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At least 19 recordsLinked to original sources
PubMed2025

Systematic Identification of Microtubule Posttranslational Modification "Readers" by Quantitative Proteomics.

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.

Protein Processing, Post-Translational↗
PubMed2026

Microtubule posttranslational modifications provide unique recognition patterns for associated proteins.

Microtubules are key components of the eukaryotic cytoskeleton involved in vital functions in virtually every cell. Among the emerging molecular mechanisms to adapt microtubules to their diverse functions is the biochemical diversification of tubulin molecules by posttranslational modifications (PTMs) and differential gene expression, a concept known as the 'tubulin code'. A key question remains whether the tubulin code has the potential to selectively control microtubule interactions of different microtubule-associated proteins (MAPs) to act as a specific signalling system. To answer this question, we used a medium-throughput in vitro approach to screen 46 proteins for their binding preferences to microtubules with altered PTM or isotype composition. We demonstrate that subsets of these MAPs have unique sensitivities to PTMs, while other proteins are not affected. As a result, PTMs, or combinations of them, differentially attract or repulse individual MAPs to microtubules. Our findings offer mechanistic proof for a key hypothesis of the tubulin code-the capacity to selectively and differentially regulate MAP-microtubule interactions.

Journal Article↗
PubMed2025

Quantitative Profiling of Histone Variants and Posttranslational Modifications by Tandem Mass Spectrometry in Arabidopsis.

Histone dynamics constitute an important layer of gene regulations associated with development and growth in multicellular eukaryotes. They also stand as key determinants of plant responses to environmental changes. Histone dynamics include the exchange of histone variants as well as post-translational modifications of their amino acid residues (such as acetylation and mono/di/trimethylation), commonly referred to as histone marks. Investigating histone dynamics with a focus on combinatorial changes occurring at their residues will greatly help unravel how plants achieve phenotypic plasticity.Mass spectrometry (MS) analysis offers unequaled resolution of the abundance of histone variants and of their marks. Indeed, relative to other techniques such as western blot or genome-wide profiling, this powerful technique allows quantifying the relative abundances of histone forms, as well as revealing coexisting marks on the same histone molecule. Yet, while MS-based histone analysis has proven efficient in several animals and other model organisms, this method stands out as more challenging in plants. One major challenge is the isolation of sufficient amounts of pure, high-quality histones, likely rendered difficult by the presence of the cell wall, for sufficiently deep and resolutive identification of histone species.In this chapter, we describe a straightforward MS-based proteomic method, implemented to characterize histone marks from Arabidopsis thaliana seedling tissues and cell culture suspensions. After acid extraction of histones, in vitro propionylation of free lysine residues, and digestion with trypsin, a treatment at highly basic pH allows obtaining sharp spectral signals of biologically relevant histone peptide forms.The method workflow described here shall be used to measure changes in histone marks between Arabidopsis thaliana genotypes, along developmental time-courses, or upon various stresses and treatments.

Histones↗
PubMed1979

Studies of esterase 6 in Drosophila melanogaster. I. The genetics of a posttranslational modification.

A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.

Alleles↗
PubMed2026

A posttranslational modification of fimbriae drives pathogenicity in Klebsiella pneumoniae.

Antimicrobial resistance is a severe public health burden. Especially concerning are multidrug resistant (MDR) infections, which restrict treatment options and significantly increase mortality risk. A major cause of MDR infections worldwide is carbapenem-resistant Klebsiella pneumoniae (CRKp). The predominant CRKp sequence type worldwide is ST258. However, the factors underlying ST258's epidemic success are not well defined. Genomic analyses of clinical isolates of CRKp have found that the two-component regulatory system CrrAB is a genomic feature of ST258, suggesting that it may contribute to its global dominance. Despite this, the molecular details underpinning CrrAB's contribution to ST258 Kp biology and pathogenicity are poorly understood. We used RNA-sequencing to identify the regulon of CrrA and found that CrrAB induces the expression of a gene, encoding Crr-regulated fimbriae modifying protein (CfmP), that is essential for pathogenesis driven by this two-component system. We performed mass spectrometry analyses of fimbriae purified from Kp expressing or lacking cfmP and found that CfmP induces a novel oxidation to a histidine residue in the major pilin subunit of fimbriae, FimA. We demonstrate that this oxidation significantly increases host cell adhesion and high bacterial loads within the host. CrrAB also drives high antibiotic resistance in CRKp. Thus, our results place CrrAB at the intersection of pathogenicity and antibiotic resistance supporting its function as an important regulatory system driving the global dominance of ST258.

