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PubMed2005

The 'interactome' of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae.

The integrity of the Saccharomyces cerevisiae cell wall requires a functional Pkc1-Slt2 MAP kinase pathway that contributes to transient growth arrest, enabling coordination of cell division with cell wall remodelling. How this coordination takes place is still an open question. Recently, we brought evidence that Knr4 protein, whose absence leads to several cell wall defects, may play a role in this function. Here, we show that Knr4 is a monomeric protein that exhibits an aberrant mobility on a SDS-gel electrophoresis and a non-globular structure. Furthermore, Knr4 is an unstable protein that is degraded as cells enter the stationary phase of growth, while its corresponding gene is constitutively expressed. In exponentially growing cells on glucose, Knr4 appeared to be present in a protein complex that migrates with an apparent Mw superior to 250 kDa. Using the TAP-tag methodology, nine potential partners of Knr4 were identified, which could be distributed into three biological processes. A first group consisted of Slt2 and Pil1, two proteins dedicated to cell wall maintenance and biogenesis. The second group comprised four proteins (Bud6, Act1, Cin8 and Jnm1) implicated in the establishment of cell polarity and bud integrity during mitosis. The last group contained four proteins (Asc1, Ubc1, Hsc82 and Gvp36) that probably deal with the stability/degradation of proteins. Deletion analysis revealed that the domain of interaction covered 2/3 of the Knr4 sequence on the N-terminal side. Moreover, the replacement of the two in vivo phosphorylated Ser(200) and Ser(203) by alanines led to a mutated protein with reduced protein interactions and a weaker complementation ability towards knr4 null mutant phenotypes. These results together with previous data from genome scale two-hybrid and synthetic interaction screens support the notion that Knr4 is a regulatory protein that participates in the coordination of cell wall synthesis with bud emergence, and that this function may be modulated by phosphorylation of this protein.

Base Sequence↗
PubMed2006

The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling.

G protein-coupled receptor kinases (GRKs) and arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization as well as in the modulation of important intracellular signaling cascades by GPCR. Novel studies have revealed a phosphorylation-independent desensitization mechanism operating through their RGS-homology (RH) domain and the recent determination of the crystal structures of GRK2 and GRK6 has uncovered interesting details on the structure-function relationships of these kinases. Emerging evidence indicates that the activity of GRKs is tightly modulated by mechanisms including phosphorylation by different kinases and interaction with several cellular proteins such as calmodulin, caveolin or RKIP. In addition, GRKs are involved in multiple interactions with non-receptor proteins (PI3K, Akt, GIT or MEK) that point to novel GRK cellular roles. In this article, our purpose is to describe the ever increasing map of functional interactions for GRK proteins as a basis to better understand its contribution to cellular processes.

Animals↗
PubMed2026

Mapping the covalent cysteine interactome of Ebselen reveals high-sensitivity target engagement and redox proteome remodeling.

Ebselen is a covalent organoselenium compound with broad pharmacological activity, yet its cellular cysteine targets and downstream proteomic consequences remain incompletely defined. Here, we integrated competitive gel-based activity-based protein profiling, reactivity-dependent tandem orthogonal proteolysis-activity-based protein profiling, and TMT-based quantitative proteomics to map Ebselen-induced cysteine engagement and proteome remodeling in living cancer cells. Ebselen exhibited dose-dependent cytotoxicity and markedly perturbed intracellular thiol-redox balance, as reflected by glutathione depletion and altered reactive oxygen species-associated fluorescence readouts. Competitive gel-based profiling confirmed concentration-dependent engagement of protein cysteine residues in live cells. Quantitative rdTOP-ABPP further identified hundreds of dose-responsive cysteine sites in HeLa and HepG2 cells and revealed a preference for cysteine microenvironments enriched with basic residues. Cross-cell-line comparison highlighted CDK5 Cys53, SMU1 Cys298, and RPSA2 Cys163 as conserved covalent nodes, among which CDK5 Cys53 showed high sensitivity to Ebselen treatment, a finding validated by competitive labeling and MS-based site assignment. Global TMT proteomics revealed extensive remodeling of redox-related and cell-survival-associated pathways, including compensatory upregulation of selenoproteins such as TXNRD1 and GPX family members. Together, these results define a chemical proteomic atlas of Ebselen-cysteine interactions and provide a framework for understanding and optimizing covalent organoselenium therapeutics.

