = radical
• Reperfusion injury – xanthine oxidase
Properties of free radicals
•
Hydroxy radical (.OH)
– The most reactive radical known in chemistry
– Diffuses a short distance, 3 nm, short-lived (10-10 sec)
•
• So active that it doesn’t travel far
• Must be made close to DNA; H2O2 is the latent form that gets close to DNA and then is
converted to hydroxy radical through the fenton reaction
• Damages deoxyribose, all four DNA bases, phosphate backbone, lipids
.-
Superoxide anion (O2
)
– Diffuses a short distance, short-lived
– Affects guanine selectively
•
•
Alkoxyl (RO.)(10-6 sec) and peroxyl (ROO.)(17 seconds)
Nitric oxide (NO.)/peroxynitrite (ONOO- or ONO2)
– ONOO- diffuses a longer distance, 9 um, long-lived (minutes)
• Can deaminate or nitrate (add nitrogen) to nucleotides
• Especially damaging to guanine
– Results in formation of 8-oxo-deoxyguanine (easy to detect)
» ONOO- is more damaging to 8-oxodeoxyguanine than guanine
– Also induces formation of 8-nitro-deoxyguanine (difficult to detect)
•
Non radicals, also a short livespan (seconds to hours)
27
Often non-radicals are formed and if radicals are formed, they are quickly converted to
non-radicals. However, non-radicals themselves can eventually contribute to oxidative
stress
Jones, D. P. Am J Physiol Cell Physiol 295: C849-C868 2008;
doi:10.1152/ajpcell.00283.2008
Copyright ©2008 American Physiological Society
Exogenous and endogenous sources
• Exogenous sources:
– Gamma radiation, UV, ultrasound, food,
drugs,
g p
pollutants, xenobiotics, toxins
• Endogenous sources:
– below
Exogenous sources
• Reactive oxygen species (ROS) sources:
–
–
–
–
–
–
–
–
–
–
Ionizing and non-ionizing radiation
Phorbol esters
Bleomycin, paraquat (these induce fibrosis)
Organic peroxides
Heavy metals (next slide)
Asbestos (fibers)
Smoke
Silica
Nanoparticles
Gases
• Ozone
• di-oxygen (O2) (oxygen itself)
– and many others
28
Metals and free radical formation
• Metals generate free radicals via the Fenton reaction
• Reduction/oxidation-active metals
– Iron, copper, chromium, and cobalt
• Increase ROS through a Fenton-like reaction
• Reduction/oxidation-inactive metals
– Lead, cadmium, mercury, nickel
• Deplete cells of antioxidants
– Especially thiol-containing antioxidants (glutathione reductase) through binding of
these metals to SH groups
• Metals induce mutagenic lipid products.
– Lipid peroxides, formed from ROS action on phospholipids (more later),
further react with metals to produce mutagenic lipids (malondialdehyde,
4-hydroxynonenal, exocyclic DNA adducts (etheno adducts)).
Endogenous location of free radical
formation
• Endoplasmic reticulum (rER, sER)
– sER biometabolism
• Cytochrome P450, conversion of carbon tetrachloride to radical CCl4 to
CCl3
• Mitochondria
• Ubiquinone
• Cytosol
– Fenton reaction, xanthine oxidase, U.V.
• Peroxisome
– oxidases
• Leukocyte granules
– NADPH oxidase
• Extracellular matrix
– Lysyl oxidase (cross links collagen)
Principal locations of radical formation in cells
Cytosol:
Nitric oxide synthetase
UV: hydroxy radical
Fenton reaction
Reperfusion injury
Granules:
NADPH oxidase
sER:
Cytochrome p450
-biotransformation
Mit h d i
Mitochondria:
Oxidative phosphorylation
Peroxisomes:
Catalase, oxidases
Extracellular matrix:
Lysyl oxidase
29
Reperfusion injury
• Reperfusion injury occurs in sites of
ischemia that once again receive
oxygen
yg
– Examples:
• myocardial infarction
• stroke
• dissolution of a thrombus (clot)
Reperfusion injury
• Exuberant free radical formation occurs
with reperfusion due to:
– Xanthine oxidase pathway
– Mitochondrial electron transport chain
– Conversion of NOS to produce superoxide
(rather than NO)
– NADPH oxidase from infiltrating leukocytes
30
Free radical formation during reperfusion:
xanthine oxidase
Cell ischemia/loss of oxygen leads to ATP degradation. If perfusion is
re-established, oxidative radicals are formed.
ATP
ISCHEMIA
ADP
Xanthine dehydrogenase
AMP
Adenosine
Inosine
Xanthine oxidase
O2
O2-
Xanthine oxidase
Xanthine
Hypoxanthine
SOD
Uric acid
H2O2
Fe++
REPERFUSION
Tissue injury
.
OH
Proximity of muscle and other cells to
endothelial cells in reperfusion injury
• Direct contact between myofibers and
other cells to endothelial cells allows
passage
p
g of nucleotides via nucleotide
transport proteins (NTP) that can be
used in the xanthine oxidase pathway
Muscle or other cell
ATP-ADP-AMP-Adenosine-Inosine
NTP
NTP
Adenosine-Inosine
X.O. pathway
Endothelial cell
Mitochondrial activity contribution to ROS
in reperfusion injury
• Distal to NADPH dehydrogenase in the
mitochondrial electron transport chain
– Ubiquinone (CoQ) increases ROS formation
• ROS induce mitochondrial permeability
transition (MPT)
• These contribute to ROS formation and
reperfusion injury
31
Increased superoxide generation from
NOS during reperfusion injury
• All three NOS (nNOS, eNOS and iNOS) can switch
from NO to superoxide generation
– This occurs with depletion of NOS substrate (arginine and
BH4)
• Both arginine and BH4 are depleted by ROS (vicious cycle)
• The increase superoxide production contributes to
ROS damage and reperfusion injury
Leukocyte infiltration with reperfusion injury
• Hypoxic tissues, including myocytes and
endothelial cells increase expression of
leukocyte adhesion molecules
– Enhanced leukocyte (neutrophil) infiltration with
reperfusion
– Increased activity of NADPH oxidase by
infiltrating leukocytes
• NADPH oxidase produces additional ROS
Reperfusion injury therapy
• Therapies
– Needed to reduce ROS damage but yet allowing
perfusion and return to normoxia
p
32
ROS lipoperoxidation
• ROS commonly cause lipoperoxidation
– Linoleic acid, the most common polyunsaturated fatty acid in
cells
– One radical reaction can oxidize 60 molecules of linoleic
acid
– One radical reaction can oxidize 200 arachidonic acid
molecules
• Lipoperoxidated fatty acids are short lived
– Reduced by glutathione peroxidase to a non-radical, or
– Interact with a metal to form:
• Aldehydes: malondialdehyde and 4-hydroxynonenal (HNE)
• 4-HNE can interact with a metal to form an epoxide
• Predilection sites of radical formation/damage
• Mitochondria
– ETS-major site of free radical formation
• Phagosomes, peroxisomes
• Enzymes with divalent cations
– Cu2+, Zn2+, Fe2+
– Via Fenton-like reaction, damage occurs at the enzymatic active site
• Endoplasmic reticulum membranes
• Damage to DNA
– OHOH formed in nucleus when H2O2, a latent form, diffuses to nucleus where it can
be converted to hydroxy radical by the Fenton reaction
– Peroxynitrite, diffuses longer distances and can cause DNA damage
• Lipid
• Lipoperoxidation, fatty acid loss
• Protein and glycoconjugates
• Oxidizes SH groups to S-S and crosslinks often at cysteine or methionine
residues
• NO
– Nitration: NO binding to an amino acid
– Nitrosation NO binding to SH, amine, or hydroxyl group
• Oxidized proteins:
– Reduced binding of transcription factors, p53, Rb, and enzymes such as kinases,
phosphatases, and DNA repair enzymes.
Cysteine (thiol) oxidation
• Thiol are converted to:
– Disulfide (SS) (most common and stable)
– Sulfenate (SO-)
– Sulfinate (SO2-)
(SO2 )
– Sulfonate (SO3-)
– Methionine (thiolether) is also oxidized
• Often there are multiple cysteines and thiol
groups
33
Protein cysteine (thiol) oxidation
• Oxidation can shut down the protein’s activity
• Enzymes with cysteine residues in active sites:
– Caspases, kinases, phosphatases, proteases
• Includes g
glutathione transferases, cytochrome
y
P450,
thioredoxins, peroxiredoxins
• Other proteins affected are numerous
– Actin, zinc fingers of transcription factors, actin, 143-3, alpha Iib/betaIII integrin of platelets, others
Oxidative effects on cell signaling
Free Radical Scavengers
• Enzymatic
– Superoxide dismutase, SOD
• Converts (O2.-) superoxide anion to H2O2
• SOD1, cytoplasm (zinc/copper)
• SOD2, mitochondria (mangenese)
• SOD3, extracellular
34
Free radical scavengers
– Enzymatic, continued
– Glutathione (GSH), three amino acids (cysteine, glumatic
acid, glycine)
• High abundance in cells
• Synthesized by GSH synthetase and glutamate cysteine ligase
• Conjugated to toxins in phase II biotransformation (to make them
more water soluble)
• Coenzyme in detoxification and hormone synthesis
– Alcohol dehydrogenase, prostaglandin synthesis
• Added to proteins by Glutaredoxin
– Nfkappa B, Caspase 3, PDGF, actin, EGFR
• Reactant for Gluthathione peroxidase (requires selenium)
– Converts H2O2 to H20
– GSH convereted to GSSG
– GSSG converted back to GSH by Glutathione reductase
• Enzymatic, continued
• Thioredoxin-1, 2 (Trx-1, 2)
– Repairs S-S
– Small p
proteins,, low levels than glutathione
g
– Oxidized thioredoxins are reduced by thioredoxin
reductase
– Trx1 and Trx 2 also:
• Bind apoptosis signal regulating factor
• Bind VitD 3 binding protein in the cytoplasm
• Reduces redox factor -1 (REF-1) which is AP endonuclease (DNA repair
enzyme) in nucleus
• Reduces Cys residues in transcription factors (AP-1, p53, HIF1)
• Enzymatic continued,
• Peroxiredoxins
– Reduce H2O2, organic peroxides and peroxynitrite
– Expressed
E
d att hi
high
h llevels
l iin cells
ll
• Catalase
– Converts H2O2 to H20
35
ROS scavengers
• Nonenzymatic (scavenge radicals)
–
–
–
–
–
Alpha tocopherol (vitamin E)
Ascorbic acid (vitamin C)
Retinoic acids (vitamin A)
Lycopenes (Vitamin A-like)
Flavenoids, genistein, resperpins, resveratrol, quercetins—electron
donors, chlorogenic acid, naringenin, anthocyanins (tart cherries)
– Ferritin, lactoferrin, ceruloplasm, hemoglobin, metallothionen, uric
acid, histidine residues
– Bind divalent cations or serve as electron donors
– Glutathione (glutamic acid-cysteine-glycine), melatonin (an –NH
group), histidine dipeptides (caronsine, anserine)
Nonenzymatic scavengers
• Nonenzymatic scavengers
– Also termed low molecular weight antioxidants
(LMWA)
– Donate an electron to the radicals but not reactive
themselves
• A wide spectrum of activity--nonspecific
– Better permeability throughout the cells than
enzymes
• Can penetrate cell membranes
• Allows radical scavenging in more areas of the cell
Repair of lipoperoxidation of phospholipids:
Vitamin E and C
PL
Fatty acyl CoA
TOC-OH
Lipoperoxidation
O-ASC-OH
PL-OO
HO-ASC-OH
O=ASC=O
lysophospholipid
PL-OOH
2GR SS
2GR-SS
2GSH
GR-[SH2]
GSSG
TOC-O
GRO-SS
FA-OOH
GPX
FA-OH
2GSH
GSSG
Phospholipid membrane
2GR-SS
GR-[SH2]
NADPH+ + H+
PL phospholipid
PL-OO
phospholipid that is oxidized (lipoperoxidation)
TOC alpha tocopherol, Vitamin E
ASC ascorbic acid, Vitamin C
GRO-[SH2]
NADP+
GRO glutaredoxin
GSH glutathione (GSSG [oxidized])
GR glutathione reductase
GPX glutathione peroxidase
36
Free radicals and the “penumbra zone”
• Penumbra zone
– A region around an area of necrosis,
infarction that is hypoxic
yp
– Occurs stroke, myocardial infarction or
other inflammatory disease
• The tissue in the penumbra zone may or may
or may not die, depending upon the amount of
hypoxia and level of free radicals
Penumbra zone
Free radical theory of aging
• Harman Denhan
– 1956
– Developed the “free
free radical theory of
aging”
• A concept that tissue damage caused by free
radicals results in a “wear and tear” type of cell
injury that is prolonged and over the years,
leads to aging changes in cells
37
Oxidative stress versus reductive stress
• Redox (reduction/oxidation) potential
– The balance between reductants and oxidants
– The “redox state” is tightly regulated in the cell, like pH
• Free radical theory of aging and suggests that all oxidative
stress is damaging
– But this is not always true, some oxidative stress is needed
for cellular physiology
• Oxidation is needed for thyroglobulin binding to iodine and thus
thyroxine activity
• Oxidation needed for neutrophil/leukocyte killing and lysosomes
– Many studies to prevent cancer and disease by use of antioxidant nutrients do not show great improvement
• Reductive stress, the opposite of oxidative stress can also be
overdone
• A balance in oxidative and reductive stress is likely optimal
– Eat a balanced diet and you’ll be ok
•
“Everything in moderation, including moderation itself”
– Michael Tassler, 1984
Cell signaling and oxidative changes
• GTP-binding protein families
– RAS and RHO
• RHO
– Induces actin and cytoskeletal changes
• RAS
– Active when bound to GTP
– Hras, Nras and Kras, three subtypes
» Kras most ubiquitous expression
– Farnesyltransferase modifies RAS post-translationally
» Allows RAS to be come functional
» Farnesyltransferase is targeted for therapies to reduce RAS
activity
– Epidermal growth factor (EGF) induces RAS activity
» Leads to: RAF/MEK/ERK/C-jun/AP-1 (more following)
» Also leads to: PI3K/AKT and inhibition of BAD (cell survival)
» Also leads to: FALGDS and PLC/protein kinase C (cell
prolifeartion)
» All of the above increase cell survival, cell cycle progression
and calcium signaling
RAS
• RAS/RAF/MEK/ERK and RAS/MEK/JNK activate
cJun pathway
– C-Jun a part of AP-1 (activated protein 1)
• AP-1 is a dimeric basic region-leucine zipper transcription
factor
– AP-1 is composed of c-jun and c-fos
» Other
Oth such
h transcription
t
i ti factors
f t
are Maf
M f and
d ATF
• AP-1 and these transcription factors bind
– TPA response elements (TRE) and c-AMP response elements (CRE)
– TRE and CRE are promoter regions of genes that encode:
» Other transcription factors, matrix degrading proteins, cyclins, cell
adhesion molecules, and cytokines.
