Redox homeostasis in the Endoplasmic Reticulum

Transcription

1 Redox homeostasis in the Endoplasmic Reticulum Dmitri Fomenko Redox Biology Center University of Nebraska - Lincoln

2 Endoplasmic reticulum The endoplasmic reticulum (ER) is organelle that forms an interconnected network of tubules, vesicles, and cisternae within cells. Rough endoplasmic reticulum is responsible for proteins synthesis. Smooth endoplasmic reticulum is responsible for synthesize lipids and steroids, metabolize carbohydrates, regulate calcium concentration and drug detoxification. ER is a major place of protein folding in the cell ER retention C-terminal peptides KDEL/HDEL

3 Outline 1. Oxidative protein folding 2. Redox control of protein glycosylation 3. Glycoprotein folding quality control link to redox processes 4. Redox control of ER to cytosol protein translocation

4 The Saccharomyces cerevisiae thiol redoxome. This thiol redoxome has 45 proteins

5 Homo sapiens Thiol Redoxome (135 proteins Cys-110 Sec-25) Protein Number Localization Phosducin 6 Arsenic methyltransferase 1 Glutathione S-transferase 2 MoeB-like 5 Peroxiredoxin 3 Rhodanese-like 1 Methionine sulfoxide reductase A 1 Gutaredoxin-like 3 Thioredoxin-like 10 Glutathione reductase 1 Peroxiredoxin 1 Thioredoxin reductase 2 Selenoprotein R 1 Selenoprotein W 1 Selenoprotein W 1 Glutathione peroxidase 3 ERO1 2 Glutathione peroxidase 2 Methionine R sulfoxide reductase 1 Anterior gradient protein 3 Protein disulfide isomerase 9 OST complex thiol oxidoreductases 2 Thioredoxin-like 6 Ouiescin Q6 sulfhydryl oxidase 1 15kDa Selenoprotein 1 Selenoprotein M 1 Deiodinase 1 Selenorprotein N 1 Selenoprotein K 1 Selenoprotein S 1 Selenoprotein T 1 Cytosol (42) Cys (34) Sec (8) ER (33) Cys (26) Sec (7)

6 Thioredoxin (β α β α β β α) Major representatives: Thioredoxins Glutharedoxins Peroxiredoxins Gluthatione peroxidases Protein disulfide isomerases (PDI) More then 60% of known thiol oxidoreductases are thioredoxin fold proteins

7 Oxidative protein folding disulfide bonds formation in the ER S S PDI Ero1 O 2 Protein PDI Erv2 O 2 QSOX O 2 Ero1 is present in all eukaryotes Erv2 is typical for fungi QSOX is typical metazoans, plants and protists

8 Oxidative protein folding disulfide bonds formation in the ER Ero1 and Erv2 2 R-SH + O 2 R-S-S-R + H 2 O 2

9 Ero1 is ER-localized disulphide-generating oxidase Flavine dependant enzyme Ero1 provides oxidizing equivalents for disulfide bond formation in the ER by relaying the oxidizing power from molecular oxygen to the reduced PDI. Ero1 directly oxidizes the active site of PDI by thioldisulfide exchange reactions with the oxidized shuttle Cys pair, the reduced form of which is reoxidized by the activesite Cys pair.

10 Humans have two Ero1 homologues, Ero1α and Ero1β. Both Ero1 isoforms are transcriptionally upregulated under conditions of ER stress. Ero1α protein is expressed in most cell types, detectable protein levels of Ero1β are found only in several tissues Ero1 activity can be regulated by intramolecular disulphide switches, to prevent cellular hyperoxidation. Grey likely structural cysteines, black - active site or red - regulatory function. The thick orange line at Cys166 indicates the connection to a likely (but unidentified) disulphide partner.

11 Mechanism of Humans Ero1α redox control Appenzeller-Herzog, Nature 2008

12 Quiescin sulfhydryl oxidases The Quiescin-sulfhydryl oxidase is family of flavoenzymes catalyzes the direct of disulfide bonds into unfolded reduced proteins with concomitant reduction of oxygen to hydrogen peroxide. QSOX 2 R-SH + O 2 R-S-S-R + H 2 O 2 Heckler et al, Biochim Biophys Acta. 2008

13 ER environment is strongly oxidizing 1. Each disulfide bond formation is associated with H 2 O 2 production 2. GSSG/GSH ratio is 5-100x higher compare to cytosol 3. ER reducing equivalent provider is not known NADPH GR GSH GSH TR GSH GPXs,? S S Protein

14 Redox control of N-linked protein glycosylation OST N33, IAP Cytosol Sep15, SelM ER SelS, SelK

15 The enzyme that catalyzes this process is a multimeric membrane-complex, called Oligosaccharyl transferase(ost). OST3/6- like proteins are membrane linked thiol oxidoreductases characterized by thioredoxin-fold and CxxC redox motifs in the active site.

