GPS-SNO: Computational Prediction of Protein S-Nitrosylation Sites with a Modified GPS Algorithm

As one of the most important and ubiquitous post-translational modifications (PTMs) of proteins, S-nitrosylation plays important roles in a variety of biological processes, including the regulation of cellular dynamics and plasticity. Identification of S-nitrosylated substrates with their exact sites is crucial for understanding the molecular mechanisms of S-nitrosylation. In contrast with labor-intensive and time-consuming experimental approaches, prediction of S-nitrosylation sites using computational methods could provide convenience and increased speed. In this work, we developed a novel software of GPS-SNO 1.0 for the prediction of S-nitrosylation sites. We greatly improved our previously developed algorithm and released the GPS 3.0 algorithm for GPS-SNO. By comparison, the prediction performance of GPS 3.0 algorithm was better than other methods, with an accuracy of 75.80%, a sensitivity of 53.57% and a specificity of 80.14%. As an application of GPS-SNO 1.0, we predicted putative S-nitrosylation sites for hundreds of potentially S-nitrosylated substrates for which the exact S-nitrosylation sites had not been experimentally determined. In this regard, GPS-SNO 1.0 should prove to be a useful tool for experimentalists. The online service and local packages of GPS-SNO were implemented in JAVA and are freely available at: http://sno.biocuckoo.org/.

Related papers:

title: Protein S-nitrosylation in health and disease: a current perspective.
abstract: Protein S-nitrosylation constitutes a large part of the ubiquitous influence of nitric oxide on cellular signal transduction and accumulating evidence indicates important roles for S-nitrosylation both in normal physiology and in a broad spectrum of human diseases. Here we review recent findings that implicate S-nitrosylation in cardiovascular, pulmonary, musculoskeletal and neurological (dys)function, as well as in cancer. The emerging picture shows that, in many cases, pathophysiology correlates with hypo- or hyper-S-nitrosylation of specific protein targets rather than a general cellular insult due to loss of or enhanced nitric oxide synthase activity. In addition, it is increasingly evident that dysregulated S-nitrosylation can not only result from alterations in the expression, compartmentalization and/or activity of nitric oxide synthases, but can also reflect a contribution from denitrosylases, including prominently the S-nitrosoglutathione (GSNO)-metabolizing enzyme GSNO reductase. Finally, because exogenous mediators of protein S-nitrosylation or denitrosylation can substantially affect the development or progression of disease, potential therapeutic agents that modulate S-nitrosylation could well have broad clinical utility.

title: S-nitrosylation in health and disease.
abstract: S-nitrosylation is a ubiquitous redox-related modification of cysteine thiol by nitric oxide (NO), which transduces NO bioactivity. Accumulating evidence suggests that the products of S-nitrosylation, S-nitrosothiols (SNOs), play key roles in human health and disease. In this review, we focus on the reaction mechanisms underlying the biological responses mediated by SNOs. We emphasize reactions that can be identified with complex (patho)physiological responses, and that best rationalize the observed increase or decrease in specific classes of SNOs across a spectrum of disease states. Thus, changes in the levels of various SNOs depend on specific defects in both enzymatic and non-enzymatic mechanisms of nitrosothiol formation, processing and degradation. An understanding of these mechanisms is crucial for the development of an integrated model of NO biology, and for effective treatment of diseases associated with dysregulation of NO homeostasis.

title: Regulation and specificity of S-nitrosylation and denitrosylation.
abstract: Signal transduction from nitric oxide (NO) is important in physiological processes such as smooth muscle relaxation and neurotransmission. NO signaling occurs through a variety of mechanisms, including S-nitrosylation (SNO) of sulfhydryl groups in protein. A paper in this issue of the journal dissects the regulatory system involved in SNO of caspase-3 and highlights the need for more direct investigations of the complex biological regulation by NO signaling.

title: S-nitrosylation: spectrum and specificity
abstract: Nitric oxide regulates a broad functional spectrum of proteins by S-nitrosylation. Specificity is conferred by acid–base and hydrophobic motifs that target critical cysteine residues and by protein–protein interactions that confine the signals in space.

