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Myrf is a key transcription factor for oligodendrocyte differentiation and central nervous system myelination. We and others have previously shown that Myrf is generated as a membrane protein in the endoplasmic reticulum (ER), and that it undergoes auto-processing to release its N-terminal fragment from the ER, which enters the nucleus to work as a transcription factor. These previous studies allow a glimpse into the unusual complexity behind the biogenesis and function of the transcription factor domain of Myrf. Here, we report that Myrf N-terminal fragments assemble into stable homo-trimers before ER release. Consequently, Myrf N-terminal fragments are released from the ER only as homo-trimers. Our re-analysis of a previous genetic screening result in Caenorhabditis elegans shows that homo-trimerization is essential for the biological functions of Myrf N-terminal fragment, and that the region adjacent to the DNA-binding domain is pivotal to its homo-trimerization. Further, our computational analysis uncovered a novel homo-trimeric DNA motif that mediates the homo-trimeric DNA binding of Myrf N-terminal fragments. Importantly, we found that homo-trimerization defines the DNA binding specificity of Myrf N-terminal fragments. In sum, our study elucidates the molecular mechanism governing the biogenesis and function of Myrf N-terminal fragments and its physiological significance.

Mdm2 is a critical negative regulator of the tumor suppressor protein p53. Mdm2 is an E3 ligase whose overexpression leads to functional inactivation of p53. Mdm2 protein stability is regulated by several mechanisms including RING (Really Interesting New Gene) domain-mediated autoubiquitination. Here we report biochemical identification of NEDD4-1 as an E3 ligase for Mdm2 that contributes to the regulation of Mdm2 protein stability in cells. NEDD4-1 was identified from Jurkat cytosolic fractions using an enzyme-dead Mdm2 mutant protein as a substrate for in vitro E3 ligase assays. We show that lysates from Nedd4-1 knockout (KO) mouse embryonic fibroblasts (MEFs) have significantly diminished E3 ligase activity toward Mdm2 compared with lysates from wild-type (WT) MEFs. Recombinant NEDD4-1 promotes Mdm2 ubiquitination in vitro in a concentration- and time-dependent manner. In cells, NEDD4-1 physically interacts with Mdm2 via the RING domain of Mdm2. Overexpression of NEDD4-1, but not an enzyme-dead NEDD4-1CS mutant, increases ubiquitination of Mdm2. NEDD4-1 catalyzes the formation of K63-type polyubiquitin chains on Mdm2 that are distinct from K48-type polyubiquitination chains mediated by the Mdm2/MdmX complex. Importantly, K63-type polyubiquitination by NEDD4-1 competes with K48-type polyubiquitination on Mdm2 in cells. As a result, NEDD4-1-mediated ubiquitination stabilizes Mdm2. NEDD4-1 knockdown reduces the t1/2 (half-life) of endogenous Mdm2 from 20 to 12 min in U2OS cells. Nedd4-1 KO MEFs manifest increased p53 levels and activity, a more robust DNA damage response and increased G1 arrest compared with WT MEFs. Similarly, NEDD4-1 knockdown in WT-p53-bearing cells increases basal p53 levels and activity in an Mdm2-dependent manner, causes stronger p53 responses to DNA damage and results in p53-dependent growth inhibition compared with corresponding NEDD4-1-proficient control cells. This study identifies NEDD4-1 as a novel component of the p53/Mdm2 regulatory feedback loop that controls p53 activity during stress responses.

BackgroundPancreatic cancer is a deadly disease with a very low 5-year patient survival rate of 6–8%. The major challenges of eliminating pancreatic cancer are treatment resistance and stromal barriers to optimal drug access within the tumor. Therefore, effective molecular targeting drugs with high intra-tumor access and retention are urgently needed for managing this devastating disease in the clinic.MethodsThis study has used the following in vitro and in vivo techniques for the investigation of exceptional anticancer drug FL118’s efficacy in treatment of resistant pancreatic cancer: cell culture; immunoblotting analysis to test protein expression; DNA sub-G1 flow cytometry analyses to test cell death; MTT assay to test cell viability; pancreatic cancer stem cell assays (fluorescence microscopy tracing; matrigel assay; CD44-positive cell colony formation assay); human luciferase-labeled pancreatic tumor orthotopic animal model in vivo imaging; pancreatic cancer patient-derived xenograft (PDX) animal models; and toxicology studies with immune-competent BALB/cj mice and beagle dogs.ResultsOur studies found that FL118 alone preferentially killed cisplatin-resistant cancer cells, while a combination of FL118 with cisplatin synergistically killed resistant pancreatic cancer cells and reduced spheroid formation of treatment-resistant pancreatic cancer stem-like cells. Furthermore, using in vivo-imaging, we found that FL118 in combination with cisplatin strongly inhibited both drug-resistant pancreatic xenograft tumor growth and metastasis. In PDX model, we demonstrated that FL118 alone effectively eliminated PDX tumors, while FL118 in combination with gemcitabine eliminated PDX tumors that showed relative resistance (less sensitivity) to treatment with FL118. These FL118 efficacy results are consistent with our molecular-targeting data showing that FL118 inhibited the expression of multiple antiapoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and ERCC6, a critical regulator of DNA repair, in treatment-resistant pancreatic stem-like cancer cells. Furthermore, FL118 toxicity studies in BALB/cj mice and beagle dogs indicated that FL118 exhibits favorable hematopoietic and biochemical toxicities.ConclusionTogether, our studies suggest that FL118 is a promising anticancer drug for further clinical development to effectively treat drug-resistant pancreatic cancer alone or in combination with other pancreatic cancer chemotherapeutic drugs.