Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation.

Protein S-acylation is a reversible post-translational modification that modulates the localization and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC (Asp-His-His-Cys) domain-containing (zDHHC) proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyze S-acylation in a two-step process involving "autoacylation" of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. Here, we developed and implemented a novel FRET-based high-throughput assay for the discovery of compounds that interfere with autoacylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. Our screen of >350,000 compounds identified two related tetrazole-containing compounds (TTZ-1 and TTZ-2) that inhibited both zDHHC2 autoacylation and substrate S-acylation in cell-free systems. These compounds were also active in human embryonic kidney 293T cells, where they inhibited the S-acylation of two substrates (SNAP25 and PSD95 [postsynaptic density protein 95]) mediated by different zDHHC enzymes, with some apparent isoform selectivity. Furthermore, we confirmed activity of the hit compounds through resynthesis, which provided sufficient quantities of material for further investigations. The assays developed provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating cellular activities of zDHHC enzymes in S-acylation.

Mechanisms and inhibition of Porcupine-mediated Wnt acylation.

Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis1-3, and aberrant Wnt signalling is frequently associated with cancers4. Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine5-7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling8,9. Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials10; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.

Colonic Medium-Chain Fatty Acids Act as a Source of Energy and for Colon Maintenance but Are Not Utilized to Acylate Ghrelin.

The capacity of microbiota to produce medium-chain fatty acids (MCFA) and related consequences for the gastrointestinal (GI) tract have never been reported before. We verified the impact of nutrition-related factors on fatty acid (FAs) production and found that caloric restriction decreased levels of most of MCFAs in the mouse cecum, whereas overnight fasting reduced the levels of acetate and butyrate but increased propionate and laurate. A diet high in soluble fibre boosted the production of short-chain fatty acids (SCFA) and caproate whereas a high-cellulose diet did not have an effect or decreased the levels of some of the FAs. Rectal infusion of caprylate resulted in its rapid metabolism for energy production. Repeated 10-day MCFA infusion impacted epididymal white adipose tissue (eWAT) weight and lipid accumulation. Repeated infusion of caprylate rectally tended to increase the concentration of active ghrelin in mice plasma; however, this increase was not statistically significant. In Caco-2 cells, caprylate increased the expression of Fabp2, Pdk4, Tlr3, and Gpr40 genes as well as counteracted TNFα-triggered downregulation of Pparγ, Occludin, and Zonulin mRNA expression. In conclusion, we show that colonic MCFAs can be rapidly utilized as a source of energy or stored as a lipid supply. Further, locally produced caprylate may impact metabolism and inflammatory parameters in the colon.

Development of an Acrylamide-Based Inhibitor of Protein S-Acylation.

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.

Acylation of the Incretin Peptide Exendin-4 Directly Impacts Glucagon-Like Peptide-1 Receptor Signaling and Trafficking.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and ß-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed ∼2.5-fold bias toward G protein recruitment and a ∼60% reduction in ß-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a ∼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. SIGNIFICANCE STATEMENT: Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.

High Fat Activates O-GlcNAcylation and Affects AMPK/ACC Pathway to Regulate Lipid Metabolism.

A high-fat diet often leads to excessive fat deposition and adversely affects the organism. However, the mechanism of liver fat deposition induced by high fat is still unclear. Therefore, this study aimed at acetyl-CoA carboxylase (ACC) to explore the mechanism of excessive liver deposition induced by high fat. In the present study, the ORF of ACC1 and ACC2 were cloned and characterized. Meanwhile, the mRNA and protein of ACC1 and ACC2 were increased in liver fed with a high-fat diet (HFD) or in hepatocytes incubated with oleic acid (OA). The phosphorylation of ACC was also decreased in hepatocytes incubated with OA. Moreover, AICAR dramatically improved the phosphorylation of ACC, and OA significantly inhibited the phosphorylation of the AMPK/ACC pathway. Further experiments showed that OA increased global O-GlcNAcylation and agonist of O-GlcNAcylation significantly inhibited the phosphorylation of AMPK and ACC. Importantly, the disorder of lipid metabolism caused by HFD or OA could be rescued by treating CP-640186, the dual inhibitor of ACC1 and ACC2. These observations suggested that high fat may activate O-GlcNAcylation and affect the AMPK/ACC pathway to regulate lipid synthesis, and also emphasized the importance of the role of ACC in lipid homeostasis.

