Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ~116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.
Tat-interacting protein of 60 kDa (TIP60) is an essential lysine acetyltransferase implicated in transcription, DNA damage response and apoptosis. TIP60 protein expression is reduced in cancers. In cervical cancers, human papillomavirus (HPV) E6 oncogene targets cellular p53, Bak and some of the PDZ domain-containing proteins for proteasome-mediated degradation through E6AP ligase. Recently, E6 oncogene from high-risk and low-risk categories was also shown to target TIP60. However, the molecular mechanisms and whether destabilization of TIP60 contributes to HPV E6-mediated transformation remain unanswered. Our proteomic analyses revealed EDD1 (E3 identified by differential display), an E3 ligase generally overexpressed in cancers as a novel interacting partner of TIP60. By investigating protein turnover and ubiquitination assays, we show that EDD1 negatively regulates TIP60's stability through the proteasome pathway. Strikingly, HPV E6 uses this function of EDD1 to destabilize TIP60. Colony-formation assays and soft agar assays show that gain of function of TIP60 or depletion of EDD1 in HPV-positive cervical cancer cells significantly inhibits cell growth in vitro. This phenotype is strongly supported by the in-vivo studies where re-activation of TIP60 in cervical cancer cells dramatically reduces tumor formation. In summary, we have discovered a novel ligase through which E6 destabilizes TIP60. Currently, in the absence of an effective therapeutic vaccine for malignant cervical cancers, cervical cancer still remains to be a major disease burden. Hence, our studies implying a distinct tumor suppressor role for TIP60 in cervical cancers show that reactivation of TIP60 could be of therapeutic value.
Hematopoietic stem cells (HSC) have the potential to replenish the blood system for the lifetime of the organism. Their two defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Here, using conditional gene knockout models, we demonstrate a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance both in the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 co-localizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell-cycle and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin and Tip60 deletion induced a robust reduction in the acH2A.Z / H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance at least in part through Tip60 dependent H2A.Z acetylation to activate Myc target genes.
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