Biology subjects

Cravatt, B.

Publications and source records attributed to Cravatt, B..

12 recordsLinked to original sources

bioRxiv2026

Lipid acyl chain remodeling governs KRAS nanoclustering and oncogenic functions through membrane composition control

KRAS signaling is spatially organized within the dynamic proteolipid nanoclusters on the plasma membrane (PM) that selectively concentrate anionic phospholipids, yet the molecular determinants governing this lipid sorting remain incompletely defined. Here we identify phospholipid acyl chain composition as a critical regulator of KRAS spatial organization and signal output. Using genetic and pharmacological perturbation of lysophosphatidylcholine acyltransferase 3 (LPCAT3), a key enzyme that incorporates polyunsaturated fatty acids into phospholipids, we demonstrate that remodeling of membrane acyl chain unsaturation alters the phosphatidylserine (PS) lipidome without changing total PS abundance. Loss of LPCAT3 depletes polyunsaturated PS species, disrupts KRAS PM association and nanoclustering, and attenuates downstream MAPK signaling and oncogenic activity across multiple KRAS mutant contexts. Acute restoration of polyunsaturated, but not saturated, lipids rescue KRAS nanoclustering, establishing a direct requirement for lipid unsaturation in sustaining signaling-competent assemblies. Mechanistically, these findings define membrane acyl chain composition as a determinant of proteolipid organization that encodes KRAS signaling capacity. Consistent with this model, LPCAT3 inhibition selectively impairs KRAS-dependent proliferation and sensitizes tumor cells to direct KRAS inhibition. Together, this work uncovers lipid acyl chain remodeling as a fundamental regulator of membrane-associated signaling and identifies membrane composition as an exploitable vulnerability in oncogenic KRAS systems.

cancer biology↗

bioRxiv2025

Inactivation of peptidoglycan remodeling promotes antibiotic susceptibility in vancomycin-resistant Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated infections globally and demands new approaches for treatment. Here we show that genetic and pharmacological inactivation of a highly conserved NlpC/P60 peptidoglycan hydrolase, secreted antigen A (SagA), enhanced vancomycin susceptibility of VREfm ex vivo and in vivo. Notably, genetic deletion of sagA impaired VREfm peptidoglycan remodeling, growth and increased the activity of vancomycin. We then identified first-in-class covalent NlpC/P60 peptidoglycan hydrolase inhibitors and demonstrated that pharmacological inactivation of SagA activity also impaired peptidoglycan remodeling and increased the efficacy of vancomycin across genetically distinct VREfm clinical isolates. Our study reveals peptidoglycan hydrolases are druggable targets whose inactivation improves the efficacy of vancomycin against VREfm.

microbiology↗

bioRxiv2025

Erucamide regulates retinal neurovascular crosstalk

Neurovasculoglial crosstalk is critical in establishing and maintaining a functional neurovascular unit. Breakdown in the unit is central to many neurodegenerative disorders of the CNS of which the retina is a component. A growing literature indicated that primary fatty acid amides (PFAMs) can regulate this crosstalk between vasculature and neuronal tissues. In this study we describe a central role for erucamide, a 22:1 mono-unsaturated omega-9 fatty acid amide, in degenerating retinal tissues. Using high-resolution global mass spectrometry-based metabolomics, we cataloged metabolites in murine models of retinal degeneration and show that while PFAMs, in general, are highly dysregulated, erucamide is the one most significantly diminished during photoreceptor atrophy. Using rodent models of retinal degeneration and novel organosilane-modified porous silicon nanoparticles (pSiNPs) for the in vivo delivery of erucamide, we demonstrate that erucamide activates CD11b+ myeloid cells, leading to the upregulation of angiogenic and neurotrophic cytokines that stabilize retinal degeneration. We identified TMEM19 as a novel binding protein for erucamide that is crucial for human iPSC-derived macrophage precursor cells activation and subsequent neurotrophic and angiogenic factor production. These findings reveal a previously unknown PFAM pathway that is modulated during retinal degenerative diseases, demonstrating that erucamide or functional analogues and their action through TMEM19 may be useful as a therapeutic alternative to neuroprotective and stem cell-based approaches for the treatment of retinal degenerative diseases.

neuroscience↗

bioRxiv2025

Ophiobolin A selectively alters mitochondria, metabolism and redox biology in breast cancer and mammary epithelial cells which have undergone epithelial to mesenchymal transition

