Subunit‐Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases

Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin‐binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site‐specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N‐terminal glycine is unveiled after protease‐mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin‐specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit‐specific labeling of ubiquitin chains when studying how DUBs process these chains.     

Role of Virally-Encoded Deubiquitinating Enzymes in Regulation of the Virus Life Cycle

The ubiquitin (Ub) proteasome system (UPS) plays a pivotal role in regulation of numerous cellular processes, including innate and adaptive immune responses that are essential for restriction of the virus life cycle in the infected cells. Deubiquitination by the deubiquitinating enzyme, deubiquitinase (DUB), is a reversible molecular process to remove Ub or Ub chains from the target proteins. Deubiquitination is an integral strategy within the UPS in regulating survival and proliferation of the infecting virus and the virus-invaded cells. Many viruses in the infected cells are reported to encode viral DUB, and these vial DUBs actively disrupt cellular Ub-dependent processes to suppress host antiviral immune response, enhancing virus replication and thus proliferation. This review surveys the types of DUBs encoded by different viruses and their molecular processes for how the infecting viruses take advantage of the DUB system to evade the host immune response and expedite their replication.     

Halogen Substituents in the Isoquinoline Scaffold Switches the Selectivity of Inhibition between USP2 and USP7

Deubiquitinases are important components of the protein regulatory network and, hence, constitute a tempting drug target. We report herein structure–activity relationship studies to develop halogen-substituted isoquionoline-1,3-dione-based inhibitors of the deubiquitinase USP2. In contrast to our previous reports, the best compound discovered was found to act through a reactive oxygen species independent, uncompetitive mechanism with an IC₅₀ of 250 nm . We show the crucial role of halogens in the common scaffold to provide potency and selectivity of our compound, where the introduction of the fluorine atom completely switches the selectivity of the inhibitor between USP2 and USP7. Our cellular studies highlight the potential applicability of the reported compound for in vivo experiments. The discovery of the isoquinoline-1,3-dione core and the knowledge obtained with regard to halogen substituents provide a platform towards understanding USP2 inhibition and the development of highly selective next-generation deubiquitinase inhibitors.     

The Roles of the Ubiquitin–Proteasome System in the Endoplasmic Reticulum Stress Pathway

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells, which is essential for synthesis, processing, sorting of protein and lipid metabolism. However, the cells activate a defense mechanism called endoplasmic reticulum stress (ER stress) response and initiate unfolded protein response (UPR) as the unfolded proteins exceed the folding capacity of the ER due to the environmental influences or increased protein synthesis. ER stress can mediate many cellular processes, including autophagy, apoptosis and senescence. The ubiquitin-proteasome system (UPS) is involved in the degradation of more than 80% of proteins in the cells. Today, increasing numbers of studies have shown that the two important components of UPS, E3 ubiquitin ligases and deubiquitinases (DUBs), are tightly related to ER stress. In this review, we summarized the regulation of the E3 ubiquitin ligases and DUBs in ER stress.     

Chemoenzymatic Synthesis of Bifunctional Polyubiquitin Substrates for Monitoring Ubiquitin Chain Remodeling

Covalent attachment of ubiquitin to target proteins is one of the most pervasive post‐translational modifications in eukaryotes. Target proteins are often modified with polymeric ubiquitin chains of defined lengths and linkages that may further undergo dynamic changes in composition in response to cellular signals. Biochemical characterization of the enzymes responsible for building and destroying ubiquitin chains is often thwarted by the lack of methods for preparation of the appropriate substrates containing probes for biochemical or biophysical studies. We have discovered that a yeast ubiquitin C‐terminal hydrolase (Yuh1) also catalyzes transamidation reactions that can be exploited to prepare site‐specifically modified polyubiquitin chains produced by thiol‐ene chemistry. We have used this chemoenzymatic approach to prepare dual‐functionalized ubiquitin chains containing fluorophore and biotin modifications. These dual‐functionalized ubiquitin chains enabled the first real‐time assay of ubiquitin chain disassembly by a human deubiquitinase (DUB) enzyme by single molecule fluorescence microscopy. In summary, this work provides a powerful new tool for elucidating the mechanisms of DUBs and other ubiquitin processing enzymes.     

Ubiquitination in T-Cell Activation and Checkpoint Inhibition: New Avenues for Targeted Cancer Immunotherapy

The advent of T-cell-based immunotherapy has remarkably transformed cancer patient treatment. Despite their success, the currently approved immunotherapeutic protocols still encounter limitations, cause toxicity, and give disparate patient outcomes. Thus, a deeper understanding of the molecular mechanisms of T-cell activation and inhibition is much needed to rationally expand targets and possibilities to improve immunotherapies. Protein ubiquitination downstream of immune signaling pathways is essential to fine-tune virtually all immune responses, in particular, the positive and negative regulation of T-cell activation. Numerous studies have demonstrated that deregulation of ubiquitin-dependent pathways can significantly alter T-cell activation and enhance antitumor responses. Consequently, researchers in academia and industry are actively developing technologies to selectively exploit ubiquitin-related enzymes for cancer therapeutics. In this review, we discuss the molecular and functional roles of ubiquitination in key T-cell activation and checkpoint inhibitory pathways to highlight the vast possibilities that targeting ubiquitination offers for advancing T-cell-based immunotherapies.     

Insight into Bortezomib Focusing on Its Efficacy against P-gp-Positive MDR Leukemia Cells

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.