Activity-Based Proteomic Profiling of Deubiquitinating Enzymes in Salmonella-Infected Macrophages Leads to Identification of Putative Function of UCH-L5 in Inflammasome Regulation

Abstract

Although protein ubiquitination has been shown to regulate multiple processes during host response to Salmonella enterica serovar Typhimurium infection, specific functions of host deubiquitinating enzymes remain unknown in this bacterial infection. By using chemical proteomics approach, in which deubiquitinating enzymes were labeled by an active-site probe and analyzed by quantitative proteomics, we identified novel deubiquitinases in chicken macrophages based on their reactivity with the probe. Also, we detected down-regulation of UCH-L3, and USP4 as well as up-regulation of USP5 and UCH-L5 deubiquitinating enzymes in macrophages infected with Salmonella Typhimurium. We showed that decrease in either UCH-L5 activity, or in UCH-L5 protein amount in chicken and human macrophages infected or stimulated with LPS/nigericin, led to decreased IL-1β release. These data point towards a putative role of UCH-L5 in inflammasome regulation during Salmonella infection. Because inflammasome activation is important in innate resistance to these bacteria, one would expect that naturally occurring or therapeutically induced alteration in UCH-L5 activation would influence disease outcome and could represent a target for new therapeutic approaches.

Salmonella enterica Serovar Typhimurium Alters the Extracellular Proteome of Macrophages and Leads to the Production of Proinflammatory Exosomes

“…Overall, the host ubiquitination pathway is widely involved in response to S . Typhimurium infection ( 9 , 41 , 42 ), but the involvement of ubiquitination in targeting proteins to exosomes during this infection is unknown. Another secreted protein of interest was OTUB1, the protein level of which was increased in cell culture medium of infected cells ( Table 1 and Fig.…”

“…The uninfected control cells were cultured in the same way but without the addition of bacteria. For infection times of >2 h, 100 μg/ml gentamicin was used to kill extracellular bacteria, as described previously ( 41 ). As a control, bacteria were inactivated with paraformaldehyde before addition to cells.…”

“…The protein concentration was measured by using the modified Lowry method (DC kit; Bio-Rad). Samples containing 200 μg of protein were precipitated by using chloroform and methanol as described earlier ( 41 ). Protein samples were reduced and alkylated, followed by tryptic digestion as previously described ( 41 ).…”

Illuminating host-mycobacterial interactions with functional genomic screening to inhibit mycobacterial pathogenesis

“…Many novel host genes were also identified in our screens, including: YPEL5 (yippee-like 5), which may interact with TBK1 (TANK binding kinase 1) (Moreira-Teixeira et al, 2018); WDR26 (WD repeat domain 26), which regulates apoptosis, ubiquitination, signal transduction, and other cellular processes; UCHL5 (ubiquitin C-terminal hydrolase L5), which mediates ubiquitination and protein degradation (Kummari et al, 2015); AHR (aryl hydrocarbon receptor) and ARNT (AHR nuclear translocator), sensors that regulate metabolism and immune functions (Rothhammer and Quintana, 2019); plus many hits without known functions ( Tables S2 and S3 ). Thus, our genome-wide CRISPR-Cas9 knockout and CRISPRi screens identified known and novel host genes that alter macrophage survival after M. bovis BCG infection.…”

Activity‐based probes for the ubiquitin conjugation–deconjugation machinery: new chemistries, new tools, and new insights

“…Kummari et al . used HA‐Ub‐VS to profile DUB activity in Salmonella‐infected chicken macrophages, and identified several DUBs that change substantially in activity following infection. UCHL3 activity decreased (although protein levels remained similar), while the proteasome‐associated DUB UCHL5 increased in both activity and protein level.…”