Breast cancer progression is facilitated by the epithelial to mesenchymal transition (EMT), generating cancer cells with enhanced metastatic capacity and resistance to chemotherapeutics. EMT is known to impart changes in metabolic pathways and mitochondrial function. Here, we show a natural product possesses EMT-specific cytotoxic activity via alterations in metabolic and mitochondrial functions. The fungus-derived sesterterpenoid, ophiobolin A (OpA), possesses nanomolar cytotoxic activity and a high therapeutic index, although its target and mechanism of action remain unknown. Here, we utilized a model of mammary epithelial cells and breast cancer cell lines with and without EMT features to characterize the mechanism of selectivity towards EMT(+) cells by OpA and to identify novel targets for the treatment of EMT-enriched breast cancer. Proteins interacting with OpA in EMT(+) cells were identified through proteomic studies. We utilized trans-mitochondrial cybrids to determine that mitochondria mediate sensitivity to OpA. Furthermore, we report effects on glycolysis, oxidative metabolism, and disruption of metabolite abundance in the TCA cycle. Alterations to mitochondrial iron concentrations and glutathione production, mediated partly by OpA engagement with the metabolic proteins CISD3 and SLC25A40, were also detected in EMT(+) cells. Antioxidant mechanisms are activated by OpA in EMT(+) cells via the NRF2-ARE pathway, verified by decreased cytotoxicity in EMT(+) cells pretreated with the NRF2 activator CDDO. Collectively, we conclude that OpA selectivity toward EMT is mediated by the mitochondria, and at sub-cytotoxic levels, generates a metabolic shift leading to cell death countered by antioxidant mechanisms.

cancer biology↗

bioRxiv2025

In vivo validation of the palmitoylation cycle as a therapeutic target in NRAS-mutant cancer

Ras proteins are functionally dependent on one or more lipid modifications.1,2 The dynamic palmitoylation of N-Ras by DHHC palmitoyl acyltransferases and depalmitoylation by ABHD17 serine hydrolases is essential for the growth of NRAS-mutant acute myeloid leukemia (AML) cells.3-6 Here we show that ABD778, an in vivo-active ABHD17 inhibitor, selectively reduces the growth of NRAS-mutant AML and melanoma cell lines and is synergistic with the MEK inhibitor PD0325901 (PD901; mirdametinib). Mechanistically, ABD778 and PD901 induce deep and durable suppression of mitogen activated protein kinase (MAPK) pathway activation. Co-treatment extended the survival of mice transplanted with NrasG12D AMLs, which acquired by-pass mutations at relapse that conferred drug resistance and restored MAPK activation. ABD778 augmented the anti-leukemia activity of PI3 kinase, pan-Ras tri-complex, and FLT3 inhibitors, and restored gilteritinib sensitivity in a patient-derived xenograft model of FLT3 inhibitor resistance. These studies validate the palmitoylation cycle as a therapeutic target in NRAS-mutant cancers.

cancer biology↗

bioRxiv2025

ABHD11 inhibition drives sterol metabolism to modulate T cell effector function and alleviate autoimmunity

Chronic inflammation in autoimmunity is driven by T cell hyperactivation. This unregulated response to self is fuelled by heightened metabolic programmes, which offers a promising new direction to uncover novel treatment strategies. /{beta}-hydrolase domain-containing protein 11 (ABHD11) is a mitochondrial hydrolase that maintains the catalytic function of -ketoglutarate dehydrogenase (-KGDH), and its expression in CD4+ T cells has been linked to remission status in rheumatoid arthritis (RA). However, the importance of ABHD11 in regulating T cell metabolism and function - and thus, the downstream implication for autoimmunity - is yet to be explored. Here, we show that pharmacological inhibition of ABHD11 dampens cytokine production by human and mouse T cells. Mechanistically, the anti-inflammatory effects of ABHD11 inhibition are attributed to increased 24,25-epoxycholesterol (24,25-EC) biosynthesis and subsequent liver X receptor (LXR) activation, which arise from a compromised TCA cycle. The impaired cytokine profile established by ABHD11 inhibition is extended to two patient cohorts of autoimmunity. Importantly, using a murine model of accelerated type 1 diabetes (T1D), we show that targeting ABHD11 suppresses cytokine production in antigen-specific T cells and delays the onset of diabetes in vivo. Collectively, our work provides pre-clinical evidence that ABHD11 is an encouraging drug target in T cell-mediated autoimmunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/643996v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@bb3fcborg.highwire.dtl.DTLVardef@1594fc8org.highwire.dtl.DTLVardef@84e078org.highwire.dtl.DTLVardef@1ad023e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Proteomic Ligandability Maps of Phosphorus(V) Stereoprobes Iden-tify Covalent TLCD1 Inhibitors

Activity-based protein profiling (ABPP) of stereoisomerically defined sets of electrophilic compounds ( stereoprobes) offers a versatile way to discover covalent ligands for proteins in native biological systems. Here we report the synthesis and chemical proteomic characterization of stereoprobes bearing a P(V)-oxathiaphospholane (OTP) reactive group. ABPP experiments identified numerous proteins in human cancer cells that showed stereoselective reactivity with OTP stereoprobes, and we confirmed several of these liganding events with recombinant proteins. OTP stereoprobes engaging the poorly characterized transmembrane protein TLCD1 impaired the incorporation of monounsaturated fatty acids into phosphatidylethanolamine lipids in cells, a lipidomic phenotype that mirrored genetic disruption of this protein. Using AlphaFold2, we found that TLCD1 structurally resembles the ceramide synthase and fatty acid elongase families of coenzyme A-dependent lipid processing enzymes. This structural similarity included conservation of catalytic histidine residues, the mutation of which blocked the OTP stereoprobe reactivity and lipid remodeling activity of recombinant TLCD1. Taken together, these data indicate that TLCD1 acts as a lipid acyltransferase in cells, and that OTP stereoprobes function as inhibitors of this enzymatic activity. Our findings thus illuminate how the chemical proteomic analysis of electrophilic compounds can facilitate the functional annotation and chemical inhibition of a key lipid metabolic enzyme in human cells.

biochemistry↗

bioRxiv2024

Chemical tools to expand the ligandable proteome: diversity-oriented synthesis-based photoreactive stereoprobes

Chemical proteomics enables the global assessment of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, been limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically-defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these photo-stereoprobes interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible nanoBRET assays. Integrated phenotypic analysis and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and discovering and characterizing bioactive small molecules by cell-based screening.

biochemistry↗

bioRxiv2023

Attenuating ABHD17 enhances S-palmitoylation, membrane localization and signal transduction of NOD2 and Crohn's disease-associated variants.

BACKGROUND AND AIMSNOD2 is an intracellular innate immune receptor that detects bacterial peptidoglycan fragments. Although nominally soluble, some NOD2 is associated with the plasma membrane and endosomal compartments for microbial surveillance. This membrane targeting is achieved through post-translational S- acylation of NOD2 by the protein acyltransferase ZDHHC5. Membrane attachment is necessary to initiate a signaling cascade in response to cytosolic peptidoglycan fragments. Ultimately, this signaling results in the production of antimicrobial peptides and proinflammatory cytokines. In most cases, S-acylation is a reversible post- translational modification with removal of the fatty acyl chain catalyzed by one of several acyl protein thioesterases. Deacylation of NOD2 by such an enzyme will displace it from the plasma membrane and endosomes, thus preventing signaling. METHODSTo identify the enzymes responsible for NOD2 deacylation, we used engineered cell lines with RNA interference and small-molecule inhibitors. These approaches were combined with confocal microscopy, acyl-resin-assisted capture, immunoblotting, and cytokine multiplex assays. RESULTSWe identified /{beta}-hydrolase domain-containing protein 17 isoforms (ABHD17A, ABHD17B, and ABHD17C) as the acyl protein thioesterases responsible for NOD2 deacylation. Inhibiting ABHD17 increased the plasma membrane localization of wild-type NOD2 and a subset of poorly acylated Crohns disease-associated variants. This enhanced NOD2 activity, increasing NF-{kappa}B activation and pro-inflammatory cytokine production in epithelial cells. CONCLUSIONSThese findings demonstrate that ABHD17 isoforms are negative regulators of NOD2. The results also suggest that targeting ABHD17 isoforms could restore functionality to specific Crohns disease-associated NOD2 variants, offering a potential therapeutic strategy. Grant SupportThis work was supported by a Project Grant from the Canadian Institutes of Health Research (grant no.: PJT166010; to G.D.F.), an Innovator Award from the Kenneth Rainin Foundation, and a grant from the National Institutes of Health, (R01CA193994 to B.F.C). A Tier 1 Canada Research Chair supports G.D.F. in Multiomics of Lipids and Innate Immunity. C.L.D. was supported by a Breakthrough Accelerator Fellowship from the Dalhousie Medical Research Foundation/Medical Research Development Office. N.M is a recipient of a graduate scholarship from the I3V Wave and the Dalhousie Medical Research Development Office. DisclosuresThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. SYNOPSISThe peptidoglycan sensor NOD2 requires post-translational S-acylation to associate with cellular membranes and transduce signals. This study identified the ABHD17 family of thioesterases as responsible for NOD2 deacylation and inactivation. Inhibiting or silencing ABHD17 isoforms increases S-acylation and functionality of NOD2 and a subset of Crohns disease-associated variants.

immunology↗

bioRxiv2022

Proteomic discovery of chemical probes that perturb protein complexes in human cells

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

biochemistry↗

bioRxiv2022

Assigning functionality to cysteines by base editing of cancer dependency genes

Chemical probes are lacking for most human proteins. Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cell proliferation. We show that the resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, correctly predicts the essentiality of cysteines targeted by cancer therapeutics and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines on >110 cancer dependency proteins. We further demonstrate how measurements of reactivity in native versus denatured proteomes can discriminate essential cysteines amenable to chemical modification from those buried in protein structures, providing a valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

systems biology↗

bioRxiv2019

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

immunology↗