Humans↗
PubMed2005

A skeleton of the human protein interactome.

In this issue of Cell, Wanker and colleagues (Stelzl et al., 2005) present a large-scale two-hybrid map of more than 3000 putative human protein-protein interactions. These new data will serve as an important source of information regarding individual protein partners and offer preliminary insight into the global molecular organization of human cells.

Humans↗
PubMed2006

Biological fingerprinting analysis of the interactome of a kinase inhibitor in human plasma by a chemiproteomic approach.

In this study, a gel free chemiproteomic method based on chromatography was developed and applied for the biological fingerprinting analysis of complex biological system. p-Aminobenzamidine (ABA), an inhibitor of trypsin-like serine proteases, was immobilized for characterizing their interacting proteins in human plasma. By the proteomic analysis method, 214 proteins were identified with obvious affinity to the immobilized ABA. By searching the sequences of above proteins with consensus patterns of the two active sites, seven proteins belong to trypsin-like serine protease group were found. Based on the Gene Ontology annotation, the identified trypsin-like serine proteases have the function of catalytic activity and calcium ion binding, and are mainly involved in the biological process of blood coagulation. Eight more other proteins related to calcium ion binding and blood coagulation were found. Nearly all of these proteins cannot be identified by directly analyzing the plasma sample demonstrating the chemiproteomics a useful approach to characterize interacting proteins in the low abundance range.

Adult↗
PubMed2005

A proteomic snapshot of the human heat shock protein 90 interactome.

Heat shock protein 90 (Hsp90) is a molecular chaperone which modulates several signalling pathways within a cell. By applying co-immunoprecipitation with endogeneous Hsp90, we were able to identify 39 novel protein interaction partners of this chaperone in human embryonic kidney cells (HEK293). Interestingly, levels of DNA-activated protein kinase catalytic subunit, an Hsp90 interaction partner found in this study, were found to be sensitive to Hsp90 inhibitor treatment only in HeLa cells but not in HEK293 cells referring to the tumorgenicity of this chaperone.

DNA-Activated Protein Kinase↗
PubMed2006

Overrepresentation of interactions between homologous proteins in interactomes.

It is well proved that the probability that a protein interacts with itself is higher than that it interacts with another protein. It has been recently shown that the probability of interaction is also higher for proteins with significant sequence similarity. In this paper we show that proteins sharing identical PFAM domains interact more often than expected by chance in Saccharomyces cerevisiae and Escherichia coli. We also analyze the variety of domain interfaces used by homologous proteins to interact and show that the overrepresentation of interactions between homological proteins is not caused by small number of pairs of identical "sticky domains" shared between interacting proteins.

Escherichia coli↗
PubMed2005

Assessing protein co-evolution in the context of the tree of life assists in the prediction of the interactome.

The identification of the whole set of protein interactions taking place in an organism is one of the main tasks in genomics, proteomics and systems biology. One of the computational techniques used by many investigators for studying and predicting protein interactions is the comparison of evolutionary histories (phylogenetic trees), under the hypothesis that interacting proteins would be subject to a similar evolutionary pressure resulting in a similar topology of the corresponding trees. Here, we present a new approach to predict protein interactions from phylogenetic trees, which incorporates information on the overall evolutionary histories of the species (i.e. the canonical "tree of life") in order to correct by the expected background similarity due to the underlying speciation events. We test the new approach in the largest set of annotated interacting proteins for Escherichia coli. This assessment of co-evolution in the context of the tree of life leads to a highly significant improvement (P(N) by sign test approximately 10E-6) in predicting interaction partners with respect to the previous technique, which does not incorporate information on the overall speciation tree. For half of the proteins we found a real interactor among the 6.4% top scores, compared with the 16.5% by the previous method. We applied the new method to the whole E.coli proteome and propose functions for some hypothetical proteins based on their predicted interactors. The new approach allows us also to detect non-canonical evolutionary events, in particular horizontal gene transfers. We also show that taking into account these non-canonical evolutionary events when assessing the similarity between evolutionary trees improves the performance of the method predicting interactions.

