Mohit Singh1, Bhawna Kumari1, Umesh C S Yadav2. 1. Metabolic Disorders and Inflammatory Pathologies Laboratory, School of Life Sciences, Central University of Gujarat, Sector-30, Gandhinagar, 382030, Gujarat, India. 2. Metabolic Disorders and Inflammatory Pathologies Laboratory, School of Life Sciences, Central University of Gujarat, Sector-30, Gandhinagar, 382030, Gujarat, India. umeshyadav@cug.ac.in.
Abstract
OBJECTIVE: Oxidized Low-Density Lipoprotein (oxLDL) is a well-established pro-inflammatory marker that activates the NLRP3 inflammasome. Ubiquitination plays an important role in modulating the stability and functions of various proteins. BRCC36 is a ubiquitin-modifying enzyme that plays a crucial role in protein stabilization and activation in the cytosol, but its role in OxLDL-induced NLRP3 inflammasome activation is not known. Here, we have investigated the role of deubiquitinating enzyme BRCC36 in regulating NLRP3 inflammasome during oxLDL stimulation. METHODS: Raw 264.7 murine macrophages were stimulated with oxLDL and effect of BRCC36 deubiquitination activity was assessed by fluorometric assay, and protein expression was assessed by Western blotting. The level of IL-1β measured by ELISA and LDH activity as pyroptotic cell death marker was assessed by fluorometric assay. RESULTS: The results showed that oxLDL increased the level of NLRP3 in macrophages and also the level of active caspase-1 and IL-1β. It also modulated the expression of deubiquitinating enzymes and caused pyroptotic cell death as indicated by LDH release. Inhibiting the proteasomal activity by MG132 and siRNA-mediated silencing of BRCC36 in macrophages potentially suppressed oxLDL-induced NLRP3 inflammasome activation and IL-1β secretion. Furthermore, the inhibition of proteasomal deubiquitinating activity with specific BRCC36 inhibitor G5 also reduced the inflammatory cell death. CONCLUSION: Taken together, our study suggests that deubiquitinating enzyme BRCC36 inhibition could potentially suppress oxLDL-induced inflammatory process by inhibiting NLRP3 activation and resultant IL-1β secretion.
OBJECTIVE: Oxidized Low-Density Lipoprotein (oxLDL) is a well-established pro-inflammatory marker that activates the NLRP3 inflammasome. Ubiquitination plays an important role in modulating the stability and functions of various proteins. BRCC36 is a ubiquitin-modifying enzyme that plays a crucial role in protein stabilization and activation in the cytosol, but its role in OxLDL-induced NLRP3 inflammasome activation is not known. Here, we have investigated the role of deubiquitinating enzyme BRCC36 in regulating NLRP3 inflammasome during oxLDL stimulation. METHODS: Raw 264.7 murine macrophages were stimulated with oxLDL and effect of BRCC36 deubiquitination activity was assessed by fluorometric assay, and protein expression was assessed by Western blotting. The level of IL-1β measured by ELISA and LDH activity as pyroptotic cell death marker was assessed by fluorometric assay. RESULTS: The results showed that oxLDL increased the level of NLRP3 in macrophages and also the level of active caspase-1 and IL-1β. It also modulated the expression of deubiquitinating enzymes and caused pyroptotic cell death as indicated by LDH release. Inhibiting the proteasomal activity by MG132 and siRNA-mediated silencing of BRCC36 in macrophages potentially suppressed oxLDL-induced NLRP3 inflammasome activation and IL-1β secretion. Furthermore, the inhibition of proteasomal deubiquitinating activity with specific BRCC36 inhibitor G5 also reduced the inflammatory cell death. CONCLUSION: Taken together, our study suggests that deubiquitinating enzyme BRCC36 inhibition could potentially suppress oxLDL-induced inflammatory process by inhibiting NLRP3 activation and resultant IL-1β secretion.
Authors: Elton Zeqiraj; Lei Tian; Christopher A Piggott; Monica C Pillon; Nicole M Duffy; Derek F Ceccarelli; Alexander F A Keszei; Kristina Lorenzen; Igor Kurinov; Stephen Orlicky; Gerald D Gish; Albert J R Heck; Alba Guarné; Roger A Greenberg; Frank Sicheri Journal: Mol Cell Date: 2015-09-03 Impact factor: 17.970