Understanding the molecular function and substrate specificity of palmitoyltransferases in regulating behavior - SUMMARY To optimize behavior, animals must balance responding to important stimuli with an ability to ignore less critical stimuli. Behavioral thresholds provide a mechanism for this: animals respond to stimuli that exceed a particular threshold and ignore stimuli below the threshold. Palmitoyltransferases (or PATs, of which there are 24 in mammals) catalyze a reversible post-translational lipid modification and are emerging as key molecular regulators that tune behavioral responses. That they are important for human health is exemplified by their association with multiple disorders impacting behavior: schizophrenia, Alzheimer’s disease, and Huntington’s disease to name a few. Despite their ubiquity and importance, major questions regarding PAT function in vivo remain unanswered. Using larval zebrafish, we recently showed that the PAT enzyme Hip14 regulates behavioral thresholds for acoustic stimuli and the ability to tune thresholds through plasticity mechanisms such as habituation. This provides a tractable system to address how Hip14 specifically, and PATs more broadly, function in vivo to regulate behavior and ideally positions us to address the following open questions: How does PAT canonical enzymatic function contribute to behavioral plasticity? How PATs contribute to behavior has not been systematically examined in vertebrates. We will use high-throughput assays to rapidly identify which PATs are crucial for regulating behavior and behavioral plasticity in vivo using the larval zebrafish. In parallel, we will examine which palmitoylation substrates act downstream by generating point mutations in key palmitoylated residues and examining behavior. How is PAT substrate specificity regulated? Most PATs are expressed in the brain, but each has a unique expression pattern. Moreover, PATs exhibit substrate specificity, but the underlying mechanisms are not known. We will investigate the extent to which PAT protein sequence versus localization contribute to substrate specificity using structure-function and tissue-specific overexpression experiments. In parallel, we will use BioID to probe how localization and domain structure influence binding partners. Do PATs have enzymatic-independent functions in vivo? The best understood molecular function for PATs, including Hip14, is catalyzing the post-translational attachment of fatty acids to target proteins. However, non-enzymatic functions for Hip14 and other PATs have been identified in vitro. For example, Hip14 can function as a Mg2+ channel, and our pilot data indicate that Hip14 can regulate behavior even when its catalytic (palmitoyltransferase) domain is mutated. We will explore non-enzymatic functions for Hip14 and interrogate whether other PATs function in vivo as cation channels to regulate behavior. To answer these questions, we use zebrafish behavior as a readout. Successful completion of these projects will uncover basic mechanisms through which palmitoyltransferases function in vivo and provide new insights into how these key enzymes regulate behavior.
