May 20, 2009Elias Arnér Selenoprotein reactivity Elias Arnér, MD PhD Division of Biochemistry...

May 20, 2009Elias Arnér

Selenoprotein reactivity

Elias Arnér, MD PhD

Division of Biochemistry

Medical Biochemistry and Biophysics

Karolinska Institutet

Stockholm, Sweden

[email protected]


H H | |H3N—C—COO

- H3N—C—COO-

| | CH2 CH2 | | Se- SH

Selenocysteine Cysteine

Selenocysteine (Sec, U)

21st amino acid

25 human selenoprotein genes

Low pKa (≈5.3), nucleophilic and highly reactive

Sec incorporated at UGA codons

+ +


Calf: -Arg-Ser-Gly-Gly-Asn-Ile-Leu-Gln-Thr-Gly-Cys-Sec-Gly-COOHRat: -Arg-Ser-Gly-Gly-Asp-Ile-Leu-Gln-Ser-Gly-Cys-Sec-Gly-COOHHuman: -Arg-Ser-Gly-Ala-Ser-Ile-Leu-Gln-Ala-Gly-Cys-Sec-Gly-COOH

Tamura & Stadtman (1996) PNAS 93, 1006-1011Gladyshev et al. (1996) PNAS 93, 6146-6151

Lee et al. (1999) J. Biol. Chem. 274, 4722-412; Sun et al. (1999) J. Biol. Chem. 274, 24522-24530; Miranda-Vizuete, A. (1999) Eur. J. Biochem. 261, 405-412

Zhong, et al. (1998) J. Biol. Chem., 273, 8581-8591

Mammalian TrxR’s are selenoproteins

Human selenoprotein TrxR reported in 1996

Conserved selenocysteine, essential for catalytic activity

Selenocysteine-containing motif present in other TrxR isoenzymes

(mitochondrial: TrxR2, testis: TGR)


Johansson et al. (2006) FRBM, 41:649-56.

Arscott, et al. (1997) PNAS, 94, 3621-3626Zhong, et al. (2000) PNAS, 97, 5854-5859Lee, et al (2000) PNAS, 97, 2521-2526Gromer et al (2003) PNAS, 100: 12618-16623 …

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Rat TrxR1 (GCCG mutant, dimer)Sandalova, et al.(2001) PNAS, 98, 9533-9538



Mouse TrxR2 (GCCG mutant, monomer)Biterova et al (2005) PNAS, 102:15018-23

Human TrxR1 (GCGG mutant, dimer)Debreczeni et al.(2006) SGC, unpublished



Catalytic mechanism of TrxR

Cheng et al. (2009) JBC, 284:3998-4008

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-Gly-Cys-Sec-Gly-COOH

-Gly-Cys-Cys-Gly-COOH

≈ 1% specific activity

Zhong and Holmgren (2000) J. Biol. Chem., 275, 18121-18128Lee, et al (2000) Proc Natl Acad Sci U S A. 97, 2521-2526 Fujiwara et al (2001) J Biochem (Tokyo). 129, 803-812Bar-Noy et al (2001) Free Radic Biol Med. 30, 51-61

Sec-to-Cys in TrxR yields very low activity


Activity of DmTrxR surprisingly high

Kanzok et al (2000) J Biol Chem. 275:40180-6

-Gly-Cys-Sec-Gly-COOH -Ser-Cys-Cys-Ser-COOH




Production in E. coli of recombinant thioredoxin reductase with Sec

Arnér, et al. (1999) J. Mol. Biol. 292, 1003-1016Johansson et al (2004) Nat. Meth. 1, 61-66Johansson et al (2005) FEBS J. 272, 3449-3460Cheng et al (2006) Nat. Protocols. 1, 604-613


The role of the C-terminal motif in TrxR assessed using recombinant variants of DmTrxR

First set of mutants – Are the flanking Serines important ?

• DmTrxR-SCCS (wt)• DmTrxR-SCCG• DmTrxR-GCCS• DmTrxR-GCCG (“Sec-to-Cys” variant of mammalian TrxR)

Second set of mutants – What is the importance of selenium ?

• DmTrxR-SCUS (Cys-to-Sec mutant)• DmTrxR-SCUG• DmTrxR-GCUS• DmTrxR-GCUG (“mammalian” motif)

Gromer et al (2003) PNAS; 100:12618-12623



0

2

4

6

8

10

12

14

16

18

20

Km [µM]

GCCG SCCG GCCS SCCS GCUG SCUG GCUS SCUS

Km for Dm-Trx was not affected

Gromer et al (2003) PNAS; 100:12618-12623



02468

101214161820

Kcat [s-1]

GCCG SCCG GCCS SCCS

02468

101214161820

Kcat [s-1]

GCUG SCUG GCUS SCUS

Gromer et al (2003) PNAS; 100:12618-12623

Effects on Kcat


Neighboring Ser residues may perhaps activate the Cys residues to reach Sec in reactivity?

Ser488Ser488

Ser488Ser488

Cys489Cys489

Cys489Cys489

Ala487Ala487

Pro486Pro486

Ser488Ser488

Ser488Ser488

Cys489Cys489

Ala487Ala487

Pro486Pro486

Cys489Cys489

Gromer et al (2003) PNAS; 100:12618-12623

Substrate(ox)

Product(red)

NADPH + H+

NADP+


But - the same does not work in mammalian TrxR

Rat TrxR1-GCUG 100%Rat TrxR1-SCCS < 0.5%

Main reason for the low activity was avery slow oxidative half-reaction

L. Johansson et al. FRBM (2006) 41, 649–656


Gromer et al (2003) PNAS; 100:12618-12623

— Could another pH-optimum profile be the answer?

