The human proteome project: current state and future direction.

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

The human proteome project: Current state and future direction.

After successful completion of the Human Genome Project (HGP), HUPO has recently officially launched a global Human Proteome Project (HPP) which is designed to map the entire human protein set. Given the presence of about 30% undisclosed proteins out of 20,300 protein gene products, a systematic global effort is necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP groups employ the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge base. The HPP participants will take advantage of the output and cross-analyses from the ongoing HUPO initiatives and a chromosome-based protein mapping strategy, termed C-HPP with many national teams currently engaged. In addition, numerous biologically-driven projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents and tools for protein studies and analyses, and a stronger basis for personalized medicine. HUPO urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

Standard guidelines for the chromosome-centric human proteome project.

The objective of the international Chromosome-Centric Human Proteome Project (C-HPP) is to map and annotate all proteins encoded by the genes on each human chromosome. The C-HPP consortium was established to organize a collaborative network among the research teams responsible for protein mapping of individual chromosomes and to identify compelling biological and genetic mechanisms influencing colocated genes and their protein products. The C-HPP aims to foster the development of proteome analysis and integration of the findings from related molecular -omics technology platforms through collaborations among universities, industries, and private research groups. The C-HPP consortium leadership has elicited broad input for standard guidelines to manage these international efforts more efficiently by mobilizing existing resources and collaborative networks. The C-HPP guidelines set out the collaborative consensus of the C-HPP teams, introduce topics associated with experimental approaches, data production, quality control, treatment, and transparency of data, governance of the consortium, and collaborative benefits. A companion approach for the Biology and Disease-Driven HPP (B/D-HPP) component of the Human Proteome Project is currently being organized, building upon the Human Proteome Organization's organ-based and biofluid-based initiatives (www.hupo.org/research). The common application of these guidelines in the participating laboratories is expected to facilitate the goal of a comprehensive analysis of the human proteome.

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion.

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.

The thioredoxin reductase-glutaredoxins-ferredoxin crossroad pathway for selenate tolerance in Synechocystis PCC6803.

Most organisms use two systems to maintain the redox homeostasis of cellular thiols. In the thioredoxin (Trx) system, NADPH sequentially reduces thioredoxin reductases (NTR), Trxs and protein disulfides. In the glutaredoxin (Grx) system, NADPH reduces the glutathione reductase enzyme occurring in most organisms, glutathione, Grxs, and protein disulfides or glutathione-protein mixed disulfides. As little is known concerning these enzymes in cyanobacteria, we have undertaken their analysis in the model strain Synechocystis PCC6803. We found that Grx1 and Grx2 are active, and that Grx2 but not Grx1 is crucial to tolerance to hydrogen peroxide and selenate. We also found that Synechocystis has no genuine glutathione reductase and uses NTR as a Grx electron donor, in a novel integrative pathway NADPH-NTR-Grx1-Grx2-Fed7 (ferredoxin 7), which operates in protection against selenate, the predominant form of selenium in the environment. This is the first report on the occurrence of a physical interaction between a Grx and a Fed, and of an electron transfer between two Grxs. These findings are discussed in terms of the (i) selectivity of Grxs and Feds (Synechocystis possesses nine Feds), (ii) crucial importance of NTR for cell fitness and (iii) resistance to selenate, in absence of a Thauera selenatis-like selenate reductase.

The minimum information required for reporting a molecular interaction experiment (MIMIx).

A wealth of molecular interaction data is available in the literature, ranging from large-scale datasets to a single interaction confirmed by several different techniques. These data are all too often reported either as free text or in tables of variable format, and are often missing key pieces of information essential for a full understanding of the experiment. Here we propose MIMIx, the minimum information required for reporting a molecular interaction experiment. Adherence to these reporting guidelines will result in publications of increased clarity and usefulness to the scientific community and will support the rapid, systematic capture of molecular interaction data in public databases, thereby improving access to valuable interaction data.

Strategies Used by Musicians to Identify Notes' Pitch: Cognitive Bricks and Mental Representations.

