nht sc2

macro
AUTOPHAGY
Upstream Autophagy Events - Induction
*1, Eroshkin A1, Mountzouris J2, Mizushima N3, Hochstrasser M4, Smith JW1, Cuervo AM5 & Klionsky DJ6
Grb
SOS
IRS1
STRAD
Ras
AMP
P
PDK1
LC3
LC3
LC3
LC3
Beclin
Beclin
P62
APG7
APG5
APG5
APG16
APG12
GAIP
P
P
ERK1/2
Akt/PKB
P+
TSC1
AMP
Metformin
AICAR
Beclin
VPS15
P
P
P
P
P+
GDP
GTP
Rheb
Rheb
P
P
P
P
P
Rapamycin
RAD-001
CCi-779
Raptor
mTOR
FkBP12
Immunosuppression
G L
Autophagy is a major self-degradative process in eukaryotic cells, with fundamental roles
in cellular and organismal homeostasis. Dysregulation of autophagy has recently been
implicated in severe human pathologies such as cancer, neurodegenerative disorders,
metabolic and muscular diseases, and viral infections. Structures targeted for autophagic
destruction are sequestered into newly emerging double-membrane vesicles called autophagosomes, and delivered for lysosomal degradation. ATG proteins form four main
multiprotein complexes: phosphatidylinositol 3-kinase (PI3K) complex, ATG13/ATG1
kinase complex, ATG9 complex, and ubiquitin-like protein conjugation systems. Current
research is aimed at understanding how these complexes interact to promote autophagy.
P
14-3-3
P+
PRAS
40
P
Rap
Beclin
Sequestration
in HD neurons
P+
P
TSC1
AMPK
Target
PRAS Raptor P
40
mTOR
Rictor
TORC1
P+
mLST
mTOR
TORC2
G L
drug
Sin1
G L
P+
mLST
P
mLST
P P P
P P P P P
PKCα
Rho
Autophagy Phases
4E-BP1 eIF4E
S6K
a
s t)
AM1800a
AP1801a
AP1802a
AP1806a
AM1818a
AP1818b
AP2183b
AP1813b
AP1812a
AP1812b
AP1817b
AP1816a
AP1820b
P
P+
Htt-p(Q)
VPS34
MEK1/2
TSC2
α
(1) Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037
(2) Abgent Inc., 6310 Nancy Ridge Dr. Ste 106, San Diego, CA 92121
(3) Department of Physiology & Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519
(4) Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520
(5) Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461
(6) Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109
(*) corresponding author: [email protected]
Nutrients
P+
Raf
P+
PTEN
PIP3
P
P+
P
Cat#
cI-PI3K
LKB1
100+ additional Ab
at www.abgent.com
Amino acids (Leu)
TSC2
www.abgent.com
RTKs
Energy stress
Wortmannin
Perifosin
LY294002*
TSC2
Glucose
MO25
ptec .com
INS/IGF
Environmental stress
www.abgent.com
www.wuxiapptec .com
g
Autopha
ATG24
P
ATG11 ATG20
ATG31
ATG29
ATG17 Cis1
ATG1
Autophagy
Antibodies
P
ATG13
V8
P
P
y Ind
on
u c ti
(ye
eIF4B
Raptor
P
P P P
mRNA translation
Cell growth
4E-BP1 eIF4E mTOR
P-
ATG13
P+
P P P
P
P eIF4B eIF3
P
V8
1.
1.
P+
Induction
Induction
(yeast)
(mammals)
G L
Preautophagosomal Structure
4.
(PAS) (yeast)
2.
P
Nucleation
7.
Retrieval
Efflux
2.
B.
C.
D.
Breakdown
3.
5.
