Sestrin2-mediated signaling in autophagy induction and oxidative metabolism

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1 Sestrin2-mediated signaling in autophagy induction and oxidative metabolism May 16, 2017 Graduate Course 2214/BioC 998 SEUNG-HYUN RO Redox Biology Center University of Nebraska - Lincoln 1

2 Part I : ULK1-Sestrin2 signaling in autophagy induction Part II : Sestrin2 regulation of oxidative metabolism 2

3 Yoshinori Ohsumi Nobel Prize winner in Physiology or Medicine - Dr. Ohsumi's work provided the basis for understanding autophagy, or how cells degrade and recycle their molecular trash I don t feel comfortable competing with many people, and instead I find it more enjoyable doing something nobody else is doing. In a way, that s what science is all about, and the joy of finding something inspires me. Dr. Yoshinori Ohsumi from Tokyo Institute of Technology 3

4 Christian de Duve - Rockfeller University, Nobel Prize (1974) 1955 Discovery of Lysosome by EM analysis 1962 Invented scientific name, Autophagy Auto (Self) Phagy (Eating) Lysosome vacuole in yeast 4

5 Autophagy Catabolic process induced by starvation, whereby cells digest their own constituents as a mechanism of cell survival or cell death 5

6 Physiological functions of Autophagy Defense mechanism for starvation and metabolic stress Cellular energy homeostasis control Cell survival, growth and differentiation Defense mechanism for diseases and aging Levine et al, Cell, 2008 Nobelförsamlingen at Karoliska,

7 7

8 Mitophagy 1. LC3 associated (Ubiquitinindependent) mitochondrial autophagy 2. Pink1/Parkin associated mitophagy Nix (Bnip3 like protein) Bnip3 (BCL2 interacting protein 3) FUNDC1 (Fun14 domain containing protein1) Pink1 (PTEN induced putative kinase 1) Parkin (E3 ubiquitin protein ligase) Saito et al, Cir Res,

9 Lipophagy occurs in 3T3-L1 adipocyte in response to starvation +Serum -Serum (2h) - lysotracker + lysotracker Green-Lipid Blue-DAPI 9

10 Autophagic vacuoles - Electron microscopy in HeLa cancer cells 10 mm Dr. Richard Kessin lab at Columbia U. Hosokawa et al. (2009) MBC, from Noboru Mizushima lab 10

11 Human diseases associated with defective autophagy Morel et al, Annu Rev Pharmacol Toxicol,

12 Autophagy and mitophagy signaling www. cellsignal.com 12

13 Autophagy signaling Beth Levine, UT Southwestern 13

14 Autophagy proteins required for autophagosome formation Choi et al, N Engl J Med,

15 Autophagy has conserved pathways among eukaryotes (yeast to animal to plant) (Arabidopsis Thaliana) Yoshinori Ohsumi 15

16 mtor suppresses autophagy under nutrient rich conditions 16

17 Autophagy induction by rapamycin in cancer cells -rapamycin +rapamycin Immunostaining of endogenous LC3 in autophagosomes (HeLa cells) 17

18 The ULK-Atg13 complexes mediate mtor signaling to the autophagy machinery S757 Jung et. al. MBC,

19 The ULK is an autophagy initiation kinase that is conserved in metazoan (Positive regulator of Hedgehog pathway) (Neural stem cell regulation) Jung et. al. FEBS letter,

20 ULK kinase is a molecular sensor in autophagy/mitophagy induction by integrating AMPK and mtor pathway - ULK1 plays critical role in autophagic clearance of mitochondria in reticulocytes (Kundu et al, Blood, 2008) - AMPK phosphorylates Ser317 and Ser 777 of ULK1 to activate autophagy in glucose starvation (Kim et al, Nature Cell Biol, 2011) - AMPK phosphorylate Ser555 of ULK1 to induce mitophagy (Egan et al, Science, 2011) S317, S777 S555 S757 Egan et al, Science,

21 ULK kinase translocate into mitochondria to induce mitophagy upon stress - S555 phosphorylation of ULK1 by AMPK kinase is critical for translocation of ULK1 into mitochondria and mitophagy induction upon hypoxia (Tian et al, FEBS Letters, 2015) - ULK1 translocate into mitochondria upon hypoxia or FCCP mito stress and phosphorylate mitophagy receptor Fundc1 at S17 to induce mitophagy process (Wu et al, EMBO Rep, 2014) 21

