Application of mass spectrometry to the identification and quantification of histone post-translational modifications

The core histones are the primary protein component of chromatin, which is responsible for the packaging of eukaryotic DNA. The NH(2)-terminal tail domains of the core histones are the sites of numerous post-translational modifications that have been shown to play an important role in the regulation of chromatin structure. In this study, we discuss the recent application of modern analytical techniques to the study of histone modifications. Through the use of mass spectrometry, a large number of new sites of histone modification have been identified, many of which reside outside of the NH(2)-terminal tail domains. In addition, techniques have been developed that allow mass spectrometry to be effective for the quantitation of histone post-translational modifications. Hence, the use of mass spectrometry promises to dramatically alter our view of histone post-translational modifications.     

Physical methods used to study core histone tail structures and interactions in solution.

The core histone tail domains are key regulatory elements in chromatin. The tails are essential for folding oligonucleosomal arrays into both secondary and tertiary structures, and post-translational modifications within these domains can directly alter DNA accessibility. Unfortunately, there is little understanding of the structures and interactions of the core histone tail domains or how post-translational modifications within the tails may alter these interactions. Here we review NMR, thermal denaturation, cross-linking, and other selected solution methods used to define the general structures and binding behavior of the tail domains in various chromatin environments. All of these methods indicate that the tail domains bind primarily electrostatically to sites within chromatin. The data also indicate that the tails adopt specific structures when bound to DNA and that tail structures and interactions are plastic, depending on the specific chromatin environment. In addition, post-translational modifications, such as acetylation, can directly alter histone tail structures and interactions.     

Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions

Although post-translational modifications such as phosphorylation mediate fundamental biological processes within the cell, relatively few methods exist that allow proteome-wide identification of proteins that interact with these modifications. We constructed a yeast surface-displayed human cDNA library and utilized it to identify protein fragments with affinity for phosphorylated peptides derived from the major tyrosine autophosphorylation sites of the epidermal growth factor receptor or focal adhesion kinase. We identified cDNAs encoding the Src homology 2 domains from adapter protein APS, phosphoinositide 3-kinase regulatory subunit 3, SH2B, and tensin, demonstrating the effectiveness of this approach. Our results suggest that large libraries of functional human protein fragments can be efficiently displayed on the yeast surface. In addition to the analysis of post-translational modifications, yeast surface-displayed human cDNA libraries have many potential applications, including identifying targets and defining potential cross-reactive proteins for small molecules or drugs.     

Histone proteomics and the epigenetic regulation of nucleosome mobility

Chromatin structure plays a vital role in the transmission of heritable gene expression patterns. The recent application of mass spectrometry to histone biology provides several striking insights into chromatin regulation. The continuing identification of new histone post-translational modifications is revolutionizing the ways in which we think about how access to genomic DNA is controlled. While post-translational modifications of the flexible histone tails continue to be an active area of investigation, the recent discovery of multiple modifications in the structured globular domains of histones provides new insights into how the nucleosome works. Recent experiments underscore the importance of a subgroup of these modifications: those that regulate histone-DNA interactions on the lateral surface of the nucleosome. This information highlights an emerging new paradigm in chromatin control, that of the epigenetic regulation of nucleosome mobility.     

The tale beyond the tail: histone core domain modifications and the regulation of chromatin structure

Histone post-translational modifications occur, not only in the N-terminal tail domains, but also in the core domains. While modifications in the N-terminal tail function largely through the regulation of the binding of non-histone proteins to chromatin, based on their location in the nucleosome, core domain modifications may also function through distinct mechanisms involving structural alterations to the nucleosome. This article reviews the recent developments in regards to these novel histone modifications and discusses their important role in the regulation of chromatin structure.     

Intrinsic disorder in the kinesin superfamily

Kinesin molecular motors perform a myriad of intracellular transport functions. While their mechanochemical mechanisms are well understood and well-conserved throughout the superfamily, the cargo-binding and regulatory mechanisms governing the activity of kinesins are highly diverse and, in general, incompletely characterized. Here we present evidence from bioinformatic predictions indicating that most kinesin superfamily members contain significant regions of intrinsically disordered (ID) residues. ID regions can bind to multiple partners with high specificity and are highly labile to post-translational modification and degradation signals. In kinesins, the predicted ID regions are primarily found in areas outside the motor domains, where primary sequences diverge by family, suggesting that the ID may be a critical structural element for determining the functional specificity of individual kinesins. To support this concept, we present a systematic analysis of the kinesin superfamily, family by family, for predicted ID regions. We combine this analysis with a comprehensive review of kinesin-binding partners and post-translational modifications. We find two key trends across the entire kinesin superfamily. First, ID residues tend to be in the tail regions of kinesins, opposite the superfamily-conserved motor domains. Second, predicted ID regions correlate to regions that are known to bind to cargoes and/or undergo post-translational modifications. We therefore propose that the ID residue is a structural element utilized by the kinesin superfamily in order to impart functional specificity to individual kinesins.     

Genome-wide histone modifications: gaining specificity by preventing promiscuity

More than 20 residues within the four core histone proteins of the nucleosome are potential sites of post-translational modifications, such as methylation, acetylation, ubiquitination and phosphorylation. It has been hypothesized that specific patterns of these modifications on the nucleosome facilitate recruitment of non-histone proteins to chromatin. When such modifications are restricted to particular regions of the genome, they seem to play an important role in creating specific chromatin domains. However, more recent results suggest that some histone modifications, particularly those that exist on a genome-wide scale, act to reduce nonspecific binding by chromatin proteins involved in silencing. This decrease of promiscuous binding ensures that the silent chromatin proteins are not titrated away from their normal locations on chromosomes. We suggest that preventing such promiscuous binding of chromatin proteins is an important part of generating specificity to create chromatin domains and overall chromosome organization.     

