Assessing predictors for new post translational modification sites: Acase study on hydroxylation.

Post-translational modifications (PTMs) play very important roles in various cell signaling pathways and biological process. Due to PTMs' extremely important roles, many major PTMs have been studied, while the functional and mechanical characterization of major PTMs is well documented in several databases. However, most currently available databases mainly focus on protein sequences, while the real 3D structures of PTMs have been largely ignored. Therefore, studies of PTMs 3D structural signatures have been severely limited by the deficiency of the data. Here, we develop PRISMOID, a novel publicly available and free 3D structure database for a wide range of PTMs. PRISMOID represents an up-to-date and interactive online knowledge base with specific focus on 3D structural contexts of PTMs sites and mutations that occur on PTMs and in the close proximity of PTM sites with functional impact. The first version of PRISMOID encompasses 17145 non-redundant modification sites on 3919 related protein 3D structure entries pertaining to 37 different types of PTMs. Our entry web page is organized in a comprehensive manner, including detailed PTM annotation on the 3D structure and biological information in terms of mutations affecting PTMs, secondary structure features and per-residue solvent accessibility features of PTM sites, domain context, predicted natively disordered regions and sequence alignments. In addition, high-definition JavaScript packages are employed to enhance information visualization in PRISMOID. PRISMOID equips a variety of interactive and customizable search options and data browsing functions; these capabilities allow users to access data via keyword, ID and advanced options combination search in an efficient and user-friendly way. A download page is also provided to enable users to download the SQL file, computational structural features and PTM sites' data. We anticipate PRISMOID will swiftly become an invaluable online resource, assisting both biologists and bioinformaticians to conduct experiments and develop applications supporting discovery efforts in the sequence-structural-functional relationship of PTMs and providing important insight into mutations and PTM sites interaction mechanisms. The PRISMOID database is freely accessible at http://prismoid.erc.monash.edu/. The database and web interface are implemented in MySQL, JSP, JavaScript and HTML with all major browsers supported.

Potential Conformational Heterogeneity of p53 Bound to S100B(ββ)

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.

PTMcosmos is a comprehensive database with an interactive web portal designed to catalog and visualize post-translational modifications (PTMs) in humans. It contains 469,183 experimentally-validated PTM sites and their supporting evidence from UniProt Knowledge Base, PhosphoSitePlus, and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). PTMcosmos summarizes the entire spectrum of CPTAC proteomics data on human cancer patients, including protein and PTM peptide abundance data from 10 different cancer types. Additionally, PTMcosmos contains cancer somatic mutations from The Cancer Genome Atlas (TCGA), thus allowing for the collective integration and analysis of different data types. In PTMcosmos, we have built an ensemble of interactive visualization tools that allow researchers to investigate altered PTM functions due to genetic alterations in close proximity. The database is live at https://ptmcosmos.wustl.edu. We used PTMcosmos to investigate PTM regulation across cancer types. First, we examined the differential abundance of the PTM sites of cancer driver genes, focusing primarily on phosphorylation of the tumor suppressor retinoblastoma protein (encoded by RB1) and acetylation of the histone acetyltransferase E1A Binding Protein P300 (EP300) across cancer types. We analyzed the association between these PTM events and downstream targets, as well as with tumor subtypes, significantly mutated gene (SMG) mutation status, and clinical features. Second, we investigated the association of the protein abundance of cancer driver genes with ubiquitylsites in lung squamous cell carcinoma (LSCC) to nominate potentially novel modes of regulation of these proteins’ activities. We further analyzed the tumor subtype specificity and tumor-normal abundance changes of these ubiquitylsites and their corresponding substrate proteins, identifying several EGFR ubiquitylsites which may regulate EGFR abundance in LSCC. Finally, we identified the linear and spatial clustering of mutations and PTM sites, identifying multiple mutation-PTM clusters in cancer related genes, including TP53, PIK3CA, CTNNB1, EGFR, and IDH1. We envision that PTMcosmos will serve both the CPTAC consortium and the wider research community to better understand the role of PTMs in cancer. Citation Format: Liang-Bo Wang, Akshay Govindan, Song Cao, Li Ding. PTMcosmos: A web portal of post-translational modifications and proteogenomic resources in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5027.

