Although our knowledge of the biochemical and biological fun
Although our knowledge of the biochemical and biological functions of E3 ubiquitin ligases has increased in B-cell malignancies, a deeper characterization of cell-context dependent substrate regulation and biological relevance is needed. The recent approval of a general proteasome inhibitor, VELCADE or bortezomib, has demonstrated a great efficacy for the treatment of MM and MCL , , . However, toxicity and the relevant side effects, which include anemia, neuropathy, and thrombocytopenia, in addition to bortezomib resistance, make this drug imperfect , , . Recent discoveries have shown that thalidomide-based drugs target Cereblon (CRBN) , a member of the CULLIN4 E3 ubiquitin ligase complex, to promote proteasomal degradation of specific targets , . This promising area of research has shown the achievement of higher drug potency, resulting in the rapid destabilization of targets via ubiquitin-dependent proteasomal degradation.
Disclosure of potential conflicts of interest
Acknowledgments This work was supported in part by grant R00-CA166181-04, R01-CA207513-01 from the National Cancer Institute and Gilead Sciences Research Scholars Program in Hematology/Oncology to L.B
Introduction Autophagy is generally believed to be a process in which nonspecific degradation of cytoplasmic content, including organelles, occurs . Recent studies have revealed that there is specificity in autophagy that allows the degradation of specific cargo , . Such a selective type of autophagy includes mitophagy, the process whereby damaged Menadione are removed from the cell , . Mitophagy is crucial for many physiological processes such as development and differentiation, and its deregulation has been implicated in Parkinson\'s disease, cardiovascular disease, as well as various infections and cancers , . Mitophagy has been shown to be mediated predominantly by the E3 ubiquitin ligase parkin, which is recruited to the mitochondria by PINK1 (PTEN-induced putative kinase protein 1) , , , . Recent studies have identified that another mitochondrial E3 ubiquitin ligase, Mulan/Hades/GIDE (mitochondrial ubiquitin ligase activator of NF-κB; hereafter referred to as Mulan), is involved in mitophagy , , . Our studies have identified Mulan as a specific substrate of the mitochondrial Omi/HtrA2 and showed that inactivation of Omi/HtrA2 protease leads to the accumulation of the Mulan protein and increased mitophagy . Mulan is one of three E3 ubiquitin ligases that are present in the mitochondria along with MARCH5/MITOL (membrane associated RING-CH5) and RNF185 , , , . Mulan is a 352-residue polypeptide that crosses the mitochondrial outer membrane (OMM) twice, its amino-terminus, which includes the RING finger domain, is located in the cytoplasm and a large domain is present in the inter-membrane space (IMS) of the mitochondria. Both the mitochondrial localization signal as well as, the RING-finger domain have been shown to be necessary for Mulan\'s function . Mulan\'s location suggests that its substrates will be proteins that are either integral or associated with the mitochondrial membrane. Mulan has been shown to directly or indirectly regulate various proteins including NF-κB, JNK, p53, Akt and Mfn2, suggesting a potential role in cell growth, apoptosis and mitophagy , , , , . In order to investigate the mechanism by which Mulan performs its normal function, we set out to identify E2 ubiquitin conjugating enzymes that form specific complexes with Mulan. We used the cytoplasmic domain of Mulan (amino acids 259–352) that includes its RING finger domain in a yeast two-hybrid screen, and identified four E2 conjugating enzymes, namely Ube2E2, Ube2E3, Ube2G2 and Ube2L3, as specific interactors. We then used fusion baits consisting of Mulan\'s RING finger domain linked to the full length of each of the four specific E2\'s that were identified. These fused baits were used to screen for interactors of the heterodimeric Mulan259–352–E2 recombinant protein. In this screen, several distinct interactors were identified for each of the Mulan259–352–E2 fusion baits. The identity of these interactors confirms Mulan\'s diverse functions that are determined by the nature of the E2 conjugating enzyme.