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Immunodeficiencies and pathologies associated with ... · PDF file Immunodeficiencies and Pathologies Associated with Mutations in STIM/ORAI Thierry Capiod, PhD INSERM U807 Hôpital

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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, PhD INSERM U807

    Hôpital Necker – Enfants Malades Université 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 Gill Nature Chemical Biology 7, 488–492 (2011)

    5

    Store-independent calcium entry

    6

    Icrac Iarc

    1

    Tetramer Orai1 Pentamer Orai1/Orai3

    3 1 31113

    31311

    ICRAC IARC

    Inward Rectification

    -1.4

    -1

    -0.6

    -0.2

    0.2

    -100-80-60-40-20 0

    20 40

    pA/pF

    mV

    -1.4

    -1

    -0.6

    -0.2

    0.2

    -100-80-60-40-20 0

    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

    Ex tr

    ac el

    lu la

    r Cy

    to so

    l Lu

    m in

    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

    49/56 Ca2+ 76-87

    Luminal Cytoplasmic

    684 685 TRPC

    CAD 342 448

    CDI 470 491

    SOAR 344 442

    C 448441

    EB1 392 652

    CRACR2A 250 400

    CRACR2A 600 685

    S P cEF hEF SAM

    T M CC1

    C C 2

    C C 3

    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

    R429C CC

  • 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

    CAD 342 448

    S P cEF hEF SAM

    T M CC1

    C C 2

    C C 3

    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

    CAD 342 448

    S P cEFhEF SAM

    T M CC1

    C C 2

    C C 3

    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

    S P cEF hEF SAM

    T M

    C C 2

    P/H/E K

    53/60 Ca2+

    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 kDa Orai1 has a glycosylation site (N223)

    Adapted from Varnai et al., TIPS, 2009

    26 M H P E P A

    L P

    PP

    E P S S R S

    STTS G

    G S P P

    G D G

    S R

    RRSRRSG

    P

    P P P P A G P P E

    E

    R

    AS

    S Y

    SQGIWDPY T

    V

    H N L M V

    E E

    S S

    P

    V V

    H EE

    K

    QQ

    A

    S

    S

    I C

    K

    L

    L

    A

    F

    T IS

    M AL

    V

    L VH

    A LV

    T CT

    I LL A

    A FS

    T

    L WA

    A SF

    F

    V GI

    T LL

    K

    L EA

    C VW L

    V LV

    YK WL

    A Q

    M

    R

    R

    S

    A L

    LL

    S

    K K

    A SS

    L SL

    M G

    AF

    Q

    V MA

    V VE

    A

    R ST

    L

    VF I

    IF

    A

    G PF

    T AS T

    V IM

    I AA

    N

    N

    I

    N

    P

    P

    P

    A A

    V

    V

    T

    M

    I

    N

    S

    S

    H

    H

    R

    R

    E

    H

    L

    L

    G

    F

    K K

    P

    P

    P P I

    T

    S S

    S

    Q

    G

    G

    G

    T NA

    P

    R

    P

    H

    Q

    L

    FL

    F

    L

    D

    D

    VHF A

    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

    COOH NH2

    P P

    H

    DL

    D D A

    G

    Y

    External

    Cytosol

    Plasma membraneTMD1 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-term 70-91 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 an

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