Biology subjects

Fairn, G. D.

Publications and source records attributed to Fairn, G. D..

13 recordsLinked to original sources

bioRxiv2026

A minimal functional ZDHHC5 defines distinct requirements for NOD2 binding and S-acylation

Protein S-acylation by ZDHHC5 promotes membrane recruitment of the innate immune receptors NOD1 and NOD2. ZDHHC5 contains an extended cytosolic tail implicated in substrate recruitment, accessory-protein interactions, and enzyme trafficking, but its contribution to NOD1 and NOD2 modification remains unclear. Here, we used mutagenesis and complementation of ZDHHC5 knockout cells to define the regions required for NOD association, S-acylation, and plasma membrane localization. Substitution of three S-acylated cysteines in the tail preserved ZDHHC5 localization and its ability to restore NOD2 membrane recruitment. Progressive truncation revealed that most of the cytosolic tail was dispensable, with a construct comprising residues 1-230 retaining activity towards both NOD1 and NOD2. Further truncation to amino acids 1-218 impaired NOD S-acylation and membrane recruitment without eliminating NOD2 association. Thus, substrate association and productive modification have distinct requirements. C-terminal tail removal also differentially affected accessory-protein association: full-length ZDHHC5 co-immunoprecipitated with both GOLGA7A and GOLGA7B, whereas only GOLGA7B was detectably associated with the 1-230 construct. Together, these findings define a minimal functional ZDHHC5 that supports NOD S-acylation and distinguish the requirements for substrate association, catalytic activity, and accessory-protein interactions. They establish that the extended tail is dispensable for NOD modification under the conditions examined, while identifying differential requirements for GOLGA7A and GOLGA7B association with ZDHHC5.

cell biology↗

bioRxiv2026

Quaternary structure and activity of glutamate dehydrogenase are regulated by reversible S-palmitoylation and mitochondrial acyl-protein thioesterases.

Glutamate dehydrogenase (GDH) is a key mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to -ketoglutarate, thereby linking amino acid and carbohydrate metabolism. GDH forms catalytically active hexamers and is regulated by various allosteric modulators, including ADP and GTP. Here, we demonstrate that GDH undergoes auto-palmitoylation in the presence of palmitoyl-CoA, leading to a dose-dependent inhibition of enzymatic activity. Using acyl-PEG exchange assays and mass spectrometry, we show that GDH monomers are predominantly mono-palmitoylated, with modification detected at multiple cysteine residues, including Cys55, Cys115, and Cys197, among the six cysteines in the mature enzyme. Blue Native PAGE analysis revealed that palmitoylation disrupts the native hexameric assembly of mammalian GDH, which is organized as a dimer-of-trimers, promoting dissociation into dimers. Importantly, this modification is reversible, as incubation with mitochondrial acyl-protein thioesterases 1 (APT1) and, to a lesser extent, /{beta} hydrolase domain 10 (ABHD10) restores both the hexameric structure and enzymatic activity. The modified Cys55 residues are positioned near the trimer interface, providing a mechanism by which palmitoylation could prevent hexamer formation, whereas Cys115 and 197 may destabilize individual trimers. These findings establish S-palmitoylation as a novel regulatory mechanism for GDH, linking mitochondrial lipid metabolism to the reversible control of a central metabolic enzyme.

biochemistry↗

bioRxiv2026

Membrane contact site resident PTP1B limits superoxide production by suppressing a Syk-Shc1-Phagocyte Oxidase relay.

