Transcript Selenoprotein reactivity Elias Arnér

Selenoprotein reactivity
Elias Arnér, MD PhD
Division of Biochemistry
Medical Biochemistry and Biophysics
Karolinska Institutet
Stockholm, Sweden
[email protected]
Elias Arnér
June 17, 2010
Berzelius and selenium
Jöns Jacob Berzelius 17791848
Elias Arnér
Berzeli park, Stockholm
June 17, 2010
Berzelius and selenium
Gripsholm chemical factory
 Where Berzelius discovered selenium in 1817-1818
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June 17, 2010
Berzelius and selenium
“[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.”
Elias Arnér
From pp. 49-50:
June 17, 2010
Selenium is closely related to oxygen and sulfur
Elias Arnér
June 17, 2010
Selenocysteine (Sec, U)
H
|
+H3N—C—COO|
CH2
|
SeSelenocysteine
Elias Arnér
H
|
+H3N—C—COO|
CH2
|
SH
Cysteine

21st amino acid

25 human
selenoprotein
genes

Low pKa (≈5.3),
nucleophilic and
highly reactive

Sec incorporated
at UGA codons
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Chemical properties of Sec vs. Cys
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June 17, 2010
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
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June 17, 2010
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
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
Elias Arnér
June 17, 2010
Nucleophilicity is more than what
is solely reflected by pKa values
Nulecophile-assisted catalysis:
u
n
c
a
t
a
l
y
s
e
d
+
Y
RY
R
X
-X
+
N
u
+
Y
RNu
RY
n
u
c
l
e
o
p
h
i
l
i
c
c
a
t
a
l
y
s
e
d
R
X
-X
- Nu
Nucleophile has to react
faster with R-X than Y(facilitated by Nu- having
high nucleophilicity)
Elias Arnér
Nucleophile has to allow rapid
reaction of R-Nu with Y(facilitated by Nu- being a
good leaving group)
June 17, 2010
So what selenoproteins are there
- and what functions do they catalyze…?
Elias Arnér
June 17, 2010
The Human Selenoproteome
(Vadim Gladyshev and collaborators)
Kryukov et al, Science. 2003;300:1439-1443
Known antioxidant enzymes
 Many more and other
selenoproteins in other
organisms!
 Plants and yeast are
examples of organisms not
having selenoproteins
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June 17, 2010
Glutathione peroxidases
These enzymes detoxify peroxides (ROOH) by a seleniumdependent redox cycling mechanism, involving Sec in the active site
and consuming glutathione:
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Elias Arnér
June 17, 2010
Effects of selenium on
cancer are dependent
upon context, type of
selenium compound,
duration and
concentration
Se compounds
and
selenoproteins
SeO32-
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June 17, 2010
Expanding the genetic code
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June 17, 2010
Expanding the genetic code
Sec
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June 17, 2010
Mechanism of selenocysteine
insertion in eukaryotes
Allmang, C. et al (2009). BBA 1790, 1415-1423.
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June 17, 2010
Mechanism of selenocysteine
insertion in bacteria (E. coli)
Yoshizawa & Böck (2009). BBA 1790, 1404-1414.
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June 17, 2010
Requirements for co-translational
selenoprotein synthesis
 tRNASec that is charged with Sec
 An in-frame UGA codon
 A secondary structure in the selenoprotein mRNA, called a
Sec Insertion Sequence (SECIS) element
 A dedicated elongation factor for co-translational Sec insertion,
interacting with both the tRNASec and the SECIS
element(directly or indirectly)
 Accessory factors to produce the Sec-tRNASec and to facilitate
the Sec insertion at the UGA codon
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June 17, 2010
Differences between eukaryotic
and bacterial SECIS elements
Eukaryotic
* In 3’-UTR
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Bacterial
* Within ORF
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Can mammalian selenoproteins be expressed in E. coli?
Bacterial selenoprotein mRNA:
5’
bacterial-type SECIS
Bacterial selenoprotein ORF
3’
UGA
Mammalian selenoprotein mRNA:
5’
mammalian-type SECIS
Mammalian selenoprotein ORF
3’
UGA
Elias Arnér
June 17, 2010
Production in E. coli of recombinant
thioredoxin reductase with Sec at the
C-terminus
FDH-H:
11 nt
UGA codon
for s el enocy s t ei ne
i ns ert i on
{
GU
G
C
A U
C G
G C
U
U A
G C
G C
C
A
U A
A U
C
C
C G
C G
G U
G C
C G
A
C G
A UA
G
A
U
U
C G
U G
G C
U G
G C
C A
}
New SECIS:
S el B
bi ndi ng
G
G
A
C
G
UU
G
G
C
U
A
A
U
A
A
U
C
G
G
A
G
U
C
G
U
A
G
G
U
C
U
G
C
A
C
C
A
A
U
C
G
U
U
A
G
C
C
U
A
U
G
C
G
G
C
C
Arnér, et al. (1999) J. Mol. Biol. 292, 1003-1016
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June 17, 2010
The thioredoxin glutathione reductase
of Schistoma mansoni
A collaboration with David L. Williams,
Illinois State University, USA
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June 17, 2010
Schistosoma mansoni



