Deciphering the mechanisms regulating itaconate production and signaling in macrophages

PROJECT SUMMARY Macrophages are innate immune cells that serve diverse roles in host defense, tissue repair, and homeostasis which are crucial to systemic physiology in all tissues. Therefore, dysregulation of macrophage function can contribute to a wide range of pathologies, including chronic inflammation, metabolic diseases, and cancer. Mitochondrial metabolites have been identified as important regulators of macrophage function with one key regulatory mode involved in post-translational modification (PTM) of cysteine residues on protein targets, which invokes changes in protein structures and functions. Within the past decade, itaconate has emerged as a crucial immunomodulatory metabolite in macrophages. Itaconate is uniquely produced in myeloid cells, most notably in macrophages, by a mitochondrial enzyme named aconitate decarboxylase 1 (ACOD1) and can directly modify protein targets in macrophages via a cysteine PTM called alkylation. However, the breadth of the cysteine targets subjected to this PTM at the proteome level in macrophages is unknown. No study to date has identified the mechanism by which ACOD1 enzyme activity is regulated. Through extensive preliminary studies, I have stoichiometrically defined the macrophage cysteine proteome undergoing itaconate-mediated alkylation by utilizing a high-throughput redox proteomic platform called mass spectrometry-based cysteine-reactive phosphate tag (CPT-MS). From this cysteine proteomic dataset, I’ve discovered a cysteine site on ACOD1 that is potently modified by itaconate. Through preliminary in vitro ACOD1 enzyme activity assays, I’ve demonstrated that the identified cysteine site is important for ACOD1 enzyme activity and that itaconate can inhibit its own production by antagonizing ACOD1 activity. Building on these preliminary data, I hypothesize that itaconate production is involved in a feedback mechanism by which itaconate alkylates crucial cysteine site(s) on ACOD1 to control enzyme activity by invoking structural changes. Using a combination of crystallography, protein biochemistry, and in vivo/in vitro CRISPR editing, I aim to 1) decipher this self-regulatory mechanism underpinning itaconate production and 2) determine the physiological importance of the ACOD1 cysteine residue identified from my CPT-MS data. The findings from this proposed work will uncover a unique mechanism of itaconate production and signaling in macrophages. Moreover, the results of this proposal will lay the foundation for the design of the first-in-class ACOD1 inhibitor/activator and other molecular effectors by targeting key cysteines to manipulate macrophage function in all macrophage-related disease contexts. Additionally, I propose a detailed training plan under this fellowship to provide me with the practical and conceptual skills that will help me complete this project and benefit my future career goals to be an independent scientist. This project involves a wide range of interdisciplinary science to which the excellent environment at Dana-Farber Cancer Institute and Harvard Medical School can provide me with the opportunity to collaborate with many experts in the fields who can give me the necessary support and training to complete my proposed aims.