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SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

Chami M, Gozuacik D, Lagorce D, Brini M, Falson P, Peaucellier G, Pinton P, Lecoeur H, Gougeon ML, le Maire M, Rizzuto R, Bréchot C, Paterlini-Bréchot P - J. Cell Biol. (2001)

Bottom Line: Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b.Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis.These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The French Institute of Health and Medical Research Institut National de la Santé et de la Recherche Médicale (INSERM/Pasteur U370)/Necker Faculty Institute of Medicine, 75015 Paris, France.

ABSTRACT
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

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S1T proteins reverse SERCA1- and SERCA2b-induced increases of [Ca2+]er. HuH7 (A and B) and HeLa (C) cells were cotransfected with erAEQ and SERCA1 (A and C) or SERCA2b (B). Cotransfection of erAEQ, SERCA1, or SERCA2b and S1T constructs was carried out as described in Materials and Methods. Quantitative assessment of [Ca2+]er was performed as described in the legend to Fig. 5.
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Figure 6: S1T proteins reverse SERCA1- and SERCA2b-induced increases of [Ca2+]er. HuH7 (A and B) and HeLa (C) cells were cotransfected with erAEQ and SERCA1 (A and C) or SERCA2b (B). Cotransfection of erAEQ, SERCA1, or SERCA2b and S1T constructs was carried out as described in Materials and Methods. Quantitative assessment of [Ca2+]er was performed as described in the legend to Fig. 5.

Mentions: To investigate the effect of S1T in a context of increased ER calcium pumping, we overexpressed SERCA1, or the ubiquitous SERCA2b, in HuH7 and HeLa cells. HuH7 cells overexpressing SERCA1 or SERCA2b proteins showed a significantly higher steady state ER calcium level than controls (448 ± 81 μM, n = 13, p-value < 0.01, and 687 ± 105 μM, n = 6, p-value < 0.01, respectively, vs. 330 ± 72 μM, n = 14) (Fig. 6A and Fig. B, and Table ). Cotransfection of SERCA1 and S1T+4 or S1T−4 significantly reduced [Ca2+]er to 297 ± 78 μM, n = 11, p-value < 0.01, and 343 ± 99 μM, n = 10, p-value = 0.02), respectively (Table ). Cotransfection of SERCA2b and S1T+4 or S1T−4 also significantly reduced [Ca2+]er to 434 ± 131 μM, n = 4, p-value < 0.01, and 445 ± 61 μM, n = 4, p-value < 0.01, respectively (Table ). Similar results were obtained in HeLa cells transfected with SERCA1 (446 ± 59 μM, n = 8) and SERCA1+S1T+4 (329 ± 55 μM, n = 4, p-value < 0.05) or SERCA1+S1T−4 (327 ± 59 μM, n = 4, p-value < 0.05) (Fig. 6 C and Table ).


SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

Chami M, Gozuacik D, Lagorce D, Brini M, Falson P, Peaucellier G, Pinton P, Lecoeur H, Gougeon ML, le Maire M, Rizzuto R, Bréchot C, Paterlini-Bréchot P - J. Cell Biol. (2001)

S1T proteins reverse SERCA1- and SERCA2b-induced increases of [Ca2+]er. HuH7 (A and B) and HeLa (C) cells were cotransfected with erAEQ and SERCA1 (A and C) or SERCA2b (B). Cotransfection of erAEQ, SERCA1, or SERCA2b and S1T constructs was carried out as described in Materials and Methods. Quantitative assessment of [Ca2+]er was performed as described in the legend to Fig. 5.
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Related In: Results  -  Collection

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Figure 6: S1T proteins reverse SERCA1- and SERCA2b-induced increases of [Ca2+]er. HuH7 (A and B) and HeLa (C) cells were cotransfected with erAEQ and SERCA1 (A and C) or SERCA2b (B). Cotransfection of erAEQ, SERCA1, or SERCA2b and S1T constructs was carried out as described in Materials and Methods. Quantitative assessment of [Ca2+]er was performed as described in the legend to Fig. 5.
Mentions: To investigate the effect of S1T in a context of increased ER calcium pumping, we overexpressed SERCA1, or the ubiquitous SERCA2b, in HuH7 and HeLa cells. HuH7 cells overexpressing SERCA1 or SERCA2b proteins showed a significantly higher steady state ER calcium level than controls (448 ± 81 μM, n = 13, p-value < 0.01, and 687 ± 105 μM, n = 6, p-value < 0.01, respectively, vs. 330 ± 72 μM, n = 14) (Fig. 6A and Fig. B, and Table ). Cotransfection of SERCA1 and S1T+4 or S1T−4 significantly reduced [Ca2+]er to 297 ± 78 μM, n = 11, p-value < 0.01, and 343 ± 99 μM, n = 10, p-value = 0.02), respectively (Table ). Cotransfection of SERCA2b and S1T+4 or S1T−4 also significantly reduced [Ca2+]er to 434 ± 131 μM, n = 4, p-value < 0.01, and 445 ± 61 μM, n = 4, p-value < 0.01, respectively (Table ). Similar results were obtained in HeLa cells transfected with SERCA1 (446 ± 59 μM, n = 8) and SERCA1+S1T+4 (329 ± 55 μM, n = 4, p-value < 0.05) or SERCA1+S1T−4 (327 ± 59 μM, n = 4, p-value < 0.05) (Fig. 6 C and Table ).

Bottom Line: Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b.Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis.These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The French Institute of Health and Medical Research Institut National de la Santé et de la Recherche Médicale (INSERM/Pasteur U370)/Necker Faculty Institute of Medicine, 75015 Paris, France.

ABSTRACT
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

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Related in: MedlinePlus