Advances in collagen cross-link analysis

The combined application of ion-trap mass spectrometry and peptide-specific antibodies for the isolation and structural analysis of collagen cross-linking domains is illustrated with examples of results from various types of collagen with the emphasis on bone and cartilage. We highlight the potential of such methods to advance knowledge on the importance of post-translational modifications (e.g., degrees of lysine hydroxylation and glycosylation) and preferred intermolecular binding partners for telopeptide and helical cross-linking domains in regulating cross-link type and placement.     

Synthesis and assembly of functional high molecular weight adiponectin multimers in an engineered strain of Escherichia coli

Adiponectin has many beneficial effects on cardiovascular and obesity-related disorders. It is part of a class of proteins that contains short collagenous domains, along with surfactant proteins A and D, and complement protein C1q. This class of biomacromolecules requires post-translational modifications to form biologically active assemblies. By introducing a set of post-translational modifying enzymes into Escherichia coli , we have created a prokaryotic expression system that functionally assembles adiponectin, as assessed by the ability of produced adiponectin multimers to suppress human endothelial cell apoptosis. This study represents the first example of the assembly of functional high order multimers of any member of this class of proteins outside of eukaryotic cells. Furthermore, the results give fundamental insight into the process of assembly such as the necessity and sufficiency of various post-translational steps for functional assembly. We expect that fine-tuning of the expression system will allow for efficient production and functional assembly of biomolecules that assemble via short collagenous domains.     

Ubiquitinated Proteome: Ready for Global?

Ubiquitin (Ub) is a small and highly conserved protein that can covalently modify protein substrates. Ubiquitination is one of the major post-translational modifications that regulate a broad spectrum of cellular functions. The advancement of mass spectrometers as well as the development of new affinity purification tools has greatly expedited proteome-wide analysis of several post-translational modifications (e.g. phosphorylation, glycosylation, and acetylation). In contrast, large-scale profiling of lysine ubiquitination remains a challenge. Most recently, new Ub affinity reagents such as Ub remnant antibody and tandem Ub binding domains have been developed, allowing for relatively large-scale detection of several hundreds of lysine ubiquitination events in human cells. Here we review different strategies for the identification of ubiquitination site and discuss several issues associated with data analysis. We suggest that careful interpretation and orthogonal confirmation of MS spectra is necessary to minimize false positive assignments by automatic searching algorithms.     

Accurate annotation of peptide modifications through unrestrictive database search

Proteins are extensively modified after translation due to cellular regulation, signal transduction, or chemical damage. Peptide tandem mass spectrometry can discover post-translational modifications, as well as sequence polymorphisms. Recent efforts have studied modifications at the proteomic scale. In this context, it becomes crucial to assess the accuracy of modification discovery. We discuss methods to quantify the false discovery rate from a search and demonstrate how several features can be used to distinguish valid modifications from search artifacts. We present a tool, PTMFinder, which implements these methods. We summarize the corpus of post-translational modifications identified on large data sets. Thousands of known and novel modification sites are identified, including site-specific modifications conserved over vast evolutionary distances.     

Evolution of distinct EGF domains with specific functions

EGF domains are extracellular protein modules cross-linked by three intradomain disulfides. Past studies suggest the existence of two types of EGF domain with three-disulfides, human EGF-like (hEGF) domains and complement C1r-like (cEGF) domains, but to date no functional information has been related to the two different types, and they are not differentiated in sequence or structure databases. We have developed new sequence patterns based on the different C-termini to search specifically for the two types of EGF domains in sequence databases. The exhibited sensitivity and specificity of the new pattern-based method represents a significant advancement over the currently available sequence detection techniques. We re-annotated EGF sequences in the latest release of Swiss-Prot looking for functional relationships that might correlate with EGF type. We show that important post-translational modifications of three-disulfide EGFs, including unusual forms of glycosylation and post-translational proteolytic processing, are dependent on EGF subtype. For example, EGF domains that are shed from the cell surface and mediate intercellular signaling are all hEGFs, as are all human EGF receptor family ligands. Additional experimental data suggest that functional specialization has accompanied subtype divergence. Based on our structural analysis of EGF domains with three-disulfide bonds and comparison to laminin and integrin-like EGF domains with an additional inter-domain disulfide, we propose that these hEGF and cEGF domains may have arisen from a four-disulfide ancestor by selective loss of different cysteine residues.     

Structure and function of WD40 domain proteins

The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.     

Surface accessibility of protein post-translational modifications

Protein post-translational modifications are crucial to the function of many proteins. In this study, we have investigated the structural environment of 8378 incidences of 44 types of post-translational modifications with 19 different approaches. We show that modified amino acids likely to be involved in protein-protein interactions, such as ester-linked phosphorylation, methylarginine, acetyllysine, sulfotyrosine, hydroxyproline, and hydroxylysine, are clearly surface associated. Other modifications, including O-GlcNAc, phosphohistidine, 4-aspartylphosphate, methyllysine, and ADP-ribosylarginine, are either not surface associated or are in a protein's core. Artifactual modifications were found to be randomly distributed throughout the protein. We discuss how the surface accessibility of post-translational modifications can be important for protein-protein interactivity.