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Mitochondrial Protein Complexes and Disease
Dr Matthew McKenzie (Head)
Chern Lim (Postdoctoral Researcher)
Abena Nsiah-Sefaa (PhD Student)
Monash Institute of Medical Research (MIMR) +
Prince Henry’s Institute (PHI)
= MIMR-PHI Institute of Medical Research
Mitochondria: Powerhouses of the Cell
• How do the mitochondria function?
• ~1,100 proteins involved
• What goes wrong in mito?
• Design of new therapies / drugs to fix the
defects
Ferrari engine
1,078 parts
Mitochondria burn sugars and fats to generate ATP
• Metabolism of sugars requires 5
protein complexes:
• I, III, IV and V
• Oxidative Phosphorylation
(OXPHOS)
• Each complex is made of multiple
subunits
• Defects in OXPHOS cause Mito
Complex I
(44 subunits)
How are the Complexes Assembled?
Separate the protein complexes on a gel
(Blue Native Polyacrylamide Gel Electrophoresis: BN-PAGE)
Isolated mitochondria
from patient skin cells
I II III IV
Separate by size on
gel for analysis
Use detergent to
make a protein
‘mixture’
I
V
V
OXPHOS Complexes
III
IV
II
We can detect all 5
OXPHOS Complexes
Loss of Complex I in Mitochondrial Disease
CI
Prof. Mike Ryan
Monash University
CV
CIII
CIV
Prof. David Thorburn
MCRI
Patient Study
• Siblings (male and
female)
• Leigh Syndrome
• Examine patient
mitochondria on BNPAGE
C=control
P=patient
C
P
C
P
C
P
C
P
We can determine which complexes are affected in Mito Disease
How are the Complexes Assembled?
• Complex I assembly is complex!
• Sub-complexes (green) are assembled together via a number of discrete stages
• Requires additional proteins (colour) that help the assembly process (assembly
factors) – 12 known
• Defects in the subunits (18) or assembly factors (9) can cause Mito disease
Mitochondria burn sugars and fats to generate ATP
• Metabolism of fats requires at least
25 proteins
• Mitochondrial fatty acid b-oxidation
(FAO)
• Defects in FAO can cause Mito
Medium Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency
Prof. Akira Ohtake
Saitama University, Japan
•
Apnea from birth
•
vomiting, disturbed consciousness at 15 months of age
•
Lymphocyte (white blood cell) MCAD activity 3.2% of
controls
*
MCAD protein is missing
Medium Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency
MCAD Deficiency: Are the OXPHOS complexes affected?
BN-PAGE
FAO protein defect results in OXPHOS Complex I Defect; Important Interactions
between two pathways
New Models of FAO Disease
Take mito patient skin cells: generate induced pluripotent stem (iPS) cells
FAO patient skin cells
FAO patient iPS cells
Pluripotent markers
Prof. Jus St John
MIMR-PHI
Oct4, Sox2
c-Myc, Klf-4
Oct4
Differentiate into neuronal cell types
Nestin
neural rosette
bIII-Tubulin
early neurons
GFAP
astrocytes
MAP2
mature neurons
Mitochondrial calcium buffering capacity
Mitochondrial
membrane potential
(Dym):
TMRM (red)
Calcium:
Fluo-4 (green)
Tra-160
Differentiate into cardiomyocytes
Mouse Models of Mitochondrial Disease
A/Prof. Ian Trounce
CERA
• Make mouse models of Mito disease
• Understanding of how the disease affects specific organs /
tissues
• Design and testing of new therapies
• Swap mitochondrial DNA (mtDNA) in mouse embryonic stem
(ES) cells
• Grow mice from ES cells with new, swapped mtDNA
Making Mito Mouse Models
ES cells with
‘new’ mtDNA
Mouse embryo
with 100s cells
B
A
B
A
Figure 3.4.8: A-Founder female chimeric Mus dunni xenocybrid mouse Figure 3.4.8: A-Founder female chimeric Mus dunni xenocybrid mouse
(#1, generated from Mus dunni xenocybrid ES clone 7) used for (#1, generated from Mus dunni xenocybrid ES clone 7) used for
breeding. This mouse was determined to be ~90% chimeric by coat breeding. This mouse was determined to be ~90% chimeric by coat
colour. B-Wild type Mus musculus C57BL/6 mouse.
colour. B-Wild type Mus musculus C57BL/6 mouse.
*
Mitochondrial Defects in Mito Mouse Model
•
Age-related Disease Model
•
Loss of Substantia Nigra dopaminergic
neurons (as in Parkinson’s disease)
•
Subtle motor impairment with aging
Mitochondrial Oxygen
Consumption in Mouse Brain
Use technique to generate new mice with
other forms of Mito Disease
(Funding from AMDF)
Accelerated decline in Mito mice brain
respiration at 24 months of age
Acknowledgements
McKenzie Research Group
Chern Lim
Abena Nsiah-Sefaa
Kirsty Carey
Complex I Assembly
Michael Ryan (Monash University)
David Thorburn (MCRI)
Xenomouse Models
Ian Trounce (CERA)
Carl Pinkert (Auburn)
Zane Andrews (Monash)
FAO Disorders
Justin St. John (MIMR-PHI)
Avihu Boneh (MCRI)
Akira Ohtake (Saitama University)
Kei Murayama (Chiba Children’s
Hospital)
Funding
AMDF
Australian Research Council
Monash University
William Buckland Foundation
Ophthalmic Research Institute Of
Australia (ORIA)
Carbohydrate metabolism: Glycolysis
Carbohydrate: amylase digestion
(saliva, small intestine)
Monosacharides: transported across
intestinal wall: glycogen or
metabolism
Glycolysis
1 glucose molecule - 2 ATP
(~30 from OXPHOS)
Mitochondrial fatty acid b-oxidation (FAO)
•
Fats stored as triacylglycerol in adipose tissue
–
metabolized by lipases (activated by glucagon, epinephrine or β-corticotropin via cAMP and PKA)
palmitic acid (C16:0)
oleic acid (C18:1)
b
alpha-linolenic acid (C18:3)
glycerol
Fatty acids
-Free fatty acids enter circulation; bound to albumin
-enter cells via fatty acid transporters (FATs, eg. FAT/CD36)
-activated in cytoplasm to a fatty-acyl CoA ester
-palmitate yields ~129 ATP
Hardie D G , Sakamoto K Physiology 2006;21:48-60
Metabolic changes induced by AMP-activated protein kinase (AMPK) in muscle, including
stimulation of glucose and fatty acid uptake, fatty acid oxidation, mitochondrial biogenesis,
inhibition of glycogen synthesis and muscle hypertrophy via inhibition of TOR
Glucose-Fatty Acid (Randle) Cycle
malonyl-CoA decarboxylase
(ACC) acetyl-CoA
carboxylase
(ACL) ATPcitrate lyase
phosphofructokinase-1
PDH kinases
Ketone synthesis
•
Acetyl-CoA generated in the liver can be converted to ketones: acetoacetate, βhydroxybutyrate, and acetone
only in liver
not in liver!
Lactic Acidosis in mitochondrial disease
Glycolysis requires NAD+
glucose
pyruvate
NAD+
TCA
cycle
acetylCoA
Pyruvate
dehydrogenase
NADH
NADH
NAD+
NADH
NAD+
lactate
NADH
[NADH]
NADH oxidized to NAD+ by
complex I. If a defect is
present then NADH
accumulates
X
X
NAD+
Excess NADH oxidized to
NAD+ by reduction of
pyruvate to lactate
Complex I
H+
Fatty acid oxidation defects…Lorenzo’s Oil
•
Lorenzo Odone (1979-2008):
– Adrenoleukodystrophy (ALD)
– Progressive damage to brain, peripheral
nervous system, adrenal glands (myelin
damage)
– loss of hearing, speech, sight, paralysis
– over-accumulation of very-long chain fatty acids
(VLCFAs, C25-C30)
– X-linked: ABCD1 gene; peroxisome b-oxidation
of VLCFAs (transporter)
– Augusto and Michaela Odone devised
treatment! (Hugo Moser)
– Treat with triacylglycerides of oleic acid (C18:1)
and erucic acid (22:1)