Lysine methylation: beyond histones

Posttranslational modifications (PTMs) of histone proteins, such as acetylation, methylation, phosphorylation, and ubiquitylation, play essential roles in regulating chromatin dynamics. Combinations of different modifications on the histone proteins, termed 'histone code' in many cases, extend the information potential of the genetic code by regulating DNA at the epigenetic level. Many PTMs occur on non-histone proteins as well as histones, regulating protein-protein interactions, stability, localization, and/or enzymatic activities of proteins involved in diverse cellular processes. Although protein phosphorylation, ubiquitylation, and acetylation have been extensively studied, only a few proteins other than histones have been reported that can be modified by lysine methylation. This review summarizes the current progress on lysine methylation of non-histone proteins, and we propose that lysine methylation, like phosphorylation and acetylation, is a common PTM that regulates proteins in diverse cellular processes.     

Changes in the nuclear distribution of cyclin (PCNA) during S-phase are not triggered by post-translational modifications that are expected to moderately affect its charge

Indirect immunofluorescence studies of HeLa cells using PCNA autoantibodies specific for cyclin have revealed striking changes in the nuclear localization of this protein during S-phase. Two-dimensional gel electrophoretic analysis of the [32P]orthophosphate and [35S]methionine labelled proteins from synchronized cells showed that phosphorylation, or other post-translational modifications that are expected to moderately affect the charge of cyclin (acetylation, glycosylation, sialylation, etc.) are not likely part of the mechanism(s) triggering the migration of this protein.     

Synthesis of proteins with defined posttranslational modifications using the genetic noncanonical amino acid incorporation approach

Posttranslational modifications modulate the activities of most eukaryotic proteins and play a critical role in all aspects of cellular life. Understanding functional roles of these modifications requires homogeneously modified proteins that are usually difficult to purify from their natural sources. An emerging powerful tool for synthesis of proteins with defined posttranslational modifications is to genetically encode modified amino acids in living cells and incorporate them directly into proteins during the protein translation process. Using this approach, homogenous proteins with tyrosine sulfation, tyrosine phosphorylation mimics, tyrosine nitration, lysine acetylation, lysine methylation, and ubiquitination have been synthesized in large quantities. In this review, we provide a brief introduction to protein posttranslational modifications and the genetic noncanonical amino acid (NAA) incorporation technique, then discuss successful applications of the genetic NAA incorporation approach to produce proteins with defined modifications, and end with challenges and ongoing methodology developments for synthesis of proteins with other modifications.     

Brain Actin Synthesized In Vitro Undergoes Two Different and Sequential Posttranslational Modifications

Abstract: We have studied the posttranslational processing of actin molecules synthesized in a cell-free system. The results of these experiments indicate that during the in vivo synthesis of the actins from rat brain the primary translational products undergo two different and sequential posttranslational modifications. These modifications are accompanied by slight changes in the isoelectric points of the proteins and can be detected by isoelectric focusing analysis. The same posttranslational modifications can be detected during the in vitro synthesis of chick embryo skeletal muscle actin. The evidence presented suggest that the first posttranslational modification may correspond to the methylation of a histidine residue, and the second modification most likely corresponds to the acetylation of the NH₂-terminal amino acid residues of actin molecules.     

Effect of biotin on phosphorylation,acetylation, methylation of rat liver histones

Biotin deficient rat liver histones showed decreased phosphorylation and methylation, and increased acetylation rates as compared to normal rat liver histones: these alterations may be related to the observed lower stability of the interactions between histones and DNA. The modifications of the metabolic process might be the consequence of an alteration of the synthesis of the enzymes involved in histone phosphorylation, acetylation and methylation mechanisms and are presumably related to a biotin effect upon the synthesis of RNA and proteins.     

SUMO化和磷酸化的相互作用和共同修饰

翻译后修饰如磷酸化、乙酰化、甲基化、泛素化和SUMO化调节不同蛋白质的不同功能。磷酸化可能是最常见的修饰之一,蛋白质磷酸化通过一系列的激酶和磷酸酶催化,从而改变蛋白质功能。SUMO修饰是一种类泛素化修饰。SUMO修饰包括活化、结合、连接和解离,涉及多个酶多个步骤的催化过程。SUMO化可调节蛋白质相互作用、亚细胞定位、蛋白质稳定性和转录活性。关于磷酸化和SUMO化的蛋白质翻译后修饰,已有广泛研究报道。但很少关注于磷酸化和SUMO化之间的相互作用,以及它们对蛋白质的共同修饰。本文综述了蛋白质磷酸化和SUMO化之间的相互作用,以及共同修饰对细胞生理和肿瘤的影响。     

High mobility group proteins and their post-translational modifications

The high mobility group (HMG) proteins, including HMGA, HMGB and HMGN, are abundant and ubiquitous nuclear proteins that bind to DNA, nucleosome and other multi-protein complexes in a dynamic and reversible fashion to regulate DNA processing in the context of chromatin. All HMG proteins, like histone proteins, are subjected to extensive post-translational modifications (PTMs), such as lysine acetylation, arginine/lysine methylation and serine/threonine phosphorylation, to modulate their interactions with DNA and other proteins. There is a growing appreciation for the complex relationship between the PTMs of HMG proteins and their diverse biological activities. Here, we reviewed the identified covalent modifications of HMG proteins, and highlighted how these PTMs affect the functions of HMG proteins in a variety of cellular processes.     

