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    • Mulan protein level is regulated

    2020-01-22

    Mulan protein level is regulated with conditions that stimulate mitophagy such as treatment of ache inhibitor with CCCP [12]. We found that exogenously expressed GFP-GABARAP and RFP-Mulan could co-precipitate and co-localize in mammalian cells and the degree of co-localization increased when the cells were treated with CCCP. In summary, we have identified four different E2 conjugating enzymes that interact with Mulan to form E2/E3 heterodimers. Each heterodimer in turn has unique specificity against various target proteins. In addition, our results suggest a mechanism of how Mulan participates in mitophagy. Mulan and Parkin are both E3 ubiquitin ligases but Mulan resides in the mitochondria whereas Parkin is recruited to the mitochondria by PINK1 during conditions of mitophagy [9], [10], [67]. Our present study shows that Mulan directly interacts with GABARAP that is different from the mechanism used by Parkin (Fig. 6B). In addition, both Mulan and Parkin are known to regulate Mfn2 whose downregulation is necessary for mitophagy to occur [13], [23]. Whether these two pathways leading to mitophagy (Mulan versus Parkin) work synergistically or are independent from one another needs to be determined. The Mulan pathway potentially compensates the absence of Parkin in knock-out (KO) animals that show only subtle phenotypes [68], [69], [70]. It will also be interesting to investigate if the Mulan pathway is compromised in Parkinson\'s disease or other neurodegenerative disorders. Our previous studies showed the Mulan pathway to mitophagy is overactive when Omi/HtrA2 protease is absent or inactive in mutant mice [12]. This results in increased mitophagy that causes neurodegeneration, premature aging, and a plethora of defects in the mutant mice [25], [27]. These observations suggest that, not only loss of mitophagy but also uncontrolled and excessive mitophagy can lead to neurodegeneration and potentially several other disorders.
    Conclusions Mulan is a mitochondrial E3 ubiquitin ligase with diverse roles in cell growth, cell death, and mitophagy. To better understand the mechanism of Mulan\'s function we used a modified yeast two-hybrid system to isolate proteins that interact with its cytoplasmic RING domain (amino acids 259–352). Four different E2 ubiquitin conjugating enzymes were isolated as specific interactors. We constructed fusion baits between Mulan259–352 and each one of the E2 proteins. These fusion baits were used in a secondary screen to isolate interactors that bound to the Mulan259–352–E2 fusion but not to Mulan259–352 or the E2 alone. One such interactor was the GABARAP protein known to be involved in mitophagy. GABARAP was able to interact with Mulan259–352–Ube2E3 though an LIR motif present in the Mulan RING domain. In addition, the presence of Ube2E3 in the complex with Mulan was also required. Our data provide a plausible mechanism by which Mulan when in complex with Ube2E3 participates in mitophagy. This mechanism is different, potentially independent, and could be operating in parallel with Parkin, another E3 ubiquitin ligase with a well-characterized role in mitophagy and Parkinson\'s disease.
    Authors\' contributions
    Acknowledgements This work was supported by grant 2KB05 to A.S.Z. by James and Esther King Biomedical Research Program, Florida Department of Health. We thank all members of Dr. Zervos\' lab for their comments and suggestions.
    Introduction Hepatocellular carcinoma (HCC) is a kind of malignant tumor that causes a considerable health burden globally with China accounting for about 50% of the worldwide incidence and mortality each year [1]. Ubiquitination is important in various biological processes such as signal transduction, intracellular transportation and protein degradation. There is increasing evidence that ubiquitination plays a critical role in the development of cancer. UBE2S is a member of the ubiquitin-conjugating enzyme family and activates ubiquitin by forming thiol ester bonds with this molecule in an enzyme-dependent manner. Specifically, UBE2S has been shown to extend the ubiquitin chain on the substrate of ubiquitin ligase to promote exit from the mitotic cell cycle [2]. Importantly, immunohistochemistry demonstrated that UBE2S is highly expressed in breast cancer and knock-down of UBE2S expression can inhibit breast cancer cell migration and invasion [3]. Western blot and immunohistochemistry results was used to reveal that UBE2S was overexpressed in oral squamous cell carcinoma and was significantly associated with the primary tumor size [4]. Another previous study found that Akt1 could phosphorylate UBE2S and enhance its stability. Additionally knockdown of UBE2S expression could inhibit NHEJ-mediated DNA repair and increase the sensitivity of glioblastoma cells to chemotherapy [5]. However, the role of UBE2S in hepatocellular carcinoma has not been previously reported.