To this day, the study of the substratum of thought and its implied mechanisms is rarely directly addressed. Nowadays, systemic approaches based on introspective methodologies are no longer fashionable and are often overlooked or ignored. Most frequently, reductionist approaches are followed for deciphering the neuronal circuits functionally associated with cognitive processes. However, we argue that systemic studies of individual thought may still contribute to a useful and complementary description of the multimodal nature of perception, because they can take into account individual diversity while still identifying the common features of perceptual processes. We propose to address this question by looking at one possible task for recognition of a "signifying sound", as an example of conceptual grasping of a perceptual response. By adopting a mixed approach combining qualitative analyses of interviews based on introspection with quantitative statistical analyses carried out on the resulting categorization, this study describes a variety of mental strategies used by musicians to identify notes' pitch. Sixty-seven musicians (music students and professionals) were interviewed, revealing that musicians utilize intermediate steps during note identification by selecting or activating cognitive bricks that help construct and reach the correct decision. We named these elements "mental anchorpoints" (MA). Although the anchorpoints are not universal, and differ between individuals, they can be grouped into categories related to three main sensory modalities - auditory, visual and kinesthetic. Such categorization enabled us to characterize the mental representations (MR) that allow musicians to name notes in relationship to eleven basic typologies of anchorpoints. We propose a conceptual framework which summarizes the process of note identification in five steps, starting from sensory detection and ending with the verbalization of the note pitch, passing through the pivotal role of MAs and MRs. We found that musicians use multiple strategies and select individual combinations of MAs belonging to these three different sensory modalities, both in isolation and in combination.

InteroPORC: automated inference of highly conserved protein interaction networks.

MOTIVATION: Protein-protein interaction networks provide insights into the relationships between the proteins of an organism thereby contributing to a better understanding of cellular processes. Nevertheless, large-scale interaction networks are available for only a few model organisms. Thus, interologs are useful for a systematic transfer of protein interaction networks between organisms. However, no standard tool is available so far for that purpose. RESULTS: In this study, we present an automated prediction tool developed for all sequenced genomes available in Integr8. We also have developed a second method to predict protein-protein interactions in the widely used cyanobacterium Synechocystis. Using these methods, we have constructed a new network of 8783 inferred interactions for Synechocystis. AVAILABILITY: InteroPORC is open-source, downloadable and usable through a web interface at http://biodev.extra.cea.fr/interoporc/.

Cadmium triggers an integrated reprogramming of the metabolism of Synechocystis PCC6803, under the control of the Slr1738 regulator.

BACKGROUND: Cadmium is a persistent pollutant that threatens most biological organisms, including cyanobacteria that support a large part of the biosphere. Using a multifaceted approach, we have investigated the global responses to Cd and other relevant stresses (H2O2 and Fe) in the model cyanobacterium Synechocystis PCC6803. RESULTS: We found that cells respond to the Cd stress in a two main temporal phases process. In the "early" phase cells mainly limit Cd entry through the negative and positive regulation of numerous genes operating in metal uptake and export, respectively. As time proceeds, the number of responsive genes increases. In this "massive" phase, Cd downregulates most genes operating in (i) photosynthesis (PS) that normally provides ATP and NADPH; (ii) assimilation of carbon, nitrogen and sulfur that requires ATP and NAD(P)H; and (iii) translation machinery, a major consumer of ATP and nutrients. Simultaneously, many genes are upregulated, such as those involved in Fe acquisition, stress tolerance, and protein degradation (crucial to nutrients recycling). The most striking common effect of Cd and H2O2 is the disturbance of both light tolerance and Fe homeostasis, which appeared to be interdependent. Our results indicate that cells challenged with H2O2 or Cd use different strategies for the same purpose of supplying Fe atoms to Fe-requiring metalloenzymes and the SUF machinery, which synthesizes or repairs Fe-S centers. Cd-stressed cells preferentially breakdown their Fe-rich PS machinery, whereas H2O2-challenged cells preferentially accelerate the intake of Fe atoms from the medium. CONCLUSION: We view the responses to Cd as an integrated "Yin Yang" reprogramming of the whole metabolism, we found to be controlled by the Slr1738 regulator. As the Yin process, the ATP- and nutrients-sparing downregulation of anabolism limits the poisoning incorporation of Cd into metalloenzymes. As the compensatory Yang process, the PS breakdown liberates nutrient assimilates for the synthesis of Cd-tolerance proteins, among which we found the Slr0946 arsenate reductase enzyme.

The HUPO PSI's molecular interaction format--a community standard for the representation of protein interaction data.