16 N33 and IAP - Role of thiol/disulfide oxidoreductases in protein glycosylation Protein glycosylation Folding Quality control OST Chaperones UGT, Sep15, SelM Retrotranslocation Cytosolic Degradation N33, IAP ER Export Secretory pathway

17 General properties of OST3/6 proteins Homozygous deletion of N33 correlates with metastatic prostate cancer and its allelic deletion is associated with human colorectal and pancreatic cancers. This observation suggests a possible tumor suppressor function of N33. There are two known cases of a natural knockout of N33 in humans which are associated with nonsyndromic mental retardation. Deletion of corresponding genes in yeast system cause ER-stress and strongly affect synthesis of glycosylated transmembrane proteins These proteins behave as strong reductases redox potential is in range mV

18 Glycoprotein folding quality control link to redox processes OST N33, IAP Cytosol Sep15, SelM ER SelS, SelK

19 Human 15 kda selenoprotein (ER protein) MAAGQGGWLRPALGLRLLLATAFQAASALGAEFASEACRELGFSSNLLCSSCDLLGQFNLLPLDPVCRGC CQEEAQFETKKLYAGAILEVCGUKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEE LSILKWNTDSVEEFLSEKLERI Signal Peptide UGT-binding domain Redox-domain CGU Active site Human SelM protein (ER protein) MSILLSPPSLLLLLAALVAPATSTTNYRPDWNRLRGLARGRVETCGGUQLNRLKEVKAFVTEDIQLYHNLVMK HLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQELGFYRKSAPEAQVPPEYLWAPAKPPEEASEHDDL Signal Peptide Redox- domain CGGU Active site LEVCG-UKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSE +E CG C+L R +V+AFV D +L+ L +K++ G+DP L LL N E D + + E VETCGGUQLNRLKEVKAFVTED-IQLYHNLVMKHLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQE

20 Function of UDP-Glc:glycoprotein glucosyltransferase (UGGT) Freeze et al, Essentials of Glycobiology 2009

21 Proposed roles of Sep15 in the quality control mechanism of protein folding. 1) Sep15 may function as a thiol-disulfide oxidoreductase that catalyzes reduction, isomerization, or oxidation of disulfide bonds in misfolded proteins recognized by UGT; 2) Sep15 may be required for recognition of glycoprotein substrates of UGT with incorrectly formed disulfide bonds and may influence the enzymatic activity of UGT towards substrates with incorrectly formed disulfide bonds.

22 Selenoprotein M (β α β β β α) MSILLSPPSLLLLLAALVAPATSTTNYRPDWNRLRGLARGRVETCGGUQLNRLKEVKAFVTEDIQLYHNLVMKHLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQELGFYRKS EEEEE HHHHH EEEE EEEEEE EEEEEE HHHHHHHHH 15 kda Protein (β α β β β α) LDQQPAAQRTYAKAILEVCTUKFRAYPQIQAFIQSGRPAKFPNLQIKYVRGLDPVVKLLDASGKVQETLSITKWNTDTVEEFFETHLAKDGAGKNS EEEEEEE HHHHHHHHH EEEEEEE EEEEEE EEEE HHHHHHHHH Trx SelM Sep15

23 Thiol retention and ER processes Many glycoproteins expressed as oligomers and quality control system should monitor folding and oligomerisation status of multimerizing proteins. Thiol retention is quality control system that monitoring the redox state of specific cysteine residues in proteins. This system is also involved in retaining unassembled proteins in the ER until specific cysteines formed intersubunits disulfide bonds. Exact mechanism of thiol retention remain elusive. Thiol retention was discovered in studies of the assembly of complex oligomeric glycoproteins- immunoglobulin M.

