Discovering motif pairs at interaction sites from protein sequences on a proteome-wide scale

MOTIVATION: Protein-protein interaction, mediated by protein interaction sites, is intrinsic to many functional processes in the cell. In this paper, we propose a novel method to discover patterns in protein interaction sites. We observed from protein interaction networks that there exist a kind of significant substructures called interacting protein group pairs, which exhibit an all-versus-all interaction between the two protein-sets in such a pair. The full-interaction between the pair indicates a common interaction mechanism shared by the proteins in the pair, which can be referred as an interaction type. Motif pairs at the interaction sites of the protein group pairs can be used to represent such interaction type, with each motif derived from the sequences of a protein group by standard motif discovery algorithms. The systematic discovery of all pairs of interacting protein groups from large protein interaction networks is a computationally challenging problem. By a careful and sophisticated problem transformation, the problem is solved using efficient algorithms for mining frequent patterns, a problem extensively studied in data mining. RESULTS: We found 5349 pairs of interacting protein groups from a yeast interaction dataset. The expected value of sequence identity within the groups is only 7.48%, indicating non-homology within these protein groups. We derived 5343 motif pairs from these group pairs, represented in the form of blocks. Comparing our motifs with domains in the BLOCKS and PRINTS databases, we found that our blocks could be mapped to an average of 3.08 correlated blocks in these two databases. The mapped blocks occur 4221 out of total 6794 domains (protein groups) in these two databases. Comparing our motif pairs with iPfam consisting of 3045 interacting domain pairs derived from PDB, we found 47 matches occurring in 105 distinct PDB complexes. Comparing with another putative domain interaction database InterDom, we found 203 matches. AVAILABILITY: http://research.i2r.a-star.edu.sg/BindingMotifPairs/resources. SUPPLEMENTARY INFORMATION: http://research.i2r.a-star.edu.sg/BindingMotifPairs and Bioinformatics online.     

Protein-specific imaging of posttranslational modifications

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Systematic functional prioritization of protein posttranslational modifications

Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory "hot spots" that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.     

Mass spectrometric analysis of histone posttranslational modifications

The uses of tandem and Fourier transform mass spectrometric methodologies for assignment of the posttranslational sites and occupancies of histones and their isoforms is described employing several illustrative examples. A comparison of information that can be obtained from intact protein sequencing and proteolytic digestion is presented.     

Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein

Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.     

The dynamics of the proteome: strategies for measuring protein turnover on a proteome-wide scale.

Quantitative proteomics captures the steady-state amount of a protein in a cell but does not explain how a change in protein amount is manifest -- whether through a change in synthesis or a change in degradation. If we are to understand the changes in the proteome, we will need to define such processes. In this brief review, strategies for the determination of intracellular protein dynamics on a proteome-wide scale are discussed.     

An atlas of posttranslational modifications on RNA binding proteins

RNA structure and function are intimately tied to RNA binding protein recognition and regulation. Posttranslational modifications are chemical modifications which can control protein biology. The role of PTMs in the regulation RBPs is not well understood, in part due to a lacking analysis of PTM deposition on RBPs. Herein, we present an analysis of posttranslational modifications (PTMs) on RNA binding proteins (RBPs; a PTM RBP Atlas). We curate published datasets and primary literature to understand the landscape of PTMs and use protein–protein interaction data to understand and potentially provide a framework for understanding which enzymes are controlling PTM deposition and removal on the RBP landscape. Intersection of our data with The Cancer Genome Atlas also provides researchers understanding of mutations that would alter PTM deposition. Additional characterization of the RNA–protein interface provided from in-cell UV crosslinking experiments provides a framework for hypotheses about which PTMs could be regulating RNA binding and thus RBP function. Finally, we provide an online database for our data that is easy to use for the community. It is our hope our efforts will provide researchers will an invaluable tool to test the function of PTMs controlling RBP function and thus RNA biology.     

Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature” forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Applications of diagonal chromatography for proteome-wide characterization of protein modifications and activity-based analyses

Numerous gel-free proteomics techniques have been reported over the past few years, introducing a move from proteins to peptides as bits of information in qualitative and quantitative proteome studies. Many shotgun proteomics techniques randomly sample thousands of peptides in a qualitative and quantitative manner but overlook the vast majority of protein modifications that are often crucial for proper protein structure and function. Peptide-based proteomic approaches have thus been developed to profile a diverse set of modifications including, but not at all limited, to phosphorylation, glycosylation and ubiquitination. Typical here is that each modification needs a specific, tailor-made analytical procedure. In this minireview, we discuss how one technique - diagonal reverse-phase chromatography - is applied to study two different types of protein modification: protein processing and protein N-glycosylation. Additionally, we discuss an activity-based proteome study in which purine-binding proteins were profiled by diagonal chromatography.     