Algorithms↗
PubMed2006

Towards a comprehensive analysis of the protein phosphatase 1 interactome in Drosophila.

Protein phosphatase type 1 (PP1) is one of the major classes of serine/threonine protein phosphatases, and has been found in all eukaryotic cells examined to date. Metazoans from Drosophila to humans have multiple genes encoding catalytic subunits of PP1 (PP1c), which are involved in a wide range of biological processes. Different PP1c isoforms have pleiotropic and overlapping functions; this has complicated the analysis of their biological roles and the identification of specific in vivo substrates. PP1c isoforms are associated in vivo with regulatory subunits that target them to specific locations and modify their substrate specificity and activity. The PP1c-binding proteins are therefore the key to understanding the role of PP1 in particular biological processes. The existence of isoform specific PP1c-binding subunits may also help to explain the unique roles of different PP1c isoforms. Here we report the identification of 24 genes encoding Drosophila PP1c-binding proteins in the yeast two-hybrid system. Sequence analysis identified a minimal interacting fragment and putative PP1c-binding motif for each protein, delimiting the region involved in binding to PP1c. Further two-hybrid analysis showed that virtually all of the interactors were capable of binding all Drosophila PP1c isoforms. One of the novel interactors, CG1553, was examined further and shown to interact with multiple isoforms by co-immunoprecipitation from Drosophila extracts and functional interaction with PP1c isoforms in vivo. Bioinformatic analyses implicate the putative PP1c-associated subunits in a diverse array of intracellular processes. Our identification of a large number of PP1c-binding proteins with the potential for directing PP1c's specific functions in Drosophila represents a significant step towards a full understanding of the range of PP1 complexes and function in animals.

Amino Acid Sequence↗
PubMedDate not supplied

Comparison of normal and breast cancer cell lines using proteome, genome, and interactome data.

Normal and cancer cell line proteomes were profiled using high throughput mass spectrometry techniques. Application of protein-level and peptide-level sample fractionation combined with LC-MS/MS analysis enabled identification of 2235 unmodified proteins representing a broad range of functional and compartmental classes. An iterative multistep search strategy was used to identify post-translational modifications, revealing several proteins that are preferentially modified in cancer cells. Information regarding both unmodified and modified protein forms was combined with publicly available gene expression and protein-protein interaction data. The resulting integrated dataset revealed several functionally related proteins that are differentially regulated between normal and cancer cell lines.

Amino Acid Sequence↗
PubMed2007

Interactome of transforming growth factor-beta type I receptor (TbetaRI): inhibition of TGFbeta signaling by Epac1.

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. Type I TGFbeta receptor (TbetaRI) is the key receptor for initiation of intracellular signaling by TGFbeta. Here we report proteomics-based identification of proteins that form a complex with TbetaRI. Using 2D-GE and MALDI TOF mass spectrometry, we identified 16 proteins that specifically interacted with a GST-fused TbetaRI Thr204Asp construct with constitutively active serine/threonine kinase. We confirmed interactions of the receptor with cAMP regulated guanine nucleotide exchange factor 1 (Epac1), beta-spectrin, PIASy, and beta-catenin proteins using immunoblotting. Interaction of the receptor with Epac1 required intact kinase activity of TbetaRI but was not affected by deletion of cAMP-binding domain of Epac1. TGFbeta1-induced C-terminal phosphorylation of Smad2 was inhibited in vivo and in vitro in the presence of Epac1. Epac1 inhibited also TGFbeta1/TbetaRI-dependent transcriptional activation, as evaluated by luciferase reporter assays. We observed that expression of Epac1 counteracted TGFbeta/TbetaRI-dependent decrease of cell adhesion and TGFbeta/TbetaRI-induced stimulation of cell migration. Thus, we have reported novel TRI-interacting proteins and have shown that Epac1 inhibited TGFbeta-dependent regulation of cell migration and adhesion.

Animals↗
PubMed2005

Phosphotyrosine interactome of the ErbB-receptor kinase family.