24 Redox control of ER - cytosol protein translocation OST N33, IAP Cytosol Sep15, SelM ER SelS, SelK

25 Selenoprotein K and Selenoprotein S >gi gb AAH Selenoprotein K [Homo sapiens] MVYISNGQVLDSRSQSPWRLSLITDFFWGIAEFVVLFFKTLLQQDVKKRRSYGNSSDSRY DDGRGPPGNPPRRMGRINHLRGPSPPPMAGGUGR >gi ref NP_ Selenoprotein S [Homo sapiens] MERQEESLSARPALETEGLRFLHTTVGSLLATYGWYIVFSCILLYVVFQKLSARLRALRQ RQLDRAAAAVEPDVVVKRQEALAAARLKMQEELNAQVEKHKEKLKQLEEEKRRQKIEMWD SMQEGKSYKGNAKKPQEEDSPGPSTSSVLKRKSDRKPLRGGGYNPLSGEGGGACSWRPGR RGPSSGGUG Proline and glycine reach proteins involved in translocation of misfolded proteins form ER to cytosol for proteosomal degradation

26 Shchedrina et al, ARS 2010 SelS is a component of the ER-Associated Protein Degradation (ERAD) system. ERAD is a pathway which protects cells from accumulation of misfolded proteins by transferring these proteins from the ER to cytosol for subsequent ubiquitination and proteasomal degradation. SelS was proposed as a mediator in the interaction of p97 ATPase and the ER membrane integral protein Derlin-1 forming a new type of retrotranslocation channel

27 Selenoprotein T ER protein with unknown function >gi SelT Homo sapiens MRLLLLLLVAASAMVRSEASANLGGVPSKRLKMQYATGPLLKFQ ICVSUGYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYRHIASF LSVFKLVLIGLIIVGKDPFAFFGMQAPSIWQWGQENKVYACMMV FFLSNMIENQCMSTGAFEITLNDVPVWSKLESGHLPSMQQLVQI LDNEMKLNVHMDSIPHHRS SelT deficiency alters cell adhesion and enhances the expression of oxidoreductase genes, while decreasing the expression of genes involved in cell structure organization, suggesting the involvement of SelT in redox regulation. Furthermore, loss of SelT elevates expression of another selenoprotein - selenoprotein W

