Anti-Ubiquitin polyclonal antibody

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.

The NF-kappa B/Rel family of transcription factors that play critical roles in a variety of cellular processes. Their production in the cell and physiological activation are tightly regulated. The proteasomal processing of inactive NF-kappa B1/p105 to active p50, with an anti-inflammatory role, is not well characterized. Here we show that ubiquitin ligase TRUSS is a mediator of transcriptional activation of anti-inflammatory cytokine IL-10 gene. Enforced expression of TRUSS led to enhanced IL-10 expression that could be inhibited in the presence of chemical inhibitors of NF-kappa B [BAY11-7082] and PI3K/Akt [LY249002] or after p65 overexpression. p50 was actively recruited on IL10 promoter in the presence of TRUSS but competed by p65 for binding. TRUSS facilitated the ubiquitination of NF-kappa B1/p105 and promoted its proteolytic processing to generate excess of p50. Our immune-histochemical studies confirmed enhanced expression of p105/p50 in the human HCC tumors. Further, the hepatic tumors of HCC patient as well as transgenic mice showed decreased levels of p50 as well as TRUSS and accumulation of p105. Thus, enhanced expression of IL-10 gene in the presence of TRUSS and regulation of NF-kappa B1/p105 processing could be an important regulatory mechanism for inflammatory response and tumorgenic transformation.