Motif-All: discovering all phosphorylation motifs

Background: Phosphorylation motifs represent common patterns around the phosphorylation site. The discovery of such kinds of motifs reveals the underlying regulation mechanism and facilitates the prediction of unknown phosphorylation event. To date, people have gathered large amounts of phosphorylation data, making it possible to perform substrate-driven motif discovery using data mining techniques. Results: We describe an algorithm called Motif-All that is able to efficiently identify all statistically significant motifs. The proposed method explores a support constraint to reduce search space and avoid generating random artifacts. As the number of phosphorylated peptides are far less than that of unphosphorylated ones, we divide the mining process into two stages: The first step generates candidates from the set of phosphorylated sequences using only support constraint and the second step tests the statistical significance of each candidate using the odds ratio derived from the whole data set. Experimental results on real data show that Motif-All outperforms current algorithms in terms of both effectiveness and efficiency. Conclusions: Motif-All is a useful tool for discovering statistically significant phosphorylation motifs. Source codes and data sets are available at: http://bioinformatics.ust.hk/MotifAll.rar

Human Protein Reference Database—2009 update

Human Protein Reference Database (HPRD—http://www.hprd.org/), initially described in 2003, is a database of curated proteomic information pertaining to human proteins. We have recently added a number of new features in HPRD. These include PhosphoMotif Finder, which allows users to find the presence of over 320 experimentally verified phosphorylation motifs in proteins of interest. Another new feature is a protein distributed annotation system—Human Proteinpedia (http://www.humanproteinpedia.org/)—through which laboratories can submit their data, which is mapped onto protein entries in HPRD. Over 75 laboratories involved in proteomics research have already participated in this effort by submitting data for over 15 000 human proteins. The submitted data includes mass spectrometry and protein microarray-derived data, among other data types. Finally, HPRD is also linked to a compendium of human signaling pathways developed by our group, NetPath (http://www.netpath.org/), which currently contains annotations for several cancer and immune signaling pathways. Since the last update, more than 5500 new protein sequences have been added, making HPRD a comprehensive resource for studying the human proteome

A comprehensive manually curated reaction map of RANKL/RANK-signaling pathway

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a member of tumor necrosis factor (TNF) superfamily that plays a key role in the regulation of differentiation, activation and survival of osteoclasts and also in tumor cell migration and bone metastasis. Osteoclast activation induced by RANKL regulates hematopoietic stem cell mobilization as part of homeostasis and host defense mechanisms thereby linking regulation of hematopoiesis with bone remodeling. Binding of RANKL to its receptor, Receptor activator of nuclear factor-kappa B (RANK) activates molecules such as NF-kappa B, mitogen activated protein kinase (MAPK), nuclear factor of activated T cells (NFAT) and phosphatidyl 3-kinase (PI3K). Although the molecular and cellular roles of these molecules have been reported previously, a systematic cataloging of the molecular events induced by RANKL/RANK interaction has not been attempted. Here, we present a comprehensive reaction map of the RANKL/RANK-signaling pathway based on an extensive manual curation of the published literature. We hope that the curated RANKL/RANK-signaling pathway model would enable new biomedical discoveries, which can provide novel insights into disease processes and development of novel therapeutic interventions

Understanding the Role of PknJ in Mycobacterium tuberculosis: Biochemical Characterization and Identification of Novel Substrate Pyruvate Kinase A