38
Mechanistic basis of free radical activity of
signalling
• Oxidative radicals can increase signaling pathways
• Any step in these pathways can become mutated and
increase activity
– Activation of growth factor receptor (listed above)
– Activate NFKappa B
» OH- can induce release of NFKappaB inhibitory subunit
– increased phosphorylation of an enzyme (kinase)
– decreased dephosphorylation (decreased phosphatase activity)
» ONOO- can nitrate (add N) to tyrosine (on tyrosine kinases) and
block phosphorylation
– Damage to cysteine residue on active enzymatic sites of regulatory
phosphotases such as PTEN (inhibits PI3K)
– Activate oncogenes
» C-jun is activated by H2O2 as is MAPK
Chronic cell injury caused by intracellular
accumulations of metals, bilirubin,
porphyrins
VPTH 655
Ackermann
Lipid accumulation
Protein accumulation
Lysosomal storage diseases
Iron, copper, bilirubin, porphyria
accumulation
Other: hyperglycosylation
39
Intracellular accumulation of metals
• Iron (Fe2+) and Copper (Cu2+)
• Accumulation of one or both results in cellular
degeneration
– Commonly hepatic degeneration/cirrhosis
– Mechanism of toxicity:
• Induction of free radical formation
– Fenton reaction
– Occurs in the region of the ion
This region is often an important site for enzymatic
activity, protein structure/conformation, signaling
activity
• Absorption and regulation
– Iron body stores are regulated at the level of
absorption by enterocyte (cells sloughed)
– Copper body stores are regulated at the level of
excretion liver (excretion into bile)
Iron absorption
Intestinal lumen
Site of enterocyte exfoliation
Intestinal wall
Exfoliation of enterocyte
Intestinal enterocytes
Intestinal lumen
Intestinal villi
Intestinal crypt
Intestinal villi-higher magnification
Iron absorption
Intestinal lumen containing dietary Iron (10-20 mg in diet/day);
Only 1-2 mg required for absorption
Fe++
Fe++
DMT-1 (divalent metal transporter)
Enterocyte
Labile iron
(Ferritin)
Nucleus
Hepicidin-ferroportin complex degraded
In lysosome-leads to reduced iron absorption
Ferroportin-produced in enterocyte
transferrin protein-carries absorbed iron (1-2 mg/day)
Hepicidin-produced in liver
40
Iron absorbed
Reduced iron absorption
Reduced iron release
Iron released
Key iron proteins
•
Transferrin
•
Ferritin
•
DMT 1
•
Ferroportin
•
Hepcidin
– Carries iron in blood
– Stores iron in cell
– Transfers iron across the cell
– Efflux of iron out of cell into the blood
• The only efflux mechanism for iron
– Binds ferroportin and induces degradation
•
Hemojuvelin
– Increases hepcidin synthesis
• HFE gene
– MHC I protein that interacts physically with DMT 1. Also, hepatocyte iron
sensor upstream regulator of hepicidin. Loss of HFE = hereditary
hemochromatosis
•
Iron regulatory protein 1 and 2
– Bind iron regulatory elements (IRE 3’ and 5’)
– IRE 3’ increases transcription of transferrin, ferritin, DMT1 and ferroportin
– IRE5’ decreases transcription
Types of hemochromatosis in man
• Hereditary hemochromatosis (HH)
– Due to mutation in HFE
• Non-HFE hemochromatosis
– Juvenile
• Mutations in hemojuvelin or hepcidin
– Adult
• Mutations in transferrin receptor 2 or ferroportin
41
Iron sources:
From duodenum
Scenescent rbcs
Liver cells
Syncyiotrophoblasts
Sy
cy o op ob as s
for passage to fetus
Fig. 4. Major iron flows are controlled by hepcidin–ferroportin interactions. Hepcidin
Blocks outflow of iron from duodenum, liver, macrophages (and placenta)
Hepcidin
• Increased hepcidin
– Decreased ferroportin
– Decreased iron absorption
– Hepcidin increased with inflammation
• Anemia of chronic inflammation
• Keeps iron from microbes
• Decreased hepcidin
– Secondary to loss of HFE gene in hereditary
hemochromatosis
– Increased ferroportin
– Increased iron absorption accumulation
– Hemochromatosis
• Hepatocellular damage and cirrhosis
42
Iron accumulation
• Normally
– 1-2 mg/iron/day absorbed
– Transferrin 30% saturated
• If the body senses enough iron
– Then transferrin does not pick up iron from the enterocyte
– The enterocyte exfoliates
• Iron stays in feces
• With Hereditary hemochromatosis
– 3-4 mg/iron/day absorbed
– Transferrin 100% saturated
Robbins and Contran Pathologic basis of disease, 7th Edition, 2005
Causes of excessive iron accumulation
• Primary
– Hereditary hemochromatosis (adult and juvenile forms)
• Juvenile form most severe
– Man, Minah Birds, Birds of Paradise, Lampreys, Rock
hydraxes
• Secondary to another disease
– Chronic liver disease
• Alcoholic cirrhosis
cirrhosis, chronic viral hepatitis
hepatitis, shunts
shunts,
porphyria
– Congenital atransferrinemia
– Ineffective erythropoiesis (dyserythropoiesis; iron absorbed
but not used)
• Beta thallasemia, aplastic anemia, pyruvate kinase deficiency
(Basenji dogs)
– Idiopathic excessive parental iron injection
• RBC transfusions, iron-dextran injection (sideroblastic
anemia)
– long term dialysis
43
Chronic Iron accumulation in liver
• Increased iron increases the Fenton reaction
• Increased ROS formation
– Lipoperoxidation, protein damage, DNA damage
• Hepatocellular damage
• Fibrosis
• Inflammation
– Lymphocytes and plasma cells
Treatment for iron storage
• Hemochromatosis
– Blood letting
– Hepcidin
• Costly
• Thalassemias
– Hepcidin?
Copper absorption
Intestinal lumen containing dietary copper (1.5-4 mg/Cu++/day)
Cu++
Enterocyte
Liver
H
Hepatocyte
t
t
Cu++ albumin
Hepatocyte- copper bound to
metallothionen and
If excess in lysosomes
Cu++ ceruloplasminSystemic delivery
lysosome
Protein for copper
excretion into bile
Biliary excretion- copper is excreted into bile and eliminated (2-4 mg/day)
44
Copper homeostasis
Cu-MT
CMT 1 = Cu membrane transporter 1
MT = metallothionen
Cu AT OX-1 = Cu chaperone
ATP7A= Cu ATPase in enterocyte
ATP7A
TGN = Trans-golgi network
ATP7B = Cu ATPase in hepatocyte
CP = ceruloplasmin
Excess Cu in bile
http://en.wikipedia.org/wiki/Wilson%27s_disease
Cu-MT
MENKES disease
ATP7A defective
Low Copper in the body
Excess Cu in bile
Cu-MT
Wilson’ss disease
Wilson
ATP7B defective
High Copper in the body
Excess Cu in bile
45
Copper accumulation
• Primary
– Wilson’s disease
• Defective Cu ATPase pump
» Impairs biliary excretion of bile by inhibiting
copper transport into the Trans Golgi Network
Ceruloplasmin
Serum copper
Urinary copper
Liver copper
Wilson’s disease
0-200
19-64
100-1000
>250
Normal
200-350
70-152
<40
20-50
Wilson’s disease
Hepatic damage secondary to copper
accumulation
•
•
•
•
•
Copper enhances ROS formation
This leads to cell injury
Hepatocellular necrosis
Fibrosis
Inflammatory cell infiltration
– Lymphocytes, plasma cells, macrophages
46
Copper accumulation
• Primary
– Wilson’s disease (previous slide)
– Bedlingham terriers (BT)
• Primary accumulation of copper
– Metallothionen of BT remains neonatal-like
• Secondary
• In other breeds, copper may accumulate in liver
secondary to bile stasis following liver damage
BT
10000
Cu
In
liver
Dalmation
7500
Doberman
5000
Skye Terrier
3000
WHWT
Cirrhosis
2000
1000
500
250
0
Copper accumulation in sheep
• Sheep on inbalanced diets can store
excessive copper in liver
– Sudden release is often seen
• Copper damages hemoglobin
– Free radical formation?
• RBC’s lyse
– Lost of deformability
– Heinz body formation
Metallothionien: Intracellular regulation of
divalent cations
• Metallothionien
– Has 20 cysteine residues
• SH groups can bind metals
– Capable of binding (or releasing)
• 12 copper ions
• 7 zinc ions
• 20 nitric oxide ions
– MT binds physiological (Zn, Cu, Fe, Se) and xenobiotic (Cd, Hg,
Ag) metals
• Zinc
– Vital for activity of some enzymes
– Vital for inactivation of some enzymes
– Vital for activation of binding of some DNA transcription factors
47
Metallothionein regulation by zinc and anti-oxidative activity
Cytokines
Glucocorticoid
GRE
STAT
MRE
Methylation
H2O2
Electrophiles
ARE
:Up regulation
:Down regulation
Structural gene
MTF-1
Zn pool
Apo MT
Apo-MT
Zinc Finger Protein
Inactive Protein
Zn7-MT
Apo-MT
Oxidative Stress
Degeneration
Overview of metallothionein (MT) gene regulation
The MT promoter has many elements that up-regulate transcription. These include the following: 1)metal
response elements (MRE), which are activated by the metal-responsive transcription factor (MTF-1) after zinc
occupancy; 2) glucocorticoid response elements (GRE); 3) elements activated by STAT (signal transducers
and activators of transcription) proteins through cytokine signaling, and 4) the antioxidant (or electrophile)
response elements (ARE) activated in response to redox status. Davis and Cousins, 2000.
Inherited copper deficiency (Menke’s)
• Fatal neurodegenerative disease in
infants
Bilirubin toxicity
• Kernicterus and dental enamel hypoplasia
– Unconjugated bilirubin can enter the brain and
cause toxicity, especially in infants (>20mg/dL)
• Blood-brain barrier is less developed in neonates
• Seen with erythroblastosis fetalis
• Other causes of elevated unconjugated bilirubin:
– Hemolytic anemias, resorption of blood from internal
hemorrhage, ineffective erythropoiesis (pernicious anemia,
thalassemia), redduced hepatic uptake (Gilbert syndrome),
Impaired conjugation (juandice of newborn (reduced UGT1A1
enzyme activity)), breast milk juandice, Crigler-Najjar syndromes
I and II, viral hepatitis, drugs, cirrhosis
– Brain enlarged, edematous, yellow
• Cerebral palsy, hearing loss, gaze
– Neurons and odontoblasts affected
48
Bilirubin damage to cells
•
•
•
•
Unconjugated bilirubin (UCB)
– Formed by catabolism of heme by splenic macrophages
– not water soluble
• Therefore, transported by albumin
– UCB is conjugated with glucuronide by hepatocytes
• Increases water solubility
• Most other cells lack the ability to conjugate bilirubin
– Hyperbilirubinemia in infants can occur with:
• Inherited deficiencies of glucuronidation enzymes
• Delayed development of glucuronidation enzymes
– Premature
P
t
birth
bi th
• “Bili lights” UV light exposure in infants with elevated bilirubin levels
– Bilirubin levels in serum increase with red blood cell lysis, bile stasis
UCB readily enters cells
UCB is antioxidant at modest elevations
– Absorbs electrons
UCB can induce injury at high levels
Bilirubin damage
• Concentrates in
– Plasma membrane, mitochondria and ER
membranes
• How? What it does? Not understood
• Neuronal toxicity, excitoxicity (glutamate
release and NMDA activation), decreased
mitochondrial ATP production, increased
Ca2+ concentration
• Ca2+ activation of phospholipases, proteolytic
enzymes, apoptosis/necrosis
Bilirubin neurotoxicity
Ostrow JD, et al Trends in Mol Med 10 (2):65-69, 2004
49
Regulation of UCB levels in CNS tissue
• UCB transportor across the blood brain barrier and choroid
plexus
• Protective
– Organic anion transport proteins (OATP)
• Transports UCB two directions
– Into cells and into blood
– Multidrug resistance receptors (p-glycoprotein)
(p glycoprotein)
• Transports UCB one direction
– Into blood in BBB
– Into blood and into CSF in choriod plexus
» Could increased CSF UCB transport be detrimental?
• These pumps likely explain why UCB accumulates in specific
regions of brain
– E.g. UCB remains in those regions lacking active pumps
– CSF accumulation?
• May allow increased levels around CSF
UCB transporters
Ostrow JD, et al Trends in Mol Med 10 (2):65-69, 2004
Porphyria
• Porphyrias
– A group of seven inherited disorders of
heme biosynthesis
y
• Greek derivation (purple)
• Enzyme deficiencies in heme synthesis occur
for each disease
• Porphyrins
– Heme intermediates
– Often toxic to certain tissues
50
History of Porphyria
1889, B.J. Stokovis, MD – first clinical
description of acute porphyria
1930, Hans Fischer, Nobel laureate,
described heme as the agent that
makes blood red
1960’s – all types have been identified
1980’s-1990’s – identification of the
molecular defects (gene mutations)
Heme Biology
Heme is an essential component of
proteins and enzymes:
hemoglobin myoglobin
hemoglobin,
myoglobin, catalase
catalase,
peroxidase, nitric oxide synthetase,
and cytochromes
Bone marrow & liver are the main tissue
sites for heme biosynthesis
Porphrins are directly toxic or
photodynamic
•
Not all porphyrias are genetic
•
Attacks of the disease can be triggered by drugs (e.g., barbiturates, alcohol, sulfa
drugs, oral contraceptives, antibiotics), other chemicals and certain foods. Fasting can
also trigger attacks.
•
They are broadly classified according to development as hepatic porphyrias or
erythropoietic porphyrias, based on the site of the overproduction and mainly
accumulation of the porphyrins (or their chemical precursors). Clinically, disease is
classified as:
–
–
–
–
–
•
•
•
Occur in patients with liver disease.
In fact, acute intermitent p
porphyria
p y ((AIP)) is the most common and severe form but 90% of
patients do not develop disease unless triggered as above
skin problems (cutaneous)
neurological complications (neurovisceral)
or occasionally both, (mixed).
Porphyrins are precursors of heme and heme is essential for hemoglobin, myoglobin,
catalase, peroxidase, cytochrome P450 (liver).
The principal problem in these deficiencies is the accumulation of porphyrins, the heme
precursors, which are toxic to tissue in high concentrations.
The chemical properties of these intermediates determine in which tissue they
accumulate, whether they are photosensitive, directly toxic and how the compound is
excreted (in the urine or feces).
51
Enzymes key to heme synthesis
Enzyme defect and the type of porphyria
Enzyme
Location of
enzyme
Associated porphyria
Type of
porphyria
δ-aminolevulinate (ALA) synthase
δ-aminolevulinate (ALA) dehydratase
Mitochondrium
X-linked sideroblastic anemia
(XLSA)
Erythropoietic
Cytosol
Doss porphyria/ALA dehydratase
deficiency
Hepatic
hydroxymethylbilane (HMB) synthase (or PBG
deaminase)
Cytosol
acute intermittent porphyria (AIP)
Hepatic
uroporphyrinogen (URO) synthase
Cytosol
Congenital erythropoietic
porphyria (CEP)
Erythropoeitic
uroporphyrinogen (URO) decarboxylase
Cytosol
Porphyria cutanea tarda (PCT)
Hepatic
coproporphyrinogen (COPRO) oxidase
Mitochondrium
Hereditary coproporphyria (HCP)
Hepatic
protoporphyrinogen (PROTO) oxidase
Mitochondrium
Variegate porphyria (VP)
Mixed
Ferrochelatase
Mitochondrium
Erythropoietic protoporphyria
(EPP)
Erythropoietic
Heme Biosynthesis & Porphrias
3 clinical
categories of symptoms
-Neurovisceral
-Cutaneous
-Combined
Inheritance patterns
-All autosomal
-Most dominant, but I
incomplete penetrance
(asymptomatic carriers)
52
Neurovisceral Porphyrias
General symptoms
include:
abdominal pain
dark urine
vomiting
muscle weakness
mental deterioration
hypertension
Acute Intermittent
Porphyria
ALAD (aminolaevulinate dehydratase
deficiency) Porphyria –
Very rare (6 reported cases)
Autosomal recessive, but heterozygotes
are highly susceptible to lead poisoning
AIP (Acute Intermittent Porphyria)
Most common & severe form (1 per 10,000)
Autosomal dominant – 120 known mutations
90% never develop clinical signs (unless
precipitated by external factors, such as
drug exposure – ethanol, oral
contraceptives, sulfonamides, etc.)
More prevalent in females, usually expressed
after puberty
Increased risk of hepatocellular carcinoma
53
Cutaneous Porphyrias
General symptoms include:
Fragile skin – prone to blistering, scarring,
and infections
caused by circulating porphyrins that when
expose to light reach an excited state,
becomes a free radical and releases energy
in chemical reactions – peroxidation of cell
membranes
Increased body hair growth
Hyperpigmentation
Cutaneous Porphyrias--Combined
Congenital
Erythropoietic
Porphyria
Variegate porphyria (no photo)
Porphyria Cutanea Tarda
Erythropoietic porphyria (no photo)
Hereditary Coproporphyria
Cutaneous Porphyrias
CEP (Congenital Erythropoietic Porphyria)
Also known as Gunther disease
Rare autosomal recessive (200 cases)
B i fform identical
Bovine
id ti l tto CEP
VP (Variegate Porphyria)
Autosomal dominant
Most common form in S. Africa
EPP (Erythropoietic Porphyria)
Autosomal dominant
Less severe skin photosensitivity – swelling, itching,
and redness
54
Combined Porphyrias
Manifest both nerovisceral and cutaneous symptoms
PCT (Porphyria Cutanea Tarda)
Autosomal dominant ((1 per
p 25,000)
,
)
Acquired form – Turkey, late 1950’s, 4,000 developed
PCT due to contaminated wheat
HCP (Hereditary Coproporphyria)
Autosomal dominant
Increased risk of liver cancer
Treatment
Mainly preventative –
Avoid sun exposure & chemicals or medications that
exacerbate the condition
IV injection of Hematin – (heme alternative) decreases
the heme synthesis pathway by feedback inhibition
Bone marrow transplant – for erythropoietic forms
show promise in reversing enzyme defects
Historical Figures
King George III of England
(ruled 1760 – 1811)
infamous for episodes
of “madness” w/
abdominal
bd i l pain
i , lilimb
b
weakness, and
darkened urine
Vincent van Gogh –
chemicals present in
artists’ pigments and
solvents induced a form
of the disease
Variegate
Porphyria
Or
Acute Intermittent
Porphyria
Acute Intermittent
Porphyria
55
Myth & Folklore
Symptoms often seen in
afflicted individuals such as:
photosensitivity, skin sores
and scars
scars, excessive hair
hair,
discolored skin, and desire
for blood
Possible explanation for myths
about vampires and
werewolves
Congenital
Erythropoietic
Porphyria
Effects of lipids and sugars on cellular
degeneration
Ackermann
VPTH 655
Factors that mobilize fat
• Intense lactation
• Pregnancy toxemia
• cattle, sheep, guinea pigs
•
•
•
•
•
•
•
•
•
Hyperlipidemia, ponies; hepatic lipidosis, cats
Starvation
Diabetes, hypothyroidism, Hyperadrenalcortisim
Alcoholism
Nephrotic syndrome
Choline deficiency
Anemia
Fatal fasting syndrome, old world primates
Hyperlipidemia diseases (more later); Schnauzers
56
Fatty liver of pregnancy
Cellular energy: required for ATP
Glucose/glycogen
Proteins
Pyruvate
Fatty acid
Fatty Acyl Co A
ACETYL CO A
KREBS (produces NADH, FADH2 for Oxidative phosphorylation)
OXIDATIVE PHOSPHORYLATION
+ NADH
+02
=ATP
Mitochondria
ATP for:
ATPase pumps, signaling, etc.
