Vadim Gladyshev. Brigham and Women s Hospital, Harvard Medical School

Transcription

1 Selenium and Redox Systems Vadim Gladyshev Center for Redox Medicine Brigham and Women s Hospital, Harvard Medical School June 13, 2011

2 Genome Transcriptome Metabolome Ionome Proteome

3 Trace Elements Metals Other elements

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5 Selenium is essential for human health Selenium deficiency may cause Increased risk of cancer Increased risk of AIDS in HIV-infected individuals Male reproduction problems Increased risk of viral infection Keshan, Kashin-Beck diseases

6 Selenocysteine Cysteine

7 Se is present in proteins in the form of 21st amino acid - selenocysteine Selenocysteine is encoded by UGA; UGA is also a stop signal mrna structure, selenocysteine insertion sequence (SECIS) element, is essential for insertion of selenocysteine

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10 Selenocysteine insertion in eukaryotes Sec EFsec L30 SBP2 AUG UGA UAA/UAG

11 Selenocysteine versus Cysteine Selenocysteine UGA codon pka = 5.2 Cysteine UGU, UGC-codons pka = 8.3 Se - S S S - S S Sec serves redox function!

12 Searches for selenoprotein genes Selenoprotein Initiation of translation AUG Selenocysteine UGA STOP SECIS 5 AAAAAAA 3 Cys-containing protein Initiation of translation AUG UGC UGU UAA UAG UGA STOP 5 AAAAAAA 3 Cysteine UAA UAG UGA

13 SECISearch on-line

14 Selenoproteome Eukaryotes: 0-57 (Aureococcus) Bacteria: 0-57 (worm symbiont) Archaea: Viruses: 0-9 (M. maripaludis) 0-1 (fowlpox virus) Model organisms Organism Selenoproteome Mouse, rat 24 Zebrafish 37 Drosophila 3 C. elegans 1 S. cerevisiae, Arabidopsis 0

15 Selenium 34 Se Sec machinery Se H CH 2 H 3 N + - C COO H Selenocysteine (21 st amino acid) - SelA (prokaryotic Sec synthase) - SelB (Sec specific elongation factor) - SelC (Sec trna) - SelD (selenophosphate synthetase) - SecS (eukaryotic Sec synthase) - PSTK (phosphoserine trna kinase) - SECISBP2 Selenoprotein Eukaryotes: >30 families - Deiodinase: DI, DII and DIII - Glutathione peroxidase:gpx1-4, GPx6 - Thioredoxin reductase: TR1, TGR, TR3 - MsrB1 - SPS2 - MsrA - Protein disulfide isomerase - SelH, SelI, SelJ, SelK, SelL, SelN, SelP, SelS, SelW, etc. Prokaryotes: >60 families - Formate dehydrogenase FdhA - SelD - FrhA and FrhD - GrdA and GrdB - Peroxiredoxin - Thioredoxin - Glutaredoxin - Proline reductase - Rhodanase-related protein - Arsenate reductase - HesB-like - GST-like - GPx-like, SelW-like, DsbA-like - Various Prx/Trx/Grx-like protein families

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17 dbteu dbteu (v. 1.0) includes transporters, maintenance proteins (e.g., repressors and chaperones), and user proteins for five elements: Cu, Mo, Ni, Co and Se. The current database contains ~17,000 proteins from more than 700 organisms across the three domains of life. Main features of dbteu: Browse Search trace element, organism, protein category, protein family, keyword, etc. BLAST search Real-time statistics Sequence download

18 Overview of trace element utilization Bacteria Phylum Mo Cu Ni Co(B 12 ) Se(Sec) Archaea Total 401(74%) 432(80%) 319(59%) 410(76%) 139(26%) Eukarya Total 46(98%) 26(55%) 39(83%) 45(96%) 6(13%) Total 105(66%) 154(96%) 51(32%) 49(31%) 76(48%)

19 Selenoprotein GPx1 GPx2 GPx3 GPx4 GPx6 SelP DI1 DI2 DI3 SelW SelV SelT SelH SelM Sep15 TR1 TGR TR3 SelS SelK SelO SelI SelN MsrB1 SPS2 Sec redox motif UxxT UxxT UxxT UxxT UxxT UxxC SxxU SxxU SxxU CxxU CxxU CxxU CxxU CxxU CxU CU CU CU U U U U CU U UxC Secondary structure and location of Sec Kryukov et al., Science 300, 1439 (2003)

20 GPx1 SelS GPx4 GPx1+SelS GPx1+GPx4 GPx4+SelS GPx1+GPx4+SelS Control sirna

21 Selenium in Tissues Cells? Tissues

22 Elemental Profiling in Tissues Cells Tissues Methods for Trace Elemental Analysis Atomic Absorption Spectroscopy (AAS) Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Neutron Activation Analysis (NAA) X-Ray Fluorescence Microscopy (XFM)

