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Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, but its pathogenesis is unknown. Current researches suggested that the disease may be influenced by a variety of factors, including epigenetic and environmental factors. In recent years, the incidence of PCOS has been increasing, leading to a decline in the quality of life of patients. Therefore, it is necessary to actively explore new treatments. Post-translational modification (PTM) is an important way to regulate protein function. Most studies have found that phosphorylation is closely related to insulin resistance of PCOS, acetylation is mainly focused on the quality of PCOS oocytes, and methylation is closely related to PCOS lipid metabolism. Exploring the role of PTM in the occurrence and development of PCOS may provide new ideas for targeted treatment of this disease. [ABSTRACT FROM AUTHOR]

The number and variety of identified histone post-translational modifications (PTMs) are continually increasing. However, the specific consequences of each histone PTM remain largely unclear, primarily due to the lack of methods for selectively and rapidly introducing a desired histone PTM in living cells without genetic engineering. Here, we report the development of a cell-permeable histone acetylation catalyst, BAHA-LANA-PEG-CPP44, which selectively enters leukemia cells, binds to chromatin, and acetylates H2BK120 of endogenous histones in a short reaction time. Time-course analyses of this in-cell catalytic reaction revealed that H2BK120 acetylation attenuates the chromatin binding of negative elongation factor E (NELFE), an onco-transcription factor. This H2BK120 acetylation-mediated removal of NELFE from chromatin reshapes transcription, slows leukemia cell viability, and reduces their tumorigenic potential in mice. Therefore, this histone acetylation catalyst provides a unique tool for elucidating the time-resolved consequences of histone PTMs and may offer a modality for cancer chemotherapy., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Understanding chromatin organization requires integrating measurements of genome connectivity and physical structure. It is well established that cohesin is essential for TAD and loop connectivity features in Hi-C, but the corresponding change in physical structure has not been studied using electron microscopy. Pairing chromatin scanning transmission electron tomography with multiomic analysis and single-molecule localization microscopy, we study the role of cohesin in regulating the conformationally defined chromatin nanoscopic packing domains. Our results indicate that packing domains are not physical manifestation of TADs. Using electron microscopy, we found that only 20% of packing domains are lost upon RAD21 depletion. The effect of RAD21 depletion is restricted to small, poorly packed (nascent) packing domains. In addition, we present evidence that cohesin-mediated loop extrusion generates nascent domains that undergo maturation through nucleosome posttranslational modifications. Our results demonstrate that a 3D genomic structure, composed of packing domains, is generated through cohesin activity and nucleosome modifications.

We take a wide variety of antioxidants, including polyphenols, daily from our diet. They are generally considered to be beneficial for our health. However, the intrinsic function of antioxidants in biological systems remain unknown. On the other hand, antioxidants in general are sensitive to oxidation, generating their oxidized intermediates. Intriguingly, these intermediates are highly reactive to proteins. Although the specific cellular targets and response mechanism remain unclear, protein modification by oxidized antioxidants may represent the intrinsic "moonlight" function of antioxidants by taking on a secondary role beyond their traditional activity. This minireview summarizes recent findings on antioxidants, with a particular focus on the interactions of antioxidant-modified proteins with histones., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Glycation, a non-enzymatic post-translational modification occurring on proteins, can be actively reversed via site-specific phosphorylation of the fructose-lysine moiety by FN3K kinase, to impact the cellular function of the target protein. A regulatory axis between FN3K and glycated protein targets has been associated with conditions like diabetes and cancer. However, the molecular basis of this relationship has not been explored so far. Here, we determined a series of crystal structures of HsFN3K in the apo-state, and in complex with different nucleotide analogs together with a sugar substrate mimic to reveal the features important for its kinase activity and substrate recognition. Additionally, the dynamics in sugar substrate binding during the kinase catalytic cycle provide important mechanistic insights into HsFN3K function. Our structural work provides the molecular basis for rational small molecule design targeting FN3K., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The term "undruggable" refers to proteins or other biological targets that have been historically challenging to target with conventional drugs or therapeutic strategies because of their structural, functional, or dynamic properties. Drugging such undruggable targets is essential to develop new therapies for diseases where current treatment options are limited or nonexistent. Thus, investigating methods to achieve such drugging is an important challenge in medicinal chemistry. Among the numerous methodologies for drug discovery, covalent modification of therapeutic targets has emerged as a transformative strategy. The covalent attachment of diverse functional molecules to targets provides a powerful platform for creating highly potent drugs and chemical tools as well the ability to provide valuable information on the structures and dynamics of undruggable targets. In this review, we summarize recent examples of chemical methods for the covalent modification of proteins and other biomolecules for the development of new therapeutics and to overcome drug discovery challenges and highlight how such methods contribute toward the drugging of undruggable targets. In particular, we focus on the use of covalent chemistry methods for the development of covalent drugs, target identification, drug screening, artificial modulation of post-translational modifications, cancer specific chemotherapies, and nucleic acid-based therapeutics.

Enzymes play a pivotal role in orchestrating complex cellular responses to external stimuli and environmental changes through signal transduction pathways. Despite their crucial roles, measuring enzyme activities is typically indirect and performed on a smaller scale, unlike protein abundance measured by high-throughput proteomics. Moreover, it is challenging to derive the activity of enzymes from proteome-wide post-translational modification (PTM) profiling data. To address this challenge, we introduce enzyme activity inference with structural equation modeling under the JUMP umbrella (JUMPsem), a novel computational tool designed to infer enzyme activity using PTM profiling data. We demonstrate that the JUMPsem program enables estimating kinase activities using phosphoproteome data, ubiquitin E3 ligase activities from the ubiquitinome, and histone acetyltransferase (HAT) activities based on the acetylome. In addition, JUMPsem is capable of establishing novel enzyme-substrate relationships through searching motif sequences. JUMPsem outperforms widely used kinase activity tools, such as IKAP and KSEA, in terms of the number of kinases and the computational speed. The JUMPsem program is scalable and publicly available as an open-source R package and user-friendly web-based R/Shiny app. Collectively, JUMPsem provides an improved tool for inferring protein enzyme activities, potentially facilitating targeted drug development., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Aspartimidylation is a post-translational modification found in multiple families of ribosomally synthesized and post-translationally modified peptides (RiPPs). We recently reported on the imiditides, a new RiPP family in which aspartimidylation is the class-defining modification. Imiditide biosynthetic gene clusters encode a precursor protein and a methyltransferase that methylates a specific Asp residue, converting it to aspartimide. A subset of imiditides harbor a tetracysteine motif, so we have named these molecules cysimiditides. Here, using genome mining, we show that there are 56 putative cysimiditides predicted in publicly available genome sequences, all within actinomycetota. We successfully heterologously expressed two examples of cysimiditides and showed that the major products are aspartimidylated and that the tetracysteine motif is necessary for protein stability. Cysimiditides bind a Zn 2+ ion, presumably at the tetracysteine motif. Using in vitro reconstitution of the aspartimidylation reaction, we show that Zn 2+ is required for the methylation and subsequent aspartimidylation of the precursor protein. An AlphaFold 3 model of the cysimiditide from Thermobifida cellulosilytica shows a hairpin structure anchored by the Zn 2+ -tetracysteine motif with the aspartimide site in the hairpin loop. An AlphaFold 3 model of this cysimiditide in complex with its cognate methyltransferase suggests that the methyltransferase recognizes the Zn 2+ -tetracysteine motif to correctly dock the precursor protein. Cysimiditides expand the set of experimentally confirmed RiPPs harboring aspartimides and represent the first RiPP class that has an obligate metal ion.