A transporter is required for:
Fatty Acyl Co A, pyruvate and ATP
Cell plasma membrane
Fats and cholesterol
• Are in the blood (as insoluble fatty forms) at
low amounts:
• Triglycerides, free fatty acids, glycerol (draw glycerol,
mono di,
mono,
di and triglyceride)
• Cholesterol, cholesterol esters
• Phospholipids (draw; phosphatidylcholine)
• Vitamins A, D, E, and K
• Also transported as lipoproteins (LDL, VLDL,
etc.) that contain apolipoproteins (Apo E, B,
etc.)
57
Lipid transport in blood
• Lipoproteins
•
•
•
•
Chylomicron, LDL, VLDL, HDL, IDL
Center is lipid part
Surface is protein part
Classified by molecular size, electrophoretic mobility, and
densities
• Density:
– Chylomicron (least dense), then VLDL, LDL, HDL (most
dense)
– Decreases with triglcyerides
– Increases with protein and phospholipid content
» Chylomicron has 83% triglyceride/2% protein
» HDL has 8% triglyceride/33% protein
Apolipoproteins
• Bind lipids and become lipoproteins. Function in LDL’s,
VLDL’s, chylmicrons, HDL’s, IDL’s.
– If they are free of lipid, then called apoplipproteins
• Serve as ligands for receptors
• Apolipoprotein A
– Major amount on: HDL’s
– Apo A-I, A-II, A-IV, A-V
• Apolipoproteins B
– Major amounts on: chylomicrons, VLDL and LDL’s
– Apo B 48, B100
• Apolipoprotein C
– Major amounts on: chylomicrons, VLDL, HDL
– Apo C-I, C-II, C-III, C-IV
• Apolipoprotein E
– Major amounts on: VLDL and HDL, some on chylomicron
– Three forms differ by one amino acid
» E3 is the common form
» E2 is associated with high triglycerides/cholesterol
» E4 is associated with high cholesterol
B-48
E, C transfer
Apo E, A, C
HDL3
LCAT
HDL2
Lipoprotein
lipase
(Triglyceride release)
Cholesterol
75% of LDL
Other tissues
25%
Macrophage R:
LDL R
SR-A
CD36
1%
58
•
•
•
•
•
•
•
Lipoproteins
Chylomicron
– B 48, picks up E and C
VLDL synthesized in hepatocyte
• B 100, E and C
• Dog also has B48
Nascent VLDL receives Apo E and C2 from HDL
• This mature VLDL transports lipids
LDL
• B 100
• B 100 B 48 in dogs
HDL1
• E, A, C
Note: HDL 1, 2, 3 are
• Dogs only; no HDL1 in man
ultracentrifuge products
Alpha HDLs (discussed later)
HDL2
are electrophoretic HDLs and
• E, A, C
probably more precise
• Dogs and man
HDL3
• E, A, C
• Dogs and man
Loss of Apoprotein C III
• Protective against cardiovascular
disease in Older Order Amish
• Science 322: 1702: 2008
Lipoprotein and hepatic lipase
• Lipoprotein lipase
• Tissue capillaries
• Activated by Apo C2
• Hydrolyzes triglycerides from VLDL
• Hepatic lipase
• Removes triglycerides in liver
59
Contents of LDL’s
•
•
•
•
•
Apolipoproteins and cholesterol
Phosphatidylcholine (Phospholipid)
Lysophophatidylcholine
Sphingomyelin
Fatty acids
• Linoleic acid, palmitic acid, stearic acid, free
fatty acids, triglycerides and trans fatty acids
Oxidation/acetylation of LDL’s
– Increases scavenger uptake by foam cells
• 3-10 fold increase
• Non-oxidized/acetylated (native) LDL’s are taken up by LDLR
on hepatocyte and macrophage
» Macrophage uptake of native LDL is slow and downregulated
– LDL’s “trapped” by proteoglycans
– Lipoperoxidation of phospholipids
phospholipids, fatty acids
acids, and Apo
B
• Occurs by lipoxygenases, MPO, iNOS, ROS
– Self propagation of the reaction
– Decomposition to of fats to aldehydes, ketones
– Apo B
• Lysine residue oxidized
• This inhibits binding of ApoB to LDLR
Repair of lipoperoxidation of phospholipids:
Vitamin E and C
PL
Fatty acyl CoA
TOC-OH
Lipoperoxidation
O-ASC-OH
PL-OO
HO-ASC-OH
O=ASC=O
lysophospholipid
PL-OOH
2GR SS
2GR-SS
2GSH
GR-[SH2]
GSSG
TOC-O
GRO-SS
FA-OOH
GPX
FA-OH
2GSH
GSSG
Phospholipid membrane
2GR-SS
GR-[SH2]
NADPH+ + H+
PL phospholipid
PL-OO
phospholipid that is oxidized (lipoperoxidation)
TOC alpha tocopherol, Vitamin E
ASC ascorbic acid, Vitamin C
GRO-[SH2]
NADP+
GRO glutaredoxin
GSH glutathione (GSSG [oxidized])
GR glutathione reductase
GPX glutathione peroxidase
60
Foam cell formation
• Acetylated LDL’s
• Rapidly taken up by macrophage LDLR (SRA-scavenger
receptor A)
– This receptor not down regulated by the cell
• Oxidized LDL’s
• Rapidly taken up by AGE receptors LDLR (SRA), CD36
and
d other
th receptors
t
– These receptors not down regulated by the cell
• AGE receptors take up oxLDL’s and AGE
• AGE (Advanced glycosylation endproducts)
» RAGE (receptor for AGE), 80 K-H, OST-48, galectin-3,
macrophage scavenger receptors type I and II (SR-A)
» CD36 is a class B scavenger receptor, but like SR-A,
takes up oxidized LDL’s and AGE
Foam cell
Endothelial cells, smooth muscle
cells, or macrophages, (or Cu++)
Acetyl LDL
Native LDL
Fast
ox LDL
slow
Fast
Native LDL R
(down regulated)
Acetylated LDL R (SR-A)
CD36, SR-A and
(not down regulated )
Other ox LDL receptors
(not down regulated)
Steinberg: Nature medicine 8:1214, 2002
Fatty acid-binding proteins (FABP)
• FABP bind fatty acids in the cell and act as
chaperones for fatty acid transport
– FABP are especially important in adipocytes and
macrophages
• Historically, FABP may have facilitied fatty acid
storage to use later in lean times (starvation)
– In adipocytes for release systemically
– In macrophages for energy of the immune system
61
FABP
• Mice with FABP KO
– Have no phenotypic change
– Have increased plasma triglyceride levels
– do not develop diabetes, atherosclerosis, etc. on
high
g fat diets
– Liver fat favored metabolism rather than storage
– Macrophages have decreased, cytokine responses,
COX2 and iNOS activity and reduced foam cell
formation
• Synthetic inhibitors of FABP-A have been
developed
Furuhashi: Nat Rev Dr Disc 7:489, 2008
FABP activity in cells
• FABP trafficking
– To rER, lipid vacuoles, mitochondria, peroxisome,
ER, cytosol, and nucleus
• Additional FABP activity in adipocytes
– Interacts with hormone-sensitive lipase (release of
fatty acids)
– May increase PPAR, and affect JNK/IKK signaling
downstream of insulin, IGF, TNF, TLR, ER function
• Additional FABP activity in macrophages
– Inhibit PPAR and liver X receptor responses
(later), regulate cholesterol efflux, regulate
JNK/IKK
62
Foam cells fight back: Mechanisms to
eliminate fat
• OxLDL induce oxysterols in the
cytoplasm
• This activates transcription of
»
»
»
»
»
»
»
A E ffor HDLs
ApoE
HDL
ABCA1- for cholesterol channel
Fatty acid synthetase, for forming fats
Fatty acids are made into free cholesterol
Cholesterol bound to ApoA1
Cholesterol/ApoA1 goes to HDL
Increase cholesterol outflow increases removal
of esterified cholesterols also
63
Cellular release of free cholesterol
Ox LDL
Apo E
-carries FC out
FC
Oxysterols
CE
lysosome
LXR RXR
Apo E
ABCA1
SREBP1c
FAS
LXR RXR RE’s
ABCA1
-transports FC
FC
FFA
HDL
Apo A
-picks up FC
CE
Lipid droplet
LXR = liver X receptor
RXR = Retinoic acid receptor
LIVER
CE = cholesterol ester
FC = free cholesterol, ABCA1 = transporter, FAS = fatty acid synthesis, FFA = free fatty acid
Joy Nat Rev Drug Disc 7:143, 2008
Reverse cholesterol transport (RCT)
– APOA1 from liver binds PL, pre-beta-1 HDL formed
• Pre-beta-1 HDL binds free cholesterol (FC)
• Foam cells release FC via ABCA1 channel
– Alpha 4 HDL formed
– LCAT enzyme esterifies FC; CE move to core
• Alpha 4 HDL also combines with recycled PL, FC, APOA
– Alpha 2 and alpha 3 HDLs formed
• Interact with triglyceride-rich lipoproteins
– Transfer of cholesterol esters from HDL to LDL by CETP
– Alpha-1 HDL and pre-alpha-1 HDL formed
• Hepatic lipase, endothelial lipase and sPLA2 release PL
– Alpha-2 HDL formed, releases APOA1
• Hepatic SCRB1 receptor picks up CE (cholesterol esters)
64
Inhibiting CETP to increase HDLs
• Seems like a good idea, but not fully effective
• Torcetrapib, CETP inhibitor
– Increased HDL in clinical trials but did not
improve clinical endpoints in patients with
established atherosclerosis; increased mortality
New therapies to increase HDLs
• New therapies:
– Niacin (increases HDL, reduces TG and Cholesterol
• Skin flushing/vasodilation
– Fibric acid (stimulates PPAR alpha)
– reconstituted HDL
– Phospholipid liposomes
• Promote cholesterol efflux
– APOA1 peptide (D4F)
– APOA1 Milano recombinant protein
– Diet and exercise
Proatherogenic activity of oxidized LDL’s
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Increase foam cells (increased scavenger uptake)
Products are chemotactic to monocytes, T cells
Cytotoxic, apoptosis
Mitogenic for smooth muscle cells
Increase scavenger receptor expression
Increase PPAR actvity (gene fx, apoptosis)
I
Immunogenic;
i autoantibody
t
tib d and
d T cellll activation
ti ti
Increases LDL aggregation
Substrate of spingomyelinase; aggregates LDL’s
Procoagulant, increases tissue factor, platelet activating factor
Increased monocyte CSF, MCF, IL-1, IL-8, chemokine
receptors,NO
Increased calcium and Nfkappa B
Increased expression of matrix metalloproteinases
Increased adhesion molecule expression
Reduce vasomotor activity and vascular contractility
65
Trans fatty acids
• A double bond between carbons in a fatty acid that changes the
conformation from cis to trans
– Oleic and elaidic acid are 18 carbon
• Oleic is cis; elaidic is trans
• Trans fatty acids
– IIncrease LDLs,
LDL d
decrease HDL
HDLs, iincrease cholesterol:HDL
h l t l HDL ratio
ti and
d
increase blood triglyceride levels
– Increase B100 size, secretion, lipid content
– Decrease LDLapoB100 catabolism (increasing LDLs)
– Increase cholestrol ester transfer from HDLs to LDLs
• Thus decreasing HDL removal of cholesterol
– Increase ICAM-1, VCAM-1 and E selectin on endothelial cells
• Pro-inflammatory promoting atherosclerosis
Atheroma formation
• Diagram
• Stages:
–
–
–
–
–
–
–
–
–
Fatty streaks in children
Isolated foam cells in adults
Multiple foam cells
Extracellular lipid accumulation
Confluent lipid core
Fibrous cap with smc proliferation
Surface ulcer, thrombus
Calcification
Fibrosis
Can regress
Ab
Above
thi
this liline
66
Factors leading to atherosclerosis
• Cigarette smoking
• Two fold increase in atherosclerosis
• Hypertension
• Higher incidence blacks; 3% of Americans
Americans, 75% of those
over 75 years of age
• Diabetes mellitus
• 14 million americans
• Serum cholesterol levels
• Familial hypercholesterolemia
– Increased cholesterol, types on next page
• 50% of Americans (24-70 years) have >200 cholesterol
levels
67
LDL Receptor
• LDL R family
• Apo E R2, VLDL R, megalin, LDLR, LDL
receptor-related protein
• Genetic mutation can reduce expression
– Multiple types of mutations identified in man
• Results in increased circulating LDL’s,
hypercholesterolemia and foam cell formation
Genetic Causes of Hyperlipoproteinemias
• I increased chylomicron
• IIa increased LDL*
• IIIa increases LDL/VLDL*
• III increased chylomicrons
• IV increased VLDL*
• V increased VLDL/chylo
*10, 40, 45% respectively
• Others
mutations in lp lipase
mutation of LDL or Apo B
mutation of LDL or Apo B
mutation of Apo E
mutation of lp lipase
mut. Lpl or C2
• Decreased HDL; mutation in Apo AI
• Tangier’s dz, mutation in cholesterol transport
• Loss of HMG-CoA reductase
Schnauzers, Cats, Cows, Ewes
• Schnauzers develop hyperlipidemia
• Cats, cows and ewes, develop fatty
livers
– Cats, L-Carnitine defect?
– Impaired ability to carry fats into the mitochondria
and out of the hepatocyte
• Cows and ewes
– Often related to
pregnancy/parturition/lactation
68
Lipid disorders in dogs and cats
Dogs
Primary disorder
Idiopathic hyperlipidemia
hypercholesterolemia
Secondary disorder
high fat diet
pancreatitis
hypothyroidism
yp y
cholestasis
hyperadrenocorticism
nephrotic syndrome
Cats
Primary disorders
inherited
hyerchylomicronemia
primary
hypercholesterolemia
Increased TG, cholesterol pancreatitis
Increased cholesterol
retinal degen
increased cholesterol
increased TG
increased TG, cholesterol
increased TG, cholesterol
pancreatitis?
pancreatitis
atheroma
insulin resistance
increased TG, cholesterol Decreased LPL
xanthomas
TG = triglycerides
Bauer JE: JAVMA 224:668-675, 2004
Appetite regulation
• Arcuate nucleus of the hypothalamus, two parts:
– 1. AgRP/NPY—increases appetite and metabolism
» Activated by: Ghrelin R (GhR) (Ghrelin; stomach),
Agouti-related peptide (AgRP), NPY
» Inhibited by: PYY, YY (gut), leptin (adipose), insulin,
amylin, pancreatic polypeptide (pancreas)
– 2. POMC/CART—decreases appetite and metabolism
» Activated by leptin (adipose), insulin (pancreas), cocaineamphetamine pro-opiomelanocortin
amphetamine,
pro opiomelanocortin POMC
• Nucleus tratus solitarius (NTS), medulla
• Receives second order neurons from the arcuate nucleus
– Also regulated directly by cholecystikinin (liver) and vagus
nerve (for sensing stomach stretching)
– Sends signals to the rest of the body
• The hypothalamus arcuate nucleus and NTS
regulate feed centers and behavior in the
hypothalamus nuclei: ventromedial,
dorsomedial, and lateral
Leptin
• Leptin will decrease appetite in those
with mutant leptin, but not normal
individuals
• In other words, excess leptin doesn’t decrease
appetite in an average person
• Leptin protects against weight loss/starvation
– There is decreased leptin with decreased fat,
therefore increased eating
– As above, excessive leptin doesn’t decrease intake
69
A few obesity targets
•
Inhibiting hunger
–
–
–
Cannibas receptor inhibitor (preventing hunger “munchies”)
PYY peptide (decreasing appetite)
SlimFast (bulky fiber? poorly degraded?)
•
Preventing food absorption
•
Delay gastric emptying
•
Enhance fat burning
–
–
–
–
•
Amylin
Human growth hormone, amphetamines
Degradation of fats
–
•
Alizyme, prevents fat absorption in gut
Olestra-WOW! Chips, a fat not absorbed
Lipases
Altering fat metabolism
–
Acetyl Co-Enzyme A carboxylase-induces fatty acid synthesis
•
•
•
TRB3 induces degradation of ACC thus decreasing synthesis
Nutrisystem, Nurrisystem for men (dan marino), Jenny Craig, Atkins Diet,
Scarsdale diet, Grapefruit diet, Slimfast, Trimspa, Houdia, LA weight loss,
Inches Away, weight watchers,
Exercise
PPAR
• PPAR alpha
– Fatty acid oxidation in liver (heart, muscle, kidney artery)
• Tissues with high fatty acid oxidation
• PPAR gamma
– Lipid storage in liver and adipose tissue
– Activated by TZD (Thiazolidinedione), an anti-diabetic drug
• Increases skeletal muscle and liver sensitivity to insulin via activation of
PPAR gamma
• Also reduces inflammation
• Weight gain side effect and sodium retention
– Widespread and present in many tissues
• PPAR beta/delta
– Fatty acid oxidation and energy decoupling in adipocytes
• Only weakly activated
PPAR
70
Cholesterol synthesis
• Cholesterol
• 27 carbons, synthesized in liver from acetyl CoA
• Acyl transferase mediates acetyl CoA formation
• Three acetyl CoA’s join to make HMG CoA
– HMG-CoA
» Can make acetoacetate and ketone bodies
» Can make nevalonate to form cholesterol
• High cholesterol levels in hepatocyte
– HMG CoA reductase is inhibited and ketones form
– No LDL receptors are made (to decrease hepatocellular
uptake of LDL and thereby reduce cholesterol intake)
• Low cholesterol levels in hepatocyte
– HMG-CoA reductase makes nevalonate
– Increased LDL receptor to increase uptake of cholesterol
• Cholesterol forms:
– Bile salts
– Steroid hormones
Steinberg 359:1426, 2008
71
Drugs that lower cholesterol
• Cholestyramia
• Bile acid exchange resin. Binds cholesterol.
