Redox homeostasis in the Endoplasmic Reticulum Dmitri Fomenko Dmitri Fomenko Redox Biology Center Redox Biology Center University of Nebraska University of Nebraska - Lincoln Lincoln Dmitri Fomenko Dmitri Fomenko
Redox homeostasis in theEndoplasmic Reticulum
Dmitri FomenkoDmitri Fomenko
Redox Biology CenterRedox Biology CenterUniversity of Nebraska University of Nebraska -- LincolnLincoln
Dmitri FomenkoDmitri Fomenko
Endoplasmic reticulum
The endoplasmic reticulum (ER) is organelle thatforms an interconnected network of tubules, vesicles,and cisternae within cells.
Rough endoplasmic reticulum is responsible forproteins synthesis.
Smooth endoplasmic reticulum is responsible forsynthesize lipids and steroids, metabolizecarbohydrates, regulate calcium concentration and drugdetoxification.detoxification.
ER is a major place of protein folding in the cell
ER retention C-terminal peptides KDEL/HDEL
Outline
1. Oxidative protein folding
2. Redox control of protein glycosylation
3. Glycoprotein folding quality control – link to redox processes 3. Glycoprotein folding quality control – link to redox processes
4. Redox control of ER to cytosol protein translocation
The Saccharomyces cerevisiae thiol redoxome. This thiol redoxome has 45 proteins
Homo sapiens Thiol Redoxome (135 proteins Cys-110 Sec-25)
Protein Number Localization Phosducin 6
Cytosol (42) Cys (34) Sec (8)
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 Selenoprotein W 1
Glutathione peroxidase 3
ERO1 2
ER (33) Cys (26) Sec (7)
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
Thioredoxin (ββββ−−−−αααα−−−−ββββ−−−−αααα−−−−ββββ−−−−ββββ−−−−αααα))))
Major representatives:
ThioredoxinsGlutharedoxinsPeroxiredoxinsGluthatione peroxidasesProtein disulfide isomerases (PDI)
More then 60% of known thiol oxidoreductases are thioredoxin fold proteinsare thioredoxin fold proteins
Oxidative protein folding – disulfide bonds formation in the ER
Protein
S S PDIEro1 O2
PDI Erv2 O2
QSOX O2
Ero1 is present in all eukaryotesErv2 is typical for fungi QSOX is typical metazoans, plants and protists
Oxidative protein folding – disulfide bonds formation in the ER
Ero1 and Erv2 2 R-SH + O2 R-S-S-R + H2O2
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 thiol-
disulfide exchange reactions with the oxidized shuttle Cys
pair, the reduced form of which is reoxidized by the active-
site Cys pair.
Ero1 is ER-localized disulphide-generating oxidase
Ero1 activity can be regulated by intramolecular disulphide switches, toprevent cellular hyperoxidation.
Humans have two Ero1 homologues, Ero1α and Ero1β. Both Ero1isoforms are transcriptionally upregulated under conditions of ER stress.Ero1α protein is expressed in most cell types, detectable protein levels ofEro1β are found only in several tissues
Grey – likely structural cysteines, black - active site or red - regulatoryfunction. The thick orange line at Cys166 indicates the connection to alikely (but unidentified) disulphide partner.
Mechanism of Humans Ero1α redox control
Appenzeller-Herzog, Nature 2008
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 + O2 R-S-S-R + H2O2
Heckler et al, Biochim Biophys Acta. 2008
ER environment is strongly oxidizing
1. Each disulfide bond formation is associated
with H2O2 production
2. GSSG/GSH ratio is 5-100x higher compare to cytosol
3. ER reducing equivalent provider is not known
NADPH GRGSH
TRGSH GPXs,
?GSH Protein
S S
OST
N33, IAP
Redox control of N-linked protein glycosylation
Sep15, SelM
SelS, SelK
ERCytosol
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.