28 Selenoprotein W EEEEEEE HHHHHHHHHHHHHH EEEEEEE EEEEE EEEEEE HHHHHHHHHHHH 2FA8 AGROBACTERIUM TUMEFACIENS PRIAIRYCTQCN-WLLRAGWMAQEILQTFASDIGEVSLIPST--GGLFEITVD-----GTIIWERKRDG-----GFPG----PKELKQRIRDLID SelW PSEUDOMONAS FLUORESCENS PEIVITYCTQCQ-WLLRAAWLAQELLSTFGDDLGKVSLVPGT--GGIFHITCN-----DVQIWERKADG-----GFPE----AKVLKQRVRDQID SelW VIBRIO CHOLERAE AQIEIYYCRQCN-WMLRSAWLSQELLHTFSEEIEYVALHPDT--GGRFEIFCN-----GVQIWERKQEG-----GFPE----AKVLKQRVRDLID SelW BOS TAURUS VVVRVVYCGAUG-YKPKYLQLKKKLEDEFPSR-LDICGEGTPQVTGFFEVFVA-----GKLVHSKKGGD-----GYVDTESKFLKLVAAIKAALA SelW MUS MUSCULUS LAVRVVYCGAUG-YKPKYLQLKEKLEHEFPGC-LDICGEGTPQVTGFFEVTVA-----GKLVHSKKRGD-----GYVDTESKFRKLVTAIKAALA SelW SEA URCHIN VIVKVIYCGGUG-YGPRYRRLKQELKDEFGDD-VDMAGESTPGTTGWLEVXVN-----GKLIHSKKNGD-----GYIDSESKLKKIVNAVSAAM- Selenoprotein V SelV MUS MUSCULUS ILIRVMYCGLUS-YGLRYIILKRTLEHQFPNL-LEFEEERATQVTGEFEVFVD-----GKLIHSKKKGD-----GFVD-ESGLKKLVGAIDEEIK SelV RATTUS NORVEGICUS ILIRVMYCGLUS-YGLRYILLKKTLEHQFPNL-LEFEEERATQVTGEFEVFVD-----GKLIHSKKKGD-----GFVD-ETSLKKLVGAIDEEIK SelV HOMO SAPIENS VLIRVTYCGLUS-YSLRYILLKKSLEQQFPNH-LLFEEDRAAQATGEFEVFVN-----GRLVHSKKRGD-----GFVN-ESRLQKIVSVIDEEIK Selenoprotein H SelH MUS MUSCULUS ATVVIEHCTSURVYGRHAAALSQALQLEAPE--LPVQVNPSKPRRGSFEVTLLRSDNSRVELWTGIKKGPPRKLKFPE----PQEVVEELKKYLS SelH HOMO SAPIENS ATVVIEHCTSURVYGRNAAALSQALRLEAPE--LPVKVNPTKPRRGSFEVTLLRPDGSSAELWTGIKKGPPRKLKFPE----PQEVVEELKKYLS SelH BOS TAURUS PSVVIEHCTSURVYGRNAAALSQALRLQAPE--LTVKVNPARPRRGSFEVTLLRADGSSAELWTGLKKGPPRKLKFPE----PHVVLEELKKYLS SelH DANIO RERIO LRVVIEHCKSURVYGRNAVVVREALADSHPE--LKVMINPHNPRRNSFEITLMDG-ERADVLWSGIKKGPPRKLKFPE----PAEVVTALKQALE Rdx12 Rdx12 SUS SCROFA VRIVVEYCEPCG-FEATYLELASAVKEQYPG--IEIESRLGG--TGAFEIEIN-----GQLVFSKLENG-----GFPY----EKDFIEAIRRASN Rdx12 HOMO SAPIENS VRIVVEYCEPCG-FEATYLELASAVKEQYPG--IEIESRLGG--TGAFEIEIN-----GQLVFSKLENG-----GFPY----EKDLIEAIRRASN Rdx12 GALLUS GALLUS VHIMVEYCEPCG-FGATYEELASAVREEYPD--IEIESRLGG--TGAFEIEIN-----GQLVFSKLENG-----GFPY----EKDLIEAIRRARN Rdx12 DANIO RERIO 2A VQIKVEYCGGUG-YEPRYQELKRVVTAEFTD--ADVSGFVGR--QGSFEIEIN-----GQLIFSKLETS-----GFPY----EDDIMGVIQRAYD Rdx12 DANIO RERIO 2 VKVKIEYCGAUG-YEPRFQELKREICGNCPD--AEVSGFVGR--RGCFEIQIN-----DFLVFSKLESG-----GFPY----SEDIIEAVVKAKD Selenoprotein T EEEEEEEEE HHHHHHHHHHHHHH EEEEEE EEEEE EEEEE HHHHHHHHHHH SelT BOS TAURUS PLLKFQICVSUG-YRRVFEEYMRVISQRYPD--IRIEGENYL TGAFEITLN-----DVPVWSKLESG-----HLPS----MQQLVQILDNEMK SelT SUS SCROFA PLLKFQICVSUG-YRRVFEEYMRVISQRYPD--IRIEGENYL TGAFEITLN-----DVPVWSKLESG-----HLPS----MQQLVQILDNEMK SelT MUS MUSCULUS PLLKFQICVSUG-YRRVFEEYMRVISQRYPD--IRIEGENYL TGAFEITLN-----DVPVWSKLESG-----HLPS----MQQLVQILDNEMK SelT ANOPHELES GAMBIAE ATMTFLYCYSCG-YRKAFDDYHNLILEKYPE--ITIRGSNYD SGAFEITLN-----DVPVWSKLETG-----RFPA----PQEMFQIIDNHLQ SelT SOLANUM TUBEROSUM NTVTIDFCSSCS-YRGTAVTMKNMLDNQFPG--IHVVLANYP SGAFEVYCN-----GELVFSKLKEN-----RFPG----ELELKDLVGRKIA SelT MEDICAGO TRUNCATULA NTVSIDFCTSCS-YKGNAVSVKNTLESLFPG--INVVLANYP SGAFEVYFN-----GELVFSKLKEN-----RFPG----EFELKELIGRRIG HHHHHHHHHHHHHHHHHHHHHHH HHHH HHHHH HHHHHHHHHHHHHHHHHHHHHHH SelT BOS TAURUS PQPIYRHIASFLSVFKLVLIGLIIVGKDPFAFFGMQ-APSIWQWG-QENKVYACMMVFFLSNMIENQCMS SelT SUS SCROFA PQPIYRHIASFLSVFKLVLIGLIIVGKDPFAFFGMQ-APSIWQWG-QENKVYACMMVFFLSNMIENQCMS SelT MUS MUSCULUS PQPIYRHIASFLSVFKLVLIGLIIVGKDPFAFFGMQ-APSIWQWG-QENKVYACMMVFFLSNMIENQCMS SelT ANOPHELES GAMBIAE PSGVNMLLSKVLLVTKLLLIAALMSNYDIGRYIGNP-FAGWWQWC-FNNKLYASMMIFFLGNTLEAQLIS SelT SOLANUM TUBEROSUM PPLPKRLLGKVVPVFQFGVIGLVMAGEQIFPRLGIAVPPPWFYQL-RANRFGTMATTWLLGNFFQSMLQS SelT MEDICAGO TRUNCATULA PPLPKRALSKVVPVLQTGAIIAITAGDQIFPRLGVT-PPQLYYSL-RANKFGSIASIWLLSNFVQSFLQS

