Regulation Of Mitochondrial Function And Apoptosis Through Degradation Of MCL1 By Ubiquitin E3 Ligase TRIM11

Apoptosis is a kind of positive programmed cell death (PCD) which is very important for multicellutar organism to control their growth, development and dealing with the external environment change. Imbalance of apoptosis can lead to many diseases, such as alzheimer's, cancer, AIDS, and heart disease, etc. There are many protein families are involved in the process of apoptosis, such as the caspase family and BCL2 family. BCL2 family proteins can promote or inhibit the mitochondrial membrane permeability and then regulate the cell apoptosis process. In this study, we found a neurological-development-related ubiquitin E3 ligase TRIM11 can interact with and degradate MCL1, member of the BCL2 family proteins, and resulting in cell apoptosis. The main works of this papers are as follows:1. In this study, we found TRIM11 can significantly reduce the proliferation ability of HeLa and 293T cell. We further found that TRIM11 can more efficiently inhibit cell proliferation under serum starvation conditions. Propidium iodide dyeing were used to detect influence of TRIM11 on cell cycle, however, TRIM11 did not significantly affect the cell cycle of 293T cell. Annexin V/PI double dyeing were used to detect influence of TRIM11 on apoptosis and we found that TRIM11 significantly promote early apoptosis of HeLa cell.2. Using nucleus and cytoplasm separation kit and immunofluorescence staining, we found TRIM11 are mainly distributed in the cytoplasm. And then through the protein sequence alignment, we found high similarities between Humanin and BH1 domain of BCL2 family proteins. And eventually we detected interaction of TRIM11 and MCL1 using co-immunoprecipitation. Using immunofluorescence detection we found that both proteins are co-localized in the cytoplasm.3. We tested the effects of full-length and RING domain missing TRIM11 mutant on cell proliferation and apoptosis, and found the missing RING domain TRIM11 has no effect on cell proliferation and apoptosis, which means that RING domain of TRIM11 is needed in promoting cell apoptosis. Then we tested the influence of TRIM11 on exogenous MCL1 protein level, and found that TRIM11 can obviously reduce the protein levels of exogenous MCL1, which depends on the proteasome systems. Meanwhile, we found that TRIM11 can obviously reduce half-life of exogenous MCL and endogenous MCL1. Finally, we found TRIM11 can significantly improve ubiquitination of MCL1 using co-immunoprecipitation.4. Using the mitochondria fluorescent Marker JC-1, we detected the influence of TRIM11 on mitochondrial membrane potential of HeLa cell and 293T cells, and found TRIM11 can significantly reduce the mitochondrial membrane potential, which revealed that TRIM11 may affect cell apoptosis through affect mitochondrial function. Mitochondria separation Kit are used to isolate Mitochondria from 293T cell, and then we detected the distribution of MCL1, TRIM11 in mitochondria and cytoplasm. We detacted obviously enrichment of MCL1 on mitochondria when transfected with TRIM11, which further illustrates the influence of TRIM11 on mitochondria. At the same time, we detected distribution of TRIM11 on mitochondria.ontents of the abstract. Times New Roman.