title: Nitrosylation The Prototypic Redox-Based Signaling Mechanism
abstract: posttranslational modifications of proteins operate by of Medicine and Biochemistry shifting a dynamic equilibrium between inactive and ac-in particular has served as a model of how protein modi-2 Centro de Investigaciones Biologicas fication triggers conformational changes that regulate Instituto " Reina Sofia " de Investigaciones Nefrologicas signal transduction. These fundamental concepts of al-Consejo Superior de Investigaciones Cientificas losteric regulation in fact originate in studies of the post-Velazquez 144 translational modification of hemoglobin by small di-28006 Madrid atomic ligands. Thus, whereas phosphorylation clearly Spain lies at the heart of many signal transduction pathways, has been expanded re-translational modification of proteins, are conserved cently by the discovery of an enzymatic function for throughout evolution and influence most aspects of cel-hemoglobin, operating within the confines of a mem-lular physiology—one is phosphorylation and the other brane-localized signaling module by binding to its target is redox based. Both exemplify dynamic regulation of protein and introducing NO groups (much as a kinase protein function by reversible modification, and they introduces phosphates) (Pawloski et al., 2001). In this govern many of the same signal transduction pathways case, a redox-based signal that is initiated through an through overlapping sets of cellular targets. They are O 2-induced allosteric transition in hemoglobin is propa-also prone to malfunction in human disease. However, gated by S-nitrosylation of an anion exchanger to sub-while many basic principles of signal transduction have serve intercellular communication. emerged from studies of phosphorylation, the redox-Two key themes have emerged from these studies of based mechanism has remained far more enigmatic. A hemoglobin: (1) the importance of allostery in posttrans-major difficulty has been to comprehend how specificity lational modification of proteins by NO, and (2) the role of action is achieved. Recent advances in understanding of O 2 /redox as an allosteric effector. The ryanodine re-how nitric oxide (NO) regulates protein function are now ceptor/calcium release channel (RyR), which is con-ogy to hemoglobin, in terms of an equilibrium between 6/675/DC1). Second, the majority of these proteins are two alternative structures that is governed by O 2 tension. regulated by S-nitrosylation of a single critical cysteine In this model, a redox-driven conformational change in residue within an acid-base or hydrophobic structural the channel provides a hydrophobic compartment that motif, which may also be subject to oxygen-or glutathi-concentrates NO and O 2 , thus generating nitrosylating one-dependent modification. Thus, S-nitrosylation equivalents. The observation that S-nitrosylation acti-emerges as a prototypic redox-based signal. …

title: S-nitrosylation of XIAP compromises neuronal survival in Parkinson's disease
abstract: Inhibitors of apoptosis (IAPs) are a family of highly-conserved proteins that regulate cell survival through binding to caspases, the final executioners of apoptosis. X-linked IAP (XIAP) is the most widely expressed IAP and plays an important function in regulating cell survival. XIAP contains 3 baculoviral IAP repeats (BIRs) followed by a RING finger domain at the C terminal. The BIR domains of XIAP possess anticaspase activities, whereas the RING finger domain enables XIAP to function as an E3 ubiquitin ligase in the ubiquitin and proteasomal system. Our previous study showed that parkin, a protein that is important for the survival of dopaminergic neurons in Parkinson's disease (PD), is S-nitrosylated both in vitro and in vivo in PD patients. S-nitrosylation of parkin compromises its ubiquitin E3 ligase activity and its protective function, which suggests that nitrosative stress is an important factor in regulating neuronal survival during the pathogenesis of PD. In this study we show that XIAP is S-nitrosylated in vitro and in vivo in an animal model of PD and in PD patients. Nitric oxide modifies mainly cysteine residues within the BIR domains. In contrast to parkin, S-nitrosylation of XIAP does not affect its E3 ligase activity, but instead directly compromises its anticaspase-3 and antiapoptotic function. Our results confirm that nitrosative stress contributes to PD pathogenesis through the impairment of prosurvival proteins such as parkin and XIAP through different mechanisms, indicating that abnormal S-nitrosylation plays an important role in the process of neurodegeneration.

title: Cisplatin-induced nitrosylation of p53 prevents its mitochondrial translocation.
abstract: The cellular response to DNA damage has been reported to involve rapid transcription-independent translocation of p53 to mitochondria. We show here that the DNA-damaging cisplatin-derived anticancer agent oxaliplatin induced both mitochondrial translocation and subsequent Bcl-xL interaction, whereas cisplatin did neither. The differential response was due to nitrosative modification of p53. Thus, cisplatin, but not oxaliplatin, induced increased expression of inducible nitric oxide synthase (iNOS). Cisplatin treatment in the presence of an iNOS inhibitor (1400W) allowed p53 mitochondrial translocation. Conversely, oxaliplatin-induced translocation of p53 was prevented by cotreatment with an exogenous NO donor. In cisplatin-treated cells, nuclear but not mitochondrial p53 showed nitrotyrosinylation that was inhibitable by 1400W. We conclude that nitrosative protein modification is more prominent in the response to cisplatin than oxaliplatin and that nitrosative modification of p53 is a major determinant of p53 subcellular location.