Klebsiella pneumoniae↗
PubMed1977

Posttranslational covalent modification of proteins.

A search for derivatized amino acids in proteins has shown that the extent of posttranslational modification of proteins is quite substantial. While only 20 primary amino acids are specified in the genetic code and are involved as monomer building blocks in the assembly of the polypeptide chain, about 140 amino acids and amino acid derivatives have been identified as constituents of different proteins in different organisms. A brief consideration of the questions about where and when the derivatization reactions occur, how the specificity of the reactions is established, and how the posttranslational modifications can facilitate biological processes, reveal a need for more information on all these points. Answers to these questions should represent significant contributions to our understanding of biochemistry and cell biology.

Amino Acid Sequence↗
PubMed2026

An integrated proteomics and transcriptomics analysis highlights concordance between protein turnover and carbohydrate transport and metabolism as key functional categories during the growth of Trichophyton rubrum.

Dermatophytes are a class of keratinophilic skin fungi that invade host skin, hair, and nails to acquire nutrients. An integrated multi-omics approach utilizing liquid chromatography-tandem mass spectrometry and RNA-seq after growth in a protein-rich soy medium was employed to capture the major subset of secreted protein families of Trichophyton rubrum. The secretome consisted mainly of proteases and cell wall-degrading enzymes, with subtilisins (Sub6 and Sub7), metallopeptidase (LAP2), and chitinase having the most abundant peptides. Transcriptional profiling indicated fungal adaptation in protein-rich media to process the protein nutrients through modulation of metabolism and general cellular function pathways. Correlation analysis between proteomics and transcriptomics data using functional KOG categories shows high concordance of KOG categories O (posttranslational modification, protein turnover, and chaperones), P (inorganic ion transport and metabolism), and G (carbohydrate transport and metabolism), as per cosine similarity analysis.IMPORTANCEDermatophytes are keratinophilic skin fungal pathogens that invade host skin, hair, and nails to acquire nutrients. There is an epidemic-like increase in infections, as well as an increase in antimicrobial resistance among dermatophytes, as witnessed over the last decade. There is hence a need to understand the key pathways and virulence factors required during growth and infection. We present an integrated multi-omics analysis (proteomics and transcriptomics data) using a vector-based similarity approach to show high concordance of KOG functional categories belonging to posttranslational modification, protein turnover, carbohydrate transport, and metabolism.

Proteomics↗
PubMed1975

Posttranslational protein modifications, with special attention to collagen and elastin.

It is apparent that significant progress has been made in our understanding of the biosynthesis, modifications, and maturation of collagen and elastin. We now recognize and partially understand special reactions involved in hydroxylations within the cell and complex cross-linking processes occurring outside the cell. Recent experiments (191) have shown that in human diploid fibroblast cultures of limited doubling potential (191) the hydroxylation of collagen prolyl residues appears to be "age" or passage-level dependent. With increasing passage level of these cultures, both the ascorbate requirements and the extent of collagen hydroxylation decrease. "Young" cell cultures have a strong requirement for complete hydroxylation and without ascorbate there is only about 50% of the normal level. "Middle-aged" cultures show higher hydroxylation without and full hydroxylation with ascorbate, whereas "old" (or cultures close to "senescence") are incapable of full hydroxylation with or without ascorbic acid. Although the overall system may show some deterioration with increasing passage levels, it appears that with increasing passage levels other components in the cell replace the ascorbate dependence of the hydroxylase system to a greater exten. In some ways, aging WI-38 cultures begin to resemble some transformed cells in their biochemical reactions, although they continue to remain diploid and eventually lose the ability to replicate. It is not yet known whether old animals can produce collagen, which may now be underhydroxylated, perhaps contributing to certain senescent changes. Careful examination of the hydroxylation index of collagen produced in organoid cultures of tissue biopsies as a function of donor age might be informative, particularly if one looks at the quality of collagen by employing collagenase and other proteolytic digests with collagen (191). One could comare the levels of frequent and characteristic peptide triplet sequences such as Gly-Pro-Hyp to Gly-Pro-Pro, Gly-Ala-Hyp to Gly-Ala-Pro, or Gly-Pro-Hyl to Gly-Pro-Lys and others for evaluation of hydroxylation throughout the entire molecule or at selected sequences.

