Crosstalk between SUMOylation and other post-translational modifications in breast cancer.

Breast cancer represents the most prevalent tumor type and a foremost cause of mortality among women globally. The complex pathophysiological processes of breast cancer tumorigenesis and progression are regulated by protein post-translational modifications (PTMs), which are triggered by different carcinogenic factors and signaling pathways, with small ubiquitin-like modifier (SUMOylation) emerging as a particularly pivotal player in this context. Recent studies have demonstrated that SUMOylation does not act alone, but interacts with other PTMs, such as phosphorylation, ubiquitination, acetylation, and methylation, thereby leading to the regulation of various pathological activities in breast cancer. This review explores novel and existing mechanisms of crosstalk between SUMOylation and other PTMs. Typically, SUMOylation is regulated by phosphorylation to exert feedback control, while also modulates subsequent ubiquitination, acetylation, or methylation. The crosstalk pairs in promoting or inhibiting breast cancer are protein-specific and site-specific. In mechanism, alterations in amino acid side chain charges, protein conformations, or the occupation of specific sites at specific domains or sites underlie the complex crosstalk. In summary, this review centers on elucidating the crosstalk between SUMOylation and other PTMs in breast cancer oncogenesis and progression and discuss the molecular mechanisms contributing to these interactions, offering insights into their potential applications in facilitating novel treatments for breast cancer.

Native ChIP: Studying the Genome-Wide Distribution of Histone Modifications in Cells and Tissue.

For the genome-wide mapping of histone modifications, chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing remains the benchmark method. While crosslinked ChIP can be used for all kinds of targets, native ChIP is predominantly used for strong and direct DNA interactors like histones and their modifications. Here we describe a native ChIP protocol that can be used for cells and tissue material.

Single-Cell Histone Modification Profiling with Cell Enrichment Using sortChIC.

Histone post-translational modifications (PTMs) influence the overall structure of the chromatin and gene expression. Over the course of cell differentiation, the distribution of histone modifications is remodeled, resulting in cell type-specific patterns. In the past, their study was limited to abundant cell types that could be purified in necessary numbers. However, studying these cell type-specific dynamic changes in heterogeneous in vivo settings requires sensitive single-cell methods. Current advances in single-cell sequencing methods remove these limitations, allowing the study of nonpurifiable cell types. One complicating factor is that some of the most biologically interesting cell types, including stem and progenitor cells that undergo differentiation, only make up a small fraction of cells in a tissue. This makes whole-tissue analysis rather inefficient. In this chapter, we present a sort-assisted single-cell Chromatin ImmunoCleavage sequencing technique (sortChIC) to map histone PTMs in single cells. This technique combines the mapping of histone PTM location in combination with surface staining-based enrichment, to allow the integration of established strategies for rare cell type enrichment. In general terms, this will enable researchers to quantify local and global chromatin changes in dynamic complex biological systems and can provide additional information on their contribution to lineage and cell-type specification in physiological conditions and disease.

Epigenetic Regulation and Molecular Mechanisms of Burn Injury-Induced Nociception in the Spinal Cord of Mice.

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.

Emerging toolkits for decoding the co-occurrence of modified histones and chromatin proteins.

In eukaryotes, DNA is packaged into chromatin with the help of highly conserved histone proteins. Together with DNA-binding proteins, posttranslational modifications (PTMs) on these histones play crucial roles in regulating genome function, cell fate determination, inheritance of acquired traits, cellular states, and diseases. While most studies have focused on individual DNA-binding proteins, chromatin proteins, or histone PTMs in bulk cell populations, such chromatin features co-occur and potentially act cooperatively to accomplish specific functions in a given cell. This review discusses state-of-the-art techniques for the simultaneous profiling of multiple chromatin features in low-input samples and single cells, focusing on histone PTMs, DNA-binding, and chromatin proteins. We cover the origins of the currently available toolkits, compare and contrast their characteristic features, and discuss challenges and perspectives for future applications. Studying the co-occurrence of histone PTMs, DNA-binding proteins, and chromatin proteins in single cells will be central for a better understanding of the biological relevance of combinatorial chromatin features, their impact on genomic output, and cellular heterogeneity.