title: S-nitrosylation of beta-arrestin regulates beta-adrenergic receptor trafficking.
abstract: Signal transduction through G protein-coupled receptors (GPCRs) is regulated by receptor desensitization and internalization that follow agonist stimulation. Nitric oxide (NO) can influence these processes, but the cellular source of NO bioactivity and the effects of NO on GPCR-mediated signal transduction are incompletely understood. Here, we show in cells and mice that beta-arrestin 2, a central element in GPCR trafficking, interacts with and is S-nitrosylated at a single cysteine by endothelial NO synthase (eNOS), and that S-nitrosylation of beta-arrestin 2 is promoted by endogenous S-nitrosogluthathione. S-nitrosylation after agonist stimulation of the beta-adrenergic receptor, a prototypical GPCR, dissociates eNOS from beta-arrestin 2 and promotes binding of beta-arrestin 2 to clathrin heavy chain/beta-adaptin, thereby accelerating receptor internalization. The agonist- and NO-dependent shift in the affiliations of beta-arrestin 2 is followed by denitrosylation. Thus, beta-arrestin subserves the functional coupling of eNOS and GPCRs, and dynamic S-nitrosylation/denitrosylation of beta-arrestin 2 regulates stimulus-induced GPCR trafficking.

title: S-nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons
abstract: Brain-derived neurotrophic factor (BDNF) and other neurotrophins have a vital role in the development of the rat and mouse nervous system by influencing the expression of many specific genes that promote differentiation, cell survival, synapse formation and, later, synaptic plasticity. Although nitric oxide (NO) is known to be an important mediator of BDNF signalling in neurons, the mechanisms by which neurotrophins influence gene expression during development and plasticity remain largely unknown. Here we show that BDNF triggers NO synthesis and S-nitrosylation of histone deacetylase 2 (HDAC2) in neurons, resulting in changes to histone modifications and gene activation. S-nitrosylation of HDAC2 occurs at Cys 262 and Cys 274 and does not affect deacetylase activity. In contrast, nitrosylation of HDAC2 induces its release from chromatin, which increases acetylation of histones surrounding neurotrophin-dependent gene promoters and promotes transcription. Notably, nitrosylation of HDAC2 in embryonic cortical neurons regulates dendritic growth and branching, possibly by the activation of CREB (cyclic-AMP-responsive-element-binding protein)-dependent genes. Thus, by stimulating NO production and S-nitrosylation of HDAC2, neurotrophic factors promote chromatin remodelling and the activation of genes that are associated with neuronal development.

title: Protein S-nitrosylation: a physiological signal for neuronal nitric oxide
abstract: Nitric oxide (NO) has been linked to numerous physiological and pathophysiological events that are not readily explained by the well established effects of NO on soluble guanylyl cyclase. Exogenous NO S-nitrosylates cysteine residues in proteins, but whether this is an important function of endogenous NO is unclear. Here, using a new proteomic approach, we identify a population of proteins that are endogenously S-nitrosylated, and demonstrate the loss of this modification in mice harbouring a genomic deletion of neuronal NO synthase (nNOS). Targets of NO include metabolic, structural and signalling proteins that may be effectors for neuronally generated NO. These findings establish protein S-nitrosylation as a physiological signalling mechanism for nNOS.

title: The Biotin Switch Method for the Detection of S-Nitrosylated Proteins
abstract: Many of the effects of nitric oxide are mediated by the direct modification of cysteine residues resulting in an adduct called a nitrosothiol. Here, we describe a novel method for detecting proteins that contain nitrosothiols. In this three-step procedure, nitrosylated cysteines are converted to biotinylated cysteines. Biotinylated proteins can then be detected by immunoblotting or can be purified by avidin-affinity chromatography. We include examples of the detection of S-nitrosylated proteins in brain lysates after in vitro S-nitrosylation, as well as the detection of endogenous S-nitrosothiols in selected neuronal proteins.

title: Human spermatozoa contain multiple targets for protein S‐nitrosylation: An alternative mechanism of the modulation of sperm function by nitric oxide?
abstract: Nitric oxide (NO) enhances human sperm motility and capacitation associated with increased protein phosphorylation. NO activates soluble guanylyl cyclase, but can also modify protein function covalently via S‐nitrosylation of cysteine. Remarkably, this mechanism remains unexplored in sperm although they depend on post‐translational protein modification to achieve changes in function required for fertilisation. Our objective was to identify targets for S‐nitrosylation in human sperm. Spermatozoa were incubated with NO donors and S‐nitrosylated proteins were identified using the biotin switch assay and a proteomic approach using MS/MS. 240 S‐nitrosylated proteins were detected in sperm incubated with S‐nitroso‐glutathione. Minimal levels were observed in glutathione or untreated samples. Proteins identified consistently based on multiple peptides included established targets for S‐nitrosylation in other cells e.g. tubulin, GST and HSPs but also novel targets including A‐kinase anchoring protein (AKAP) types 3 and 4, voltage‐dependent anion‐selective channel protein 3 and semenogelin 1 and 2. In situ localisation revealed S‐nitrosylated targets on the postacrosomal region of the head and throughout the flagellum. Potential targets for S‐nitrosylation in human sperm include physiologically significant proteins not previously reported in other cells. Their identification will provide novel insight into the mechanism of action of NO in spermatozoa.

title: Shear flow increases S-nitrosylation of proteins in endothelial cells.
abstract: Endothelial cells (ECs) constantly exposed to shear flow increase nitric oxide production via the activation of endothelial nitric oxide synthase. Nitric oxide-mediated S-nitrosylation has recently been identified as an important post-translational modification that may alter signalling and/or protein function. S-nitrosylation of endothelial proteins after shear flow treatment has not been fully explored. In this study, the CyDye switch method was utilized to examine S-nitrosylated proteins in ECs after exposure to shear flow.



