One‐Pot Semisynthesis of Exon 1 of the Huntingtin Protein: New Tools for Elucidating the Role of Posttranslational Modifications in the Pathogenesis of Huntington’s Disease

The natural enzymes involved in regulating many of the posttranslational modifications (PTMs) within the first 17 residues (Nt17) of Huntingtin exon 1 (Httex1) remain unknown. A semisynthetic strategy that allows the site‐specific introduction of PTMs within Nt17 by using expressed protein ligation (EPL) was developed. This strategy was used to produce untagged wild‐type (wt) and T3‐phosphorylated (pT3) Httex1 containing 23 glutamine residues (Httex1‐23Q). Our studies show that pT3 significantly slows the oligomerization and fibrillization of Httex1‐23Q and that Httex1 variants containing polyQ repeats below the pathogenic threshold readily aggregate and form fibrils in vitro. These findings suggest that crossing the polyQ pathogenic threshold is not essential for Httex1 aggregation. The ability to produce wt or site‐specifically modified tag‐free Httex1 should facilitate determining its structure and the role of N‐terminal PTMs in regulating the functions of Htt in health and disease.     

Comprehensive Profiling for Histone H4of Human Liver Cells Using High Resolution LTQ-Orbitrap Mass Spectrometry

The post translational modifications of histone variants are playing an important role in the structure of chro‐ matin, the regulation of gene activities and the diagnosis of diseases, and conducting in‐depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ‐Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH₂‐terminal tails were also identified after the LC‐MS/MS analysis of trypsin, Arg‐C, Glu‐C and chymotrypsin digests.     

Structure and binding of the H4 histone tail and the effects of lysine 16 acetylation

The H4 histone tail plays a critical role in chromatin folding and regulation--it mediates strong interactions with the acidic patch of proximal nucleosomes and its acetylation at lysine 16 (K16) leads to partial unfolding of chromatin. The molecular mechanism associated with the H4 tail/acidic patch interactions and its modulation via K16 acetylation remains unknown. Here we employ a combination of molecular dynamics simulations, molecular docking calculations, and free energy computations to investigate the structure of the H4 tail in solution, the binding of the H4 tail with the acidic patch, and the effects of K16 acetylation. The H4 tail exhibits a disordered configuration except in the region Ala15-Lys20, where it exhibits a strong propensity for an α-helical structure. This α-helical region is found to dock very favorably into the acidic patch groove of a nucleosome with a binding free energy of approximately -7 kcal mol(-1). We have identified the specific interactions that stabilize this binding as well as the associated energetics. The acetylation of K16 is found to reduce the α-helix forming propensity of the H4 tail and K16's accessibility for mediating external interactions. More importantly, K16 acetylation destabilizes the binding of the H4 tail at the acidic patch by mitigating specific salt bridges and longer-ranged electrostatic interactions mediated by K16. Our study thus provides new microscopic insights into the compaction of chromatin and its regulation via posttranslational modifications of histone tails, which could be of interest to chromatin biology, cancer, epigenetics, and drug design.     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular dynamics simulation on HP1 protein binding by histone H3 tail methylation and phosphorylation

Trimethylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Phosphorylation of histone H3 has been linked with mitotic chromatin condensation. During mitosis in vivo, H3 lysine 9 methylation and serine 10 phosphorylation can occur concomitantly on the same histone tail, whereas the influence of phosphorylation to trimethylation H3 tail recruiting HP1 remains controversial. In this work, molecular dynamics simulation of HP1 complexed with both trimethylated and phosphorylated H3 tail were performed and compared with the results from the previous methylated H3‐HP1 trajectory. It is clear from the 10‐ns dynamics simulation that two adjacent posttranslational modifications directly increase the flexibility of the H3 tail and weaken HP1 binding to chromatin. A combinatorial readout of two adjacent posttranslational modifications—a stable methylation and a dynamic phosphorylation mark—establish a regulatory mechanism of protein–protein interactions. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

A Versatile Approach for Site‐Specific Lysine Acylation in Proteins

Using amber suppression in coordination with a mutant pyrrolysyl‐tRNA synthetase‐tRNA^Pyl pair, azidonorleucine is genetically encoded in E. coli . Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site‐specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su‐H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post‐translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.     

Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

A range of isoxazole‐containing amino acids was synthesized that displaced acetyl‐lysine‐containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4‐mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole‐containing peptides are comparable to those of a hyperacetylated histone H4‐mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole‐based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4‐mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl‐lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3.     

Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin—immunity protein complex

To understand how proteins perform their function, knowledge about their structure and dynamics is essential. Here we use a combination of an efficient chemical lysine acetylation reaction and nanoLC-MALDI tandem mass spectrometry to probe the accessibility of every lysine residue in a protein complex. To demonstrate the applicability of this approach, we studied the interaction between the DNase domain of Colicin E9 (E9) and its immunity protein Im9. Free E9 and E9 in complex with Im9 were rapidly acetylated, followed by proteolytic digestion and analysis by LC-MALDI-TOF/TOF MS/MS. Acetylated peptides could be filtered out of the complex peptide mixtures using selective ion chromatograms of the specific immonium marker ions. Additionally, isobaric acetylated peptides, acetylated at different sites, could be separated by their LC retention times. The combination of LC and MALDI-TOF/TOF MS/MS provided information about the amount of acetylation on each individual lysine even for peptides containing several lysine residues. In general, our data agree well with those derived from the crystal structure of E9 and the E9:Im9 complex. Interestingly, next to in the binding interface expected lysines, K89 and K97, two from the crystal structure data unexpected lysines, K81 and K76, were observed to become less exposed upon Im9 binding. Moreover, K55 and K63, positioned in the predicted DNA binding region, were also found to be less accessible upon Im9 binding. These findings may illustrate some of the described differences in the solution-phase structure of the E9:Im9 complex compared with the crystal structure.     

A Genetically Encoded Allysine for the Synthesis of Proteins with Site‐Specific Lysine Dimethylation

Using the amber suppression approach, N^ϵ‐(4‐azidobenzoxycarbonyl)‐δ,ϵ‐dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site‐specific lysine dimethylation. Using this approach, dimethyl‐histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.     

Global proteomic analysis of lysine acetylation in zebrafish (Danio rerio) embryos

Lysine acetylation is an important post‐translational modification (PTM). Since the development of MS‐based proteomics technology, important roles of lysine acetylation beyond histones have focused on chromatin remodeling during the cell cycle and regulation of nuclear transport, metabolism, and translation. Zebrafish (Danio rerio) is a widely used vertebrate model in genetics and biologic studies. Although studies in several mammalian species have been performed, the mechanism of lysine acetylation in D. rerio embryos is incompletely understood. Here, we investigated the global acetylome in D. rerio embryos by using an MS‐based proteomics approach. We identified 351 acetylated peptides and 377 nonredundant acetylation sites on 189 lysine‐acetylated proteins in 5‐day postfertilization (hpf) embryos of D. rerio. Among lysine‐acetylated peptides, 40.2% indicated three motifs: (ac)KxxxK, (ac)KxxxxK, and Lx(ac)K. Of 190 acetylated proteins, 81 (42.6%) were mainly distributed in the cytoplasm. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that lysine acetylation in D. rerio was enriched in metabolic pathways. Additionally, 17 of 30 acetylated ribosomal proteins were evolutionarily conserved between zebrafish and humans. Our results indicate that acetyllysine might have regulatory effects on ribosomal proteins involved in protein biosynthesis.     

Simultaneous detection of protein phosphorylation and acetylation by high-resolution NMR spectroscopy

Post-translational protein modifications (PTMs) such as phosphorylation and acetylation regulate a large number of eukaryotic signaling processes. In most instances, it is the combination of different PTMs that "encode" the biological outcome of these covalent amendments in a highly dynamic and cell-state-specific manner. Most research tools fail to detect different PTMs in a single experiment and are unable to directly observe dynamic PTM states in complex environments such as cell extracts or intact cells. Here we describe in situ observations of phosphorylation and acetylation reactions by high-resolution liquid-state NMR spectroscopy. We delineate the NMR characteristics of progressive lysine acetylation and provide in vitro examples of joint phosphorylation and acetylation events and how they can be deciphered on a residue-specific basis and in a time-resolved and quantitative manner. Finally, we extend our NMR investigations to cellular phosphorylation and acetylation events in human cell extracts and demonstrate the unique ability of NMR spectroscopy to simultaneously report the establishment of these PTMs by endogenous cellular enzymes.     

