Redox regulation, Oxidative Stress and Selenoproteins. Redox and Protein Folding. Danyelle M. Townsend May 18 22, 2015 Charleston, SC

Transcription

1 Redox regulation, Oxidative Stress and Selenoproteins Redox and Protein Folding Danyelle M. Townsend May 18 22, 2015 Charleston, SC

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5 Endoplasmic Reticulum (ER) Albert Claude 1945 Lace work structure Rough ER Protein synthesis: Secreted, membrane bound, digestive enzymes, hormones and antibodies Translocon docking sites for ribosome GSH/ GSSG ~10:1 Smooth ER: Lipid / phospholipid and steroid synthesis Calcium ion storage Sarcoplasmic ER: Calcium storage: major role in excitation / contraction coupling

6 Demand exceeds capacity..

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8 Agents/Treatments that induce ER stress response Conditions temperature shifts (e.g., heat-shock) heavy metals, arsenite, iodoacetamide anoxia, hydrogen peroxide, superoxide ions, free radicals proteasome inhibitors amytal, azide, dinitrophenol, ionophores antimycin Probable effects protein denaturation protein aggregation reaction with sulfhydryl goups, conformational changes in protein oxygen toxicity, free radical fragmentation of proteins inhibition of proteolysis inhibition of oxidative phosphorylation, changes in redox state, covalent modification of proteins hydroxylamine ethanol amino acid analogs, puromycin cleavage of asparagine-glycine bonds translation errors abnormal proteins

9 Grek and Townsend (2013): ER stress and Cancer Biology:

10 ERAD" Misfolded protein" Retrograde transport " through the translocon " Ubiquitination" Sec proteins and ER export Degradation in the" 26S proteasome"

11 Adapted from

12 Three UPR transducers and one master regulator GRP78 / BiP: Glucose related protein / Binding Immunoglobin Protein Zhang, K. et al. J. Biol. Chem. 2004

13 UPR signaling mediated by PERK protein kinase RNA-like endoplasmic reticulum kinase Zhang, K. et al. J. Biol. Chem. 2004;279:

14 UPR signaling mediated by IRE1 and ATF6 Zhang, K. et al. J. Biol. Chem. 2004

15 Nature Medicine: Iwawaki et al., (2004) 10:1014 Transgenic mouse model for monitoring endoplasmic reticulum stress in vivo

16 Wek et al., Nature Medicine (2010) 16:

17 Duration and amplitude of ER stress determines cell fate

18 Unfolded and misfolded proteins magically appear?

19 GSH/ Grx / Srx SH Sulfhydryl ROS RNS N 2 O 3 GSH / Trx SSG S-glutathionylation 2 S. Cysteinyl radical ROS RNS NO GSNO 2 SNO S-nitrosylation SOH Sulfenic acid GSH J. Uys, P. Mulholand, D.M. Townsend (2014) Molecular Pharmacology

20 GSTP S-glutathionylates ER resident proteins Mass Spectrometry Identification of S-glutathionylated ER proteins Protein ID MW (kda) Calnexin 97 Calreticulin 60 Endoplasmin 94 SERCA2A 114 GRP78, Bip 78 Protein disulfide isomerase (A5) 59

21 GSTP is a resident Endoplasmic Reticulum (ER) protein DAPI - nuclear Calnexin GSTP Merge - TRIO DAPI - nuclear BIP GSTP Merge - TRIO

22 GSTP is a resident ER Protein ER Cyto Nuc SERCA2 Calnexin GAPDH Histone H3 GSTP Gstp1/p

23 GSTP regulates ER Calcium homeostasis ER Calcium homeostasis (% of control) 120% 100% 80% 60% 40% 20% 0% Thapsigargin Gstp1/p2 +/+ Gstp1/p2+/+ Gstp1/p2-/ Time (second)

24 GSTP interacts with ER protein and enhances S-glutathionylation GSTP Calnexin GSTP BIP GSTP Calreticulin GSTP Total IP IP IgG IP: Calnexin IP: BIP IP: Calreticulin IP: PDI Relative Pr-SSG level * * WT Gstp1/p2 +/+ Gstp1/p2-/- -/- * PDI Disulfiram (10µM)

