Understanding the role of ubiquitination-like processes in bacterial antiviral immunity

/ABSTRACT Eukaryotic ubiquitination involves the conjugation of one protein to another through the sequential activity of three enzymes, E1, E2, and E3. These enzymes catalyze the transfer of ubiquitin or ubiquitin-like proteins onto target protein substrates. Ubiquitination contributes to the regulation of a wide variety of eukaryotic cellular processes, including protein degradation, gene transcription, DNA repair, and cell cycle control. Another important role for ubiquitin or ubiquitin-like protein conjugation is in the human innate antiviral immune response. During infection, the innate immune system serves as the first line of defense against viral threats. By recognizing the foreign, or “non-self”, components of the virus, the immune system activates various pathways to prevent widespread infection. Two examples of antiviral innate immune pathways that are known to be regulated by canonical ubiquitin or ubiquitin-like protein conjugation are cGAS-STING and ISG15, but many of the mechanistic details of these pathways have yet to be uncovered. Recently, operons in bacteria have been discovered that encode E1 and/or E2 ubiquitin transferase domains, and, in some instances, canonical ubiquitin-like proteins. These operons have been defined as bacterial innate immune systems that function through protein conjugation mechanisms and are evolutionarily related to the human cGAS-STING and ISG15 immune pathways. Here, I have proposed a multidisciplinary strategy to understand the molecular mechanisms of protein conjugation used by bacterial defense systems. In one of these systems, an E1-E2 ubiquitin transferase enzyme, Cap2, conjugates a non-ubiquitin-like protein, a signaling enzyme called bacterial cGAS, onto an unknown target molecule to protect bacteria from phage infection. I will use mass spectrometry-based proteomics, mechanistic biochemistry, and phage infection assays to explore how phage defense is achieved through conjugation of a non-ubiquitin-like protein (cGAS) in this system. I will also explore the mechanisms of the bacterial ISG15-like (Bil) and two other ubiquitin protein-containing defense systems using mechanistic biochemistry and mass spectrometry. Specifically, I will determine how the conjugation of canonical ubiquitin-like proteins confers defense against phage infection. Deciphering the details of ubiquitination-like processes in bacterial innate immunity will inform our understanding of the underlying mechanisms of conserved human innate immunity pathways, including cGAS-STING and ISG15. Gaining new insights into these mechanisms could pave the way for developing novel therapeutics that modulate the human immune response during viral infection. Project Number: 1F31AI186492-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Ashley Sullivan | Institution: UNIVERSITY OF COLORADO, Boulder, CO | Award Amount: $42,843 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F07A-M (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F31AI18649201A1