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Dynamic ADP-ribosylation signalling is a crucial pathway that controls fundamental cellular processes, in particular, the response to cellular stresses such as DNA damage, reactive oxygen species and infection. In some pathogenic microbes the response to oxidative stress is controlled by a SirTM/zinc-containing macrodomain (Zn-Macro) pair responsible for establishment and removal of the modification, respectively. Targeting this defence mechanism against the host's innate immune response may lead to novel approaches to support the fight against emerging antimicrobial resistance. Earlier studies suggested that Zn-Macros play a key role in the activation of this defence. Therefore, we used phylogenetic, biochemical, and structural approaches to elucidate the functional properties of these enzymes. Using the substrate mimetic asparagine-ADP-ribose as well as the ADP-ribose product, we characterise the catalytic role of the zinc ion in the removal of the ADP-ribosyl modification. Furthermore, we determined structural properties that contribute to substrate selectivity within the different Zn-Macro branches. Together, our data not only give new insights into the Zn-Macro family but also highlight their distinct features that may be exploited for the development of future therapies.

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Epigenetic mechanisms, including histone post-translational modifications (PTMs), play a critical role in regulating pain perception and the pathophysiology of burn injury. However, the epigenetic regulation and molecular mechanisms underlying burn injury-induced pain remain insufficiently explored. Spinal dynorphinergic (Pdyn) neurons contribute to heat hyperalgesia induced by severe scalding-type burn injury through p-S10H3-dependent signaling. Beyond p-S10H3, burn injury may impact various other histone H3 PTMs. Double immunofluorescent staining and histone H3 protein analyses demonstrated significant hypermethylation at H3K4me1 and H3K4me3 sites and hyperphosphorylation at S10H3 within the spinal cord. By analyzing Pdyn neurons in the spinal dorsal horn, we found evidence of chromatin activation with a significant elevation in p-S10H3 immunoreactivity. We used RNA-seq analysis to compare the effects of burn injury and formalin-induced inflammatory pain on spinal cord transcriptomic profiles. We identified 98 DEGs for burn injury and 86 DEGs for formalin-induced inflammatory pain. A limited number of shared differentially expressed genes (DEGs) suggest distinct central pain processing mechanisms between burn injury and formalin models. KEGG pathway analysis supported this divergence, with burn injury activating Wnt signaling. This study enhances our understanding of burn injury mechanisms and uncovers converging and diverging pathways in pain models with different origins.

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Nitric oxide (NO) has been firmly established as a key signaling molecule in plants, playing a significant role in regulating growth, development and stress responses. Given the imperative of sustainable agriculture and the urgent need to meet the escalating global demand for food, it is imperative to safeguard crop plants from the effects of climate fluctuations. Plants respond to environmental challenges by producing redox molecules, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), which regulate cellular, physiological, and molecular processes. Nitric oxide (NO) plays a crucial role in plant stress tolerance, acting as a signaling molecule or free radical. NO is involved in various developmental processes in plants through diverse mechanisms. Exogenous NO supplementation can alleviate the toxicity of abiotic stresses and enhance plant resistance. In this review we summarize the studies regarding the production of NO in peroxisomes, and how its molecule and its derived products, (ONOO-) and S-nitrosoglutathione (GSNO) affect ROS metabolism in peroxisomes. Peroxisomal antioxidant enzymes including catalase (CAT), are key targets of NO-mediated post-translational modification (PTM) highlighting the dynamic metabolism of ROS and RNS in peroxisomes.

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Glycation is a protein post-translational modification that can occur on lysine and arginine residues as a result of a non-enzymatic process known as the Maillard reaction. This modification is irreversible, so the only way it can be removed is by protein degradation and replacement. Small reactive carbonyl species, glyoxal and methylglyoxal, are the primary glycating agents and are elevated in several conditions associated with an increased risk of cardiovascular disease, including diabetes, rheumatoid arthritis, smoking, and aging. Thus, how protein glycation impacts the cardiomyocyte is of particular interest, to both understand how these conditions increase the risk of cardiovascular disease and how glycation might be targeted therapeutically. Glycation can affect the cardiomyocyte through extracellular mechanisms, including RAGE-based signaling, glycation of the extracellular matrix that modifies the mechanical environment, and signaling from the vasculature. Intracellular glycation of the cardiomyocyte can impact calcium handling, protein quality control and cell death pathways, as well as the cytoskeleton, resulting in a blunted contractility. While reducing protein glycation and its impact on the heart has been an active area of drug development, multiple clinical trials have had mixed results and these compounds have not been translated to the clinic-highlighting the challenges of modulating myocyte glycation. Here we will review protein glycation and its effects on the cardiomyocyte, therapeutic attempts to reverse these, and offer insight as to the future of glycation studies and patient treatment.