23 X-ray fluorescence microscopy (XFM)

24 Role of Selenium in Testes?? Liver Testes Sperm??

25 Selenium Distribution in Mouse Testes H&E H&E Selenium 20 μm Low High Se in mouse testes localizes to elongating spermatids

26 Selenium Transport to Mouse Testes Selenoprotein P (SelP) Synthesized in liver Major plasma selenoprotein

27 Selenium Transport to Mouse Testes Selenoprotein P (SelP) Synthesized in liver Major plasma selenoprotein H&E Selenium SelP is the main Se delivery protein to mouse testes J. Mol. Biol. (2009) 389,

28 Selenium Utilization in Testes Glutathione Peroxidase 4 (GPx4) Highly expressed in mouse testes Has mitochondrial (mgpx4) and nuclear (ngpx4) forms

29 Selenium Utilization in Testes Glutathione Peroxidase 4 (GPx4) Highly expressed in mouse testes Has mitochondrial (mgpx4) and nuclear (ngpx4) forms mgp x4 +/+ -/- mgpx4 KO decreased Se in spermatocytes by ~ 50% J. Mol. Biol. (2009) 389,

30 Selenium Utilization in Testes Glutathione Peroxidase 4 (GPx4) Highly expressed in mouse testes Has mitochondrial (mgpx4) and nuclear (ngpx4) forms mgp x4 ngpx 4 +/+ +/+ -/- -/- J. Mol. Biol. (2009) 389,

31 Selenium Utilization in Sperm J. Mol. Biol. (2009) 389,

32 Role of Selenium in Testes SelP? Elongating spermatids mgpx4? Liver Testes Sperm? midpiece? Liver SelP is the main source of Se Se is distributed to elongating spermatids in mouse testes Se is utilized by mitochondrial form of GPx4 Se is localized to midpiece in sperm cells

33 Selenoproteins: a subset of thiol oxidoreductases ROS detoxification Metabolic processes Signal transduction Thiol Redoxome a set of thiol oxidoreductases in an organism Transcription control Protein modification Oxidative folding Protein degradation

34 High-throughout identification of catalytic redox-active cysteine residues Fomenko et al., Science 315, (2007)

35 Essentially all known thiol oxidoreductases could be detected by the Sec/Cys approach Thioredoxin Glutaredoxin AhpD DsbA Protein disulfide isomerase Thioredoxin reductase Methionine-S-sulfoxide reductase Methionine-R-sulfoxide reductase Glutathione peroxidase Peroxiredoxin Arsenate reductase Ohr (OsmC)

36 Functions of Cys in proteins 1. Redox catalysis (e.g., thiol oxidoreductases) 2. Regulatory cysteines (e.g., kinases, phosphatases) 3. Structural disulfides (e.g., secretory proteins) 4. Metal-coordinating cysteines (e.g., zinc fingers) 5. Non-redox catalysis (e.g., certain proteases) 6. Sites of posttranslational modifications (e.g., membrane targeting) 7. Other functions

37 Thiophosphate (SPO 3 ) affects selenoprotein expression 75 Se labeling WB NIH 3T3 cells

38 Cys can be synthesized from sulfide and P-Ser on trna [Ser]Sec In vitro synthesis of Cys on O-phosphoseryl-tRNA [Ser]Sec by SecS from SPO 3 S II - O P O - I O - Se II - O P O - I O - SPO 3 SePO 3

39 Can Cys replace Sec? Experimental evidence +SPO TR1 28 Se diets

40 Sec is replaced with Cys in TR1 in thiophosphate-treated cells control 1 mm SPO 3 Source of TRs Protein Peptide sequences Sec, % Cys, % NIH 3T3, control NIH 3T3, SPO 3 treated TR1 TR1 TR1 TR1 R.SGGDILQSGCUG R.SGGDILQSGCCG R.SGGDILQSGCUG R.SGGDILQSGCCG

41 Cys is inserted in place of Sec in TR1 under Se deficiency 0 ppm Se 0.1 ppm Se 2 ppm Se Se deficient selenoproteins Se adequate selenoproteins ~ Se rich selenoproteins Source of TR1 Protein Peptide sequences Sec, % Cys, % Liver, 0 ppm Se TR1 R.SGGDILQSGCUG 49 TR1 R.SGGDILQSGCCG 51 Liver, 0.1 ppm Se TR1 TR1 R.SGGDILQSGCUG R.SGGDILQSGCCG 91 9 Liver, 2 ppm Se TR1 R.SGGDILQSGCUG R.SGGDILQSGCCG 100 0

42 Cys insertion in place of Sec: Under Se deficiency, Cys is inserted in place of Sec Regular diet: 10% of Sec sites may be occupied by Cys New Cys biosynthesis pathway in mammals Biological function of thiophosphate