ATG4
LC3/ R
ATG8
Calpain-1,2
tATG5
Bcl-XL
ATG7
LC3/
ATG8 *
ATG12
ATG12
*
Apoptosis
tATG5 Bcl-XL
ATG5
Autolysosome/ vacuole
Bcl-2
Bcl-2
Beclin
Beclin
Autophagy
Bcl-2/XL Inhibit Autophagy
via Beclin-1(ATG6) Binding
-
LC3/ PE
ATG8
membrane
recruitment
Cell Death Homeostasis Cell Death
ATG12
ATG10
ATG3
Hydrolases:
Ape1/Ams1 (yeast)
Thr-193
ATG5
LC3/
ATG8
Fusion
Autophagosome
Autophagy Expansion & Apoptosis
C.
# AP1817b).
ATG12 ATG5
ATG5 ATG12
ATG16 ATG16
ATG4
LC3/
ATG8
ATG12
ATG16 ATG16
ATG5
ATG5
Autophagosome
ATG12
ATG and APG are synonymous references to autophagy pathway proteins; i.e. ATG8 and APG8 name the same molecule
ATG8 Conjugation Cycle
ATG4:ATG8 Cleavage Event
ATG8
Activation by E1 enzyme (ATG7)
pro-ATG8
ATG7
α4
ATG7
G
G
+ATP
G
_
_
G
ATG4 Dual Protease
=O
=O
C-term
1
Cys -S- C
Cys -SH C -AMP
=O
C -O -
3
2
AUTL1
ATG8
118-TFG-MKLSV
pro-GATE16
114-TFG-F
pro-GBRAP
G
cleavage
GBRAP
=O
114-VYG-L
G
=O
G
O
-O
=O
=O
-O
O
O
=O
=O
=O
Ubl (Atg8) Family
0.05
SP Name (syn)
UBCP1
UFM1
HERPU
ATG12
ATG12
ATG8 (Aut7, Cvt5)
GBRL2 (GATE16)
GBRAP
GBRL3
904
GBRL1
MLP3C (LC3c)
MLP3B (LC3b)
MLP3A (LC3a)
URM1
HUB1
UBTD2
TMUB2
TMUB1
UBD (FAT10)
UBIM (FUBI)
UCRP (ISG15)
NEDD8
RUB1
UBIQ
UBIQ
SMT3
SUMO1 (Sentrin)
SUMO4
SUMO3
1000 SUMO2
Bootstrap Consensus Tree
453
401
582
249
991
999
999
996
1000
UBCP1
399
526 584
1000
260
363
504
296
901
407
910
1000
564
1000
1000
Species
H.s.
H.s.
H.s.
S.c.
H.s.
S.c.
H.s.
H.s.
H.s.
H.s.
H.s.
H.s.
H.s.
S.c.
S.c.
H.s.
H.s.
H.s.
H.s.
H.s.
H.s.
H.s.
S.c.
S.c.
H.s.
S.c.
H.s.
H.s.
H.s.
H.s.
PAM250(%)
62.4
64.8
59.3
57.7
63.1
62.6
63.3
60
60.8
61
60.4
60.7
61.5
66.6
70.4
74.2
70.6
68.7
74.6
78.3
77.8
89
83.7
99.5
100
67.9
70.7
68.8
70.2
70.2
170
180
190
200
210
E I L NP P R E G K K L L V L DV DY T L F DHR S C A E T G V E L MR P Y L HE F L T S A Y E DY D I V
MS K V S F K I T L T S - DP R L P Y K V L S V P E S T P F T A V L K F A A E E F K V P A A T S A I I
Y T P Y G W L Q L S W F Q Q I Y A R Q Y Y MQ Y L A A T A A S G A F V P P P S A Q E I P V V S A P A P A P
DE K N I Q K I Q I K F Q P I G S I G Q L K P S V C K I S MS Q S F AMV I L F L K R R L K MDH V Y C Y
D T K K K I D I L L K A V G D T P I MK T K K WA V E R T R T I Q G L I D F I K K F L K L V A S E Q L F I
NR I P V I C E K A E K S D - I P E I DK R K Y L V P A DL T VGQ F V Y V I R K R I ML P P E K A I F I
D R V P V I V E K V S G S Q - I V D I D K R K Y L V P S D I T V A Q F MW I I R K R I Q L P S E K A I F L