As a crucial post-translational modification (PTM), protein ubiquitination mediates the breakdown of particular proteins, which plays a pivotal role in a large number of biological processes including plant growth, development, and stress response. The ubiquitin-proteasome system (UPS) consists of ubiquitin (Ub), ubiquitinase, deubiquitinating enzyme (DUB), and 26S proteasome mediates more than 80% of protein degradation for protein turnover in plants. For the ubiquitinases, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), the FBK (F-box Kelch repeat protein) is an essential component of multi-subunit E3 ligase SCF (Skp1-Cullin 1-F-box) involved in the specific recognition of target proteins in the UPS. Many FBK genes have been identified in different plant species, which regulates plant growth and development through affecting endogenous phytohormones as well as plant tolerance to various biotic and abiotic stresses associated with changes in secondary metabolites such as phenylpropanoid, phenolic acid, flavonoid, lignin, wax, etc. The review summarizes the significance of the ubiquitination modification of protein, the role of UPS in protein degradation, and the possible function of FBK genes involved in plant growth, development, secondary metabolism, and stress response, which provides a systematic and comprehensive understanding of the mechanism of ubiquitination and potential function of FBKs in plant species.

Heat shock proteins (HSPs) are essential molecular chaperones that protect cells by aiding in protein folding and preventing aggregation under stress conditions. Small heat shock proteins (sHSPs), which include members from HSPB1 to HSPB10, are particularly important for cellular stress responses. These proteins share a conserved α-crystallin domain (ACD) critical for their chaperone function, with flexible N- and C-terminal extensions that facilitate oligomer formation. Phosphorylation, a key post-translational modification (PTM), plays a dynamic role in regulating sHSP structure, oligomeric state, stability, and chaperone function. Unlike other PTMs such as deamidation, oxidation, and glycation-which are often linked to protein destabilization-phosphorylation generally induces structural transitions that enhance sHSP activity. Specifically, phosphorylation promotes the disaggregation of sHSP oligomers into smaller, more active complexes, thereby increasing their efficiency. This disaggregation mechanism is crucial for protecting cells from stress-induced damage, including apoptosis, inflammation, and other forms of cellular dysfunction. This review explores the role of phosphorylation in modulating the function of sHSPs, particularly HSPB1, HSPB4, and HSPB5, and discusses how these modifications influence their protective functions in cellular stress responses.

Melanosome transport is regulated by major proteins, including Rab27a, Melanophilin (Mlph), and Myosin Va (Myo-Va), that form a tripartite complex. Mutation of these proteins causes melanosome aggregation around the nucleus. Among these proteins, Mlph is a linker between Rab27a and Myo-Va. There are some studies about the regulation of Mlph transcriptional expression. However, its regulation by post-translational modifications remains unclear. In this study, inhibition of HDACs by SAHA and TSA disrupted melanosome transport, causing melanosome aggregation. Specifically, we identified a novel mechanism in which HDAC5 regulates Mlph expression via Sp1. Knockdown of HDAC5 increased the acetylation of Sp1 and the binding to the Mlph promoter, thereby modulating its expression. This study highlights the crucial role of HDAC5 in melanosome transport through its interaction with Sp1. These findings suggest that HDAC5-mediated deacetylation is pivotal in the post-translational modification of melanosome transport, providing insights into the molecular mechanisms underlying this process., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The modification of proteins and other biomolecules with the small protein ubiquitin has enthralled scientists from many disciplines for decades, creating a broad research field. Ubiquitin research is particularly rich in molecular and mechanistic understanding due to a plethora of (poly)ubiquitin structures alone and in complex with ubiquitin machineries. Furthermore, due to its favorable properties, ubiquitin serves as a model system for many biophysical and computational techniques. Here, we review the current knowledge of ubiquitin signals through a ubiquitin-centric, structural biology lens. We amalgamate the information from 240 structures in the Protein Data Bank (PDB), combined with single-molecule, molecular dynamics, and nuclear magnetic resonance (NMR) studies, to provide a comprehensive picture of ubiquitin and polyubiquitin structures and dynamics. We close with a discussion of the latest frontiers in ubiquitin research, namely the modification of ubiquitin by other post-translational modifications (PTMs) and the notion that ubiquitin is attached to biomolecules beyond proteins., Competing Interests: Declaration of interests D.K. is a founder, shareholder, and SAB member of Entact Bio and Proxima Bio., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Significant effort has been spent attempting to unravel the causal relationship between histone post-translational modifications and fundamental DNA processes, including transcription, replication, and repair. However, less attention has been paid to understanding the reciprocal influence-that is, how DNA processes, in turn, shape the distribution and patterns of histone modifications and how these changes convey information, both temporally and spatially, from one process to another. Here, we review how histone modifications underpin the widespread bidirectional crosstalk between different DNA processes, which allow seemingly distinct phenomena to operate as a unified whole., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

N-terminal acetylation is a highly abundant protein modification in eukaryotic cells. This modification is catalysed by N-terminal acetyltransferases acting co- or post-translationally. Here, we review the eukaryotic N-terminal acetylation machinery: the enzymes involved and their substrate specificities. We also provide an overview of the impact of N-terminal acetylation, including its effects on protein folding, subcellular targeting, protein complex formation, and protein turnover. In particular, there may be competition between N-terminal acetyltransferases and other enzymes in defining protein fate. At the organismal level, N-terminal acetylation is highly influential, and its impairment was recently linked to cardiac dysfunction and neurodegenerative diseases., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