• Lovastatin
• Inhibits HMG-CoA reductase. Prevents cholesterol
formation
• Bezafibrate
• Stimulates lipoprotein lipase. Triggers cholesterol
degradation.
• Probucol
• Lowers triglycerides, mechanism uncertain
Utilization of energy
• Brown fat
• Fidgeting
– Levine JA: Nonexercise activity thermogenesis (NEAT): environment
and biology. Am J Physiol Endocrinol Metab. 2004 May;286(5):E67585:
• Nonexercise activity thermogenesis (NEAT) is the energy
expended
d d ffor everything
thi th
thatt iis nott sleeping,
l
i
eating,
ti
or sports-like
t lik
exercise. It includes the energy expended walking to work, typing,
performing yard work, undertaking agricultural tasks, and fidgeting.
• The variability in NEAT might be viewed as random, but human
and animal data contradict this. It appears that changes in NEAT
subtly accompany experimentally induced changes in energy
balance and are important in the physiology of weight change.
Inadequate modulation of NEAT plus a sedentary lifestyle may thus
be important in obesity. It then becomes intriguing to dissect
mechanistic studies that delineate how NEAT is regulated into
neural, peripheral, and humoral factors.
• Exercise
White fat progenitor cells
• White fat adipocyte progenitor cells form in
pre- and peri-natal timeperoids
– Continues through adulthood
• The proliferative and resting (stem) pool of
these cells are present in the wall of blood
vessels of white adipose tissue
– These can proliferate to make fat
Science 322:583, 2008
72
Brown fat utilization and thermogenesis
• Cold increases
– PGC1 (a powerful transcriptional coactivator) for PPAR
gamma (peroxisome proliferator activator receptor)
– TR (thyroid hormone receptor)
– RAR (retinoic acid receptor)
– ER (estrogen receptor)
• PGC1
PGC1, TR
TR, RAR and
d ER activate
ti t UCP ((uncoupling
li proteins)
t i ) and
d
genes of mitochondrial respiratory chain for ATP synthesis
(ATPase and cytochrome oxidase C)
• The UCP (uncoupling proteins) are in the inner mitochondrial
membrane along with F0/F1 ATPase.
• The UCP allow H+ passage with loss of gradient resulting in heat
with reduced ATP.
– PGC1 also activates NRF 1and 2 (nuclear respiratory
factors) for mitochondrial biosynthesis and increases
conversion of type I to type II muscle fibers
• Increased mitochondria for more heat
Glycogen accumulation
• Hepatic accumulation with diabetes mellitus
• Glycogenosis
– Acccumulation of glycogen with enzyme
deficiencies
• Acid maltase deficiency (cattle)
Hyperglycosylation and hyperglycemic
vasculopathy
• Hyperglycemic vasculopathy in diabetes mellitus
– Persistent levels of blood glucose leads to glycosylation
of vessels
– Glucose
Gl
attaches
tt h to
t amino
i groups off proteins
t i
• Schiff base is formed and is reversible
• Amadori complex
– A more stable adherence of glucose to the protein
• Advanced glycosylation end products (AGEs)
– Irreversible glycosylation
73
Mediation of glycosylation damage
• Glycosylation of LDL’s, hemoglobin, albumin,
extracellular matrix proteins, and basement
membrane (thickens)
• AGEs bind RAGE receptors on macrophages
– Induction of IL-1, TNF, inflammation
• Glycosylation of amino groups in DNA
nucleotides
– Altered transcription and increased strand breaks
Intracellular glucose damage
• Occurs in tissues that do not require insulin for
glucose entry (in diabetics)
– Lens, neurons, blood vessels
• Converted to sorbitol by aldose reductase and then
sorbitol to fructose by sorbitol dehydrogenase
• Sorbitol and fructose induce oncotic pressue, influx of
water and swelling
– Sorbitol also reduces ATPase pumps in schwann cells (of
nerve fibers) and retinal capillary endothelial cells
– Also, increased protein kinase C and diacylglycerol can
occur
Equine polysaccharide storage myopathy
• Quarter horses, paints, draughts, and 50 other
breeds
• Accumulation of glycogen in muscle type II
fibers
– Accumulates with ubiquitin
ubiquitin, amylopectin
amylopectin,
polyglucosan)
– Muscle fiber size variation, muscle atrophy,
PAS stained glycogen
in skeletal muscle
Leman Neuromusc dis 4:277, 2008
74
Size variation
Normal
PAS + fibers with altered
myofiber direction at edges
Valentine JVDI 20:572, 2008
Equine glycogen branching enzyme
deficiency (glycogenosis type IV)
• Quarter and Paint horses
– Missense mutation of glycogen
debranching
g enzyme
y
1 ((GBE1))
– Premature stop codon
– Cannot store and mobilize glycogen
– Fatal in foals
Leman Neuromusc dis 4:277, 2008
Protein aggregates and conformational
disease
75
Ubiquitin degradation of proteins damaged beyond
repair
Ubiquitin
Ubiquitin
Ubiquitin
Damaged protein
E1-ubquitin activating enzyme
Deubiquitizing enzyme (DUBs)
E2
ubquitin conjugating enzyme
E2-ubquitin
E3-ubquitin ligase
Ubiquitin
Ubiquitin
Ubiquitin
H
-C-N-Lysine-damaged protein
O
26S proteosome and ATP:
Degradation of damaged protein
Nalepa et al:
Nat Rev
Drug Disc
5, 596-613
Ubiquitin-proteasome system (UPS)
a. Ubiquitin activation
– E1 (ubiquitin-activating enzyme) adds phosphorus from
ATP
b. Building ligase-substrate complex
– E2 (ubiquitin-conjugating enzyme) replaces E1
– E2 bi
binds
d E3 ((ubiquin
bi i liligase)) and
dS
Substrate
b t t protein
t i
c. Ubiquitin transfer to lysine of substrate protein and formation
of polyubiquitin chain
d. Release of polyubiquitylated substrate from E3 by
deubiquitylating enzymes (DUBs)
e. Proteasome unfolds substrate with ATP and ubiquitins
released
76
Protein facts
• Nearly all proteins except 13 coded by
mitochondrial DNA are nuclear-encoded and
translated in the cytosol
• Newly translated unfolded proteins have
hydrophobic ends that can aggregate
Protein facts
• Chaperone proteins
– Functional class of unrelated proteins
– Assist in non-covalent assembly of proteins
– Not a component of the final protein complex
– Bind hydrophobic regions of proteins and this
requires ATP
• Except for lectin-chaperones
– These bind carbohydrate units
Protein facts
• Ubiquitization
– Cytosol and mitochondria have systems for protein
degradation
g
– ER
• Misfolded proteins go to the Golgi and then are
retrogradely translocated to the cytosol
– Peroxisome and nucleus
• These proteins are handled in the cytosol if misfolded
77
Protein facts
• “Normal” proteins should be flexible
– If not flexible they can aggregate to beta sheets
– Beta sheet aggregations is exacerbated by
• pH, temperature, high salt and macromolecular crowding
Gregersen N, J Inherit Metab Dis 29:456, 2006
Protein facts
• Abberent proteins that incorrectly fold can
result from:
– Transcription/translation/post-translational
Transcription/translation/post translational
modification errors
• Post-translational modification include
– Acetylation, glycosylation, ubiquitylation, nitration,
phosphorylation, farnesylation
– Amino acid substitutions
– Oxidative damage
Protein aggregates increase with age
– People are living beyond 30 years of age
– With time, proteins can aggregate
•
Theories for protein aggregation with age:
– Mutation in protein
– Beta sheet structure, undegradable
– Misfolded and lacks enzymatic
y
site
– Abberant endoproteolytic cleavage
– Example: altered presenilin site in gamma secretase of AD
– Translation of protein can exceed foldase activity
– Foldases make cis/trans isomeres and SH bonds
– Reduced foldase and other substrates available
– Defective ubiquitin
– Deletions in ubiquitin results in loss of C-terminal glycine of the ubiquitin protein and loss of activity
» Loss of proteosome degradation and accumulation of proteins
78
Accumulation of proteins in the rER
• rER stress induced by:
– Reduced redox (reduces disulfide bond
formation in the rER)
– Reduced glucose (reduces N-linked protein
glycosylation)
– Reduced calcium (reduced protein folding)
– Viral infections (overloads ER with proteins)
– High fat diet (liver)
Unfolded protein response (UPR) of the rER
• Induces foldases
• Increases HSP expression
• Initiation of ER-assisted degradation (ERAD) of proteins
– Reduced RNA translation
– Retrograde translocation of unfolded proteins to the cytosol for
ubiquitin degradation
• Foldase activity, HSP expression and ERAD are activated
by transmembrane proteins in the rER that sense protein
accumlation
– PERK (PRKR-like ER kinase), IRE1 (inositol-requiring kinase 1),
ATF6 (activating transcription factor 6)
Kim et al Nat Rev Drug Disc 7:1013-1023, 2008
Transmembrane proteins mediating UPR
79
Transmembrane proteins mediating UPR
Misfolded proteins cause cell damage by:
• Unfolded proteins in the rER
– Can lead to apoptosis or autophagy
• Inhibition of ubiquitylation
– Proteins cannot be degraded and aggregate
• Chaperone sequestration
– Huntingtin protein, Lewy bodies (alpha synuclein) and beta
y
amyloid
• Bind chaperone proteins and make aggregates
• Transcription factor sequestration
– Huntingtin protein has polyglutamine groups
• bind transcription factors (TATA binding protein) and CREB binding
protein (CBP)
• Mitochondrial dysfunction and oxidative stress
• Pore formation
– soluble oligomeric aggregates for pores
• E.g., pores are formed by alpha synuclein and beta amyloid
• Calcium and glutamate levels can change
Kim et al Nat Rev
Drug Disc 7:10131023, 2008
80
Types of diseases with protein aggregates
•
•
•
•
Lead
Viral inclusion bodies
Hyaline droplets (rat kidney)
Intermediate filament accumulations
•
•
•
•
•
Tauopathies
Prions
Alpha synuclein
Neuroserpin
Glutamine repeats
– Inherited mutations, Mallory bodies with alcohol, Rosenthal fibers with
Alexander’s disease
– Huntington’s Disease
• Amyloid
• Alzheimer’s proteins
Lead
• Binds SH groups
• Decreases Fe++ in hemoglobin
• Increases RBC denaturation
– Mercury can act similarly
81
Other protein inclusions
• Viral inclusion bodies
– Composed of viral proteins
• Also can contain heat shock p
proteins,, ubiquitin
q
• Hyaline droplet nephropathy, male rats
– Occur spontaneously
– Can increase with certain drugs
• A consideration for pharmaceutical industry and drug
testing
Intermediate filament defects
• “Filaments/tubules” that aggregate in the cytosol
• Autosomal dominant defect
– Microfilaments
• actin
– Intermediate filaments
• Keratin, desmin, glial fibrillary acid protein (GFAP)-a neurofilament,
vimentin, internexin, nuclear lamins (Lamin A and C, lamin B1 and
B2)
– Microtubules
• Tubulin
Intermediate filaments
• Can be modified and undergo:
– Farnesylation, phosphorylation, glycosylation,
tranglumate cross-linking
• Loss of filaments formation or modification
can lead to:
– Cellular fragility
• Loss of structure
– Decreased transcription
• Loss of DNA and nuclear support
82
Intermediate filament disorders
• Keratin disorders, continued
– Epidermolysis bullosa simplex
• Keratin 5, 14
– Loose
L
anagen syndrome
d
• Sparse, short hair
• Keratin 6hf
– Monilethrix
• Alopecia
• Keratins 1, 6
– Many other keratin disorders
• Lamin disorders
– Dilated cardiomyopathy
• Lamins A and C
– Werners
W
syndrome
d
((aging)
i )
• Lamins A and C
– Hutchinson-Gilford progeria (aging)
• Lamins A and C
– Forms of muscular dystrophy (Emery-Dreifuss)
• Lamins A and C
• Alexander disease
– GFAP aggregate
• Amyotrophic
y
p
lateral sclerosis
– Neurofilaments, peripherin aggregates
• Juvenile cataracts
– Phakinin, filensin aggregates
• Dilated cardiomyopathies
– Desmin aggregates
83
• Keratin-related disorders (e.g., disorders in which
keratin accumulates)
– Cirrhosis
• Increased accumulation secondary to disease development
– Alcoholic cirrhosis, nonalcoholic steatohepatitis and hepatic
neoplasia:
» Mallory bodies form
» Mallory bodies are composed of keratins 8 and 18
» Not degraded by proteosome
– Pancreatitis
– Inflammatory bowel disease
Neurodegenerative diseases with aggregated
proteins
Disease
Protein
Normal
Structure
Aggregate/
Inclusion
Location
Prion dz
Prion
Alpha helix,
random coil
Beta pleated sheet,
proteinase K resistant
Extracellular
Alzheimers dz
Amyloid
precursor
protein (APP)
Alpha helix,
random coil
Beta pleated sheet,
fragment of APP
Extracellular
Tauopathies and
Alzheimers
Tau (microtubule
Binding protein)
3 and 4 repeat
isoforms
Hyperphosphorylated
aggregated protein
Intracellular
Parkinsons dz
Alpha synuclein
Random coil
repeats
Aggregated
Lewy bodies
Cytoplasmic
Multiple system
Atrophy
Alpha synuclein
Random coil
repeats
Aggregated, glial
cytoplasmic
inclusions
Cytoplasmic
Huntington dz
Huntingtin
Trinucleotide
Repeats
(polyglutamines)
Insoluble aggregates
Nuclear
Neuroserpins
Serpins
Beta sheet and loops
Insoluble
Er
Spinocerebellar
Ataxia
Ataxins
Trinucleotide repeats
Insoluble aggregates
Nuclear
Components of intracellular aggregates
•
•
•
•
•
The protein (Huntingtin for example)
Ubiquitin
Chaperone
E3 ligase
19S and 20S components of the 26 proteosome
84
Prions
• 1732 Scrapie first reported in Germany and France;
recognized earlier
• M’ Gowan JP: Investigation into the disease of sheep called
“Scrapie.” William Blackwood and Sons, Edinburgh, 1914
• 1959 Hadlow connected scrapie/kuru in
• Hadlow William J. Lancet ii, 289, 1959
– Characterized spongioform disease in mink
• 1986 BSE recognized
• 1996 CJDv recognized
Prion disease spectrum
• Ovine spongioform encephalopathy (scrapie)-sheep
– Dr. Bill Hadlow, veterinary pathologist
•
•
•
•
Bovine spongioform encephalopathy (BSE-”Mad Cow”)
Feline spongioform encephalopathy--cats
Chronic Wasting Disease-elk,
Chronic-Wasting
Disease elk deer
Transmissible mink encephalopathy-mink
•
•
•
•
•
Kuru-man
Creutzfeldt-Jakob disease -man
CJD variant-man
Gerstmann-Strauessler-Schleinker-man
Fatal familial insomnia (FFI)-man
Normal function or PrpC
Aguzzi et al: Annu Rev Neurosci 31:439, 2008
85
Mechanisms of PrpC function: three
models
models
TM = transmembrane protein
models
86
Aguzzi et al: Annu
Rev Neurosci 31:439,
2008
Aguzzi et al: Annu
2008
Aguzzi et al: Annu
2008
87
Prion PrpC
• Prion isoform PrpC– rER to endosome to plasma membrane
• A glycoprotein associated with glycophosphatidylinositol membrane protein
(GPI).
– Thus, prion-GPI protein association which anchors PrpC to membranes.
•
•
•
•
•
•
•
Encoded by PRNP gene
208 amino acids
glycosylated
Alpha helical and beta sheet structure
Octapeptide repeats (OR) perhaps responsible for normal function
Some folded incorrectly and reverts to ER and proteosome
Function not fully determined
– Postively regulates neural precursor proliferation
– But depletion does not result in neurodegeneration
• Three types of glycosylation on PrpC
• Mice lacking PrpC have limited problems
• Mice lacking PrpC do not develop disease when given PrpSC
– Develop cattle lacking PrpC to reduce BSE fears?
Prions
• Prp Sc
– Misfolded, resistant to protease K digestion and acid
– 43% of the PrpSc is in beta sheet structure
– Induces misfolding of PrpC
• Misfolded Prp (Sc)
– Goes to cytosol, proteosome, aggregate not degraded and
toxic
Prion lesions
• Cytoplasmic vacuoles
– Content and cause unknown
• Glial cell reaction
88
Spongiform change in cerebral neurons
Normal neuron with normal prion isoform
Neuron with scrapie isoform prion causing a cytoplasmic vacuole
Creutzfeldt-Jakob disease
Obex-area from
U.S. BSE case:
A
“The billion dollar
diagnosis”
Histopathology: H&E stain
B
Immunohistochemistry: anti-PrP Ab
John P. Kluge, Arthur J. Davis, and
Hall (NVSL-APHIS-USDA),
unpublished results
89
Mutations in PRNP gene
90
Tauopathies
• Tau proteins function in microtubule assembly
• Tau proteins accumulate in:
– Alzheimer’s disease, amyotrophic lateral sclerosis,
Creutzfeld-Jakob Dz, Down’s Syndrome
• Alzheimer’s
Al h i
’ di
disease
– Neurofibrillary tangles in neurons
• Form after amyloid beta is deposited
• Tau becomes hyperphosphorylated
• HyperP tau plus ubiquitin---forms aggregate of hyperphosphorylated
Tau
– Nonfunctional microtubules
– toxicity
•
Other conformational CNS-related
diseases
Alzheimers disease
– Neurofibrillary tangles (tau protein)
– Amyloid (amyloid precursor protein)
– More to follow
•
Alpha synuclein
•
Neuroserpins
•
Glutamine repeats in Huntingtin protein
• Present in Lewy bodies in neurons of parkinsonism and Alzheimer disease-Lewy body variant
• Dopamine-producing cells are affected
• Familial encephalopathy
– Collins bodies in neurons
• Huntington’s dz
• Mutated Huntingtin contains polyglutamines that bind other proteins and forms aggregates
– Binds TATA transcription factor and CREB binding protein (CBP)
• Rhes a guanine nucleotide binding protein, identified in 2009, binds the
Hungtington and induces sumoylation that is toxic.
