Functional Analysis Of A Novel Protein Deubiquitinase

Objective: Initially,histone chaperones were deemed as the carrier of histone,as more research going on,it was pass as play critical roles at all stages of histone transactions.Certain histone chaperones have been assigned to promote specific nucleosome assembly pathways,a critical step towards chromatin restoration on newly synthesized or repaired DNA.The histone H3-H4 chaperone anti-silencing function 1(ASF1)plays a critical role in chromatin assembly.Although histone chaperone ASF1 A has been reported to be dysregulated in multiple tumours,the underlying molecular mechanism that how the abundance and function of ASF1 A are regulated remains unclear.The ubiquitin-specific peptidase 52/poly(A)nuclease 2(USP52/PAN2),a member of the ubiquitin-specific protease(USP)superfamily,contains a WD40 repeat domain at the N terminus,a ubiquitin C-terminal hydrolase(UCH)domain,and a C-terminal RNase domain of the DEDD superfamily.USP52 has been well characterized as a poly(A)nuclease in the PAN2/PAN3 deadenylation complex,and a recent study reported that USP52 is a key component of P-body(processing body)and functions to prevent HIF1 A m RNA degradation.Yet,whether USP52 is capable of removing ubiquitin linkages remains an open question,although crystal structure analysis of its yeast or fungi orthologue indicated that the UCH domain lacks catalytic residues required for protease activity and is incompatible with catalysis.In this study,we aimed to examine whether USP52 is able to biochemically oppose protein ubiqutination and investigate the role of deubiquitinase USP52 in breast carcinogenesis.Also,we aimed to understand that how the abundance and function of ASF1 A are regulated.Results: 1.Histone chaperone ASF1 A is physically associated with USP52.ASF1 A interacts with USP52 through the N-terminal region of ASF1 A and the WD40 repeat domain of USP52.ASF1 A and USP52 co-exist in a protein complex without PAN3 and Histone H3.2.USP52 is a bona fide deubiquitinase.Eukaryote cells-purified recombinant USP52 was able to trim K6-,K11-,K48-,K63-,and M1-linked ubiquitin chains,thus suggest that USP52 is capable of cleaving specific types of ubiquitin linkages and it is a bona fide deubiquitinase.3.USP52 deubiquitinates ASF1 A.A series of deubiquitination assays and Mass spectrometry analysis of ASF1 A ubiquitin conjugation sites revealed that USP52 was able to efficiently remove K48-linked poly-ubiquitin linkages of wild type ASF1 A and USP52 targets ASF1A/K129 for deubiquitination.4.USP52 stabilizes ASF1 A.Western blotting analysis with MG132 suggest that ASF1 A is a substrate of USP52.Cycloheximide(CHX)chase assays revealed that USP52 depletion was clearly associated with a decreased half-life of ASF1 A.Together,these results strongly support the notion that USP52 controls the stability of ASF1 A.5.USP52 promotes chromatin assembly through stabilizing ASF1 A.MNase digestion resistance results and EDU staining favor the argument that USP52-promoted ASF1 A stabilization regulates DNA synthesis-coupled chromatin assembly through ASF1A-mediated deposition of H3K56 Ac and USP52-promoted ASF1 A stabilization is required for proper S phase progression.6.USP52/ASF1 A signaling is implicated in breast carcinogenesis.IHC staining showed that the expression levels of USP52 and ASF1 A were highly expressed in various types of breast carcinoma samples,and the levels of their expression strongly correlated with each other.Colony formation assays showed that USP52/ASF1 A axis is required for breast cancer cell proliferation.The rusults of xenotransplantation of breast cancer cells onto athymic mice point a role of the USP52-promoted ASF1 A stabilization in promoting breast carcinogenesis.7.USP52/ASF1 A signaling confers DNA damage tolerance.A series of cell apoptosis assays induced by IR or CPT revealed that USP52-promoted ASF1 A stabilization confers cellular resistance of breast cancer cells to genotoxic insults.Conclusion: Here we report that ASF1 A is physically associated with USP52,which is previously identified as a pseudo-deubiquitinase.Through a body of work,we discovered that USP52,prurified from higher organism but not prokaryotic cells,is able to remove ubiquitins either from specific types of poly-conjugated ubiquitin chains or K48-linked polyubiquitinated ASF1 A and stabilize ASF1 A.Additionally we find that USP52/ASF1 A increases the level of H3K56 Ac,in ASF1 A dependented manner,promotes deposition of histones and chromatin assembly through DNA replication,that’s important to genomic stability.Moreover,IHC analysis clinical specimens we revealed that USP52 and ASF1 A were high expressed and positive correlation in various kinds of breast tumors.Cellular cytotoxicity analysis revealed the improtent role of USP52/ASF1 A signaling axis confers DNA damage tolerance.Accroding to these evidences,our study support the pursuit of USP52/ASF1 A as a potential targets for breast cancer intervention.