25 Thapsigargin and Velcade are more toxic to Gstp1/p2-/- BMDDCs Viability (IC 50 value) Thapsigargin (nm) Velcade (nm) Gstp1/p2 +/ Gstp1/p2 -/ ratio

26 ER dysfunction in liver disease

27 J Pharmacol Exp Ther Jan;344(1): doi: /jpet Epub 2012 Oct 23. Mouse liver protein sulfhydryl depletion after acetaminophen exposure. Yang X, Greenhaw J, Shi Q, Roberts DW, Hinson JA, Muskhelishvili L, Davis K, Salminen WF. Source Division of Systems Biology, National Center for Toxicological Research, Jefferson, Arkansas, USA. Protein SH depleted regions correlated with areas exhibiting histopathologic injury NOT GLOBAL DECREASE IN LIVER

28 J Pharmacol Exp Ther Feb;340(2): doi: /jpet Epub 2011 Nov 1. Changes in mouse liver protein glutathionylation after acetaminophen exposure. Yang X, Greenhaw J, Ali A, Shi Q, Roberts DW, Hinson JA, Muskhelishvili L, Beger R, Pence LM, Ando Y, Sun J, Davis K, Salminen WF. 1h = Increased P-SSG 24h = Decreased P-SSG at site of necrosis PROTEIN OXIDATION 10x > APAP-Conjugation

29 Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi Colin J. Henderson*, C. Roland Wolf* Neil Kitteringham,Helen Powell, Diana Otto*, and B. Kevin Park NAPQI + GSH Detox (NOT related to GSTP) NAPQI + Protein adduct equivalent GSTP +/+ decreased GSH/GSSG * Toxic role of GSTP downstream of protein conjugation of NADPQI

30 Decreased S-glutathionylation of BiP in GSTP1P2 knockout mouse liver BiP GAPDH GSTp

31 BiP is s-glutathionylated on C41 and C BiP (M+2H-129) DLGTTYSC(glut)VGVFK 46 MH + = Da M+2H + = Da y y PSS G Merge (M+2H-129-H 2 O) y H 2 O b y y b y b 5 -H 2 O y (y ) y (M+2H-H 2 O-NH 3 ) y y y y b y b y b y b m/z

32 HEPG2 Cells pre-treated with NAC (and treated with 15mM APAP for 16hrs) have 28% increase in GSH RESULTS: Relative GSH concentration * *

33 BiP-SSG is concurrent with APAP induced liver toxicity BiP: GSTpi dissociates when under ER stress GSTpi KO mice have 42% less SSG-BiP than GSTpi WT mice NAC treatment increases GSH / decreases BiP-SSG and reduces apoptosis

34 Protein Disulfide Isomerase 55 kda Homodimer 14 PDI isoforms in ER Two UPRE in promotor (Kox = 1mM) Widely distributed across eukaryotic Tian tissues et al., 2006: Cell: 14(1): mm Concentrations 1% total pr. Content of cells Decreased PDI leads to protein aggregation (ie Antichaperone)

35 Disulfide Formation SH SH Sulfydryl Disulfide Isomerizations S------S SH SH PDI S-S PDI S S SH SH PDI S-----S Disulfide S S S S

36 Townsend et al., Mol Pharm 2006 Townsend et al., Cancer Research 2009 Xiong et al., I J Cell Biology 2012 Grek et al., ER stress and Cancer 2014 PDI-SSG is concurrent with UPR activation PABA/NO IP:α-PDI IP:IgG DTT WB:PSSG WB:PDI

37 PDI-SSG Alters Structure / Function Circular Dichroism Intrinsic Fluorescence C GST INTACT GSTSG GSTSG GST Intact Molecular ellip Wavelength, nm Emission, au Wavelength, nm W38 Percent Activity μm PABA/NO

38 UPR-Induced Cell Death 20 um PABA/NO (h) WB:Bip WB:Chop WB:LC3 WB:Actin 25 um PABA/NO 0.45 um TG (h) WB:P-JNK WB:JNK1/2 WB: P-PERK WB: PERK WB: P-eIF2 WB: eif2

