Proteolytic deubiquitinating enzymes bridge a gap in substrate recognition through complex regulatory mechanisms. A growing portion of these are accomplished through proteoforms that uniquely control association and diverse sets of cleavage capabilities that relay distinct physiological outcomes. This study describes substrate biasing governed by UCHL5 proteoforms. It demonstrates that N-terminal ubiquitination activates the enzyme towards monoubiquitin substrates, a feature that is conserved across UCHL5 homologs. Crystallographic and spectroscopic data suggest that the N-terminal ubiquitin binds intramolecularly in an allosteric binding site and inhibits branched chain substrate cleavage. Association with Rpn13/Adrm1 relieves this inhibition and reestablishes its ability to debranch, potentially controlling nonspecific debranching compared to retention of needed activity on the 26S proteasome. Collectively, this study describes the molecular basis for substrate selectivity in a deubiquitinating enzyme, an unexplored area in the enzymes that counteract ubiquitin E3 ligases.
Rishi S. Patel, Nipuni M. Pannala, Chih-Hsuan Lai et al.· bioRxiv· 0 citations
Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in piglets, imposing significant economic burdens on swine production worldwide. Despite its impact, detailed characterization of intestinal colonization and comprehensive genomic profiling of virulent neonatal porcine ETEC strains remain limited. In this study, we isolated a β‐hemolytic ETEC strain, PT4357, from a diarrheic neonatal piglet and conducted whole‐genome sequencing (WGS) coupled with experimental infection in neonatal piglets to define its pathogenicity. The PT4357 belonged to serotype O8:H7 and sequence type ST2521 and harbored multiple virulence and antimicrobial resistance (AMR) genes, including the plasmid‐borne colistin resistance gene mcr-1. Interestingly, a novel chaperone‐usher (CU) assembly pathway pilus has a homolog of the α family usher gene that was identified in the chromosome of PT4357. Compared with the CS1 pilus on ETEC, structural superposition of the CU pathway pilus of PT4357, namely, CS33, showed strong conservation in the major subunit and chaperone and was relatively divergent in the N‐terminal of usher and minor subunits while retaining essential core folds. Oral inoculation confirmed villi attachment of the ETEC PT4357, causing watery diarrhea in neonatal piglets without mucosal damage. Furthermore, the adherence of the ETEC PT4357 on porcine IPEC cells but not human Caco‐2 cells suggests limited human tropism. Our results indicate that Taiwan porcine ETECs carried AMR genes and alternative CU pathway pilus. The role of the CS33 pilus on the pathogenicity or virulence of the pathogen requires further investigation and surveillance.
Yi-Fang Lai, Yen-Chen Chang, Wei-Ting Lee et al.· Transboundary and Emerging D...· 0 citations