Reversible Post-translational Modification Of Proteins Research Articles

Dual-Role Ubiquitination Regulation Shuttling the Entire Life Cycle of the Flaviviridae.

Ubiquitination is a reversible protein post-translational modification that regulates various pivotal physiological and pathological processes in all eukaryotes. Recently, the antiviral immune response is enhanced by the regulation of ubiquitination. Intriguingly, Flaviviridae viruses can ingeniously hijack the ubiquitination system to help them survive, which has become a hot topic among worldwide researchers. The Flaviviridae family members, such as HCV and CSFV, can cause serious diseases of humans and animals around the world. The multiple roles of ubiquitination involved in the life cycle of Flaviviridae family would open new sight for future development of antiviral tactic. Here, we discuss recent advances with regard to functional roles of ubiquitination and some ubiquitin-like modifications in the life cycle of Flaviviridae infection, shedding new light on the antiviral mechanism research and therapeutic drug development.

Recent Development of Machine Learning Methods in Sumoylation Sites Prediction.

Sumoylation of proteins is an important reversible post-translational modification of proteins and mediates a variety of cellular processes. Sumo-modified proteins can change their subcellular localization, activity, and stability. In addition, it also plays an important role in various cellular processes such as transcriptional regulation and signal transduction. The abnormal sumoylation is involved in many diseases, including neurodegeneration and immune-related diseases, as well as the development of cancer. Therefore, identification of the sumoylation site (SUMO site) is fundamental to understanding their molecular mechanisms and regulatory roles. In contrast to labor-intensive and costly experimental approaches, computational prediction of sumoylation sites in silico has also attracted much attention for its accuracy, convenience, and speed. At present, many computational prediction models have been used to identify SUMO sites, but their contents have not been comprehensively summarized and reviewed. Therefore, the research progress of relevant models is summarized and discussed in this paper. We have briefly summarized the development of bioinformatics methods for sumoylation site prediction by mainly focusing on the benchmark dataset construction, feature extraction, machine learning method, published results, and online tools. We hope that this review will provide more help for wet-experimental scholars.

Comparative O-GlcNAc Proteomic Analysis Reveals a Role of O-GlcNAcylated SAM68 in Lung Cancer Aggressiveness.

Simple SummaryLung cancer claims the most lives annually among cancers; to date, invasion and metastasis still pose challenges to effective treatment. O-GlcNAcylation, an enzymatic modification of proteins after biosynthesis, modulates the functions of many proteins. Aberrant O-GlcNAcylation is linked to pathogenic mechanisms of cancer, including invasion and metastasis. However, little is known about the profile of O-GlcNAcylated proteins involved in cancer aggressiveness. Here, by comparing profiles of O-GlcNAcylated proteins from two lung cancer cell lines different in their invasive potential, we identified candidates for O-GlcNAcylated proteins that may be involved in cancer aggressiveness. One of these candidates, SAM68, was further characterized. Results confirmed O-GlcNAcylation of SAM68; functional analyses on SAM68 with mutations at O-GlcNAcylation sites suggested a role of O-GlcNAcylated SAM68 in modulating lung cancer cell migration/invasion. Future elucidation of the functional significance of differential O-GlcNAcylation of proteins identified in this study may provide new insights into mechanisms of lung cancer progression.O-GlcNAcylation is a reversible and dynamic post-translational protein modification catalyzed by O-GlcNAc transferase (OGT). Despite the reported association of O-GlcNAcylation with cancer metastasis, the O-GlcNAc proteome profile for cancer aggressiveness remains largely uncharacterized. Here, we report our comparative O-GlcNAc proteome profiling of two differentially invasive lung adenocarcinoma cell lines, which identified 158 down-regulated and 106 up-regulated candidates in highly invasive cells. Among these differential proteins, a nuclear RNA-binding protein, SAM68 (SRC associated in mitosis of 68 kDa), was further investigated. Results showed that SAM68 is O-GlcNAcylated and may interact with OGT in the nucleus. Eleven O-GlcNAcylation sites were identified, and data from mutant analysis suggested that multiple serine residues in the N-terminal region are important for O-GlcNAcylation and the function of SAM68 in modulating cancer cell migration and invasion. Analysis of clinical specimens found that high SAM68 expression was associated with late cancer stages, and patients with high-OGT/high-SAM68 expression in their tumors had poorer overall survival compared to those with low-OGT/low-SAM68 expression. Our study revealed an invasiveness-associated O-GlcNAc proteome profile and connected O-GlcNAcylated SAM68 to lung cancer aggressiveness.