22 Sestrin : Stress-inducible protein family A hsesn1 hsesn2 hsesn3 msesn1 msesn2 msesn3 dsesn csesn B h/msesn2 C h/msesn1 h/msesn3 dsesn csesn e.g. Cumene Hydroperoxide Kim, An and Ro et al, Nature Commun,

23 Sestrins : Stress-inducible proteins Transcriptionally induced by hypoxia, genotoxic stress or oxidative stress (Budanov et al, Oncogene 2002) Sestrin1/2 prevent oxidative damage by activating Nrf2/Keap1 pathway (Bae et al, Cell Metab. 2013) Sesn3 can activate Akt via mtorc2 to regulate hepatic insulin sensitivity and glucose metabolism (Tao et al, Diabetes 2015) Sestrins interact with GATOR2 and negatively regulate mtorc1 in amino acid sensing pathway (Chantranupong et al, Cell Rep. 2015) - GATOR2 complex (WDR24, WDR59, Mios, Sec13 and Seh1L) - GATOR1 complex (DEPDC5, Nprl2 and Nprl3) Parmigiani et al, Int Rev Cell Mol Biol,

24 Sestrins : Stress-inducible proteins Sestrin induces autophagy by activating AMPK and inhibiting mtorc1 in Drosophila and mammals (Lee et al, Science, 2010, Lee et al, Cell Metabolism, 2012) Ho et al, Trends Biochem Sci,

25 ULK1 phosphorylates p62 at S403 and Sestrin2 promotes ULK1 mediated p62 phosphorylation Ro et al, FEBS J,

26 ULK1 phosphorylates Sestrin2 and inhibit mtorc1 through enhancing Sestrin2-GATOR2 binding Kimball et al, Cell Signal,

27 GFP-LC3 transgenic mouse [B6, D2-Tg(CAG-GFP/LC3)53Nmi] Is a useful tool for autophagy research Tg: GFP-LC3 mouse Live imaging of autophagosome in pancreatic tissues of GFP-LC3 mouse RIKEN BRC Experimental Animal Division Kuma et al, Nature 2004 Cao et al, PLoS ONE

28 Sesn2 induces autophagosome formation and increases mitophagy activity Sesn2 +/+ GFP-LC3 mice Sesn2 -/- GFP-LC3 mice Kim et al, Autophagy,

29 Adipose tissue - energy reservoir & metabolic homeostasis control (Stopping of blood flow) Modified from Trayhurn, Acta Physiol Scand.,

30 Inhibition of mammalian target of rapamycin (mtor) Induces autophagy in differentiated adipocytes -rapamycin +rapamycin Immunostaining of LC3, an autophagosomal marker 30

31 ULK1/2 are important for basal and rapamycininduced autophagy in 3T3-L1 adipocytes Ro et al, Autophagy,

32 ULK1/2 are important for basal and rapamycininduced autophagy in 3T3-L1 adipocytes Ro et al, Autophagy,

33 ULK1-Sestrin2 signaling in adipose autophagy and metabolism 33

34 - Autophagy signaling Summary : Part I 1. Autophagy (Self-eating) is induced by stress and requires 18 Atg proteins for autophagosome formation 2. mtorc1 suppresses autophagy by phosphorylating ULK1- Atg13-Fip200 complex under nutrient rich conditions 3. AMPK induce autophagy/mitophagy by inhibiting mtorc1 and activating ULK1 autophagy kinase 4. Sestrins can directly activate AMPK and suppress mtor through GATOR complexes 5. Sestrin2 promotes ULK1 mediated p62 phosphorylation at Ser ULK1-Sestrin2 induce autophagy in adipose tissue and consequently contribute on insulin/glucose and fat metabolism 34

35 Part I : ULK1-Sestrin2 signaling in autophagy induction Part II : Sestrin2 regulation of oxidative metabolism 35

36 Sestrins regulate Oxidative Metabolism Sestrins mtorc1 ULK1/2 Atg13 AMPK PGC1 Keap1 Nrf2 Prx Autophagy Mitochondria Contents ROS Balance of Oxidative Metabolism 36