Phagocytosis is a specialized endocytic process used by macrophages and dendritic cells to engulf particles, which requires coordinated signaling cascades, cytoskeletal remodeling, and assembly of antimicrobial machinery to eliminate pathogens. During Fc {gamma} receptor (Fc{gamma}R)-mediated phagocytosis, dynamic actin depolymerization at the base of the phagocytic cup creates permissive conditions for endoplasmic reticulum-plasma membrane (ER-PM) membrane contact sites (MCS) to form. We demonstrate that the ER-resident protein tyrosine phosphatase PTP1B localizes to newly formed or expanded ER-PM MCS during phagocytosis and dephosphorylates Syk. Using TIRF microscopy with MCS residents, including MAPPER, STIM1, and E-Syts, we show that actin clearance allows ER proteins to approach the plasma membrane. PTP1B colocalizes with Fc{gamma}Rs in actin-cleared zones and physically interacts with Syk, a critical mediator of phagocytic signaling. Loss of PTP1B led to sustained Syk hyperphosphorylation without affecting phagocytosis. However, the PTP1B-deficient cells showed a {asymp}3-fold increase in NADPH oxidase 2 (NOX2)-mediated superoxide production. Using unbiased proteomics, we identified the adapter protein Shc1 as a critical intermediate linking Syk phosphorylation to NOX2 activation. Shc1 phosphorylation during phagocytosis is dependent on Src family kinases and Syk, while genetic ablation of SHC1 reduced superoxide production by {asymp}40%. Proximity ligation assays reveal enhanced Shc1-p47phox interactions in PTP1B-deficient cells during phagocytosis. These findings establish an SFK-Syk-Shc1-NOX2 signaling axis that PTP1B negatively regulates at MCS between the ER and the forming phagosome, providing new mechanistic insights into antimicrobial responses during phagocytosis.

cell biology↗

bioRxiv2025

Acyl-protein thioesterase 1 (LYPLA1) activity promotes the growth of MDA-MB-468 triple-negative breast cancer cells.

Protein S-acylation is a lipid-based, often reversible post-translational modification that can regulate many aspects of protein behavior, including subcellular localization, protein-interactions, and activity. Emerging evidence has identified roles for individual protein acyltransferases encoded by the ZDHHC in cancers, yet the roles of de-S-acylation enzymes are less clear. Recent evidence suggests that acyl-protein thioesterase (APT1)/LYPLA1 can impact epithelial-mesenchymal transition and metastasis. This study integrates patient datasets, CRISPR dependency data, and in vitro assays to find APT1 as a context-dependent vulnerability in triple-negative breast cancer (TNBC). Despite the highest protein abundance in luminal MCF7 cells, basal-like MDA-MB-468 cells exhibited the most prominent specific APT1 activity, reflecting subtype-specific regulation. Inhibition of APT1 with ML348 increased S-acylation of nuclear and mitochondrial proteins without altering global acylation. Functionally, APT1 inhibition reduced cell proliferation while inducing minimal apoptosis, consistent with cytostatic growth arrest. Cell-cycle analysis revealed G1 accumulation and reduced S/G2 transition, linking proteomic changes to impaired replication. These findings establish APT1 as a regulator of TNBC proliferation through dynamic de-S-acylation of cell-cycle and mitochondrial proteins, highlighting it as a potential therapeutic vulnerability in aggressive breast cancers.

cancer biology↗

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

Apolipoprotein A1 and high-density lipoprotein limit low-density lipoprotein transcytosis by binding scavenger receptor B1

Atherosclerosis results from the deposition and oxidation of low-density lipoprotein (LDL) and immune cell infiltration in the sub-arterial space leading to arterial occlusion. Numerous studies have shown that transcytosis transports circulating LDL across endothelial cells lining the blood vessels. LDL transcytosis is initiated by binding to either Scavenger Receptor B1 (SR-B1) or Activin A receptor-like kinase 1 (ALK1) on the apical side of endothelial cells leading to its transit and release on the basolateral side. Individuals with elevated levels of circulating high-density lipoprotein (HDL) are partly protected from atherosclerosis due to its ability to remove excess cholesterol and act as an antioxidant. Apolipoprotein A1 (APOA1), an HDL constituent, can bind to SR-B1, raising the possibility that APOA1/HDL may also compete with LDL for SR-B1 binding and thereby limit LDL deposition in the sub-arterial space. To examine this possibility, we used in vitro approaches to quantify the internalization and transcytosis of fluorescent LDL in coronary endothelial cells. Using microscale thermophoresis and affinity capture, we find that SR-B1 and APOA1 directly interact, and that binding is enhanced when using the cardioprotective variant of APOA1 termed Milano (APOA1-Milano). In a murine model, transiently increasing the levels of HDL reduced the acute deposition of fluorescently labeled LDL in the atheroprone inner curvature of the aorta. Reduced LDL deposition was also observed when increasing circulating wild-type APOA1 or the APOA1-Milano variant, with a more robust inhibition from the APOA1-Milano. The results suggest that HDL may limit SR-B1-mediated LDL transcytosis and deposition, adding to the mechanisms by which it can act as an atheroprotective particle.

