An emerging field: Post-translational modification in microbiome

Post-translational modifications (PTMs) play an essential role in most biological processes. PTMs on human proteins have been extensively studied. Studies on bacterial PTMs are emerging, which demonstrate that bacterial PTMs are different from human PTMs in their types, mechanisms and functions. Few PTM studies have been done on the microbiome. Here, we reviewed several studied PTMs in bacteria including phosphorylation, acetylation, succinylation, glycosylation, and proteases. We discussed the enzymes responsible for each PTM and their functions. We also summarized the current methods used to study microbiome PTMs and the observations demonstrating the roles of PTM in the microbe-microbe interactions within the microbiome and their interactions with the environment or host. Although new methods and tools for PTM studies are still needed, the existing technologies have made great progress enabling a deeper understanding of the functional regulation of the microbiome. Large-scale application of these microbiome-wide PTM studies will provide a better understanding of the microbiome and its roles in the development of human diseases.     

Spatiotemporal regulation of posttranslational modifications in the DNA damage response

A timely and accurate cellular response to DNA damage requires tight regulation of the action of DNA damage response (DDR) proteins at lesions. A multitude of posttranslational modifications (PTMs) of chromatin and chromatin‐associated proteins coordinates the recruitment of critical proteins that dictate the appropriate DNA repair pathway and enable the actual repair of lesions. Phosphorylation, ubiquitylation, SUMOylation, neddylation, poly(ADP‐ribosyl)ation, acetylation, and methylation are among the DNA damage‐induced PTMs that have taken center stage as important DDR regulators. Redundant and multivalent interactions of DDR proteins with PTMs may not only be a means to facilitate efficient relocalization, but also a feature that allows high temporal and spatial resolution of protein recruitment to, and extraction from, DNA damage sites. In this review, we will focus on the complex interplay between such PTMs, and discuss the importance of their interconnectivity in coding DNA lesions and maintaining the integrity of the genome.     

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

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

Progress in epigenetic histone modification analysis by mass spectrometry for clinical investigations

Chromatin biology and epigenetics are scientific fields that are rapid expanding due to their fundamental role in understanding cell development, heritable characters and progression of diseases. Histone post-translational modifications (PTMs) are major regulators of the epigenetic machinery due to their ability to modulate gene expression, DNA repair and chromosome condensation. Large-scale strategies based on mass spectrometry have been impressively improved in the last decade, so that global changes of histone PTM abundances are quantifiable with nearly routine proteomics analyses and it is now possible to determine combinatorial patterns of modifications. Presented here is an overview of the most utilized and newly developed proteomics strategies for histone PTM characterization and a number of case studies where epigenetic mechanisms have been comprehensively characterized. Moreover, a number of current epigenetic therapies are illustrated, with an emphasis on cancer.     

Top-down Proteomics: Technology Advancements and Applications to Heart Diseases

Introduction: Heart diseases are a leading cause of morbidity and mortality for both men and women worldwide, and impose significant economic burdens on the healthcare systems. Despite substantial effort over the last several decades, the molecular mechanisms underlying diseases of the heart remain poorly understood.

Areas covered: Altered protein post-translational modifications (PTMs) and protein isoform switching are increasingly recognized as important disease mechanisms. Top-down high-resolution mass spectrometry (MS)-based proteomics has emerged as the most powerful method for the comprehensive analysis of PTMs and protein isoforms. Here, we will review recent technology developments in the field of top-down proteomics, as well as highlight recent studies utilizing top-down proteomics to decipher the cardiac proteome for the understanding of the molecular mechanisms underlying diseases of the heart.

Expert commentary: Top-down proteomics is a premier method for the global and comprehensive study of protein isoforms and their PTMs, enabling the identification of novel protein isoforms and PTMs, characterization of sequence variations, and quantification of disease-associated alterations. Despite significant challenges, continuous development of top-down proteomics technology will greatly aid the dissection of the molecular mechanisms underlying diseases of the hearts for the identification of novel biomarkers and therapeutic targets.     