DR V P V I V E K AP K AR - I G DL DK K K Y L V P S DL T VGQ F Y F L I R K R I HL R AE DAL F F
D R V P L I V E K A P K A R - V P D L D R R K Y L V P S D L T DG Q F Y L L I R K R I H L R P E D A L F F
DR V P V I V E K AP K AR - V P DL DK R K Y L V P S DL T VGQ F Y F L I R K R I HL R P E DAL F F
NK I P V V V E R Y P R E T F L P P L DK T K F L V P Q E L T MT Q F L S I I R S R MV L R A T E A F Y L
T K I P V I I E R Y K G E K Q L P V L D K T K F L V P D H V NM S E L I K I I R R R L Q L N A NQ A F F L
S K I P V I I E R Y K G E K Q L P V L D K T K F L V P D H V NM S E L V K I I R R R L Q L N P T Q A F F L
- - - R V HK I K MDK E DP V T VG DL I DH I V S T M I NNP NDV S I F I E DDS I R P G I I T L I
M I E V V V NDR L G K K V R V K C L AE DS VG DF K K V L S L Q I G TQ P NK I V L Q
E P P P N S G Y E C Q L R L R L S T G K D L K L V V R S T D T V F HM K R R L H A A E G V E P G S Q R WF
T C L P P S P G L I T V R L K F L NDT E E L A V A R P E DT VG A L K S K Y F P - - G - Q E S QMK L I
- - - P Q E P - - L V L R L K F L N D S E Q V A R AWP H D T I G S L K R T Q F P - - G - R E Q Q V R L I
- P S DE E L P L F L V E S G DE A K R H L L Q V R R S S S V AQ V K AM I E T K T G I I P E T Q I V T
MQ L F V R - - A Q E L H T F E V T G Q E T V A Q I K A H V A S L E G I A P E D Q V V L
- - V D K C D E P L S I L V R N N K G R S S T Y E V R L T Q T V A H L K Q Q V S G L E G V Q D D L F WL T
ML I K V K T L T G K E I E I D I E P T DK V E R I K E R V E E K E G I P P QQQ R L I
M I V K V K T L T G K E I S V E L K E S DL V Y H I K E L L E E K E G I P P S QQ R L I
MQ I F V K T L T G K T I T L E V E S S D T I D N V K S K I Q D K E G I P P D Q Q R L I
MQ I F V K T L T G K T I T L E V E P S D T I E N V K A K I Q D K E G I P P D Q Q R L I
- - E V K P E T H I N L K V S - DG S S E I F F K I K K T T P L R R L M E A F A K R Q G K E M D S L R F L
DK K E G E Y I K L K V I G Q DS S E I HF K V K MT T HL K K L K E S Y CQ R QG V P MNS L R F L
T E N N - N H I N L K V A G Q DG S V V Q F K I K R Q T P L S K L M K A Y C E P R G L S M K Q I R F R
T E N - - D H I N L K V A G Q DG S V V Q F K I K R H T P L S K L M K A Y C E R Q G L S M R Q I R F R
T E N N - D H I N L K V A G Q DG S V V Q F K I K R H T P L S K L M K A Y C E R Q G L S M R Q I R F R
220
I WS A T NM K W I T N DG I G I N P - I H NQ F P A E NQ P