Evolutionarily Established Palmitoylation-Dependent Regulatory Mechanisms of the Vertebrate Glutamatergic Synapse and Diseases Caused by Their Disruption.

Glutamate is the major excitatory neurotransmitter in the vertebrate brain and various modifications have been established in the glutamatergic synapses. Generally, many neuronal receptors and ion channels are regulated by S-palmitoylation, a reversible post-translational protein modification. Genome sequence databases show the evolutionary acquisition and conservation concerning vertebrate-specific palmitoylation of synaptic proteins including glutamate receptors. Moreover, palmitoylation of some glutamate receptor-binding proteins is subsequently acquired only in some mammalian lineages. Recent progress in genome studies has revealed that some palmitoylation-catalyzing enzymes are the causative genes of neuropsychiatric disorders. In this review, I will summarize the evolutionary development of palmitoylation-dependent regulation of glutamatergic synapses and their dysfunctions which are caused by the disruption of palmitoylation mechanism.

Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes-a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS.

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope-labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated, enabling accurate relative quantification of site-specific lysine acetylation dynamics in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics.

Bull Sperm Capacitation Is Accompanied by Redox Modifications of Proteins.

The ability to fertilise an egg is acquired by the mammalian sperm during the complex biochemical process called capacitation. Capacitation is accompanied by the production of reactive oxygen species (ROS), but the mechanism of redox regulation during capacitation has not been elucidated. This study aimed to verify whether capacitation coincides with reversible oxidative post-translational modifications of proteins (oxPTMs). Flow cytometry, fluorescence microscopy and Western blot analyses were used to verify the sperm capacitation process. A fluorescent gel-based redox proteomic approach allowed us to observe changes in the level of reversible oxPTMs manifested by the reduction or oxidation of susceptible cysteines in sperm proteins. Sperm capacitation was accompanied with redox modifications of 48 protein spots corresponding to 22 proteins involved in the production of ROS (SOD, DLD), playing a role in downstream redox signal transfer (GAPDHS and GST) related to the cAMP/PKA pathway (ROPN1L, SPA17), acrosome exocytosis (ACRB, sperm acrosome associated protein 9, IZUMO4), actin polymerisation (CAPZB) and hyperactivation (TUBB4B, TUB1A). The results demonstrated that sperm capacitation is accompanied by altered levels of oxPTMs of a group of redox responsive proteins, filling gaps in our knowledge concerning sperm capacitation.

Prediction of protein ubiquitination sites via multi-view features based on eXtreme gradient boosting classifier

Ubiquitination is a common and reversible post-translational protein modification that regulates apoptosis and plays an important role in protein degradation and cell diseases. However, experimental identification of protein ubiquitination sites is usually time-consuming and labor-intensive, so it is necessary to establish effective predictors. In this study, we propose a ubiquitination sites prediction method based on multi-view features, namely UbiSite-XGBoost. Firstly, we use seven single-view features encoding methods to convert protein sequence fragments into digital information. Secondly, the least absolute shrinkage and selection operator (LASSO) is applied to remove the redundant information and get the optimal feature subsets. Finally, these features are inputted into the eXtreme gradient boosting (XGBoost) classifier to predict ubiquitination sites. Five-fold cross-validation shows that the AUC values of Set1-Set6 datasets are 0.8258, 0.7592, 0.7853, 0.8345, 0.8979 and 0.8901, respectively. The synthetic minority oversampling technique (SMOTE) is employed in Set4-Set6 unbalanced datasets, and the AUC values are 0.9777, 0.9782 and 0.9860, respectively. In addition, we have constructed three independent test datasets which the AUC values are 0.8007, 0.6897 and 0.7280, respectively. The results show that the proposed method UbiSite-XGBoost is superior to other ubiquitination prediction methods and it provides new guidance for the identification of ubiquitination sites. The source code and all datasets are available at https://github.com/QUST-AIBBDRC/UbiSite-XGBoost/.

