Straight A's: protein acylation in the S-activation and autophagic degradation of NOD-like receptors.

Over the past decade, S-acylation has emerged as a crucial regulator of several innate immune signaling pathways, with new insights continually being gained. S-acylation, a reversible post-translational modification, involves the attachment of fatty acyl chains to cysteine residues, influencing protein localization, function, and stability. In this mini-review, we examine the accumulating evidence of the role of S-acylation in regulating nucleotide oligomerization domain (NOD)-like receptors.

Effect of statins on IL-10 signaling and production by chronic lymphocytic leukemia cells.

The cytokine-signaling inhibitor ruxolitinib causes disease flares of chronic lymphocytic leukemia (CLL). This tumor-promoting activity correlates with its ability to inhibit interleukin (IL)-10 production by CLL B cells that have been activated with IL-2 and the Toll-like receptor 7 (TLR7) agonist resiquimod (called 2S cells) in vitro. In TLR-activated normal human B cells, IL-10 production is regulated by cholesterol biosynthesis and can be inhibited by statins.

Sphingosine-1-phosphate signalling activates E-Syt1 to facilitate HDL-derived cholesterol transport.

Cholesterol derived from high-density lipoprotein (HDL) is rapidly redistributed to intracellular compartments in steroidogenic and bile-producing cells, but the molecular mechanisms governing this essential transport process remain poorly understood. Here we uncover a signalling cascade coordinating HDL-derived cholesterol transport through membrane contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM). We find that HDL-resident sphingosine-1-phosphate (S1P) activates S1P receptor 3 and its associated G protein αq, leading to phospholipase-C-β3-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate and an elevation in cytosolic calcium.

Attenuating ABHD17 Isoforms Augments the S-acylation and Function of NOD2 and a Subset of Crohn's Disease-associated NOD2 Variants.

Background & Aims: NOD2 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.

Caspase-8: Arbitrating Life and Death in the Innate Immune System.

The canonical function of caspase-8 is to control timely cellular apoptosis to maintain tissue homeostasis and clear dysfunctional cells; however, emerging findings reveal novel, non-canonical roles of caspase in addition to regulating cellular apoptosis, including inflammatory response regulation, immune function, and cell differentiation. Furthermore, the functional versatility of caspase-8 is reported to be contingent on the presence and dimerization of various isoforms, which are produced through alternative splicing, altering its function and protein-protein interactions. Equally important are post-translational modifications, including phosphorylation and ubiquitination, which can act as a nexus to control caspase-8 activity and cellular localization.

Using the Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis.

Macrophages are known for engulfing and digesting pathogens and dead cells through a specialized form of endocytosis called phagocytosis. Unfortunately, many macrophage cell lines are refractory to most reagents used for transient transfections. Alternative transient approaches, such as electroporation or transduction with lentiviral vectors, typically cause cell death (electroporation) or can be time-consuming to generate numerous lentivirus when using different genes of interest.

acylation of PNPLA2/ATGL: a necessity for triacylglycerol lipolysis and lipophagy in hepatocytes.

The intricate balance between lipolysis and lipophagy in cellular lipid homeostasis has fascinated researchers for years. A growing body of evidence highlights the critical roles of PNPLA2/ATGL (patatin like phospholipase domain containing 2) in both lipolysis and lipophagy. Here, we discuss our recent study, which revealed that PNPLA2 must be S-acylated on Cys15 for its robust catalytic activity.

S-acylation of ATGL is required for lipid droplet homoeostasis in hepatocytes.

Lipid droplets (LDs) are organelles specialized in the storage of neutral lipids, cholesterol esters and triglycerides, thereby protecting cells from the toxicity of excess lipids while allowing for the mobilization of lipids in times of nutrient deprivation. Defects in LD function are associated with many diseases. S-acylation mediated by zDHHC acyltransferases modifies thousands of proteins, yet the physiological impact of this post-translational modification on individual proteins is poorly understood.

Phosphatidylinositol 3,4-bisphosphate: Out of the shadows and into the spotlight.

Phosphoinositide 3-kinases regulate many cellular functions, including migration, growth, proliferation, and cell survival. Early studies equated the inhibition of Class I PI3Ks with loss of; phosphatidylinositol 3,4,5-trisphosphate (PIP3), but over time, it was realised that these; treatments also depleted phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). In recent years, the; use of better tools and an improved understanding of its metabolism have allowed for the; identification of specific roles of PI(3,4)P2.

Apolipoprotein A1 and high-density lipoprotein limit low-density lipoprotein transcytosis by binding SR-B1.