• Rhes protein is present only in corpa striatum neurons and thus is the reason
for toxicity in this location
Subramaniam et al, Science 324:1327, 2009
Lewy bodies
91
Additional conformational diseases
• Eosinophilic (acidophilic) pneumonia of mice
– Accumulation of Ym1, Ym2 protein in macrophages
of older mice and 86-moth eaten mouse strain
• YM-1 is a chitinase-like lectin protein
J. Biol. Chem., Vol.
277, Issue 7, 54685475, February 15,
2002
Serpin defects
• Alpha-1 anti-trypsin
– Cirrhosis
• Accumulation of protein in hepatocyte ER
• Protein aggregate not degraded due to lack of chaperone recognition
– Emphysema
• Secondary to increased neutrophil elastase activity
– Elastase activity unregulated
– Mutation opens beta sheet allowing insertion of next sheet
• C1 inhibitor
– Angioedema secondary to uncontrolled complement
• Antithrombin
– Thrombosis secondary to increased thrombin activity
– Mutation of hinge region
• Antiplasmim
– Hemorrhage and fibrinolysis secondary to increased plasmin activity
– Mutation adds an amino acid lengthening loop structure of protein
• Neuroserpin
Protein accumulation in neuron ER similar to alpha one antitrypsin
Additional conformation diseases
• Cystic fibrosis
– CFTR (cystic fibrosis transmembrane regulator)
– Translated and processed in rER
– Trafficks in the cytosol via help with HSC70/HSP70
• This trafficking does not occur with CFTR delta-Phe508
variant
– Glycosylated CFTR is bound by calnexin, a lectin
chaperone
• 75% of CFTR protein is rejected here (normally) and
degraded
– Thus, only 25% makes it to the plasma membrane (normally)
• Undegraded CFTR forms in a perinuclear “aggresome”
contain CFTR, ubiquitin, and intermediate filaments
Gregersen N, J Inherit Metab Dis 29:456, 2006
92
Amyloid
• Extracellular deposition of an insoluble
proteinaceous material
– Proteinaceous material in beta sheet
– More than 20 different proteins are substrates
– Admixed with:
• P component (similar to C reactive protein)
– A pentraxin protein
– Resistant to degradation
– Once in amyloid, even more resistant to degradation
• GAG’s: Heparan sulfate, chondroitin sulfate, etc.
Amyloid
Examples of Amyloid
•
•
•
•
Secondary systemic amyloid
Primary systemic amyloid
Islet associated amyloid
y
peptide
p p
((IAAP))
Transthyretin
– Senile systemic
– Familial polyneuropathy
• Alzheimer’s disease
• Familial nonneuropathic: lysozyme
93
Examples of Amyloid
• Hereditary cerebral amyloid angiopathy
• Cystatin
•
•
•
•
•
•
Dialysis: beta 2 microglobin
Finnish type: gelosin
Familial amyloid polyneuropathy: Apolipoprotein A1
Hereditary renal amyloid: fibrinogen
Pituitary: prolactin
Atrial amyloid: atrial naturetic factor
Clinical entity
Associated
Diseases
Major fibril
Protein
Precursor protein
Muliple Myeloma
(monoclonal B cell
proliferation)
AL
Immunoglobulin light
chains
Systemic (generalized)
amyloidosis
Immunocyte dyscrasias
(primary amyloidosis)
Reactive systemic
amyloidosis (secondary amyloidosis)
Chronic inflammation
AA
SAA (serum associated
Amyloid)
Hemodialysis-associated amyloid
Chronic renal failure
A B2M
Beta 2 microglobin
Familial Mediteranean fever
AA
SAA
Familial Amyloidtic Neuropathies
ATTR
Transthyretin
Systemic senile amyloidosis
ATTR
Transthyretin
Abeta
APP (amyloid precursor)
A Cal
Calcitonin
Hereditary amyloidosis
Localized Amyloidosis
Senile cerebral
Alzheimers disease
Endocrine
Medullary Ca of the thyroid
Islets of langerhans
Type II diabetes
Isolated atrial amyloid
Prion diseases
CNS prions
AIAPP
Islet amyloid peptide
AANF
Atrial natriuretic factor
Prp-Sc
Normal prion protein
Secondary systemic amyloid (SAA)
• Liver cell produces SAA secondary to inflammation
– IL-1, TNF incite SAA release
• SAA released
– An acute phase protein
– Function unknown
• Macrophage RAGE receptor uptake (takes up glycosylated
proteins also)
• Altered endoproteolytic cleavage of SAA
– Cleaved at the N-terminus
• SAA amyloid forms with P component, GAG’s and other matrix
material
94
Primary Systemic Amyloid
• B cell/plasma cell (plasmacytoma)
– Released Ig light chain to excess or altered
– Altered processing results in amyloidgenic
fragments
– Amyloid
Islet associated amyloid peptide
• Co-secreted with insulin
• Deposited pancreatic islets of type II diabetics
• Occurs in humans and cats
• Decreased beta cell activity
• What is the cause?
» Compression, obliteration of vessels/blood flow, toxicity?
Transthyretin
• Carrier of thyroid hormones and vitamin A
• Senile systemic amyloid
• Wild type tranthyretin
• Altered proteolysis
– Transthyretin fragments form amyloid
• Familial polyneuropathy
• TTR variants (polymorphisms/mutations)
– Altered TTR and/or fragments forms amyloid
95
Therapies for amyloid
• Eprodisate
– GAG mimetics (eprodisate is an example)
• Binds GASs binding site on amyloid fibrils
• GAGS (e.g., heparan sulfate) promote fibril assembly
• Inhibition of the underlying inflammation
– anticytokines antibodies to:
• TNF, IL-1 beta, IL-6
• Inhibition of causes of inflammation
– antibiotics
Alzheimers Disease
• Amyloid precursor protein (APP)
•
•
•
•
A membrane protein
Cleaved by alpha, beta and gamma secretases
Accumulates around vessels
Preceeds tau accumulation in neurons
• Alpha secretase pathway
• Does not result in amyloid
• Beta/gamma secretase pathway
• Results in Amyloid beta (Ab) of 42 aa length
• Gamma is a membrane enzyme containing presenilin which can
also mutate
• Amyloid beta (Ab)
• Released following beta/gamma secretase activity
• Affects synapses, increases microglial and astrocyte reaction,
increases cytokines and complement.
• Altered neuron ion and ROS activity
96
Alzheimers induced atrophy
100th year anniversary 2006
Amyloid core with dystrophic neuritis
Increased neuronal activity around
plaques
• Dementia may be explained, in part, by
increased activity of neurons around plaques
– Reduced synaptic transmission to these
surrounding neurons
– Increased activity
Science 321:1686, 2008
97
Treatments for AD
• Antibodies to Amyloid beta
– Potential effective, but serious hypersensitivity reactions
• Decrease activity of beta and gamma secretases
• Using specific secretase inhibitors, NSAIDS
• NSAIDS
• Decreases gamma secretase, decrease inflammatory reaction
• Decrease cholesterol
• Increased APOE4 associated with increased cholesterol levels and
AD
• Increased cholesterol may increase gamma secretase
• Decrease Cu/Zn with chelators
• Decreases amyloid beta aggregates
• Decrease Amyloid beta toxicity (how?)
• GAG mimetics
New therapy: proof-of-principal in a mouse model
• Inhibitor to beta secretase
– Anchored to membrane by a sterol
– Allows the beta secretase inhibitor to be functional
in the endosome where beta secretase is active
– Other inhibitors do make make it to endosome and
less effective
Science 320:520, 2008
Hepatoencephalopathy
• A cognitive disorientation secondary to
hepatic disease
• Seen with:
– Extrahepatic or intrahepatic shunts
– Chronic liver disease
– Cirrhosis, often secondary to:
• Alcohol, copper accumulation, hemochromatosis
98
Hepatoencephalopathy
• Lack of hepatic function leads to:
– Increased ammonia
• Enteric bacterial metabolism of urea
– Dietary proteins
– Glutamine
• Skeletal proteins
– Increased/altered endogenous levels of:
• Benzodiazipine receptors
• Neurotransmitter activity:
–
–
–
–
GABA (increased)
Glutamine (decreased)
Increased aromatic amino acids
Decreased branched chained amino acids
» Amino acids affect neurotransmission
• Circulating mercaptans
– Enteric derived sulfur groups
• All of the above affect neuronal and glial activity.
Other accumulations
• Fibrin
– Fibrinoid necrosis
• Urate crystals
– Uric acid and urate crystals
– Accumulate
A
l t with
ith altered
lt d purine
i metabolism
t b li
• Gout
• Articular goat in man
• Visceral goat in avian and reptiles
• Pyrophosphate crystals
– Pseudogout
– Man and dogs (periarticular)
Other accumulations
• Mineralization
– Accumulation of calcium - phosphate
– Dystrophic
• occurs in areas of necrosis
– Also calcinosis circumscripta, heart (with age)
• Calcium-phosphate in a form of apatite
– With pump failure, Ca-P binds phospholipids that make a nidus
– Metastatic
• Secondary to high calcium levels
– Renal secondary hyperphosphatemia, PTH release, vitamin D
toxicosis (plants, rodenticides)
– Lung, stomach, kidney
» Areas with altered pH
99
Melanime and cyanuric acid
• Tainted dog and cat food
• Tainted baby formula
Pigment accumulation
• Carbon (anthracosis), melanin, lipofuscinceroid, hemoglobin, myoglobin
Cell Death and Apoptosis
100
Necrosis
Necrosis (from first lecture)
Apoptosis
• Occurs physiologically in:
– Embryogenesis, hormone-induced
involution (p
(prostate secondary
y to
castration), breast tissue after lactation,
cell deletion (gut crypts, thymic selectin of
lymphocytes), tumor cells, cytotoxic T cells,
secondary to toxins and viruses
101
Apoptosis: cellular features
Apoptosis
Cells shrinkage, chromatin condensation,
cytoplasmic blebs, phagocytosis, caspase
activity, transglutaminase activity,
DNA degradation dependent on calcium
and magnesium, DNA ladders, annexin V
expression,
i
phosphatidylserine
h
h tid l i expression
i
on outer membrane leaflet, lack of neutrophil
infiltration
Necrosis
Plasma membrane damage,
dilation of cytocavitary network,
peripheralization of chromatin,
infiltration of neutrophils, release of
Inflammatory mediators (see later slide)
Apoptosis
Apoptosis
102
Apoptosis: 180-200 bp ladder
A. Control
B. Apoptosis
C. Necrosis
Apoptotic Thymocytes
Susan Elomore: Toxic Pathol 35:495-516, 2007
Cytoplasmic Budding
103
Tingible Body Macrophage
Apoptosis: initiation/prevention
• Initiators of apoptosis:
– TNF, nitric oxide, fas ligand, granzyme, viral infection,
radiation, corticosteroids, DNA damage
• Extrinsic
E t i i pathway
th
• Intrinsic pathway
• Inhibition of apoptosis:
– Growth factors, differentiation factors, adequate
intracellular nutrition, insulin, others
– Activates “survival signaling pathway”
p53 activation
CK1
CK2
DNA damage induces activation of:
ATM (ataxia telangiectasia)
ATR (ATM and Rad3-related protein kinases)
DNA-PK (DNA protein kinase)
p53
p53 phosphorylated*
P53 phosphorylation
at a.a. site 20 allows binding
to MDM2 but prevents export
to the cytoplasm where
degradation normally occurs.
Activated p53
ARF
MDM2
Ubiquitylation of
p53 by MDM2 (a
Ubiquitin ligase) and
increased cell proliferation
GADD45alpha
(DNA repair)
p21
Inhibition of cell proliferation
Apoptosis
Repair Senescence
*P53 levels are normally
low in cells and bound
to MDM2 (a ubiquitin ligase).
Phosphorylation of p53 at
site 15, 37 greatly enhances
transcriptional activity.
ARF – induced by c Myc, reduces
MDM-2 mediated degradation of p53,
thereby inhibiting
proliferation and promoting repair or
apoptosis
104
Transcription factor regulation of apoptosis
• Myc
– c-Myc, n-Myc, and L-myc
– Leucine zipper transcription factors
– Exacerbate or reduce gene expression
• Rather than turning “on or off”
– If growth conditions are good, Myc promotes proliferation
(see cell proliferation)
– If growth conditions are poor, cells do not achieve the
“survival threshold” and Myc enhances apoptosis
Myc enhancement of apoptosis
• Myc can enhance apoptosis by:
– Activation of the intrinsic death pathway (Cytochrome C
release)
• Through:
– R
Reduced
d
d BCL2 expression
i (BCL2 iis antiapoptotic)
ti
t ti )
– Enhanced PUMA expression (PUMA is proapoptotic)
– Synergy with death receptor signalling
– Generation of ROS
– Increased ARF gene expression
• ARF inhibits MDM2 allowing increased p53 activity,
increased p21 and other proteins that inhibit of cell
proliferation
– Allows p53 to induce apoptosis (above)
105
Extrinsic/intrinsic apoptosis signaling
• Perforin/Granzyme pathway
– Granzyme A and B (serine proteases)
• B activates Caspase 10, ICAD and cleaves Bid
• A DNA nicking
• Extrinsic signaling
– Ligand/receptors
• TNF alpha/TNFR1, Fas/CD95L(Fas Ligand)), Apo3L/DR3,
Apo2L/DR2, Apo2L/DR5
• Death domains of 80 amino acids
– Activates Procaspase 8 to caspase 8 (active)
– Inhibited by cFLIP which binds adaptor protein FADD and caspase 8
and Toso which blocks the Fas pathway
• Intrinsic signaling
– DNA damage, UV damage, viral infection, toxins,
hyperthermia, free radicals, cell injury
• ATM/ATR-p53 activation
– Direct activation of BAX
– Puma, Noka inhibition of BCL-2
Apoptosis
TNF alpha
APO-2,3L
Trail receptor 1-4
DR 2,3,5
Cytotoxic T cell expressing
FasL (CD95 Ligand)
Fas (CD95)
TNF receptor 1
DNA damage/cell damage
Procaspase 8
ATM/ATR
Granzyme B
perforin
Cytotoxic T cell:
Perforin,
Granzyme A, B
p53
Caspase 10, BID
Caspase 8
Caspase 12
Puma,
Noxa
Nucleus
Initiator caspases:
2, 8, 9, 10, 12
BAD
(binds BCL2)
Mitochondria
Caspase 12 Cytochrome C
AIF, CAD,
Endo G
ICAD
CAD mammalian endonuclease
Endonuclease G
AIF
DNA degradation
SET
Survival Signaling:
EGF, PDGF, IL-2, 3,
Insulin
14-3-3
(AKT phosphorylates
BAD, BAD-P binds
14-3-3 releases BCL-2)
BAX
Caspases 3, 6, 7
“Effector caspases”
Transglutaminase X-link proteins
Cytokeratins, PARP, Gelosin (actin cleavage)
Fodrin, NuMA
Granzyme A
PI3K
BID
Endoplasmic
reticulum
perforin
PETN
PDK1,2 AKT
BCL 2
released
BAX (forms pore)
SMAC,
Diablo Apaf-1
Effector caspases:
3, 6, 7
ATP
Procaspase 9
Apaf-1 Aven
IAP
Survivin
Caspase 9
Components of the “apoptosome”
ATP
Cytochrome C
Denotes inhibition
Denotes activity
or transition
Denotes movement
Apaf-1
Procaspase 9
Caspases
•
Caspases
– Precursor-pro-caspases
• Removal of N-terminal domain during activation
– Caspase 1, 4, 5
• Proteolytic activation of IL-1b, not as active with apoptosis
• Involved with inflammation
– Caspases 2, 3, 6-11, 12, 14
• Cysteine proteases involved with apoptosis
– Caspase 12
• Endoplasmic reticulum-released apoptotic factor initiated by amyloid beta
• Released from endoplasmic reticulum and induces activation of effector caspases
– Caspase 13
• Bovine gene
– Caspase 14
• Embryonic tissues
– Initiator caspases
• 2, 8, 9, 10, 12
– Effector caspases
• 3, 6, 7
•
There is no one key enzyme, protein, or signal responsible for the ultimate death
and lethal blow to a cell.