Why selenoprotein?

6.0 6.5 7.0 7.5 8.0 8.5 9.0

Per

cent

age

of a

ctiv

ityde

term

ined

at p

H 7

.4

0

20

40

60

80

100

120

140

GCCG SCCG GCCS SCCS

6.0 6.5 7.0 7.5 8.0 8.5 9.00

20

40

60

80

100

120

140

GCUG SCUG GCUS SCUS

Per

cent

age

of a

ctiv

ityde

term

ined

at p

H 7

.4


Gromer et al (2003) PNAS; 100:12618-12623

— Could reactions with electrophilic agents be the answer?

0

10

20

30

40

50

60

70

80

90

100

Inhibition [%]

GCCG SCCG GCCS SCCS GCUG SCUG GCUS SCUS

Form of DmTrxR

Effect of 1 µM auranofin

Why selenoprotein?


NADPH + H+ NADP+

Electrophilic drugs inactivating TrxR1:

Electrophilic compounds easily target the selenocysteine residue in TrxR

S Se

SH

Se-

SH

Se

DNCB, DNFB, ...iodoacetic acid, iodoacetamide, 4-vinylpyridine, …Aurothioglucose, auranofin, …Nitrosoureas, other alkylating anticancer agentsquinone derivativeselectrophilic prostaglandin derivativesplatinum compounds (cisplatin, oxaliplatin)…and many more….


Effects of targeting Sec in TrxR1?

Anestål et al (2008) PLoS ONE, e1846; Arnér (2009) BBA, In press

Cell growthRedox regulation

Antioxidant defensep53 function

Increased cell proliferation, modulation of cell function, or induction of apoptosis, depending upon additional stimuli and growth conditions

ROSproduction

• Rapid process

• Caspase involvement

• Membrane integrity lost

• Not requiring protein synthesis

SS S

Se

SecTRAPs

Apoptosis and/or necrosis due to lethal prooxidant gain of function in selenium

compromised forms of TrxR

SS

SeS

Cell death, cell cycle arrest or other cellular effects due to an impaired thioredoxin

system

Complete active site inhibited- similar effect as TrxR knockdown Only Sec residue targeted

(Induction of TrxR1 through an Nrf2 response)Electrophilic compounds

targeting TrxR

Intact TrxR/Trx system


How about Sec properties compared to Cys if introduced into any type of protein?


The concept of a Sel-tag

TrxR ORF

Engineered SECIS

rTrxR(...-Gly-Cys-Sec-Gly-COOH)

peptide/protein ORF

Engineered SECIS

Sel-tagged peptide or protein(...-Gly-Cys-Sec-Gly-COOH)


Properties of a Sel-tag

is a small modification of parent peptide

is redox active

has unique biochemical characteristics due to the Sec residue and selenolthiol motif

Johansson et al (2004) Nat. Meth. 1, 61-66Johansson et al (2005) FEBS J. 272, 3449-3460Cheng et al (2006) Nat. Protocols. 1, 604-613

Sel-tag


Using the Sel-tag for purification

One-step purification over PAO Sepharose

N As

Sep

har

ose

mat

rix

Se

S

Sec

Cys

Gly

GlyCOOH

Protein

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Cheng et al (2006) Nat. Protocols. 1, 604-613


Mammalian TrxR: -Gly-Cys-Sec-Gly-COOH

Drosophila TrxR: -Ser-Cys-Cys-Ser-COOH

Which “Sel-tag”?


-Gly-Cys-Sec-Gly (GCUG)

-Ser-Cys-Sec-Ser (SCUS)

-Gly-Cys-Cys-Gly (GCCG)

-Ser-Cys-Cys-Ser (SCCS)

Dithiol variants

Selenolthiol variants

Fel d 1

Fel d 1, one of the allergens from the domestic cat (Felis domesticus).

Which “Sel-tag”?


Protein Purification w/ PAO sepharose

Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981


Pre-reduced with 2 mM DTT React with 5-IAF, 5 min Addition of ß-mercaptoethanol SDS-PAGE Fluorescence documentation

under UV light Coomassie staining

Protein labeling – Fluorescent labeling

Non-specific labeling through six structural cysteines

Short labeling time Lower reaction pH



Selenolate-targeted radiolabeling with a positron emitter

S|Se

SH

Se-

DTT

SH

Se-[11C]CH3

+ I-

[11C]CH3I


Selenolate-targeted radiolabeling with a positron emitter





Carbon-11 labeling and MicroPET imaging of Annexin A5

Before NAP-5 (~30%) After NAP-5 (>99%)


Carbon-11 labeling and MicroPET imaging


PET imaging of apoptosis ─ Tumor model

Mice with tumor xenograft (multiple myeloma) i.v. injection of drug 19 hours later, i.v. injection of 11C-Annexin V One hour MicroPET scanning


Summary of Sec reactivity

Oxidoreductases may evolve to use either Cys or Sec

Sec may substitute Cys in DmTrxR, but Cys can not substitute Sec in rat TrxR even if “activated” by neighboring Ser residues - importance of the active site microenvironment

Sec is clearly more reactive with electrophilic compounds than “activated Cys”, and Sec furthermore gives a broader pH-range of reactivity

The Sec reactivity can be used for some biotech applications where Cys is not useful

– How about the differences between Sec and Cys in Nature….?

May 20, 2009Elias Arnér Selenoprotein reactivity Elias Arnér, MD PhD Division of Biochemistry...

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