immunodeficiencies and pathologies associated with ... · immunodeficiencies and pathologies...

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

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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


Thierry CAPIOD, PhD


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• 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)

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Store-independent calcium entry

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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


Thierry CAPIOD, PhD


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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


Thierry CAPIOD, PhD


10 Adapted fromStathopulos et al, Cell 2008

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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


Thierry CAPIOD, PhD


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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

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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



Thierry CAPIOD, PhD


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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

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Mutations in STIM1 and cancer

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Mutations in STIM1 and cancer (2)


Thierry CAPIOD, PhD


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STIM2

SP cEF hEF SAM T

MCC2

P/H/E K

53/60Ca2+

80-91

Luminal Cytosol

Predicted molecular weight 84 kDa


CC1

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ICRAC inhibition

STIM2 (2)

Adapted from Soboloff et al., Curr Biol, 2006

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Biphasic effects of STIM2 over-expression

STIM2 (3)

Adapted from Brandman et al., Cell, 2007


Thierry CAPIOD, PhD


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Resting cytosolic Ca2+ concentration

STIM2 (4)

Adapted from Brandman et al, Cell, 2007

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Mutations in STIM2 and cancer

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Orai1, immunodeficiency and cancer


Thierry CAPIOD, PhD


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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

QQ

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


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• SOAR (STIM-Orai Activating Region): 344-442

• CAD (Calcium-Activating Domain): 342-448

• Orai1:254-301 C-term70-91 N-term

L273S


Thierry CAPIOD, PhD


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Orai1 mutations and immunodeficiency

Adapted from Feske, Ann NY Acad Sci, 2011

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A88SfsX25

• Lymphocytes from one patient lacked CRAC channel currents and SOCE, resulting in a severe T cell activation defect

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L194P and A103E

• The patient lacks the sustained phase of the Ca2+ response which is due to the activation of SOCE, hence of Icrac


Thierry CAPIOD, PhD


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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


Thierry CAPIOD, PhD


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Mutations in Orai1 and cancer

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Orai2


Adapted from Varnai et al., TIPS, 2009

36



Thierry CAPIOD, PhD


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Orai3


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

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• 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)



Thierry CAPIOD, PhD


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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

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Conclusion and perspectives

42

Summary of known diseases related to Orai and STIM mutations

Adapted from Feske, Ann NY Acad Sci, 2011


Thierry CAPIOD, PhD


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• 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:

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• 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:

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