Identification of the sites of post-translational modifications (PTMs) in protein, RNA, and DNA sequences is currently a very hot topic. This is because the information thus obtained is very useful for in-depth understanding the biological processes at the cellular level and for developing effective drugs against major diseases including cancers as well. Although this can be done by means of various experimental techniques, it is both time-consuming and costly to determine the PTM sites purely based on experiments. With the avalanche of biological sequences generated in the post-genomic age, it is highly desired to develop bioinformatics tools for rapidly and effectively identifying the PTM sites. In the last few years, many efforts have been made in this regard, and considerable progresses have been achieved. This review is focused on those prediction methods that have the following two features. (1) They have been developed by strictly observing the 5-steps rule so that they each have a user-friendly web-server for the majority of experimental scientists to easily get their desired data without the need to go through the detailed mathematics involved. (2) Their cornerstones have been based on Pseudo Amino Acid Composition (PseAAC) or Pseudo K-tuple Nucleotide Composition (PseKNC), and hence the prediction quality is generally higher than most of the other PTM prediction methods.

Post-translational modification (PTM) plays a crucial role in biological functions and corresponding disease developments. Discovering disease-associated non-synonymous SNPs (nsSNPs) altering PTM sites can help to estimate the various PTM candidates involved in diseases, therefore, an integrated analysis between SNPs, PTMs and diseases is necessary. However, only a few types of PTMs affected by nsSNPs have been studied without considering disease-association until now. In this study, we developed a new database called PTM-SNP which contains a comprehensive collection of human nsSNPs that affect PTM sites, together with disease information. Total 179,325 PTM-SNPs were collected by aligning missense SNPs and stop-gain SNPs on PTM sites (position 0) or their flanking region (position -7 to 7). Disease-associated SNPs from GWAS catalogs were also matched with detected PTM-SNP to find disease associated PTM-SNPs. Our result shows PTM-SNPs are highly associated with diseases, compared with other nsSNP sites and functional classes including near gene, intron and so on. PTM-SNP can provide an insight about discovering important PTMs involved in the diseases easily through the web site. PTM-SNP is freely available at http://gcode.kaist.ac.kr/ptmsnp.

Actin filament assembly contributes to the endocytic pathway pleiotropically, with active roles in clathrin-dependent and clathrin-independent endocytosis as well as subsequent endosomal trafficking. Endocytosis comprises a series of dynamic events, including the initiation of membrane curvature, bud invagination, vesicle abscission and subsequent vesicular transport. The ultimate success of endocytosis requires the coordinated activities of proteins that trigger actin polymerization, recruit actin-binding proteins (ABPs) and organize endocytic proteins (EPs) that promote membrane curvature through molecular crowding or scaffolding mechanisms. A particularly interesting phenomenon is that multiple EPs and ABPs contain a substantial percentage of intrinsically disordered regions (IDRs), which can contribute to protein coacervation and phase separation. In addition, intrinsically disordered proteins (IDPs) frequently contain sites for post-translational modifications (PTMs) such as phosphorylation, and these modifications exhibit a high preference for IDR residues [Groban ES et al. (2006) PLoS Comput Biol 2, e32]. PTMs are implicated in regulating protein function by modulating the protein conformation, protein-protein interactions and the transition between order and disorder states of IDPs. The molecular mechanisms by which IDRs of ABPs and EPs fine-tune actin assembly and endocytosis remain mostly unexplored and elusive. In this review, we analyze protein sequences of budding yeast EPs and ABPs, and discuss the potential underlying mechanisms for regulating endocytosis and actin assembly through the emerging concept of IDR-mediated protein multivalency, coacervation, and phase transition, with an emphasis on the phospho-regulation of IDRs. Finally, we summarize the current understanding of how these mechanisms coordinate actin cytoskeleton assembly and membrane curvature formation during endocytosis in budding yeast.

Both Carboxy-Terminal Tails of α- and β-Tubulin Are Essential, but Either One Will Suffice

InterPro is a new integrated documentation resource for protein families, domains and functional sites, developed initially as a means of rationalising the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Merged annotations from PRINTS, PROSITE and Pfam form the InterPro core. Each combined InterPro entry includes functional descriptions and literature references, and links are made back to the relevant parent database(s), allowing users to see at a glance whether a particular family or domain has associated patterns, profiles, fingerprints, etc. Merged and individual entries (i.e. those that have no counterpart in the companion resources) are assigned unique accession numbers. Release 1.2 of InterPro (June 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification (PTMs) encoded by 6581 different regular expressions, profiles, fingerprints and Hidden Markov Models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1000000 hits from 264333 different proteins out of 384572 in SWISS-PROT and TrEMBL).