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O-GlcNAc transferase (OGT) is the sole enzyme responsible for the post-translational modification of O-GlcNAc on thousands of target nucleocytoplasmic proteins. To date, nine variants of OGT that segregate with OGT Congenital Disorder of Glycosylation (OGT-CDG) have been reported and characterized. Numerous additional variants have been associated with OGT-CDG, some of which are currently undergoing investigation. This disorder primarily presents with global developmental delay and intellectual disability (ID), alongside other variable neurological features and subtle facial dysmorphisms in patients. Several hypotheses aim to explain the etiology of OGT-CDG, with a prominent hypothesis attributing the pathophysiology of OGT-CDG to mutations segregating with this disorder disrupting the OGT interactome. The OGT interactome consists of thousands of proteins, including substrates as well as interactors that require noncatalytic functions of OGT. A key aim in the field is to identify which interactors and substrates contribute to the primarily neural-specific phenotype of OGT-CDG. In this review, we will discuss the heterogenous phenotypic features of OGT-CDG seen clinically, the variable biochemical effects of mutations associated with OGT-CDG, and the use of animal models to understand this disorder. Furthermore, we will discuss how previously identified OGT interactors causal for ID provide mechanistic targets for investigation that could explain the dysregulated gene expression seen in OGT-CDG models. Identifying shared or unique altered pathways impacted in OGT-CDG patients will provide a better understanding of the disorder as well as potential therapeutic targets.

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Upon infection of host cells the Legionella pneumophila bacterium releases a multitude of effector enzymes into the host's cytoplasm that manipulate cellular host pathways, including the host-ubiquitination pathways. The effectors belonging to the SidE-family are involved in non-canonical phosphoribosyl serine ubiquitination (PR-ubiquitination) of host substrate proteins. This results in the recruitment of ER-remodeling proteins and the formation of a Legionella-containing vacuole which is crucial in the onset of legionnaires disease. PR-ubiquitination is a dynamic process reversed by other Legionella effectors called Dups. During PR-Ubiquitin phosphodiester hydrolysis Dups form a covalent intermediate with the phosphoribosyl ubiquitylated protein using its active site His67 residue. We envisioned that covalent probes to target Legionella effectors could be of value to study these effectors and contribute to deciphering the complex biology of Legionella infection. Hence we effectively installed a photo-activatable pyridinium warhead on the 5'-OH of triazole-linked ribosylated ubiquitin allowing crosslinking of the probe to the catalytic histidine residues in Legionella SidE or Dup enzymes. In vitro tests on recombinantly expressed DupA and SdeAPDE revealed that the probe was able to capture the enzymes covalently upon photo-activation.

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Ubiquitination is an essential post-translational modification mechanism involving the ubiquitin protein's bonding to a substrate protein. It is crucial in a variety of physiological activities including cell survival and differentiation, and innate and adaptive immunity. Any alteration in the ubiquitin system leads to the development of various human diseases. Numerous researches show the highly reversibility and dynamic of ubiquitin system, making the experimental identification quite difficult. To solve this issue, this article develops a model using a machine learning approach, tending to improve the ubiquitin protein prediction precisely. We deeply investigate the ubiquitination data that is proceed through different features extraction methods, followed by the classification. The evaluation and assessment are conducted considering Jackknife tests and 10-fold cross-validation. The proposed method demonstrated the remarkable performance in terms of 100 %, 99.88 %, and 99.84 % accuracy on Dataset-I, Dataset-II, and Dataset-III, respectively. Using Jackknife test, the method achieves 100 %, 99.91 %, and 99.99 % for Dataset-I, Dataset-II and Dataset-III, respectively. This analysis concludes that the proposed method outperformed the state-of-the-arts to identify the ubiquitination sites and helpful in the development of current clinical therapies. The source code and datasets will be made available at Github.

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Protein lysine lactylation, a recently discovered post-translational modification (PTM), is prevalent across tissues and cells of diverse species, serving as a regulator of glycolytic flux and biological metabolism. The yak (Bos grunniens), a species that has inhabited the Qinghai-Tibetan Plateau for millennia, has evolved intricate adaptive mechanisms to cope with the region's unique geographical and climatic conditions, exhibiting remarkable energy utilization and metabolic efficiency. Nonetheless, the specific landscape of lysine lactylation in yaks remains poorly understood. Herein, we present the first comprehensive lactylome profile of the yak, effectively identifying 421, 308, and 650 lactylated proteins in the heart, muscles, and liver, respectively. These lactylated proteins are involved in glycolysis/gluconeogenesis, the tricarboxylic acid cycle, oxidative phosphorylation, and metabolic process encompassing carbohydrates, lipids, and proteins during both anaerobic and aerobic glucose bio-oxidation, implying their crucial role in material and energy metabolism, as well as in maintaining homeostasis in yaks.