Protein acylation by saturated very long chain fatty acids and endocytosis are involved in necroptosis.

Necroptosis is a form of cell death characterized by receptor-interacting protein kinase activity and plasma membrane permeabilization via mixed-lineage kinase-like protein (MLKL). This permeabilization is responsible for the inflammatory properties of necroptosis. We previously showed that very long chain fatty acids (VLCFAs) are functionally involved in necroptosis, potentially through protein fatty acylation. Here, we define the scope of protein acylation by saturated VLCFAs during necroptosis. We show that MLKL and phosphoMLKL, key for membrane permeabilization, are exclusively acylated during necroptosis. Reducing the levels of VLCFAs decreases their membrane recruitment, suggesting that acylation by VLCFAs contributes to their membrane localization. Acylation of phosphoMLKL occurs downstream of phosphorylation and oligomerization and appears to be, in part, mediated by ZDHHC5 (a palmitoyl transferase). We also show that disruption of endosomal trafficking increases cell viability during necroptosis, possibly by preventing recruitment, or removal, of phosphoMLKL from the plasma membrane.

Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation.

Although cancer cells are frequently faced with a nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anticancer strategy. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) is downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low-glucose conditions. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo uridine triphosphate synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation, promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation, counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.

Emerging roles of protein O-GlcNAcylation in cardiovascular diseases: Insights and novel therapeutic targets.

Cardiovascular diseases (CVDs) are the leading cause of death globally. While the major focus of pharmacological and non-pharmacological interventions has been on targeting disease pathophysiology and limiting predisposing factors, our understanding of the cellular and molecular mechanisms underlying the pathogenesis of CVDs remains incomplete. One mechanism that has recently emerged is protein O-GlcNAcylation. This is a dynamic, site-specific reversible post-translational modification of serine and threonine residues on target proteins and is controlled by two enzymes: O-linked ß-N-acetylglucosamine transferase (OGT) and O-linked ß-N-acetylglucosaminidase (OGA). Protein O-GlcNAcylation alters the cellular functions of these target proteins which play vital roles in pathways that modulate vascular homeostasis and cardiac function. Through this review, we aim to give insights on the role of protein O-GlcNAcylation in cardiovascular diseases and identify potential therapeutic targets in this pathway for development of more effective medicines to improve patient outcomes.

O-linked N-acetylglucosamine transferase (OGT) regulates pancreatic α-cell function in mice.

The nutrient sensor O-GlcNAc transferase (OGT) catalyzes posttranslational addition of O-GlcNAc onto target proteins, influencing signaling pathways in response to cellular nutrient levels. OGT is highly expressed in pancreatic glucagon-secreting cells (α-cells), which secrete glucagon in response to hypoglycemia. The objective of this study was to determine whether OGT is necessary for the regulation of α-cell mass and function in vivo. We utilized genetic manipulation to produce two α-cell specific OGT-knockout models: a constitutive glucagon-Cre (αOGTKO) and an inducible glucagon-Cre (i-αOGTKO), which effectively delete OGT in α-cells. Using approaches including immunoblotting, immunofluorescent imaging, and metabolic phenotyping in vivo, we provide the first insight on the role of O-GlcNAcylation in α-cell mass and function. αOGTKO mice demonstrated normal glucose tolerance and insulin sensitivity but displayed significantly lower glucagon levels during both fed and fasted states. αOGTKO mice exhibited significantly lower α-cell glucagon content and α-cell mass at 6 months of age. In fasting, αOGTKO mice showed impaired pyruvate stimulated gluconeogenesis in vivo and reduced glucagon secretion in vitro. i-αOGTKO mice showed similarly reduced blood glucagon levels, defective in vitro glucagon secretion, and normal α-cell mass. Interestingly, both αOGTKO and i-αOGTKO mice had no deficiency in maintaining blood glucose homeostasis under fed or fasting conditions, despite impairment in α-cell mass and function, and glucagon content. In conclusion, these studies provide a first look at the role of OGT signaling in the α-cell, its effect on α-cell mass, and its importance in regulating glucagon secretion in hypoglycemic conditions.