Posttranslationally modified self-peptides promote hypertension in mouse models.

Posttranslational modifications can enhance immunogenicity of self-proteins. In several conditions, including hypertension, systemic lupus erythematosus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here, we show that the murine class I major histocompatibility complex (MHC-I) variant H-2Db uniquely presents isoLG-modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins, including sodium glucose transporter 2, cadherin 16, Kelch domain-containing protein 7A, and solute carrier family 23, that are recognized by CD8+ T cells in tissues of hypertensive mice, induce T cell proliferation in vitro, and prime hypertension after adoptive transfer. Finally, we find patterns of isoLG-adducted antigen restriction in class I human leukocyte antigens that are similar to those in murine analogs. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.

The role of protein post-translational modifications in prostate cancer.

Involving addition of chemical groups or protein units to specific residues of the target protein, post-translational modifications (PTMs) alter the charge, hydrophobicity, and conformation of a protein, which in turn influences protein function, protein-protein interaction, and protein aggregation. These alterations, which include phosphorylation, glycosylation, ubiquitination, methylation, acetylation, lipidation, and lactylation, are significant biological events in the development of cancer, and play vital roles in numerous biological processes. The processes behind essential functions, the screening of clinical illness signs, and the identification of therapeutic targets all depend heavily on further research into the PTMs. This review outlines the influence of several PTM types on prostate cancer (PCa) diagnosis, therapy, and prognosis in an effort to shed fresh light on the molecular causes and progression of the disease.

HSP90 multi-functionality in cancer.

The 90-kDa heat shock proteins (HSP90s) are molecular chaperones essential for folding, unfolding, degradation and activity of a wide range of client proteins. HSP90s and their cognate co-chaperones are subject to various post-translational modifications, functional consequences of which are not fully understood in cancer. Intracellular and extracellular HSP90 family members (HSP90α, HSP90ß, GRP94 and TRAP1) promote cancer by sustaining various hallmarks of cancer, including cell death resistance, replicative immortality, tumor immunity, angiogenesis, invasion and metastasis. Given the importance of HSP90 in tumor progression, various inhibitors and HSP90-based vaccines were developed for the treatment of cancer. Further understanding of HSP90 functions in cancer may provide new opportunities and novel therapeutic strategies for the treatment of cancer.

Dissecting the functional behavior of the differentially phosphorylated prolyl isomerase, Pin1.

Protein post-translational modifications (PTMs) play an intricate role in a diverse range of cellular processes creating a complex PTM code that governs cell homeostasis. Understanding the molecular build-up and the critical factors regulating this PTM code is essential for targeted therapeutic design whereby PTM mis-regulation is prevalent. Here, we focus on Pin1, a peptidyl-prolyl cis-trans isomerase whose regulatory function is altered by a diverse range of PTMs. Through employing advanced mass spectrometry techniques in combination with fluorescence polarization and enzyme activity assays, we elucidate the impact of combinatorial phosphorylation on Pin1 function. Moreover, two phosphorylation sites were identified whereby Ser71 phosphorylation preceded Ser16 phosphorylation, leading to the deactivation of Pin1's prolyl isomerase activity before affecting substrate binding. Together, these findings shed light on the regulatory mechanisms underlying Pin1 function and emphasize the importance of understanding PTM landscapes in health and disease.

A protein O-GlcNAc glycosyltransferase regulates the antioxidative response in Yersinia pestis.

Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins is commonly associated with a variety of stress responses and cellular processes in eukaryotes, but its potential roles in bacteria are unclear. Here, we show that protein HmwC acts as an O-GlcNAc transferase (OGT) responsible for O-GlcNAcylation of multiple proteins in Yersinia pestis, a flea-borne pathogen responsible for plague. We identify 64 O-GlcNAcylated proteins (comprising 65 sites) with differential abundance under conditions mimicking the mammalian host (Mh) and flea vector (Fv) environments. Deletion of hmwC, encoding a putative OGT, structurally distinct from any existing member of the GT41 family, results in reduced O-GlcNAcylation, reduced growth, and alterations in virulence properties and survival under stress. Purified HmwC can modify target proteins in vitro using UDP-GlcNAc as sugar donor. One of the target proteins, OsdY, promotes Y. pestis survival under oxidative stress conditions. Thus, our results support that regulation of antioxidative responses through O-GlcNAcylation may be a conserved process shared by prokaryotes and eukaryotes.

Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle.

Protein post-translational modifications (PTMs) are crucial for cancer cells to adapt to hypoxia; however, the functional significance of lysine crotonylation (Kcr) in hypoxia remains unclear. Herein we report a quantitative proteomics analysis of global crotonylome under normoxia and hypoxia, and demonstrate 128 Kcr site alterations across 101 proteins in MDA-MB231 cells. Specifically, we observe a significant decrease in K131cr, K156cr and K220cr of phosphoglycerate kinase 1 (PGK1) upon hypoxia. Enoyl-CoA hydratase 1 (ECHS1) is upregulated and interacts with PGK1, leading to the downregulation of PGK1 Kcr under hypoxia. Abolishment of PGK1 Kcr promotes glycolysis and suppresses mitochondrial pyruvate metabolism by activating pyruvate dehydrogenase kinase 1 (PDHK1). A low PGK1 K131cr level is correlated with malignancy and poor prognosis of breast cancer. Our findings show that PGK1 Kcr is a signal in coordinating glycolysis and the tricarboxylic acid (TCA) cycle and may serve as a diagnostic indicator for breast cancer.

Mechanistic insights into lanthipeptide modification by a distinct subclass of LanKC enzyme that forms dimers.

Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a "negative cooperativity" mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes.

Glycosylation: mechanisms, biological functions and clinical implications.

Protein post-translational modification (PTM) is a covalent process that occurs in proteins during or after translation through the addition or removal of one or more functional groups, and has a profound effect on protein function. Glycosylation is one of the most common PTMs, in which polysaccharides are transferred to specific amino acid residues in proteins by glycosyltransferases. A growing body of evidence suggests that glycosylation is essential for the unfolding of various functional activities in organisms, such as playing a key role in the regulation of protein function, cell adhesion and immune escape. Aberrant glycosylation is also closely associated with the development of various diseases. Abnormal glycosylation patterns are closely linked to the emergence of various health conditions, including cancer, inflammation, autoimmune disorders, and several other diseases. However, the underlying composition and structure of the glycosylated residues have not been determined. It is imperative to fully understand the internal structure and differential expression of glycosylation, and to incorporate advanced detection technologies to keep the knowledge advancing. Investigations on the clinical applications of glycosylation focused on sensitive and promising biomarkers, development of more effective small molecule targeted drugs and emerging vaccines. These studies provide a new area for novel therapeutic strategies based on glycosylation.

The role of acetylation in obesity-induced cardiac metabolic alterations.

Obesity is a growing public health problem, with its prevalence rate having tripled in the last five decades. It has been shown that obesity is associated with alterations in cardiac energy metabolism, which in turn plays a significant role in heart failure development. During obesity, the heart becomes highly dependent on fatty acid oxidation as its primary source of energy (ATP), while the contribution from glucose oxidation significantly decreases. This metabolic inflexibility is associated with reduced cardiac efficiency and contractile dysfunction. Although it is well recognized that alterations in cardiac energy metabolism during obesity are associated with the risk of heart failure development, the molecular mechanisms controlling these metabolic changes are not fully understood. Recently, posttranslational protein modifications of metabolic enzymes have been shown to play a crucial role in cardiac energy metabolic changes seen in obesity. Understanding these novel mechanisms is important in developing new therapeutic options to treat or prevent cardiac metabolic alteration and dysfunction in obese individuals. This review discusses posttranslational acetylation changes during obesity and their roles in mediating cardiac energy metabolic perturbations during obesity as well as its therapeutic potentials.

