The correlation between cellular O-GlcNAcylation and sensitivity to O-GlcNAc inhibitor in colorectal cancer cells.

The upregulation of O-GlcNAc signaling has long been implicated in the development and progression of numerous human malignancies, including colorectal cancer. In this study, we characterized eight colorectal cancer cell lines and one non-cancerous cell line for O-GlcNAc-related profiles such as the expression of OGT, OGA, and total protein O-GlcNAcylation, along with their sensitivity toward OSMI-1 (Os), an OGT inhibitor (OGTi). Indeed, Os dose-dependently suppressed the viability of all colorectal cancer cell lines tested. Among the three O-GlcNAc profiles, our results revealed that Os IC50 exhibited the strongest correlation with total protein O-GlcNAcylation (Pearson Correlation Coefficient r = -0.73), suggesting that total O-GlcNAcylation likely serves as a better predictive marker for OGTi sensitivity than OGT expression levels. Furthermore, we demonstrated that Os exhibited a synergistic relationship with regorafenib (Re). We believed that this synergism could be explained, at least in part, by the observed Re-mediated increase of cellular O-GlcNAcylation, which was counteracted by Os. Finally, we showed that the Os:Re combination suppressed the growth of NCI-H508 tumor spheroids. Overall, our findings highlighted OGTi as a potential anticancer agent that could be used in combination with other molecules to enhance the efficacy while minimizing adverse effects, and identified total cellular O-GlcNAcylation as a potential predictive marker for OGTi sensitivity.

The histone acylation reader ENL/AF9 regulates aging in Drosophila melanogaster.

Histone acylation plays a pivotal role in modulating gene expression, ensuring proper neurogenesis and responsiveness to various signals. Recently, the evolutionary conserved YAF9, ENL, AF9, TAF41, SAS5 (YEATS) domain found in four human paralogs, has emerged as a new class of histone acylation reader with a preference for the bulkier crotonyl group lysine over acetylation. Despite advancements, the role of either histone crotonylation or its readers in neurons remains unclear. In this study, we employed Drosophila melanogaster to investigate the role of ENL/AF9 (dENL/AF9) in the nervous system. Pan-neuronal dENL/AF9 knockdown not only extended the lifespan of flies but also enhanced their overall fitness during aging, including improved sleep quality and locomotion. Moreover, a decreased activity of dENL/AF9 in neurons led to an up-regulation of catalase gene expression which combined with reduced levels of malondialdehyde (MDA) and an enhanced tolerance to oxidative stress in aging flies. This study unveiled a novel function of histone crotonylation readers in aging with potential implications for understanding age-related conditions in humans.

O-GlcNAcylation mediates Wnt-stimulated bone formation by rewiring aerobic glycolysis.

Wnt signaling is an important target for anabolic therapies in osteoporosis. A sclerostin-neutralizing antibody (Scl-Ab), that blocks the Wnt signaling inhibitor (sclerostin), has been shown to promote bone mass in animal models and clinical studies. However, the cellular mechanisms by which Wnt signaling promotes osteogenesis remain to be further investigated. O-GlcNAcylation, a dynamic post-translational modification of proteins, controls multiple critical biological processes including transcription, translation, and cell fate determination. Here, we report that Wnt3a either induces O-GlcNAcylation rapidly via the Ca2+-PKA-Gfat1 axis, or increases it in a Wnt-ß-catenin-dependent manner following prolonged stimulation. Importantly, we find O-GlcNAcylation indispensable for osteoblastogenesis both in vivo and in vitro. Genetic ablation of O-GlcNAcylation in the osteoblast-lineage diminishes bone formation and delays bone fracture healing in response to Wnt stimulation in vivo. Mechanistically, Wnt3a induces O-GlcNAcylation at Serine 174 of PDK1 to stabilize the protein, resulting in increased glycolysis and osteogenesis. These findings highlight O-GlcNAcylation as an important mechanism regulating Wnt-induced glucose metabolism and bone anabolism.

Functions of Coenzyme A and Acyl-CoA in Post-Translational Modification and Human Disease.