Atherosclerosis results from the deposition and oxidation of LDL and immune cell infiltration in the sub-arterial space leading to arterial occlusion. Studies have shown that transcytosis transports circulating LDL across endothelial cells lining blood vessels. LDL transcytosis is initiated by binding to either scavenger receptor B1 (SR-B1) or activin A receptor-like kinase 1 on the apical side of endothelial cells leading to its transit and release on the basolateral side.

Attenuating ABHD17 isoforms augments the -acylation and function of NOD2 and a subset of Crohn's disease-associated NOD2 variants.

Background And Aims: NOD2 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 -acylation of NOD2 by the protein acyltransferase ZDHHC5.

Multifaceted roles and regulation of nucleotide-binding oligomerization domain containing proteins.

Nucleotide-binding oligomerization domain-containing proteins, NOD1 and NOD2, are cytosolic receptors that recognize dipeptides and tripeptides derived from the bacterial cell wall component peptidoglycan (PGN). During the past two decades, studies have revealed several roles for NODs beyond detecting PGN fragments, including activation of an innate immune anti-viral response, NOD-mediated autophagy, and ER stress induced inflammation. Recent studies have also clarified the dynamic regulation of NODs at cellular membranes to generate specific and balanced immune responses.

Super-Resolution Spinning-Disk Confocal Microscopy Using Optical Photon Reassignment (SoRa) to Visualize the Actin Cytoskeleton in Macrophages.

Macrophages are motile, morphologically plastic cells that undergo substantial cytoskeletal remodeling to facilitate their roles in innate and adaptive immunity. Macrophages are adept at producing a variety of specialized actin-driven structures and processes including the formation of podosomes and the ability to engulf particles through phagocytosis and sample large amounts of extracellular fluid via micropinocytosis. Here, we describe techniques for immunostaining proteins and transfecting macrophages with plasmids for use with either fixed or live cell imaging.

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.

HER2 expression defines unique requirements for flotillin and c-Src in 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 phosphoinositide-3-kinase (PI3K)-Akt pathway. Although some studies have indicated that EGFR signaling may be controlled by signal enrichment within various membrane rafts, such as flotillin nanodomains, others have found a limited effect of disruption of these nanodomains on EGFR signaling, suggesting that specific factors may define context-specific control of EGFR signaling.

Endoplasmic reticulum-Phagosome contact sites from the cradle to the grave.

Phagocytosis is a key component of the innate immune system used to ingest apoptotic cells and microorganisms for their destruction and recycling of macromolecules and the presentation of antigens to adaptive immune system cells. The newly formed vacuole or nascent phagosome undergoes a maturation process reminiscent of the classical endocytic maturation process, reaching a highly degradative phagolysosome stage before its tubulovesicular breakdown into lysosomes. The process is highly regulated and can be disrupted by various pathogenic organisms.

PtdIns(3,4)P2, Lamellipodin, and VASP coordinate actin dynamics during phagocytosis 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 remodeling 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.

FASN inhibitor TVB-3166 prevents S-acylation of the spike protein of human coronaviruses.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses mediates host cell entry and is S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes have been reported to post-translationally modify spike proteins; however, strategies to exploit this modification are lacking. Using resin-assisted capture MS, we demonstrate that the spike protein is S-acylated in SARS-CoV-2-infected human and monkey epithelial cells.

DJ-1 binds to Rubicon to Impair LC-3 Associated Phagocytosis.

The ability to effectively clear infection is fundamental to host survival. Sepsis, defined as dysregulated host response to infection, is a heterogenous clinical syndrome that does not uniformly clear intact bacterial or sterile infection (i.e.

Kinase-independent synthesis of 3-phosphorylated phosphoinositides by a phosphotransferase.

Despite their low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P and PtdIns(3,4)P 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 in host organisms.

Fyn and TOM1L1 are recruited to clathrin-coated pits and regulate Akt signaling.

The epidermal growth factor (EGF) receptor (EGFR) controls many aspects of cell physiology. EGF binding to EGFR elicits the membrane recruitment and activation of phosphatidylinositol-3-kinase, leading to Akt phosphorylation and activation. Concomitantly, EGFR is recruited to clathrin-coated pits (CCPs), eventually leading to receptor endocytosis.

Monitoring Transmembrane and Peripheral Membrane Protein Interactions by Förster Resonance Energy Transfer Using Fluorescence Lifetime Imaging Microscopy.

Caveolae are bulb-shaped invaginations of the plasma membrane that are enriched in specific lipids including cholesterol, phosphatidylserine and sphingolipids. Caveolae have many described cellular roles and functions, including endocytic transport, transcytosis, mechanosensing, and serving as a buffer against plasmalemmal stress. Caveola are formed through interactions between integral membrane proteins (Caveolin) and a cavin family of peripheral proteins (Cavins).

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

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases.