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Autophagy Events - Induction Macro www.abcepta.com Upstream Autophagy Events - Induction macro INS/IGF Environmental stress Wortmannin Glucose Perifosin Amino acids (Leu) Energy stress R LY294002* TKs AUTOPHAGY *1, Eroshkin A1, Mountzouris 2J, Mizushima N3, Hochstrasser M4, Smith JW1, Cuervo AM5 & Klionsky DJ6 Grb cI-PI3K PTEN SOS (1) Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 MO25 STRAD IRS1 Ras Nutrients (2) Abcepta Inc., 10320 Camino Santa Fe Ste G, San Diego, CA 92121 LKB1 PIP3 (3) Department of Physiology & Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519 (4) Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520 P Htt-p(Q) (5) Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 AMP P+ Raf P+ Beclin Beclin (6) Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109 P P+ (*) corresponding author: [email protected] P P VPS34 Sequestration α MEK1/2 PDK1 VPS15 in HD neurons P+ P+ utophagy is a major self-degradative process in eukaryotic cells, with fundamental roles 100+ additional Ab P P A at www.abgent.com AMPK ERK1/2 Akt/PKB in cellular and organismal homeostasis. Dysregulation of autophagy has recently been AMP Metformin implicated in severe human pathologies such as cancer, neurodegenerative disorders, Cat# Target P P P AICAR P+ P+ P P SC2 SC1 SC2 SC1 SC2 T T T AM1800a LC3 T T P P metabolic and muscular diseases, and viral infections. Structures targeted for autophagic P P P P AP1801a LC3 destruction are sequestered into newly emerging double-membrane vesicles called auto- 14-3-3 AP1802a LC3 GDP GTP P+ Rheb Rheb phagosomes, and delivered for lysosomal degradation. ATG proteins form four main AP1806a LC3 PRAS multiprotein complexes: phosphatidylinositol 3-kinase (PI3K) complex, ATG13/ATG1 AM1818a Beclin P 40 Raptor Rapamycin Raptor Rictor Sin1 AP1818b Beclin Rap RAD-001 PRAS P kinase complex, ATG9 complex, and ubiquitin-like protein conjugation systems. Current CCi-779 40 AP2183b P62 FkBP12 mTOR mTOR mTOR research is aimed at understanding how these complexes interact to promote autophagy. TORC1 TORC2 AP1813b APG7 Immunosuppression G L G L G L AP1812a APG5 drug mLST P+ mLST P+ mLST P AP1812b APG5 Rho PKC α P P P P P P P P AP1817b APG16 S6K 4E-BP1 eIF4E ) st AP1816a APG12 ea (y Autophagy Phases ion AP1820b GAIP uct Ind phagy eIF4B Auto P+ Raptor P P ATG20 ATG24 1. 1. ATG11 P P P mRNA translation Induction ATG31 4E-BP1 eIF4E mTOR Induction ATG29 Cis1 Cell growth (mammals) (yeast) P ATG17 G L Autophagy P ATG1 P eIF4B eIF3 P ATG13 P- P+ ATG13 Preautophagosomal Structure V8 P P P P V8 P Antibodies P 2. 4. (PAS)(yeast) 7. Nucleation Retrieval Efflux 2. Permeases A. Mouse cerebellar Nucleation cell lines stably express- Isolation Membrane A. B. ing human GFP-LC3 fu- 6. sion protein evaluated or Phagophore Breakdown by immunocytochem- istry using LC3 anti- 3. 