Coenzyme A (CoA) is synthesized from pantothenate, L-cysteine and adenosine triphosphate (ATP), and plays a vital role in diverse physiological processes. Protein acylation is a common post-translational modification (PTM) that modifies protein structure, function and interactions. It occurs via the transfer of acyl groups from acyl-CoAs to various amino acids by acyltransferase. The characteristics and effects of acylation vary according to the origin, structure, and location of the acyl group. Acetyl-CoA, formyl-CoA, lactoyl-CoA, and malonyl-CoA are typical acyl group donors. The major acyl donor, acyl-CoA, enables modifications that impart distinct biological functions to both histone and non-histone proteins. These modifications are crucial for regulating gene expression, organizing chromatin, managing metabolism, and modulating the immune response. Moreover, CoA and acyl-CoA play significant roles in the development and progression of neurodegenerative diseases, cancer, cardiovascular diseases, and other health conditions. The goal of this review was to systematically describe the types of commonly utilized acyl-CoAs, their functions in protein PTM, and their roles in the progression of human diseases.

Current computational tools for protein lysine acylation site prediction.

As a main subtype of post-translational modification (PTM), protein lysine acylations (PLAs) play crucial roles in regulating diverse functions of proteins. With recent advancements in proteomics technology, the identification of PTM is becoming a data-rich field. A large amount of experimentally verified data is urgently required to be translated into valuable biological insights. With computational approaches, PLA can be accurately detected across the whole proteome, even for organisms with small-scale datasets. Herein, a comprehensive summary of 166 in silico PLA prediction methods is presented, including a single type of PLA site and multiple types of PLA sites. This recapitulation covers important aspects that are critical for the development of a robust predictor, including data collection and preparation, sample selection, feature representation, classification algorithm design, model evaluation, and method availability. Notably, we discuss the application of protein language models and transfer learning to solve the small-sample learning issue. We also highlight the prediction methods developed for functionally relevant PLA sites and species/substrate/cell-type-specific PLA sites. In conclusion, this systematic review could potentially facilitate the development of novel PLA predictors and offer useful insights to researchers from various disciplines.

Ping, pong, and freeze: Structural insights into the inhibition of ceramide synthase by Fumonisin B1.

Fumonisin B1 (FB1) targets sphingolipid biosynthesis, inhibiting ceramide synthases. In this issue of Structure, Zhang et al.1 determined the cryoelectron microscopic structures of yeast ceramide synthase in complex with FB1 and its acylated derivative, acyl-FB1, revealing a two-step "ping-pong" mechanism for the N-acylation of FB1 and how it inhibits ceramide synthase.

S-acylation of NLRP3 provides a nigericin sensitive gating mechanism that controls access to the Golgi.

NLRP3 is an inflammasome seeding pattern recognition receptor activated in response to multiple danger signals which perturb intracellular homeostasis. Electrostatic interactions between the NLRP3 polybasic (PB) region and negatively charged lipids on the trans-Golgi network (TGN) have been proposed to recruit NLRP3 to the TGN. In this study, we demonstrate that membrane association of NLRP3 is critically dependant on S-acylation of a highly conserved cysteine residue (Cys-130), which traps NLRP3 in a dynamic S-acylation cycle at the Golgi, and a series of hydrophobic residues preceding Cys-130 which act in conjunction with the PB region to facilitate Cys-130 dependent Golgi enrichment. Due to segregation from Golgi localised thioesterase enzymes caused by a nigericin induced breakdown in Golgi organisation and function, NLRP3 becomes immobilised on the Golgi through reduced de-acylation of its Cys-130 lipid anchor, suggesting that disruptions in Golgi homeostasis are conveyed to NLRP3 through its acylation state. Thus, our work defines a nigericin sensitive S-acylation cycle that gates access of NLRP3 to the Golgi.

A vacuolar protein MaSCPL1 mediates anthocyanin acylation modifications in blue-flowered grape hyacinth.

The grape hyacinth is renowned for its profuse blue flowers, which confer substantial scientific and ornamental significance as well as considerable potential for industrial applications. The serine carboxypeptidase-like acyltransferases (SCPL-ATs) family is crucial for the blue flower coloration. To elucidate SCPL-ATs involved in anthocyanin modification in grape hyacinth, we performed a transcriptomic analysis of grape hyacinth SCPL-ATs. Through gene expression profiling, we identified a promising candidate gene, MaSCPL1, whose expression patterns corresponded with variations in anthocyanin content throughout petal coloration. Subsequently, the functional role of the MaSCPL1 gene was validated using the native petal regeneration system, and the silencing of MaSCPL1 led to a decreased total anthocyanin content and Dp3MG content in grape hyacinth petals. Furthermore, we employed yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase assays to explore the regulatory interactions between the anthocyanin biosynthesis transcription factor MaMybA and the MaSCPL1 promoter. Our findings indicate that MaMybA can bind to the MaSCPL1 promoter and significantly activate its expression. Furthermore, the MaMybA-RNAi resulted in a substantial multifold reduction in the expression of MaSCPL1, implying that the regulation of MaSCPL1 expression is mediated by MaMybA. This study revealed the MaSCPL1 gene has been associated with anthocyanin acylated modification in grape hyacinth and elucidated the important role of the MaMybA-MaSCPL1 module in colouration grape hyacinth.

Activity-Based Acylome Profiling with N-(Cyanomethyl)-N-(phenylsulfonyl)amides for Targeted Lysine Acylation and Post-Translational Control of Protein Function in Cells.

Lysine acylations are ubiquitous and structurally diverse post-translational modifications that vastly expand the functional heterogeneity of the human proteome. Hence, the targeted acylation of lysine residues has emerged as a strategic approach to exert biomimetic control over the protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents and lack methods to quantify the magnitude of specific acylations on a global level. In this study, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific introduction and proteome-wide mapping of posttranslational lysine acylations in human cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish a structure-activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify paralog-selective chemical probes that acetylate conserved lysines within interferon-stimulated antiviral RNA-binding proteins, generating de novo proteoforms with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in the protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy in biomimetic control over protein function through targeted delivery and global profiling of endogenous and artificial lysine acylations, potentially advancing therapeutic modalities and our understanding of biological processes orchestrated by these post-translational modifications.

Lysophospholipid remodeling mediated by the LplT and Aas protein complex in the bacterial envelope.

Lysophospholipid transporter LplT and acyltransferase Aas consist of a lysophospholipid-remodeling system ubiquitously found in gram-negative microorganisms. LplT flips lysophospholipid across the inner membrane which is subsequently acylated by Aas on the cytoplasmic membrane surface. Our previous study showed that the proper functioning of this system is important to protecting Escherichia coli from phospholipase-mediated host attack by maintaining the integrity of the bacterial cell envelope. However, the working mechanism of this system is still unclear. Herein, we report that LplT and Aas form a membrane protein complex in E. coli which allows these two enzymes to cooperate efficiently to move lysophospholipids across the bacterial membrane and catalyze their acylation. The direct interaction of LplT and Aas was demonstrated both in vivo and in vitro with a binding affinity of 2.3 µM. We found that a cytoplasmic loop of LplT adjacent to the exit of the substrate translocation pathway plays an important role in maintaining its interaction with Aas. Aas contains an acyl-acyl carrier protein synthase domain and an acyl-transferase domain. Its interaction with LplT is mediated exclusively by its transferase domain. Mutations within the three loops near the putative catalytic site of the transferase domain, respectively, disrupt its interaction with LplT and lysophospholipid acylation activity. These results support a hypothesis of the functional coupling mechanism, in which LplT directly interacts with the transferase domain of Aas for specific substrate membrane migration, providing synchronization of substrate translocation and biosynthetic events.

Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.

Exploring acylated anthocyanin-based extracts as a natural alternative to synthetic food dyes: Stability and application insights.

This work explores the potential of anthocyanin-based extracts (hibiscus calyxes - HC, red cabbage - RC, and butterfly pea flower - BPF) as natural alternatives to synthetic dyes in the food industry. Analyses in a pH range for food applications revealed higher color stability for the BPF extract, keeping vibrant colors over the 7 days at room temperature. At pH 3 and 100 °C, the BPF was more stable, losing half of its anthocyanin concentration after 14 h, while RC and HC lost half of their color after 7 and 2 h, respectively. The bisulfite bleaching followed a second-order reaction for HC and RC, and a first-order reaction for BPF, suggesting a minor effect of the bisulfite on this extract. Incorporating these extracts into porcine protein and agar-agar gelatin formulations produced consistent products with appealing hues, particularly the blue and purple colors for BPF and RC, dependent on the pH.

Innovative bamboo-plastic composites interfacial compatibility design approach: Self-assembled crosslinked structure of polydopamine with acylated chitin fibers.

Achieving interfacial compatibility through sustainable methods is a key objective in natural fiber-plastic composites research, aimed at optimizing mechanical performance. This study introduced an innovative organic bamboo-plastic composite (BPC) interfacial layer, incorporating O-acylated chitin fibers densely coated with polydopamine (PDA) via a mild and facile self-assembly method. Chitin nanofibers were acylated with dodecenylsuccinic anhydride in a deep eutectic solvent in a one-pot process. The resulting BPCs exhibited significantly enhanced mechanical properties, with tensile strength, flexural strength, modulus, and impact strength increased by 73.64 %, 39.19 %, 15.42 %, and 63.57 %, respectively, compared to untreated BPCs. This improvement highlights the effectiveness of tailoring cross-linked networks across heterogeneous interfaces in providing strength, dissipating strain, and promoting interfacial compatibility. Furthermore, these modified BPCs demonstrated enhanced thermal stability, crystallization behavior, and moderate hydrophobicity. This surface treatment strategy offers a distinctive approach to producing high-performance, eco-friendly BPCs, also facilitating the processing and utilization of marine biological resources on a wide scale.

Enzymatic acylation of cyanidin-3-O-glucoside with aromatic and aliphatic acid methyl ester: Structure-stability relationships of acylated derivatives.

Anthocyanins are water-soluble pigments, but they tend to be unstable in aqueous solutions. Modification of their molecular structure offers a viable approach to alter their intrinsic properties and enhance stability. Aromatic and aliphatic acid methyl esters were used as acyl donors in the enzymatic acylation of cyanidin-3-O-glucoside (C3G), and their analysis was conducted using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). The highest conversion rate achieved was 96.41 % for cyanidin-3-O-(6″-feruloyl) glucoside. Comparative evaluations of stability revealed that aromatic acyl group-conjugated C3G exhibited superior stability enhancement compared with aliphatic acyl group derivatives. The stability of aliphatic C3G decreased with increasing carbon chain length. The molecular geometries of different anthocyanins were optimized, and energy level calculations using density functional theory (DFT) identified their sites with antioxidant activities. Computational calculations aligned with the in vitro antioxidant assay results. This study provided theoretical support for stabilizing anthocyanins and broadened the application of acylated anthocyanins as food colorants and nutrient supplements.

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. Here, we show that zDHHC11 regulates LD catabolism by modifying adipose triacylglyceride lipase (ATGL), the rate-limiting enzyme of lipolysis, both in hepatocyte cultures and in mice. zDHHC11 S-acylates ATGL at cysteine 15. Preventing the S-acylation of ATGL renders it catalytically inactive despite proper localization. Overexpression of zDHHC11 reduces LD size, whereas its elimination enlarges LDs. Mutating ATGL cysteine 15 phenocopies zDHHC11 loss, causing LD accumulation, defective lipolysis and lipophagy. Our results reveal S-acylation as a mode of regulation of ATGL function and LD homoeostasis. Modulating this pathway may offer therapeutic potential for treating diseases linked to defective lipolysis, such as fatty liver disease.

Protocol for evaluating S-acylated protein membrane affinity using protein-lipid conjugates.

S-acylation of proteins allows their association with membranes. Here, we present a protocol for establishing a platform for membrane affinity evaluation of S-acylated proteins in vitro. We describe steps for preparing lipid-maleimide compounds, mCherry-p62 recombinant proteins, and total cellular membranes. We then detail procedures for synthesizing protein-lipid conjugates using lipid-maleimide compounds and recombinant proteins and evaluating the membrane affinity of protein-lipid conjugates. For complete details on the use and execution of this protocol, please refer to Huang Xue et al.1.

Recruitment, regulation, and release: Control of signaling enzyme localization and function by reversible S-acylation.

An ever-growing number of studies highlight the importance of S-acylation, a reversible protein-lipid modification, for diverse aspects of intracellular signaling. In this review, we summarize the current understanding of how S-acylation regulates perhaps the best-known class of signaling enzymes, protein kinases. We describe how S-acylation acts as a membrane targeting signal that localizes certain kinases to specific membranes, and how such membrane localization in turn facilitates the assembly of signaling hubs consisting of an S-acylated kinase's upstream activators and/or downstream targets. We further discuss recent findings that S-acylation can control additional aspects of the function of certain kinases, including their interactions and, surprisingly, their activity, and how such regulation might be exploited for potential therapeutic gain. We go on to describe the roles and regulation of de-S-acylases and how extracellular signals drive dynamic (de)S-acylation of certain kinases. We discuss how S-acylation has the potential to lead to "emergent properties" that alter the temporal profile and/or salience of intracellular signaling events. We close by giving examples of other S-acylation-dependent classes of signaling enzymes and by discussing how recent biological and technological advances should facilitate future studies into the functional roles of S-acylation-dependent signaling.