I NN - S F AP S P Y V NQ S F A P S P F V N - DT L P P T F VD - K T VPQS F V N - NV I P P T F V N - NT I P P T F V N - NT I P P T L V NNK S L V SM L V NG H S M V S V L V NQ H S M V S V N D T DWE L E G - KGG - S V L K D - F SG -RPL T D - YQG - R L L Q DP YQG - Q L L G DD C NG - K R L E D - L AG - A P L E D - F EG -KPL E D - Y S G - K QMND - F QG - KQ I DD - F AG - K Q L E D - F AG - K Q L E D - Y DG - I R I Q A - F EG -QR I AD - FGG -QP I SG - F DG - Q P I N E - F DG - Q P I N E - -
7
12
6
4
B
PL
His-280
160
8
D
10 H
Asp-278
cleavage
UBCP1_HUMAN
RRVKE YKV
UFM1_HUMAN
HERPU_HUMAN
G LG PG F SG
ATG12_YEAST
T S KQK S HK
ATG12_HUMAN
- - - - - - - - ATG8_YEAST
- - - - - - - - GBRL2_HUMAN
- - - - - - - - GBRAP_HUMAN
- - - - - - - - GBRL3_HUMAN
- - - - - - - - GBRL1_HUMAN
- - - - - - - - MLP3C_HUMAN
- - - - - - - - MLP3B_HUMAN
- - - - - - - - MLP3A_HUMAN
- - - - - - - - URM1_YEAST
- - - - - - - - HUB1_YEAST
UBTD2_HUMAN
S D I E T L D I P
TMUB2_HUMAN
E AP L R S E DS
TMUB1_HUMAN
- - - - - AP DS
UBD_HUMAN
KVVK - - - - UBIM_HUMAN
UCRP_HUMAN
- - - - - - - - NEDD8_HUMAN
RUB1_YEAST
UBIQ_YEAST
UBIQ_HUMAN
SMT3_YEAST
- - - - - - - - SUMO1_HUMAN
SUMO4_HUMAN
SUMO3_HUMAN
SUMO2_HUMAN
9
ATG7 (E1)
delipidation
Pre-autophagosome structure (PAS) formation & Autophagosome
292
11
Cleavage
PL
PE-Ligation
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San Diego, CA 92124, USA
Tel: 858.622.0099
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114-VYG
NH 2
O-P-O
=O
=O
N-
O-P-O
O
114-TFG
ATG3 (E2)
-O
N-
_
=O
C
O-P-O
118-TFG
ATG4B
ATG4B
=O
=O
GATE16
ATG8-PE
ATG8-PE
O
LC3
proteolytic
C-t
erm
pro-LC3
ATG3
Cys -S- C _
G
Papain-like domain
ATG4A
G
C
α3
N
ATG8 orthologs
_
ATG4B
α1
4
Proteases
O
interactive surface
α2
ATG4B
G
G
6.
Expansion
B.
Western blot
analysis of LC3 antibody
(Abgent, Cat. # AP1801a)
in rat brain lysate.
Lipidated (arrow, II) and
non-lipidated LC3 (arrow,
I) detected in membrane
fraction (P); only nonlipidated APG8a detected
in soluble fraction (S).
D. Mouse leukaemic monocyte macrophage cells treated with U18666A, a drug that causes cholesterol and lipid storage in cells, thereby block
ing fusion between late endosomes and lysosomes. LC3 antibody (Abgent, Cat. # AP1806a) detected punctuate staining indicative of autophagic
vacuole/phagosomal structures.
Nucleation
Isolation Membrane
or Phagophore
Cell Survival
A.
A. Mouse cerebellar
cell lines stably express ing human GFP-LC3 fu sion protein evaluated
by immunocytochem istry using LC3 anti body (Abgent, Cat. #
AM1800a). Punctuate
autophagic cell vesicles
, with numerous GFP-LC3
vesicles are displayed.
Permeases
230
240
250
260
- - E A K MK E L G V S T NA NY K I T F ML DS A AM I T V H T P R R G
- - AQ T AG N V F L K HG S E L R I I P R D R V - - - - G S C
A NQ N A A P Q V V V N P G A NQ N L R M N A Q G G P I V E E D D E I N
- - Q Q N I G E L WMQ F K T N D E L I V S Y C A S V A F G
- - D Q E V G T L Y E C F G S D G K L V L H Y C K S Q A WG
- - A A L M S A I Y Q E H K D K DG F L Y V T Y S - - - - G E N T F G R
- - S L T MG Q L Y E K E K D E D G F L Y V A Y S - - - - G E N T F G F
- - S A T MG Q L Y Q E H H E E D F F L Y I A Y S - - - - D E S V Y G L
- - S A T MG Q L Y E D S H E E D D F L Y V A Y S - - - - N E S V Y G K
- - S A T MG Q L Y E D N H E E D Y F L Y V A Y S - - - - D E S V Y G K
- - S A T M A E I Y R D Y K D E DG F V Y M T Y A - - - - S Q E T F G C L
- - S T P I S E V Y E S E K D E DG F L Y M V Y A - - - - S Q E T F G M K
- - S T P I A D I Y E Q E K D E DG F L Y M V Y A - - - - S Q E T F G F
- - - - - E K D Y I L E DG D I I S F T S T L HG - - - - G
- - H I S L E D Y E V H DQ T N L E L Y Y L
- - K MK F E E L K I P K DY V VQ V I V S Q P VQ NP T P V E N
- - AR T L R S L N I T DNC V I HC HR S P PG S AV PG P S AS L A
- - TQT L G S L HL P P NC V L HC HV S T R - - - - VG P P NP P C
- - G K MM A D Y G I R K G N L L F L A S Y C I G - - - - G
- - E A T L G Q CG V E A L T T L E V AG R ML G - - - - G
- - Q L P L G E Y G L K P L S T V F MNL R L R G - - - - GG T E P G
- - E K T AADY K I L GG S V L HL V L AL RG - - - -GGG L RQ
- - K L T V T D A H L V E G MQ L H L V L T L R G - - - - G N
- -G R T L S DY N I Q K E S T L HL V L R L RG - - - -G
- -G R T L S DY N I Q K E S T L HL V L R L RG - - - -G
- - DQ T P E D L DM E D N D I I E A H R E Q I G - - - - G A T Y
- - NHT P K E L GME E E DV I E V Y Q E Q T G - - - - G HS T V
- - T DK P AQ L E ME DE DT I DV F QQ P T G - - - - G V Y
- - T DT P AQ L E ME DE DT I DV F QQQ T G - - - - G V P E S S L
- - T DT P AQ L E ME DE DT I DV F QQQ T G - - - - G V Y
ATG8 Orthologs
UBIQ Orthologs
H.s. - Homo sapiens (Human)
S.c. - Saccharomyces cerevisiae (Baker’s yeast)
PAM250 (Accepted Point Mutations), Mutational matrix corresponding to a level of evolutionary change, measured by the frequency of substitutions/mutations for proteins, which have diverged 250% (250 mutations per 100 amino acids) [Ref.25].
Bootstrap
reliable from a statistical point of view [Ref. 26]. The bootstrap values on each interior branch of this tree presented at the above show the number of trees matching out 1,000 possible trees.
Cys-74
3
C
C
N-term
Y
A
1
2
E
D
13
Tyr-54
Catalytic triad
C-term
I
5
F
H
Fingers domain (HAUSP-like domain)
Abbreviations (Synonyms):
References:
AKT, RAC-alpha serine/threonine-protein kinase (C-AKT, RAC-PK-alpha, Protein kinase B, PKB);
AMP, Adenosine monophosphate (5’-adenylic acid); ATP, Adenosine 5’-triphosphate (5-(6aminopurin-9-yl)-3,4-dihydroxy-oxolan-2-yl methoxy-hydroxy-phosphoryloxy-hydroxy-phosphoryl
oxyphosphonic acid)); AMPK, 5’-AMP-activated protein kinase catalytic subunit alpha-1 (AMPK
alpha-1 chain); ATG4B, Cysteine protease ATG4B (Autophagin-1, hAPG4B, AUTL1); ATG8,
BCL2, Apoptosis regulator
Bcl-2; eIF4B, Eukaryotic translation initiation factor 4B; ERK1, Extracellular signal-regu lated kinase 1 (MAPK3, MAPK 1, p44-MAPK, ERT2, p44-ERK1, PRKM3); FkBP12, Immunophilin
FKBP12.6 (12.6 kDa FKBP, Rotamase 1B, FK506-binding protein 1B, FKBP12.6, FKBP1L, FKBP9, OTK4);
GBRAP, Gamma-aminobutyric acid receptor-associated protein (GABARAP, MM46); GBRL2,
Gamma-aminobutyric acid receptor-associated protein-like 2 (GEF-2, FLC3A, GATE16); GDP, Guanosine diphosphate (Guanosine 5’-(trihydrogen diphosphate)); Grb, Growth factor receptor-bound
protein; GTP, Guanosine 5’-triphosphate (2-amino-6-oxo-purine-5’-triphosphate); IGF, Insulin
growth factor; Htt, Huntington (HD) disease protein; MLP3C, Microtubule-associated proteins
1A/1B light chain 3C (Autophagy-related protein LC3 C); mTOR, Mammalian target of rapamy cin (FRAP, RAPT1); NEDD8, Neddylin (Ubiquitin-like protein Nedd8); P+, monophosphate;
PKC, Protein kinase C; PRAS40, Proline-rich AKT1 substrate 1 (40 kDa AKT1S1); PP2A,
Serine/threonine-protein phosphatase 2A; Raf, RAF proto-oncogene serine/threonine-protein ki nase (Raf-1, cRaf); RAPTOR, Regulatory-associated protein of mTOR; Rheb, Ras homolog
enriched in brain (GTP-binding protein Rheb); S6K, Ribosomal protein S6 kinase alpha-1 (S6Kalpha 1, p90S6K, RSK-1, p90-RSK 1); SUMO1
Ubiquitin-like protein SMT3C, GAP-modifying protein 1, UBL1, PIC1, GMP1); TSC1, Hamartin
(Tuberous sclerosis 1 protein); TSC2, Tuberin (Tuberous sclerosis 2 protein); UBIQ, Ubiquitin;
VPS34, Vacuolar protein sorting-associated protein 34 (END12, PEP15, VPL7, VPT29); VPS15,
Vacuolar protein sorting-associated protein 15 (GRD8, VAC4, VPL19).
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3. Cuervo AM (2004) Trends Cell Biol. 14(2):70-7 Review
4. Levine B & Klionsky DJ (2004) Dev Cell. 6(4):463-77 Review
5. Klionsky DJ (2005) J Cell Sci. 118(Pt 1):7-18 Review
6. Hochstrasser M (2000) Nat Cell Biol. 2(8):E153-7 Review
7. Kerscher O et al. (2006) Annu Rev Cell Dev Biol. 22:159-80 Rev Ubl
8. Haar EV et al. (2007) Nat Cell Biol. Feb 4 [Epub ahead of print] PRAS40
9. Arsham AM & Neufeld TP (2006) Curr Opin Cell Biol. 18(6):589-97 Rev TOR
10. De Virgilio C & Loewith R (2006) Int J Biochem Cell Biol. 38(9):1476-81 Rev TOR
11. Mikalsen T et al. (2006) Biotechnol Annu Rev. 12:153-223 Rev TOR-inh
12. Proud CG (2004) DNA Repair (Amst). 3(8-9):927-34. Rev TOR
13. Krymskaya VP (2003) Cell Signal. 15(8):729-39 Rev TSC
14. Reggiori F & Klionsky DJ (2002) Eukaryot Cell. 1(1):11-21 Rev Ape/Ams
15. Furuya N et al. (2005) Autophagy. 1(1):46-52 Vps/Beclin
16. Kanazawa T et al. (2004) J Biol Chem. 279(9):8452-9 TOR-AA
17. Pattingre S. et al. (2005) Cell 122(6):927-39 (Beclin).
18. Kumanomidou T. et al. (2006) J Mol Biol. 355(4):612-8 (Atg4b).
19. Sugawara K. et al., (2005) J Biol Chem. 280(48):40058-65 (Atg4b).
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