37 Sestrins may play a role in the regeneration of overoxidized forms of Prxs Obesity Inflammation UV Radiation Chemicals O 2 SOD O 2 H 2 O 2 Fenton react. (Fe, Cu) Haber-Weiss react. Mitochondria OH ROS Scavengers Glutathione Lipoic acid Tocopherols Ascorbic acid GPx Prx H 2 O Prx-SH Prx S S Prx I regeneration ROS Prx-SOH excess H 2 O 2 Srx Sestrins Sestrins: ATP- dependent cysteine sulfinyl reductase Prx-SO 2 H Cell injury (Lipids, DNA, Proteins) Still controversial Woo et al, Antioxid Redox Signal 2009 (Sue Goo Rhee group) Sestrins are involved in the regulation of redox balance in breast and colon cancer cells, fibroblasts and macrophages Budanov et al, Science,

38 Sestrin2 has an alkylhydroperoxide reductase activity through cysteine No alkylhydroperoxidase activity despite structure similarity A B (alkylhydroperoxidase) Ferrous oxidation xylenol orange (FOX) assay (AhpD: M. tuberculosis alkylhydroperoxidase) C Cumene hydroperoxide hsesn2-c125 Kim, An and Ro et al, Nature Commun,

39 LD size (mm) LD size (mm) PG PG-Sn2 PG PG-Sn2 PG PG-Sn2 PG PG-Sn2 PG PG-Sn2 Transgenic Sestrin2 expression increases fat accumulation in BAT A C57BL/6 B Liver Muscle BAT ewat swat tet-sesn2 Pparg-tTA (PG) Pparg-tTA/ tet-sesn2 (PG-Sn2) hsesn2 Actin Adipose Tissue (AT) Non-AT Pparg (active) tta tta Sesn2 tet (active) Sesn2 tta Pparg (inactive) Sesn2 tet (inactive) No significant change in GTT or ITT under HFD Slight improvement in GTT under LFD C PG PG-Sn BAT *** *** ewat LFD Sestrin2 reduces lipogenic gene expression and increases mitochondrial contents in BAT HFD Ro et al, PNAS,

40 1 o -ISA: CM-H 2 DCFDA CM-DCF intensity GFP Ucp1 mrna expression Sestrin2 inhibits Ucp1 expression by suppressing ROS in primary brown adipocytes A 1 o -ISA: Protein Sn2 WT Sn2 CS B Sesn2 Pparg Ucp1 Actin - Sn2 CS : Redox- deficient C125S mutant C Con Sesn2 WT D *** 1 o -ISA ns GFP Sn2 WT Sn2 CS 1 o -ISA *** *** *** Sesn2 CS NAC Con Sn2 WT Sn2 CS NAC Ro et al, PNAS,

41 UCP1 induced by cold and adrenergic receptor activation triggers heat generation in mitochondria Forskolin (FSK) Isoproterenol (ISO) 41

42 Ucp2 mrna p-p38/p38 Ucp1 mrna PG PG-Sn2 PG Ucp1/Actin PG-Sn2 Sestrin2 inhibits Ucp1 expression by suppressing ROS and p38 Sestrin2 E LFD-BAT: mrna PG PG-Sn2 ** * RT Cold F Ucp1 Actin Sesn2 p-p38 p38 LFD-BAT: Protein RT Cold G LFD-BAT: Protein PG RT * * PG-Sn2 ns ns Cold ROS p38 UCP1 Ro et al, PNAS,

43 Antioxidants suppress Ucp1 expression - BHA : butylated hydroxyanisole - NAC : N-acetylcysteine - SB : p38 MAPK inhibitor Antioxidants ROS p38 UCP1 Ro et al, PNAS,

44 Sestrin2 inhibits Ucp1 expression through suppressing ROS in BAT thermogenesis 44

45 Summary : Part II -Oxidative metabolism 1. Sestrins are involved in the regeneration of Prxs 2. Sestrin2 is an alkylhydroperoxidase forming sulfenylation intermediate of Cys125 when induced by cumene hydroperoxides 3. Sestrin2 Tg mice developed the whitening of BAT due to decreased UCP1 although with increased mitochondria contents 4. Sestrin2 as well as antioxidants inhibits UCP1 expression through suppressing ROS in BAT 5. ROS plays critical role in UCP1 expression and thermogenesis 6. Maintaining physiological level of Sestrin2 and ROS is important for adipocyte metabolism 45