Selenium and Redox SystemsSelenium and Redox Systems

Vadim Gladyshev

Center for Redox MedicineCenter for Redox Medicine

Brigham and Women’s Hospital, Harvard Medical School

June 13, 2011

Genome

TranscriptomeTranscriptome

Proteome

Metabolome IonomeIonome

Trace Elements

Metals Other elements

Selenium is essential for

human healthSelenium deficiency may cause

• Increased risk of cancer• Increased risk of AIDS in HIV-infected • Increased risk of AIDS in HIV-infected

individuals• Male reproduction problems• Increased risk of viral infection• Keshan, Kashin-Beck diseases

Cysteine Selenocysteine

• Se is present in proteins in the form of 21st amino acid - selenocysteine

• Selenocysteine is encoded by UGA;UGA is also a stop signalUGA is also a stop signal

• mRNA structure, selenocysteine insertion sequence (SECIS) element, is essential for insertion of selenocysteine

SBP2

EFsec

Sec

Selenocysteine insertion in eukaryotesSelenocysteine insertion in eukaryotes

L30

AUG UAA/UAGUGA

L30

Cysteine Selenocysteine

Selenocysteine versus Cysteine

Cysteine SelenocysteineUGU, UGC-codonsUGA codon

pKa = 8.3pKa = 5.2

S-

S

SSe-

S

S

Sec serves redox function!

AUGUGA

STOP

5’ AAAAAAA 3’

SECIS

Initiation oftranslation

Selenocysteine

UAAUAGUGA

Selenoprotein

Searches for selenoprotein genes

AUG

UGCUGU

STOP

5’ AAAAAAA 3’

Initiation oftranslation

CysteineUAAUAGUGA

Cys-containing protein

SECISearch on-line

Selenoproteome

Eukaryotes: 0-57 (Aureococcus)

Bacteria: 0-57 (worm symbiont)

Archaea: 0-9 (M. maripaludis)

Viruses: 0-1 (fowlpox virus) Viruses: 0-1 (fowlpox virus)

Model organismsOrganism Selenoproteome

Mouse, rat 24

Zebrafish 37

Drosophila 3

C. elegans 1

S. cerevisiae, Arabidopsis 0

CH2

+ -

Se H

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

Selenium

34

SeCOOCH3N

+ -

H

Selenocysteine (21st amino acid)

• 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

Se78.96

• Sec machinery- SelA (prokaryotic Sec synthase)

- SelB (Sec specific elongation factor)

- SelC (Sec tRNA)

- SelD (selenophosphate synthetase)

- SecS (eukaryotic Sec synthase)

- PSTK (phosphoserine tRNA kinase)

- SECISBP2

http://gladyshevlab.bwh.harvard.edu/trace_element

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

– Searchtrace element, organism, protein category, protein family, keyword, etc.

– BLAST search

– Real-time statistics

– Sequence download

Mo Ni Co(B12)Cu Se(Sec)Phylum

Total 401(74%) 432(80%) 319(59%) 410(76%) 139(26%)

Overview of trace Overview of trace element utilizationelement utilization

Bacteria

Total 46(98%) 26(55%) 39(83%) 45(96%) 6(13%)

Total 105(66%) 154(96%) 51(32%) 49(31%) 76(48%)

Archaea

Eukarya

SelenoproteinSec redox

motifSecondary structure and location of Sec

GPx1 UxxT

GPx2 UxxT

GPx3 UxxT

GPx4 UxxT

GPx6 UxxT

SelP UxxC

DI1 SxxU

DI2 SxxU

DI3 SxxU

SelW CxxU

SelV CxxU

SelT CxxUSelT CxxU

SelH CxxU

SelM CxxU

Sep15 CxU

TR1 CU

TGR CU

TR3 CU

SelS U

SelK U

SelO U

SelI U

SelN CU

MsrB1 U

SPS2 UxC

Kryukov et al., Science

300, 1439 (2003)

GP

x1

SelS

GP

x4

GP

x1+

SelS

GP

x1+

GP

x4

GP

x4+

SelS

GP

x1+

GP

x4+

SelS

Co

ntr

ol

siRNA

Selenium in Tissues

Cells Tissues?