29 Selenoprotein N >gi SELN_HUMAN MGRARPGQRGPPSPGPAAQPPAPPRRRARSLALLGALLAAAAAAAVRVCARHAEAQAAARQELALKTLGT DGLFLFSSLDTDGDMYISPEEFKPIAEKLTGSCSVTQTGVQWCSHSSLQPQLPWLNUSSCLSLLRSTPAA SCEEEELPPDPSEETLTIEARFQPLLPETMTKSKDGFLGVSRLALSGLRNWTAAASPSAVFATRHFQPFL PPPGQELGEPWWIIPSELSMFTGYLSNNRFYPPPPKGKEVIIHRLLSMFHPRPFVKTRFAPQGAVACLTA ISDFYYTVMFRIHAEFQLSEPPDFPFWFSPAQFTGHIILSKDATHVRDFRLFVPNHRSLNVDMEWLYGAS ESSNMEVDIGYIPQMELEATGPSVPSVILDEDGSMIDSHLPSGEPLQFVFEEIKWQQELSWEEAARRLEV AMYPFKKVSYLPFTEAFDRAKAENKLVHSILLWGALDDQSCUGSGRTLRETVLESSPILTLLNESFISTW SLVKELEELQNKQENSSHQKLAGLHLEKYSFPVEMMICLPNGTVVHHINANYFLDITSVKPEEIESNLFS FSSTFEDPSTATYMQFLKEGLRRGLPLLQP SelN defficiensy is assosiated with classical form of rigid spine muscular dystrophy. Recent studies have identified SelN as a key protein in cell protection against oxidative stress and redox-related calcium homeostasis. SelN work reductase in the regulation of ryanodine receptor calcium channel activity.

30 Methionine sulfoxide reductase MsrB3 >gi gb AAH MSRB3 protein [Homo sapiens] MSAFNLLHLVTKSQPVALRACGLPSGSCRDKKNCKVVFSQQELRKRLTPLQYHVTQEKGTESAFEGEYTH HKDPGIYKCVVCGTPLFKSETKFDSGSGWPSFHDVINSEAITFTDDFSYGMHRVETSCSQCGAHLGHIFD DGPRPTGKRYCINSAALSFTPADSSGTAEGGSGVASPAQADKAEL Methionine sulfoxide reduction is an important process, by which cells regulate biological processes and cope with oxidative stress. MsrA and MsrB are enzymes involved in the reduction of methionine sulfoxides in proteins

31 Anterior gradient proteins gi ref NP_ anterior gradient protein 2 MEKIPVSAFLLLVALSYTLARDTTVKPGAKKDTKDSRPKLPQTLSRGWGDQLIWTQTYEEALYKSKT SNKPLMIIHHLDECPHSQALKKVFAENKEIQKLAEQFVLLNLVYETTDKHLSPDGQYVPRIMFVDPS LTVRADITGRYSNRLYAYEPADTALLLDNMKKALKLLKTEL >gi gb AAL anterior gradient protein 3 MMLHSALGLCLLLVTVSSNLAIAIKKEKRPPQTLSRGWGDDITWVQTYEEGLFYAQKSKKPLMVIHH LEDCQYSQALKKVFAQNEEIQEMAQNKFIMLNLMHETTDKNLSPDGQYVPRIMFVDPSLTVRADIAG RYSNRLYTYEPRDLPLLIENMKKALRLIQSEL AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Anterior Gradient family proteins are implicated in the formation of the cement gland and the induction of forebrain fate. The human homologs, hagr2 and hagr3, are proteins associated with estrogen-positive breast tumors. hagr2 has also been implicated in prostate cancer.

32 Summary ER is a second cell compartment with largest number of thiol oxidoreductases and intensive thiol related processes ER environment is strongly oxidizing; however, exact oxidizing status is unknown Reductive processes in the ER are remain unclear. Existing ROS sensors are not suitable for measurement of ER ROS level Function of the 30% of ER thiol oxidoreductases is unknown