Readers of PCNA modifications

The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), acts as a central coordinator of DNA transactions by providing a multivalent interaction surface for factors involved in DNA replication, repair, chromatin dynamics and cell cycle regulation. Posttranslational modifications (PTMs), such as mono- and polyubiquitylation, sumoylation, phosphorylation and acetylation, further expand the repertoire of PCNA’s binding partners. These modifications affect PCNA’s activity in the bypass of lesions during DNA replication, the regulation of alternative damage processing pathways such as homologous recombination and DNA interstrand cross-link repair, or impact on the stability of PCNA itself. In this review, we summarise our current knowledge about how the PTMs are “read” by downstream effector proteins that mediate the appropriate action. Given the variety of interaction partners responding to PCNA’s modified forms, the ensemble of PCNA modifications serves as an instructive model for the study of biological signalling through PTMs in general.     

Post-translational Modification in Control of SIRT1 Stability during DNA Damage Response

SIRT1 (silent mating type information regulation 2 homolog 1), a class III histone deacetylase, is known to participate in multiple steps of the DNA damage response (DDR) by deacetylating several key DDR proteins. At present, the mechanisms regulating SIRT1 protein stability upon DNA damage have yet to be fully elucidated. In this study, we reveal that, under severe DNA damage, SIRT1 undergoes two forms of post-translational modifications (PTMs): (i) increased polyubiquitination and proteasomal degradation mediated by TRIM28 (tripartite motif-containing protein 28), a RING-domain E3 ligase; and (ii) cleavage at C-terminal mediated by caspases. Importantly, there is reciprocal effects between these forms of PTMs: while suppression of proteasome reduces caspases-mediated cleavage, the cleaved SIRT1 has enhanced interaction with TRIM28, thus facilitating the ubiquitination and proteasomal degradation of SIRT1. Functionally, SIRT1 works as an anti-apoptotic protein in DDR, and the above-mentioned PTMs of SIRT1 subsequently enhances cell death induced by DNA damage agents. Thus, our study has uncovered a pivotal role of SIRT1 post-translational regulation in determining cell fate in DDR.     

组蛋白泛素化修饰及其在DNA损伤应答中的作用

泛素化修饰是真核生物细胞内重要的翻译后修饰类型,通过调节蛋白质活性、稳定性和亚细胞定位广泛参与细胞内各项信号传导与代谢过程,对维持正常生命活动具有重要意义。组蛋白作为染色质中主要的蛋白成分,与DNA复制转录、修复等行为密切相关,是研究翻译后修饰的热点。DNA损伤后,组蛋白泛素化修饰通过调节核小体结构、激活细胞周期检查点、影响修复因子的招募与装配等诸多途径参与损伤应答。同时,组蛋白泛素化修饰还能调节其他位点翻译后修饰,并通过这种串扰(crosstalk)作用调节DNA损伤应答。本文介绍了组蛋白泛素化修饰的主要位点和相关组分(包括E3连接酶、去泛素化酶与效应分子),以及这些修饰作用共同编译形成的信号网络在DNA损伤应答中的作用,最后总结了目前该领域研究所面临的一些问题,以期为科研人员进一步探索组蛋白密码在DNA损伤应答中的作用提供参考。     

Proteomic analysis of phosphorylation, oxidation and nitrosylation in signal transduction

Signal transduction pathways control cell fate, survival and function. They are organized as intricate biochemical networks which enable biochemical protein activities, crosstalk and subcellular localization to be integrated and tuned to produce highly specific biological responses in a robust and reproducible manner. Post translational Modifications (PTMs) play major roles in regulating these processes through a wide variety of mechanisms that include changes in protein activities, interactions, and subcellular localizations. Determining and analyzing PTMs poses enormous challenges. Recent progress in mass spectrometry (MS) based proteomics have enhanced our capability to map and identify many PTMs. Here we review the current state of proteomic PTM analysis relevant for signal transduction research, focusing on two areas: phosphorylation, which is well established as a widespread key regulator of signal transduction; and oxidative modifications, which from being primarily viewed as protein damage now start to emerge as important regulatory mechanisms.     