A major goal of proteomics is the complete description of the protein interaction network underlying cell physiology. A large number of small scale and, more recently, large-scale experiments have contributed to expanding our understanding of the nature of the interaction network. However, the necessary data integration across experiments is currently hampered by the fragmentation of publicly available protein interaction data, which exists in different formats in databases, on authors' websites or sometimes only in print publications. Here, we propose a community standard data model for the representation and exchange of protein interaction data. This data model has been jointly developed by members of the Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organization (HUPO), and is supported by major protein interaction data providers, in particular the Biomolecular Interaction Network Database (BIND), Cellzome (Heidelberg, Germany), the Database of Interacting Proteins (DIP), Dana Farber Cancer Institute (Boston, MA, USA), the Human Protein Reference Database (HPRD), Hybrigenics (Paris, France), the European Bioinformatics Institute's (EMBL-EBI, Hinxton, UK) IntAct, the Molecular Interactions (MINT, Rome, Italy) database, the Protein-Protein Interaction Database (PPID, Edinburgh, UK) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, EMBL, Heidelberg, Germany).

Prediction, assessment and validation of protein interaction maps in bacteria.

High-throughput proteomics technologies, especially the yeast two-hybrid system, produce large volumes of protein-protein interaction data organized in networks. The complete sequencing of many genomes raises questions about the extent to which such networks can be transferred between organisms. We attempted to answer this question using the experimentally derived Helicobacter pylori interaction map and the recently described interacting domain profile pair (IDPP) method to predict a virtual map for Escherichia coli. The extensive literature concerning E.coli was used to assess all predicted interactions and to validate the IDPP method, which clusters protein domains by sequence and connectivity similarities. The IDPP method has a much better heuristic value than methods solely based on protein homology. The IDPP method was further applied to Campylobacter jejuni to generate a virtual interaction map. An in-depth comparison of the chemotaxis pathways predicted in E.coli and C.jejuni led to the proposition of new functional assignments. Finally, the prediction of protein-protein interaction maps across organisms enabled us to validate some of the interactions on the original experimental map.

Twenty years of protein interaction studies for biological function deciphering.

Intensive methodological developments and technology innovation have been devoted to protein-protein interaction studies over 20years. Genetic indirect assays and sophisticated large scale biochemical analyses have jointly contributed to the elucidation of protein-protein interactions, still with a lot of drawbacks despite heavy investment in human resources and technologies. With the most recent developments in mass spectrometry and computational tools for studying protein content of complex samples, the initial goal of deciphering molecular bases of biological functions is now within reach. Here, we described the various steps of this process and gave examples of key milestones in this scientific story line. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

"4D Biology for health and disease" workshop report.

The "4D Biology Workshop for Health and Disease", held on 16-17th of March 2010 in Brussels, aimed at finding the best organising principles for large-scale proteomics, interactomics and structural genomics/biology initiatives, and setting the vision for future high-throughput research and large-scale data gathering in biological and medical science. Major conclusions of the workshop include the following. (i) Development of new technologies and approaches to data analysis is crucial. Biophysical methods should be developed that span a broad range of time/spatial resolution and characterise structures and kinetics of interactions. Mathematics, physics, computational and engineering tools need to be used more in biology and new tools need to be developed. (ii) Database efforts need to focus on improved definitions of ontologies and standards so that system-scale data and associated metadata can be understood and shared efficiently. (iii) Research infrastructures should play a key role in fostering multidisciplinary research, maximising knowledge exchange between disciplines and facilitating access to diverse technologies. (iv) Understanding disease on a molecular level is crucial. System approaches may represent a new paradigm in the search for biomarkers and new targets in human disease. (v) Appropriate education and training should be provided to help efficient exchange of knowledge between theoreticians, experimental biologists and clinicians. These conclusions provide a strong basis for creating major possibilities in advancing research and clinical applications towards personalised medicine.

Shroom2, a myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions.

Defects in myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.

The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes.

Bacteria of Shigella spp. are responsible for shigellosis in humans. They use a type III secretion system to inject effector proteins into host cells and induce their entry into epithelial cells or trigger apoptosis in macrophages. We present evidence that the effector OspG is a protein kinase that binds various ubiquitinylated ubiquitin-conjugating enzymes, including UbcH5, which belongs to the stem cell factor SCF(beta-TrCP) complex promoting ubiquitination of phosphorylated inhibitor of NF-kappaB type alpha (phospho-IkappaBalpha). Transfection experiments indicated that OspG can prevent phospho-IkappaBalpha degradation and NF-kappaB activation induced by TNF-alpha stimulation. Infection of epithelial cells by the S. flexneri wild-type strain, but not an ospG mutant, led to accumulation of phospho-IkappaBalpha, consistent with OspG inhibiting SCF(beta-TrCP) activity. Upon infection of ileal loops in rabbits, the ospG mutant induced a stronger inflammatory response than the wild-type strain. This finding indicates that OspG negatively controls the host innate response induced by S. flexneri upon invasion of the epithelium.