Distinctive core histone post-translational modification patterns in Arabidopsis thaliana

Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants.     

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.     

Rapid global characterization of immunoglobulin G1 following oxidative stress

ABSTRACT

Although peroxide and leachable metal-induced chemical modifications are among the most important quality attributes in bioprocess development, there is no mainstream characterization method covering all common modifications theoretically possible on therapeutic proteins that also gives consistent results quickly. Here, we describe a method for rapid and consistent global characterization of leachable metals- or peroxide-stressed immunoglobulin (Ig) G1 monoclonal antibodies (mAbs). Using two independent protease digestions, data-independent acquisition and data-dependent acquisition liquid chromatography high-resolution mass spectrometry, we monitored 55 potential chemical modifications on trastuzumab, a humanized IgG1 mAb. Processing templates including all observed peptides were developed on Skyline to consistently monitor all modifications throughout the stress conditions for both enzymatic digestions. The Global Characterization Data Processing Site, a universal automated data processing application, was created to batch process data, plot modification trends for peptides, generate sortable and downloadable modification tables, and produce Jmol code for three-dimensional structural models of the analyzed protein. In total, 53 sites on the mAb were found to be modified. Oxidation rates generally increased with the peroxide concentration, while leachable metals alone resulted in lower rates of modifications but more oxidative degradants. Multiple chemical modifications were found on IgG1 surfaces known to interact with Fc?RIII, complement protein C1q, and FcRn, potentially affecting activity. The combination of Skyline templates and the Global Characterization Data Processing Site results in a universally applicable assay allowing users to batch process numerous modifications. Applying this new method to stability studies will promote a broader and deeper understanding of stress modifications on therapeutic proteins.     

Mapping post-translational modifications of the histone variant MacroH2A1 using tandem mass spectrometry

Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although post-translational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys(115) is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the epsilon amino group of lysine residues Lys(17), Lys(122), and Lys(238) and phosphorylated on Thr(128). Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.     

Providing cellular signposts--post-translational modifications of intermediate filaments

Intermediate filaments are dynamically regulated by their post-translational modifications. Initially these modifications were found to regulate filament dynamics and organization. In the last few years, their roles have extended significantly to facilitating, for example, the recruitment and sequestration of signaling molecules that regulate a wide range of cellular functions. While phosphorylation has been established as the principal post-translational modification regulating intermediate filament function, other modifications with co-operative roles are emerging, adding a further dimensions to intermediate filament-mediated signaling.     

Improved RNA isolation from cells in tissue culture using a commercial nucleic acid extractor.

The Applied Biosystems 340A Nucleic Acid Extractor automates isolation of either DNA or RNA from tissue or cells in culture. We have found that several modifications to the manufacturer's recommended protocol greatly improve the quality of RNA that can be routinely isolated from cells in culture. These modifications include lysis of monolayer cells directly on plates, centrifuging samples after homogenization to remove precipitable RNase contaminants and purging the instrument's reagent lines with 0.1% diethyl pyrocarbonate. These simple modifications enhance both RNA quality and reproducibility of yield.     

Development of rabbit monoclonal and polyclonal antibodies for detection of site-specific histone modifications and their application in analyzing overall modification levels

In addition to DNA sequence information,site-specific histone modifications are another important determinant ofgene expression in a eukaryotic organism.We selected four modification sites in common histones that are known tosignificantly impact chromatin function and generated monoclonal or polyclonal antibodies that recognize each of thosesite-specific modifications.We used these antibodies to demonstrate that the site-specific histone modification levelsremain relatively constant in different organs of the same organism.We also compared the levels of selected histonemodifications among several representative organisms and found that site-specific modifications are highly variable amongdifferent organisms,providing new insight into the evolutionary divergence of specific histone modifications.     