Elias Arnér
An infectious worm
May cause schistosomiasis (bilharzia), with fever, portal
hypertension, abdominal symptoms, death due to fatigue,
diarrhea or CNS symptoms
About 200 million people infected, more than 200,000 deaths
annually
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The “TGR” of S. mansoni
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June 17, 2010
The “TGR” of S. mansoni
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June 17, 2010
Substrate spectrum of pure recombinant SmTGR
K m ( µM )
Vmax ( µM /min)
K cat ( s -1 )
Kcat /K m ( M -1 s-1 )
Di sulfide proteins
SmTrx1
SmPrx
6.37 ± 0.9
NA
36 ± 1.7
NA
30
NA
4.7 x 10
NA
6
Low MW di sulfides
DTN B
114 ± 9.0
19.2 ± 1.6
16
1.4 x 10
5
GSSG
71.5 ± 11.8
26 ± 3.5
21.7
3.0 x 10
5
HED
1867 ± 92
21.4 ± 0.33
17.8
9.6 x 10
3
GSH
248.6 ± 7.8
24.1 ± 3.9
20.1
8.1 x 10
4
Se substrates
Na 2SeO 3
66.7 ± 6.9
2.54 ± 0.046
2.1
3.1 x 10
4
Peroxi des
H 2O 2
68,000 ± 5000
11.3 ± 0.46
9.4
1.4 x 10
2
48,550 ± 2000
1140 ± 220
12.8 ± 0.33
29.5 ± 4.5
10.7
24.6
2.2 x 10
2.2 x 10
2
Other subsrates
t- Butyl -OOH
all oxan
li poic acid
li poami de
2610 ± 300
1342 ± 11.4
2.03 ± 0.135
14.2 ± 0.46
1.7
11.8
6.5 x 10
8.8 x 10
2
Coupled
Grx assay
DH AA
ubiq ui none