S-acylation of P2K1 mediates extracellular ATP-induced immune signaling in Arabidopsis

S-acylation is a reversible protein post-translational modification mediated by protein S-acyltransferases (PATs). How S-acylation regulates plant innate immunity is our main concern. Here, we show that the plant immune receptor P2K1 (DORN1, LecRK-I.9; extracellular ATP receptor) directly interacts with and phosphorylates Arabidopsis PAT5 and PAT9 to stimulate their S-acyltransferase activity. This leads, in a time-dependent manner, to greater S-acylation of P2K1, which dampens the immune response. pat5 and pat9 mutants have an elevated extracellular ATP-induced immune response, limited bacterial invasion, increased phosphorylation and decreased degradation of P2K1 during immune signaling. Mutation of S-acylated cysteine residues in P2K1 results in a similar phenotype. Our study reveals that S-acylation effects the temporal dynamics of P2K1 receptor activity, through autophosphorylation and protein degradation, suggesting an important role for this modification in regulating the ability of plants in respond to external stimuli.

Glutathione S-transferases and their implications in the lung diseases asthma and chronic obstructive pulmonary disease: Early life susceptibility?

Our lungs are exposed daily to airborne pollutants, particulate matter, pathogens as well as lung allergens and irritants. Exposure to these substances can lead to inflammatory responses and may induce endogenous oxidant production, which can cause chronic inflammation, tissue damage and remodeling. Notably, the development of asthma and Chronic Obstructive Pulmonary Disease (COPD) is linked to the aforementioned irritants. Some inhaled foreign chemical compounds are rapidly absorbed and processed by phase I and II enzyme systems critical in the detoxification of xenobiotics including the glutathione-conjugating enzymes Glutathione S-transferases (GSTs). GSTs, and in particular genetic variants of GSTs that alter their activities, have been found to be implicated in the susceptibility to and progression of these lung diseases. Beyond their roles in phase II metabolism, evidence suggests that GSTs are also important mediators of normal lung growth. Therefore, the contribution of GSTs to the development of lung diseases in adults may already start in utero, and continues through infancy, childhood, and adult life. GSTs are also known to scavenge oxidants and affect signaling pathways by protein-protein interaction. Moreover, GSTs regulate reversible oxidative post-translational modifications of proteins, known as protein S-glutathionylation. Therefore, GSTs display an array of functions that impact the pathogenesis of asthma and COPD.In this review we will provide an overview of the specific functions of each class of mammalian cytosolic GSTs. This is followed by a comprehensive analysis of their expression profiles in the lung in healthy subjects, as well as alterations that have been described in (epithelial cells of) asthmatics and COPD patients. Particular emphasis is placed on the emerging evidence of the regulatory properties of GSTs beyond detoxification and their contribution to (un)healthy lungs throughout life. By providing a more thorough understanding, tailored therapeutic strategies can be designed to affect specific functions of particular GSTs.

S‐acyltransferase zDHHC9 Promotes Pathogenic Signaling in the Heart

Heart disease remains a leading cause of mortality, contributing to over 600,000 deaths per year in the United States. Notwithstanding, the molecular mechanisms underlying the pathophysiology of heart disease remain largely elusive. Receptors, ion channels, and signaling proteins that regulate cardiac contractility and physiology are modified by post-translational modifications such as phosphorylation. Recent research into the roles of S-acylation or palmitoylation, the reversible post-translational modification of proteins with fatty acids, has demonstrated critical functions in the regulation of intracellular signaling in physiology and disease. Palmitoylation, which is catalyzed by the zinc finger Asp-His-His-Cys (zDHHC) family of S-acyltransferases, has been implicated in trafficking, stability, sorting and binding of proteins to cofactors. However, the roles of palmitoylation in cardiac signal transduction and cardiac pathogenesis are not well understood. Our preliminary work indicates that cardiac-specific transgenic overexpression of the Golgi-specific acyl transferase zDHHC9 (TgZdhhc9) results in cardiac hypertrophy and severe cardiomyopathy at six and eight months of age respectively in mice. Furthermore, using unbiased palmitoyl proteomics, we discovered that zDHHC9 palmitoylates Rab3 GTPase-activating protein catalytic subunit (Rab3gap1) that hydrolyzes the active GTP-bound form of Rab3 GTPase protein (Rab3) and inactivates Rab3 function. More so, mice subjected to transverse aortic constriction surgery, a pressure-overload model of pathological cardiac hypertrophy, showed increased palmitoylation of Rab3gap1, compared to sham controls. In addition, we observed that overexpression of zDHHC9 resulted in retention of Rab3gap1 at the Golgi and post-Golgi vesicles in cardiomyocytes and TgZdhhc9 hearts showed increased levels of Rab3a-GTP prior to disease onset, suggesting enhancement of cardiac Rab3a activity. Ongoing research will identify which hypertrophic signaling effectors are modulated by the zDHHC9/ Rab3gap1/Rab3a cascade in the heart. Altogether, these data suggest zDHHC9-mediated palmitoylation of Rab3gap1 may contribute to hypertrophic signaling and heart disease.