Preparation of acylated pectin with gallic acid through enzymatic method and their emulsifying properties, antioxidation activities and antibacterial activities.

In this study, native pectin (Na-Pe) was acylated with gallic acid through enzymatic method. UV-Vis, Fourier transform infrared spectroscopy and proton NMR analyses demonstrated that the phenolic hydroxyl group on gallic acid attacked the carbomethoxy of Na-Pe and replaced the methoxy group to form a new ester group under catalysis. The galloyl content of acylated pectin prepared via 24-h reaction (Ac1-Pe) was 16.8%, while that prepared via 48-h reaction (Ac2-Pe) reached 20.7%. The emulsifying properties, antioxidation activities and antibacterial activities of acylated pectin was significantly improved compared with those of Na-Pe. The emulsion activity and emulsion stability of the pectin emulsion improved from 1.08% and 56.13% (Na-Pe) to 1.57% and 88.27% (Ac1-Pe) and 1.71% and 93.3% (Ac2-Pe), respectively. The DPPH clearance of the pectin improved from 2.68% (Na-Pe) to 68.92% (Ac1-Pe) and 76.98% (Ac2-Pe) and the inhibition ratio in the ß-carotene bleaching assay of the pectin increased from 3.15% (Na-Pe) to 73.02% (Ac1-Pe) and 78.96% (Ac2-Pe). The inhibition rate of the pectin against Escherichia coli and Staphylococcus aureus also improved from 2.93% and 8.92% (Na-Pe) to 26.95% and 42.18% (Ac1-Pe) and 31.56% and 47.87% (Ac2-Pe), respectively.

Elevated O-GlcNAcylation Promotes Malignant Phenotypes of Hypopharyngeal Squamous Cell Carcinoma by Stabilizing Nrf2 through Regulation of the PI3K/Akt Pathway.

BACKGROUND AND PURPOSE: O-GlcNAcylation is a significant protein posttranslational modification with O-linked ß-N-acetylglucosamine (GlcNAc) for intracellular signaling. Elevated O-GlcNAcylation contributes to cell proliferation, cell migration, cell apoptosis and signal transduction in various cancers. However, the expression level and functional role of O-GlcNAcylation in Hypopharyngeal Squamous Cell Carcinoma (HSCC) is not clearly elucidated. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a master transcriptional factor that has been found to be aberrantly activated in HSCC. Here, we provide a molecular rationale between O-GlcNAcylation and Nrf2 in HSCC patients. METHODS: The protein levels of O-GlcNAcylation and Nrf2 in HSCC tissues were detected by immunohistochemistry technique and western blot analysis. Then, O-GlcNAcylation knockdown HSCC cells were applied in this study. Cell proliferation was detected by CCK8, colony-forming analysis, and cell cycle assays. Cell migration and invasion ability was evaluated by transwell assays. Cell apoptosis was measured by TUNEL analysis. RESULTS: O-GlcNAcylation was obviously up-regulated in HSCC tissues, which correlated with tumor size and lymph node metastasis. In addition, the protein level of Nrf2 was found to positively correlate with the expression of O-GlcNAcylation both in vivo and in vitro. Knockdown of O-GlcNAcylation significantly inhibited HSCC cell growth, suppressed cell migration, and promoted cell apoptosis, whereas overexpression of Nrf2 reversed these phenotypes. Mechanismly, the upregulation of O-GlcNAcylation promoted the phosphorylation of Akt, leading to the stabilization of Nrf2; this could be attenuated by inhibition of the PI3K/Akt signaling pathway. CONCLUSION: Here, we provide a molecular association between O-GlcNAcylation and Nrf2 in HSCC patients, thus providing valuable therapeutic targets for the disease.