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Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

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Colorectal cancer is a leading cause of cancer-related mortality worldwide. Traditionally, colorectal cancer has been recognized as a disease caused by genetic mutations. However, recent studies have revealed the significant role of epigenetic alterations in the progression of colorectal cancer. Epithelial-mesenchymal transition, a critical step in cancer cell metastasis, has been found to be closely associated with the tumor microenvironment and immune factors, thereby playing a crucial role in many kinds of biological behaviors of cancers. In this review, we explored the impact of N6-methyladenosine and post-translational modifications (like methylation, acetylation, ubiquitination, SUMOylation, glycosylation, etc.) on the process of epithelial-mesenchymal transition in colorectal cancer and the epigenetic regulation for the transcription factors and pathways correlated to epithelial-mesenchymal transition. Furthermore, we emphasized that the complex regulation of epithelial-mesenchymal transition by epigenetics can provide new strategies for overcoming drug resistance and improving treatment outcomes. This review aims to provide important scientific evidence for the prevention and treatment of colorectal cancer based on epigenetic modifications.

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It is commonly known that different macrophage phenotypes play specific roles in different pathophysiological processes. In recent years, many studies have linked the phenotypes of macrophages to their characteristics in different metabolic pathways, suggesting that macrophages can perform different functions through metabolic reprogramming. It is now gradually recognized that lactate, previously overlooked as a byproduct of glycolytic metabolism, acts as a signaling molecule in regulating multiple biological processes, including immunological responses and metabolism. Recently, lactate has been found to mediate epigenetic changes in macrophages through a newfound lactylation modification, thereby regulating their phenotypic transformation. This novel finding highlights the significant role of lactate metabolism in macrophage function. In this review, we summarize the features of relevant metabolic reprogramming in macrophages and the role of lactate metabolism therein. We also review the progress of research on the regulation of macrophage metabolic reprogramming by lactylation through epigenetic mechanisms.

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Due to their capacity to mediate repetitive protein interactions, intrinsically disordered regions (IDRs) are crucial for the formation of various types of protein-RNA complexes. The functions of IDRs are strongly modulated by post-translational modifications (PTMs). Phosphorylation is the most common and well-studied modification of IDRs, which can alter homomeric or heteromeric interactions of proteins and impact their ability to phase separate. Moreover, phosphorylation can influence the RNA-binding properties of proteins, and recent studies demonstrated its selective impact on the global profiles of protein-RNA binding and regulation. These findings highlight the need for further integrative approaches to understand how signalling remodels protein-RNA networks in cells.

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In the vast landscape of human health, head and neck cancer (HNC) poses a significant health burden globally, necessitating the exploration of novel diagnostics and therapeutics. Metabolic alterations occurring within tumor microenvironment are crucial to understand the foundational cause of HNC. Post-translational modifications (PTMs) have recently emerged as a silent foe exerting a significantly heightened influence on various aspects of the biological processes associated with the onset and advancement of cancer, particularly in the context of HNC. There are numerous targets involved in HNC but recently, the enzyme pyruvate kinase M2 (PKM2) has come out as a hot target due to its involvement in glycolysis resulting in metabolic reprogramming of cancer cells. Various PTMs have been reported to affect the structure and function of PKM2 by modulating its activity. This review aims to investigate the impact of PTMs on the interaction between PKM2 and several signaling pathways and transcription factors in the context of HNC. These interactions possess significant ramification for cellular proliferation, apoptosis, angiogenesis and metastasis. This review primarily explores the role of PTMs influencing PKM2 and its involvement in tumor development. While acknowledging the significance of PKM2 interactions with other tumor regulators, the emphasis lies on dissecting PTM-related mechanisms rather than solely scrutinizing individual regulators. It lays the framework for the development of more sophisticated diagnostic tools and uncovers exciting possibilities for precision medicine essential for effectively addressing the complexity of this malignancy in a precise and focused manner.

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Processing food and feed challenges official control e.g. by modifying proteins, which leads to significant underestimation in targeted, MS-based protein quantification. Whereas numerous studies identified processing-induced changes on proteins in various combinations of matrices and processing conditions, studying their impact semi-quantitatively on specific protein sequences might unveil approaches to improve protein quantification accuracy. Thus, 335 post-translational modifications (e.g. oxidation, deamidation, carboxymethylation, Amadori, acrolein adduction) were identified by bottom-up proteomic analysis of 37 bovine materials relevant in food and feed (meat, bone, blood, milk) with varying processing degrees (raw, spray-dried, pressure-sterilized). To mimic protein recovery in a targeted analysis, peak areas of marker and reference peptides were compared to those of their modified versions, which revealed peptide-specific recoveries and variances across all samples. Detailed analysis suggests that incorporating two modified versions additionally to the unmodified marker may significantly improve quantification accuracy in targeted MS-based food and feed control in processed matrices.

