The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems. Optimized inhibitors showed high selectivity for DAGLβ over other serine hydrolases, including DAGLα (∼60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGLβ inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLβ is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

Serine hydrolases are a diverse enzyme class representing ∼1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.

The nutrient- and growth factor-responsive kinase mTOR complex 1 (mTORC1) regulates many processes that control growth, including protein synthesis, autophagy, and lipogenesis. Through unknown mechanisms, mTORC1 promotes the function of SREBP, a master regulator of lipo- and sterolgenic gene transcription. Here, we demonstrate that mTORC1 regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase. Dephosphorylated, nuclear, catalytically active lipin 1 promotes nuclear remodeling and mediates the effects of mTORC1 on SREBP target gene, SREBP promoter activity, and nuclear SREBP protein abundance. Inhibition of mTORC1 in the liver significantly impairs SREBP function and makes mice resistant, in a lipin 1-dependent fashion, to the hepatic steatosis and hypercholesterolemia induced by a high-fat and -cholesterol diet. These findings establish lipin 1 as a key component of the mTORC1-SREBP pathway.

Hepatic glucose production (HGP) is required to maintain normoglycemia during fasting. Glucagon is the primary hormone responsible for increasing HGP; however, there are many additional hormone and metabolic factors that influence glucagon sensitivity. In this study we report that the bioactive lipid lysophosphatidic acid (LPA) regulates hepatocyte glucose production by antagonizing glucagon-induced expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Treatment of primary hepatocytes with exogenous LPA blunted glucagon-induced PEPCK expression and glucose production. Similarly, knockout mice lacking the LPA-degrading enzyme phospholipid phosphate phosphatase type 1 (PLPP1) had a 2-fold increase in endogenous LPA levels, reduced PEPCK levels during fasting, and decreased hepatic gluconeogenesis in response to a pyruvate challenge. Mechanistically, LPA antagonized glucagon-mediated inhibition of STAT3, a transcriptional repressor of PEPCK. Importantly, LPA did not blunt glucagon-stimulated glucose production or PEPCK expression in hepatocytes lacking STAT3. These data identify a novel role for PLPP1 activity and hepatocyte LPA levels in glucagon sensitivity via a mechanism involving STAT3.

Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation (unlike newborn lung alveoli), selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis via autophagy and mitochondrial fusion and promotes basal tearing. Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic α-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis.

Adipose tissue is a critical regulator of energy balance that must rapidly shift its metabolism between fasting and feeding to maintain homeostasis. Adenosine has been characterized as an important regulator of adipocyte metabolism primarily through its actions on A1 adenosine receptors (A1R). We sought to understand the role A1R plays specifically in adipocytes during fasting and feeding to regulate glucose and lipid metabolism.

Hypermetabolism following severe burn injuries is associated with adipocyte dysfunction, elevated beige adipocyte formation, and increased energy expenditure. The resulting catabolism of adipose leads to detrimental sequelae such as fatty liver, increased risk of infections, sepsis, and even death. While the phenomenon of pathological white adipose tissue (WAT) browning is well-documented in cachexia and burn models, the molecular mechanisms are essentially unknown. Here, we report that adipose triglyceride lipase (ATGL) plays a central role in burn-induced WAT dysfunction and systemic outcomes. Targeting adipose-specific ATGL in a murine (AKO) model resulted in diminished browning, decreased circulating fatty acids, and mitigation of burn-induced hepatomegaly. To assess the clinical applicability of targeting ATGL, we demonstrate that the selective ATGL inhibitor atglistatin mimics the AKO results, suggesting a path forward for improving patient outcomes.

Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis.

Lipids exert key structural, metabolic, and signaling functions in cells. Lipid diversity found in cells and tissues is regulated principally by metabolic enzymes whose activity is modulated posttranslationally to shape head group and fatty acyl composition of membrane lipids. Methodologies capable of monitoring in vivo changes in the lipidome are needed to assign substrate specificity of metabolic enzymes, which represents a key step toward understanding structure-function of lipids in living systems. The resulting lipid annotations also serve as important biomarkers for understanding mode of action for pharmacological agents targeting metabolic enzymes in cells and animal models. In this chapter, we describe a general metabolomics workflow to complement (chemo)proteomic efforts to modulate lipid pathways for basic science and translational applications.