NA
NA
NA
NA
NA
NA
4
3
NA
NA
SmTGR, in essence, combines the activities
of mammalian TrxR’s, GR’s and Grx’s
Kuntz et al. (2007) PLoS Med 4(6): e206
Elias Arnér
June 17, 2010
Finding inhibitors of SmTGR?
gold comp le xes
14-15
cis -platinum complexes 16
Mannich ba ses17
1,4-naph thoquinones 18-20
auranofin
aurothioglucose
aurothiomalate
RMA19
RMA35
3-DAP
CDE4
CDE16-2
menadione
plumbagin
naphthazarin
JD155
JD159
M5
LS766
LS852
LS1121
LS908
LS1108
LS1114
LS1105
LS808
LS826
100
100
100
80
98
100
99
98
82
85
100
70
100
63
24
26
29
30
41
44
63
74
95
(Incubation with 50 µM inhibitor 15 min in presence of NADPH, then DTNB assay)
Kuntz et al. (2007) PLoS Med 4(6): e206
Elias Arnér
June 17, 2010
Auranofin inhibits SmTGR in larvae
GR activity
25
control
20
15
10
5
+ 10 µM auranofin
0
0
2
4
6
0
Time (hr)
TrxR activity
60
control
Activity
(nmol/min/mg)
60
50
40
30
20
10
+ 10 µM auranofin
0
0
2
Time (hr)
6
4
2
Time (hr)
70
Activity
(nmol/min/mg)
LDH activity
160
140
120
100
80
60
40
20
0
Activity
(nmol/min/mg)
Activity
(nmol/min/mg)
30
4
6
GPx activity
50
40
30
20
10
0
0
2
4
6
Time (hr)
 SmTGR was specifically inhibited in cultured
Schistosoma mansoni by treatment with auranofin
Kuntz et al. (2007) PLoS Med 4(6): e206
Elias Arnér
June 17, 2010
GR activity
70
TrxR activity
60
25
Activity
(nmol/min/mg)
Activity
(nmol/min/mg)
30
20
15
10
5
50
40
30
20
10
0
0
0
2
4
6
0
Time (hr)
2
Time (hr)
4
6
GSH:GSSG ratio
25
20
15
10
5
0
0
2
4
6
Time (hr)
 Inhibition of SmTGR coincides with oxidation of GSH
 83% of the treated worms were dead after 6 hrs
Kuntz et al. (2007) PLoS Med 4(6): e206
Elias Arnér
June 17, 2010
Is SmTGR an essential protein?
TGR
C
GAPDH
C
RNAi
RNAi
100
% Survival
80
60
40
20
0
1
2
Days
3
4
 SmTGR is essential for Schistosoma mansoni survival in
culture, under both aerobic and anaerobic conditions
Kuntz et al. (2007) PLoS Med 4(6): e206
Elias Arnér
June 17, 2010
Can inhibition of SmTGR cure
schistosomiasis?
•
Mice were infected with 100 S. mansoni cercariae
Injected i.p. with 6 mg auranofin/kg body weight (safe dose for healthy
mice), twice daily for nine days, beginning 7 days post infection
One week after final dose, the mice were perfused and worms counted
Worm counts
•
•
50
40
30
20
10
0
**
**
Control
AF
Treatment
 Treatment of infected mice with auranofin gave
significantly lower worm burden (p<0.015, n=7)
Kuntz et al. (2007) PLoS Med 4(6): e206
Elias Arnér
June 17, 2010
Mammalian thioredoxin reductase
- kinetic properties
- importance for cancer therapy
Elias Arnér
June 17, 2010
The mammalian thioredoxin system
ASK-1 Apoptosis
induction
Dehydroscorbic acid
Trx-S2
NADPH + H+
Ascorbic acid
Lipid hydroperoxides
Alcohols
Secreted Trx as cocytokine
Converted to Trx80 as
secreted chemokine
TrxR
Ribonucleotide
reductase
ASK-1 Apoptosis
repression
GSSG
α-Lipoic Acid
DHLA
2 GSH
NADP+
P53 maturation
and function
Trx-(SH)2
H2O2
H2O+O2
GPxox
AP-1 activation
NF-κB repression
(in cytosol)
Prxox
GPxred
Prxred
Nordberg J. & Arnér E.S.J. (2001). Free Rad. Biol. Med.31,1287-1312.
Elias Arnér
June 17, 2010
Catalytic mechanism of TrxR
Rat TrxR1 (GCCG mutant, dimer)
Sandalova, et al.(2001) PNAS, 98, 9533-9538
Human TrxR1 (GCGG mutant, dimer)
Debreczeni et al.(2006) SGC, unpublished