Human umbilical vein ECs were subjected to shear flow for 30 min, and S-nitrosylated proteins were detected by the CyDye switch method. In principle, free thiols in proteins become blocked by alkylation, the S-nitrosylated bond is reduced by ascorbate, and then CyDye labels proteins. Proteins that separately labelled with Cy3 or Cy5 were mixed and subjected to two-dimensional gel electrophoresis for further analysis. More than 100 S-nitrosoproteins were detected in static and shear-treated ECs. Among these, 12 major proteins of heterogeneous function showed a significant increase in S-nitrosylation following shear flow. The S-nitrosylated residues in tropomyosin and vimentin, which were localized in the hydrophobic motif of each protein, were identified as Cys170 and Cys328, respectively.



Post-translational S-nitrosylation of proteins in ECs can be detected by a reliable CyDye switch method. This flow-induced S-nitrosylation of endothelial proteins may be essential for the adaptation and remodelling of ECs under flow conditions.

title: Proteomic Identification of S-Nitrosylated Proteins in Arabidopsis1[w]
abstract: Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins. Strikingly, many of these proteins have been identified previously as targets of S-nitrosylation in animals. At the enzymatic level, a case study demonstrated NO-dependent reversible inhibition of plant glyceraldehyde-3-phosphate dehydrogenase, suggesting that this enzyme could be affected by S-nitrosylation. The results of this work are the starting point for further investigation to get insight into signaling pathways and other cellular processes regulated by protein S-nitrosylation in plants.

title: Unbiased identification of cysteine S-nitrosylation sites on proteins
abstract: Covalent addition of nitric oxide (NO) to Cys-sulfur in proteins, or S-nitrosylation, plays pervasive roles in the physiological and pathophysiological modulation of mammalian protein functions. Knowledge of the specific protein Cys residues that undergo NO addition in different biological settings is fundamental to understanding NO-mediated signal transduction. Here, we describe in detail an MS-based proteomic protocol for facile, high-throughput and unbiased discovery of SNO-Cys residues in proteins from complex biological samples. The approach, termed SNOSID (SNO-Cys site identification), can be used to identify endogenous and chemically induced S-nitrosylation sites in proteins from tissues or cells. Identified SNO-Cys sites may provide insights into novel mechanisms and proteins that mediate NO bioactivities in health and disease. SNOSID builds on the biotin-switch method for covalent addition of disulfide-linked biotin at S-nitrosylation sites on proteins. Biotinylated proteins are then subjected to trypsinolysis and the resulting biotin-tagged peptides are affinity-captured on streptavidin-agarose. After selective elution with β-mercaptoethanol, the peptides are sequenced using nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS). Validation that identified peptide ions as originating from authentic NO-Cys-containing precursor proteins can be provided by establishing that these peptide ions are absent from control samples where S–NO bonds were subjected to prior photolysis, using a UV transilluminator. The protocol requires approximately 2 days for sample processing, including the incubation time for proteolysis. An additional 1–2 days is needed for sample analysis by nLC-MS/MS and data analysis/interpretation.

title: SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures.
abstract: Reversible addition of NO to Cys-sulfur in proteins, a modification termed S-nitrosylation, has emerged as a ubiquitous signaling mechanism for regulating diverse cellular processes. A key first-step toward elucidating the mechanism by which S-nitrosylation modulates a protein's function is specification of the targeted Cys (SNO-Cys) residue. To date, S-nitrosylation site specification has been laboriously tackled on a protein-by-protein basis. Here we describe a high-throughput proteomic approach that enables simultaneous identification of SNO-Cys sites and their cognate proteins in complex biological mixtures. The approach, termed SNOSID (SNO Site Identification), is a modification of the biotin-swap technique [Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P. & Snyder, S. H. (2001) , comprising biotinylation of protein SNO-Cys residues, trypsinolysis, affinity purification of biotinylated-peptides, and amino acid sequencing by liquid chromatography tandem MS. With this approach, 68 SNO-Cys sites were specified on 56 distinct proteins in S-nitrosoglutathione-treated (2-10 microM) rat cerebellum lysates. In addition to enumerating these S-nitrosylation sites, the method revealed endogenous SNO-Cys modification sites on cerebellum proteins, including alpha-tubulin, beta-tubulin, GAPDH, and dihydropyrimidinase-related protein-2. Whereas these endogenous SNO proteins were previously recognized, we extend prior knowledge by specifying the SNO-Cys modification sites. Considering all 68 SNO-Cys sites identified, a machine learning approach failed to reveal a linear Cys-flanking motif that predicts stable transnitrosation by S-nitrosoglutathione under test conditions, suggesting that undefined 3D structural features determine S-nitrosylation specificity. SNOSID provides the first effective tool for unbiased elucidation of the SNO proteome, identifying Cys residues that undergo reversible S-nitrosylation.

