elias arnér , md phdgenomics.unl.edu/rbc_2011/index_files/mon4.pdf · 2011. 6. 14. · h h...

Selenoprotein reactivity

Elias Arnér, MD PhD

Division of Biochemistry

June 13, 2011Elias Arnér

Division of Biochemistry

Medical Biochemistry and Biophysics

Karolinska Institutet

Stockholm, Sweden

[email protected]

Berzelius and selenium

Berzeli park, Stockholm


Jöns Jacob Berzelius 1779-

1848



Gripsholm chemical factory

� Where Berzelius discovered selenium in 1817-1818


From pp. 49-50:

“[the element]…which I, to mark its akin

properties with tellurium, have named Selenium,

from Σελήνη, moon (goddess). What is more, it is

in this regard, midway between sulfur and

tellurium, and has almost more characters of

sulfur than of tellurium.”


H H

Selenocysteine (Sec, U)

� 21st amino acid

� 25 human selenoprotein genes


H H | |H3N—C—COO

- H3N—C—COO-

| | CH2 CH2 | | Se- SH

Selenocysteine Cysteine

� Low pKa (≈5.3), nucleophilic and highly reactive

� Sec incorporated at UGA codons

+ +

Chemical properties of Sec vs. Cys

Elias Arnér June 13, 2011

Chemical properties of Sec vs. Cys

High Sec reactivity at pH ≈ 2.0 (!)

with IAA or IAM, much more

reactive than Cys


Sec pKa ≈ 5.2

Nucleophilicity is more than what is solely reflected by pKa values

� Nucleophilicity is governed by a number of factors, including:

• pKa• basicity vs. acidity (Lewis or Brønstedt theories)

• polarizability

• electronegativity


• electronegativity

• atomic radius

• solvation energy (solvent)

• chemical bond strength between reacting atoms

• …

� Sec is clearly much more nucleophilic than Cys

Arnér ES. Exp Cell Res. 2010, 316:1296-303

Nucleophilicity is more than what is solely reflected by pKa values

Nulecophile-assisted catalysis:

uncatalysed R-X+ Y

- XR-Y

nucleophilic catalysed R-X+ Nu

R-Nu+ Y

R-Y


nucleophilic catalysed R-X+ Nu

- XR-Nu

+ Y

- NuR-Y

Nucleophile has to react

faster with R-X than Y-

(facilitated by Nu- having

high nucleophilicity)

Nucleophile has to allow rapid

reaction of R-Nu with Y-

(facilitated by Nu- being a

good leaving group)

These enzymes detoxify peroxides (ROOH) by a selenium-

dependent redox cycling mechanism, involving Sec in the active site

and consuming glutathione:

Glutathione peroxidases


Mammalian thioredoxin reductase

- kinetic properties- high Sec reactivity


- high Sec reactivity- importance for cancer therapy

The mammalian thioredoxin system

TrxR

NADPH + H+ Trx-S2Dehydroscorbic acid

Ascorbic acid

Lipid hydroperoxides

Alcohols

ASK-1��

ASK-1�� Apoptosis induction

Apoptosis repression

Secreted Trx as co-cytokine

Converted to Trx80 as secreted chemokine

Ribonucleotide reductase


NADP+ Trx-(SH)2

Alcohols

α-Lipoic Acid

DHLA

GSSG

2 GSH

H2O2

H2O+O2GPxox

GPxredPrxox

Prxred

NF-κB repression (in cytosol)

AP-1 activation

P53 maturation and function

reductase

Nordberg J. & Arnér E.S.J. (2001). Free Rad. Biol. Med.31,1287-1312.

Catalytic cycle(s) of TrxR1

� Both subunits required for normal Sec-dependent Trx reducing activity

� One single subunit half-active


� One single subunit half-active site is still redox active, and may redox cycle with certain substrates if the Sec residue becomes destroyed

-Gly-Cys-Sec-Gly-COOH

≈ 1% specific activity

Sec-to-Cys in TrxR yields very low activity


-Gly-Cys-Cys-Gly-COOH

≈ 1% specific activity

Zhong and Holmgren (2000) J. Biol. Chem., 275, 18121-18128

Lee, et al (2000) Proc Natl Acad Sci U S A. 97, 2521-2526

Fujiwara et al (2001) J Biochem (Tokyo). 129, 803-812

Bar-Noy et al (2001) Free Radic Biol Med. 30, 51-61

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

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)


• 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


12

14

16

18

20

Km

[µ

M]

Km for Dm-Trx was not affected


0

2

4

6

8

10

12

Km

[µ

M]

GC

CG

SCC

G

GC

CS

SCC

S

GC

UG

SCU

G

GC

US

SCU

S

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


12

14

16

18

20

Kc

at

[s-1

]

12

14

16

18

20

Kc

at

[s-1

]

Effects on Kcat


0

2

4

6

8

10

12

Kc

at

[s-1

]

GC

CG

SCC

G

GC

CS

SCC

S

0

2

4

6

8

10

12

Kc

at

[s-1

]

GC

UG

SCU

G

GC

US

SCU

S

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

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

Why selenoprotein?P

erc

enta

ge o

f activity

dete

rmin

ed a

t pH

7.4

60

80

100

120

140

GCCG

60

80

100

120

140

Perc

enta

ge o

f activity

dete

rmin

ed a

t pH

7.4


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

— Could another pH-optimum profile be the answer?

