Abstract Covalent post-translational protein modifications (PTMs) contribute to all aspects of cell physiology and are a primary source of protein functional diversity in mammalian cells. Our overarching goal is to better understand the role of protein methylation signaling in the regulation of diverse biological functions and how disruption in these mechanisms contributes to cancer and other disease pathologies. While the most commonly studied methylation events occur on lysine and arginine residues ? glutamine, cysteine, and histidine residues are also methylated ? though these modification events are thought to occur on only a limited number of proteins and relatively little is known about the enzymes that catalyze these chemical reactions. In particular, while modification of histidine residues by methylation is thought to be a rare event on proteins, the methylation of actin at histidine 73 (actin-H73me) is a canonical modification in mammals that was identified more than fifty years ago. However, the function of actin-H73me is enigmatic and the enzyme/s generating this abundant modification event are not known. In preliminary work we have identified SETD3 as the first known metazoan protein histidine methyltransferase (PHMT). SETD3, a little studied cytoplasmic protein that belongs to the SET domain family of enzymes, is implicated in processes involved in muscle function and cancer. However, a clear function for SETD3 is not known. Our initial data identified SETD3 as a critical host factor required for infection by a broad class of enteroviruses. We also have evidence for SETD3 regulation of smooth muscle physiology in cells and in vivo. In humans, beyond actin, virtually nothing is known about the molecular, signaling and biological consequences associated with histidine methylation. Our central hypothesis is that histidine methylation of actin and other proteins by enzymes such as SETD3 have an underappreciated and significant role in signal transduction, cell biology, and disease pathogenesis. We propose to use biochemical, cellular, genetic, and proteomic approaches to elucidate the molecular, biological and pathological functions of histidine methylation, with a focus on the biology surrounding SETD3 and SETD3-catalyzed modification of actin. In Aim 1 we will perform experiments to gain a molecular level understanding of the consequence of actin H73 methylation by SETD3. The goal of Aim 2 is to investigate SETD3 cellular functions. Experiments are planned to identify the role of SETD3?s enzymatic activity in actin- related cellular functions and cancer cell phenotypes. We will also explore the molecular mechanisms by which actin is paired with SETD3 for methylation. The goal of Aim 3 is to expand our knowledge of histidine methylation signaling in humans beyond that of SETD3 and actin. We will use computational, proteomic, and biochemical strategies to identify and validate novel human histidine methylated proteins and to discover new histidine methyltransferases.