Elemental Profiling in Tissues

Cells Tissues

Methods for Trace Elemental AnalysisMethods for Trace Elemental Analysis

• Atomic Absorption Spectroscopy (AAS)

• Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

• Neutron Activation Analysis (NAA)

• X-Ray Fluorescence Microscopy (XFM)

X-ray fluorescence microscopy (XFM)

Role of Selenium in Testes

Liver Testes Sperm

??

??

Selenium Distribution in Mouse Testes

H&EH&E

20 μm Low High

Selenium

Se in mouse testes localizes to elongating spermatids

Selenoprotein P (SelP) Synthesized in liver

Major plasma selenoprotein

Selenium Transport to Mouse Testes

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 testesJ. Mol. Biol. (2009) 389, 808–818

Selenium Utilization in TestesGlutathione Peroxidase 4 (GPx4)

Highly expressed in mouse testes

Has mitochondrial (mGPx4) and nuclear (nGPx4) forms

Selenium Utilization in TestesGlutathione 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, 808–818

Selenium Utilization in TestesGlutathione Peroxidase 4 (GPx4)

Highly expressed in mouse testes

Has mitochondrial (mGPx4) and nuclear (nGPx4) forms

mGP

x4

nGPx

4

+/+

-/-

+/+

-/-

J. Mol. Biol. (2009) 389, 808–818

Selenium Utilization in Sperm

J. Mol. Biol. (2009) 389, 808–818

Role of Selenium in Testes

Liver Testes Sperm

• Liver SelP is the main source of Se

??

??

SelP Elongating

spermatids

mGPx4

midpiece

• Se is distributed to elongating spermatids in mouse testes

• Liver SelP is the main source of Se

• Se is localized to midpiece in sperm cells

• Se is utilized by mitochondrial form of GPx4

Metabolic processes Signal

transduction

ROSdetoxification

Selenoproteins:a subset of thiol oxidoreductases

Thiol Redoxome – a set of

Transcription control

Protein degradation

Protein modification

Oxidative folding

Thiol Redoxome – a set of thiol oxidoreductases in an organism

High-throughout identification of catalytic redox-active cysteine residues

Fomenko et al., Science 315, 387-389 (2007)

Essentially all known thiol oxidoreductases could be detected by the Sec/Cys approach

Thioredoxin Methionine-S-sulfoxide reductase

Glutaredoxin Methionine-R-sulfoxide reductase

AhpD Glutathione peroxidase

DsbA Peroxiredoxin

Protein disulfide isomerase Arsenate reductase

Thioredoxin reductase Ohr (OsmC)Thioredoxin reductase Ohr (OsmC)

1. Redox catalysis (e.g., thiol oxidoreductases)

2. Regulatory cysteines (e.g., kinases, phosphatases)

Functions of Cys in proteins

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

Thiophosphate (SPO3) affects selenoprotein expression

75Se

NIH 3T3 cells

Se labeling

WB

In vitro synthesis of Cys on

O-phosphoseryl-tRNA[Ser]Sec

by SecS from SPO3

Cys can be synthesized from sulfide and P-Ser on tRNA[Ser]Sec

S SeSII

-O – P – O-

IO-

SeII

-O – P – O-

IO-

SPO3 SePO3

98

62

4938

TR1

+SPO3

Can Cys replace Sec? Experimental evidence

28

17

14

6

3

Se diets

Sec is replaced with Cys in TR1 in thiophosphate-treated cells

control 1 mM SPO3

Source of TRs Protein Peptide sequences Sec, % Cys, %

NIH 3T3, control TR1

TR1

R.SGGDILQSGCUG

R.SGGDILQSGCCG

90

10

NIH 3T3, SPO3

treated

TR1

TR1

R.SGGDILQSGCUG

R.SGGDILQSGCCG

4

96

0 ppm Se 0.1 ppm Se 2 ppm Se

Se deficientselenoproteins

Se adequateselenoproteins ~

Se richselenoproteins

Cys is inserted in place of Sec in TR1 under Se deficiency

Liver, 0 ppm Se TR1

TR1

R.SGGDILQSGCUG

R.SGGDILQSGCCG49

51

Liver, 0.1 ppm Se TR1

TR1

R.SGGDILQSGCUG

R.SGGDILQSGCCG91

9

Liver, 2 ppm Se TR1 R.SGGDILQSGCUG

R.SGGDILQSGCCG100

0

Source of TR1 Protein Peptide sequences Sec, % Cys, %

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