Noah Brookes’s research while affiliated with University of Chicago and other places

Extracellular signal-regulated kinases (ERK1/2) are key effector proteins of the mitogen-activated protein kinase pathway, choreographing essential processes of cellular physiology. Here, we discover that ERK1/2 are subject to S-acylation, a reversible lipid modification of cysteine residues, at C271/C254. The levels of ERK1/2 S-acylation are modulated by epidermal growth factor (EGF) signaling, mirroring its phosphorylation dynamics, and acylation-deficient ERK2 displays altered phosphorylation patterns. We show that ERK1/2 S-acylation is mediated by “writer” protein acyl transferases (PATs) and “eraser” acyl protein thioesterases (APTs) and that chemical inhibition of either lipid addition or removal alters ERK1/2’s EGF-triggered transcriptional program. Finally, in a mouse model of metabolic syndrome, we find that ERK1/2 lipidation levels correlate with alterations in ERK1/2 lipidation writer/eraser expression, solidifying a link between ERK1/2 activity, ERK1/2 lipidation, and organismal health. This study describes how lipidation regulates ERK1/2 and offers insight into the role of dynamic S-acylation in cell signaling more broadly.

As the "writer" enzymes of protein S-acylation, a dynamic and functionally significant post-translational modification (PTM), DHHC family proteins have emerged in the past decade as both key modulators of cellular homeostasis and as drivers of neoplastic, autoimmune, metabolic, and neurological pathologies. Currently, biological and clinical discovery is hampered by the limitations of existing DHHC family inhibitors, which possess poor physicochemical properties and off-target profiles. However, progress in identifying new inhibitory scaffolds has been meager, in part due to a lack of robust in vitro assays suitable for high-throughput screening (HTS). Here, we report the development of palmitoyl transferase probes (PTPs), a novel family of turn-on pro-fluorescent molecules that mimic the palmitoyl-CoA substrate of DHHC proteins. We use the PTPs to develop and validate an assay with an excellent Z'-factor for HTS. We then perform a pilot screen of 1687 acrylamide-based molecules against zDHHC20, establishing the PTP-based HTS assay as a platform for the discovery of improved DHHC family inhibitors.

The extracellular signal-regulated kinases (ERK1/2) are key effector proteins of the mitogen activated protein kinase pathway, choreographing essential processes of cellular physiology. Critical in regulating these regulators are a patchwork of mechanisms, including post-translational modifications (PTMs) such as MEK-mediated phosphorylation. Here, we discover that ERK1/2 are subject to S-palmitoylation, a reversible lipid modification of cysteine residues, at C271/C254. Moreover, the levels of ERK1/2 S-acylation are modulated by epidermal growth factor (EGF) signaling, mirroring its phosphorylation dynamics, and palmitoylation-deficient ERK2 displays altered phosphorylation patterns at key sites. We find that chemical inhibition of either lipid addition or removal significantly alters ERK1/2's EGF-triggered transcriptional program. We also identify a subset of "writer" protein acyl transferases (PATs) and an "eraser" acyl protein thioesterase (APT) that drive ERK1/2's cycle of palmitoylation and depalmitoylation. Finally, we examine ERK1/2 S-acylation in a mouse model of metabolic syndrome, correlating changes in its lipidation levels with alterations in writer/eraser expression and solidifying the link between ERK1/2 activity, ERK1/2 lipidation, and organismal health. This study not only presents a previously undescribed mode of ERK1/2 regulation and a node to modulate MAPK pathway signaling in pathophysiological conditions, it also offers insight into the role of dynamic S29 palmitoylation in cell signaling more generally.

p>Protein S -acylation is a dynamic lipid post-translational modification that can modulate the localization and activity of target proteins. In humans, the installation of the lipid onto target proteins is catalyzed by a family of 23 Asp-His-His-Cys domain-containing protein acyltransferases (DHHC-PATs). DHHCs are increasingly recognized as critical players in cellular signaling events and in human disease. However, progress elucidating the functions and mechanisms of DHHC “writers” has been hampered by a lack of chemical tools to perturb their activity in live cells. Herein, we report the synthesis and characterization of cyano-myracrylamide (CMA) , a broad-spectrum DHHC family inhibitor with similar potency to 2-bromopalmitate (2BP), the most commonly used DHHC inhibitor in the field. Possessing an acrylamide warhead instead of 2BP’s α-halo fatty acid, CMA inhibits DHHC family proteins in cellulo while demonstrating decreased toxicity and avoiding inhibition of the S- acylation eraser enzymes – two of the major weaknesses of 2BP. Our studies show that CMA engages with DHHC family proteins in cells, inhibits protein S- acylation, and disrupts DHHC-regulated cellular events. CMA represents an improved chemical scaffold for untangling the complexities of DHHC-mediated cell signaling by protein S -acylation.</p

p>Protein S -acylation is a dynamic lipid post-translational modification that can modulate the localization and activity of target proteins. In humans, the installation of the lipid onto target proteins is catalyzed by a family of 23 Asp-His-His-Cys domain-containing protein acyltransferases (DHHC-PATs). DHHCs are increasingly recognized as critical players in cellular signaling events and in human disease. However, progress elucidating the functions and mechanisms of DHHC “writers” has been hampered by a lack of chemical tools to perturb their activity in live cells. Herein, we report the synthesis and characterization of cyano-myracrylamide (CMA) , a broad-spectrum DHHC family inhibitor with similar potency to 2-bromopalmitate (2BP), the most commonly used DHHC inhibitor in the field. Possessing an acrylamide warhead instead of 2BP’s α-halo fatty acid, CMA inhibits DHHC family proteins in cellulo while demonstrating decreased toxicity and avoiding inhibition of the S- acylation eraser enzymes – two of the major weaknesses of 2BP. Our studies show that CMA engages with DHHC family proteins in cells, inhibits protein S- acylation, and disrupts DHHC-regulated cellular events. CMA represents an improved chemical scaffold for untangling the complexities of DHHC-mediated cell signaling by protein S -acylation.</p