Reversible protein phosphorylation is a prevalent signaling mechanism which modulates cellular metabolism in response to changing environmental conditions. In this study, we focus on previously uncharacterized Mycobacterium tuberculosis Ser/Thr protein kinase (STPK) PknJ, a putative transmembrane protein. PknJ is shown to possess autophosphorylation activity and is also found to be capable of carrying out phosphorylation on the artificial substrate myelin basic protein (MyBP). Previous studies have shown that the autophosphorylation activity of M. tuberculosis STPKs is dependent on the conserved residues in the activation loop. However, our results show that apart from the conventional conserved residues, additional residues in the activation loop may also play a crucial role in kinase activation. Further characterization of PknJ reveals that the kinase utilizes unusual ions (Ni2+, Co2+) as cofactors, thus hinting at a novel mechanism for PknJ activation. Additionally, as shown for other STPKs, we observe that PknJ possesses the capability to dimerize. In order to elucidate the signal transduction cascade emanating from PknJ, the M. tuberculosis membrane-associated protein fraction is treated with the active kinase and glycolytic enzyme Pyruvate kinase A (mtPykA) is identified as one of the potential substrates of PknJ. The phospholabel is found to be localized on serine and threonine residue(s), with Ser37 identified as one of the sites of phosphorylation. Since Pyk is known to catalyze the last step of glycolysis, our study shows that the fundamental pathways such as glycolysis can also be governed by STPK-mediated signaling

Akt-Induced Phosphorylation of N-CoR at Serine 1450 Contributes to Its Misfolded Conformational Dependent Loss (MCDL) in Acute Myeloid Leukemia of the M5 Subtype

10.1371/journal.pone.0070891PLoS ONE88-POLN

Discovering Sequence Motifs with Arbitrary Insertions and Deletions

Biology is encoded in molecular sequences: deciphering this encoding remains a grand scientific challenge. Functional regions of DNA, RNA, and protein sequences often exhibit characteristic but subtle motifs; thus, computational discovery of motifs in sequences is a fundamental and much-studied problem. However, most current algorithms do not allow for insertions or deletions (indels) within motifs, and the few that do have other limitations. We present a method, GLAM2 (Gapped Local Alignment of Motifs), for discovering motifs allowing indels in a fully general manner, and a companion method GLAM2SCAN for searching sequence databases using such motifs. glam2 is a generalization of the gapless Gibbs sampling algorithm. It re-discovers variable-width protein motifs from the PROSITE database significantly more accurately than the alternative methods PRATT and SAM-T2K. Furthermore, it usefully refines protein motifs from the ELM database: in some cases, the refined motifs make orders of magnitude fewer overpredictions than the original ELM regular expressions. GLAM2 performs respectably on the BAliBASE multiple alignment benchmark, and may be superior to leading multiple alignment methods for “motif-like” alignments with N- and C-terminal extensions. Finally, we demonstrate the use of GLAM2 to discover protein kinase substrate motifs and a gapped DNA motif for the LIM-only transcriptional regulatory complex: using GLAM2SCAN, we identify promising targets for the latter. GLAM2 is especially promising for short protein motifs, and it should improve our ability to identify the protein cleavage sites, interaction sites, post-translational modification attachment sites, etc., that underlie much of biology. It may be equally useful for arbitrarily gapped motifs in DNA and RNA, although fewer examples of such motifs are known at present. GLAM2 is public domain software, available for download at http://bioinformatics.org.au/glam2

Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites

Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin αIIbβ3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin αIIbβ3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin αIIbβ3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future

Differential Proteomic Analysis of Mammalian Tissues Using SILAM

Differential expression of proteins between tissues underlies organ-specific functions. Under certain pathological conditions, this may also lead to tissue vulnerability. Furthermore, post-translational modifications exist between different cell types and pathological conditions. We employed SILAM (Stable Isotope Labeling in Mammals) combined with mass spectrometry to quantify the proteome between mammalian tissues. Using 15N labeled rat tissue, we quantified 3742 phosphorylated peptides in nuclear extracts from liver and brain tissue. Analysis of the phosphorylation sites revealed tissue specific kinase motifs. Although these tissues are quite different in their composition and function, more than 500 protein identifications were common to both tissues. Specifically, we identified an up-regulation in the brain of the phosphoprotein, ZFHX1B, in which a genetic deletion causes the neurological disorder Mowat–Wilson syndrome. Finally, pathway analysis revealed distinct nuclear pathways enriched in each tissue. Our findings provide a valuable resource as a starting point for further understanding of tissue specific gene regulation and demonstrate SILAM as a useful strategy for the differential proteomic analysis of mammalian tissues