– In other words, apoptosis factors occur simultaneously
106
BCL-2 families
• Anti-apoptotic
– Contain all BCL-2 homology (BH) domains, BH1-4
• Includes membrane anchoring, channel formation, and
regulation domains
• BCL2 prevents Bax formation of a pore
– Reduces release of Cytochrome C and reduces apoptosome formation
– Members:
• Bcl-2, Bcl-xl, Bcl-w, Mcl-1, Boo/Diva
• Pro-apoptotic
– Fewer BH domains
– Members:
• Bax, Bak, Bok/Mtd, Bcl-xs (BH3, BH1, BH2 domains)
• Blk, Bad, Bmf, Bid, Puma, Noxa (BH3 only domains)
– BH3 proteins sense cell damage and act only through BAX and Bak
Uptake of apoptotic cells
• Appears to contribute to killing
– Tingle-body macrophages form
• These are macrophages with abudant cytoplasm and
internalized portions of apoptotic cells
• Apoptotic cells express
– Phosphatidylcholine (PC) and phosphatidylserine
(PS) on the outer surface
• PC allows detection of apoptotic cells (“find me”) by
macrophages
• PS allows internalizatoin (“eat me”) by macrophages
a. “Find me”
b. “Eat me”
LPC = lipophosphatidylcholine. PtdSer = Phosphatidylserine, ox LDL = oxidized low density lipoprotein; CD36 = scavenger
Receptor; BAI1 = brain angiogenesis inhibitor; ICAM-3 = intercellular adhesion molecule -3; CD14 = Lipopolysaccharide
binding protein receptor; alpha v beta 3 intergrin; MFGE8 = milk fat globule EGF factor 8; MER = receptor tyrosine kinase;
GAS6 = growth arrest-specific 6;
Nature Rev: Immunol 7:964-973, 2007
107
Non-apoptotic cell death
• Resistance to apoptosis is a hallmark of
cancer cells
• Defects in non-apoptotic cell death are
associated with cancer
• Non-apoptotic cell death:
– Senescence
– Necrosis
– Autophagy
– Mitotic catastrophe
Senescence
• From previous slide during cell cycle lecture:
• Senescent cells, especially stem cells, reduces proliferation and therefore,
neoplastic transformation
• Occurs by:
–
–
–
–
Telomere shortening
p53 activated and reduces cell proliferation
p16 (INK 4a)/pRB expressed (in response to oncogenes such as Ras, BRAF)
inhibition of cell proliferation
• Also, new today:
– Senescent cells have:
• Flattened cytoplasm, increased granularity, changes in metabolism
• Induction of senescence-associated beta galactosidase (SA-beta Gal).
Necrosis
• Unregulated cell death with release of intracellular
components
– membrane enlargement (stretching) due to swelling
– cell swelling
• dilation of cytocavitary network
– vacuoles, nuclear membrane, sER, rER, mitochondria
– fragmentation of the nuclear chromatin ultrastructurally
– Failure of ion transport, ATP production, and pH balance
– Inflammation (neutrophils, inflammatory mediators) and
damage of surrounding tissue
108
Necrosis
• Inflammatory mediators released:
•
•
•
•
•
•
uric acid
adenine phosphate
purine metabolites
heat shock proteins
IL-1, TNF
HMGB1 (high mobility group box 1 protein) released from nucleus
• HMGB1 binds macrophage RAGE receptor and also TLR 2 an d 4.
Autophagy
•
Type I programmed cell death
•
Type II programmed cell death
– Apoptosis
– Autophagosomes and autophagolysosomes accumulate
• Occurs with
–
–
–
–
–
–
–
–
Growth factor withdrawl
Differentiation and developmental triggers
Massive cell elimination
When phagocytes do not take up dying cells
Caspase independent
Caspase-independent
Increased lysosomal activity
Protein translocation to autophagosomes
Signalling involves phosphotidylinositol 3-kinase (PI3K) and target of
rapamycin (TOR)
• Defects in this signaling pathway lead to cancer
• Defects in beclin 1 gene, which works with PI3K to induce autophagy lead to
cancer
– Cell survival
• Nutrients for times of stress
– Can occur concurrently with apoptosis (type 1)
– Can induce cell death without apoptosis (type 1)
Autophagy
• A normal cellular process
• Contributes to cellular homeostasis for turnover of organelles
and superfluous proteins
• Maintains an amino acid p
pool for g
gluconeogenesis
g
and p
protein
synthesis during starvation
• Cell death (for when phagocytes cannot keep up)
• Anti-aging mechanism for removing substances oxidized by
free radicals
• Triggered by nutrient depletion (starvation)
109
Autophagy
• Type I programmed cell death
– Apoptosis
• Type II programmed cell death
– Autophagosomes and autophagolysosomes accumulate
• Occurs when
– massive cell elimination occurs
– When phagocytes do not take up dying cells
– Autophagsomal activity reduces the cellular “mass” making type I
apoptosis more efficient in developmental cell loss
• Type I and type II programmed cell death are not
mutually exclusive
Cellular degradation of macromolecules and organelles
• Microautophagy
– Degradative material is delivered to the lysosomes by
membrane invaginations of cytoplasm into the lysosome
• Macroautophagy
– Large organelles (especially mitochondria,
mitochondria endoplasmic
reticulum and peroxisomes) within a double membrane vesicle
that are degraded in autophagosomes that fuse with
lysosomes
• Chaperone-mediated autophagy
– Degradation of specific proteins that are bound to
chaperonens and internalized into the lysosome
• Degraded by calcium-dependent cysteine proteases, calpains, proteasomes
Autophagy and apoptosis
Nat Rev Mol Biol 8:741, 2007
110
AVi-initial autophagosomal vacuole, contains a mitochondria and rER
rER around the autophagosome
Degraded material
Partially degraded rER
Kelekar: Ann NY Acad Sci 1066:259-271, 2005
Mechanisms of autophagosomal assembly
• LC3-microtubule associated light chain arranges, structurally, the initial
endosome
• LAMP1/2 (lysosome-associated membrane protein) adhere to the
phagosomal membrane as it matures and fuses with lysosome
• Acid phosphatases and cathespins contribute to degradation.
• Inhibition of autophagosomes
– Growth factors that activate TOR
– TOR inhibits autophagosome formation
• Induction of autophagosomes
– Nutrient and amino acid starvation
• Activate Beclin-1, p150 and Class III PI-3Kinase
• Also activated ERK ½ and GAIP (which initiates GTP degradation to GDP); GDP
induces autophagosome formation
Autophagosome formation: nucleation (Beclin-1), and
elongation (LC3 PE)
Cell 15:334, 2008
111
Another view of nucleation and elongation
Nat Rev Mol Biol 8:741, 2007
112
Autophagy and aging
• Cells with limited mitotic activity
• Accumulate more cell degradation products
(garbage)
– Can reduce cell adaptability, viability, and function
• Cells can undergo apoptosis
– Lipofuscin-is a main “waste” substance
• Derived principally from incompletely degraded mitochondria
– Mitochondria become damaged with time due to ROS production that
injures mitochondrial DNA, mitochondrial proteins, and mitochondrial
processes like fission/turnover
• Lipofuscin and ceroid become difficult to degrade in
lysosomes/autophagosomes and therefore accumulates
Autophagy in disease
• Especially important in diseases of non-dividing cells of the
nervous system, muscle or when protein turnover is critical
– Vacuolar myopathies
• X-linked myopathy, inclusion body myositis, Marinesco-Sjorgren syndrome
– Danon disease (cardiomyopathy and retardation)
• Deficiency in LAMP-2
– Parkinson’s disease, Huntington’s disease, Alzheimer’s disease,
and spongioform enephalopathies
• Protein aggregates occur in these diseases; autophagosomes degrade the
protein aggregates to a point
Pyroptosis and pyronecrosis
• Cell death initiated by pathogen activation of
the inflammasome
• Inflammasome is triggered by NOD-like
NOD like
receptors (NLR)
– NLR induce caspase-1 activation
• IL-1beta and IL-18 are activated by caspase-1
113
Jenny et al: Nature Rev Immunol 8:372-379, 2008
Pyroptosis and pyronecrosis
• Unlike apoptosis, pyroptosis and pyronecrosis
both:
– induce inflammation, do not have mitochondrial
permeability
bilit and
d llack
k chromatin
h
ti condensation
d
ti
• Unlike apoptosis, pyronecrosis is not
dependent on caspase activity
• Like apoptosis, pyroptosis uses capase
(caspase-1; not capase 3; however)
Etosis
Wartha and Henriques-Normark Science Signalling ePub April 7, 2009
114
Etosis
• Neutrophils and mast cells undergo a special
type of cell death resulting in release of DNA
(histones and attached antimicrobial peptides).
– NETs are formed (neutrophil extracellular traps)
p bacteria
which entrap
– Following slides of NET (also covered with
Neutrophil section under inflammation)
• Etosis: the cell death process that lyses
nuclear membranes to allow release of DNA
for NET formation
– An ROS signal near NOX2 mediates Etosis
Wartha and Henriques-Normark Science Signalling ePub April 7, 2009
Neutrophil NETs
• Neutrophil NETs
– Neutrophil extracellular traps composed of DNA backbone embedded in
antimicrobial peptides and enzymes.
– These surround, entrapped and kill bacteria
– Components:
• Histones h1, h2a, h2, h4
• Primary granule contents: neutrophils elastase, cathespin G,
myeloperoxidase, bacterial permeability increasing proteins
• Secondary granutes: lactoferrin
• Tertiary granules: gelatinase, peptidoglycan recognition proteins (PGRP)
• Antimicrobial peptides: LL37 (cathelicidin), HNP2 (human neutrophil peptide;
an alpha defensin)
– NETs and pulmonary mucin
• With CF and other chronic pneumonias, neutrophil DNA can make
respiratory sections very thick and tenacious
– Neutrophil actin accumulation from degraded neutrophils also contribute
to mucin tenacity
• Pulmzyme: A DNAase that breaks down neutrophil DNA
Neutrophil NET
Some bacteria can release DNAases that degrade NETs and allow release
Many stimuli can induce NET release
Curr Opin Micro 10:52, 2007
115
Cell proliferation
Cell cycle
• Induction from Go to G1
– Growth factors
– Signaling
• Cyclins
• Checkpoints and inhibitors
– G1 to S
– G2 to M
• Checkpoint control
• Transcription and passage through G1/S
– E2F activation
Response of cells to injury
DNA injury
Dividing cells
Non-dividing cells
Inhibition of proliferation
Apoptosis/death
(Failure of repair)
Cancer
Repair
Mutations
116
Signaling pathways and receptors: some initiate cell
proliferation
•
•
•
•
•
•
PI3 kinase
MAP-kinase
IP3 pathway
cAMP
AMP pathway
h
Steroid pathway
JAK/STAT pathway
• Growth factor receptors
– PI3K
– MAPK
– IP3
• Seven transmembrane
receptor
– cAMP
– IP3
• Steroid receptor
• Cytokine receptor
7-transmembrane receptor:
Chemokines, (nor)epinephrine
glucagon, serotonin, vasopressin,
histamine, calcitonin, rhodopsin,
parathyroid hormone
Growth factor receptors:
EGF, KGF, ILGF, PDGF,
FGF, TGF alpha, VEGF,
c-Kit
PLC
PI3 K
IP3-DAG
Ras (GTP/GDP, Raf)
MKKK, MEK, ERK (MAPK)
PLC
Hormone receptor:
Thyroid hormone,
Vitamin D, retinoids
JAK JAK
G proteins-ras
Ca++
cAMP
PKC
PK A
STAT
Steroid
receptor
STAT-P
Cytoskeletal
C
t k l t l proteins
t i
Ca++/K+ pumps
Calpain
Ca++ BP-calmodulin
PKB/AKT
Cytokine receptor:
Interleukins, Interferons,
EPO, G-CSF
Ion channels,
vision, olfactory
Steroid TF
cMyc, cJun, cFos, Foxo3 PPAR
NF kappa B
Cyclins
Cell proliferation
Cell metabolism/differentiation
Inflammatory/immune responses
Abbreviations of signaling molecules
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
AKT—protein kinase B (below)
RAF—(MKKK)
MEK—(MKK)
ERK—(MK)
MAPK—mitogen activated protein kinase
IP3 inositol 1,4,5 triphosphate
PI3—phosphoinositol three kinase
DAG--diacylglycerol
PLC phospholipase C
PLC—phospholipase
PKA—protein kinase A (AKT)
PKB—protein kinase B
PKC—protein kinase C
cAMP—adenocyl 3,5, cylic
monophosphate
JAK—janus activating kinases
STAT—signal transducers and activation
of transcription
Ca++ BP—calcium binding protein
Steroid TF—transcription factor
PPAR—peroxisome proliferation activating
receptor
cMyc, c-Jun, c-Fos—transcription factors
for cell proliferation
•
•
•
•
•
•
•
•
•
•
EGF—epidermal growth factor
KGF—keratinocyte growth factor
ILGF—insulin-like growth factor
PDGF—platelet derived growth factor
FGF-fibroblast growth factor
TGF—transforming growth factor
VEGF V
VEGF—Vascular
l endothelial
d th li l growth
th
factor
C-Kit—stem cell factor
EPO—erythropoietin
G-CSF—Granulocyte-colony
stimulating factor
117
Ras gene induction of cell proliferation
Ras/Rho
• Small GTPases
• RAS subfamily
– H-ras, N-ras, E-ras, r-ras, Rap, Ral, rit, rheb
• Rho subfamily
– Rho A, Rho B, Rho C, Rac 1, Rac 2 Cdc42/G25k
• Ras mediates
– PI-3 pathway
– RAF/MEKK pathway
• These induce transcription factors that increase
transcription of Cyclin D
Transcription factor regulation of
proliferation by Myc
• Myc
– c-Myc, n-Myc, and L-myc
– Leucine zipper transcription factors
– Exacerbate or reduce gene expression
• Rather than turning “on
on or off”
off
– If growth conditions are good, Myc promote proliferation
through:
• Enhanced expression of:
– D and E cyclins
– Cyclin dependent kinase 4 (CDK 4)
• Reduced expression of:
– P21 (CIP1) and p15 (INK 4B)
– If growth conditions are poor, Myc also may enhance
apoptosis (see apoptosis)
118
Cyclins
• Proteins that complex with cyclin dependent
kinases
• When complexed and phosphorylated the
CDK’s become active serine/threonine
kinases
– CDK’s are expressed constitutively
• Cyclins rise and are degraded by the ubiquitin
pathway rapidly
• Cyclins allow cells to pass through the
Cyclinmajor
A
points of the cell cycle:
CDK 1
– Go-G1-S-G2-M
Cell cycle checkpoints
• G1 checkpoint
– Major checkpoint after cell signaling activation
• prevent cells from entering cell synthesis
• Best understood checkpoint
• Synthesis checkpoint
– Reduction
R d ti iin DNA synthesis
th i
• Regulated by ATM
• Cyclin A and E remain inactive
• Least understood checkpoint
• G2 checkpoint
– Inhibitory phosphorylations of cdc2
• Prevents cell proliferation right before chromosomal separation
• Cdc25C is complexed with 14-3-3 and unable to activate cdc2
– Cdc25C is a phosphataste inactivated by 14-3-3
• Partially understood checkpoint
– better than S, less than G1
Cell injury
The Cell Cycle
p53
G0- non-replicating cell
EGF
Cyclin B
C-Myc
CIP/KIP
CDK 1, 2
Cdc25c-14-3-3 Cdc2
FGF
+
IGF
M
Nutrients
1 hr
G1
G2/M checkpoint
8 hr
TGF B
-
Cyclin D
G2
CDK 4,6
2 hr
Cyclin E
Cyclin A
CDK 2
ATM
S
Cyclin A
CDK 2
p53
INK4
CIP/KIP
G1/S checkpoint
S checkpoint
CDK 1
Cell injury
8 hr
CIP/KIP
CIP/KIP
p53
Cell injury
p220NPAT
HiNF-p
Histone synthesis
and nucleosome
packaging
p53
119
Ras/nutrient induction of cell proliferation: Getting
cells out of Go
Nutrients
Growth factors
TOR
RAS
S6K
PI3K
S6 phosphorylation
40S
60S
TOR
eIF4E
S6 phosphorylation
40S
60S
ERK
S6K
Cyclin D
eIF4E
Cyclin D
Cyclin D
Cell proliferation
Cyclin activation of transcription and passage through
G1/S checkpoint
p130 released allowing E2F activity
p130
Transcription
and passage
through G1/S
checkpoint
DP
p130
ATP
E2F
Cyclin D
DP
CDK 4
Cyclin D
CDK 6
CD kinase activity
Cyclin E
phosphorylates RB
E2F
RB
P
P
P
RB-P
CDK 2
P
P
P
Phosphorylated RB releases E2F and
also opens chromatin structure for
transcription.
P
phosphorylation
RB
denotes retinoblastoma protein
Histone acetylase phosphorylate RB,
decrease E2F and close chromatin structure.
E2F a transcription factor that enhances cell proliferation and passage across the G1/S checkpoint
P130 inactivates E2F and prevents Go cells from undergoing proliferation
DP
a DNA binding protein
Cell activities needed for proliferation and activated by cyclin/CDK’s and
E2F transcription
•
•
•
•
Nuclear envelope breakdown
Centrosome function
Spindle assembly
Chromosome condensation
120
With cell injury: Inhibition of proliferation
to allow repair
• If cells have DNA mutations and
continue replication the mutation is fixed
and p
passed to the daughter
g
cell
• If proliferation is ceased, cells can then
attempt repair
P53: The King.
Guardian of the Genome
121
p53 activation
DNA damage induces activation of:
ATM (ataxia telangiectasia)
ATR (ATM and Rad3-related protein kinases)
DNA-PK (DNA protein kinase)
CK1
CK2
p53
p53 phosphorylated*
P53 phosphorylation
at a.a. site 20 allows binding
to MDM2 but prevents export
to the cytoplasm where
degradation normally occurs.