title: Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture
abstract: We have modified the biotin switch assay for protein S-nitrosothiols (SNOs), using resin-assisted capture (SNO-RAC). Compared with existing methodologies, SNO-RAC requires fewer steps, detects high-mass S-nitrosylated proteins more efficiently, and facilitates identification and quantification of S-nitrosylated sites by mass spectrometry. When combined with iTRAQ labeling, SNO-RAC revealed that intracellular proteins may undergo rapid denitrosylation on a global scale. This methodology is readily adapted to analyzing diverse cysteine-based protein modifications, including S-acylation.

title: Nitrosothiol reactivity profiling identifies S-nitrosylated proteins with unexpected stability.
abstract: Nitric oxide (NO) regulates protein function by S-nitrosylation of cysteine to form nitrosothiols. Nitrosothiols are highly susceptible to nonenzymatic degradation by cytosolic reducing agents. Here we show that although most protein nitrosothiols are rapidly degraded by cytosolic reductants, a small subset form unusually stable S-nitrosylated proteins. Our findings suggest that stable S-nitrosylation reflects a protein conformation change that shields the nitrosothiol. To identify stable protein nitrosothiols, we developed a proteomic method for profiling S-nitrosylation. We examined the stability of over 100 S-nitrosylated proteins, and identified 10 stable nitrosothiols. These proteins remained S-nitrosylated in cells after NO synthesis was inhibited, unlike most S-nitrosylated proteins. Taken together, our data identify a class of NO targets that form stable nitrosothiols in the cell and are likely to mediate the persistent cellular effects of NO.

title: Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells.
abstract: S-nitrosylation, the selective modification of cysteine residues in proteins to form S-nitrosocysteine, is a major emerging mechanism by which nitric oxide acts as a signaling molecule. Even though nitric oxide is intimately involved in the regulation of vascular smooth muscle cell functions, the potential protein targets for nitric oxide modification as well as structural features that underlie the specificity of protein S-nitrosocysteine formation in these cells remain unknown. Therefore, we used a proteomic approach using selective peptide capturing and site-specific adduct mapping to identify the targets of S-nitrosylation in human aortic smooth muscle cells upon exposure to S-nitrosocysteine and propylamine propylamine NONOate. This strategy identified 20 unique S-nitrosocysteine-containing peptides belonging to 18 proteins including cytoskeletal proteins, chaperones, proteins of the translational machinery, vesicular transport, and signaling. Sequence analysis of the S-nitrosocysteine-containing peptides revealed the presence of acid/base motifs, as well as hydrophobic motifs surrounding the identified cysteine residues. High-resolution immunogold electron microscopy supported the cellular localization of several of these proteins. Interestingly, seven of the 18 proteins identified are localized within the ER/Golgi complex, suggesting a role for S-nitrosylation in membrane trafficking and ER stress response in vascular smooth muscle.

title: GPS 2.0, a Tool to Predict Kinase-specific Phosphorylation Sites in Hierarchy *S
abstract: Identification of protein phosphorylation sites with their cognate protein kinases (PKs) is a key step to delineate molecular dynamics and plasticity underlying a variety of cellular processes. Although nearly 10 kinase-specific prediction programs have been developed, numerous PKs have been casually classified into subgroups without a standard rule. For large scale predictions, the false positive rate has also never been addressed. In this work, we adopted a well established rule to classify PKs into a hierarchical structure with four levels, including group, family, subfamily, and single PK. In addition, we developed a simple approach to estimate the theoretically maximal false positive rates. The on-line service and local packages of the GPS (Group-based Prediction System) 2.0 were implemented in Java with the modified version of the Group-based Phosphorylation Scoring algorithm. As the first stand alone software for predicting phosphorylation, GPS 2.0 can predict kinase-specific phosphorylation sites for 408 human PKs in hierarchy. A large scale prediction of more than 13,000 mammalian phosphorylation sites by GPS 2.0 was exhibited with great performance and remarkable accuracy. Using Aurora-B as an example, we also conducted a proteome-wide search and provided systematic prediction of Aurora-B-specific substrates including protein-protein interaction information. Thus, the GPS 2.0 is a useful tool for predicting protein phosphorylation sites and their cognate kinases and is freely available on line.