Advances in the Development Ubiquitin-Specific Peptidase (USP) Inhibitors

Ubiquitylation and deubiquitylation are reversible protein post-translational modification (PTM) processes involving the regulation of protein degradation under physiological conditions. Loss of balance in this regulatory system can lead to a wide range of diseases, such as cancer and inflammation. As the main members of the deubiquitinases (DUBs) family, ubiquitin-specific peptidases (USPs) are closely related to biological processes through a variety of molecular signaling pathways, including DNA damage repair, p53 and transforming growth factor-β (TGF-β) pathways. Over the past decade, increasing attention has been drawn to USPs as potential targets for the development of therapeutics across diverse therapeutic areas. In this review, we summarize the crucial roles of USPs in different signaling pathways and focus on advances in the development of USP inhibitors, as well as the methods of screening and identifying USP inhibitors.

Protein S-palmitoylation in immunity.

S-palmitoylation is a reversible posttranslational lipid modification of proteins. It controls protein activity, stability, trafficking and protein-protein interactions. Recent global profiling of immune cells and targeted analysis have identified many S-palmitoylated immunity-associated proteins. Here, we review S-palmitoylated immune receptors and effectors, and their dynamic regulation at cellular membranes to generate specific and balanced immune responses. We also highlight how this understanding can drive therapeutic advances to pharmacologically modulate immune responses.

PUP-Fuse: Prediction of Protein Pupylation Sites by Integrating Multiple Sequence Representations.

Pupylation is a type of reversible post-translational modification of proteins, which plays a key role in the cellular function of microbial organisms. Several proteomics methods have been developed for the prediction and analysis of pupylated proteins and pupylation sites. However, the traditional experimental methods are laborious and time-consuming. Hence, computational algorithms are highly needed that can predict potential pupylation sites using sequence features. In this research, a new prediction model, PUP-Fuse, has been developed for pupylation site prediction by integrating multiple sequence representations. Meanwhile, we explored the five types of feature encoding approaches and three machine learning (ML) algorithms. In the final model, we integrated the successive ML scores using a linear regression model. The PUP-Fuse achieved a Mathew correlation value of 0.768 by a 10-fold cross-validation test. It also outperformed existing predictors in an independent test. The web server of the PUP-Fuse with curated datasets is freely available.

Recent Advances in Predicting Protein S-Nitrosylation Sites.

Protein S-nitrosylation (SNO) is a process of covalent modification of nitric oxide (NO) and its derivatives and cysteine residues. SNO plays an essential role in reversible posttranslational modifications of proteins. The accurate prediction of SNO sites is crucial in revealing a certain biological mechanism of NO regulation and related drug development. Identification of the sites of SNO in proteins is currently a very hot topic. In this review, we briefly summarize recent advances in computationally identifying SNO sites. The challenges and future perspectives for identifying SNO sites are also discussed. We anticipate that this review will provide insights into research on SNO site prediction.

Targeting the Ubiquitin Signaling Cascade in Tumor Microenvironment for Cancer Therapy.

Tumor microenvironments are composed of a myriad of elements, both cellular (immune cells, cancer-associated fibroblasts, mesenchymal stem cells, etc.) and non-cellular (extracellular matrix, cytokines, growth factors, etc.), which collectively provide a permissive environment enabling tumor progression. In this review, we focused on the regulation of tumor microenvironment through ubiquitination. Ubiquitination is a reversible protein post-translational modification that regulates various key biological processes, whereby ubiquitin is attached to substrates through a catalytic cascade coordinated by multiple enzymes, including E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases. In contrast, ubiquitin can be removed by deubiquitinases in the process of deubiquitination. Here, we discuss the roles of E3 ligases and deubiquitinases as modulators of both cellular and non-cellular components in tumor microenvironment, providing potential therapeutic targets for cancer therapy. Finally, we introduced several emerging technologies that can be utilized to develop effective therapeutic agents for targeting tumor microenvironment.