Sporangimicins A-D, acylated maltose derivatives from a rare actinomycete of the genus Pseudosporangium.

Sporangimicins A-D (1-4), four anomeric pairs of diacyl disaccharides that represent a new metabolite class, were discovered from the culture extract of an actinomycete Pseudosporangium sp. RD061809. Compounds 1-4 caused peak separation in the HPLC chromatogram and partial duplication of the NMR resonances by anomeric interconversion of a maltose core modified at the two sugar 6-positions with an isobutanoyl and a methyl-branched long-chain dienoyl groups. A highlight of the structure elucidation was application of Ohrui-Akasaka's method to a chromatographically inseparable mixture of 3 and 4, which proved the composition ratio of 3 and 4 to be 82:18 and the R/S ratio at the anteiso-methyl bearing chiral center in 3 to be 66:34. Compounds 1-4 showed antimicrobial activity against Gram-positive bacteria and modest cytotoxicity toward P388 murine leukemia cells.

Structural Analysis of the Hepatitis B Virus RNA Encapsidation Signal ε by Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE).

RNA structure is crucial for RNA function, including in viral cis-elements such as the hepatitis B virus (HBV) RNA encapsidation signal ε. Interacting with the viral polymerase ε mediates packaging of the pregenomic (pg) RNA into capsids, initiation of reverse transcription, and it affects the mRNA functions of pgRNA. As free RNA, the 61-nucleotide (nt) ε sequence adopts a bipartite stem-loop structure with a central bulge and an apical loop. Due to stable Watson-Crick base pairing, this was already predicted by early RNA folding programs and confirmed by classical enzymatic and chemical structure probing. A newer, high-resolution probing technique exploits the selective acylation of solvent-accessible 2'-hydroxyls in the RNA backbone by electrophilic compounds such as 2-methylnicotinic acid imidazolide (NAI), followed by mapping of the modified sites by primer extension. This SHAPE principle has meanwhile been extended to numerous applications. Here we provide a basic protocol for NAI-based SHAPE of isolated HBV ε RNA which already provided insights into the impact of mutations, and preliminarily, of polymerase binding on the RNA structural dynamics. While the focus is on NAI modification, we also briefly cover target RNA preparation by in vitro transcription, primer extension using a radiolabeled primer, and analysis of the resulting cDNAs by denaturing polyacrylamide gelelectrophoresis (PAGE). Given the high tolerance of SHAPE chemistry to different conditions, including applicability in live cells, we expect this technique to greatly facilitate deciphering the conformational dynamics underlying the various functions of the ε element, especially in concert with the recently solved three-dimensional structure of the free RNA.

Insight into the Stabilization Mechanism of Succinylation Modification on Black Bean Protein Gels: Molecular Conformation, Microstructure, and Gel Properties.

The objective of this work was to investigate the effect of succinylation treatment on the physicochemical properties of black bean proteins (BBPI), and the relationship mechanism between BBPI structure and gel properties was further analyzed. The results demonstrated that the covalent formation of higher-molecular-weight complexes with BBPI could be achieved by succinic anhydride (SA). With the addition of SA at 10% (v/v), the acylation of proteins amounted to 92.53 ± 1.10%, at which point there was a minimized particle size of the system (300.90 ± 9.57 nm). Meanwhile, the protein structure was stretched with an irregular curl content of 34.30% and the greatest processable flexibility (0.381 ± 0.004). The dense three-dimensional mesh structure of the hydrogel as revealed by scanning electron microscopy was the fundamental prerequisite for the ability to resist external extrusion. The thermally induced hydrogels of acylated proteins with 10% (v/v) addition of SA showed excellent gel elastic behavior (1.44 ± 0.002 nm) and support capacity. Correlation analysis showed that the hydrogel strength and stability of hydrogels were closely related to the changes in protein conformation. This study provides theoretical guidance for the discovery of flexible proteins and their application in hydrogels.