Deciphering the dynamics: Exploring the impact of mechanical forces on histone acetylation.

Living cells navigate a complex landscape of mechanical cues that influence their behavior and fate, originating from both internal and external sources. At the molecular level, the translation of these physical stimuli into cellular responses relies on the intricate coordination of mechanosensors and transducers, ultimately impacting chromatin compaction and gene expression. Notably, epigenetic modifications on histone tails govern the accessibility of gene-regulatory sites, thereby regulating gene expression. Among these modifications, histone acetylation emerges as particularly responsive to the mechanical microenvironment, exerting significant control over cellular activities. However, the precise role of histone acetylation in mechanosensing and transduction remains elusive due to the complexity of the acetylation network. To address this gap, our aim is to systematically explore the key regulators of histone acetylation and their multifaceted roles in response to biomechanical stimuli. In this review, we initially introduce the ubiquitous force experienced by cells and then explore the dynamic alterations in histone acetylation and its associated co-factors, including HDACs, HATs, and acetyl-CoA, in response to these biomechanical cues. Furthermore, we delve into the intricate interactions between histone acetylation and mechanosensors/mechanotransducers, offering a comprehensive analysis. Ultimately, this review aims to provide a holistic understanding of the nuanced interplay between histone acetylation and mechanical forces within an academic framework.

Post-translational modification prediction via prompt-based fine-tuning of a GPT-2 model.

Post-translational modifications (PTMs) are pivotal in modulating protein functions and influencing cellular processes like signaling, localization, and degradation. The complexity of these biological interactions necessitates efficient predictive methodologies. In this work, we introduce PTMGPT2, an interpretable protein language model that utilizes prompt-based fine-tuning to improve its accuracy in precisely predicting PTMs. Drawing inspiration from recent advancements in GPT-based architectures, PTMGPT2 adopts unsupervised learning to identify PTMs. It utilizes a custom prompt to guide the model through the subtle linguistic patterns encoded in amino acid sequences, generating tokens indicative of PTM sites. To provide interpretability, we visualize attention profiles from the model's final decoder layer to elucidate sequence motifs essential for molecular recognition and analyze the effects of mutations at or near PTM sites to offer deeper insights into protein functionality. Comparative assessments reveal that PTMGPT2 outperforms existing methods across 19 PTM types, underscoring its potential in identifying disease associations and drug targets.

CHHM: a Manually Curated Catalogue of Human Histone Modifications Revealing Hotspot Regions and Unique Distribution Patterns.

Histone modification is one of the key elements in epigenetic control and plays important roles in regulation of biological processes and disease development. Currently, records of human histone modifications with various levels of confidence in evidence are scattered in various knowledgebases and databases. In the present study, a curated catalogue of human histone modifications, CHHM, was obtained by manual retrieval, evidence assessment, and integration of modification records from 10 knowledgebases/databases and 3 complementary articles. CHHM contains 6612 nonredundant modification entries covering 31 types of modifications (including 9 types of emerging modifications) and 2 types of histone-DNA crosslinks, that were identified in 11 H1 variants, 21 H2A variants, 21 H2B variants, 9 H3 variants, and 2 H4 variants. For ease of visualization and accessibility, modification entries are presented with aligned protein sequences in an Excel file. Confidence level in evidence is provided for each entry. Acylation modifications contribute to the highest number of modification entries in CHHM. This supports that cellular metabolic status plays a very important role in epigenetic control. CHHM reveals modification hotspot regions and uneven distribution of the modification entries across the histone families. Such uneven distribution may suggest that a particular histone family is more susceptible to certain types of modifications. CHHM not only serves as an important and user-friendly resource for biomedical and clinical researches involving histone modifications and transcriptional regulation, but also provides new insights for basic researches in the mechanism of human histone modifications and epigenetic control.

Methyl-CpG-binding 2 K271 lactylation-mediated M2 macrophage polarization inhibits atherosclerosis.