Oxidative stress-induced posttranslational modifications of human hemoglobin in erythrocytes

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H₂O₂/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30–100 Gy triggered a remarkable increase of intracellular ROS. However, 30 Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100 Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.     

Regulation of Bcl-2 family proteins by posttranslational modifications

Like many proteins, function and abundance of Bcl-2 family proteins are influenced by posttranslational modifications. These modifications include phosphorylation, proteolytic cleavage, ubiquitination, and proteosomal degradation. These modifications, depending on cellular context and the proteins involved, can result either in a promotion of inhibition of apoptosis. Many of these modifications are governed by the activity of enzymes. As modulation of enzymatic activity can be achieved fairly efficiently using small molecules, understanding the effects of posttranslational modifications may allow for the therapeutic inhibition or promotion of apoptosis.     

Numerous posttranslational modifications provide opportunities for the intricate regulation of metabolic enzymes at multiple levels

The metabolic plasticity displayed by plants during normal development, and in response to environmental fluctuations and stressors, is essential for their growth and survival. The capacity to regulate metabolic enzymes intricately arises in part from posttranslational modifications that can affect enzymatic activity, intracellular localization, protein-protein interactions, and stability. Protein phosphorylation and thiol/disulfide redox modulation are important modifications in plants, and it is likely that O-glycosylation and S-nitrosylation will also emerge as important mechanisms. Recent advances in the field of proteomics, in particular the development of novel and specific chemistries for the detection of a diverse number of modifications, are rapidly expanding our awareness of possible modifications and our understanding of the enzymes whose functions are likely to be regulated posttranslationally.     

Analysis of posttranslational modifications exemplified using protein kinase A

With the completion of the major genome projects, one focus in biomedical research has shifted from the analysis of the rather static genome to the highly dynamic proteome. The sequencing of whole genomes did not lead to much anticipated insights into disease mechanisms; however, it paved the way for proteomics by providing the databases for protein identification by peptide mass fingerprints. The relative protein distribution within a cell or tissue is subject to change upon external and internal stimuli. Signal transduction events extend beyond a simple change in protein levels; rather they are governed by posttranslational modifications (PTMs), which provide a quick and efficient way to modulate cellular signals. Because most PTMs change the mass of a protein, they are amenable to analysis by mass spectrometry. Their investigation adds a level of functionality to proteomics, which can be expected to greatly aid in the understanding of the complex cellular machinery involved in signal transduction, metabolism, differentiation or in disease. This review provides an overview on posttranslational modifications exemplified on the model system cAMP-dependent protein kinase. Strategies for detection of selected PTMs are described and discussed in the context of protein kinase function.     

Interferon-stimulated gene 15 enters posttranslational modifications of p53

The tumor suppressor protein p53 is a central governor of various cellular signals. It is well accepted that ubiquitination as well as ubiquitin-like (UBL) modifications of p53 protein is critical in the control of its activity. Interferon-stimulated gene 15 (ISG15) is a well-known UBL protein with pleiotropic functions, serving both as a free intracellular molecule and as a modifier by conjugating to target proteins. Initially, attentions have historically focused on the antiviral effects of ISG15 pathway. Remarkably, a significant role in the processes of autophagy, DNA repair, and protein translation provided considerable insight into the new functions of ISG15 pathway. Despite the deterministic revelation of the relation between ISG15 and p53, the functional consequence of p53 ISGylation appears somewhat confused. More important, more recent studies have hinted p53 ubiquitination or other UBL modifications that might interconnect with its ISGylation. Here, we aim to summarize the current knowledge of p53 ISGylation and the differences in other significant modifications, which would be beneficial for the development of p53-based cancer therapy.     

InSite: a computational method for identifying protein-protein interaction binding sites on a proteome-wide scale

We propose InSite, a computational method that integrates high-throughput protein and sequence data to infer the specific binding regions of interacting protein pairs. We compared our predictions with binding sites in Protein Data Bank and found significantly more binding events occur at sites we predicted. Several regions containing disease-causing mutations or cancer polymorphisms in human are predicted to be binding for protein pairs related to the disease, which suggests novel mechanistic hypotheses for several diseases.