5. body (Abgent, Cat. # Expansion Fusion AM1800a). Punctuate autophagic cell vesicles , with numerous GFP-LC3 Autophagosome vesicles are displayed. Autophagy Expansion & Apoptosis C. D. LC3/ B. Western blot R Calpain-1,2 analysis of LC3 antibody ATG8 Hydrolases: ATG4 Thr-193 (Abgent, Cat. # AP1801a) Ape1/Ams1 (yeast) in rat brain lysate. ATG5 tATG5 Lipidated (arrow, II) and Cell Death Homeostasis Cell Death Bcl-XL non-lipidated LC3 (arrow, LC3/ ATG12 I) detected in membrane ATG8 Autolysosome/vacuole fraction (P); only non- ATG7 Apoptosis al tATG5 Bcl-XL v Bcl-2 Bcl-2 Beclin vi lipidated APG8a detected ATG10 r Beclin in soluble fraction (S). LC3/ ATG12 ATG12 Cell Su C. ATG8 * * ATG5 Autophagy # AP1817b). ATG3 Bcl-2/XL Inhibit Autophagy D. Mouse leukaemic monocyte macrophage cells treated with U18666A, a drug that causes cholesterol and lipid storage in cells, thereby block- via Beclin-1(ATG6) Binding ing fusion between late endosomes and lysosomes. LC3 antibody (Abgent, Cat. # AP1806a) detected punctuate staining indicative of autophagic LC3/ PE ATG12 ATG12 vacuole/phagosomal structures. ATG8 ATG5 ATG5 ATG16 ATG16 ATG4 Autophagosome membrane ATG16 ATG16 recruitment ATG5 LC3/ ATG12 ATG5 ATG8 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 Activation by E1 enzyme (ATG7) ATG8 pro-ATG8 interactive surface α2 ATG7 ATG7 α4 G ATG4B G G G_ G G +ATP _ - - 1 C=O O Cys -SH Cys -S- C=O C=O -AMP ATG4B 3 4 α1 C-term ATG4 Dual Protease α3 Proteases 2 Papain-like domain AUTL1 ATG8 N G ATG8 orthologs ATG4A G pro-LC3 LC3 ATG3 118-TFG-MKLSV 118-TFG ATG3 pro-GATE16 proteolytic GATE16 m 11 9 8 7 12 6 114-TFG-F 114-TFG er pro-GBRAP cleavage GBRAP C-t _ G Cleavage 4 Cys -S- C=O 114-VYG-L 114-VYG ATG7 (E1) ATG8-PE ATG8-PE ATG4B D ATG3 (E2) _ ATG4B _ G G NH2 10 H 1 =O C=O C =O PL N-term O-P-O- - - O N N O delipidation =O =O O Asp-278 =O 3 Y A 2 O-P-O- O-P-O- O O cleavage PL B C O O =O O O =O =O E =O =O His-280 C D 13 5 PE-Ligation Tyr-54 160 170 180 190 200 210 220 230 240 250 260 F H UBCP1_HUMANR R V K E Y K V E I L N P P R E G K K LLV L D V D Y T L F D H R S C A E T G V E L M R P Y L H E F L T S A Y E D Y D I V I W S A T N M K W I --- E A K M K E L G V S T N A N Y K I T F M L D S A A M I T V H T P R R G Cys-74 UFM1_HUMAN M S K V S F K I T L T S - D P 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 T N D G I G I N P ---- A Q T A G N V F L K H G S E L R I I P R D R V - --- G S C Catalytic triad C-term HERPU_HUMANG L G P G F S G 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 I H N Q F P A E N Q P A N Q N A A P Q V V V N P G A N Q N L R M N A Q G G P I V E E DDE I N ATG12_YEASTT S K Q K S H K D E 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 M S Q S F A M V I L F L K R R L K M D H V Y C Y I N N - S F A P S P --- 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 ATG12_HUMAN--------- D T KKK I D I LLK A V G D T P I M K T K K W A V E R T R T I Q G L I D F I KKF L K L V A S E Q L F I Y V N Q S F A P S P --- 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 I ATG8_YEAST--------- N R I P V I C E K A E K S D - I P E I D KRKY L V P A D L T V G Q F V Y V I R K R I M L P P E K A I F I F V N - D T L P P T --- A A L M S A I Y Q E H K D K D G F L Y V T Y S - --- G E N T F G R GBRL2_HUMAN--------- D R V P V I V E K V S G S Q - I V D I D KRKY 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 