Diacylglycerol kinases (DGKs) are metabolic kinases involved in regulating cellular levels of diacylglycerol and phosphatidic lipid messengers. The development of selective inhibitors for individual DGKs would benefit from discovery of protein pockets available for inhibitor binding in cellular environments. Here we utilized a sulfonyl-triazole probe (TH211) bearing a DGK fragment ligand for covalent binding to tyrosine and lysine sites on DGKs in cells that map to predicted small molecule binding pockets in AlphaFold structures. We apply this chemoproteomics-AlphaFold approach to evaluate probe binding of DGK chimera proteins engineered to exchange regulatory C1 domains between DGK subtypes (DGKα and DGKζ). Specifically, we discovered loss of TH211 binding to a predicted pocket in the catalytic domain when C1 domains on DGKα were exchanged that correlated with impaired biochemical activity as measured by a DAG phosphorylation assay. Collectively, we provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.

Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5500 cysteine sites across ~3000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.

The serine hydrolase α/β-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, 2-arachidonoylglycerol (2-AG), and controls its availability at cannabinoid receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1(-/-) or Cnr2(-/-) mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/2 mice, a genetic model of juvenile Huntington's disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/2 mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H2O2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2.

Phosphoinositide-3 (PI-3) kinase signaling has a pervasive role in cancer. One of the key effectors of PI-3 kinase signaling is AKT, a kinase that promotes growth and survival in a variety of cancers. Genetically engineered mouse models of prostate cancer have shown that AKT signaling is sufficient to induce prostatic epithelial neoplasia (PIN), but insufficient for progression to adenocarcinoma. This contrasts with the phenotype of mice with prostate-specific deletion of Pten, where excessive PI-3 kinase signaling induces both PIN and locally invasive carcinoma. We reasoned that additional PI-3 kinase effector kinases promote prostate cancer progression via activities that provide biological complementarity to AKT. We focused on the PKN kinase family members, which undergo activation in response to PI-3 kinase signaling, show expression changes in prostate cancer, and contribute to cell motility pathways in cancer cells.

Lipid droplets (LDs) are dynamic organelles that undergo dynamic changes in response to changing cellular conditions. During nutrient depletion, LD numbers increase to protect cells against toxic fatty acids generated through autophagy and provide fuel for beta-oxidation. However, the precise mechanisms through which these changes are regulated have remained unclear. Here, we show that the small GTPase RalA acts downstream of autophagy to directly facilitate LD growth during nutrient depletion. Mechanistically, RalA performs this function through phospholipase D1 (PLD1), an enzyme that converts phosphatidylcholine (PC) to phosphatidic acid (PA) and that is recruited to lysosomes during nutrient stress in a RalA-dependent fashion. RalA inhibition prevents recruitment of the LD-associated protein perilipin 3, which is required for LD growth. Our data support a model in which RalA recruits PLD1 to lysosomes during nutrient deprivation to promote the localized production of PA and the recruitment of perilipin 3 to expanding LDs.

The diacylglycerol kinases (DGKs) are a family of enzymes responsible for the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). In addition to their primary function in lipid metabolism, DGKs have recently been identified as potential therapeutic targets in multiple cancers, including glioblastoma (GBM) and melanoma. Aside from its tumorigenic properties, DGKα is also a known promoter of T-cell anergy, supporting a role as a recently-recognized T cell checkpoint. In fact, the only significant phenotype previously observed in Dgka knockout (KO) mice is the enhancement of T-cell activity. Herein we reveal a novel, macrophage-specific, immune-regulatory function of DGKα. In bone marrow-derived macrophages (BMDMs) cultured from wild-type (WT) and KO mice, we observed increased responsiveness of KO macrophages to diverse stimuli that yield different phenotypes, including LPS, IL-4, and the chemoattractant MCP-1. Knockdown (KD) of Dgka in a murine macrophage cell line resulted in similar increased responsiveness. Demonstrating in vivo relevance, we observed significantly smaller wounds in Dgka-/- mice with full-thickness cutaneous burns, a complex wound healing process in which macrophages play a key role. The burned area also demonstrated increased numbers of macrophages. In a cortical stab wound model, Dgka-/- brains show increased Iba1+ cell numbers at the needle track versus that in WT brains. Taken together, these findings identify a novel immune-regulatory checkpoint function of DGKα in macrophages with potential implications for wound healing, cancer therapy, and other settings.