title: SysPTM: A Systematic Resource for Proteomic Research on Post-translational Modifications*
abstract: With the rapid expansion of protein post-translational modification (PTM) research based on large-scale proteomic work, there is an increasing demand for a suitable repository to analyze PTM data. Here we present a curated, web-accessible PTM data base, SysPTM. SysPTM provides a systematic and sophisticated platform for proteomic PTM research equipped not only with a knowledge base of manually curated multi-type modification data but also with four fully developed, in-depth data mining tools. Currently, SysPTM contains data detailing 117,349 experimentally determined PTM sites on 33,421 proteins involving nearly 50 PTM types, curated from public resources including five data bases and four web servers and more than one hundred peer-reviewed mass spectrometry papers. Protein annotations including Pfam domains, KEGG pathways, GO functional classification, and ortholog groups are integrated into the data base. Four online tools have been developed and incorporated, including PTMBlast, to compare a user's PTM dataset with PTM data in SysPTM; PTMPathway, to map PTM proteins to KEGG pathways; PTMPhylog, to discover potentially conserved PTM sites; and PTMCluster, to find clusters of multi-site modifications. The workflow of SysPTM was demonstrated by analyzing an in-house phosphorylation dataset identified by MS/MS. It is shown that in SysPTM, the role of single-type and multi-type modifications can be systematically investigated in a full biological context. SysPTM could be an important contribution to modificomics research. SysPTM is freely available online at www.sysbio.ac.cn/SysPTM.

title: Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of S-nitrosylated proteins.
abstract: High-throughput proteomic analysis based on a biotin switch combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS) enables simultaneous identification of S-nitrosylated sites and their cognate proteins in complex biological mixtures, which is a great help in elucidating the functions and mechanisms of this redox-based post-translational modification. However, detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 adopted in these systems, which are hard to fully remove in the subsequent MS-based analyses, can suppress the peptide signals and influence the SNO-Cys site identification and the reproducibility of the experiments. Here we developed a detergent-free biotin-switch method, which applied urea to replace detergents, and successfully combined it with LC/MS/MS in the analysis of S-nitrosylated proteins. With this approach, 44 SNO-Cys sites were specified on 35 distinct proteins in S-nitrosoglutathione (GSNO)-treated HeLa cell extracts of proteins with good reproducibility. The LC/MS performance was greatly improved as analyzed with Pep3D and the amount of samples for analysis reduced from 40 mg used in the literature to 3-5 mg. For S-nitrosylated targets detected both in the control sample and in the GSNO-treated sample, extracted ion chromatography (XIC) was employed to estimate the quantitative change of S-nitrosylation (S-nitrosation), which facilitates the judgment on 'accept or reject' of the identified targets.

title: Systematic study of protein sumoylation: Development of a site‐specific predictor of SUMOsp 2.0
abstract: Protein sumoylation is an important reversible post‐translational modification on proteins, and orchestrates a variety of cellular processes. Recently, computational prediction of sumoylation sites has attracted much attention for its cost‐efficiency and power in genomic data mining. In this work, we developed SUMOsp 2.0, an accurate computing program with an improved group‐based phosphorylation scoring algorithm. Our analysis demonstrated that SUMOsp 2.0 has greater prediction accuracy than SUMOsp 1.0 and other existing tools, with a sensitivity of 88.17% and a specificity of 92.69% under the medium threshold. Previously, several large‐scale experiments have identified a list of potential sumoylated substrates in Saccharomyces cerevisiae and Homo sapiens; however, the exact sumoylation sites in most of these proteins remain elusive. We have predicted potential sumoylation sites in these proteins using SUMOsp 2.0, which provides a great resource for researchers and an outline for further mechanistic studies of sumoylation in cellular plasticity and dynamics. The online service and local packages of SUMOsp 2.0 are freely available at: http://sumosp.biocuckoo.org/.

title: GPS: a comprehensive www server for phosphorylation sites prediction
abstract: Protein phosphorylation plays a fundamental role in most of the cellular regulatory pathways. Experimental identification of protein kinases' (PKs) substrates with their phosphorylation sites is labor-intensive and often limited by the availability and optimization of enzymatic reactions. Recently, large-scale analysis of the phosphoproteome by the mass spectrometry (MS) has become a popular approach. But experimentally, it is still difficult to distinguish the kinase-specific sites on the substrates. In this regard, the in silico prediction of phosphorylation sites with their specific kinases using protein's primary sequences may provide guidelines for further experimental consideration and interpretation of MS phosphoproteomic data. A variety of such tools exists over the Internet and provides the predictions for at most 30 PK subfamilies. We downloaded the verified phosphorylation sites from the public databases and curated the literature extensively for recently found phosphorylation sites. With the hypothesis that PKs in the same subfamily share similar consensus sequences/motifs/functional patterns on substrates, we clustered the 216 unique PKs in 71 PK groups, according to the BLAST results and protein annotations. Then, we applied the group-based phosphorylation scoring (GPS) method on the data set; here, we present a comprehensive PK-specific prediction server GPS, which could predict kinase-specific phosphorylation sites from protein primary sequences for 71 different PK groups. GPS has been implemented in PHP and is available on a www server at .

