Thiol Redox Systems June 2014 Aida Rodriguez Garcia, Brenna Zimmer, Jus:n Prigge, Shelby Bickford, Tom Reichenbach, and Rakesh Basavalingappa

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1 Thiol Redox Systems June 2014 Aida Rodriguez Garcia, Brenna Zimmer, Jus:n Prigge, Shelby Bickford, Tom Reichenbach, and Rakesh Basavalingappa

2 Introduc6on Thioredoxin func:on, isoforms, localiza:on, structure, and targets Glutaredoxin func:on, isoforms, localiza:on, structure, and targets Recycling structures, mechanisms, and localiza:on Glutathione- biosynthesis, GSH/GSSG intracellular, [glutathione], and import processes

3 THIOREDOXIN SYSTEM Maintaining cellular redox homeostasis and protec:ng cells from oxida:ve stress. Ø Reducing the disulfide in its target proteins Ø Binding to its substrate as a regulator

4 Thioredoxin func6ons An:oxidant Reduce methionine sulfoxide reductase (Msr) and peroxiredoxins (Prx). DNA synthesis Electron donor for ribonucleo:de reductase (RNR). Trx- (SH) 2 Transcrip:on Transcrip:on factor DNA binding (p53, AP- 1, NF- ƙb ) Cell Signalling Protein binding (Ref- 1, ASK1, Vit D3 )

5 Thioredoxin isoforms in humans Arner, E.S. and A. Holmgren, Physiological func/ons of thioredoxin and thioredoxin reductase. Eur J Biochem, (20): p

6 Describe the structure of thioredoxin. Structure Thioredoxin- a fold for all reasons 3(3):

7 Name at lease three protein targets of thioredoxin. An:oxid Redox Signal Apr 1;18(10): doi: /ars Epub 2012 Jun 26.

8 Glutaredoxin (GRX) Proteins Ini:ally discovered by Arne Holmgren in the mid 1970s. Proc. Natl. Acad. Sci. USA, 73 (1976), pp Small, ~10-15 KDa redox proteins. Catalyze glutathione- dependent reduc:on of dithiol protein disulfides, (similar to Thioredoxin) Unique to GRX, can also catalyze reduc:on of Glutathione- mixed disulfides with proteins or small molecules including cysteine and 2- hydroxyethyl disulfide. Hanschmann et. al., An/oxid Redox Signal Nov 1;19(13):

9 GRX Proteins Func:on as the electron donor for Ribonucleo:de Reductase. An:oxidant func:on as electron donors for methionine sulfoxide reductases which repair proteins containing oxidized methionine residues. Reduced GRX1 nega:vely regulates apoptosis by direct interac:on with ASK1, resul:ng in inhibi:on of ASK1 kinase ac:vity.

10 GRX Proteins Four isoforms exist in humans. Contain either dithiol or monothiol ac:ve sites Dithiol GRXs GRX1 (ac:ve site Cys- Pro- Tyr- Cys) GRX2, ac:ve site Cys- Ser- Tyr- Cys Monothiol GRXs GRX3/GRX5 ac:ve site, Cys- Gly- Phe- Ser

11 Human Isoforms of Glutaredoxin Glutaredoxin- 1 (GRX1). MAQEFVNCKIQPGKVVVFIKPTCPYCRRAQEILSQLPIKQGLLEFVDITATNHT NEIQDYLQQLTGARTVPRVFIGKDCIGGCSDLVSLQQSGELLTRLKQIGALQ Dithiol cataly:c ac:ve site is underlined Predominantly cytoplasmic, but can translocate to the nucleus, be exported from the cell, and localizes to the intermembrane space of the mitochondria. Sahlin, L., Mol. Hum. Reprod., 6 (2000), pp ; Lundberg, M., Biochem Biophys Res Commun Jul 2;319(3):801-9; Pai, HV., An/oxid Redox Signal Nov;9(11):

12 GRX1 GRX1 in reduced form (lem) or with bound glutathionyl moiety (right). Sun, C. et. al., J Mol Biol Jul 24;280(4): ; Yang, Y. et. al., Biochemistry Dec 8;37(49):

13 Human Isoforms of Glutaredoxin Glutaredoxin- 2 (GRX2) is encoded by three transcript variants, each of which encodes a slightly different protein. GRX2a MIWRRAALAGTRLVWSRSGSAGWLDRAAGAAGAAAAAASGMESNTSSSLENLATAPVNQIQETIS DNCVVIFSKTSCSYCTMAKKLFHDMNVNYKVVELDLLEYGNQFQDALYKMTGERTVPRIFVNGTFIG GATDTHRLHKEGKLLPLVHQCYLKKSKRKEFQ Precursor protein pep:de is in yellow. Mature mitochondrial protein lacks this region. Dithiol cataly:c ac:ve site is underlined Localizes to the mitochondrial matrix Pai, H V et al., An/oxid. Redox Signaling 9 (2007), pp Ac:ve site Serine allows for coordina:on of a [2Fe2S] cluster Uniquely enables GRX2 to receive electrons from Thioredoxin Reductase. Johansson, C et. al., J Biol Chem Feb 27;279(9): Key role in oxida:ve stress response by reduc:on of glutathionylated substrates and small molecule disulfides, including GSSG.

14 Human Isoforms of Glutaredoxin GRX2b MNPRDKQVSRFSPLKDVYTWVALAGIQRSGSPGRTRSAARRMESNTSSSLE NLATAPVNQIQETISDNCVVIFSKTSCSYCTMAKKLFHDMNVNYKVVELDLLE YGNQFQDALYKMTGERTVPRIFVNGTFIGGATDTHRLHKEGKLLPLVHQCYL KKSKRKEFQ Dithiol cataly:c ac:ve site is underlined

15 Human Isoforms of Glutaredoxin GRX2c MESNTSSSLENLATAPVNQIQETISDNCVVIFSKTSCSYCTMAKKLFHDMNVN YKVVELDLLEYGNQFQDALYKMTGERTVPRIFVNGTFIGGATDTHRLHKEGKL LPLVHQCYLKKSKRKEFQ Dithiol cataly:c ac:ve site is underlined GRX2b/c proteins were found to be expressed in tes:s and cancer cell lines. Expression appears to be limited to the nucleus and cytoplasm, due to the lack of a mitochondrial localiza:on sequence found only on the GRX2a protein. Lönn ME et. al., An/oxid Redox Signal Mar;10(3):

16 GRX2 Dimer

17 Human Isoforms of Glutaredoxin GRX3 protein MAAGAAEAAVAAVEEVGSAGQFEELLRLKAKSLLVVHFWAPWAPQCAQMNEVMAELAKELPQVS FVKLEAEGVPEVSEKYEISSVPTFLFFKNSQKIDRLDGAHAPELTKKVQRHASSGSFLPSANEHLKEDL NLRLKKLTHAAPCMLFMKGTPQEPRCGFSKQMVEILHKHNIQFSSFDIFSDEEVRQGLKAYSSWPTY PQLYVSGELIGGLDIIKELEASEELDTICPKAPKLEERLKVLTNKASVMLFMKGNKQEAKCGFSKQILEIL NSTGVEYETFDILEDEEVRQGLKAYSNWPTYPQLYVKGELVGGLDIVKELKENGELLPILRGEN Trx domain, PICOT (PKC- interac:ng cousin of TRX) subfamily is in purple; interacts with protein kinase C (PKC) theta. GRX3 inhibits the ac:va:on of c- Jun N- terminal kinase and the transcrip:on factors, AP- 1 and NF- kb, induced by PKC theta or T- cell ac:va:ng s:muli. Wi]e S. et. al., J Biol Chem Jan 21;275(3): GRX PICOT- like domains are in green. Monothiol cataly:c ac:ve sites are underlined. Both GRX and TRX domain are required for ac:vity. Coordinates [2Fe2S] clusters, and is important for response and signaling processes during oxida:ve stress.

18 GRX3 Dimer Hypothe:cal model of GRX3. Dimer binds two [2Fe 2S] clusters. Iron- Sulfur cluster may regulate ac:vity, but is not redox ac:ve. Haunhorst P et. al., Biochem Biophys Res Commun Apr 2;394(2):372-6.

19 Human Isoforms of Glutaredoxin GRX5 protein (glutaredoxin- related protein 5, mitochondrial precursor): MSGSLGRAAAALLRWGRGAGGGGLWGPGVRAAGSGAGGGGSAEQLDAL VKKDKVVVFLKGTPEQPQCGFSNAVVQILRLHGVRDYAAYNVLDDPELRQGI KDYSNWPTIPQVYLNGEFVGGCDILLQMHQNGDLVEELKKLGIHSALLDEKK DQDSK Precursor protein pep:de is in yellow. Mature mitochondrial protein lacks this region. GRX PICOT- like domain is in green Monothiol cataly:c ac:ve site is underlined Can bind a [2Fe2S] cluster.

20 GRX5 Tetramer Suggested to be required for biogenesis, transfer, and inser:on of iron- sulfur clusters into other proteins. Glutathione binding occurs.

21 Recycling Thioredoxin reductase (TrxR) Trx- S 2 + NADPH + H + TrxR FAD Se- S Trx- (SH) 2 + NADP + [3.13 from Redox Biochemistry] Mammals: TrxR1 and TrxR2 Plants: ferredoxin- thioredoxin reductase Sandalova, T., Zhong, L., Lindqvist, Y., Holmgren, A., and Schneider, G Three- dimensional structure of a mammalian thioredoxin reductase: Implica:ons for mechanisms and evolu:on of a selenocysteine- dependent enzyme. PNAS. 98:17, Sandalova, T., Zhong, L., Lindqvist, Y., Holmgren, A., and Schneider, G Three- dimensional structure of a mammalian thioredoxin reductase: Implica:ons for mechanisms and evolu:on of a selenocysteine- dependent enzyme. PNAS. 98:17,

22 Recycling Glutathione reductase (GR) Grx (SH) 2 GrxR FAD S 2 GSSR/PSSG/PSSP + GSH RSH/PSH + GSSG + H + + NADPH 2GSH + NADP + [Modified from 3.15, 3.16, 3.17 from Redox Biochemistry] Mammals: cytosol, nucleus, mitochondria Plants: cytosol, mitochondria, and chloroplast PDB: 3GRS, R= 18.6%, Res. = 1.54Å, R Free = n/a Interface Domain FAD Domain Glutathione- binding Site NADPH Domain hqp://upload.wikimedia.org/wikipedia/commons/thumb/2/24/gsr_cataly:c_cycle.png/800px- GSR_Cataly:c_Cycle.PNG

23 Glutathione biosynthesis pathway ATP ADP + Pi Glutamate + Cysteine γ- Glutamylcysteine ligase γ- Glutamylcysteine + Glycine ATP ADP + Pi Glutathione synthetase Glutathione Lushchak, V.I., J. Amino Acids. 2012: doi: /2012/ Noctor, et al., J. Experimental Botany (321), pp

24 Maintenance of intracellular GSH/GSSG ra6o Cytosol, mitochondria - >10:1 Endoplasmic reticulum - 1:1 Redox Biochemistry, Etd by R. Banerjee

25 Compartmentaliza6on of GSH Franco & Cidlowski, An/oxid Redox Signaling 2012

26 Cellular import of GSH Hgt1p/Opt1p first high- specificity, high- affinity glutathione transporter (S. cerevisiae) Glutathione import transporters in plasma membrane Name Transporter family Substrate Remarks OAT1 (SLC22A6) OAT family Acetylsalicylate, methotrexate, urate, GSH Sodium independent Organic anion exchanger OAT3 (SLC22A8) OAT family 2- OG (p- aminohippurate), GSH Sodium independent Organic anion exchanger Indirect import NaC3 (SLC13A3) SLC13 family Succinate, citrate, α- ketoglutarate, GSH Sodium dependent dicarboxylate carriers Cys- gly and glutamate Cys, gly and glutamate Bachhawat et al., Biochimica et Biophysica Acta (2013)

27 Tack! Thank You! Gracias! Danke!