Interactions between short modified peptide motifs and modular protein domains are central events in cell signal-transduction. We determined interaction partners to all cytosolic tyrosine residues of the four members of the ErbB-receptor family in an unbiased fashion by quantitative proteomics using pull-down experiments with pairs of phosphorylated and nonphosphorylated synthetic peptides. Each receptor had characteristic preferences for interacting proteins and most interaction partners had multiple binding sites on each receptor. EGFR and ErbB4 had several docking sites for Grb2, while ErbB3 was characterized by six binding sites for PI3K. We identified STAT5 as a direct binding partner to EGFR and ErbB4 and discovered new recognition motifs for Shc and STAT5. The overall pattern of interaction partners of EGFR and ErbB4 suggests similar roles during signaling through their respective ligands. Phosphorylation kinetics of several tyrosine resides was measured by mass spectrometry and correlated with interaction partner preference. Our results demonstrate that system-wide mapping of peptide-protein interactions sites is possible, and suggest shared and unique roles of ErbB-receptor family members in downstream signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Biological context networks: a mosaic view of the interactome.

Network models are a fundamental tool for the visualization and analysis of molecular interactions occurring in biological systems. While broadly illuminating the molecular machinery of the cell, graphical representations of protein interaction networks mask complex patterns of interaction that depend on temporal, spatial, or condition-specific contexts. In this paper, we introduce a novel graph construct called a biological context network that explicitly captures these changing patterns of interaction from one biological context to another. We consider known gene ontology biological process and cellular component annotations as a proxy for context, and show that aggregating small process-specific protein interaction sub-networks leads to the emergence of observed scale-free properties. The biological context model also provides the basis for characterizing proteins in terms of several context-specific measures, including 'interactive promiscuity,' which identifies proteins whose interacting partners vary from one context to another. We show that such context-sensitive measures are significantly better predictors of knockout lethality than node degree, reaching better than 70% accuracy among the top scoring proteins.

Protein Interaction Mapping↗
PubMed2006

Characterization of the interactome of the human MutL homologues MLH1, PMS1, and PMS2.

Postreplicative mismatch repair (MMR) involves the concerted action of at least 20 polypeptides. Although the minimal human MMR system has recently been reconstituted in vitro, genetic evidence from different eukaryotic organisms suggests that some steps of the MMR process may be carried out by more than one protein. Moreover, MMR proteins are involved also in other pathways of DNA metabolism, but their exact role in these processes is unknown. In an attempt to gain novel insights into the function of MMR proteins in human cells, we searched for interacting partners of the MutL homologues MLH1 and PMS2 by tandem affinity purification and of PMS1 by large scale immunoprecipitation. In addition to proteins known to interact with the MutL homologues during MMR, mass spectrometric analyses identified a number of other polypeptides, some of which bound to the above proteins with very high affinity. Whereas some of these interactors may represent novel members of the mismatch repairosome, others appear to implicate the MutL homologues in biological processes ranging from intracellular transport through cell signaling to cell morphology, recombination, and ubiquitylation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Dissecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16.

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.

Algal Proteins↗
PubMed2006

Proteome analysis of plant-virus interactome: comprehensive data for virus multiplication inside their hosts.

Known host-parasite molecular interactions are widespread among parasite families, but these interactions have to be particularly large considering that viruses generally encode few proteins. Although some particular virus-host interactions are well described, no global study has yet shown multiple and simultaneous interactions in a host-parasite biological system. To prove that these multiple interactions occur in biological conditions, the complexes formed by a plant virus (rice yellow mottle virus) and the proteins of its natural host (rice) were extracted and purified from infected tissue sample. Remarkably mass spectrometry permitted the identification of a large number of proteins from the complexes that are involved in different functions not encoded by the virus but probably essential for its biological life cycle. This recruiting of proteins was strongly confirmed by the repetition of experiments using different pairs of virus-host and the use of high salt concentration to extract the complexes. We mainly identified proteins involved in plant defense, metabolism, translation, and protein synthesis and some proteins involved in transport. This study demonstrates that viruses are able to recruit many proteins from their hosts to ensure their development. Among different pairs of virus-host, similar protein functions were identified suggesting a particular importance of these proteins for viruses. The identification of particular paralog proteins among multigenic families suggests the high specificity of the recruiting for some protein functions.

Dose-Response Relationship, Drug↗