Limits...
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.

Show MeSH

Related in: MedlinePlus

CsA inhibits chemosensitive phenotype of JC15-overexressing cells. (a) Cells were left untreated, treated with cisplatin (+Cpl) alone or supplemented with 1 nM CsA (+Cpl+CsA) and subjected to MTT assay. Data shown as mean±S.E.M. n=8, *P (two tailed) <0.0001. (b) Cells expressing wild-type JC15 were left untreated (UnT), pre-treated with CsA followed by cisplatin exposure (+Cpl+CsA) or treated with cisplatin alone (+Cpl). The opening of mitochondrial transition pore was adjudged by staining with potential-sensitive MitoTracker Red CMXRos dye. UT, untransfected cells; UnT, untreated cells. Data are represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001. (c) Relative caspase activity of cisplatin-treated JC15-expressing cells pre-exposed to CsA represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3973195&req=5

fig2: CsA inhibits chemosensitive phenotype of JC15-overexressing cells. (a) Cells were left untreated, treated with cisplatin (+Cpl) alone or supplemented with 1 nM CsA (+Cpl+CsA) and subjected to MTT assay. Data shown as mean±S.E.M. n=8, *P (two tailed) <0.0001. (b) Cells expressing wild-type JC15 were left untreated (UnT), pre-treated with CsA followed by cisplatin exposure (+Cpl+CsA) or treated with cisplatin alone (+Cpl). The opening of mitochondrial transition pore was adjudged by staining with potential-sensitive MitoTracker Red CMXRos dye. UT, untransfected cells; UnT, untreated cells. Data are represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001. (c) Relative caspase activity of cisplatin-treated JC15-expressing cells pre-exposed to CsA represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001

Mentions: Upon pre-treatment with CsA, we observed >50% suppression in drug sensitivity along with reversal of mitochondrial depolarization as indicated by the maintenance of potential (Figures 2a and b), cytochrome c release and caspase activity observed (Figure 2c and Supplementary Figure S6B) in JC15-overexpressing HEK293T cells and cancer cells (Supplementary Figures S3A–D, S5A and B). However, the significant release of cytochrome c in cisplatin-treated cells even after CsA pre-treatment might probably be attributed to lower cellular retentive nature of CsA (<3 h) and its non-specific binding to other cytosolic cyclophilins;20 hence, releasing CypD from its inhibited state and activating MPTP channel. To support our above observation, treatment of mitochondria with CsA attenuated the elevated swelling of the organelle under high JC15 levels, signifying restoration of membrane potential and integrity of the inner mitochondria membrane (Figure 1f).


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

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

CsA inhibits chemosensitive phenotype of JC15-overexressing cells. (a) Cells were left untreated, treated with cisplatin (+Cpl) alone or supplemented with 1 nM CsA (+Cpl+CsA) and subjected to MTT assay. Data shown as mean±S.E.M. n=8, *P (two tailed) <0.0001. (b) Cells expressing wild-type JC15 were left untreated (UnT), pre-treated with CsA followed by cisplatin exposure (+Cpl+CsA) or treated with cisplatin alone (+Cpl). The opening of mitochondrial transition pore was adjudged by staining with potential-sensitive MitoTracker Red CMXRos dye. UT, untransfected cells; UnT, untreated cells. Data are represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001. (c) Relative caspase activity of cisplatin-treated JC15-expressing cells pre-exposed to CsA represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: CsA inhibits chemosensitive phenotype of JC15-overexressing cells. (a) Cells were left untreated, treated with cisplatin (+Cpl) alone or supplemented with 1 nM CsA (+Cpl+CsA) and subjected to MTT assay. Data shown as mean±S.E.M. n=8, *P (two tailed) <0.0001. (b) Cells expressing wild-type JC15 were left untreated (UnT), pre-treated with CsA followed by cisplatin exposure (+Cpl+CsA) or treated with cisplatin alone (+Cpl). The opening of mitochondrial transition pore was adjudged by staining with potential-sensitive MitoTracker Red CMXRos dye. UT, untransfected cells; UnT, untreated cells. Data are represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001. (c) Relative caspase activity of cisplatin-treated JC15-expressing cells pre-exposed to CsA represented as mean±S.E.M. n=3, *P (two tailed) <0.0001, **P (two tailed) <0.001
Mentions: Upon pre-treatment with CsA, we observed >50% suppression in drug sensitivity along with reversal of mitochondrial depolarization as indicated by the maintenance of potential (Figures 2a and b), cytochrome c release and caspase activity observed (Figure 2c and Supplementary Figure S6B) in JC15-overexpressing HEK293T cells and cancer cells (Supplementary Figures S3A–D, S5A and B). However, the significant release of cytochrome c in cisplatin-treated cells even after CsA pre-treatment might probably be attributed to lower cellular retentive nature of CsA (<3 h) and its non-specific binding to other cytosolic cyclophilins;20 hence, releasing CypD from its inhibited state and activating MPTP channel. To support our above observation, treatment of mitochondria with CsA attenuated the elevated swelling of the organelle under high JC15 levels, signifying restoration of membrane potential and integrity of the inner mitochondria membrane (Figure 1f).

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