Negative regulation of FOXP3 expression by c-Rel O-GlcNAcylation.

O-GlcNAcylation is a reversible post-translational protein modification that regulates fundamental cellular processes including immune responses and autoimmunity. Previously, we showed that hyperglycemia increases O-GlcNAcylation of the transcription factor, nuclear factor kappaB c-Rel at serine residue 350 and enhances the transcription of the c-Rel-dependent proautoimmune cytokines interleukin-2, interferon gamma and granulocyte macrophage colony stimulating factor in T cells. c-Rel also plays a critical role in the transcriptional regulation of forkhead box P3 (FOXP3)-the master transcription factor that governs development and function of Treg cells. Here we show that the regulatory effect of c-Rel O-GlcNAcylation is gene-dependent, and in contrast to its role in enhancing the expression of proautoimmune cytokines, it suppresses the expression of FOXP3. Hyperglycemia-induced O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; streptozotocin-induced diabetes and spontaneous diabetes in nonobese diabetic mice. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases c-Rel binding at the FOXP3 promoter and negatively regulates FOXP3 expression. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating immunosuppressive FOXP3 expression and proautoimmune gene expression in autoimmune diabetes with potential therapeutic implications.

Controlling Kinase Activities by Selective Inhibition of Peptide Substrates.

Phosphorylation is the most common reversible post-translational modification (PTM) of proteins. Because a given kinase often has many substrates in a cell and is involved in numerous functions, traditional inhibition of the enzyme leads to unintended consequences. Here we report synthetic receptors to manipulate kinase phosphorylation precisely for the first time, utilizing the receptors' abilities to bind peptides with high affinity and specificity. The inhibition enables selective phosphorylation of peptides with identical consensus motifs in a mixture. A particular phosphosite can be inhibited while other sites in the same substrate undergo phosphorylation. The receptors may work either individually on their targeted strands or in concert to protect segments of a long sequence. The binding-derived inhibition is able to compete with protein-protein interactions within a multidomain kinase, enabling controlled PTM to be performed in a previously unavailable manner.

Site-specific deacylation by ABHD17a controls BK channel splice variant activity

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

Roles of Ubiquitination and Deubiquitination in Regulating Dendritic Cell Maturation and Function.

Dendritic cells (DCs) are specialized antigen-presenting cells that play a key role in immune homeostasis and the adaptive immune response. DC-induced immune tolerance or activation is strictly dependent on the distinct maturation stages and migration ability of DCs. Ubiquitination is a reversible protein post-translational modification process that has emerged as a crucial mechanism that regulates DC maturation and function. Recent studies have shown that ubiquitin enzymes, including E3 ubiquitin ligases and deubiquitinases (DUBs), are pivotal regulators of DC-mediated immune function and serve as potential targets for DC-based immunotherapy of immune-related disorders (e.g., autoimmune disease, infections, and tumors). In this review, we summarize the recent progress regarding the molecular mechanisms and function of ubiquitination in DC-mediated immune homeostasis and immune response.

Gas-Phase Fragmentation of ADP-Ribosylated Peptides: Arginine-Specific Side-Chain Losses and Their Implication in Database Searches.

ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and particularly of their acceptor amino acids remains a major challenge. The attachment sites of the modification are difficult to localize by mass spectrometry (MS) because of the labile nature of the linkage and the complex fragmentation pattern of the ADP-ribose in MS/MS experiments. In this study we performed a comprehensive analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine, or lysine residues. In addition to the fragmentation of the peptide backbone, various cleavages of the ADP-ribosylated amino acid side chains were investigated. We focused on gas-phase fragmentations that were specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid, or aspartic acid was the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine was relatively stable. The main cleavage site was the inner bond of the guanidine group, which resulted in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking peptide fragment ions resulting from this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches. Furthermore, the presence of diagnostic ions and of losses of fragments from peptide ions allowed us, in most cases, to distinguish between ADP-ribosylated arginine and serine residues.