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Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
1The screen versions of these slides have full details of copyright and acknowledgements
1
Immunodeficiencies and Pathologies Associated with Mutations
in STIM/ORAI
Thierry Capiod, PhDINSERM U807
Hôpital Necker – Enfants MaladesUniversité Paris Descartes
2
Role of STIM (Stromal Interaction Molecule) and Orai:Release of calcium from the endoplasmic reticulum and activation of plasma membrane calcium channels
• STIM1 - Endoplasmic reticulum and plasma membrane
• STIM2 - Endoplasmic reticulum
• Orai1, Orai2, Orai3 - Plasma membrane
• Store-operated calcium entry (SOCE)
• Store-independent calcium entry (SICE)
3 Jonathan Soboloff, Muniswamy Madesh & Donald L GillNature Chemical Biology 7, 488–492 (2011)
Resting T cells
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
2The screen versions of these slides have full details of copyright and acknowledgements
4
• Store-operated calcium entry (SOCE)• ICRAC: Calcium release activated calcium current
Activated T cells
Jonathan Soboloff, Muniswamy Madesh & Donald L GillNature Chemical Biology 7, 488–492 (2011)
5
Store-independent calcium entry
6
Icrac Iarc
1
Tetramer Orai1Pentamer Orai1/Orai3
3 1 31113
31311
ICRAC IARC
Inward Rectification
-1.4
-1
-0.6
-0.2
0.2
-100-80-60-40-200
20 40
pA/pF
mV
-1.4
-1
-0.6
-0.2
0.2
-100-80-60-40-200
20 40
pA/pF
mV
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
3The screen versions of these slides have full details of copyright and acknowledgements
7
ICRAC: Orai1 IARC: Orai1/Orai3
STIM1/STIM2
STIM1
Extr
acel
lula
rCy
toso
lLu
min
al
8
STIM1, immunodeficiency and cancer
• Human STIM1 has been implicated as one of the potential tumour suppressor genes within the 11p15.5 locus
9
STIM1
49/56Ca2+76-87
Luminal Cytoplasmic
684 685TRPC
CAD342 448
CDI470 491
SOAR344 442
C448441
EB1392 652
CRACR2A250 400
CRACR2A600 685
SP cEF hEF SAM T
M CC1CC2
CC3
P/S K
Predicted molecular weight: 77 kDa
Adapted from Le Deist and Capiod, Medecine-Sciences 2011
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
4The screen versions of these slides have full details of copyright and acknowledgements
10 Adapted fromStathopulos et al, Cell 2008
11
STIM1 mutants
EF hand domain
Punctae
Adapted from Liou et al., Curr Biol, 2005 and Baba et al., PNAS, 2006
SAM domain
Punctae
12
S/P
CC
CC
SAM
EFh
STIM1 mutations and immunodeficiency
Cytosol
ER lumen
1538-1G>A
E128RfsX9
R429CCC
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
5The screen versions of these slides have full details of copyright and acknowledgements
13
STIM1 mutation: E128RfsX9 / E136X
• 3 patients from one family were homozygous for an adenine insertion in exon 3, which results in a frameshift, premature stop codon, and termination of position 136 of the protein sequence
• A predicted 15 kDa N-terminal STIM1 fragment, however, was not observed
• STIM1 mRNA levels were greatly reduced and STIM1 protein expression was not detectable
• SOCE was totally abolished in these patients
14
STIM1 mutation: 1538-1G>A
• Patient was homozygous for a splice site mutation in exon 8 of STIM1 (1,538–1 G >A) and lacked normally spliced, full-length STIM1 mRNA transcripts
• Neither full-length nor truncated STIM1 proteins (representing abnormal STIM1 splice products) were detectable in the patient’s B cells and SOCE was totally abolished in this patient's B cells
• Expression of either wild-type STIM1 or STIM2 was able to rescue SOCE in fibroblasts from one of the STIM1-deficient patients
15
Luminal Cytoplasmic
CAD342 448
SP cEF hEF SAM T
M CC1CC2
CC3
P/S
K
• Immunodeficiency• Lymphoproliferative disease
• Eczema• Chronic diarrhea
• Arthritis
R429C
Adapted from Le Deist and Capiod, Medecine-Sciences 2011
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
6The screen versions of these slides have full details of copyright and acknowledgements
16
Luminal Cytoplasmic
CAD342 448
SP cEFhEF SAM T
M CC1CC2
CC3
P/S
K
R429C
• The mutation completely abolished Ca2+ influx in purified CD3+ T cells• The clinically asymptomatic heterozygous mother
showed a partial impairment of Ca2+ flux
17
Mutations in STIM1 and cancer
18
Mutations in STIM1 and cancer (2)
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
7The screen versions of these slides have full details of copyright and acknowledgements
19
STIM2
SP cEF hEF SAM T
MCC2
P/H/E K
53/60Ca2+
80-91
Luminal Cytosol
Predicted molecular weight 84 kDa
Adapted from Le Deist and Capiod, Medecine-Sciences 2011
CC1
20
ICRAC inhibition
STIM2 (2)
Adapted from Soboloff et al., Curr Biol, 2006
21
Biphasic effects of STIM2 over-expression
STIM2 (3)
Adapted from Brandman et al., Cell, 2007
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
8The screen versions of these slides have full details of copyright and acknowledgements
22