In recent years, it was found that lysine malonylation modification can affect biological metabolism and play an important role in plant life activities. Platycodon grandiflorus, an economic crop and medicinal plant, had no reports on malonylation in the related literature. This study qualitatively introduces lysine malonylation in P. grandiflorus. A total of 888 lysine malonylation-modified proteins in P. grandiflorus were identified, with a total of 1755 modification sites. According to the functional annotation, malonylated proteins were closely related to catalysis, binding, and other reactions. Subcellular localization showed that related proteins were enriched in chloroplasts, cytoplasm, and nuclei, indicating that this modification could regulate various metabolic processes. Motif analysis showed the enrichment of Alanine (A), Cysteine (C), Glycine (G), and Valine (V) amino acids surrounding malonylated lysine residues. Metabolic pathway and protein-protein interaction network analyses suggested these modifications are mainly involved in plant photosynthesis. Moreover, malonylated proteins are also involved in stress and defense responses. This study shows that lysine malonylation can affect a variety of biological processes and metabolic pathways, and the contents are reported for the first time in P. grandiflorus, which can provide important information for further research on P. grandiflorus and lysine malonylation's role in environment stress, photosynthesis, and secondary metabolites enrichment., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Post-translational modifications of histone H3 on lysine 9, specifically acetylation (H3K9ac) and tri-methylation (H3K9me3), play a critical role in regulating chromatin accessibility. However, the role of these modifications in lineage segregation in the mammalian blastocyst remains poorly understood. We demonstrate that di- and tri-methylation marks, H3K9me2 and H3K9me3, decrease during cavitation and expansion of the rabbit blastocyst. Notably, H3K9me3 levels are particularly low in inner cell mass cells at the onset of blastocyst formation but increase again just before gastrulation. Conversely, H3K9ac is abundant in early blastocyst stages but decreases during the transition from the inner cell mass to the epiblast. These distinct distribution patterns correlate with high expression levels of methyltransferases (EHMT1, EHMT2, SETDB1) and deacetylases (HDAC1, HDAC2, HDAC5) in expanding blastocysts. Functionally, inhibiting H3K9me2/3 through an EHMT1/2 inhibitor disrupts primitive endoderm segregation, whereas enhancing histone acetylation (including H3K9ac) using a class I HDAC inhibitor promotes epiblast expansion at the expense of the primitive endoderm. These modifications impact the expression of genes associated with pluripotency and lineage determination, underscoring the importance of H3K9 modifications in embryonic cell fate decisions., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Gene expression is regulated by chromatin DNA methylation and other features, including histone post-translational modifications (PTMs), chromatin remodelers and transcription factor occupancy. A complete understanding of gene regulation will require the mapping of these chromatin features in small cell number samples. Here we describe a novel genome-wide chromatin profiling technology, named as Nicking Enzyme Epitope targeted DNA sequencing (NEED-seq). NEED-seq offers antibody-targeted controlled nicking by Nt.CviPII-pGL fusion to study specific protein-DNA complexes in formaldehyde fixed cells, allowing for both visual and genomic resolution of epitope bound chromatin. When applied to nuclei, NEED-seq yielded genome-wide profile of chromatin-associated proteins and histone PTMs. Additionally, NEED-seq of lamin B1 and B2 demonstrated their association with heterochromatin. Lamin B1- and B2-associated domains (LAD) segregated to three different states, and states with stronger LAD correlated with heterochromatic marks. Hi-C analysis displayed A and B compartment with equal lamin B1 and B2 distribution, although methylated DNA remained high in B compartment. LAD clustering with Hi-C resulted in subcompartments, with lamin B1 and B2 partitioning to facultative and constitutive heterochromatin, respectively, and were associated with neuronal development. Thus, lamin B1 and B2 show structural and functional partitioning in mammalian nucleus., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Brief: Bovine sperm and seminal plasma (SP) proteoform atlas was characterized using top-down proteomics. Specific post-translational modifications and protein truncations correlated with semen freezability, with potential links to sperm functional processes., Abstract: Top-down proteomics was employed to construct a proteoform atlas of sperm and SP from bulls with low semen freezability (LF) and high semen freezability (HF). Sperm and seminal proteins were fractionated by tandem size exclusion chromatography (<30 kDa) and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry. This approach enabled identification of 299 SP (from 46 families) and 267 sperm proteoforms (from 139 families). Seventy proteoforms belonging to beta-defensin 10, c-type natriuretic peptide (NPPC), caltrin, seminal ribonuclease, osteopontin and binder of sperm protein (BSP) 3 families were unique to HF bulls' SP. LF seminal proteins had 77 unique proteoforms, including caltrin, NPPC, osteopontin, BSP3, serpin family A member 5 and β-NGF families. Proteoform families of SP in HF and LF bulls were related to Ca2+ uptake, capacitation, acrosome reaction, sperm protection, fertilization and proteolytic processes. Thirty-three proteoforms of NPPC, caltrin and cylicin-2 families were upregulated in HF sperm. Twenty-two proteoforms of caltrin, cylicin-2, adenosine triphosphate (ATP) synthases and malate dehydrogenase families were among those upregulated in LF sperm. Truncated and acetylated histone H2A and non-truncated and acetylated c-Myc-binding protein were prevalent in LF sperm. Cylicin-2 proteoforms were observed in HF and LF sperm, and truncated glyceraldehyde-3-phosphate dehydrogenases were observed only in HF sperm. In silico analyses indicated the enrichment of mitochondrial metabolic pathways in HF sperm, including fatty acid metabolism and TCA cycle. Our study provides an unprecedented description of the bovine SP and sperm proteoforms. Post-translational processing appears to define the bioproperties of semen proteins and their associations with sperm cryoresistance.

Lysine benzoylation (Kbz) is a newly identified post-translational modification associated with active transcription and metabolism in eukaryotes. However, whether Kbz exists in pathogenic fungi and its function remains unknown. Here, we demonstrated for the first time that Kbz is present in Aspergillus flavus and identified 60 benzoylated sites on 46 benzoylated proteins by global benzoylome analysis. Our data demonstrated that alcohol dehydrogenase B (AdhB) is regulated by benzoylation on lysine 321 (K321), and mutations of Kbz site in AdhB significantly reduced the alcohol dehydrogenase activity in vivo and in vitro . Both adhB deletion mutant and benzoylated site mutants (K321R and K321A) exhibited similar phenotype, including decreased conidiation and seed colonization, increased sclerotia formation and aflatoxin production, and more sensitive to cell wall damage stress. We also found that GcnE has benzoyltransferase activity in vitro and in vivo , and its repression leads to decreased Kbz level and enzymatic activity of AdhB. The catalytic site E139 is important for the benzoyltransferase function of GcnE. Our study uncovers a previously unknown mechanism by which benzoylation regulates AdhB activity to affect the development, secondary metabolism, pathogenicity, and stress response of A. flavus . Meanwhile, it points out the important role of Kbz in the pathogenicity of pathogenic fungi.IMPORTANCE Aspergillus flavus is a ubiquitous opportunistic pathogen of plants and animals, which produces carcinogenic and toxic secondary metabolite aflatoxin. A. flavus and aflatoxin contamination have emerged as a global food safety concern. Currently, post-translational modification plays crucial modulatory roles in the fungal development and virulence, but the role of benzoylation in fungal pathogenicity remains undetermined, which limits the development of prevention and control technique. Here, we first identified 46 benzoylated proteins in A. flavus , and found that benzoyltransferase GcnE exerted effects on pathogenicity and aflatoxin production by regulating the benzoylation of AdhB. This finding not only provided valuable information for prevention and control of A. flavus contamination, but also offered basic knowledge for investigation of the regulation mechanism of secondary metabolism in other fungi., Competing Interests: The authors declare no conflict of interest.

Acute pancreatitis (AP) represents a severe inflammatory condition of the exocrine pancreas, precipitating systemic organ dysfunction and potential failure. The global prevalence of acute pancreatitis is on an ascending trajectory. The condition carries a significant mortality rate during acute episodes. This underscores the imperative to elucidate the etiopathogenic pathways of acute pancreatitis, enhance comprehension of the disease's intricacies, and identify precise molecular targets coupled with efficacious therapeutic interventions. The pathobiology of acute pancreatitis encompasses not only the ectopic activation of trypsinogen but also extends to disturbances in calcium homeostasis, mitochondrial impairment, autophagic disruption, and endoplasmic reticulum stress responses. Notably, the realm of epigenetic regulation has garnered extensive attention and rigorous investigation in acute pancreatitis research over recent years. One of these modifications, lysine acetylation, is a reversible post-translational modification of proteins that affects enzyme activity, DNA binding, and protein stability by changing the charge on lysine residues and altering protein structure. Numerous studies have revealed the importance of acetylation modification in acute pancreatitis, and that it is a favorable target for the design of new drugs for this disease. This review centers on lysine acetylation, examining the strides made in acute pancreatitis research with a focus on the contributory role of acetylomic alterations in the pathophysiological landscape of acute pancreatitis, thereby aiming to delineate novel therapeutic targets and advance the development of more efficacious treatment modalities., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

C-Terminal cyclic imides are posttranslational modifications that can arise from spontaneous intramolecular cleavage of asparagine or glutamine residues resulting in a form of irreversible protein damage. These protein damage events are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN), indicating that these aging-related modifications may require cellular quality control mechanisms to prevent deleterious effects. However, the factors that determine protein or peptide susceptibility to C-terminal cyclic imide formation or their effect on protein stability have not been explored in detail. Here, we characterize the primary and secondary structures of peptides and proteins that promote intrinsic formation of C-terminal cyclic imides in comparison to deamidation, a related form of protein damage. Extrinsic effects from solution properties and stressors on the cellular proteome additionally promote C-terminal cyclic imide formation on proteins like glutathione synthetase that are susceptible to aggregation if the protein damage products are not removed by CRBN. This systematic investigation provides insight into the regions of the proteome that are prone to these unexpectedly frequent modifications, the effects of this form of protein damage on protein stability, and the biological role of CRBN., Competing Interests: Competing interests statement:Harvard University has filed a PCT patent application on April 13, 2022 covering the chemical structures and their use. C.M.W. and W.X. are inventors of this patent. The Woo lab receives or has received support from Merck and Ono Pharmaceuticals.

Posttranslational modifications (PTMs) of proteins play critical roles in regulating many cellular events. Antibodies targeting site-specific PTMs are essential tools for detecting and enriching PTMs at sites of interest. However, fundamental difficulties in molecular recognition of both PTM and surrounding peptide sequence have hindered the efficient generation of highly sequence-specific anti-PTM antibodies. Furthermore, the widespread use of potentially inconsistent, nonrenewable, and molecularly undefined antibodies presents experimental challenges thought to contribute to the reproducibility problem in biomedical research. In this study, we describe the binding mode-guided development of a platform that efficiently generates potent and selective recombinant antibodies to PTMs that are molecularly defined and renewable. Our platform is built on our previous discovery of an unconventional binding mode of anti-PTM antibodies, antigen clasping, where two antigen binding sites cooperatively sandwich a single antigen, creating extensive interactions with the antigen and leading to high selectivity and potency. We designed the platform that generates clasping antibodies with two distinct binding units, resulting in efficient generation of antibodies to a set of trimethylated histone H3 with high levels of specificity and affinity. Performance comparison in chromatin immunoprecipitation, a common application in epigenomics, revealed that a clasping antibody to trimethylated histone H3 at lysine 27 exhibited superior specificity to a widely used conventional antibody and captured symmetric and asymmetric nucleosomes in a less biased manner. We further generated clasping antibodies to phosphotyrosine antigens by using the same principle. These results suggest the broad applicability of our platform to generating high-performance clasping antibodies to diverse PTMs., Competing Interests: Competing interests statement:A.J.R. holds equity in EpiCypher. A.K., S.K., and T.H. are listed as inventors of a pending patent on the technology and antibodies developed in this work filed by New York University. A.K., S.K., and T.H. are inventors of US10208110B2 on recombinant histone PTM antibodies managed by the University of Chicago. A.J.R. is a co-inventor of the ICeChIP technology US10732185B2 managed by the University of Chicago and licensed to EpiCypher. A.J.R. is a scientific advisor at EpiCypher. R.N.S. has received consulting fees from EpiCypher. S.K. is a co-founder, receives consulting fees and holds equity in Aethon Therapeutics; is a co-founder and holds equity in Revalia Bio; has received research funding from Aethon Therapeutics, Argenx BVBA, Black Diamond Therapeutics, and Puretech Health, all outside of the current work.

Background: TPM3 (tropomyosin 3) is an actin-binding protein in vascular smooth muscle cells, where posttranslational modifications critically regulate its actin affinity, influencing cardiovascular function. Emerging evidence suggests that Khib (2-hydroxyisobutyrylation) plays a significant role in the cardiovascular system. Histone deacetylase 3 (HDAC3) serves as an "eraser" of Khib marks. However, the impact of TPM3 de-2-hydroxyisobutyrylation on vascular contraction remains unclear., Methods and Results: In this study, we employed mouse models and in vitro experiments to elucidate the mechanism by which phenylephrine-induced HDAC3 activation drives vasoconstriction via de-2-hydroxyisobutyrylation of TPM3. Our findings demonstrate that phenylephrine triggers HDAC3 nuclear export and promotes its interaction with TPM3, resulting in decreased Khib modification and enhanced vasoconstriction. Coimmunoprecipitation experiments confirmed that phenylephrine reduces Khib levels on TPM3 in mouse aorta. Additionally, ex vivo vascular tension assays using mouse aortic rings revealed that treatment with the Khib donor, ethyl 2-hydroxyisobutyrate, induces endothelium-independent vasodilation and ameliorates hypertensive vascular dysfunction. Molecular docking and kinetic simulations identified Lys141 of TPM3 as the primary site targeted by HDAC3-mediated de-2-hydroxyisobutyrylation. This was further validated by adenoviral transfection of isolated blood vessels with a Lys141-mutated TPM3 construct, which abolished the effects of HDAC3 on TPM3 Khib modification and vascular contractility., Conclusions: These findings underscore the critical role of TPM3 de-2-hydroxyisobutyrylation in vasoconstriction and suggest that modulating this posttranslational modification could provide a novel therapeutic strategy for hypertensive vascular dysfunction.