Activated p53
ARF
MDM2
Ubiquitylation of
p53 by MDM2 (a
Ubiquitin ligase) and
increased cell proliferation
GADD45alpha
(DNA repair)
p21
Inhibition of cell proliferation
Apoptosis
*P53 levels are normally
low in cells and bound
to MDM2 (a ubiquitin ligase).
Phosphorylation of p53 at
site 15, 37 greatly enhances
transcriptional activity.
ARF – induced by c Myc, reduces
MDM-2 mediated degradation of p53,
thereby inhibiting
proliferation and promoting repair or
apoptosis
Repair Senescence
P53 modifications
• Mutations in p53 itself
– 18,000
– Especially occur in the DNA binding portion
• Residues 98-292
• Post-translational modifications
– Phosphorylation
p y
of serine and theronine residues
– Acetylation
• Phosphorylation and acetylation “stabilizes” p53 in
the nucleus
– Can also occur on mutated p53 resulting in nuclear
accumulation
• Upon dephosphorylation and deacetylation p53
binds DNA
– Ubiquitylation, sumoylation (small ubiquitin-like
proteins)
p53 inhibition of cell proliferation: INK4 and CIP/KIP
inhibition of cyclins
• p53 enhances
– INK4 inhibitors
•
•
•
•
INK4a (p16)
b (p
(p15)
5)
INK4b
INK 4c (p18)
INK 4d (p19)
– INK inhibitors:
• Reduce activity of:
– Cyclin D (not E) by:
– Prevention of cyclin
D binding to CDK4
• p53 enhances
– CIP/KIP inhibitors
• WAF (p21)
• KIP (p27)
( 27)
• KIP2 (p57)
– CIP/KIP inhibitors:
• Reduce activity of:
– Cyclin D, E, A, B
by:
– Forming
heterodimers with
the cyclin/CDK
complex
122
Therapeutic strategies to inhibit CDK
•
•
•
•
•
•
Direct inhibitors CDK
Prevention of CDK/cyclin binding
Enhancement of CDK-I
Inhibition of CDK-I degradation
Inhibition of cyclin synthesis
Promotion of cyclin ubiquitization
(degradation)
• Inhibition of CDK activating kinases and
cdc25 phosphatases
• Stimulation of CDK-I activation
Markers of cell proliferation
Marker
BrdU
Method of detection
Flow, IHC
Sensitivity
S phase
3H thymidine
radioactivity
S phase
Ploidy
Flow
Ki-67
Flow, IHC
Other
thymidine analoge
Can’t distinguish
G2 from M phase
PCNA
Flow, IHC
all proliferative
cells
broad
Cyclins
Flow, IHC
All phases
PHK2
Flow
Sensitive
AgNOR
Light microscopy
Not precise
labile antigen
stabile antigen, DNA
lesions increase
Used in vet med?
easy
BrdU = bromodeoxyuridine; ploidy = number of chromosomes [aneuploidy, diploidy,
Tetraploidy]; PCNR = proliferating cell nuclear antigen; AgNOR = silver stain of
Nucleolar organizing region
123
Cell proliferation differences in stem cells
• Proliferating stem cells have short G1-S
phases
– Longer G1-S phases promote cell
differentiation; shorter phase reduces
differentiation
– Thus, G1 phase is a window of increased
susceptibility to differentiation signals
• Shorter phase protects the cell from activation
that will induce differentiation
Neganova I et al: J Anat 213:30, 2008
Cell proliferation differences in stem cells,
continued
• Cyclin D is constitutively expressed, not
up-regulated
• Additional regulation occurs by:
– Oct4 and Nanog proteins which enhance
cdk 4 and 6 expression (cdk’s of cyclin D)
• Oct4 and Nanog are stem cell master
pluripotency regulators
– Sox2 protein enhances cdk2 (cdk of cyclin
E)
Neganova I et al: J Anat 213:30, 2008
Cellular senescence
• Senescent cells, especially in stem cells, reduces
proliferation and therefore, neoplastic transformation
• With DNA damage
– Lymphoid cells often undergo apoptosis
– Epithelial and mesenchymal cells often undergo
senescence
• Molecular mechanisms of senescence:
– Telomere shortening
• p53 activated and reduces cell proliferation
– p16 (INK 4a)/pRB expressed (in response to
oncogenes such as Ras, BRAF)
• inhibition of cell proliferation
• Without senescence, injured cells proliferate and more easily
contribute to cancer development
124
Neosis
• Some senescence may be “leaky”
– A few cells that are senescent and destined to die by
apoptosis may escape the senescent “mitotic crisis”
• Senescent mitotic block by short telomeres, no telomerase, and of
tumor suppressor genes such p53, pRB and p16 (INK 4a)
• Neosis cells have no telomeres, but p53, pRB and p16 (INK4a) are
inhibited (allowing proliferation)
– These cells with no telomerase may get abnormal chromosomal
joining
– Telomerase may then also become active—resulting in additional
lifespan/growth
– Mutations readily increase
– Escaped cells undergo nuclear budding and cytokinesis
– Escaped cells undergo endomitosis of polyploid DNA, DNA
misrepair, chromatin modulation
• Nucleus forms small buds (karyokinesis) that end up in small
regions of cytoplasm (cytokinesis)
» “Raju cells”
Polyamines
•
Cationic amino acids
– Spermidine
• Tetramine first identified in semen by Leewenheuk
– Putrescine
• Diamine first identified in bacteria
– Ornithine decarboxylase (ODC)
• First enzyme in polyamine synthesis
• Decarboxylates arginine and ornithine to make spermidine and putrescine
– DAX is an efflux
ff
port for
f polyamines from
f
the cell
– OAZ (ODC regulator anti-enzyme) inhibits ODC
– Spermidine acetyltransferase induces polyamine catabolism
•
•
•
Present in diet and gi bacteria
Induce cell growth and proliferation
Polyamines
– Associate with nucleic acids and thus affect transcription by:
• altering chromatin structure and affecting protein DNA binding
– Polyamine enzymes that affect polyamine charge by post-translational
modifications (acetylation) could also affect histones and thus chromatin
structure and transcription
125
Cleveland and colleagues have proposed a model
in which MYC-regulated ODC contributes to
oncogenesis (see figure). The MYC family of
oncoproteins
p
are activated in up
p to 70% of human
cancers. MYC functions as a transcription factor
that dimerizes with a partner, coined MAX, and this
complex binds to the E-box sequence CACGTG to
activate the transcription of target genes268. The
gene for ODC (ODC) harbours two conserved
CACGTG elements in ODC intron 1, and MYC
activates ODC transcription by binding to these
elements in cells15, 23. ODC then decarboxylates
ornithine to produce putrescine, which is then
further converted (by respective synthases) into the
polyamines spermidine and spermine. As a net
result MYC-overexpressing cells express elevated
levels of polyamine
Polyamines
• Mutant APC
– Decreases OAZ (ODC antienzyme)
• Increases ODC
– Increases polyamines
– Increased Myc activation
• Increases ODC
– Increases polyamines
• Mutant Kras
– Loss of PPAR gamma inhibition
• Reduced spermidine acetyltransferase
(polyamine catabolism and export)
– Decreased polyamine catabolism and export by DAX
Genetic variation: SNPs.
Epigenetic events: DNA methylation, HDACs,and polyamines
(telomeres covered by Kuipel)
126
SNP’s and cancer
• Single nucleotide polymorphisms (SNP’s)
– Single-base variations in DNA between individuals tied
to traits and disease
• Code red hair, freckles, pudginess, love of chocolate
• Genetic risk for disease, including
g cancer
• There are 15 million locations in the genome where one base can
differ between individuals
– 3 million SNPs identified by HapMap
– United Kingdom has heavily investigated:
» Rheumatoid arthritis, bipolar disorder, coronary artery
disease, type 1 diabetes, and Crohn’s disease
• Other genetic variations
– Gene copy number account for 20% of differences in
gene activity; SNP’s account for many of the rest
– Chromosomal inversions, insertions, deletions
SNPs and cancer
•
SNPedia
– Website of SNPs, including cancer
– www.snpedia.com
– Visit during class
•
Science “Breakthrough of the Year”
– Human Genetic Variation
– Science 318:1842-1843, 2007
– Discuss during lecture
•
Sequenome
•
Projects
– A company in San Diego that does high-throughput SNP identification
through mass spectroscopy
– SeatlleSNPs Variation Discovery Resource
– Cancer Genome Anatomy Project
• SNP500 Cancer project
– NIH’s Pharmacogenetics Research Network
– International HapMap project
SNP’s
• SNP
– A SNP differs by a single base at a given position
in the genome with a frequency of 1% in at least
one population
– Account for 90% of the total variation in the human
genome
• Other types of genetic variation include:
– Nucleotide repeats, microsatellites, gene amplification,
chromosomal amplification
– Nonsynonymous SNP shifts from one amino acid
to another
– Synonymous SNPs have no amino acid change
127
SNPs and disease (cancer)
•
SNPs associated with cancer susceptibility
–
N-acetyltransferase 2 (NAT2)
–
Glutathione S Transferase (GSTM1)
•
•
•
–
Homozygous individuals at increased risk for bladder and colorectal adenoma
Myeloperoxidase
•
–
Slow acetylations increase risk for bladder cancer, especially in smokers
Rapid acetylations increase risk for colon cancer
SNP in the promoter at G-463A is associated with decreased lung cancer risk in Caucasians
Also SNPs in
•
•
cytochrome P4501A associated with lung cancer risk
TNF and IL-1 associated with diffuse B cell lymphoma
•
SNPs and cancer outcome
•
Pharmacogenetics and cancer therapy SNPs
–
Cytochrome P450 enzyme CYP3A4 associated with increased long term survival
–
Thiopurine S methytransferase
–
Cytochrome P450 CYP2C
–
5, 10 methylenetetrahydrofolate reductase (MTHFR)
•
•
•
–
Increased thiopurine toxicity
Metabolism of alkylating agents
Increased methotrexate toxicity
UDP-glucurosyltransferase (UGT1A1)
•
Increased irinotecan toxicity
Epigenetic inheritance
• Definition:
Cellular information, other than the DNA
sequence itself, that is heritable during
cell division.
• Types of epigenetics information:
•
•
DNA methylation.
Histone Modifications.
DNA methylation
• Occurs in cytosines that precede
guanines
• Dinucleotide CpG.
• 60-90% of all CpGs are methylated in
mammals.
• Unmethylated CpGs are grouped in
clusters called “CpG islands” that are
present in the 5' regulatory regions of
many genes.
128
DNA methylation and gene expression
• DNA methylation
– Covalent binding of a methyl group to the 5-carbon position of
cytosine/guanine dinucleotides (CpG)
• Termed m5CpG
– 60-90% of cytokine/guanine sites are methylated in repetitive
regions of DNA
– Hypomethylated sites are usually in CpG
CpG-rich
rich regions (CpG islands)
near promoter regions
• Often near the core promoter and transcription start site
• Transcriptions occurs if:
–
–
–
–
Transcription factors are present
The CpG island is unmethylated
The chromatin state is “open”
Histones are hyperacetylated (opens chromatin structure; more later)
• Many promoters do lack CpG sites
– By evolution, these were lost due to deamination of m5C and loss
– Methylation of CpG islands reduces transcription
• Inhibits binding of transcription factors
• Promotes binding of methylated DNA binding proteins
DNA Methylation Reaction Catalyzed by DNA Methyltransferase (DNMT)
DNA Hypomethylation
Some toxic carcinogens act through
methylation alterations:
• Cadmium inhibits DNMT activity .
• Arsenic induces Ras hypomethylation in
mice.
Dietary relation:
• High dietary methionine increases
methylation leading to low cancer incidence.
129
DNA Hypomethylation and mechanisms of
tumor formation
Generation of chromosomal instability.
• Hypomethylation causes recombination
and chromosomal rearrangements
g
leading
g
to deletions and translocations.
• Depletion of DNA methyl transferases
leads to aneuploidy.
DNA Hypomethylation and mechanisms of
tumor formation
Genomic Imprinting.
• It is the relative silencing of one parental
allele compared to the other parental allele
• Maintained partly by differentially
methylated regions.
• In cancer, hypomethylation disrupts
imprinting called ‘Loss of imprinting (LOI)’.
eg LOI of IGF-2 causes Wilms tumor.
DNA Hypermethylation in tumors
• Hypermethylation of CpG island in the
promoter regions of tumor suppressor genes.
• Results in silencing of gene.
• First reported in Retinoblastoma tumor
suppressor gene.
• Followed by BRCA1, VHL, p16 INK4a.
• DNA repair gene also silenced further
increasing the chances of cancer.
130
In normal cells (top) DNA methylation is concentrated in repetitive regions of the
genome and most CpG island promoters are unmethylated. In tumor cells, the
compartmentalization breaks down and repetitive DNA loses methylation while CpG
island promoters acquire it, resulting in silencing of the associated gene.
DNA methylation and cancer cells
• Tumor cells often have:
– Global hypomethylation of repetitive DNA
– Region-specific hypermethylation (esp CpG regions)
• G
Global
oba hypomethylation
ypo et y at o
– Leads to chromosomal instability/mutation
• Seen with oncogenes, c-myc, ras
– Results in activation of these genes
– Gene-specific hypomethylation also occurs
• MAGE, S100A4
• Regional hypermethylation of CpG islands
– Seen with tumor suppressor genes
• Results in decreased tumor suppressor gene expression
131
Inactivation of both alleles of a tumor suppressor gene. One allele can be inactivated by methylation and
The second inactivated by point mutation, methylation or deletion.
Gronbaek K, et al: APMIS 115: 1039-1059, 2007
DNA methylation pathway: SAM
SAM-universal methyl donor to DNA, RNA, hormones, neurotransmitters,
membrane lipids, proteins and other molecules
DNA
Methylated DNA
(DNA methyltransferase)
S adenosylhomocysteine (SAH) S adenosylmethionine (SAM)
Adenosine
Homocysteine
THF
Cobalamin (B12)
Methionine
pyridoxine (B6))
riboflavin (B2)
Tetrahydrofolate (THF)
Methyl Tetrahydrofolate (THF)
Vitamins B2, B6 and B12 and
folate (THF) involved above
Diet
Methyltransferases
• 75% of methyl groups from SAM
– Go to the formation of phosphatidylcholine
• 25% of methyl groups from SAM
– Go to methylation of DNA
• SAM:
– Is broken down to gluthathione
– Has antioxidative properties in this regard
132
DNA methylation
•
Can result in point mutations
•
Hypomethylation
•
Promoter hypermethylation
– Loss of m5C and converstion to T
– Can lead to up-regulation of non-desired genes
• Cellular proliferation, decreased apoptosis
– Reduces tumor suppressor gene activity
• Examples:
–
–
–
–
–
–
Autonomous growth (Ras, SOCS)
Enhanced proliferation (p15, p16)
Reduced apoptosis (DAPK)
Tumor invasion (CDH1, TIMP)
Angiogenesis (THBS1)
Genome instability (MGMT, LMNA, MLH1, CHFR)
•
miRNA methylation
•
Methylation can also be a biomarker (hypermethylation of the above
genes)
Histone Modifications
• Histones undergo posttranslational
modifications which alter their interaction
with DNA and nuclear proteins.
• The
Th H3 and
d H4 hi
histones
t
h
have llong ttails
il
protruding from the nucleosome which can
be covalently modified at several places.
• Modifications of the tail include
methylation, acetylation, phosphorylation,
ubiquitination etc.
Histone Modifications
• Acetylation is associated with
transcriptional activation .
• Effect of histone methylation depends
on the amino acid residue and its
position in the histone tail.
133
Transcription active
HAT adds Ac
Transcription inactive
H3L4 trimethylation
H3L9 trimethylation
MBD is a protein
that binds m5CpG
Attracts HDAC
which removes Ac
Gronbaek K, et al: APMIS 115: 1039-1059, 2007
Histone acetylases and histone deacetylases
• Histone acetyltransferases (HAT)
– Contributes to enhanced transcription of genes
• Adds Ac
• Relaxes the chromatin structure
• Increased transcription
• Histone deacetylases (HDAC)
– Contributes to reduced transcription
p
of g
genes by
y
• Ac removal
• Tightens the chromatin structure
• Decreased transcription
HDAC, HAT, and HDAC inhibitors
TF = denotes transcription factor
Kim T-Y Bang Y-J,
Robertson KD:
Epigenetics 1:1 14-23,
2006
134
Histone acetylases and histone deacetylases
•
Histones
– 146 nucleotide wrap around histones = nucleosome
– H1 not involved with acetylation
– H2A, H2B, H3, H4
• Lysine-rich tails are acetylated by HATs which removes the negative charge of lysine and
allows relaxation of interaction between negative histones and the positive DNA
– Histone acetyltransferases (HATs)
• Acetylate lysine residues of histones
– This enhances transcription
– Transcription also enhanced by methylation of lysine 9 of histone 3
• Acetylation also of E2F (key to cell proliferation)
proliferation), p53
p53, GATA
– Regulates transcription of genes for cell proliferation
– Increase cell arrest, apoptosis, and cell differentitation = decreased cancer
– Histone deacetylases (HDACs)
• Deacetylate lysine residues on histones
• Transcription reduced by methylation of lysine 4 of histone 3
– May allow inhibition of tumor suppressor gene
• Repress (decrease) gene transcription by removing the charge-neutral acetyl groups
– Enhances gene transcription of other genes
– Histone deacetylase inhibitors (HDAC-I)
• Allows acetylation and thus gene transcription
• Resultingly acts like HATs to decrease cell cycling (cell arrest), increasing apoptosis, and
differentiation thus decreasing cancer
• Later this semester: Cancer therapies; HDAC-I
HDAC inhibitors (HDACi) and transcription
• Inhibition of HDAC (with HDACi) would conceiveably
increase transcription of all genes
– Due to increased acetylation and relaxed chromatin
structure
– For cancer therapy, could up regulate expression of a
tumor suppressor gene
• 20% of all genes are affected by HDACs
• In fact, acetylation increases or decreases transcription
– The ratio of up-regulated to down-regulated genes is 1:1
• Therefore, up and down regulation is equal
• The HDACis may
– Increase tumor suppressor gene expression
– inhibit cell repair of neoplastic cells and also affect
proliferation rates
• http://www.methylgene.com/HDAC_animat.swf
Acetylation effects on the cell
•
Acetylation occurs in:
– Histones, as discussed
• FYI, proteins can be acetylated, methylated, ubiquitinated, phosphorylated, ADPribosylated and deiminated.