BackgroundThe completion of 19 insect genome sequencing projects spanning six insect orders provides the opportunity to investigate the evolution of important gene families, here tubulins. Tubulins are a family of eukaryotic structural genes that form microtubules, fundamental components of the cytoskeleton that mediate cell division, shape, motility, and intracellular trafficking. Previous in vivo studies in Drosophila find a stringent relationship between tubulin structure and function; small, biochemically similar changes in the major alpha 1 or testis-specific beta 2 tubulin protein render each unable to generate a motile spermtail axoneme. This has evolutionary implications, not a single non-synonymous substitution is found in beta 2 among 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. This raises an important question, How do tubulins evolve while maintaining their function? To answer, we use molecular evolutionary analyses to characterize the evolution of insect tubulins.ResultsSixty-six alpha tubulins and eighty-six beta tubulin gene copies were retrieved and subjected to molecular evolutionary analyses. Four ancient clades of alpha and beta tubulins are found in insects, a major isoform clade (alpha 1, beta 1) and three minor, tissue-specific clades (alpha 2-4, beta 2-4). Based on a Homarus americanus (lobster) outgroup, these were generated through gene duplication events on major beta and alpha tubulin ancestors, followed by subfunctionalization in expression domain. Strong purifying selection acts on all tubulins, yet maximum pairwise amino acid distances between tubulin paralogs are large (0.464 substitutions/site beta tubulins, 0.707 alpha tubulins). Conversely orthologs, with the exception of reproductive tissue isoforms, show little sequence variation except in the last 15 carboxy terminus tail (CTT) residues, which serve as sites for post-translational modifications (PTMs) and interactions with microtubule-associated proteins. CTT residues overwhelming comprise the co-evolving residues between Drosophila alpha 2 and beta 3 tubulin proteins, indicating CTT specializations can be mediated at the level of the tubulin dimer. Gene duplications post-dating separation of the insect orders are unevenly distributed, most often appearing in major alpha 1 and minor beta 2 clades. More than 40 introns are found in tubulins. Their distribution among tubulins reveals that insertion and deletion events are common, surprising given their potential for disrupting tubulin coding sequence. Compensatory evolution is found in Drosophila beta 2 tubulin cis-regulation, and reveals selective pressures acting to maintain testis expression without the use of previously identified testis cis-regulatory elements.ConclusionTubulins have stringent structure/function relationships, indicated by strong purifying selection, the loss of many gene duplication products, alpha-beta co-evolution in the tubulin dimer, and compensatory evolution in beta 2 tubulin cis-regulation. They evolve through gene duplication, subfunctionalization in expression domain and divergence of duplication products, largely in CTT residues that mediate interactions with other proteins. This has resulted in the tissue-specific minor insect isoforms, and in particular the highly diverse α3, α4, and β2 reproductive tissue-specific tubulin isoforms, illustrating that even a highly conserved protein family can participate in the adaptive process and respond to sexual selection.

IntroductionRhoptries proteins (ROPs) are crucial throughout different stages of the Toxoplasma gondii (T. gondii) lifecycle, playing key roles in both the invasion of host cells and their subsequent survival. ROP34 is particularly noteworthy as it significantly influences host gene expression and aids in the transition from the tachyzoite to the bradyzoite form.Materials and methodsThis research utilized various bioinformatics tools to assess physico-chemical properties, allergenic and antigenic characteristics, sites for post-translational modifications (PTMs) and protein's secondary and three-dimensional structures of the ROP34 protein. Furthermore, the study identified potential B-cell, MHC-binding, and cytotoxic T-lymphocyte (CTL) epitopes within the ROP34 sequence.ResultsThe ROP34 peptide comprised 553 amino acid residues, with a calculated average molecular weight (MW) of 61.60149 kDa, an aliphatic index of 73.98, and a GRAVY score of − 0.554. The antigenicity of the multi-epitope peptide was estimated to be 0.526563 and 0.6025 by the ANTIGENpro and VaxiJen servers, respectively, suggesting ROP34 as an immunogenic protein with no allergenic potential. Secondary structure analysis revealed a composition of 52.80% random coil, 36.17% alpha helix, and 11.03% extended strand. The Ramachandran plot for the refined model depicted that 97.46% of the residues were situated in the favored region.ConclusionThis in silico research serves as a foundation for designing effective immunization tactics to target toxoplasmosis. The present article lays the groundwork for future studies and offers perspectives for the advancement of an appropriate toxoplasmosis vaccine.Graphical