title: GPS: a novel group-based phosphorylation predicting and scoring method.
abstract: Protein phosphorylation is an important reversible post-translational modification of proteins, and it orchestrates a variety of cellular processes. Experimental identification of phosphorylation site is labor-intensive and often limited by the availability and optimization of enzymatic reaction. In silico prediction may facilitate the identification of potential phosphorylation sites with ease. Here we present a novel computational method named GPS: group-based phosphorylation site predicting and scoring platform. If two polypeptides differ by only two consecutive amino acids, in particular when the two different amino acids are a conserved pair, e.g., isoleucine (I) and valine (V), or serine (S) and threonine (T), we view these two polypeptides bearing similar 3D structures and biochemical properties. Based on this rationale, we formulated GPS that carries greater computational power with superior performance compared to two existing phosphorylation sites prediction systems, ScanSite 2.0 and PredPhospho. With database in public domain, GPS can predict substrate phosphorylation sites from 52 different protein kinase (PK) families while ScanSite 2.0 and PredPhospho offer at most 30 PK families. Using PKA as a model enzyme, we first compared prediction profiles from the GPS method with those from ScanSite 2.0 and PredPhospho. In addition, we chose an essential mitotic kinase Aurora-B as a model enzyme since ScanSite 2.0 and PredPhospho offer no prediction. However, GPS offers satisfactory sensitivity (94.44%) and specificity (97.14%). Finally, the accuracy of phosphorylation on MCAK predicted by GPS was validated by experimentation, in which six out of seven predicted potential phosphorylation sites on MCAK (Q91636) were experimentally verified. Taken together, we have generated a novel method to predict phosphorylation sites, which offers greater precision and computing power over ScanSite 2.0 and PredPhospho.

title: CSS-Palm 2.0: an updated software for palmitoylation sites prediction.
abstract: Protein palmitoylation is an essential post-translational lipid modification of proteins, and reversibly orchestrates a variety of cellular processes. Identification of palmitoylated proteins with their sites is the foundation for understanding molecular mechanisms and regulatory roles of palmitoylation. Contrasting to the labor-intensive and time-consuming experimental approaches, in silico prediction of palmitoylation sites has attracted much attention as a popular strategy. In this work, we updated our previous CSS-Palm into version 2.0. An updated clustering and scoring strategy (CSS) algorithm was employed with great improvement. The leave-one-out validation and 4-, 6-, 8- and 10-fold cross-validations were adopted to evaluate the prediction performance of CSS-Palm 2.0. Also, an additional new data set not included in training was used to test the robustness of CSS-Palm 2.0. By comparison, the performance of CSS-Palm was much better than previous tools. As an application, we performed a small-scale annotation of palmitoylated proteins in budding yeast. The online service and local packages of CSS-Palm 2.0 were freely available at: http://bioinformatics.lcd-ustc.org/css_palm.

title: Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences
abstract: In 2001 and 2002, we published two papers (Bioinformatics, 17, 282-283, Bioinformatics, 18, 77-82) describing an ultrafast protein sequence clustering program called cd-hit. This program can efficiently cluster a huge protein database with millions of sequences. However, the applications of the underlying algorithm are not limited to only protein sequences clustering, here we present several new programs using the same algorithm including cd-hit-2d, cd-hit-est and cd-hit-est-2d. Cd-hit-2d compares two protein datasets and reports similar matches between them; cd-hit-est clusters a DNA/RNA sequence database and cd-hit-est-2d compares two nucleotide datasets. All these programs can handle huge datasets with millions of sequences and can be hundreds of times faster than methods based on the popular sequence comparison and database search tools, such as BLAST.

title: Large-scale clustering of cDNA-fingerprinting data.
abstract: Clustering is one of the main mathematical challenges in large-scale gene expression analysis. We describe a clustering procedure based on a sequential k-means algorithm with additional refinements that is able to handle high-throughput data in the order of hundreds of thousands of data items measured on hundreds of variables. The practical motivation for our algorithm is oligonucleotide fingerprinting-a method for simultaneous determination of expression level for every active gene of a specific tissue-although the algorithm can be applied as well to other large-scale projects like EST clustering and qualitative clustering of DNA-chip data. As a pairwise similarity measure between two p-dimensional data points, x and y, we introduce mutual information that can be interpreted as the amount of information about x in y, and vice versa. We show that for our purposes this measure is superior to commonly used metric distances, for example, Euclidean distance. We also introduce a modified version of mutual information as a novel method for validating clustering results when the true clustering is known. The performance of our algorithm with respect to experimental noise is shown by extensive simulation studies. The algorithm is tested on a subset of 2029 cDNA clones coming from 15 different genes from a cDNA library derived from human dendritic cells. Furthermore, the clustering of these 2029 cDNA clones is demonstrated when the entire set of 76,032 cDNA clones is processed.