OST
N33, IAP
Protein glycosylation
Chaperones
Folding
UGT, Sep15, SelM
Quality control
RetrotranslocationCytosolic Degradation
ER
N33 and IAP - Role of thiol/disulfide oxidoreductases in protein glycosylation
Export Secretory pathway
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
General properties of OST3/6 proteins
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 -280 -300mV
OST
N33, IAP
Glycoprotein folding quality control link to redox processes
Sep15, SelM
SelS, SelK
ERCytosol
Human 15 kDa selenoprotein (ER protein)MAAGQGGWLRPALGLRLLLATAFQAASALGAEFASEACRELGFSSNLLCSSCDLLGQFNLLPLDPVCRGC
CQEEAQFETKKLYAGAILEVCGUKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEE
LSILKWNTDSVEEFLSEKLERI
Human SelM protein (ER protein)MSILLSPPSLLLLLAALVAPATSTTNYRPDWNRLRGLARGRVETCGGUQLNRLKEVKAFVTEDIQLYHNLVMK
Signal PeptideSignal Peptide
UGTUGT--bindingbinding
domaindomain Redox-domain
CGUActive site
MSILLSPPSLLLLLAALVAPATSTTNYRPDWNRLRGLARGRVETCGGUQLNRLKEVKAFVTEDIQLYHNLVMK
HLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQELGFYRKSAPEAQVPPEYLWAPAKPPEEASEHDDL
Signal PeptideSignal Peptide Redox- domain
CGGUActive site
LEVCG-UKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSE
+E CG C+L R +V+AFV D +L+ L +K++ G+DP L LL N E + + + D + + E
VETCGGUQLNRLKEVKAFVTED-IQLYHNLVMKHLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQE
Function of UDP-Glc:glycoprotein glucosyltransferase (UGGT)
Freeze et al, Essentials of Glycobiology 2009
Proposed roles of Sep15 in the quality control mechanismof 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.
Selenoprotein M (ββββ−−−−αααα−−−−ββββ−−−−ββββ−−−−ββββ−−−−αααα)
MSILLSPPSLLLLLAALVAPATSTTNYRPDWNRLRGLARGRVETCGGUQLNRLKEVKAFVTEDIQLYHNLVMKHLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQELGFYRKS
________________________________________EEEEE________HHHHH___________EEEE_______EEEEEE______EEEEEE___HHHHHHHHH________
15 kDa Protein (ββββ−−−−αααα−−−−ββββ−−−−ββββ−−−−ββββ−−−−αααα)
LDQQPAAQRTYAKAILEVCTUKFRAYPQIQAFIQSGRPAKFPNLQIKYVRGLDPVVKLLDASGKVQETLSITKWNTDTVEEFFETHLAKDGAGKNS
____________EEEEEEE_______HHHHHHHHH_______EEEEEEE_____EEEEEE_____EEEE________HHHHHHHHH__________
Trx SelM Sep15
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.
Thiol retention and ER processes
OST
N33, IAP
Redox control of ER - cytosol protein translocation
Sep15, SelM
SelS, SelK
ERCytosol
>gi|34783186|gb|AAH13162.2| Selenoprotein K [Homo sapiens]
MVYISNGQVLDSRSQSPWRLSLITDFFWGIAEFVVLFFKTLLQQDVKKRRSYGNSSDSRY
DDGRGPPGNPPRRMGRINHLRGPSPPPMAGGUGR
>gi|45439349|ref|NP_982298.1| Selenoprotein S [Homo sapiens]
MERQEESLSARPALETEGLRFLHTTVGSLLATYGWYIVFSCILLYVVFQKLSARLRALRQ
RQLDRAAAAVEPDVVVKRQEALAAARLKMQEELNAQVEKHKEKLKQLEEEKRRQKIEMWD
SMQEGKSYKGNAKKPQEEDSPGPSTSSVLKRKSDRKPLRGGGYNPLSGEGGGACSWRPGR
Selenoprotein K and Selenoprotein S
SMQEGKSYKGNAKKPQEEDSPGPSTSSVLKRKSDRKPLRGGGYNPLSGEGGGACSWRPGR
RGPSSGGUG
Proline and glycine reach proteins involved in translocation of
misfolded proteins form ER to cytosol for proteosomal
degradation
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
Selenoprotein T – ER protein with unknown function
>gi|18803851|SelT Homo sapiens
MRLLLLLLVAASAMVRSEASANLGGVPSKRLKMQYATGPLLKFQ
ICVSUGYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYRHIASF
LSVFKLVLIGLIIVGKDPFAFFGMQAPSIWQWGQENKVYACMMV
FFLSNMIENQCMSTGAFEITLNDVPVWSKLESGHLPSMQQLVQIFFLSNMIENQCMSTGAFEITLNDVPVWSKLESGHLPSMQQLVQI
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
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
>gi|172044683|SELN_HUMANMGRARPGQRGPPSPGPAAQPPAPPRRRARSLALLGALLAAAAAAAVRVCARHAEAQAAARQELALKTLGT
DGLFLFSSLDTDGDMYISPEEFKPIAEKLTGSCSVTQTGVQWCSHSSLQPQLPWLNUSSCLSLLRSTPAA
SCEEEELPPDPSEETLTIEARFQPLLPETMTKSKDGFLGVSRLALSGLRNWTAAASPSAVFATRHFQPFL
PPPGQELGEPWWIIPSELSMFTGYLSNNRFYPPPPKGKEVIIHRLLSMFHPRPFVKTRFAPQGAVACLTA
ISDFYYTVMFRIHAEFQLSEPPDFPFWFSPAQFTGHIILSKDATHVRDFRLFVPNHRSLNVDMEWLYGAS
ESSNMEVDIGYIPQMELEATGPSVPSVILDEDGSMIDSHLPSGEPLQFVFEEIKWQQELSWEEAARRLEV
AMYPFKKVSYLPFTEAFDRAKAENKLVHSILLWGALDDQSCUGSGRTLRETVLESSPILTLLNESFISTW
SLVKELEELQNKQENSSHQKLAGLHLEKYSFPVEMMICLPNGTVVHHINANYFLDITSVKPEEIESNLFS
Selenoprotein N
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.
>gi|25123199|gb|AAH40053.1| MSRB3 protein [Homo sapiens]
MSAFNLLHLVTKSQPVALRACGLPSGSCRDKKNCKVVFSQQELRKRLTPLQYHVTQEKGTESAFEGEYTH
HKDPGIYKCVVCGTPLFKSETKFDSGSGWPSFHDVINSEAITFTDDFSYGMHRVETSCSQCGAHLGHIFD
DGPRPTGKRYCINSAALSFTPADSSGTAEGGSGVASPAQADKAEL
Methionine sulfoxide reductase – MsrB3
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
gi|5453541|ref|NP_006399.1| anterior gradient protein 2
MEKIPVSAFLLLVALSYTLARDTTVKPGAKKDTKDSRPKLPQTLSRGWGDQLIWTQTYEEALYKSKT
SNKPLMIIHHLDECPHSQALKKVFAENKEIQKLAEQFVLLNLVYETTDKHLSPDGQYVPRIMFVDPS
LTVRADITGRYSNRLYAYEPADTALLLDNMKKALKLLKTEL
>gi|19068182|gb|AAL55402.1| anterior gradient protein 3
MMLHSALGLCLLLVTVSSNLAIAIKKEKRPPQTLSRGWGDDITWVQTYEEGLFYAQKSKKPLMVIHH
LEDCQYSQALKKVFAQNEEIQEMAQNKFIMLNLMHETTDKNLSPDGQYVPRIMFVDPSLTVRADIAG
RYSNRLYTYEPRDLPLLIENMKKALRLIQSEL
Anterior gradient proteins
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.
Summary
• ER is a second cell compartment with largest number of thioloxidoreductases 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