Blockage of O-linked GlcNAcylation induces AMPK-dependent autophagy in bladder cancer cells.

BACKGROUND: High levels of the post-translational modification O-GlcNAcylation (O-GlcNAc) are found in multiple cancers, including bladder cancer. Autophagy, which can be induced by stress from post-translational modifications, plays a critical role in maintaining cellular homeostasis and regulating tumorigenesis. The impact of O-GlcNAcylation on autophagy in bladder cancer remains unclear. Here, we evaluate the change in autophagic activity in response to O-GlcNAcylation and explore the potential mechanisms. METHODS: O-GlcNAcylation levels in bladder cancer cells were altered through pharmacological or genetic manipulations: treating with 6-diazo-5-oxo-norleucine (DON) or thiamet-G (TG) or up- and downregulation of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA). Autophagy was determined using fluorescence microscopy and western blotting. Co-immunoprecipitation (Co-IP) assays were performed to evaluate whether the autophagy regulator AMP-activated protein kinase (AMPK) was O-GlcNAc modified. RESULTS: Cellular autophagic flux was strikingly enhanced as a result of O-GlcNAcylation suppression, whereas it decreased at high O-GlcNAcylation levels. Phosphorylation of AMPK increased after the suppression of O-GlcNAcylation. We found that O-GlcNAcylation of AMPK suppressed the activity of this regulator, thereby inhibiting ULK1 activity and autophagy. CONCLUSION: We characterized a new function of O-GlcNAcylation in the suppression of autophagy via regulation of AMPK. GRAPHICAL ABSTRACT: Blockage of O-linked GlcNAcylation induces AMPK dependent autophagy in bladder cancer cells.

S-acylation modulates the function of the apical sodium-dependent bile acid transporter in human cells.

The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids. ASBT function is rapidly regulated by several posttranslational modifications. One reversible posttranslational modification is S-acylation, involving the covalent attachment of fatty acids to cysteine residues in proteins. However, whether S-acylation affects ASBT function and membrane expression has not been determined. Using the acyl resin-assisted capture method, we found that the majority of ASBT (∼80%) was S-acylated in ileal brush border membrane vesicles from human organ donors, as well as in HEK293 cells stably transfected with ASBT (2BT cells). Metabolic labeling with alkyne-palmitic acid (100 µm for 15 h) also showed that ASBT is S-acylated in 2BT cells. Incubation with the acyltransferase inhibitor 2-bromopalmitate (25 µm for 15 h) significantly reduced ASBT S-acylation, function, and levels on the plasma membrane. Treatment of 2BT cells with saturated palmitic acid (100 µm for 15 h) increased ASBT function, whereas treatment with unsaturated oleic acid significantly reduced ASBT function. Metabolic labeling with alkyne-oleic acid (100 µm for 15 h) revealed that oleic acid attaches to ASBT, suggesting that unsaturated fatty acids may decrease ASBT's function via a direct covalent interaction with ASBT. We also identified Cys-314 as a potential S-acylation site. In conclusion, these results provide evidence that S-acylation is involved in the modulation of ASBT function. These findings underscore the potential for unsaturated fatty acids to reduce ASBT function, which may be useful in disorders in which bile acid toxicity is implicated.

Relationship between metabolic syndrome and acylated/desacylated ghrelin ratio in patients with schizophrenia under olanzapine medication.