Rationale: Posttranslational modifications of proteins have not been addressed in studies aimed at elucidating the cardioprotective effect of exercise in atherosclerotic cardiovascular disease (ASCVD). In this study, we reveal a novel mechanism by which exercise ameliorates atherosclerosis via lactylation. Methods: Using ApoE-/- mice in an exercise model, proteomics analysis was used to identify exercise-induced specific lactylation of MeCP2 at lysine 271 (K271). Mutation of the MeCP2 K271 lactylation site in aortic plaque macrophages was achieved by recombinant adenoviral transfection. Explore the molecular mechanisms by which motility drives MeCP2 K271 lactylation to improve plaque stability using ATAC-Seq, CUT &Tag and molecular biology. Validation of the potential target RUNX1 for exercise therapy using Ro5-3335 pharmacological inhibition. Results: we showed that in ApoE-/- mice, methyl-CpG-binding protein 2 (MeCP2) K271 lactylation was observed in aortic root plaque macrophages, promoting pro-repair M2 macrophage polarization, reducing the plaque area, shrinking necrotic cores, reducing plaque lipid deposition, and increasing collagen content. Adenoviral transfection, by introducing a mutant at lysine 271, overexpressed MeCP2 K271 lactylation, which enhanced exercise-induced M2 macrophage polarization and increased plaque stability. Mechanistically, the exercise-induced atheroprotective effect requires an interaction between MeCP2 K271 lactylation and H3K36me3, leading to increased chromatin accessibility and transcriptional repression of RUNX1. In addition, the pharmacological inhibition of the transcription factor RUNX1 exerts atheroprotective effects by promoting the polarization of plaque macrophages towards the pro-repair M2 phenotype. Conclusions: These findings reveal a novel mechanism by which exercise ameliorates atherosclerosis via MeCP2 K271 lactylation-H3K36me3/RUNX1. Interventions that enhance MeCP2 K271 lactylation have been shown to increase pro-repair M2 macrophage infiltration, thereby promoting plaque stabilization and reducing the risk of atherosclerotic cardiovascular disease. We also established RUNX1 as a potential drug target for exercise therapy, thereby providing guidance for the discovery of new targets.

Mass spectrometry imaging of SOD1 protein-metal complexes in SOD1G93A transgenic mice implicates demetalation with pathology.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons in the central nervous system (CNS). Mutations in the metalloenzyme SOD1 are associated with inherited forms of ALS and cause a toxic gain of function thought to be mediated by dimer destabilization and misfolding. SOD1 binds two Cu and two Zn ions in its homodimeric form. We have applied native ambient mass spectrometry imaging to visualize the spatial distributions of intact metal-bound SOD1G93A complexes in SOD1G93A transgenic mouse spinal cord and brain sections and evaluated them against disease pathology. The molecular specificity of our approach reveals that metal-deficient SOD1G93A species are abundant in CNS structures correlating with ALS pathology whereas fully metalated SOD1G93A species are homogenously distributed. Monomer abundance did not correlate with pathology. We also show that the dimer-destabilizing post-translational modification, glutathionylation, has limited influence on the spatial distribution of SOD1 dimers.

An improved SNAP-ADAR tool enables efficient RNA base editing to interfere with post-translational protein modification.

RNA base editing relies on the introduction of adenosine-to-inosine changes into target RNAs in a highly programmable manner in order to repair disease-causing mutations. Here, we propose that RNA base editing could be broadly applied to perturb protein function by removal of regulatory phosphorylation and acetylation sites. We demonstrate the feasibility on more than 70 sites in various signaling proteins and identify key determinants for high editing efficiency and potent down-stream effects. For the JAK/STAT pathway, we demonstrate both, negative and positive regulation. To achieve high editing efficiency over a broad codon scope, we applied an improved version of the SNAP-ADAR tool. The transient nature of RNA base editing enables the comparably fast (hours to days), dose-dependent (thus partial) and reversible manipulation of regulatory sites, which is a key advantage over DNA (base) editing approaches. In summary, PTM interference might become a valuable field of application of RNA base editing.