Confocal imaging analysis revealed that 92% of cells expressed GF

Confocal imaging analysis revealed that 92% of cells expressed GFP NT CTMP in the cyto plasm, with a smaller amount of cells expressing GFP NT CTMP at the plasma membrane. GFP C terminal tagged CTMP were also prepared to explore the possibility that the GFP tag at the N terminus affected iments to date were inhibitor Vandetanib performed using an overexpression system, we examined the subcellular localization of endogenous CTMP in HEK293 cells. Immunoblot analy sis confirmed endogenous CTMP was localized at the mitochondria as well as in the cytoplasm. Inhibitors,Modulators,Libraries To determine the precise localization of CTMP in mitochon dria, we first isolated mitochondria fractions which were isolated under the following conditions i 2 M NaCl for mitochondria outer membrane, ii 100 mM Na2CO3for intermembrane space and or mitochondrial matrix and iii 1% Triton X 100 for mitochondria inner or outer membrane protein.

CTMP was solubilized in Na2CO3, indicating that CTMP is a soluble protein Inhibitors,Modulators,Libraries in either the inter membrane space and or the mitochon drial matrix. Mitochondrial targeting sequence mediated mitochondrial localization of CTMP is inhibited by phosphorylation event Bioinformatics analysis of CTMP sequence using MitoProt II 1. 0a4 predicted the mitochondrial signal peptide could be cleaved at amino acid position 32, closed to the identified phosphorylation site. In order to investigate the potential mitochondrial targeting sequence of CTMP, an N terminal 31 amino acid deletion mutant of CTMP was constructed based on the prediction of MitoProt II 1. 0a4. Inhibitors,Modulators,Libraries As predicted, this mutant form of CTMP did not localize to the mitochon dria.

Since Ser37 and Ser38 of CTMP were identified as in vivo phosphorylation sites, a negatively charged side group mimic CTMP Inhibitors,Modulators,Libraries mutant was generated. Con focal analysis of cells expressing this negatively charged side group mimic CTMP mutant showed that the majority of cells expressed CTMP in the cytoplasm, suggesting phosphorylation on Ser37 Ser38 is an impor tant regulatory mechanism for CTMP shuttling to the mitochondria. CTMP overexpression sensitizes the cell to apoptosis induced by staurosporine To evaluate the apoptotic role of CTMP, we overexpressed CTMP in HeLa cells for 24 h and subsequently treated the cells with 1 M staurosporine for the indicated times. Stauroporine mediated apoptosis was detected Inhibitors,Modulators,Libraries at 3 h of treatment.

Apoptosis was more pronounced in CTMP transduced compared to controls, suggesting that CTMP overexpression increases the sensitivity of cells to programmed cell death. CTMP mediated Hsp70 sequestration leads to the dissociation of enough Hsp70 and Apaf 1 Recent studies show that heat shock proteins fam ily, including Hsp90, Hsp70 and Hsp27, can influence apoptosis through direct physical interaction with key components of the apoptotic machinery. Since CTMP overexpression appears to enhance the stau rosporine induced apoptosis, the possible interaction of CTMP with these Hsp proteins was moni tored in HeLa cells expressing HA CTMP.

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