BACKGROUND: Ghrelin is a peptide hormone that mediates glucose homeostasis and lipid metabolism. Acylated ghrelin (AG) and desacylated ghrelin (DAG) are the two main forms of ghrelin, which have opposing roles in energy homeostasis. The AG/DAG ratio has been proposed to be associated with metabolic syndrome (MetS) in the general population. This study compared the relationships between MetS and ghrelin parameters in patients with schizophrenia. METHODS: Patients diagnosed with schizophrenia and under olanzapine monotherapy were recruited. Fasting blood samples were collected for the analyses of metabolic and ghrelin parameters. The serum levels of total ghrelin and AG were measured by enzyme-linked immunosorbent assay kits. DAG level was calculated by subtracting the AG level from the total ghrelin level. RESULTS: We recruited 151 subjects with schizophrenia, and classified them into those with MetS (n = 41) and those without MetS (n = 110). Subjects with MetS had a significantly higher AG/DAG ratio, as well as lower total ghrelin and DAG levels. There were no sex differences in ghrelin parameters. The AG/DAG ratio was significantly and positively correlated with weight, body mass index, waist circumference, insulin level, homeostasis model assessment of insulin resistance and number of MetS components. Multiple linear regression analysis indicated that the number of MetS components remained significantly associated with the AG/DAG ratio. CONCLUSIONS: Our results revealed that lower AG/DAG ratios were associated with better metabolic profiles in olanzapine-treated patients with schizophrenia. These observations suggest that the balance between AG and DAG plays a crucial role in the metabolic homeostasis among patients with schizophrenia.

Glucose Tolerance-Improving Activity of Helichrysoside in Mice and Its Structural Requirements for Promoting Glucose and Lipid Metabolism.

An acylated flavonol glycoside, helichrysoside, at a dose of 10 mg/kg/day per os for 14 days, improved the glucose tolerance in mice without affecting the food intake, visceral fat weight, liver weight, and other plasma parameters. In this study, using hepatoblastoma-derived HepG2 cells, helichrysoside, trans-tiliroside, and kaempferol 3-O-ß-D-glucopyranoside enhanced glucose consumption from the medium, but their aglycones and p-coumaric acid did not show this activity. In addition, several acylated flavonol glycosides were synthesized to clarify the structural requirements for lipid metabolism using HepG2 cells. The results showed that helichrysoside and related analogs significantly inhibited triglyceride (TG) accumulation in these cells. The inhibition by helichrysoside was more potent than that by other acylated flavonol glycosides, related flavonol glycosides, and organic acids. As for the TG metabolism-promoting activity in high glucose-pretreated HepG2 cells, helichrysoside, related analogs, and their aglycones were found to significantly reduce the TG contents in HepG2 cells. However, the desacyl flavonol glycosides and organic acids derived from the acyl groups did not exhibit an inhibitory impact on the TG contents in HepG2 cells. These results suggest that the existence of the acyl moiety at the 6'' position in the D-glucopyranosyl part is essential for glucose and lipid metabolism-promoting activities.

SIRT2 and Lysine Fatty Acylation Regulate the Activity of RalB and Cell Migration.

Protein lysine fatty acylation is increasingly recognized as a prevalent and important protein post-translation modification. Recently, it has been shown that K-Ras4a, R-Ras2, and Rac1 are regulated by lysine fatty acylation. Here, we investigated whether other members of the Ras superfamily could also be regulated by lysine fatty acylation. Several small GTPases exhibit hydroxylamine resistant fatty acylation, suggesting they may also have protein lysine fatty acylation. We further characterized one of these GTPases, RalB. We show that RalB has C-terminal lysine fatty acylation, with the predominant modification site being Lys200. The lysine acylation of RalB is regulated by SIRT2, a member of the sirtuin family of nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacylases. Lysine fatty acylated RalB exhibited enhanced plasma membrane localization and recruited its known effectors Sec5 and Exo84, members of the exocyst complex, to the plasma membrane. RalB lysine fatty acylation did not affect the proliferation or anchorage-independent growth but did affect the trans-well migration of A549 lung cancer cells. This study thus identified an additional function for protein lysine fatty acylation and the deacylase SIRT2.

Activity-Guided Design of HDAC11-Specific Inhibitors.

Mammalian histone deacetylases (HDACs) are a class of enzymes that play important roles in biological pathways. Existing HDAC inhibitors target multiple HDACs without much selectivity. Inhibitors that target one particular HDAC will be useful for investigating the biological functions of HDACs and for developing better therapeutics. Here, we report the development of HDAC11-specific inhibitors using an activity-guided rational design approach. The enzymatic activity and biological function of HDAC11 have been little known, but recent reports suggest that it has efficient defatty-acylation activity and that inhibiting it could be useful for treating a variety of human diseases, including viral infection, multiple sclerosis, and metabolic diseases. Our best inhibitor, SIS17, is active in cells and inhibited the demyristoylation of a known HDAC11 substrate, serine hydroxymethyl transferase 2, without inhibiting other HDACs. The activity-guided design may also be useful for the development of isoform-specific inhibitors for other classes of enzymes.

Nacubactam Enhances Meropenem Activity against Carbapenem-Resistant Klebsiella pneumoniae Producing KPC.

Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most antibiotics, making CRE infections extremely difficult to treat with available agents. Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) are predominant carbapenemases in CRE in the United States. Nacubactam is a bridged diazabicyclooctane (DBO) ß-lactamase inhibitor that inactivates class A and C ß-lactamases and exhibits intrinsic antibiotic and ß-lactam "enhancer" activity against Enterobacteriaceae In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying blaKPC-2 or blaKPC-3; meropenem-nacubactam restored susceptibility. Upon testing isogenic Escherichia coli strains producing KPC-2 variants with single-residue substitutions at important Ambler class A positions (K73, S130, R164, E166, N170, D179, K234, E276, etc.), the K234R variant increased the meropenem-nacubactam MIC compared to that for the strain producing KPC-2, without increasing the meropenem MIC. Correspondingly, nacubactam inhibited KPC-2 (apparent Ki [Ki app] = 31 ± 3 µM) more efficiently than the K234R variant (Ki app = 270 ± 27 µM) and displayed a faster acylation rate (k2/K), which was 5,815 ± 582 M-1 s-1 for KPC-2 versus 247 ± 25 M-1 s-1 for the K234R variant. Unlike avibactam, timed mass spectrometry revealed an intact sulfate on nacubactam and a novel peak (+337 Da) with the K234R variant. Molecular modeling of the K234R variant showed significant catalytic residue (i.e., S70, K73, and S130) rearrangements that likely interfere with nacubactam binding and acylation. Nacubactam's aminoethoxy tail formed unproductive interactions with the K234R variant's active site. Molecular modeling and docking observations were consistent with the results of biochemical analyses. Overall, the meropenem-nacubactam combination is effective against carbapenem-resistant K. pneumoniae Moreover, our data suggest that ß-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

Elevated O-GlcNAcylation enhances pro-inflammatory Th17 function by altering the intracellular lipid microenvironment.

Chronic, low-grade inflammation increases the risk for atherosclerosis, cancer, and autoimmunity in diseases such as obesity and diabetes. Levels of CD4+ T helper 17 (Th17) cells, which secrete interleukin 17A (IL-17A), are increased in obesity and contribute to the inflammatory milieu; however, the relationship between signaling events triggered by excess nutrient levels and IL-17A-mediated inflammation is unclear. Here, using cytokine, quantitative real-time PCR, immunoprecipitation, and ChIP assays, along with lipidomics and MS-based approaches, we show that increased levels of the nutrient-responsive, post-translational protein modification, O-GlcNAc, are present in naive CD4+ T cells from a diet-induced obesity murine model and that elevated O-GlcNAc levels increase IL-17A production. We also found that increased binding of the Th17 master transcription factor RAR-related orphan receptor γ t variant (RORγt) at the IL-17 gene promoter and enhancer, as well as significant alterations in the intracellular lipid microenvironment, elevates the production of ligands capable of increasing RORγt transcriptional activity. Importantly, the rate-limiting enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase 1 (ACC1), is O-GlcNAcylated and necessary for production of these RORγt-activating ligands. Our results suggest that increased O-GlcNAcylation of cellular proteins may be a potential link between excess nutrient levels and pathological inflammation.