Resting cytosolic Ca2+ concentration
STIM2 (4)
Adapted from Brandman et al, Cell, 2007
23
Mutations in STIM2 and cancer
24
Orai1, immunodeficiency and cancer
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
9The screen versions of these slides have full details of copyright and acknowledgements
25
Orai1
Predicted molecular weight: 33 kDaOrai1 has a glycosylation site (N223)
Adapted from Varnai et al., TIPS, 2009
26M H P E P A
L P
PP
E P S S R S
STTSG
G S P P
G DG
SR
RRSRRSG
P
P P P P A G P P E
E
R
AS
S Y
SQGIWDPYT
V
H NL M V
EE
SS
P
VV
H EE
K
A
S
S
IC
K
L
L
A
F
TIS
MAL
V
LVH
ALV
TCT
ILL A
AFS
T
LWA
ASF
F
VGI
TLL
K
LEA
CVW L
VLV
YKWL
AQ
M
R
R
S
AL
LL
S
KK
ASS
LSL
MG
AF
Q
VMA
VVE
A
RST
L
VFI
IF
A
GPF
TAS T
VIM
IAA
N
N
I
N
P
P
P
AA
V
V
T
M
I
N
S
S
H
H
R
R
E
H
L
L
G
F
KK
P
P
PP I
T
S S
S
Q
G
G
G
TNAP
R
P
H
Q
L
FL
F
L
D
D
VHFA
H
D
Y
V
E
L
L
H
Y
K
R
R
R
H
L
Q
Q
G
GT
D
T
N
E
P
S
SS
A
E
A
A
Q
COOHNH2
PP
H
DL
D DA
G
Y
External
Cytosol
PlasmamembraneTMD1 TMD2 TMD3 TMD4
R91W / A103E / L194P
E106 / D110 / D112/ D114/ E190
Orai1
Adapted from Le Deist and Capiod, Medecine-Sciences 2011
27
• SOAR (STIM-Orai Activating Region): 344-442
• CAD (Calcium-Activating Domain): 342-448
• Orai1:254-301 C-term70-91 N-term
L273S
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
10The screen versions of these slides have full details of copyright and acknowledgements
28
Orai1 mutations and immunodeficiency
Adapted from Feske, Ann NY Acad Sci, 2011
29
A88SfsX25
• Lymphocytes from one patient lacked CRAC channel currents and SOCE, resulting in a severe T cell activation defect
30
L194P and A103E
• The patient lacks the sustained phase of the Ca2+ response which is due to the activation of SOCE, hence of Icrac
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
11The screen versions of these slides have full details of copyright and acknowledgements
31
The effect of wild-type and R91W mutant Orai1 expression on CRAC channel currents
Severe Combined Immune Deficiency
• This R91W mutation disrupts CRAC channel function but does not interfere with ORAI1 expression at the plasma membrane or ORAI1 interaction with STIM1
• Current voltage relationships show inhibition of Icrac in these mutants
32 Adapted from Feske et al, Clin Immunol, 2010
33
Orai1 mutation E106Q
• This charge-neutralizing mutation has been reported to profoundly inhibit CRAC channel currents
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
12The screen versions of these slides have full details of copyright and acknowledgements
34
Mutations in Orai1 and cancer
35
Orai2
Predicted molecular weight: 29 kDa
Adapted from Varnai et al., TIPS, 2009
36
Mutations in Orai2 and cancer
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
13The screen versions of these slides have full details of copyright and acknowledgements
37
Orai3
Predicted molecular weight: 31 kDa
No mutation related to immunodeficiency detected so farAdapted from Varnai et al., TIPS, 2009
38
Orai3, SOCE and Icracin MCF7 breast cancer cell line
• Only the siRNA knockdown of Orai3 was effective in both markedly reducing store-operated Ca2+ entry and Icrac
39
• Somatic mutations in Orai3:– No mutation was observed in pancreas (22 samples),
central nervous system (22 samples), large intestine (11 samples), breast (11 samples) and biliary tract (2 samples)
Mutations in Orai3 and cancer
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
14The screen versions of these slides have full details of copyright and acknowledgements
40
Orai3 mutation
• This dominant-negative E81Q mutation has no effect on Icrac, when expressed in HEK293 cells, while it completely inhibits Iarc in the same cells
41
Conclusion and perspectives
42
Summary of known diseases related to Orai and STIM mutations
Adapted from Feske, Ann NY Acad Sci, 2011
Immunodeficiencies and pathologies associated with mutations in STIM/ORAI
Thierry CAPIOD, PhD
15The screen versions of these slides have full details of copyright and acknowledgements
43
• Picard et al., New England Journal of Medicine (2009) 360: 1971-1980
• Hogan et al., Annual review of Immunology (2010) 28: 491-533
• Feske et al., Clinical Immunology (2010) 135: 169-182
• Johnstone et al., Journal of Cellular and Molecular Medicine (2010) 14: 1890-1903
• Feske, Annals of the New York Academy of Sciences (2011) 1238: 74-90
• Byun et al., Journal of Experimental Medicine (2012) 11: 2307-2312
• Fuchs et al., The Journal of Immunology (2012) 188: 1523-1533
• Berna-Erro et al., Journal of Cellular and Molecular Medicine (2012) 16: 407-424
Short list of reviews on the topic:
44
• Olivier MignenINSERM U1078, Université Bretagne Occidentale, France
• Anne-Sophie Borowiec and Gabriel BidauxINSERM U1003, Université Lille 1, France
• Rachida Tacine, Emilie Dugon and Lucile BroncyINSERM U807, Hôpital Necker – Enfants Malades, France
Acknowledgements:
45