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Chaperoning mitochondrial permeability transition: regulation of transition pore complex by a J-protein, DnaJC15.

Sinha D, D'Silva P - Cell Death Dis (2014)

Bottom Line: DnaJC15 was found to exert its proapoptotic function through the essential component of MPTP, cyclophilin D (CypD).Our results reveal a specific role of DnaJC15 in recruitment and coupling of CypD with mitochondrial permeability transition.In summary, our analysis provides first-time insights on the functional connection between mitochondrial inner membrane protein translocation machinery-associated J-protein DnaJC15 and regulation of cell death pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, India.

ABSTRACT
Mitochondria have a central role in the intrinsic pathway of apoptosis and involve activation of several transmembrane channels leading to release of death factors. Reduced expression of a mitochondrial J-protein DnaJC15 was associated with the development of chemoresistance in ovarian cancer cells. DnaJC15 was found to be a part of mitochondrial protein-transport machinery, though its connection with cell death mechanisms is still unclear. In the present study, we have provided evidence towards a novel function of DnaJC15 in regulation of mitochondrial permeability transition pore (MPTP) complex in normal and cancer cells. Overexpression of DnaJC15 resulted in MPTP opening and induction of apoptosis, whereas reduced amount of protein suppressed MPTP activation, upon cisplatin treatment. DnaJC15 was found to exert its proapoptotic function through the essential component of MPTP, cyclophilin D (CypD). Our results reveal a specific role of DnaJC15 in recruitment and coupling of CypD with mitochondrial permeability transition. In summary, our analysis provides first-time insights on the functional connection between mitochondrial inner membrane protein translocation machinery-associated J-protein DnaJC15 and regulation of cell death pathways.

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Overexpression of JC15 leads to enhanced chemosensitivity. (a) Relative viability of cells variably expressing JC15 or JC15V133W were either left untreated or treated with cisplatin (+Cpl) and measured using MTT assay. Data are represented as mean±S.E.M. n=8, *P (two tailed) <0.0001, **P (two tailed) <0.001. (b and c) Cisplatin-treated cells with altered levels of JC15 or JC15V133W mutant were dual stained with AnnexinV–AlexaFlour 488/propidium iodide (PI) (b) or AnnexinV–AlexaFlour 488/MitoTracker Red CMXRos (c). Bars represent mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001; UnT, untreated untransfected cells; T, untransfected cells treated with cisplatin. (d) Fold caspase 3/7 activity of cells with altered wild-type or mutant JC15 expression over untransfected cells represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001; NT, non-targeting siRNA control; UT, cisplatin-treated untransfected cells. (e and f) Isolated mitochondria overexpressing JC15 and JC15V133W were induced to swell in the absence (e) or presence (f) of 1 nM cyclosporin (CsA) and represented as mean±S.E.M. n=3, P<0.0001 with reference to UT
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fig1: Overexpression of JC15 leads to enhanced chemosensitivity. (a) Relative viability of cells variably expressing JC15 or JC15V133W were either left untreated or treated with cisplatin (+Cpl) and measured using MTT assay. Data are represented as mean±S.E.M. n=8, *P (two tailed) <0.0001, **P (two tailed) <0.001. (b and c) Cisplatin-treated cells with altered levels of JC15 or JC15V133W mutant were dual stained with AnnexinV–AlexaFlour 488/propidium iodide (PI) (b) or AnnexinV–AlexaFlour 488/MitoTracker Red CMXRos (c). Bars represent mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001; UnT, untreated untransfected cells; T, untransfected cells treated with cisplatin. (d) Fold caspase 3/7 activity of cells with altered wild-type or mutant JC15 expression over untransfected cells represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001; NT, non-targeting siRNA control; UT, cisplatin-treated untransfected cells. (e and f) Isolated mitochondria overexpressing JC15 and JC15V133W were induced to swell in the absence (e) or presence (f) of 1 nM cyclosporin (CsA) and represented as mean±S.E.M. n=3, P<0.0001 with reference to UT

Mentions: Loss of JC15 expression has been previously implicated in the development of chemoresistance in ovarian carcinoma.6, 7, 8 Indeed, prolonged exposure of ovarian cancer-derived OVCAR-3 and breast cancer MCF7 cells to cisplatin led to progressive reduction of JC15 expression together with an increase in cell viability (Supplementary Figures S1A–D). However, loss of the protein expression was not limited to cisplatin alone. In support to previous observations on clinical samples,8, 19 exposure of both OVCAR-3 and MCF7 cells to doxorubicin had a similar effect on the JC15 protein levels and led to development of chemoresistance in these cells (Supplementary Figures S1E–H). Therefore, to adjudge the role of JC15 in cell death, we altered JC15 expression in HEK293T and assayed for sensitivity to the drug. We observed that loss of JC15 expression in the cells resulted in increased chemoresistance (Figure 1a). The cells showed suppression of apoptosis as suggested by lower annexinV–PI-stained population and activity of effecter caspases 3 and 7 (Figures 1b and d). JC15-depleted cells also showed better maintenance of mitochondrial membrane potential under treated conditions as compared with controls (Figure 1c). This shows that depletion of JC15 protects the cells from cisplatin-mediated apoptotic cell death.


