Liquid-liquid phase separation in innate immunity.

Intrinsic and innate immune responses are essential lines of defense in the body's constant surveillance of pathogens. The discovery of liquid-liquid phase separation (LLPS) as a key regulator of this primal response to infection brings an updated perspective to our understanding of cellular defense mechanisms. Here, we review the emerging multifaceted role of LLPS in diverse aspects of mammalian innate immunity, including DNA and RNA sensing and inflammasome activity. We discuss the intricate regulation of LLPS by post-translational modifications (PTMs), and the subversive tactics used by viruses to antagonize LLPS. This Review, therefore, underscores the significance of LLPS as a regulatory node that offers rapid and plastic control over host immune signaling, representing a promising target for future therapeutic strategies.

The logic of protein post-translational modifications (PTMs): Chemistry, mechanisms and evolution of protein regulation through covalent attachments.

Protein post-translational modifications (PTMs) play a crucial role in all cellular functions by regulating protein activity, interactions and half-life. Despite the enormous diversity of modifications, various PTM systems show parallels in their chemical and catalytic underpinnings. Here, focussing on modifications that involve the addition of new elements to amino-acid sidechains, I describe historical milestones and fundamental concepts that support the current understanding of PTMs. The historical survey covers selected key research programmes, including the study of protein phosphorylation as a regulatory switch, protein ubiquitylation as a degradation signal and histone modifications as a functional code. The contribution of crucial techniques for studying PTMs is also discussed. The central part of the essay explores shared chemical principles and catalytic strategies observed across diverse PTM systems, together with mechanisms of substrate selection, the reversibility of PTMs by erasers and the recognition of PTMs by reader domains. Similarities in the basic chemical mechanism are highlighted and their implications are discussed. The final part is dedicated to the evolutionary trajectories of PTM systems, beginning with their possible emergence in the context of rivalry in the prokaryotic world. Together, the essay provides a unified perspective on the diverse world of major protein modifications.

Lamin post-translational modifications: emerging toggles of nuclear organization and function.

Nuclear lamins are ancient type V intermediate filaments with diverse functions that include maintaining nuclear shape, mechanosignaling, tethering and stabilizing chromatin, regulating gene expression, and contributing to cell cycle progression. Despite these numerous roles, an outstanding question has been how lamins are regulated. Accumulating work indicates that a range of lamin post-translational modifications (PTMs) control their functions both in homeostatic cells and in disease states such as progeria, muscular dystrophy, and viral infection. Here, we review the current knowledge of the diverse types of PTMs that regulate lamins in a site-specific manner. We highlight methods that can be used to characterize lamin PTMs whose functions are currently unknown and provide a perspective on the future of the lamin PTM field.

The centriolar tubulin code.

Centrioles are microtubule-based cell organelles present in most eukaryotes. They participate in the control of cell division as part of the centrosome, the major microtubule-organizing center of the cell, and are also essential for the formation of primary and motile cilia. During centriole assembly as well as across its lifetime, centriolar tubulin display marks defined by post-translational modifications (PTMs), such as glutamylation or acetylation. To date, the functions of these PTMs at centrioles are not well understood, although pioneering experiments suggest a role in the stability of this organelle. Here, we review the current knowledge regarding PTMs at centrioles with a particular focus on a possible link between these modifications and centriole's architecture, and propose possible hypothesis regarding centriolar tubulin PTMs's function.

Tubulin post-translational modifications in meiosis.

Haploid gametes are produced from diploid parents through meiosis, a process inherent to all sexually reproducing eukaryotes. Faithful chromosome segregation in meiosis is essential for reproductive success, although it is less clear how the meiotic spindle achieves this compared to the mitotic spindle. It is becoming increasingly clear that tubulin post-translational modifications (PTMs) play critical roles in regulating microtubule functions in many biological processes, and meiosis is no exception. Here, I review recent advances in the understanding of tubulin PTMs in meiotic spindles, especially focusing on their roles in spindle integrity, oocyte aging, and non-Mendelian transmission.

Exploring the dynamic landscape of immunopeptidomics: Unravelling posttranslational modifications and navigating bioinformatics terrain.

Immunopeptidomics is becoming an increasingly important field of study. The capability to identify immunopeptides with pivotal roles in the human immune system is essential to shift the current curative medicine towards personalized medicine. Throughout the years, the field has matured, giving insight into the current pitfalls. Nowadays, it is commonly accepted that generalizing shotgun proteomics workflows is malpractice because immunopeptidomics faces numerous challenges. While many of these difficulties have been addressed, the road towards the ideal workflow remains complicated. Although the presence of Posttranslational modifications (PTMs) in the immunopeptidome has been demonstrated, their identification remains highly challenging despite their significance for immunotherapies. The large number of unpredictable modifications in the immunopeptidome plays a pivotal role in the functionality and these challenges. This review provides a comprehensive overview of the current advancements in immunopeptidomics. We delve into the challenges associated with identifying PTMs within the immunopeptidome, aiming to address the current state of the field.

Oxygen-regulated post-translation modifications as master signalling pathway in cells.

Oxygen is essential for viability in mammalian organisms. However, cells are often exposed to changes in oxygen availability, due to either increased demand or reduced oxygen supply, herein called hypoxia. To be able to survive and/or adapt to hypoxia, cells activate a variety of signalling cascades resulting in changes to chromatin, gene expression, metabolism and viability. Cellular signalling is often mediated via post-translational modifications (PTMs), and this is no different in response to hypoxia. Many enzymes require oxygen for their activity and oxygen can directly influence several PTMS. Here, we review the direct impact of changes in oxygen availability on PTMs such as proline, asparagine, histidine and lysine hydroxylation, lysine and arginine methylation and cysteine dioxygenation, with a focus on mammalian systems. In addition, indirect hypoxia-dependent effects on phosphorylation, ubiquitination and sumoylation will also be discussed. Direct and indirect oxygen-regulated changes to PTMs are coordinated to achieve the cell's ultimate response to hypoxia. However, specific oxygen sensitivity and the functional relevance of some of the identified PTMs still require significant research.

Post-translational Modifications of Key Machinery in the Control of Mitophagy.

Mitophagy refers to the process of selective removal of damaged or superfluous mitochondria via the autophagy/lysosome pathway. In the past decade the molecular mechanisms underlying mitophagy have been extensively studied. It is now well established that the key mitophagy machinery undergoes extensive post-translational modifications (PTMs) such as phosphorylation/dephosphorylation, ubiquitination/deubiquitination, and acetylation/deacetylation that involve an array of enzymes including protein kinases/phosphatases, E3 ligases/deubiquitinases, acetyltransferases/deacetylases. In this review we provide a systematic summary of these key PTMs, and discuss the effectors and the functional implications of such PTMs in mitophagy-related diseases. Understanding PTM of the mitophagy machinery offers a unique window of opportunity for the discovery of novel mitophagy interventional strategies and for the control of mitophagy-related diseases.

Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic.

Protein SUMOylation is a ubiquitylation-like post-translational modification (PTM) that is synthesized through an enzymatic cascade involving an E1 (SAE1:SAE2), an E2 (UBC9), and various E3 enzymes. In the final step of this process, the small ubiquitin-like modifier (SUMO) is transferred from the UBC9∼SUMO thioester onto a lysine residue of a protein substrate. This reaction can be accelerated by an E3 ligase. As the UBC9∼SUMO thioester is chemically unstable, a stable mimetic is desirable for structural studies of UBC9∼SUMO alone and in complex with a substrate and/or an E3 ligase. Recently, a strategy for generating a mimetic of the yeast E2∼SUMO thioester by mutating alanine 129 of Ubc9 to a lysine has been reported. Here, we reproduce and further investigate this approach using the human SUMOylation system and characterize the resulting mimetic of human UBC9∼SUMO1. We show that substituting lysine for alanine 129, but not for other active-site UBC9 residues, results in a UBC9 variant that is efficiently auto-SUMOylated. The auto-modification is dependent on cysteine 93 of UBC9, suggesting that it proceeds via this residue, through the same pathway as that for SUMOylation of substrates. The process is also partially dependent on aspartate 127 of UBC9 and accelerated by high pH, highlighting the importance of the substrate lysine protonation state for efficient SUMOylation. Finally, we present the crystal structure of the UBC9-SUMO1 molecule, which reveals the mimetic in an open conformation and its polymerization via the noncovalent SUMO-binding site on UBC9. Similar interactions could regulate UBC9∼SUMO in some cellular contexts.

Bidirectional regulation between AP-1 and SUMOylation pathway genes modulates inflammatory signaling during Salmonella infection.

Post-translational modifications (PTMs), such as SUMOylation, are known to modulate fundamental processes of a cell. Infectious agents such as Salmonella Typhimurium (STm), which causes gastroenteritis, utilize the PTM mechanism SUMOylation to hijack the host cell. STm suppresses host SUMO pathway genes UBC9 (also known as UBE2I) and PIAS1 to perturb SUMOylation for an efficient infection. In the present study, the regulation of SUMO pathway genes during STm infection was investigated. A direct binding of c-Fos (encoded by FOS), a component of activator protein-1 (AP-1), to promoters of both UBC9 and PIAS1 was observed. Experimental perturbation of c-Fos led to changes in the expression of both UBC9 and PIAS1. STm infection of fibroblasts with SUMOylation-deficient c-Fos (c-FOS-KOSUMO-def-FOS) resulted in uncontrolled activation of target genes, leading to massive immune activation. Infection of c-FOS-KOSUMO-def-FOS cells favored STm replication, indicating misdirected immune mechanisms. Finally, chromatin immunoprecipitation assays confirmed a context-dependent differential binding and release of AP-1 to and from target genes due to its phosphorylation and SUMOylation, respectively. Overall, our data point towards the existence of a bidirectional cross-talk between c-Fos and the SUMO pathway and highlight their importance in AP-1 function in STm infection and beyond. This article has an associated First Person interview with the first author of the paper.

Virus hijacking of host epigenetic machinery to impair immune response.

Epigenetic modifications, such as DNA hypermethylation, histone acetylation/methylation, or nucleosome positioning, result in differential gene expression. These modifications can have an impact on various pathways, including host antiviral immune responses. In this review, we summarize the current understanding of epigenetic modifications induced by viruses to counteract host antiviral immune responses, which are crucial for establishing and maintaining infection of viruses. Finally, we provide insights into the potential use of epigenetic modulators in combating viral infections and virus-induced diseases.

MLysPRED: graph-based multi-view clustering and multi-dimensional normal distribution resampling techniques to predict multiple lysine sites.

Posttranslational modification of lysine residues, K-PTM, is one of the most popular PTMs. Some lysine residues in proteins can be continuously or cascaded covalently modified, such as acetylation, crotonylation, methylation and succinylation modification. The covalent modification of lysine residues may have some special functions in basic research and drug development. Although many computational methods have been developed to predict lysine PTMs, up to now, the K-PTM prediction methods have been modeled and learned a single class of K-PTM modification. In view of this, this study aims to fill this gap by building a multi-label computational model that can be directly used to predict multiple K-PTMs in proteins. In this study, a multi-label prediction model, MLysPRED, is proposed to identify multiple lysine sites using features generated from human protein sequences. In MLysPRED, three kinds of multi-label sequence encoding algorithms (MLDBPB, MLPSDAAP, MLPSTAAP) are proposed and combined with three encoding strategies (CHHAA, DR and Kmer) to convert preprocessed lysine sequences into effective numerical features. A multidimensional normal distribution oversampling technique and graph-based multi-view clustering under-sampling algorithm were first proposed and incorporated to reduce the proportion of the original training samples, and multi-label nearest neighbor algorithm is used for classification. It is observed that MLysPRED achieved an Aiming of 92.21%, Coverage of 94.98%, Accuracy of 89.63%, Absolute-True of 81.46% and Absolute-False of 0.0682 on the independent datasets. Additionally, comparison of results with five existing predictors also indicated that MLysPRED is very promising and encouraging to predict multiple K-PTMs in proteins. For the convenience of the experimental scientists, 'MLysPRED' has been deployed as a user-friendly web-server at http://47.100.136.41:8181.

Adapt-Kcr: a novel deep learning framework for accurate prediction of lysine crotonylation sites based on learning embedding features and attention architecture.

Protein lysine crotonylation (Kcr) is an important type of posttranslational modification that is associated with a wide range of biological processes. The identification of Kcr sites is critical to better understanding their functional mechanisms. However, the existing experimental techniques for detecting Kcr sites are cost-ineffective, to a great need for new computational methods to address this problem. We here describe Adapt-Kcr, an advanced deep learning model that utilizes adaptive embedding and is based on a convolutional neural network together with a bidirectional long short-term memory network and attention architecture. On the independent testing set, Adapt-Kcr outperformed the current state-of-the-art Kcr prediction model, with an improvement of 3.2% in accuracy and 1.9% in the area under the receiver operating characteristic curve. Compared to other Kcr models, Adapt-Kcr additionally had a more robust ability to distinguish between crotonylation and other lysine modifications. Another model (Adapt-ST) was trained to predict phosphorylation sites in SARS-CoV-2, and outperformed the equivalent state-of-the-art phosphorylation site prediction model. These results indicate that self-adaptive embedding features perform better than handcrafted features in capturing discriminative information; when used in attention architecture, this could be an effective way of identifying protein Kcr sites. Together, our Adapt framework (including learning embedding features and attention architecture) has a strong potential for prediction of other protein posttranslational modification sites.

Musashi-2 potentiates colorectal cancer immune infiltration by regulating the post-translational modifications of HMGB1 to promote DCs maturation and migration.

Post-translational modifications (PTMs) of the non-histone protein high-mobility group protein B1 (HMGB1) are involved in modulating inflammation and immune responses. Recent studies have implicated that the RNA-binding protein (RBP) Musashi-2 (MSI2) regulates multiple critical biological metabolic and immunoregulatory functions. However, the precise role of MSI2 in regulating PTMs and tumor immunity in colorectal cancer (CRC) remains unclear. Here, we present data indicating that MSI2 potentiates CRC immunopathology in colitis-associated colon cancer (CAC) mouse models, cell lines and clinical specimens, specifically via HMGB1-mediated dendritic cell (DC) maturation and migration, further contributes to the infiltration of CD4+ and CD8+ T cells and inflammatory responses. Under stress conditions, MSI2 can exacerbate the production, nucleocytoplasmic transport and extracellular release of damage-associated molecular patterns (DAMPs)-HMGB1 in CRC cells. Mechanistically, MSI2 mainly enhances the disulfide HMGB1 production and protein translation via direct binding to nucleotides 1403-1409 in the HMGB1 3' UTR, and interacts with the cytoplasmic acetyltransferase P300 to upregulate its expression, further promoting the acetylation of K29 residue in HMGB1, thus leading to K29-HMGB1 nucleocytoplasmic translocation and extracellular release. Furthermore, blocking HMGB1 activity with glycyrrhizic acid (Gly) attenuates MSI2-mediated immunopathology and immune infiltration in CRC in vitro and in vivo. Collectively, this study suggests that MSI2 may improve the prognosis of CRC patients by reprogramming the tumor immune microenvironment (TIME) through HMGB1-mediated PTMs, which might be a novel therapeutic option for CRC immunotherapy.

Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis.

Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.

Beyond the Usual Suspects: Examining the Role of Understudied Histone Variants in Breast Cancer.

The incorporation of histone variants has structural ramifications on nucleosome dynamics and stability. Due to their unique sequences, histone variants can alter histone-histone or histone-DNA interactions, impacting the folding of DNA around the histone octamer and the overall higher-order structure of chromatin fibers. These structural modifications alter chromatin compaction and accessibility of DNA by transcription factors and other regulatory proteins to influence gene regulatory processes such as DNA damage and repair, as well as transcriptional activation or repression. Histone variants can also generate a unique interactome composed of histone chaperones and chromatin remodeling complexes. Any of these perturbations can contribute to cellular plasticity and the progression of human diseases. Here, we focus on a frequently overlooked group of histone variants lying within the four human histone gene clusters and their contribution to breast cancer.

Thiol redox proteomics: Characterization of thiol-based post-translational modifications.

Redox post-translational modifications on cysteine thiols (redox PTMs) have profound effects on protein structure and function, thus enabling regulation of various biological processes. Redox proteomics approaches aim to characterize the landscape of redox PTMs at the systems level. These approaches facilitate studies of condition-specific, dynamic processes implicating redox PTMs and have furthered our understanding of redox signaling and regulation. Mass spectrometry (MS) is a powerful tool for such analyses which has been demonstrated by significant advances in redox proteomics during the last decade. A group of well-established approaches involves the initial blocking of free thiols followed by selective reduction of oxidized PTMs and subsequent enrichment for downstream detection. Alternatively, novel chemoselective probe-based approaches have been developed for various redox PTMs. Direct detection of redox PTMs without any enrichment has also been demonstrated given the sensitivity of contemporary MS instruments. This review discusses the general principles behind different analytical strategies and covers recent advances in redox proteomics. Several applications of redox proteomics are also highlighted to illustrate how large-scale redox proteomics data can lead to novel biological insights.

Top-down proteomics of myosin light chain isoforms define chamber-specific expression in the human heart.

Myosin functions as the "molecular motor" of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an 'atrial' and 'ventricular' isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v (gene: MYL2), in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v (MYL3) and MLC-2a (MYL7) were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Moreover, we found elevated MLC-2 phosphorylation in male hearts compared to female hearts across each cardiac chamber. Overall, top-down proteomics allowed an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

Heme Interactions as Regulators of the Alternative Pathway Complement Responses and Implications for Heme-Associated Pathologies.

Heme (Fe2+-protoporphyrin IX) is a pigment of life, and as a prosthetic group in several hemoproteins, it contributes to diverse critical cellular processes. While its intracellular levels are tightly regulated by networks of heme-binding proteins (HeBPs), labile heme can be hazardous through oxidative processes. In blood plasma, heme is scavenged by hemopexin (HPX), albumin and several other proteins, while it also interacts directly with complement components C1q, C3 and factor I. These direct interactions block the classical pathway (CP) and distort the alternative pathway (AP). Errors or flaws in heme metabolism, causing uncontrolled intracellular oxidative stress, can lead to several severe hematological disorders. Direct interactions of extracellular heme with alternative pathway complement components (APCCs) may be implicated molecularly in diverse conditions at sites of abnormal cell damage and vascular injury. In such disorders, a deregulated AP could be associated with the heme-mediated disruption of the physiological heparan sulphate-CFH coat of stressed cells and the induction of local hemostatic responses. Within this conceptual frame, a computational evaluation of HBMs (heme-binding motifs) aimed to determine how heme interacts with APCCs and whether these interactions are affected by genetic variation within putative HBMs. Combined computational analysis and database mining identified putative HBMs in all of the 16 APCCs examined, with 10 exhibiting disease-associated genetic (SNPs) and/or epigenetic variation (PTMs). Overall, this article indicates that among the pleiotropic roles of heme reviewed, the interactions of heme with APCCs could induce differential AP-mediated hemostasis-driven pathologies in certain individuals.

iCysMod: an integrative database for protein cysteine modifications in eukaryotes.

As important post-translational modifications, protein cysteine modifications (PCMs) occurring at cysteine thiol group play critical roles in the regulation of various biological processes in eukaryotes. Due to the rapid advancement of high-throughput proteomics technologies, a large number of PCM events have been identified but remain to be curated. Thus, an integrated resource of eukaryotic PCMs will be useful for the research community. In this work, we developed an integrative database for protein cysteine modifications in eukaryotes (iCysMod), which curated and hosted 108 030 PCM events for 85 747 experimentally identified sites on 31 483 proteins from 48 eukaryotes for 8 types of PCMs, including oxidation, S-nitrosylation (-SNO), S-glutathionylation (-SSG), disulfide formation (-SSR), S-sulfhydration (-SSH), S-sulfenylation (-SOH), S-sulfinylation (-SO2H) and S-palmitoylation (-S-palm). Then, browse and search options were provided for accessing the dataset, while various detailed information about the PCM events was well organized for visualization. With human dataset in iCysMod, the sequence features around the cysteine modification sites for each PCM type were analyzed, and the results indicated that various types of PCMs presented distinct sequence recognition preferences. Moreover, different PCMs can crosstalk with each other to synergistically orchestrate specific biological processes, and 37 841 PCM events involved in 119 types of PCM co-occurrences at the same cysteine residues were finally obtained. Taken together, we anticipate that the database of iCysMod would provide a useful resource for eukaryotic PCMs to facilitate related researches, while the online service is freely available at http://icysmod.omicsbio.info.