Evolution of histone H3: emergence of variants and conservation of post-translational modification sites

Histone H3 proteins are highly conserved across all eukaryotes and are dynamically modified by many post-translational modifications (PTMs). Here we describe a method that defines the evolution of the family of histone H3 proteins, including the emergence of functionally distinct variants. It combines information from histone H3 protein sequences in eukaryotic species with the evolution of these species as described by the tree of life (TOL) project. This so-called TOL analysis identified the time when the few observed protein sequence changes occurred and when distinct, co-existing H3 protein variants arose. Four distinct ancient duplication events were identified where replication-coupled (RC) H3 variants diverged from replication-independent (RI) forms, like histone H3.3 in animals. These independent events occurred in ancestral lineages leading to the clades of metazoa, viridiplantae, basidiomycota, and alveolata. The proto-H3 sequence in the last eukaryotic common ancestor (LECA) was expanded to at least 133 of its 135 residues. Extreme conservation of known acetylation and methylation sites of lysines and arginines predicts that these PTMs will exist across the eukaryotic crown phyla and in protists with canonical chromatin structures. Less complete conservation was found for most serine and threonine phosphorylation sites. This study demonstrates that TOL analysis can determine the evolution of slowly evolving proteins in sequence-saturated datasets.     

A Comparative Analysis and Review of lysyl Residues Affected by Posttranslational Modifications

Post-translational modification is the most common mechanism of regulating protein function. If phosphorylation is considered a key event in many signal transduction pathways, other modifications must be considered as well. In particular the side chain of lysine residues is a target of different modifications; notably acetylation, methylation, ubiquitylation, sumoylation, neddylation, etc. Mass spectrometry approaches combining highly sensitive instruments and specific enrichment strategies have enabled the identification of modified sites on a large scale. Here we make a comparative analysis of the most representative lysine modifications (ubiquitylation, acetylation, sumoylation and methylation) identified in the human proteome. This review focuses on conserved amino acids, secondary structures preference, subcellular localization of modified proteins, and signaling pathways where these modifications are implicated. We discuss specific differences and similarities between these modifications, characteristics of the crosstalk among lysine post translational modifications, and single nucleotide polymorphisms that could influence lysine post-translational modifications in humans.     

Histone modifications as a platform for cancer therapy

Tumorigenesis and metastasis are a progression of events resulting from alterations in the processing of the genetic information. These alterations result from stable genetic changes (mutations) involving tumor suppressor genes and oncogenes (e.g., ras, BRAF) and potentially reversible epigenetic changes, which are modifications in gene function without a change in the DNA sequence. Mutations of genes coding for proteins that directly or indirectly influence epigenetic processes will alter the cell's gene expression program. Epigenetic mechanisms often altered in cancer cells are DNA methylation and histone modifications (acetylation, methylation, phosphorylation). This article will review the potential of these reversible epigenetic processes as targets for cancer therapies.     

The functional diversity of protein lysine methylation

Large‐scale characterization of post‐translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination, has highlighted their importance in the regulation of a myriad of signaling events. While high‐throughput technologies have tremendously helped cataloguing the proteins modified by these PTMs, the identification of lysine‐methylated proteins, a PTM involving the transfer of one, two or three methyl groups to the ε‐amine of a lysine side chain, has lagged behind. While the initial findings were focused on the methylation of histone proteins, several studies have recently identified novel non‐histone lysine‐methylated proteins. This review provides a compilation of all lysine methylation sites reported to date. We also present key examples showing the impact of lysine methylation and discuss the circuitries wired by this important PTM.     

Characterization of post-translational modifications of the linker histones H1 and H5 from chicken erythrocytes using mass spectrometry

Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified all six major isoforms of the chicken histone H1 (H101, H102, H103, H110, H11R and H11L) and, in addition, the specialist avian isoform H5. In all the histone variants, both the acetylated and nonacetylated N (alpha)-terminal peptides were identified. Mass spectrometry analysis also enabled the identification of a wide range of post-translational modifications including acetylation, methylation, phosphorylation and deamidation. Furthermore, a number of amino acids were identified that were modified with both acetylation and methylation. These results highlight the extensive modifications that are present on the linker histone proteins, indicating that, similar to the core histones, post-translational modifications of the linker histones may play a role in chromatin remodelling and gene regulation.     

有丝分裂期间蛋白质的翻译后修饰及其功能

有丝分裂期间蛋白质的翻译后修饰对于有丝分裂顺利完成以及细胞功能发挥具有重要的调控作用。常见的修饰类型包括磷酸化修饰、糖基化修饰、SUMO化修饰、乙酰化修饰、甲基化修饰。这些翻译后修饰可以维持染色体结构、促进后期染色体分离、协助末期核膜重新形成。本文对有丝分裂过程中相关蛋白质翻译后修饰的最新类型和功能进行了系统总结,以期能为肿瘤基础研究提供新的方向。