biochemistry↗

bioRxiv2022

HER2 expression defines unique requirements for flotillin and c-Src for EGFR signaling

The epidermal growth factor receptor (EGFR) controls many cellular functions. Upon binding its ligand, the receptor undergoes dimerization, phosphorylation, and activation of signals including the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. While some studies indicated that EGFR signaling may be controlled by signal enrichment within membrane raft nanodomains, others have found a limited effect of membrane raft disruption on EGFR signaling, suggesting that specific factor(s) may define context-specific control of EGFR signaling by membrane rafts. Ligand-bound EGFR can homodimerize, or instead undergo heterodimerization with the related receptor HER2 when the latter is expressed. We examined how EGFR signaling in the presence of HER2 distinctly requires membrane raft nanodomains. Induction of HER2 expression altered EGFR signaling duration consistent with EGFR/HER2 heterodimer formation. EGFR and c-Src localized within plasma membrane structures demarked by flotillin, a membrane raft protein, selectively in HER2-expressing cells. Consistently, HER2-expressing cells, but not cells lacking HER2, were dependent on flotillin and c-Src for EGFR signaling leading to Akt activation and cell proliferation. Hence, HER2 expression establishes the requirement of EGFR signaling for flotillin membrane rafts and c-Src, leading to Akt activation. Summary statementThe role of membrane rafts in EGFR signaling may be context-specific. We find that the related receptor HER2 defines unique signaling requirements for EGFR for membrane rafts, flotillin, and c-Src.

cell biology↗

bioRxiv2022

PtdIns(3,4)P2, Lamellipodin, and VASP coordinate cytoskeletal remodeling during phagocytic cup formation in macrophages.

Phosphoinositides are pivotal regulators of vesicular traffic and signaling during phagocytosis. Phagosome formation, the initial step of the process, is characterized by local membrane remodelling and reorganization of the actin cytoskeleton that leads to formation of the pseudopods that drive particle engulfment. Using genetically-encoded fluorescent probes we found that upon particle engagement a localized pool of PtdIns(3,4)P2 is generated by the sequential activities of class I phosphoinositide 3-kinases and phosphoinositide 5-phosphatases. Depletion of the enzymes responsible for this locally generated pool of PtdIns(3,4)P2 blocks pseudopod progression and ultimately phagocytosis. We show that the PtdIns(3,4)P2 effector Lamellipodin (Lpd) is recruited to nascent phagosomes by PtdIns(3,4)P2. Furthermore, we show that silencing of Lpd inhibits phagocytosis and produces aberrant pseudopodia with disorganized actin filaments. Lastly, vasodilator-stimulated phosphoprotein (VASP) was identified as a key actin-regulatory protein mediating phagosome formation downstream of Lpd. Mechanistically, our findings imply that a pathway involving PtdIns(3,4)P2, Lpd and VASP mediates phagocytosis at the stage of particle engulfment.

cell biology↗

bioRxiv2022

Confinement of unliganded EGFR by tetraspanin nanodomains gates EGFR ligand binding and signaling

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology. EGFR is activated by ligand binding, triggering receptor dimerization, activation of kinase activity, and intracellular signaling. EGFR is transiently confined within various plasma membrane nanodomains, yet how this may contribute to regulation of EGFR ligand binding is poorly understood. To resolve how EGFR nanoscale compartmentalization gates ligand binding, we developed single-particle tracking methods to track the mobility of ligand-bound and total EGFR, in combination with modeling of EGFR ligand binding. In comparison to unliganded EGFR, ligand-bound EGFR was more confined and distinctly regulated by clathrin and tetraspanin nanodomains. Ligand binding to unliganded EGFR occurred preferentially in tetraspanin nanodomains, and disruption of tetraspanin nanodomains impaired EGFR ligand binding and altered the conformation of the receptors ectodomain. We thus reveal a novel mechanism by which EGFR confinement within tetraspanin nanodomains regulates receptor signaling at the level of ligand binding.

cell biology↗

bioRxiv2021

Anisotropy of plasmalemmal sterols and cell mating require StARkin domain proteins Ysp2 and Lam4.