title: Leptin-specific patterns of gene expression in white adipose tissue.
abstract: Leptin is a hormone that regulates body weight by decreasing food intake and increasing energy expenditure. ob/ob mice carry leptin mutations and are obese and hyperphagic. Leptin administration to lean and ob/ob mice activates a novel metabolic program that depletes adipose tissue. Although this response is physiologically distinct from that evident after food restriction, the molecular nature of these differences is as yet unknown. Expression monitoring of 6500 genes using oligonucleotide microarrays in wild-type, ob/ob, and transgenic mice expressing low levels of leptin revealed that differences in ambient leptin levels have dramatic effects on the phenotype of white adipose tissue. These data identified a large number of genes that are differentially expressed in ob/ob mice. To delineate the components of the transcriptional program specifically affected by leptin, the level of the same 6500 genes was monitored in wild-type and ob/ob mice at various times after leptin treatment or food restriction. A novel application of k-means clustering identified 8 clusters of adipose tissue genes whose expression was different between leptin treatment and food restriction in ob/ob mice and 10 such clusters in wild-type experiments. One of the clusters was repressed specifically by leptin in both wild-type and ob/ob mice and included several genes known to be regulated by SREBP-1/ADD1. Further studies confirmed that leptin decreases the levels of SREBP-1/ADD1 RNA and transcriptionally active SREBP-1/ADD1 protein in white adipose tissue. Future studies of the molecular basis for the apparent coordinate regulation of the other clusters of leptin-regulated genes may reveal additional mechanisms by which leptin exerts its weight-reducing effects.

title: Temporal proteomic analysis of IGF‐1R signalling in MCF‐7 breast adenocarcinoma cells
abstract: Dysregulation of the insulin‐like growth factor 1 receptor signalling network is implicated in tumour growth and resistance to chemotherapy. We explored proteomic changes resulting from insulin‐like growth factor 1 stimulation of MCF‐7 adenocarcinoma cells as a function of time. Quantitative analysis using iTRAQ™ reagents and 2‐D LC‐MS/MS analysis of three biological replicates resulted in the identification of 899 proteins (p≤0.05) with an estimated mean false‐positive rate of 2.6%. Quantitative protein expression was obtained from 681 proteins. Further analysis by supervised k‐means clustering identified five temporal clusters, which were submitted to the FuncAssociate server to assign overrepresented gene ontology terms. Proteins associated with vesicle transport were significantly overrepresented. We further analyzed our data set for proteins showing temporal significance using the software, extraction and analysis of differential gene expression, resulting in 20 significantly and temporally changing proteins (p≤0.1). These significant proteins play roles in, among others, altered glucose metabolism (lactate dehydrogenase A and pyruvate kinase M1/M2) and cellular stress (nascent polypeptide‐associated complex subunit α and heat shock (HSC70) proteins). We used multiple reaction monitoring to validate these interesting proteins and have revealed several differences in relative peptide expression corresponding to protein isoforms and variants.

title: Clustering protein environments for function prediction: finding PROSITE motifs in 3D
abstract: BackgroundStructural genomics initiatives are producing increasing numbers of three-dimensional (3D) structures for which there is little functional information. Structure-based annotation of molecular function is therefore becoming critical. We previously presented FEATURE, a method for describing microenvironments around functional sites in proteins. However, FEATURE uses supervised machine learning and so is limited to building models for sites of known importance and location. We hypothesized that there are a large number of sites in proteins that are associated with function that have not yet been recognized. Toward that end, we have developed a method for clustering protein microenvironments in order to evaluate the potential for discovering novel sites that have not been previously identified.ResultsWe have prototyped a computational method for rapid clustering of millions of microenvironments in order to discover residues whose surrounding environments are similar and which may therefore share a functional or structural role. We clustered nearly 2,000,000 environments from 9,600 protein chains and defined 4,550 clusters. As a preliminary validation, we asked whether known 3D environments associated with PROSITE motifs were "rediscovered". We found examples of clusters highly enriched for residues that share PROSITE sequence motifs.ConclusionOur results demonstrate that we can cluster protein environments successfully using a simplified representation and K-means clustering algorithm. The rediscovery of known 3D motifs allows us to calibrate the size and intercluster distances that characterize useful clusters. This information will then allow us to find new clusters with similar characteristics that represent novel structural or functional sites.

title: Knowledge-based 3D analysis from 2D medical images
abstract: An anatomical knowledge-based system for image analysis that interprets CT/MR (computed tomography/magnetic resonance) images of the human chest cavity is reported. The approach utilizes a low-level image analysis system with the ability to analyze the data in bottom-up (or data-driven) and top-down (or model-driven) modes to improve the high-level recognition process. Several image segmentation algorithms, including K-means clustering, pyramid-based region extraction, and rule-based merging, are used for obtaining the segmented regions. To obtain a reasonable number of well-segmented regions that have a good correlation with the anatomy, a priori knowledge in the form of masks is used to guide the segmentation process. Segmentation of the brain is also considered.<<ETX>>

title: Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase.
abstract: Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.