Dissection of the physiological interactomes of histone post-translational modifications (hPTMs) is crucial for understanding epigenetic regulatory pathways. Peptide- or protein-based histone photoaffinity tools expanded the ability to probe the epigenetic interactome, but in situ profiling in native cells remains challenging. Here, we develop a nucleus-targeting histone-tail-based photoaffinity probe capable of profiling the hPTM-mediated interactomes in native cells, by integrating cell-permeable and nuclear localization peptide modules into an hPTM peptide equipped with a photoreactive moiety. These types of probes, such as histone H3 lysine 4 trimethylation and histone H3 Lysine 9 crotonylation probes, enable the probing of epigenetic interactomes both in HeLa cell and hard-to-transfect RAW264.7 cells, resulting in the discovery of distinct interactors in different cell lines. The utility of this probe is further exemplified by characterizing interactome of emerging hPTM, such as AF9 was detected as a binder of histone H3 Lysine 9 lactylation, thus expanding the toolbox for profiling of hPTM-mediated PPIs in live cells., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

A recent study reported that isomerization of aspartyl residues (Asp) occurs in various tissues and proteins in vivo. For a comprehensive analysis of post-translational modifications, the mass spectrometry (MS)-based proteomic approach is a straightforward method; however, the isomerization of Asp does not alter its molecular weight. Therefore, a unique method is required to analyse Asp isomers using MS. Herein, we present a novel strategy, isoAsp-Quest, which is a database search-oriented isoAsp identification method. isoAsp is specifically converted to 18O-labelled Lα-Asp by the enzymatic reaction of protein L-isoaspartyl-O-methyltransferase (PIMT) in 18O water with a mass shift of 2 Da, which, in principle, enables us to distinguish Asp isomers. However, in practise, a labelled Lα-Asp signal overlaps with that of endogenous Lα-Asp, making detection challenging. Therefore, degradation of the endogenous Lα-Asp peptide by AspN and subsequent removal of AspN were performed prior to the PIMT reaction. This strategy was applied to bovine lens α-crystallin. Consequently, several Asp isomerization sites, consistent with human αA-crystallin, were identified in bovine αA-crystallin, indicating that this strategy is also effective for biological proteins. Therefore, isoAsp-Quest enables the analysis of Lβ-Asp in a straightforward and rapid workflow, which may be useful for the quality control of protein products and biomarker discovery., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Various post-translational modifications (PTMs) participate in nearly all aspects of biological processes by regulating protein functions, and aberrant states of PTMs are frequently associated with human diseases. Here, we present a comprehensive database of PTMs associated with diseases (PTMD 2.0), including 342 624 PTM-disease associations (PDAs) in 15 105 proteins for 93 types of PTMs and 2083 diseases. Based on the distinct PTM states in diseases, we classified all PDAs into six categories: upregulation (U) or downregulation (D) of PTM levels, absence (A) or presence (P) of PTMs, and creation (C) or disruption (N) of PTM sites. We provided detailed annotations for each PDA and carefully annotated disease-associated proteins by integrating the knowledge from 101 additional resources that covered 13 aspects, including disease-associated information, variation and mutation, protein-protein interaction, protein functional annotation, DNA and RNA element, protein structure, chemical-target relationship, mRNA expression, protein expression/proteomics, subcellular localization, biological pathway annotation, functional domain annotation and physicochemical property. With a data volume of ∼8 GB, we anticipate that PTMD 2.0 will serve as a fundamental resource for further analysing the relationships between PTMs and diseases. The online service of PTMD 2.0 is freely available at https://ptmd.biocuckoo.cn/., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Post-translational modifications (PTMs) are essential for modulating protein function and influencing stability, activity and signaling processes. The dbPTM 2025 update significantly expands the database to include over 2.79 million PTM sites, of which 2.243 million are experimentally validated from 48 databases and over 80 000 research articles. This version integrates proteomic data from 13 cancer types, with a particular focus on phosphoproteomic data and kinase activity profiles, allowing the exploration of personalized phosphorylation patterns in tumor samples. Integrating kinase-substrate phosphorylations with E3 ligase-substrate interactions, dbPTM 2025 provides a detailed map of PTM regulatory networks, offering insights into cancer-specific post-translational regulations. This update also includes advanced search capabilities, enabling users to efficiently query PTM data across species, PTM types and modified residues. The platform's new features-interactive visualization tools and streamlined data downloads-allow researchers to access and analyze PTM data easily. dbPTM 2025 also enhances functional annotations, regulatory networks and disease associations, broadening its application for cancer research and the study of disease-associated PTMs. Through these enhancements, dbPTM 2025 is a comprehensive, user-friendly resource, facilitating the study of PTMs and their roles in cancer research. The database is now freely accessible at https://biomics.lab.nycu.edu.tw/dbPTM/., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sumoylation is a posttranslational modification essential for multiple cellular functions in eukaryotes. ULP-2 is a conserved SUMO protease required for embryonic development in Caenorhabditis elegans. Here, we revealed that ULP-2 controls germline development by regulating the PHD-SET domain protein, SET-26. Specifically, loss of ULP-2 results in sterility and a progressive elevation of global protein sumoylation. In the germline of ulp-2 null mutant, meiosis is arrested at the diplotene stage and the cells in the proximal germline acquire a somatic fate. Germline RNAseq analysis revealed the down-regulation of numerous germline genes in ulp-2 mutants, whereas somatic gene expression is up-regulated. To determine the key factors that are regulated by ULP-2, we performed a yeast two-hybrid screen and identified the histone methylation reader, SET-26 as a ULP-2 interacting protein. Loss of SET-26 enhanced the sterility of ulp-2 mutant animals. Consistently, SET-26 is sumoylated and its sumoylation levels are regulated by ULP-2. Moreover, we detected a reduction in H3K4 tri-methylation (H3K4me3) histone levels bound to SET-26 in the ulp-2 mutant background suggesting a dependence of this histone reader on balanced sumoylation. Finally, a comparative proteomics screen between WT and ulp-2 loss of activity identified the predicted methyltransferase SET-27 as a ULP-2-dependent SET-26-associated protein. SET-27 knockout genetically interacts with ULP-2 in the germline, but not with SET-26. Taken together, we revealed a SUMO protease/H3K4me3 histone reader axis which is required for the maintenance and regulation of germline development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Carvalho et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Raman Optical Activity combined with Circularly Polarized Luminescence (ROA-CPL) was used in the spectral recognition of glutathione peptide (GSH) and its model post-translational modifications (PTMs). We demonstrate the potential of ROA spectroscopy and CPL probes (EuCl 3 , Na 3 [Eu(DPA) 3 ], NaEuEDTA) in the study of unmodified peptide, i.e. GSH, and its derivatives, i.e. glutathione oxidized (GSSG), S-acetylglutathione (GSAc) and S-nitrosoglutathione (GSNO). ROA spectral features of GSH, GSSG, and GSAc were determined along with thier changes upon the different pH conditions. Apart from the ROA, induced CPL signals of Eu(III) probes also proved to be sensitive to the structural modifications of GSH-based model PTMs, enabling their spectral recognition, especially by the NaEuEDTA probe., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Deactivation of the mitochondrial pyruvate dehydrogenase complex (PDC) is important for the metabolic switching of cancer cell from oxidative phosphorylation to aerobic glycolysis. Studies examining PDC activity regulation have mainly focused on the phosphorylation of pyruvate dehydrogenase (E1), leaving other post-translational modifications largely unexplored. Here, we demonstrate that the acetylation of Lys 488 of pyruvate dehydrogenase complex component X (PDHX) commonly occurs in hepatocellular carcinoma, disrupting PDC assembly and contributing to lactate-driven epigenetic control of gene expression. PDHX, an E3-binding protein in the PDC, is acetylated by the p300 at Lys 488, impeding the interaction between PDHX and dihydrolipoyl transacetylase (E2), thereby disrupting PDC assembly to inhibit its activation. PDC disruption results in the conversion of most glucose to lactate, contributing to the aerobic glycolysis and H3K56 lactylation-mediated gene expression, facilitating tumor progression. These findings highlight a previously unrecognized role of PDHX acetylation in regulating PDC assembly and activity, linking PDHX Lys 488 acetylation and histone lactylation during hepatocellular carcinoma progression and providing a potential biomarker and therapeutic target for further development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Higher Education Press.)

This review examines the giant elastic protein titin and its critical roles in heart function, both in health and disease, as discovered since its identification nearly 50 years ago. Encoded by the TTN (titin gene), titin has emerged as a major disease locus for cardiac disorders. Functionally, titin acts as a third myofilament type, connecting sarcomeric Z-disks and M-bands, and regulating myocardial passive stiffness and stretch sensing. Its I-band segment, which includes the N2B element and the PEVK (proline, glutamate, valine, and lysine-rich regions), serves as a viscoelastic spring, adjusting sarcomere length and force in response to cardiac stretch. The review details how alternative splicing of titin pre-mRNA produces different isoforms that greatly impact passive tension and cardiac function, under physiological and pathological conditions. Key posttranslational modifications, especially phosphorylation, play crucial roles in adjusting titin's stiffness, allowing for rapid adaptation to changing hemodynamic demands. Abnormal titin modifications and dysregulation of isoforms are linked to cardiac diseases such as heart failure with preserved ejection fraction, where increased stiffness impairs diastolic function. In addition, the review discusses the importance of the A-band region of titin in setting thick filament length and enhancing Ca² + sensitivity, contributing to the Frank-Starling Mechanism of the heart. TTN truncating variants are frequently associated with dilated cardiomyopathy, and the review outlines potential disease mechanisms, including haploinsufficiency, sarcomere disarray, and altered thick filament regulation. Variants in TTN have also been linked to conditions such as peripartum cardiomyopathy and chemotherapy-induced cardiomyopathy. Therapeutic avenues are explored, including targeting splicing factors such as RBM20 (RNA binding motif protein 20) to adjust isoform ratios or using engineered heart tissues to study disease mechanisms. Advances in genetic engineering, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), offer promise for modifying TTN to treat titin-related cardiomyopathies. This comprehensive review highlights titin's structural, mechanical, and signaling roles in heart function and the impact of TTN mutations on cardiac diseases., Competing Interests: None.

Post-translational modifications are crucial in regulating biological functions across both prokaryotes and eukaryotes. In Aeromonas hydrophila , CobQ, a recently identified novel deacetylase, plays a significant role in lysine deacetylation, influencing bacterial metabolism and stress responses. The present study utilized quantitative proteomics to investigate the impact of cobQ deletion on the global protein expression profile in A. hydrophila . Through data-independent acquisition mass spectrometry, we identified 233 upregulated and 41 downregulated proteins in the cobQ deletion mutant ( ΔahcobQ ) strain compared to the wild-type (WT) strain. Key differentially expressed proteins were involved in oxidative phosphorylation, bacterial secretion, and ribosomal function. Additionally, phenotypic assays demonstrated that the ΔahcobQ strain exhibited an increased resistance to oxidative phosphorylation inhibitors, suggesting a pivotal role for AhCobQ in energy metabolism. Outer membrane proteins and efflux pumps also showed altered expression, indicating potential implications for membrane permeability and antibiotic resistance. These results suggested that AhCobQ plays a vital regulatory role in maintaining metabolic homeostasis and responding to environmental stress, highlighting its potential as a target for therapeutic interventions against A. hydrophila infections.

Bacillus altitudinis is a well-known beneficial microorganism in plant rhizosphere, capable of enhancing plant growth and salt tolerance in saline soils. However, the mechanistic changes underlying salt tolerance in B. altitudinis at the level of post-translational modifications remain unclear. Here, diverse lysine modifications including acetylation, succinylation, crotonylation, and malonylation were determined in the B. altitudinis response to salt stress by immunodetection, and the acetylation level greatly increased under salt stress. The in-depth acetylome landscape showed that 1032 proteins in B. altitudinis were differentially acetylated under salt stress. These proteins were involved in many physiological aspects closely related to salt tolerance like energy generation and conversion, amino acid synthesis and transport, cell motility, signal transduction, secretion system, and repair system. Moreover, we also identified the differential acetylation of key enzymes involved in the major osmolyte biosynthesis and conversion and antioxidant defenses. Thiol peroxidase (TPX), a key protective antioxidant enzyme, had 3 upregulated acetylation sites (K7/139/157) under salt stress. Site-specific mutations demonstrated that K7/139/157 acetylation strongly regulated TPX function in scavenging intracellular ROS, thereby impacting bacterial growth under salt stress. To our knowledge, this is the first study showing that bacteria adaptation to salt stress occurs at the level of PTMs.

Posttranslational modification (PTM) of the amyloid precursor protein (APP) plays a critical role in Alzheimer's disease (AD). Recent evidence reveals that lactylation modification, as a novel PTM, is implicated in the occurrence and development of AD. However, whether and how APP lactylation contributes to both the pathogenesis and cognitive function in AD remains unknown. Here, we observed a reduction in APP lactylation in AD patients and AD model mice and cells. Proteomic mass spectrometry analysis further identified lysine 612 (APP-K612la) as a crucial site for APP lactylation, influencing APP amyloidogenic processing. A lactyl-mimicking mutant (APPK612T) reduced amyloid-β peptide (Aβ) generation and slowed down cognitive deficits in vivo. Mechanistically, APPK612T appeared to facilitate APP trafficking and metabolism. However, lactylated APP entering the endosome inhibited its binding to BACE1, suppressing subsequent cleavage. Instead, it promoted protein interaction between APP and CD2-associated protein (CD2AP), thereby accelerating the endosomal-lysosomal degradation pathway of APP. In the APP23/PS45 double-transgenic mouse model of AD, APP-Kla was susceptible to L-lactate regulation, which reduced Aβ pathology and repaired spatial learning and memory deficits. Thus, these findings suggest that targeting APP lactylation may be a promising therapeutic strategy for AD in humans.

Potyvirids are the largest group of plant RNA viruses. Pelota, a core component of RNA quality controls (RQC), promotes the degradation of potyvirids' genomic RNA by recognizing a specific G 1-2 A 6-7 motif. Here we demonstrate that the viral RNA-dependent RNA polymerase, NIb, acts as a SUMOylation decoy to effectively reduce Pelota SUMOylation by competing with SCE1 to inhibit Pelota-mediated RQC. TuMV NIb is comprised of two functional SUMO interacting motif (SIM): SIM2 and SIM3. The former is identified as the key site for NIb's SUMOylation by SUMO3, whereas the latter is responsible for the interaction with SCE1. These two SIMs are conserved among the majority of potyvirids-encoded NIbs. Thus, virus protein-mediated SUMOylation decoy strategy to suppress host defense may be a common feature in plant virus pathosystems. These findings highlight a dynamic interplay between plant defense mechanism and viral counter-strategy by orchestrating the post-translational modifications of virus and host defense components., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Studying native protein structures at near-atomic resolution in a crowded environment presents challenges. Consequently, understanding the structural intricacies of proteins within pathologically affected tissues often relies on mass spectrometry and proteomic analysis. Here, we utilized cryoelectron microscopy (cryo-EM) and the Build and Retrieve (BaR) method to investigate protein complexes' structural characteristics such as post-translational modification, active site occupancy, and arrested conformational state in Alzheimer's disease (AD) using brain lysate from a rat model (TgF344-AD). Our findings reveal novel insights into the architecture of these complexes, corroborated through mass spectrometry analysis. Interestingly, it has been shown that the dysfunction of these protein complexes extends beyond AD, implicating them in cancer, as well as other neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and schizophrenia. By elucidating these structural details, our work not only enhances our understanding of disease pathology but also suggests new avenues for future approaches in therapeutic intervention., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Peptide sequencing via tandem mass spectrometry (MS/MS) is essential in proteomics. Unlike traditional database searches, deep learning excels at de novo peptide sequencing, even for peptides missing from existing databases. Current deep learning models often rely on autoregressive generation, which suffers from error accumulation and slow inference speeds. In this work, we introduce π-PrimeNovo, a non-autoregressive Transformer-based model for peptide sequencing. With our architecture design and a CUDA-enhanced decoding module for precise mass control, π-PrimeNovo achieves significantly higher accuracy and up to 89x faster inference than state-of-the-art methods, making it ideal for large-scale applications like metaproteomics. Additionally, it excels in phosphopeptide mining and detecting low-abundance post-translational modifications (PTMs), marking a substantial advance in peptide sequencing with broad potential in biological research., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Histones are positively charged proteins found in the chromatin of eukaryotic cells. They regulate gene expression and are required for the organization and packaging of DNA within the nucleus. Histones are extremely conserved, allowing for transcription, replication, and repair. This review delves into their complex structure and function in DNA assembly, their role in nucleosome assembly, and the higher-order chromatin structures they generate. We look at the five different types of histone proteins: H1, H2A, H2B, H3, H4, and their variations. These histones bind with DNA to produce nucleosomes, the basic units of chromatin that are essential for compacting DNA and controlling its accessibility. Their dynamic control of chromatin accessibility has important implications for genomic stability and cellular activities. We elucidate regulatory mechanisms in both normal and pathological situations by investigating their structural features, diverse interaction mechanisms, and chromatin impact. In addition, we discuss the functions of histone post-translational modifications (PTMs) and their significance in various disorders. These alterations, which include methylation, acetylation, phosphorylation, and ubiquitination, are crucial in regulating histone function and chromatin dynamics. We specifically describe and explore the role of changed histones in the evolution of cancer, neurological disorders, sepsis, autoimmune illnesses, and inflammatory conditions. This comprehensive review emphasizes histone's critical role in genomic integrity and their potential as therapeutic targets in various diseases., (Copyright © 2025. Published by Elsevier Inc.)