Acetyl-CoA carboxylase (ACC) regulates lipid synthesis; however, its role in inflammatory regulation in macrophages remains unclear. We generated mice that are deficient in both ACC isoforms in myeloid cells. ACC deficiency altered the lipidomic, transcriptomic, and bioenergetic profile of bone marrow-derived macrophages, resulting in a blunted response to proinflammatory stimulation. In response to lipopolysaccharide (LPS), ACC is required for the early metabolic switch to glycolysis and remodeling of the macrophage lipidome. ACC deficiency also resulted in impaired macrophage innate immune functions, including bacterial clearance. Myeloid-specific deletion or pharmacological inhibition of ACC in mice attenuated LPS-induced expression of proinflammatory cytokines interleukin-6 (IL-6) and IL-1β, while pharmacological inhibition of ACC increased susceptibility to bacterial peritonitis in wild-type mice. Together, we identify a critical role for ACC in metabolic regulation of the innate immune response in macrophages, and thus a clinically relevant, unexpected consequence of pharmacological ACC inhibition.

Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK-STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1 (UCP1). We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. The inability of STAT3-/- preadipocytes to differentiate can be rescued using Wnt ligand secretion inhibitors when applied during the induction stage. Through chemical inhibition and RNAi, we show that it is the canonical β-catenin pathway that is responsible for the block in differentiation; inhibition or knockdown of β-catenin can fully rescue adipogenesis and UCP1 expression in the STAT3-/- adipocytes. During the induction stage, Wnts 1, 3a, and 10b have increased expression in the STAT3-/- adipocytes, potentially explaining the increased levels and activity of β-catenin. Our results for the first time point towards an interaction between the JAK/STAT pathway and the Wnt/β-catenin pathway during the early stages of in-vitro adipogenesis.

Extracellular signal-regulated kinases (ERKs) mediate downstream signaling of RAS-RAF-MEK as key regulators of the mitogen-activated protein kinase (MAPK) pathway. Activation of ERK signaling is a hallmark of cancer and upstream MAPK proteins have been extensively pursued as drug targets for cancer therapies. However, the rapid rise of resistance to clinical RAF and MEK inhibitors has prompted interest in targeting ERK (ERK1 and ERK2 isoforms) directly for cancer therapy. Current methods for evaluating activity of inhibitors against ERK isoforms are based primarily on analysis of recombinant proteins. Strategies to directly and independently profile native ERK1 and ERK2 activity would greatly complement current cell biological tools used to probe and target ERK function. Here, we present a quantitative chemoproteomic strategy that utilizes active-site directed probes to directly quantify native ERK activity in an isoform-specific fashion. We exploit a single isoleucine/leucine difference in ERK substrate binding sites to enable activity-based profiling of ERK1 versus ERK2 across a variety of cell types, tissues, and species. We used our chemoproteomic strategy to determine potency and selectivity of academic (VX-11e) and clinical (Ulixertinib) ERK inhibitors. Correlation of potency estimates by chemoproteomics with anti-proliferative activity of VX-11e and Ulixertinib revealed that >90% inactivation of both native ERK1 and ERK2 is needed to mediate cellular activity of inhibitors. Our findings introduce one of the first assays capable of independent evaluation of native ERK1 and ERK2 activity to advance drug discovery of oncogenic MAPK pathways.

α/β-Hydrolase domain-containing 6 (ABHD6) contributes to the hydrolysis of the major endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS) and in the periphery. ABHD6 blockade has been proposed as novel strategy to treat multiple sclerosis (MS), based on the observation that the inhibitor WWL70 exerts protective anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE). According to recent data, WWL70 exhibits off-target anti-inflammatory activity in microglial cells and the potential of ABHD6 as drug target in MS remains controversial. Here we further investigated the role of ABHD6 during autoimmune demyelination by comparing the efficacy of two novel inhibitors with different CNS permeability in vivo. Preventive treatment with the systemically active inhibitor KT182 ameliorated the neurological signs of EAE during the time-course of disease. By contrast, administration of the peripherally restricted compound KT203 was ineffective in attenuating EAE symptomatology. Both inhibitors failed to improve corticospinal tract conduction latency and to attenuate inflammation at EAE recovery phase, despite being equally active at targeting brain ABHD6. Chronic administration of KT182 was associated to a partial loss of brain CB1 receptor coupling ability, suggesting the engagement of CB1 receptor-mediated mechanisms during the EAE disease progression. In cultured neurons, KT182 attenuated NMDA-stimulated excitotoxicity and mitochondrial calcium overload. However, these protective effects were not attributable to ABHD6, as they were not mimicked by the alternative inhibitors KT203, KT195 and WWL70. These results indicate that ABHD6 blockade exerts only modest therapeutic effects against autoimmune demyelination and call into question its utility as novel drug target in MS.