Novel Function Of E3 Ubiquitin Ligase Hoil-1 In Mitophagy

Autophagy is a kind of self protection and stress response mechanism when energy nutrition deficiency. Broadly speaking, autophagy means to take aggregated proteins in cytoplasm or damaged organelles, which enclosed in autophagosome, to lysosome for degradation. This process can not only produce amino acids, nucleotides, free fatty acid nutrients and other small molecules of nutrients to compensate for the lack of external nutrient supply, also can effectively remove aging and damaged organelles, such as peroxisomes, endoplasmic reticulum, mitochondria and ribosomes and so on, in order to maintain cellular homeostasis and achieve the organelles update.Mitochondria are the "power plant" of energy conversion in eukaryotic cells. Accompanied by the occurrence of oxidative phosphorylation, they could inevitably produce a large number of reactive oxygen species. Excessive ROS will cause mitochondrial imbalance, leading to mitochondrial damage, eventually triggering apoptosis. Therefore, it is very important to removal of mitochondria to maintain the stability of intracellular environment. The phenomenon that cells have the ability to selectivitely degradate mitochondria is called mitophagy.Disorders of mitophagy is often associated with neurodegenerative diseases, cardiovascular disease, neuromuscular disease, and closely related to tumor and other ailments. Recent studies have shown that the mice in PD model showed mitochondrial enlargement and edema. It suggests us that PD may be induced by disorder of mitochondrial removal.Parkin and PINK1(PTEN induced kinase gene) are two identified gene in AR JP. Studies have found that Parkin can induce mitophagy when mitochondrial depolarization, it will translocate to the damaged mitochondria, and this process relies on the recruitment of PINK1. Parkin is a member of RBR ubiquitin ligase family. When mitochondria were treated by mitochondrial uncoupler agents CCCP, the protein on mitochondrial outer membrane will be labeled by ubiquitin in order to promote degradation of damaged mitochondria.In this study, we established the CCCP stimulation induced mitophagy model and evaluation system. Then we select one ubiquitin E3 ligase, HOIL 1L, with the most similar structure of Parkin though a depth research on RBR family proteins. In order to find out whether it can be involved in the regulation of mitophagy. The main results were as follows: 1. Establish mitohagy model and detection system under CCCP treatment.1) Test the expression of Parkin and HOIL 1L in some tumor cell lines, we found that the expression of Parkin can’t be detected in breast cancer, cervical cancer and other cancer cell, meanwhile HOIL 1L express was more widespread.2) Under CCCP and Oligomycin treatment, we observed mitochondrial outer membrane protein TOM20 would fission to fragment instesd of network.3) In mitohagy model and under 24 h CCCP treatment, we overexpressed positive control molecules Parkin, mitochondria in cells expressing Parkin could be removed by mitophagy.4) Under the condition of CCCP stimulation, LC3, an autophagy marker, would accumulation and located to the mitochondria. 2.The ubiquitin ligase HOIL 1L can promote Parkin independent mitophagy.1) Using the established model of mitophagy, we found HOIL 1L could promote mitophagy in a certain extent.2) Using the established model of mitophagy, we found HOIP couldn’t promote mitophagy.3) Establish mitohagy model in mito Keima He La cells.4) Using the established model of mitophagy in mito Keima He La cells, we found HOIL 1L could raise the mitophagy index, while HOIP couldn’t.5) In addition, using Annexin V FITC/PI apoptosis kit, we figured out that in the early term of mitophagy model apoptosis wouldn’t be induced. 3. The ubiquitin ligase HOIL 1L can promote Parkin dependent mitophagy1) Though instantaneous interference HOIL 1L in He La cell, we found that in the cells expression of Parkin, mitochondrial degradation was suppressed, and show the Parkin mutant phenotypes.2) Overexpression Parkin and HOIP/ HOIL 1L or both in HEK393 T cells, it shown that Parkin could interact with HOIL 1L and HOIP. So HOIL 1L could interact with Parkin and to release its self suppression.In conclusion, our study found a RBR member of ubiquitin ligase HOIL 1L could promote Parkin independent mitophagy, and is very important to maintain the morphology and the function of the mitochondria. Further study found that HOIL 1L can interact with Parkin, and promote Parkin dependent mitophagy.