Multiple epigenetic modulations occur to chromatin rather than to DNA itself and these influence gene expression or gene silencing profoundly. Both the creation of these post-translational modifications and the mechanisms of their readout are regulated significantly by electrical forces several of which are discussed. They are also influenced by phase separation which itself is driven by electrical forces., (© 2025. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Histones are essential nuclear proteins that package eukaryotic DNA into chromosomes, play a vital role in gene regulation, DNA replication, DNA repair and chromosome condensation. Understanding histone modifications is crucial for grasping biological and disease-related processes. Specific alterations in histone modifications serve as sensitive and selective biomarkers for conditions like cancer, impacting both tumor and immune cells and affecting their interactions. Indeed, the interest in histone modifications is growing in the field of tumor immunology and immunotherapy. Different techniques have been developed to characterize histone proteins and their modifications. Here, we present a simple acid extraction protocol to identify and quantify histones. The workflow described here can be used to detect and measure histone proteins or specific residues of histone, even capturing changes resulting from treatment with epigenetic drugs (Epi-drugs) or other drugs in in different human cancer cell line models., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2025 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Lysine deacetylases (KDACs or HDACs) are metal-dependent enzymes that regulate lysine acetylation, a post-translational modification that is present on thousands of human proteins, essential for many cellular processes, and often misregulated in diseases. The selective inhibition of KDACs would allow for understanding of the biological roles of individual KDACs and therapeutic targeting of individual enzymes. Recent studies have suggested that purportedly specific KDAC inhibitors have significant off-target binding, but the biological consequences of off-target binding were not evaluated. We compared the effects of treatment with two of the reportedly most KDAC-selective inhibitors, Tubastatin A and PCI-34051, in HT1080 cells in which the endogenous KDAC6 or KDAC8 gene has been mutated to inactivate enzyme catalysis while retaining enzyme expression. Genetic inactivation results in much stronger deacetylation defects on known targets compared to inhibitor treatment. Gene expression analysis revealed that both inhibitors have extensive and extensively overlapping off-target effects in cells, even at low inhibitor doses. Furthermore, Tubastatin A treatment led to increased histone acetylation, while inactivation of KDAC6 or KDAC8 did not. Genetic inactivation of KDAC6, but not KDAC8, impaired tumor formation in a xenograft model system, in contrast to previous reports with KDAC inhibitors suggesting the reverse. We conclude that the majority of observed biological effects of treatment with KDAC inhibitors are due to off-target effects rather than the intended KDAC inhibition. Developing a truly specific KDAC6 inhibitor could be a promising therapeutic avenue, but it is imperative to develop new inhibitors that selectively mimic genetic inactivation of individual KDACs., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Posttranslational modifications (PTMs) play a crucial role in modulating the structure, function, localization, and interactions of proteins, with many PTMs being localized within supersecondary structures, such as helical pairs. These modifications can significantly influence the conformation and stability of these structures. For instance, phosphorylation introduces negative charges that alter electrostatic interactions, while acetylation or methylation of lysine residues affects the stability and interactions of alpha helices or beta strands. Given the pivotal role of supersecondary structures in the overall protein architecture, their modulation by PTMs is essential for protein functionality. This chapter explores the latest advancements in predicting sites for the five PTMs (phosphorylation, acetylation, glycosylation, methylation, and ubiquitination) known to be localized within supersecondary structures. The chapter highlights the recent advances in the prediction of these PTM sites, including the use of global contextualized embeddings from protein language models, integration of structural information, utilization of reliable positive and negative sites, and application of contrastive learning. These methodologies and emerging trends offer a roadmap for novel innovations in addressing PTM prediction challenges, particularly those linked to supersecondary structures., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Posttranslational modifications (PTMs) of proteins regulate several biological processes, and investigating their diversity is crucial for understanding the mechanisms of cell regulation. Glycosylation is one of the most complex posttranslational modifications that control fundamental cellular processes such as protein folding, protein trafficking, host-pathogen interactions, cell adhesion, and cytokine receptor signaling networks. N-linked glycosylation denotes the attachment of glycans (oligosaccharides) to a nitrogen atom of asparagine (N) residues in the consensus motif Asn-X-Ser/Thr (NXS/T), where X is any amino acid except proline. Therefore, mutations in this posttranslational modification (i.e., N-glycosylation) site cause many human genetic diseases, including cancer. In the past decade, high-throughput quantitative proteome profiling tools have significantly renewed our interest in discovering novel cancer diagnostic or prognostic biomarkers through the simultaneous examination of the enormous amount of high-quality data of thousands of proteins and genes in complex biological systems. In this chapter, we describe how aberrant N-linked glycopeptides could be selectively identified as novel single tumor markers through the use of mass spectrometry (MS)-based proteomics, also known as Solid-phase extraction of N-glycopeptides (SPEG), and reasonable hypotheses that have the potential capacity to revolutionize biomarker discovery and bring those markers to the clinic as early as possible., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Resistance to antitumor drugs, antimicrobial drugs, and antiviral drugs severely limits treatment effectiveness and cure rate of diseases. Protein post-translational modifications (PTMs) represented by glycosylation, ubiquitination, SUMOylation, acetylation, phosphorylation, palmitoylation, and lactylation are closely related to drug resistance. PTMs are typically achieved by adding sugar chains (glycosylation), small proteins (ubiquitination), lipids (palmitoylation), or functional groups (lactylation) to amino acid residues. These covalent additions are usually the results of signaling cascades and could be reversible, with the triggering mechanisms depending on the type of modifications. PTMs are involved in antitumor drug resistance, not only as inducers of drug resistance but also as targets for reversing drug resistance. Bacteria exhibit multiple PTMs-mediated antimicrobial drug resistance. PTMs allow viral proteins and host cell proteins to form complex interaction networks, inducing complex antiviral drug resistance. This review summarizes the important roles of PTMs in drug resistance, providing new ideas for exploring drug resistance mechanisms, developing new drug targets, and guiding treatment plans., Competing Interests: Declaration of Competing Interest The corresponding author Zhe-sheng Chen is the Editor-in-Chief of Drug Resistance Updates, The researchers state that this investigation was carried out without any business or monetary connections that could be interpreted as possible conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Histone posttranslational modifications (PTMs) contribute to transcriptional regulation and generate in combination a "histone code," which is largely conserved among organismal kingdoms. By binding to specific PTMs, histone reader proteins act as molecular interpreters of the histone code. These proteins play a crucial role in gene regulation and chromatin structure by recruiting other proteins to the chromatin or blocking histone-modifying enzymes from accessing chromatin. Revealing the specificity of histone readers is, therefore, fundamental to understanding their function in gene regulation. Here, we describe a protocol to identify the binding specificity of histone readers to PTMs using histone peptide arrays and in vitro pull-down assays., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Glycosylation, a crucial posttranslational modification (PTM), plays important roles in numerous biological processes and is linked to various diseases. Despite its significance, the structural complexity and diversity of glycans present significant challenges for mass spectrometry (MS)-based quantitative analysis. This review aims to provide an in-depth overview of recent advancements in labeling strategies for N-glycomics and O-glycomics, with a specific focus on enhancing the sensitivity, specificity, and throughput of MS analyses. We categorize these advancements into three major areas: (1) the development of isotopic/isobaric labeling techniques that significantly improve multiplexing capacity and throughput for glycan quantification; (2) novel methods that aid in the structural elucidation of complex glycans, particularly sialylated and fucosylated glycans; and (3) labeling techniques that enhance detection ionization efficiency, separation, and sensitivity for matrix-assisted laser desorption/ionization (MALDI)-MS and capillary electrophoresis (CE)-based glycan analysis. In addition, we highlight emerging trends in single-cell glycomics and bioinformatics tools that have the potential to revolutionize glycan quantification. These developments not only expand our understanding of glycan structures and functions but also open new avenues for biomarker discovery and therapeutic applications. Through detailed discussions of methodological advancements, this review underscores the critical role of derivatization methods in advancing glycan identification and quantification., (© 2024 The Author(s). Proteomics published by Wiley‐VCH GmbH.)

Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Microtubules, dynamic polymers assembled from α, β-tubulin dimers, contribute to myriad cellular processes. This is largely attributed to microtubule-associated proteins (MAPs). How MAPs selectively bind microtubules to carry out various functions is not known. The "Tubulin Code" theory proposes that posttranslational modifications (PTMs) of microtubules serve as signs that can be read by specific MAPs, thereby conferring specific functional properties to the microtubules. In support of this hypothesis, "reader" MAPs have been identified for various tubulin PTMs, but, until recently, no systematic screening had been performed to identify readers in an unbiased manner. We addressed this by developing a reader identification pipeline that uses quantitative mass spectrometry to interrogate the microtubule proteome of cells programmed to express specific PTMs. This pipeline can be used to identify readers for any tubulin PTM from various cell types as long as the writer enzymes are known. We also provide an alternative, complementary approach to obtain modified microtubules using a generic writer enzyme in vitro., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Forkhead box O (FOXO) transcription factors constitute a mammalian family of proteins, comprising FOXO1, FOXO3, FOXO4, and FOXO6. Originally recognized as downstream regulators within the insulin pathway, FOXO factors exhibit the ability to bind to diverse target gene promoters, thereby governing crucial facets of cellular homeostasis. These encompass cellular energy generation, resilience against oxidative stress, and the modulation of cell viability and proliferation. The dysregulation of FOXO proteins has been established as pivotal in metabolic disorders, human longevity, and the inhibition of tumorigenesis. Notably subject to posttranslational modifications for regulation, FOXO inactivation predominantly arises from excessive activation of their upstream modifying enzymes, presenting a plethora of potential avenues for pharmaceutical reinstatement of FOXO activity., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The microtubule cytoskeleton is a major driving force of neuronal circuit development. Fine-tuned remodelling of this network by selective activation of microtubule-regulating proteins, including microtubule-severing enzymes, has emerged as a central process in neuronal wiring. Tubulin posttranslational modifications control both microtubule properties and the activities of their interacting proteins. However, whether and how tubulin posttranslational modifications may contribute to neuronal connectivity has not yet been addressed. Here we show that the microtubule-severing proteins p60-katanin and spastin play specific roles in axon guidance during zebrafish embryogenesis and identify a key role for tubulin polyglutamylation in their functional specificity. Furthermore, our work reveals that polyglutamylases with undistinguishable activities in vitro, TTLL6 and TTLL11, play exclusive roles in motor circuit wiring by selectively tuning p60-katanin- and spastin-driven motor axon guidance. We confirm the selectivity of TTLL11 towards spastin regulation in mouse cortical neurons and establish its relevance in preventing axonal degeneration triggered by spastin haploinsufficiency. Our work thus provides mechanistic insight into the control of microtubule-driven neuronal development and homeostasis and opens new avenues for developing therapeutic strategies in spastin-associated hereditary spastic paraplegia., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)