Top‐down characterization of mouse core histones

Histone post‐translational modifications (PTMs) play various roles in chromatin‐related cellular processes, and comprehensive analysis of these combinatorial PTMs at the intact protein level by top‐down proteomics is the method of choice to reveal their crosstalk and biological functions. Here, we report our top‐down characterization of the core histones from mouse fibroblasts cells NIH/3T3, which is a classic model used in many kinds of research. With nanoRPLC‐MS/MS analysis and ProteinGoggle database search, 547 protein species were identified with spectrum‐level FDR ≤ 1%, where PTMs in 51 protein species were unambiguously localized with PTM scores ≥1. High‐resolution MS/MS data also allowed the unambiguous identification of acetylation instead of trimethylation. This study presents a general picture of combinatorial PTMs of mouse core histones, which serves as a basic reference for all future related biological studies.     

How Post‐Translational Modifications Influence Amyloid Formation: A Systematic Study of Phosphorylation and Glycosylation in Model Peptides

A reciprocal relationship between phosphorylation and O‐glycosylation has been reported for many cellular processes and human diseases. The accumulated evidence points to the significant role these post‐translational modifications play in aggregation and fibril formation. Simplified peptide model systems provide a means for investigating the molecular changes associated with protein aggregation. In this study, by using an amyloid‐forming model peptide, we show that phosphorylation and glycosylation can affect folding and aggregation kinetics differently. Incorporation of phosphoserines, regardless of their quantity and position, turned out to be most efficient in preventing amyloid formation, whereas O‐glycosylation has a more subtle effect. The introduction of a single β‐galactose does not change the folding behavior of the model peptide, but does alter the aggregation kinetics in a site‐specific manner. The presence of multiple galactose residues has an effect similar to that of phosphorylation.     

Tau蛋白的翻译后修饰与阿尔茨海默病

阿尔茨海默病（Alzheimer disease,AD）是一种常见的神经退行性疾病,由过度磷酸化Tau蛋白聚集形成的神经纤维缠结是该病主要的病理特征之一,Tau蛋白的异常磷酸化与Tau蛋白的聚集及AD的进程相关.越来越多的证据表明,Tau蛋白的异常聚集与Tau蛋白相关神经退行性疾病的发生和发展及Tau蛋白的其他翻译后修饰有一定的关系,如糖基化、乙酰化、截断、肽脯氨酸异构化、泛素化等.本文重点综述Tau蛋白翻译后修饰与AD相互关系的研究进展.     

The Pentablock Amphiphilic Copolymer T1107 Prevents Aggregation of Denatured and Reduced Lysozyme

Aggregation of denatured or unfolded proteins establishes a large energy barrier to spontaneous recovery of protein form and function following traumatic injury, tissue cryopreservation, and biopharmaceutical storage. Some tissues utilize small heat shock proteins (sHSPs) to prevent irreversible aggregation, which allows more complex processes to refold or remove the unfolded proteins. It is postulated that large, amphiphilic, and biocompatible block copolymers can mimic sHSP function. Reduced and denatured hen egg white lysozyme (HEWL) is used as a model aggregating protein. The poloxamine T1107 prevents aggregation of HEWL at 37 °C by three complimentary measures. Structural analysis of denatured HEWL reveals a partially folded conformation with preserved or promoted beta‐sheet structures only in the presence of T1107. The physical association of T1107 with denatured HEWL, and the ability to prevent aggregation, is linked to the critical micelle temperature of the polymer. The results suggest that T1107, or a similar amphiphilic block copolymer, can find use as a synthetic chaperone to augment the innate molecular repair mechanisms of natural cells.