Amino Acid Sequence↗
PubMed2026

Lysine iminylation derived from ω-3 polyunsaturated fatty acids.

Protein posttranslational modifications (PTMs) play a central role for regulating protein function and cellular processes, with many PTMs arising from reactions with electrophilic metabolites. Here we extend the known landscape of PTMs with the identification of "lysine C3-iminylation," the conjugation of protein lysine residues with propionaldehyde. To stabilize iminylation for mass spectrometric analyses and distinguish it from other isomeric PTMs, we developed a fixation and stable-isotope labeling approach based on parallel reduction of proteome with sodium borohydride and borodeuteride. Analyses of protein hydrolysates confirmed the presence of C3-iminylation in Caenorhabditis elegans and mouse. Additionally, proteomics results demonstrated specificity of this PTM in vitro and in vivo and revealed C3-iminylation in proteins related to critical metabolic pathways. Importantly, collective evidence from isotope tracing as well as genetic, dietary, and pharmacological manipulation experiments uncovered that C3-iminylation originates from cytochrome P450 (CYP)-mediated oxidation of omega-3 fatty acids. Correspondingly, C3-iminylation levels were elevated in C. elegans daf-2(e1370) mutants, an aging model, in which CYP activity is generally increased. These findings not only expand our understanding of the biochemical diversity of PTMs but also underscore the complex interplay between lipid metabolism and protein modifications, enabling further exploration of their biological and clinical implications.

Animals↗
PubMed2025

A method for the detection and enrichment of endogenous cereblon substrates.

C-terminal cyclic imides are posttranslational modifications (PTMs) on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. Here, we develop an antibody-like reagent, termed "cerebody," for visualizing and enriching C-terminal cyclic imide-modified proteins. We describe the engineering of CRBN derivatives to produce cerebody and use it to identify CRBN substrates by western blot and enrichment from whole-cell and tissue lysates. CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation.

Humans↗
PubMed2026

Analytical challenges for mapping non-canonical and non-protein ubiquitin/Ubl modifications by mass spectrometry.

INTRODUCTION: Covalent modification by ubiquitin via Lys isopeptide bonds is fundamental for regulating protein turnover and function. Additionally, ubiquitin esterification occurs on Ser/Thr/Tyr residues in proteins and on non-proteinaceous substrates including ribose, saccharides, lipids, and small molecule drugs. Ubiquitin posttranslational modifications may therefore be much more widespread across cell biological pathways. Recent literature (PubMed) reflects the increased interest in analytical methods for mapping of non-canonical substrates modified by ubiquitin and ubiquitin-like (UBL) proteins. AREAS COVERED: Mass spectrometry (MS)-based methodologies involve advanced proteomic techniques to identify ubiquitin modifications on amino acids other than Lys, such as Ser, Thr, Tyr and Cys as well as protein N-termini. After digestion, standard MS workflows identify canonical ubiquitination by detecting a ubiquitin C-terminal tag attached to the amine side chains of Lys residues of substrate-derived peptides suitable for MS/MS sequencing. For non-canonical modifications on proteins and substrates other than proteins, specialized strategies are required, such as using antibodies to enrich N-terminally modified peptides in combination with using high-resolution MS/MS based on softer fragmentation technologies to detect esterification and possibly other types of substrate modifications. EXPERT OPINION: Enabling such technologies will reveal a previously unrecognized angle of the ubiquitin code's complexity in cells.

Humans↗
PubMed2025

Genome-Wide Profiling of Histone Modifications in Fission Yeast Using CUT&Tag.

Eukaryotic DNA is organized in the nucleus in the form of chromatin. Nucleosomes, the fundamental unit of chromatin, are subject to many posttranslational modifications (PTMs) as well as compositional variations through incorporation of histone variants. These alterations play important roles in regulation of genome structure and activity. Genome-wide profiling of these regulatory features is essential for understanding of genome function. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) is a widely used method to assay genome-wide localization in fission yeast but suffers from the requirement for a large amount of input chromatin, antibodies, and a cumbersome experimental pipeline. New methods such as Cleavage Under Targets and Tagmentation (CUT&Tag), which combine the specificity of targeted cleavage and adapter insertion with the sensitivity of next-generation sequencing, enable identification and characterization of various epigenetic marks affording low input requirement as well as more streamlined protocols. However, these approaches have not been adapted for use in fission yeast, Schizosaccharomyces pombe. Here, we describe an adapted CUT&Tag protocol for epigenomic profiling in fission yeast using the heterochromatin-associated histone H3K9 methylation PTM for benchmarking.

Schizosaccharomyces↗
PubMed2025

ChIPmentation for Epigenomic Analysis in Fission Yeast.

Histone modifications and transcription factor-DNA interactions regulate vital processes such as transcription, recombination, repair, and accurate chromosome segregation. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been instrumental in studying genome-wide distribution of DNA-bound or chromatin-associated factors and histone posttranslational modifications (PTMs). Here, we describe a ChIPmentation protocol adapted for fission yeast, Schizosaccharomyces pombe. This method merges Tn5 mediated tagmentation with existing ChIP protocols, resulting in lower sample input requirements with significant reduction in hands-on time and sample preparation costs.

Schizosaccharomyces↗
PubMed2026

Feeding the epigenome: EZH2 as a metabolic integrator of cell fate in development and cancer.

Epigenetic regulation is intimately linked to cellular metabolism, enabling environmental and nutritional cues to shape gene expression programs through dynamic modifications of chromatin structure. This metabolism-epigenetics interface is mediated, in part, by the dependence of chromatin-modifying enzymes on key metabolites, including S-adenosylmethionine (SAM), acetyl-CoA, UDP-GlcNAc, and α-ketoglutarate, which serve as substrates or cofactors for DNA and histone modifications. Among these regulators, EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), has emerged as a key mediator linking metabolic state to epigenetic regulation by translating metabolic inputs into changes in chromatin architecture and gene expression. EZH2 governs developmental cell fate through H3K27me3-mediated gene repression and is frequently dysregulated in cancer, where it promotes dedifferentiation, tumor progression, and metabolic reprogramming. Importantly, EZH2 activity is itself modulated by cellular metabolic status through posttranslational modifications, including phosphorylation, acetylation, methylation, ubiquitination, and O-GlcNAcylation, which influence its stability, catalytic activity, and chromatin-binding capacity. These modifications are responsive to nutrient availability and signaling pathways involving glucose, SAM, NAD+, and other metabolic intermediates. Consequently, disruption of this finely tuned regulatory network can contribute to developmental abnormalities, metabolic dysfunction, and oncogenesis. In this review, we examine the molecular mechanisms governing EZH2 regulation and discuss how metabolic control of EZH2 shapes chromatin dynamics, cell fate decisions, and disease pathogenesis. Elucidating how metabolic signals modulate EZH2 activity will advance our understanding of development and disease while uncovering potential therapeutic opportunities to target metabolism-driven epigenetic dysregulation.

Humans↗
PubMed1978

Characterization and genetic mapping of modA. A mutation in the post-translational modification of the glycosidases of Dictyostelium discoideum.