In the budding yeast S. cerevisiae Cdc42 is required for polarized growth and the formation of mating projections (shmoos). Negatively charged lipids including phosphatidylserine and phosphatidylinositol 4,5-bisphosphate support a positive feedback loop that recruits Cdc42 effectors and MAP kinase scaffolds, many of which contain polybasic patches that directly interact with the membrane. Here, using genetically encoded sterol sensor ALOD4 we find that ergosterol is accumulated in the cytosolic leaflet of buds and shmoos. The accumulation of ergosterol in the plasma membrane requires both Osh and Lam proteins however cells lacking Ysp2/Lam2 and Lam4 displayed a reversal in the polarity of ergosterol. The redistribution of ergosterol impairs the polarization of phosphatidylserine and phosphatidylinositol 4,5-bisphophate which further impacts shmoo formation, MAPK signaling and mating efficiency. Our observations demonstrate that the ability of Lam proteins to deliver ergosterol from the plasma membrane to the ER helps maintain a gradient of ergosterol which in turn supports robust cell polarity. SummaryThe sterol sensor ALOD4 is enriched at sites of polarized growth. Elimination of the Osh proteins solubilized the ALOD4 whereas elimination of Ysp2 and Lam4 reversed ALOD4 polarization. Cells lacking Ysp2 and Lam4 have defects in mating and MAP kinase signaling.

cell biology↗

bioRxiv2021

Phosphatidylinositol 3-kinase-independent synthesis of PtdIns(3,4)P2 by a phosphotransferase

Despite their comparatively low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival, and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival within host organisms. We demonstrate that PtdIns(3,4)P2 is generated in host cells by effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signaling to gain entry and even provoke oncogenic transformation.

cell biology↗

bioRxiv2021

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease

The COVID-19 pandemic has led to an urgent need for the identification of new antiviral drug therapies that can be rapidly deployed to treat patients with this disease. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of COVID-19. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cellbased experimental assessment reveals several clinically-relevant repurposing drug candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

cell biology↗

bioRxiv2020

Fatty Acid Synthase inhibition prevents palmitoylation of SARS-CoV2 SpikeProtein and improves survival of mice infected with murine hepatitis virus.

The Spike protein of SARS-CoV2 and other coronaviruses mediate host cell entry and are S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes of the ZDHHC family have been reported to modify Spike proteins post-translationally. Using resin-assisted capture mass spectrometry, we demonstrate that the Spike protein is S-acylated in SARS-CoV2 infected human and monkey cells. We further show that increased abundance of the human acyltransferase ZDHHC5 results in increased S-acylation of the SARS-CoV2 Spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also find that the S-acylation of the Spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also found on the immunocaptured protein. Yet, ZDHHC5 was highly selective for palmitate, suggesting that other ZDHHC enzymes mediated the incorporation of other fatty acyl chains. Thus, since multiple ZDHHC isoforms may modify the Spike protein, we examined the ability of the fatty acid synthase inhibitor TVB-3166 to prevent the S-acylation of the Spike proteins of SARS-CoV-2 and human CoV-229E. Treating cells with TVB-3166 inhibited S-acylation of ectopically expressed SARS-CoV2 Spike and attenuated the ability of SARS-CoV2 and human CoV-229E to spread in vitro. Additionally, treatment of mice with a comparatively low dose of TVB-3166 promoted survival from an otherwise fatal murine coronavirus infection. Our findings further substantiate the necessity of CoV Spike protein S-acylation and the potential use of fatty acid synthase inhibitors.

cell biology↗