Mouse TrxR2 (GCCG mutant, monomer)
Biterova et al (2005) PNAS, 102:15018-23
Arscott, et al. (1997) PNAS, 94, 3621-3626
Zhong, et al. (2000) PNAS, 97, 5854-5859
Lee, et al (2000) PNAS, 97, 2521-2526
Gromer et al (2003) PNAS, 100: 12618-16623
…
Elias Arnér
June 17, 2010
Catalytic mechanism of TrxR
Arnér, E.S.J. (2009) BBA 1790, 495-526
Elias Arnér
June 17, 2010
The selenenylsulfide of TrxR
Cheng et al. (2009) JBC, 284:3998-4008
Elias Arnér
June 17, 2010
Catalytic cycle(s) of TrxR1
 Both subunits required for
normal Sec-dependent Trx
reducing activity
 One single subunit half-active
site is still redox active, and
may redox cycle with certain
substrates if the Sec residue
becomes destroyed
Elias Arnér
June 17, 2010
Electrophilic compounds easily target the
selenocysteine residue in TrxR
NADPH + H+
S
NADP+
SH
Se
Se-
Electrophilic drugs inactivating TrxR1:
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….
Elias Arnér
SH
Se
June 17, 2010
A549 cells
Controls/MOCK
V1/V2 siRNA
75Se:
TrxR1
Protein:
Eriksson et al (2009) FRBM, 47:1661-1671
Elias Arnér
June 17, 2010
Effects of targeting TrxR1?
TrxR1 siRNA
MOCK
 Knockdown of TrxR1 to ≈10% sensitizes cells towards DNCB and menadoione
 No difference in sensitivity towards auranofin
Eriksson et al (2009) FRBM, 47:1661-1671
Elias Arnér
June 17, 2010
Effects of targeting TrxR1?
TrxR1 siRNA
MOCK
 Knockdown of TrxR1 to ≈10% makes cells more resistant to cisplatin
 No difference in sensitivity towards oxaliplatin or juglone
Eriksson et al (2009) FRBM, 47:1661-1671
Elias Arnér
June 17, 2010
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
Intact TrxR/Trx system
(Induction of TrxR1 through an Nrf2 response)
Electrophilic compounds
targeting TrxR
S
S
S
Se
S
S
ROS
production
SecTRAPs
S
Se
• Rapid process
Complete active site inhibited
- similar effect as TrxR knockdown
Only Sec residue targeted
• Caspase involvement
• Membrane integrity lost
• Not requiring protein synthesis
Cell death, cell cycle arrest or other cellular
effects due to an impaired thioredoxin
system
Apoptosis and/or necrosis due to lethal
prooxidant gain of function in selenium
compromised forms of TrxR
Anestål et al (2008) PLoS ONE, e1846; Arnér (2009) BBA, In press
Elias Arnér
June 17, 2010
Summary of selenoprotein reactivity
 Oxidoreductases may evolve to use either Cys or Sec
 Cys-containing orthologues of selenoproteins likely to depend upon
either i) “activated Cys” residues, and ii) higher expression levels
 Sec is clearly more reactive with electrophilic compounds
than “activated Cys” residues
 The Sec reactivity in TrxR may play a role in chemotherapy - either
against parasites such as Schistosoma, or in human cancer treatment
 The Sec reactivity can be used for a number of biotech applications,
where Cys is not as useful
Elias Arnér
June 17, 2010
Elias Arnér
June 17, 2010
Acknowledgements
Hanna-Stina Ahlzén
Marcus Cebula
Qing Cheng
Victor Croitoru
Pascal Dammeyer
Sofi Eriksson
Stefanie Prast-Nielsen
Jianqiang Xu
Elias Arnér
Sharon Stone-Elander
Jan-Olov Thorell
Erik Samén
Li Lu
Stig Linder
Lars Holmgren
Stefan Ståhl
Lars Abrahmsén
Ylva Lindqvist
Tanja Sandalova
Charles Williams, Jr.
Narimantas Cenas
Stephan Gromer
June 17, 2010