– Transcription factors
• The acetylation occurs on lysine which is also the site of ubiquitin
– Therefore, can affect degradation
– Importin
• Import export of proteins across the nuclear envelop
– p53
• Regulating cell proliferation, repair, apoptosis
– HSP90
• Protein stability
– STAT3
• Cytokine signaling
– Microtubules
• Cell structure
– Ku70
• Allows Bax to be free
– Apoptosis
– HMGB1
• A cytokine-like protein involved with inflammation, fever, and nausea
135
Protein regulation by chaperone proteins
• Chaperones proteins:
– Constitutively expressed in normal cells
•
(1-2% of total cellular protein content)
– Function as complexes with adaptor
molecules and co-chaperone proteins
– Do not covalently modify the substrate or
“client molecules” on which they act
• Interact with client proteins via cyclical binding
pockets
HSP 90
HSP 70
HSP 70
HSP 60
HSP 10
136
Chaperones are required for essential housekeeping functions
• Chaperones mediate
– de novo protein folding during polypeptide-chain
synthesis
– translocation of protein across membranes
– Quality control in the endoplasmic reticulum
– Normal protein turnover
– Post-translational regulation of signaling molecules
– Assembly/disassembly of transcriptional complexes
– Processing of immunogenic peptides by the immune
system
a. Prevents aggregation
b. Intracellular trafficking
c. Maintains proteins in stable state for alterations such as ligand binding,
phosphorylation or multi-subunit assembly
d. Target proteins for degradation
Chaperones as Heat Shock Proteins (HSP)
• Upregulated in conditions of cellular stress
–
–
–
–
–
Heat
Hypoxia
Cellular Starvation
Radiation Exposure
Exposure to chemical mutagens
• Upregulated in tumor cells
– General hypoxic environment
– Rapid cell proliferation
– Function as biochemical buffers for extensive genetic
heterogeneity that is characteristic in tumors
137
Nature Reviews Cancer 5, 761-772 (2005); doi:10.1038/nrc1716
HSP90 AND THE CHAPERONING OF CANCER
Chaperone Proteins act on client proteins as complexes
Drug Discovery Today Vol.9, No. 20 October 2004
ATP driven conformational changes is needed for
chaperone/client protein interactions
Stryer, Biochemistry, Fourth Edition
138
HSP upregulation in tumor cells
• HSP90
– Maintaining cellular signaling protein stability (hormone
receptors and protein kinases)
• HSP70
– Stability of multi-protein complexes
• ER chaperones
– Folding and maturation of immunoglobulins and MHC
class I molecules
– Calreticulin, calnexin, tapaisin, protein disulfide
isomerase
Inhibiting HSP to treat cancer
• Several experimental protocols are being used to target
the chaperone proteins that are highly abundant in
tumor cells and use them as therapeutic targets
• HSP70 and HSP90 are the most abundant chaperone
proteins identified in tumor cells
cells, and the focus of most
research
• Many oncogene protein products are “clients” of HSP90
– ErbB2, EGFR, Bcr-Abl tyrosine kinase, Met tyrosine kinase, cRAf, b-Raf, androgen and estrogen receptors, HIF alpha and
telomerase
• Inhibition of HSP90 leads to degradation of the above
oncoproteins
Drug Inhibition of HSP90
139
RNA
• Much of the genome codes for mRNA
– Some of this is spliced out as introns
• 0.5% of the genome codes for rRNA
• 0.2% of the genome codes for tRNA
• 0.0?% codes for miRNA
– Thus, most of the supercoiled genome codes for mRNA
• A lot of the mRNA genes are not heavily transcribed
• Whereas,
Whereas rRNA and tRNA genes are often repeatedly
transcribed
• Of the transcription activity in a cell
• mRNA > rRNA > tRNA
– Of the transcripts that result in actual proteins:
• rRNA > tRNA > mRNA
• Thus much of mRNA is modified, spliced, or unstable and
does not result in protein in comparison to r and t RNA
– r and t RNA are more “routine” RNA’s and proteins
RNA polymerases
miRNA (more later)
DNA
RNA
Polymerase I
39%
Pre-RNA:
DNA polymerases
Pre-ribosomal RNA
RNA
Polymerase III
3%
snRNA Pre-messenger RNA (hn RNA)
1. Modification
(Sno RNA, nucleolus)
2 Processing
2.
(Sno RNA)
3. Self splicing
(group I intron)
RNA:
Gm7
5.8S
RNAase P Pre-transfer RNA
1. Modification
(poly A tail and 5’ cap)
2 Splicing
2.
(sn RNA)
3. Editing
(var.; gRNA)
4. Transport
5. Stability (ds RNA)
1. Modification
(base, ribose)
2 Processing
2.
(ribozyme)
3. Splicing
(enzymatic)
4. Editing
(enzymatic)
mRNA
rRNA
18S
DNA synthesis and
cell replication
RNA
Polymerase II
58%
tRNA
A (200)
28S
1%
17%
79%
Protein
140
RNA nomenclature
Pre-rRNA: precursor of ribosomal RNAs, 5.8s, 18s, 28s
Pre-mRNA: precursor of mRNAs, contains exons and introns
Pre-tRNA: precursor of tRNA, contains several tRNA
CAP:
Modified guanosine at the 5’ end of all RNA polymerase transcripts
A (200)
Polyadenylated tail of mRNA
RNAase P:
sn RNA:
catalytic (ribozyme) that processes the 5’ end of tRNA
small nuclear RNA, U1-U6, generally U-rich, pre-mRNA splicing
and pre-rRNA processing
sno RNA:
small nucleolar RNA, 200 different types involved with rRNA
modification and processing
Group I intron: self splicing RNA sequence in 28s ribosomal RNA
RNA modifications: RNA stability (cap, poly A tail), formation of tertiary structure,
interaction with RNA proteins (more later)
Transcription of rRNA and tRNA as precursors: ensures equal amounts of RNA
Pre-mRNA splicing: enlarges protein repertoire (more than alternative splicing), it has
evolutionary advantages
RNA polymerase II holoenzyme for mRNA generation
DNA arranged in a loop-like structure for
RNA polymerase II transcription
RTF
RTF
Adaptor
80
60
40
RTF-regulatory
transcription factors
250
30a
110
30b
RNA-Pol II
150
TBP
+TFIIB
+TFIIE
+TFIIF
+TFIIH
Holoenzyme
General transcription factors (GTF): TFIID, TFIIB, E, F, H
mRNA stability and transport after synthesis
•
•
•
mRNP (mRNA proteins) are adaptors of mRNA to allow it to
interface intracellular machinery for subcellular location, translation,
decay with miRNA, and signal transduction
– Some mRNP components are activators/others repressors
Most mRNP components (proteins and miRNA for degradation)
bind specific recognition elements in the untranslated 5 prime or 3
primer regions.
A few mRNP components (proteins) target:
– 7 methyguanosine cap, five prime end (CBC20/80)
– Poly A tail at three prime end
• PolyA binding protein nuclear
• PolyA binging protein cytoplasmic (eIF4E, PABP)
– 5’ end for transcription (eIF4G)
– Y box proteins
• Packing proteins along the length of mRNA
– EJC (exon junction complexes)
141
• mRNA export adaptor proteins
– Target mRNA to nuclear pore for exit
– Exportin 5
• mRNA export and fates in the
cytoplasm
– Exported by five prime end and
immediately engaged with ribosome
– Exported by non-five prime end, not
engaged with ribosome
• Transported to specific sites of cytoskeleton
• Co-localized with other mRNA
– Allows close interaction of protein subunits shortly
after translation
– Stored translationally silent
• eIF4E, CPEB, and EJC attached
mRNA moves to a cytoplasmic
site for translation; co-localizes
with other mRNA; promotes
assembly of protein units
NUCLEUS
--mRNA exported non- 5’ first
CYTOPLASM
mRNA circular and
translationally silent
mRNA strand
Nuclear pore
--mRNA exported 5’ first and translated
eIF4G
EJC (exons)
mRNA export
adaptor for
nuclear pore
Poly A binding
proteins eIF4E,
PABP; also CPEB,
EJC
Poly A binding
protein
CBC20/80
ribosome
142
mRNA degradation
• mRNA degradation
–
–
–
–
Polyadenylases degrade Poly A tail
5 prime cap removed by decapping enzymes
RNA bodyy degraded
g
by
y exonucleases
mRNA endonuclease cleavage
• Sequence specific
• occurs by RISC in association with miRNA
– Abberent mRNA’s
• Premature translation stop signal (PTC’s discussed last week)
• Lack a translation signal (nonstop RNA)
– These occur by mutation, missplicing, premature polyadenylation
• More comments on post-transcriptional regulation
later
• Stress cells and mRNA degradation (next slide)
• P bodies and Stress Granules
• P bodies (PB):
– Cytoplasmic processing bodies that form around
aggregates of RNP not involved in translation
– These RNP
RNP’s
s are targeted for PB
PB’ss by miRNA/RISC
• Stress granules
– Retirement homes
• Under stress, there is global translation arrest of “housekeeping”
gene transcripts
• Stress granules are composed of:
– Inactive mRNP’s, 40S ribosomal units, mRNA binding proteins
TIA-1 and TIAR
» TIA-1 and TIAR have prion like domains that self oligomerize
and promote assembly
– When stress relieved, stress granules disassemble
More on mRNA post transcriptional processing and it’s effect on
RNA stability
• mRNA structures that affect post
transcriptional processing
–5
5’ cap
– 5’ untranslated region (5’ UTR)
– Open reading frame (ORF)
– 3’ untranslated region (3’ UTR)
– 3’ poly adenine tail
143
RNA post-transcriptional modifications for stability or degradation
• 5’ Cap
– Methylation of cap regulates expression
– Cap prevents degradation
• Increases stability of mRNA
• 5’ UTR controls mRNA stability by
– It’s ribosomal entry site
– Actions on the 5’ UTR by upstream ORF
– 5’ polypyrimidine sequences
– RNA secondary structure
• ORF in mRNA regulate post transcriptional mRNA
stability and activity by
– Unusual codons
• These can slow translation
• 3
3’ UTRs affected by
– RNA binding proteins (RNA BP)
• Alter translation (can increase or decrease translation)
• Tag mRNA for degradation
– Decreased mRNA stabilithy
• Protect UTR from nucleases
– Increased mRNA stability
– RNA BP (CBC20/80, eIF4G, eIF4E, PABP) altered by
• Mutations of the RNA BP gene
• Increased UTR target sequences for the RNA BP to bind
• Decreased UTR target sequences for RNA BP binding
• Poly AAAAA tails
– Prevent degradation
– Long tails have efficient translation
– Short tails inefficient translation
144
• Signaling affects on stability
– MAPKK
– Increase RNA BP
• PABP-1 and TTP
• These reduce deadenylation of the poly AAAA
tail
• This increases stability and increases
translation
RNA silencing
• Small RNA’s from inside or outside the
cells are processed by iRNA machinery
to inhibit g
genes and p
proteins by:
y
– Cleaving mRNA’s
• siRNA fully complementary to mRNA
– Blocking protein synthesis
• miRNA partially matched with mRNA
– Inhibiting transcription (RITS)
• Complex enters nucleus and inhibits chromatin
mi and siRNAs
• iRNA’s
– Term for microRNA (miRNA), small
inhibitor RNA ((siRNA)) and intermediates
– 20-26 nucleotides in length
– Also: repeat-associated small interfering
RNA’s (rasiRNA)
145
miRNA
– MicroRNAs (miRNAs)
– RNA polymerase II makes miRNA
•
•
•
•
•
•
•
miRNA are double stranded
21-25 nucleotides long
Derived from short hair-pin precursors
1000 miRNA genes (non-protein encoding) currently known
400 different types
30% of human genes regulated by miRNA
Coded in introns from mRNA or snoRNA (small nucleolar RNA [sno RNA,
a.k.a.: ribosomal RNA])
– miRNAs are pieces of mRNA from the non-protein coding regions
• Pri-miRNA’s (folded)
– Come off RNA polymerase II with mRNA and bind Drosha and Pasha
– Double stranded (due to folding)
• Pre-miRNA
–
–
–
–
Formed after Drosha and Pasha cleavage
Bound to exportin, transported to cytoplasm
Cleaved by dicer
Double stranded
CYTOPLASM
NUCLEUS
RNA poly II
miRNA gene
Pri-miRNA
Pre-miRNA
(Tails released by Drosha)
Dicer
miRNA
• Dicer protein/enzyme
– Converts pre-miRNA to mi RNA by
cleaving
g off the p
pre-miRNA loop
p end
– Then eliminates one RNA strand
• 5 prime end preserved and enters RISC
complex
– Less stable, unwinds more easily
146
siRNA
• Short-intefering or silencing RNA
– Long ds RNA and mi RNA
• si RNA sources of formation:
– Endogenous
• Not fully defined
– By synthesis
– By some viruses
• Delivered for therapy by:
– Gene (viral) vectors
– Synthetic delivery
Wikipedia.com
si and mi RNA activity on gene silencing
Long ds RNA
Occurs spontaneously
Occurs with viral infection
Can occur experimentally
Argonaut proteins
Dicer
R2D2
ATP
micro ds RNA
Encoded in the genome
Single stranded si RNA
RISC is composed of:
Argonaut protein and ss RNA
Enters nucleus and inhibits
transcription
mRNA degradation if si RNA
is fully complementary
Inhibition of translation/protein synthesis if mRNA
is not fully (only partially) complementary
147
RISC
• RISC complex
– RNA induced silencing complex (RISC)
• Composed of:
– Argonaut
A
t protein
t i and
d smallll single
i l strand
t d off RNA
• Mediates:
– mRNA cleavage
» si RNA fully complementary
– Protein synthesis inhibition
» mi RNA partially mismatched
– Transcriptional gene silencing
» RITS
» A complex that enters the nucleus and affects
chromatin
•
mRNA cleavage by RISC
– RISC (siRNA and argonaut protein) plus mRNA
• siRNA is sequence specific to that mRNA
• mRNA is destroyed
•
Blockage of protein synthesis
– RISC ((mi RNA and argonaut
g
p
protein p
plus mRNA))
• mi RNA is only partially matched with mRNA
• mRNA unable to enter ribosome for translation
•
Transcriptional gene silencing
– RITS complex (RNA-induced transcriptional silencing (RITS) complex)
• Composed of:
– RISC (si RNA and argonaut protein)
– Chp1 and Tas 3 proteins
• RITS transported to the nucleus
• siRNA within the RITS
– Matches up with chromatin nucleotides
– Also binds methylated lysine 9 histones
• RITS/chromatin complex enlarges (other proteins bind)
– TRANSCRIPTION (RNA polymerase binding) prevented
How do iRNAs affect cancer?
•
miRNAs can enhance or repress mRNA translation
– miR369-3 stimulates translation during arrested cells (non-proliferating)
– miR369-3 reduces translation in dividing cells
• Science 318:1877, 2007
•
Improper activation of mRNA regulation in cancerous cells
– Oncogenic miRNAs (oncomiRNAs)
• miR-155 enhances cell proliferation
• miR17-92
iR17 92 reduces
d
c-myc apoptosis
t i actions
ti
• miR-21 inhibits TPM and apoptosis
• miRNAs may alter transcription factors, enhance
oncogenes, reduces tumor suppressor genes, and
increase angiogenesis in tumors
• American J Pathol 171:728, 2007
• Some miRNAs inhibit cancer
– miR-15 and miR-16 inhibit BCL-2 allowing apoptosis
– Let-7a inhibits RAS
148
iRNA as a therapy
•
Therapy
– siRNA delivery
• High pressure tail vein
– Recent work used low volume and normal pressure
– Problem: excretion
» There is rapid excretion
g
is less of an issue
» Degradation
• Vector delivery
– Retrovirus, adenovirus, adeno-associated virus (AAV) with various promotors
» Problems with non-specific delivery to unwanted sites, interferon reactions to the
hairpin RNA
• Benefit
– Knocking down genes with precision
» Other genes unaffected
– siRNAs are more potent and longer-lasting than oligonucleotides (antisense DNA) and
ribozymes
» siRNAs have a greater potency in turning off genes
• Hurdles:
– Delivery to the correct neoplastic cell (“either” deleted)
» This is true for systemic delivery or vector delivery
– Continued activity over time (repeated administration needed possibly)
149

Mechanisms of Cellular injury

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