F V D - K T V P Q S --- 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 GBRAP_HUMAN--------- D R V P V I V E K A P K A R - I G D L D KKKY L V P S D L T V G Q F Y F L I R K R I H L R A E D A L FFF V N - N V I P P T --- S A T MG Q L Y Q E H H E E D FFL Y I A Y S ---- D E S V Y G L GBRL3_HUMAN--------- D R V P L I V E K A P K A R - V P D L D RRKYL V P S D L T D G Q F Y L L I R K R I H L R P E D A L FFF V N - N T I P P T --- S A T MG Q L Y E D S H E E DDF L Y V A Y S ---- N E S V Y G K GBRL1_HUMAN--------- D R V P V I V E K A P K A R - V P D L D KRKY L V P S D L T V G Q F Y F L I R K R I H L R P E D A L FFF V N - N T I P P T --- 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 MLP3C_HUMAN--------- N K I P V V V E R Y P R E TFL P P L D K T K F L V P Q E L T M T Q F L S I I R S R M V L R A T E A F Y LLV N N K S L V S M --- S A T M A E I Y R D Y K D E D G F V Y M T Y A ---- S Q E T F G C L Pre-autophagosome structure (PAS) formation & Autophagosome MLP3B_HUMAN--------- 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 N M S E L I K I I R R R L Q L N A N Q A F F LLV N G H S M V S V --- S T P I S E V Y E S E K D E D G F L Y M V Y A ---- S Q E T F G M K Fingers domain (HAUSP-like domain) MLP3A_HUMAN--------- 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 N M S E L V K I I R R R L Q L N P T Q A F F LLV N Q H S M V S V --- S T P I A D I Y E Q E K D E D G F L Y M V Y A ---- S Q E T F G F URM1_YEAST------------ R V H K I K M D K E D P V T V G D L I D H I V S T M I N N P N D V S I F I E DDS I R P G I I T L I N D T D W E L E G ------- E K D Y I L E D G D I I S F T S T L H G - --- G HUB1_YEAST M I E V V V N D R L G KKV R V K C L A E D S V G D F KKV L S L Q I G T Q P N K I V L Q K G G - S V L K D ---- H I S L E D Y E V H D Q T N L E L YYL UBTD2_HUMANS D I E T L D I P 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 H M K R R L H A A E G V E P G S Q R W FFS G - R P L T D ---- K M K F E E L K I P K D Y V V Q V I V S Q P V Q N P T P V E N TEMUB2_HUMANA P L R S E D S T C L P P S P G L I T V R L K F L N D T E E L A V A R P E D T V G A L K S K Y F P - - G - Q E S Q M K L I Y Q G - R L L Q D P --- A R T L R S L N I T D N C V I H C H R S P P G S A V P G P S A S L A TMUB1_HUMAN----- A P D S --- P Q E P - - L V L R L K F L N D S E Q V A R A W P H D T I G S L K R T Q F P - - G - R E Q Q V R L I Y Q G - Q L L G D D --- T Q T L G S L H L P P N C V L H C H V S T R - - - - V G P P N P P C UBD_HUMANK V V K ------ P S D E E L P L F L V E S G D E A K R H L L Q V R R S SSV A Q V K A M I E T K T G I I P E T Q I V T C N G - K R L E D ---- G K MM A D Y G I R K G N LLF L A S Y C I G ---- G UBIM_HUMAN 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 LLA G - A P L E D ---- E A T L G Q C G V E A L T T L E V A G R M L G - --- G UCRP_HUMAN----------- 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 DDL F W L T F E G - K P L E D ---- Q L P L G E Y G L K P L S T V F M N L R L R G - --- G G T E P G NEDD8_HUMAN M L I K V K T L T G K E I E I D I E P T D K V E R I K E R V E E K E G I P P QQQ R L I Y S G - K
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