We have isolated a mutant of Dictyostelium discoideum, M31, which produces a reduced number of alpha-mannosidase-1 molecules per cell during the developmental program of the organism. We find that several of the glycosidases, a group of lysosomal proteins produced by D. discoideum, are altered in strain M31 and that this strain produces a reduced level of at least three of these activities. These enzymes do not share a common protein subunit but are known to share a common antigenic determinant which is, in part, carbohydrate in nature. In the wild type parent of M31, alpha-mannosidase-1 is modified by the addition of mannose and glucosamine (probably as N-acetylglucosamine) in the molar ratio of 5:2. alpha-Mannosidase-1 was also found to contain phosphoserine/phosphothreonine residues. alpha-Mannosidase-1 and other glycosidases are electrophoretically less negative when isolated from strain M31 than when isolated from wild type cells. The mutation present in M31, modA, appears to affect posttranslational modification, modA is a recessive mutation which we map onto linkage group I.

Amino Acids↗
PubMed2024

Mass Spectrometry-Based Proteomics for Assessing Epitranscriptomic Regulations.

Epitranscriptomics is a rapidly evolving field that explores chemical modifications in RNA and how they contribute to dynamic and reversible regulations of gene expression. These modifications, for example, N6-methyladenosine (m6A), are crucial in various RNA metabolic processes, including splicing, stability, subcellular localization, and translation efficiency of mRNAs. Mass spectrometry-based proteomics has become an indispensable tool in unraveling the complexities of epitranscriptomics, offering high-throughput, precise protein identification, and accurate quantification of differential protein expression. Over the past two decades, advances in mass spectrometry, including the improvement of high-resolution mass spectrometers and innovative sample preparation methods, have allowed researchers to perform in-depth analyses of epitranscriptomic regulations. This review focuses on the applications of bottom-up proteomics in the field of epitranscriptomics, particularly in identifying and quantifying epitranscriptomic reader, writer, and eraser (RWE) proteins and in characterizing their functions, posttranslational modifications, and interactions with other proteins. Together, by leveraging modern proteomics, researchers can gain deep insights into the intricate regulatory networks of RNA modifications, advancing fundamental biology, and fostering potential therapeutic applications.

Proteomics↗
PubMed1976

Intracellular enzymes of collagen biosynthesis in rat kidney in streptozotocin diabetes.

The activities of the four enzymes catalyzing intracellular post-translational modifications of the collagen polypeptide chains were assayed in the kidneys of rats with streptozotocin diabetes. When the changes in the four enzyme activities were expressed per milligram of protein in the 15,000 X g supernatant of the kidney homogenates, there were no changes in any of the enzyme activities at four weeks and only slight increases in the prolyl and lysyl hydroxylase activities at 12 weeks after the induction of diabetes. When the changes were expressed as total enzyme activities per two kidneys, again no changes were found in any enzyme activity at four weeks, but at 12 weeks significant increases were found in all four enzyme activities, namely prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase, and collagen glucosyltransferase. The data would be consistent with an increased collagen synthesis in diabetic kidneys, but they do not support the hypothesis that there might be specific changes in some of these enzyme activities or in the level of certain posttranslational modifications of the collagen polypeptide chains in this disease.

Animals↗
PubMed1979

Tyrosinase maturation and pigment expression in B16 melanoma: relation to theophylline treatment and intracellular cyclic AMP.

We have studied the effects of theophylline treatment on pigmentation characteristics and growth of two B16 melanoma cell lines, HFH-18 and P/140. Cell counts of control and theophylline-treated cultures confirmed that the drug inhibits cell growth. Light and electron microscope cytochemistry with the L-dopa reaction indicated that the two cell lines differ in their ability to transfer Golgi-associated tyrosinase to developing premelanosomes. The results of these experiments, considered with results of electrophoretic analyses and activity measurements by the Pomerantz method, also provide evidence that increased tyrosinase synthesis occurs in response to theophylline treatment. In addition, results indicate that theophylline induces changes in the rate of synthetic or degradative posttranslational modification of tyrosinase. Measurements of intracellular cyclic AMP levels by radioimmunoassay in control cultures and in theophylline- and alpha-MSH-treated cultures were made. Although the hormone induced spectacular increases in cyclic AMP levels, theophylline produced no detectable change. These results indicate that theophylline differs from alpha-MSH because theophylline-induced changes in pigmentation may not require the participation of intracellular cyclic AMP.

Animals↗