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Human DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is crucial to the maintenance of genome integrity. The extreme infidelity of Polι and the fact that both its up- and down-regulation correlate with various cancers indicate that Polι expression and access to the replication fork should be strictly controlled. Here, we identify RNF2, an E3 ubiquitin ligase, as a new interacting partner of Polι that is responsible for Polι stabilization in vivo. Interestingly, while we report that RNF2 does not directly ubiquitinate Polι, inhibition of the E3 ubiquitin ligase activity of RNF2 affects the cellular level of Polι thereby protecting it from destabilization. Additionally, we indicate that this mechanism is more general, as DNA polymerase η, another Y-family polymerase and the closest paralogue of Polι, share similar features.

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APE2 plays important roles in the maintenance of genomic and epigenomic stability including DNA repair and DNA damage response. Accumulating evidence has suggested that APE2 is upregulated in multiple cancers at the protein and mRNA levels and that APE2 upregulation is correlative with higher and lower overall survival of cancer patients depending on tumor type. However, it remains unknown how APE2 protein abundance is maintained and regulated in cells. Here, we provide the first evidence of APE2 regulation via the posttranslational modification ubiquitin. APE2 is poly-ubiquitinated via K48-linked chains and degraded via the ubiquitin-proteasome system where K371 is the key residue within APE2 responsible for its ubiquitination and degradation. We further characterize MKRN3 as the E3 ubiquitin ligase for APE2 ubiquitination in cells and in vitro. In summary, this study offers the first definition of the APE2 proteostasis network and lays the foundation for future studies pertaining to the posttranslational modification regulation and functions of APE2 in genome integrity and cancer etiology/treatment.

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Inflammasomes serve as critical sensors for disruptions to cellular homeostasis, with inflammasome assembly leading to inflammatory caspase activation, gasdermin cleavage, and cytokine release. While the canonical pathways leading to priming, assembly, and pyroptosis are well characterized, recent work has begun to focus on the role of post-translational modifications (PTMs) in regulating inflammasome activity. A diverse array of PTMs, including phosphorylation, ubiquitination, SUMOylation, acetylation, and glycosylation, exert both activating and inhibitory influences on members of the inflammasome cascade through effects on protein-protein interactions, stability, and localization. Dysregulation of inflammasome activation is associated with a number of inflammatory diseases, and evidence is emerging that aberrant modification of inflammasome components contributes to this dysregulation. This review provides insight into PTMs within the NLRP3 inflammasome pathway and their functional consequences on the signaling cascade and highlights outstanding questions that remain regarding the complex web of signals at play.

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Aberrant expression of the pluripotency-associated transcription factor Sox2 is associated with poor prognosis in colorectal cancer (CRC). We investigated the regulatory roles of major post-translational modifications in Sox2 using two CRC cell lines, SW480 and SW620, derived from the same patient but with low and high Sox2 expression, respectively. Acetylation of K75 in the Sox2 nuclear export signal was relatively increased in SW480 cells and promotes Sox2 nucleocytoplasmic shuttling and proteasomal degradation of Sox2. LC-MS-based proteomics analysis identified HDAC4 and p300 as binding partners involved in the acetylation-mediated control of Sox2 expression in the nucleus. Sox2 K75 acetylation is mediated by the acetyltransferase activity of CBP/p300 and ACSS3. In SW620 cells, HDAC4 deacetylates K75 and is regulated by miR29a. O-GlcNAcylation on S246, in addition to K75 acetylation, also regulates Sox2 stability. These findings provide insights into the regulation of Sox2 through multiple post-translational modifications and pathways in CRC.

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Post-translational modifications (PTMs) of transcription factors regulate transcriptional activity and play a key role in essentially all biological processes and generate indispensable insight towards biological function including activity state, subcellular localization, protein solubility, protein folding, substrate trafficking, and protein-protein interactions. Amino acids modified chemically via PTMs, function as molecular switches and affect the protein function and characterization and increase the proteome complexity. Krüppel-like transcription factors (KLFs) control essential cellular processes including proliferation, differentiation, migration, programmed cell death and various cancer-relevant processes. We investigated the interactions of KLF group-2 members with their binding partners to assess the role of acetylation and phosphorylation in KLFs on their binding affinity. It was observed that acetylation and phosphorylation at different positions in KLFs have a variable effect on binding with specific partners. KLF2-EP300, KLF4-SP1, KLF6-ATF3, KLF6-JUN, and KLF7-JUN show stabilization upon acetylation or phosphorylation at variable positions. On the other hand, KLF4-CBP, KLF4-EP300, KLF5-CBP, KLF5-WWP1, KLF6-SP1, and KLF7-ATF3 show stabilization or destabilization due to acetylation or phosphorylation at variable positions in KLFs. This provides a molecular explanation of the experimentally observed dual role of KLF group-2 members as a suppressor or activator of cancers in a PTM-dependent manner.

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