Post-translational modifications (PTMs) represent an important regulatory instrument that modulates structure, dynamics and function of proteins. The large number of PTM sites in the Hsp90 proteins that are scattered throughout different domains indicated that synchronization of multiple PTMs through a combinatorial code can be invoked as an important mechanism to orchestrate diverse chaperone functions and recognize multiple client proteins. In this study, we have combined structural and coevolutionary analysis with molecular simulations and perturbation response scanning analysis of the Hsp90 structures to characterize functional role of PTM sites in allosteric regulation. The results reveal a small group of conserved PTMs that act as global mediators of collective dynamics and allosteric communications in the Hsp90 structures, while the majority of flexible PTM sites serve as sensors and carriers of the allosteric structural changes. This study provides a comprehensive structural, dynamic and network analysis of PTM sites across Hsp90 proteins, identifying specific role of regulatory PTM hotspots in the allosteric mechanism of the Hsp90 cycle. We argue that plasticity of a combinatorial PTM code in the Hsp90 may be enacted through allosteric coupling between effector and sensor PTM residues, which would allow for timely response to structural requirements of multiple modified enzymes.

MusiteDeep is an online resource providing a deep-learning framework for protein post-translational modification (PTM) site prediction and visualization. The predictor only uses protein sequences as input and no complex features are needed, which results in a real-time prediction for a large number of proteins. It takes less than three minutes to predict for 1000 sequences per PTM type. The output is presented at the amino acid level for the user-selected PTM types. The framework has been benchmarked and has demonstrated competitive performance in PTM site predictions by other researchers. In this webserver, we updated the previous framework by utilizing more advanced ensemble techniques, and providing prediction and visualization for multiple PTMs simultaneously for users to analyze potential PTM cross-talks directly. Besides prediction, users can interactively review the predicted PTM sites in the context of known PTM annotations and protein 3D structures through homology-based search. In addition, the server maintains a local database providing pre-processed PTM annotations from Uniport/Swiss-Prot for users to download. This database will be updated every three months. The MusiteDeep server is available at https://www.musite.net. The stand-alone tools for locally using MusiteDeep are available at https://github.com/duolinwang/MusiteDeep_web.

While tandem mass spectrometry can detect post-translational modifications (PTM) at the proteome scale, reported PTM sites are often incomplete and include false positives. Computational approaches can complement these datasets by additional predictions, but most available tools use prediction models pre-trained for single PTM type by the developers and it remains a difficult task to perform large-scale batch prediction for multiple PTMs with flexible user control, including the choice of training data. We developed an R package called PTMscape which predicts PTM sites across the proteome based on a unified and comprehensive set of descriptors of the physico-chemical microenvironment of modified sites, with additional downstream analysis modules to test enrichment of individual or pairs of PTMs in protein domains. PTMscape is flexible in the ability to process any major modifications, such as phosphorylation and ubiquitination, while achieving the sensitivity and specificity comparable to single-PTM methods and outperforming other multi-PTM tools. Applying this framework, we expanded proteome-wide coverage of five major PTMs affecting different residues by prediction, especially for lysine and arginine modifications. Using a combination of experimentally acquired sites (PSP) and newly predicted sites, we discovered that the crosstalk among multiple PTMs occur more frequently than by random chance in key protein domains such as histone, protein kinase, and RNA recognition motifs, spanning various biological processes such as RNA processing, DNA damage response, signal transduction, and regulation of cell cycle. These results provide a proteome-scale analysis of crosstalk among major PTMs and can be easily extended to other types of PTM.

Protein 3D structures, determined by their amino acid sequences, are the support of major crucial biological functions. Post-translational modifications (PTMs) play an essential role in regulating these functions by altering the physicochemical properties of proteins. By virtue of their importance, several PTM databases have been developed and released in decades, but very few of these databases incorporate real 3D structural data. Since PTMs influence the function of the protein and their aberrant states are frequently implicated in human diseases, providing structural insights to understand the influence and dynamics of PTMs is crucial for unraveling the underlying processes. This review is dedicated to the current status of databases providing 3D structural data on PTM sites in proteins. Some of these databases are general, covering multiple types of PTMs in different organisms, while others are specific to one particular type of PTM, class of proteins or organism. The importance of these databases is illustrated with two major types of in silico applications: predicting PTM sites in proteins using machine learning approaches and investigating protein structure-function relationships involving PTMs. Finally, these databases suffer from multiple problems and care must be taken when analyzing the PTMs data.