Mapping of peroxyl radical induced damage on genomic DNA

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative base modifications were observed to occur to a greater extent than strand breaks at every concentration measured. The sequence-specific distribution of peroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine positions in the examined sequences was found to be significantly oxidized. The order of reactivity of DNA bases toward oxidation by peroxyl radicals was found to be G > C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by peroxyl radicals depends on the exact specification of 5' and 3' flanking bases in a polarity dependent manner. Every guanine in the 5'XGC3' motif was found to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' purine flanks drastically reduced the extent of peroxyl radical G oxidation. The pattern of base modification and the influence of nearest neighbors differs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequences.     

Interfacing protein lysine acetylation and protein phosphorylation: ?Ancient modifications meet on ancient proteins

Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature.     

7-Substituted 2-phenyl-benzofurans as ER beta selective ligands

A series of 2-(4-hydroxy-phenyl)-benzofuran-5-ols with relatively lipophilic groups in the 7-position of the benzofuran was prepared and the affinity and selectivity for ER beta was measured. Many of the analogues were found to be potent and selective ER beta ligands. Additional modifications at the benzofuran 4-position as well as at the 3'-position of the 2-phenyl group were found to further increase selectivity. Such modifications led to compounds with <10 nM potency and >100-fold selectivity for ER beta.     

Chemical modification of critical catalytic residues of lysine, arginine, and tryptophan in human glucose phosphate isomerase

Human glucose phosphate isomerase was subjected to a series of chemical modifications aimed at identifying residues essential for catalytic activity. A specific lysine was found to stoichiometrically react with pyridoxal 5'-phosphate forming a reversible Schiff base which could be reduced with NaBH4. The covalently modified enzyme was specifically cleaved with hydroxylamine at three labile Asn-Gly sequences yielding a series of peptides which were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis. The modified lysine was located in the COOH-terminal peptide. A critical arginine residue/subunit was found to be stoichiometrically modified with either 2,3-butadione or cyclohexadione. At high concentrations of butadione, an irreversible nonspecific modification of essentially all arginines occurred. An essential tryptophan residue was found to be stoichiometrically modified with N-bromosuccinimide in a similar fashion. Each of the chemical modifications of these three residues followed pseudo-first order and rate saturation kinetics and the modifications were prevented by the presence of substrates or competitive inhibitors. Circular dichroic spectral studies and analytical gel filtration indicated that these modifications have no effect on the quarternary structure and little effect on the secondary and tertiary structures of the enzyme. However, the extensive modification of arginine with butadione caused a dissociation of the enzyme into monomers and significant changes in tertiary structure. These studies provide new insights into functional aspects of isomerization and also provide an effective method for evaluating structural consequences of chemical or genetic modification of the enzyme.     

Modified alpha A crystallin in the retina: altered expression and truncation with aging

Crystallins are small heat shock proteins with chaperone function that prevent heat- and oxidative stress-induced aggregation of proteins. This is the first report describing modifications of alphaA crystallin in the sensory retina, including altered content and truncation with aging. Proteins from adult, middle age, and old Fischer 344 Brown Norway rats were compared. Western immunoblotting was used to evaluate alphaA crystallin content and identify protein spots on two-dimensional gels containing alphaA crystallin. The type and site of multiple post-translational modifications were identified by mass spectrometry. We found the content of alphaA crystallin was significantly decreased in the oldest rats. On two-dimensional gels, retinal crystallins resolved into multiple spots with altered migration, indicative of changes in intrinsic charge and/or truncation. Post-translational modifications that were identified included oxidation, phosphorylation, deamidation, acetylation, and truncation. In samples from rats of all ages, a highly modified N-terminus containing these modifications was found. We also observed an age-dependent difference in the extent of N- and C-terminal truncation. These results suggest that protection against stress-induced protein aggregation is compromised in the aged retina.     

PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state

Histone posttranslational modifications (PTMs) and sequence variants regulate genome function. Although accumulating evidence links particular PTM patterns with specific genomic loci, our knowledge concerning where and when these PTMs are imposed remains limited. Here, we find that lysine methylation is absent prior to histone incorporation into chromatin, except at H3K9. Nonnucleosomal H3.1 and H3.3 show distinct enrichments in H3K9me, such that H3.1 contains more K9me1 than H3.3. In addition, H3.3 presents other modifications, including K9/K14 diacetylated and K9me2. Importantly, H3K9me3 was undetectable in both nonnucleosomal variants. Notably, initial modifications on H3 variants can potentiate the action of enzymes as exemplified with Suv39HMTase to produce H3K9me3 as found in pericentric heterochromatin. Although the set of initial modifications present on H3.1 is permissive for further modifications, in H3.3 a subset cannot be K9me3. Thus, initial modifications impact final PTMs within chromatin.     

SeMoP: a new computational strategy for the unrestricted search for modified peptides using LC-MS/MS data

A novel computational approach, termed Search for Modified Peptides (SeMoP), for the unrestricted discovery and verification of peptide modifications in shotgun proteomic experiments using low resolution ion trap MS/MS spectra is presented. Various peptide modifications, including post-translational modifications, sequence polymorphisms, as well as sample handling-induced changes, can be identified using this approach. SeMoP utilizes a three-step strategy: (1) a standard database search to identify proteins in a sample; (2) an unrestricted search for modifications using a newly developed algorithm; and (3) a second standard database search targeted to specific modifications found using the unrestricted search. This targeted approach provides verification of discovered modifications and, due to increased sensitivity, a general increase in the number of peptides with the specific modification. The feasibility of the overall strategy has been first demonstrated in the analysis of 65 plasma proteins. Various sample handling induced modifications, such as beta-elimination of disulfide bridges and pyrocarbamidomethylation, as well as biologically induced modifications, such as phosphorylation and methylation, have been detected. A subsequent targeted Sequest search has been used to verify selected modifications, and a 4-fold increase in the number of modified peptides was obtained. In a second application, 1367 proteins of a cervical cancer cell line were processed, leading to detection of several novel amino acid substitutions. By conducting the search against a database of peptides derived from proteins with decoy sequences, a false discovery rate of less than 5% for the unrestricted search resulted. SeMoP is shown to be an effective and easily implemented approach for the discovery and verification of peptide modifications.     

The additivity principle in the reaction of angiotensin II and its analogs with antibodies and receptors of glomerular cells from the rat adrenal cortex

The binding of angiotensin II and its analogues (13) to rabbit antibodies and glomerular cell receptors from rat adrenal cortex was studied, using the radioimmunoassay method and radioreceptor analysis. Double modifications introduced into the angiotensin structure were found to increase in an additive fashion its binding to the antibodies and renal cell receptors. The relative binding activity of the analogues carrying a double modification can be assessed if the activities of the analogues with the appropriate single modifications are known. It was concluded that the testing of modifications in the peptide structure for their additivity may provide some insight into the conformational properties of peptides during their binding to the protein.     

Co- and post-translational modifications in Rubisco: unanswered questions

Both the large (LS) and small (SS) subunits of Rubisco are subject to a plethora of co- and post-translational modifications. With the exceptions of LS carbamylation and SS transit sequence processing, the remaining modifications, including deformylation, acetylation, methylation, and N-terminal proteolytic processing of the LS, are still biochemically and/or functionally undefined although they are found in nearly all forms of Rubisco from vascular plants. A collection of relatively unique enzymes catalyse these modifications, and several have been characterized in other organisms. Some of the observed modifications in the LS and SS clearly suggest novel changes in enzyme specificity and/or activity, and others have common features with other co- and post-translationally modifying enzymes. With the possible exception of Lys14 methylation in the LS, processing of both the LS and SS of Rubisco is by default an ordered process sequentially leading up to the final forms observed in the holoenzyme. An overview of the nature of structural modifications in the LS and SS of Rubisco is presented, and, where possible, the nature of the enzymes catalysing these modifications (either through similarity with other known enzymes or through direct enzymological characterization) is described. Overall, there are a distinct lack of functional and mechanistic observations for modifications in Rubisco and thus represent many potentially productive avenues for research.