Search for: All records

Abstract Target validation is key to the development of protein degrading molecules such as proteolysis‐targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin‐proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3‐catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN‐pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP‐HaloTag fusion with higher activities than VHL‐engaging HaloPROTACs at sub‐micromolar concentrations of the compound. The CRBN‐engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell. 

Abstract While extensive investigations have been devoted to the study of genetic pathways related to fatty liver diseases, much less is known about epigenetic mechanisms underlying these disorders. DNA methylation is an epigenetic link between environmental factors (e.g., diets) and complex diseases (e.g., non‐alcoholic fatty liver disease). Here, it is aimed to study the role of DNA methylation in the regulation of hepatic lipid metabolism. A dynamic change in the DNA methylome in the liver of high‐fat diet (HFD)‐fed mice is discovered, including a marked increase in DNA methylation at the promoter of Beta‐klotho (Klb), a co‐receptor for the biological functions of fibroblast growth factor (FGF)15/19 and FGF21. DNA methyltransferases (DNMT) 1 and 3A mediate HFD‐induced methylation at theKlbpromoter. Notably, HFD enhances DNMT1 protein stability via a ubiquitination‐mediated mechanism. Liver‐specific deletion ofDnmt1or3aincreasesKlbexpression and ameliorates HFD‐induced hepatic steatosis. Single‐nucleus RNA sequencing analysis reveals pathways involved in fatty acid oxidation inDnmt1‐deficient hepatocytes. Targeted demethylation at theKlbpromoter increasesKlbexpression and fatty acid oxidation, resulting in decreased hepatic lipid accumulation. Up‐regulation of methyltransferases by HFD may induce hypermethylation of theKlbpromoter and subsequent down‐regulation ofKlbexpression, resulting in the development of hepatic steatosis. 

Abstract Di‐ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB‐binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage‐specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA)N^ϵ‐L‐thiaprolyl‐L‐Lys (L‐ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA‐mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage‐specific UB‐based probes to decipher the biological signals mediated by protein ubiquitination. 

Abstract The E6 protein of the human papillomavirus (HPV) underpins important protein interaction networks between the virus and host to promote viral infection. Through its interaction with E6AP, a host E3 ubiquitin (UB) ligase, E6 stirs the protein ubiquitination pathways toward the oncogenic transformation of the infected cells. For a systematic measurement of E6 reprogramming of the substrate pool of E6AP, we performed a proteomic screen based on “orthogonal UB transfer (OUT)” that allowed us to identify the ubiquitination targets of E6AP dependent on the E6 protein of HPV‐16, a high‐risk viral subtype for the development of cervical cancer. The OUT screen identified more than 200 potential substrates of the E6‐E6AP pair based on the transfer of UB from E6AP to the substrate proteins. Among them, we verified that E6 would induce E6AP‐catalyzed ubiquitination of importin proteins KPNA1‐3, protein phosphatase PGAM5, and arginine methyltransferases CARM1 to trigger their degradation by the proteasome. We further found that E6 could significantly reduce the cellular level of KPNA1 that resulted in the suppression of nuclear transport of phosphorylated STAT1 and the inhibition of interferon‐γ‐induced apoptosis in cervical cancer cells. Overall, our work demonstrates OUT as a powerful proteomic platform to probe the interaction of E6 and host cells through protein ubiquitination and reveals a new role of E6 in down‐regulating nuclear transport proteins to attenuate tumor‐suppressive signaling.