Modification-specific proteomics in plant biology

Post-translational modifications (PTMs) are involved in the regulation of a wide range of biological processes, and affect e.g. protein structure, activity and stability. Several hundred PTMs have been described in the literature, but relatively few have been studied using mass spectrometry and proteomics. In general, methods for PTM characterization are developed to study yeast and mammalian biology and later adopted to investigate plants. Our point of view is that it is advantageous to enrich for PTMs on the peptide level as part of a quantitative proteomics strategy to not only identify the PTM, but also to determine the functional relevance in the context of regulation, response to abiotic stress etc. Protein phosphorylation is the only PTM that has been studied extensively at the proteome wide level in plants using mass spectrometry based methods.     

Early butyrate induced acetylation of histone H4 is proteoform specific and linked to methylation state

Histone posttranslational modifications (PTMs) help regulate DNA templated processes; however, relatively little work has unbiasedly explored the single-molecule combinations of histone PTMs, their dynamics on short timescales, or how these preexisting histone PTMs modulate further histone modifying enzyme activity. We use quantitative top down proteomics to unbiasedly measure histone H4 proteoforms (single-molecule combinations of PTMs) upon butyrate treatment. Our results show that histone proteoforms change in cells within 10 minutes of application of sodium butyrate. Cells recover from treatment within 30 minutes after removal of butyrate. Surprisingly, K20me2 containing proteoforms are the near-exclusive substrate of histone acetyltransferases upon butyrate treatment. Single-molecule hierarchies of progressive PTMs mostly dictate the addition and removal of histone PTMs (K16ac > K12ac ≥ K8ac > K5ac, and the reverse on recovery). This reveals the underlying single-molecule mechanism that explains the previously reported but indistinct and unexplained patterns of H4 acetylation. Thus, preexisting histone PTMs strongly modulate histone modifying enzyme activity and this suggests that proteoform constrained reaction pathways are crucial mechanisms that enable the long-term stability of the cellular epigenetic state.     

Engineering chromatin states: Chemical and synthetic biology approaches to investigate histone modification function

Patterns of histone post-translational modifications (PTMs) and DNA modifications establish a landscape of chromatin states with regulatory impact on gene expression, cell differentiation and development. These diverse modifications are read out by effector protein complexes, which ultimately determine their functional outcome by modulating the activity state of underlying genes. From genome-wide studies employing high-throughput ChIP-Seq methods as well as proteomic mass spectrometry studies, a large number of PTMs are known and their coexistence patterns and associations with genomic regions have been mapped in a large number of different cell types. Conversely, the molecular interplay between chromatin effector proteins and modified chromatin regions as well as their resulting biological output is less well understood on a molecular level. Within the last decade a host of chemical approaches has been developed with the goal to produce synthetic chromatin with a defined arrangement of PTMs. These methods now permit systematic functional studies of individual histone and DNA modifications, and additionally provide a discovery platform to identify further interacting nuclear proteins. Complementary chemical- and synthetic-biology methods have emerged to directly observe and modulate the modification landscape in living cells and to readily probe the effect of altered PTM patterns on biological processes. Herein, we review current methodologies allowing chemical and synthetic biological engineering of distinct chromatin states in vitro and in vivo with the aim of obtaining a molecular understanding of histone and DNA modification function. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.     

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.     

组蛋白翻译后修饰与DNA损伤响应蛋白招募的调控

组蛋白翻译后修饰是细胞DNA损伤早期应答反应的重要内涵,一方面是松弛、开放染色质结构的必要分子调节事件,以便DNA损伤响应蛋白能接近DNA损伤位点;另一方面直接参与DNA损伤修复蛋白招募过程的调控。综述了在DNA损伤信号激发下,发生的组蛋白主要修饰类型,异组蛋白H2AX、H2A.Z在DNA损伤部位与组蛋白置换,及其对DNA损伤响应蛋白招募的调节作用和机制。     

In-depth analysis of the Sirtuin 5-regulated mouse brain malonylome and succinylome using library-free data-independent acquisitions

Post-translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry-based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data-independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label-free quantification. In this study, we present a powerful approach that combines antibody-based enrichment with comprehensive DIA acquisitions and spectral library-free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, while offering a fully automated workflow. We analyzed brains from wild-type and Sirtuin 5 (SIRT5)-knock-out mice, and discovered and quantified 466 malonylated and 2211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 164 malonylated and 578 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 common SIRT5-regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome.