Mitochondrial deubiquitinase USP30 regulates cell metabolism-mediated miRNA biogenesis and microvascular inflammation - Abstract Acute inflammatory diseases are life-threatening health conditions that are most often caused by bacterial or viral infection such as Pseudomonas aeruginosa- or SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) and sepsis. ARDS and sepsis-related mortality remains at unexpectedly high levels due to lack of effective pharmacotherapies. Hence, a new therapeutic strategy for ARDS and sepsis is needed. Microvascular inflammation and barrier disruption play critical roles in the pathogenesis of acute inflammatory diseases. A mitochondrial de-ubiquitinating enzyme, USP30, plays a vital role in regulation of mitochondrial outer membrane protein homeostasis. USP30 has been considered as a potential target for treating Parkinson’s disease and cancers; however, the role of USP30 in microvascular endothelial cells (ECs) and acute inflammatory diseases has not been reported. In our preliminary data, we discovered that inhibiting USP30 diminished EC dysfunction and reduced the severity of experimental lung injury. Mechanistically, we discovered a non-canonical pathway of USP30 that links MAT2A stability, the S- adenosylmethionine (SAM) cycle, DNA methylation, miRNA-30a-5p synthesis. Based on our comprehensive preliminary data, we hypothesize that inhibiting USP30 preserves EC function by modulating intracellular signaling cascades implicated in MAT2A stability, SAM production, DNA methylation, and miR-30a-5p expression. We propose testing the hypothesis with the following Specific Aims: Aim 1 is to determine if USP30 in the endothelium is a potential target for treatment of acute lung injury. We will determine if inhibiting EC USP30 diminishes leukocyte cell adhesion to EC and transendothelial migration and preserves EC barrier integrity. Further, we will determine if depletion of USP30 in endothelial cells reduces severity of pseudomonas aeruginosa- or sepsis-induced experimental lung injury. Aim 2 is to determine the molecular mechanisms by which inhibiting USP30 destabilizes MAT2A, decreases the SAM production, and increases miR-30a-5p. We will determine if inhibiting USP30- induced miR-30a-5p occurs through modulation of MAT2A stability in the SAM cycle, reduction of SAM production and pri-miR-30 promoter methylation. Further, we will determine if miR-30a-5p regulates USP30 inhibition-mediated MDM2, MLC, and NFAT5 downregulation. Aim 3 is to determine if the protective effect of USP30 inhibition occurs through modulating miR-30a-5p expression. We will determine if USP30 inhibition preserves EC function by modulating miR-30a-5p expression in HLMVECs. Further, we will determine if EC USP30 depletion reduces severity of experimental lung injury through modulating miR- 30a-5p expression by using a cutting-edge RNA nanoparticle technology. Comprehensive understanding of mitochondrial de-ubiquitinating enzyme inhibition-induced changes of cell metabolisms and EC function is important for development of new therapeutic targets for treatment of acute inflammatory diseases.
