Project Grant F31AI202774

DETERMINING THE ROLE OF U9-ORF IN ENDOTHELIAL BARRIER INTEGRITY DURING SEPSIS - PROJECT SUMMARY IMMUNE DYSREGULATION IN RESPONSE TO INFECTION CAN LEAD TO SEPSIS, A CONDITION THAT RESULTS IN ABNORMAL PHYSIOLOGIC AND BIOCHEMICAL PROCESSES THAT CAN LEAD TO LIFE-THREATENING ORGAN DYSFUNCTION. DESPITE THE PREVALENCE OF THIS SYNDROME, WITH AT LEAST 1.7 MILLION ADULTS IN THE UNITED STATES DEVELOPING SEPSIS PER YEAR, MUCH REMAINS TO BE UNDERSTOOD SURROUNDING THE INITIATION, PROGRESSION, AND REGULATION OF THIS CONDITION. WE DETERMINED THAT THE MURINE GENE U90296 IS HIGHLY UPREGULATED IN MACROPHAGES AND IS PROTECTIVE IN A BACTERIAL LIPOPOLYSACCHARIDE (LPS) MODEL OF SEPSIS. ORIGINALLY ANNOTATED AS A LONG NON-CODING RNA, WE DISCOVERED THAT U90926 ENCODES AN 87 AMINO ACID OPEN READING FRAME (U9-ORF), WHICH CONTAINS AN N-TERMINAL SECRETION SIGNAL SEQUENCE. AFTER CONFIRMING U9-ORF'S SECRETION FROM MACROPHAGES, WE FOUND THAT A C-TERMINAL RGD MOTIF MEDIATES BINDING TO INTEGRIN V3, WHICH IS UPREGULATED ON ENDOTHELIAL CELLS DURING INFLAMMATION. CONSIDERING THAT VASCULAR LEAKAGE AND ENDOTHELIAL DYSFUNCTION ARE HALLMARKS OF SEPSIS PATHOPHYSIOLOGY, WE HYPOTHESIZE THAT U9-ORF IS SECRETED FROM MACROPHAGES AND BINDS INTEGRIN V3 ON ENDOTHELIAL CELLS TO PROMOTE ENDOTHELIAL BARRIER INTEGRITY. THE FOCUS OF THE PROPOSED STUDIES IS TO 1) DETERMINE IF THE MICROPROTEIN U9-ORF STABILIZES THE ENDOTHELIUM AND MINIMIZES VASCULAR LEAK, AND 2) DETERMINE IF U9-ORF IS SUFFICIENT TO PROTECT AGAINST SEPSIS-ASSOCIATED MORTALITY. USING A COMBINATION OF IN VITRO AND IN VIVO APPROACHES, WE WILL ASSESS THE EFFECTS OF U9-ORF ON INTRACELLULAR SIGNALING IN, AND MACROMOLECULAR FLUX ACROSS, THE ENDOTHELIAL BARRIER TO REVEAL HOW U9-ORF PROTECTS DURING ENDOTOXIC SHOCK. COMPLETION OF THE PROPOSED RESEARCH WILL CONTRIBUTE TO THE GROWING FIELD OF MICROPROTEIN BIOLOGY BY REVEALING THE FUNCTIONAL IMPORTANCE OF THE PROTEOME "HIDDEN" WITHIN "NON-CODING" TRANSCRIPTS, WHILE UNCOVERING NEW PROTECTIVE PATHWAYS DURING EXCESSIVE INFLAMMATION. THE PROPOSED RESEARCH IS COMPLEMENTED WITH A TRAINING PLAN TO ENHANCE EXPERTISE IN IMMUNOLOGY AND CELL BIOLOGY, SCIENTIFIC COMMUNICATION SKILLS, BIOMEDICAL GRANTSMANSHIP, EXPERIMENTAL RIGOR, AND TIME-MANAGEMENT SKILLS IN PREPARATION FOR A CAREER AS AN ACADEMIC PRINCIPAL INVESTIGATOR. THE UNIVERSITY OF VERMONT IS AN OPTIMAL TRAINING ENVIRONMENT WITH STATE-OF-THE-ART FACILITIES AND A NETWORK OF HIGHLY COLLABORATIVE AND SUPPORTIVE RESEARCH FACULTY AND STAFF.