39 Generation of S-glutathionylation refractory PDI (conversion of basic a.a. adjacent to catalytic cysteines to neutral) PDI-WT a domain WCGHCK a domain WCGHCK PDI-FLFL WCGFCL WCGFCL Histidine (H) Phenylalanine (F) Alanine (A) Lysine (K) Leucine (L)

40 PDI FLFL is refractory to S-glutathionylation In Vitro PABA/NO PDI WT PDI FLFL Rotenone PDI WT PDI FLFL WB: P-SSG WB: PDI In Cells Ctr FLAG- PDI WT FLAG- PDI FLFL Rotenone (µm) PABA/NO WB: P-SSG IP: FLAG WB: PDI- PSSG WB: PDI WB: PDI

41 Mean O.D. S-glutathionylation refractory PDI 1) Blunts the toxic effects of rotenone 2) Diminishes activation of the UPR in PC12 neuronal cells Rotenone (um) PDI mrna fold increase BIP mrna fold increase CHOP mrna fold increase PDI WT PDI FLFL PDI WT PDI WT PDI FLFL PDI FLFL

42 Ueharal et al., Nature (2006) 441, S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration Takashi Uehara1,4, Tomohiro Nakamura1, Dongdong Yao1, Zhong-Qing Shi1, Zezong Gu1, Yuliang Ma2, Eliezer Masliah3, Yasuyuki Nomura4 and Stuart A. Lipton1,3

43 Grek, C and Townsend, D.M. (2013)

44 PDI is a chaperone for ERα What are the consequences of S- glutahtionylation of PDI on ERα stability and transcriptional activation?

45 PDI: Chaperone for Estrogen Receptor alpha Xiong, Y et al, Int J Cell Biol. 2012

46 ERα: hormone binding transcription factor Ligand binding domain DNA binding domain DNA activation Ross Weatherman: 2006: Nature Chemical Biology, 2, Lin et al. Genome Biology (2004: 5:R6

47 PDI-SSG blunts chaperone activity with ERα PABA/NO (h) PABA/NO (h) PSSG PDI PDI ERα 50 IP: PDI 50 IP: ERα Xiong et al., IJCB

48 RNS induce proteasomal degradation of ERα A B (h) ERα ERβ Actin C PABA/NO DMSO MG132 PS ERα Actin Relative (%) ERα mrna n= h 8h 50 0 Xiong, Y et al, Int J Cell Biol. 2012

49 PDI-SSG has anti-estrogen effects E2 (20 nm) (h) ERα ERβ c-myc cyclin D1 p21 WAF1/CIP1 Actin PABA/NO (20 µm) (h) ERα ERβ cyclin D1 p21 WAF1/CIP1 Actin Cyclin D1 mrna and protein were down-regulated by anti-estrogen ICI (Faslodex). ICI also increased the expression of the CDK inhibitor p21 WAF1/ CIP1 (Watts CK, et al. 1995)

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51 Grek, C and Townsend, D.M. (2013)

52 ER stress is worth stressing over ER stress has been linked to the following medical conditions: Huntington s Parkinson s Alzheimer's Heart Disease A G E

53 Acknowledgements Townsend Lab Ying Xiong, Ph.D. Zhiwei Ye, Ph.D. Jordan Frederick, Ph.D. Lin He Tiffany Ancrum Yefim Manevich, Ph.D. Joachim Uys, Ph.D. Novelos Therapeutics, Inc. Christopher Pazoles, Ph.D. Harry Palmin, M.B.A. NIH Funding: NIEHS: R56 ES NIGM: GM Kenneth Tew, Ph.D., D.Sc. Jie Zhang, Ph.D. Robert Bowers, Ph.D. Christina Grek, Ph.D. Yian Chen, Ph.D. National Cancer Institute Larry Keefer, Ph.D. Joseph Saavedra, Ph.D. Mahdol University, Thailand Chonticha Saisawang, Ph.D. Albert Ketterman, Ph.D.