May 20, 2009 Elias Arnér Selenoprotein reactivity Elias Arnér, MD PhD Division of Biochemistry Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden [email protected]
Jan 29, 2016
May 20, 2009Elias Arnér
Selenoprotein reactivity
Elias Arnér, MD PhD
Division of Biochemistry
Medical Biochemistry and Biophysics
Karolinska Institutet
Stockholm, Sweden
May 20, 2009Elias Arnér
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
+ +
May 20, 2009Elias Arnér
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)
May 20, 2009Elias Arnér
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
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Mouse TrxR2 (GCCG mutant, monomer)Biterova et al (2005) PNAS, 102:15018-23
Human TrxR1 (GCGG mutant, dimer)Debreczeni et al.(2006) SGC, unpublished
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Catalytic mechanism of TrxR
Cheng et al. (2009) JBC, 284:3998-4008
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May 20, 2009Elias Arnér
-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
May 20, 2009Elias Arnér
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
May 20, 2009Elias Arnér
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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
May 20, 2009Elias Arnér
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
May 20, 2009Elias Arnér
The role of the C-terminal motif in TrxR assessed using recombinant variants of DmTrxR
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
May 20, 2009Elias Arnér
The role of the C-terminal motif in TrxR assessed using recombinant variants of DmTrxR
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
May 20, 2009Elias Arnér
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+
May 20, 2009Elias Arnér
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
May 20, 2009Elias Arnér
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
May 20, 2009Elias Arnér
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?
May 20, 2009Elias Arnér
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….
May 20, 2009Elias Arnér
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
May 20, 2009Elias Arnér
How about Sec properties compared to Cys if introduced into any type of protein?
May 20, 2009Elias Arnér
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)
May 20, 2009Elias Arnér
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
May 20, 2009Elias Arnér
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
May 20, 2009Elias Arnér
Mammalian TrxR: -Gly-Cys-Sec-Gly-COOH
Drosophila TrxR: -Ser-Cys-Cys-Ser-COOH
Which “Sel-tag”?
May 20, 2009Elias Arnér
-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”?
May 20, 2009Elias Arnér
Protein Purification w/ PAO sepharose
Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981
May 20, 2009Elias Arnér
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
Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981
May 20, 2009Elias Arnér
Selenolate-targeted radiolabeling with a positron emitter
S|Se
SH
Se-
DTT
SH
Se-[11C]CH3
+ I-
[11C]CH3I
May 20, 2009Elias Arnér
Selenolate-targeted radiolabeling with a positron emitter
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Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981
May 20, 2009Elias Arnér
Carbon-11 labeling and MicroPET imaging of Annexin A5
Before NAP-5 (~30%) After NAP-5 (>99%)
May 20, 2009Elias Arnér
Carbon-11 labeling and MicroPET imaging
May 20, 2009Elias Arnér
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
May 20, 2009Elias Arnér
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….?