6.0 6.5 7.0 7.5 8.0 8.5 9.0

Perc

enta

ge o

f activity

dete

rmin

ed a

t pH

7.4

0

20

40GCCG SCCG GCCS SCCS

6.0 6.5 7.0 7.5 8.0 8.5 9.00

20

40 GCUG

SCUG GCUS SCUS

Perc

enta

ge o

f activity

dete

rmin

ed a

t pH

7.4

40

50

60

70

80

90

100In

hib

itio

n [

%]

Effect of 1 µM auranofin

Why selenoprotein?


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

— Could reactions with electrophilic agents be the answer?

0

10

20

30

In

hib

itio

n [

%]

GCCG SCCG GCCS SCCS GCUG SCUG GCUS SCUS

Form of DmTrxR

NADPH + H+ NADP+

Electrophilic compounds easily target the selenocysteine residue in TrxR

S Se

SH

Se-


Electrophilic drugs inactivating TrxR1:

SH

Se

DNCB, DNFB, ...

iodoacetic acid, iodoacetamide, 4-vinylpyridine, …

Aurothioglucose, auranofin, …

Nitrosoureas, other alkylating anticancer agents

quinone derivatives

electrophilic prostaglandin derivatives

platinum compounds (cisplatin, oxaliplatin)

…and many more….

TrxR1

Controls/MOCK V1/V2 siRNA

A549 cells

75Se:


Protein:

Eriksson et al (2009) FRBM, 47:1661-1671

TrxR1 siRNA

MOCK

Effects of targeting TrxR1?


� Knockdown of TrxR1 to ≈10% sensitizes cells towards DNCB and menadoione� No difference in sensitivity towards auranofin


TrxR1 siRNA

MOCK

Effects of targeting TrxR1?


� Knockdown of TrxR1 to ≈10% makes cells more resistant to cisplatin� No difference in sensitivity towards oxaliplatin or juglone


Effects of targeting Sec in TrxR1?

Cell growth

Redox regulation

Antioxidant defense

p53 function

Increased cell proliferation, modulation of cell

function, or induction of apoptosis, depending

upon additional stimuli and growth conditions

ROS

(Induction of TrxR1 through an Nrf2 response)Electrophilic compounds

targeting TrxR

Intact TrxR/Trx system


Anestål et al (2008) PLoS ONE, e1846; Arnér (2009) BBA, 1790, 495-526

ROS

production

• 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

Properties of a Sel-tag

� is a small modification of parent peptide

� is redox active

Elias Arnér

� is redox active

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

Johansson et al (2004) Nat. Meth. 1, 61-66

Johansson et al (2005) FEBS J. 272, 3449-3460

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

Sel-tag

May 31, 2010

Selenolate-targeted fluorescence labeling using the Sel-tag

S

|

Se

DTT

5-IAF or other thiolate

(selenolate) targeting

fluorescent probe

R

Elias Arnér

SH

Se-

DTT

SH

Se

+ R-

May 31, 2010

� -Gly-Cys-Sec-Gly (GCUG)

� -Ser-Cys-Sec-Ser (SCUS)

� -Gly-Cys-Cys-Gly (GCCG)

Selenolthiol variants

Fel d 1

Which “Sel-tag”?

Elias Arnér

� -Gly-Cys-Cys-Gly (GCCG)

� -Ser-Cys-Cys-Ser (SCCS)

Dithiol variants

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

May 31, 2010

Protein labeling – Fluorescent labeling

Elias Arnér

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

May 31, 2010

Selenolate-targeted radiolabeling with a positron emitter

S

|

Se[11C]CH3I

Elias Arnér

SH

Se-

DTT

SH

Se-[11C]CH3

+ I-

[ C]CH3I

May 31, 2010

Selenolate-targeted radiolabeling with a positron emitter

Elias Arnér

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

May 31, 2010

Selenolate-targeted radiolabeling of Sel-tagged Annexin V

[11C]CH3I

Elias Arnér

DTT

[ C]CH3I

May 31, 2010

+ I-

11C-labeling and microPET imaging

Elias Arnér May 31, 2010

11C-labeled Annexin in microPET imaging of a healthy mouse


PET imaging of apoptosis in liver after anti-Fas using11C-labeled Sel-tagged Annexin A5


PET imaging of apoptosis in cancer using 11C-labeled Sel-tagged Annexin A5 with treated xenograft tumor (24 hrs after doxorubicin)


Sec vs. Cys, in Nature???


A Sec-to-Cys variant of TrxR1 is made in vivoupon selenium deficiency!


A Sec-to-Cys variant of GPx4 can save the lethal phenotype of GPx4-/-


Summary of Sec vs. Cys reactivity

� Oxidoreductases may evolve to use either Cys or Sec – “why”?

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

� Sec is clearly more reactive with electrophiles than “activated Cys”, due to higher nucleophilicity and beyond the sole impact of different pKa

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

Elias Arnér

� The Sec reactivity can be used for biotech applications where Cys is not useful, such as radiolabeling of protein ligands for PET imaging

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

June 13, 2011

elias arnér , md phdgenomics.unl.edu/rbc_2011/index_files/mon4.pdf · 2011. 6. 14. · h h...

Documents