Chaperoning mitochondrial permeability transition: regulation of transition pore complex by a J-protein, DnaJC15.

Sinha D, D'Silva P - Cell Death Dis (2014)

Overexpression of JC15 leads to enhanced chemosensitivity. (a) Relative viability of cells variably expressing JC15 or JC15V133W were either left untreated or treated with cisplatin (+Cpl) and measured using MTT assay. Data are represented as mean±S.E.M. n=8, *P (two tailed) <0.0001, **P (two tailed) <0.001. (b and c) Cisplatin-treated cells with altered levels of JC15 or JC15V133W mutant were dual stained with AnnexinV–AlexaFlour 488/propidium iodide (PI) (b) or AnnexinV–AlexaFlour 488/MitoTracker Red CMXRos (c). Bars represent mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001; UnT, untreated untransfected cells; T, untransfected cells treated with cisplatin. (d) Fold caspase 3/7 activity of cells with altered wild-type or mutant JC15 expression over untransfected cells represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001; NT, non-targeting siRNA control; UT, cisplatin-treated untransfected cells. (e and f) Isolated mitochondria overexpressing JC15 and JC15V133W were induced to swell in the absence (e) or presence (f) of 1 nM cyclosporin (CsA) and represented as mean±S.E.M. n=3, P<0.0001 with reference to UT
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3973195&req=5

fig1: Overexpression of JC15 leads to enhanced chemosensitivity. (a) Relative viability of cells variably expressing JC15 or JC15V133W were either left untreated or treated with cisplatin (+Cpl) and measured using MTT assay. Data are represented as mean±S.E.M. n=8, *P (two tailed) <0.0001, **P (two tailed) <0.001. (b and c) Cisplatin-treated cells with altered levels of JC15 or JC15V133W mutant were dual stained with AnnexinV–AlexaFlour 488/propidium iodide (PI) (b) or AnnexinV–AlexaFlour 488/MitoTracker Red CMXRos (c). Bars represent mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001; UnT, untreated untransfected cells; T, untransfected cells treated with cisplatin. (d) Fold caspase 3/7 activity of cells with altered wild-type or mutant JC15 expression over untransfected cells represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001; NT, non-targeting siRNA control; UT, cisplatin-treated untransfected cells. (e and f) Isolated mitochondria overexpressing JC15 and JC15V133W were induced to swell in the absence (e) or presence (f) of 1 nM cyclosporin (CsA) and represented as mean±S.E.M. n=3, P<0.0001 with reference to UT
Mentions: Loss of JC15 expression has been previously implicated in the development of chemoresistance in ovarian carcinoma.6, 7, 8 Indeed, prolonged exposure of ovarian cancer-derived OVCAR-3 and breast cancer MCF7 cells to cisplatin led to progressive reduction of JC15 expression together with an increase in cell viability (Supplementary Figures S1A–D). However, loss of the protein expression was not limited to cisplatin alone. In support to previous observations on clinical samples,8, 19 exposure of both OVCAR-3 and MCF7 cells to doxorubicin had a similar effect on the JC15 protein levels and led to development of chemoresistance in these cells (Supplementary Figures S1E–H). Therefore, to adjudge the role of JC15 in cell death, we altered JC15 expression in HEK293T and assayed for sensitivity to the drug. We observed that loss of JC15 expression in the cells resulted in increased chemoresistance (Figure 1a). The cells showed suppression of apoptosis as suggested by lower annexinV–PI-stained population and activity of effecter caspases 3 and 7 (Figures 1b and d). JC15-depleted cells also showed better maintenance of mitochondrial membrane potential under treated conditions as compared with controls (Figure 1c). This shows that depletion of JC15 protects the cells from cisplatin-mediated apoptotic cell death.

Bottom Line: DnaJC15 was found to exert its proapoptotic function through the essential component of MPTP, cyclophilin D (CypD).Our results reveal a specific role of DnaJC15 in recruitment and coupling of CypD with mitochondrial permeability transition.In summary, our analysis provides first-time insights on the functional connection between mitochondrial inner membrane protein translocation machinery-associated J-protein DnaJC15 and regulation of cell death pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, India.

ABSTRACT
Mitochondria have a central role in the intrinsic pathway of apoptosis and involve activation of several transmembrane channels leading to release of death factors. Reduced expression of a mitochondrial J-protein DnaJC15 was associated with the development of chemoresistance in ovarian cancer cells. DnaJC15 was found to be a part of mitochondrial protein-transport machinery, though its connection with cell death mechanisms is still unclear. In the present study, we have provided evidence towards a novel function of DnaJC15 in regulation of mitochondrial permeability transition pore (MPTP) complex in normal and cancer cells. Overexpression of DnaJC15 resulted in MPTP opening and induction of apoptosis, whereas reduced amount of protein suppressed MPTP activation, upon cisplatin treatment. DnaJC15 was found to exert its proapoptotic function through the essential component of MPTP, cyclophilin D (CypD). Our results reveal a specific role of DnaJC15 in recruitment and coupling of CypD with mitochondrial permeability transition. In summary, our analysis provides first-time insights on the functional connection between mitochondrial inner membrane protein translocation machinery-associated J-protein DnaJC15 and regulation of cell death pathways.

Show MeSH
Related in: MedlinePlus