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Representation of molecule sets i.e. cases where a participant may be one of a list of molecules (use case 1.3i). ( https://github.com/HUPO-PSI/miXML/blob/master/3.0/pub/Appendix 10.docx ). (DOCX 25Â kb)

Representation of a negative feature range (use case 1.3a). ( https://github.com/HUPO-PSI/miXML/blob/master/3.0/pub/Appendix 3.docx ). (DOCX 27Â kb)

Representation of the systematic capture of the stoichiometry of molecules within an interaction (use case 1.3j). ( https://github.com/HUPO-PSI/miXML/blob/master/3.0/pub/Appendix 11.docx ). (DOCX 31Â kb)

Representation of a cooperative interaction in PSI-MI XML3.0.0 (Use case 1.3Â h). ( https://github.com/HUPO-PSI/miXML/blob/master/3.0/pub/Appendix 9.docx ). (DOCX 28Â kb)

Representation of multiple feature detection methods and feature roles (use case 1.3c, use case 1.3d). ( https://github.com/HUPO-PSI/miXML/blob/master/3.0/pub/Appendix 5.docx ). (DOCX 68Â kb)

Representation of an abstracted interaction, a manually curated protein complex, in PSI-MI XML3.0.0 (use case 1.3Â g). ( https://github.com/HUPO-PSI/miXML/blob/master/3.0/pub/Appendix 8.docx ). (DOCX 48Â kb)

Representation of the sequence change caused by introduction of a mutation (use case 1.3b). ( https://github.com/HUPO-PSI/miXML/blob/master/3.0/pub/Appendix 4.docx ). (DOCX 38Â kb)

Representation of variable conditions (dynamic interactions) in an experiment (use case 1.3f). ( https://github.com/HUPO-PSI/miXML/blob/master/3.0/pub/Appendix 7.docx ). (DOCX 43Â kb)

Protein phosphatase 2A (PP2A) is one of the most abundant serine–threonine phosphatases in mammalian cells. PP2A is a hetero-trimeric holoenzyme participating in a variety of physiological processes whose deregulation is often associated to cancer. The specificity and activity of this phosphatase is tightly modulated by a family of regulatory B subunits that dock the catalytic subunit to the substrates. Here we characterize a novel and unconventional molecular mechanism controlling the activity of the tumor suppressor PP2A. By applying a mass spectrometry-based interactomics approach, we identified novel PP2A interacting proteins. Unexpectedly we found that a significant number of RAB proteins associate with the PP2A scaffold subunit (PPP2R1A), but not with the catalytic subunit (PPP2CA). Such interactions occur in vitro and in vivo in specific subcellular compartments. Notably we demonstrated that one of these RAB proteins, RAB9, competes with the catalytic subunit PPP2CA in binding to PPP2R1A. This competitive association has an important role in controlling the PP2A catalytic activity, which is compromised in several solid tumors and leukemias.

Cell physiology is governed by an intricate mesh of physical and functional links among proteins, nucleic acids and other metabolites. The recent information flood coming from large-scale genomic and proteomic approaches allows us to foresee the possibility of compiling an exhaustive list of the molecules present within a cell, enriched with quantitative information on concentration and cellular localization. Moreover, several high-throughput experimental and computational techniques have been devised to map all the protein interactions occurring in a living cell. So far, such maps have been drawn as graphs where nodes represent proteins and edges represent interactions. However, this representation does not take into account the intrinsically modular nature of proteins and thus fails in providing an effective description of the determinants of binding. Since proteins are composed of domains that often confer on proteins their binding capabilities, a more informative description of the interaction network would detail, for each pair of interacting proteins in the network, which domains mediate the binding. Understanding how protein domains combine to mediate protein interactions would allow one to add important features to the protein interaction network, making it possible to discriminate between simultaneously occurring and mutually exclusive interactions. This objective can be achieved by experimentally characterizing domain recognition specificity or by analyzing the frequency of co-occurring domains in proteins that do interact. Such approaches allow gaining insights on the topology of complexes with unknown three-dimensional structure, thus opening the prospect of adopting a more rational strategy in developing drugs designed to selectively target specific protein interactions.

Cell physiology is governed by an intricate mesh of physical and functional links among proteins, nucleic acids and other metabolites. The recent information flood coming from large-scale genomic and proteomic approaches allows us to foresee the possibility of compiling an exhaustive list of the molecules present within a cell, enriched with quantitative information on concentration and cellular localization. Moreover, several high-throughput experimental and computational techniques have been devised to map all the protein interactions occurring in a living cell. So far, such maps have been drawn as graphs where nodes represent proteins and edges represent interactions. However, this representation does not take into account the intrinsically modular nature of proteins and thus fails in providing an effective description of the determinants of binding. Since proteins are composed of domains that often confer on proteins their binding capabilities, a more informative description of the interaction network would detail, for each pair of interacting proteins in the network, which domains mediate the binding. Understanding how protein domains combine to mediate protein interactions would allow one to add important features to the protein interaction network, making it possible to discriminate between simultaneously occurring and mutually exclusive interactions. This objective can be achieved by experimentally characterizing domain recognition specificity or by analyzing the frequency of co-occurring domains in proteins that do interact. Such approaches allow gaining insights on the topology of complexes with unknown three-dimensional structure, thus opening the prospect of adopting a more rational strategy in developing drugs designed to selectively target specific protein interactions.

One of the goals of functional genomics is the description of reliable and complete protein interaction networks. To facilitate ligand discovery from complex protein mixtures, we have developed an improved approach that is affected by a negligible fraction of false positives. We have combined a novel technique based on the display of cDNA libraries on the capsid of bacteriophage lambda and an efficient plaque assay to reveal phage displaying ligands that are enriched after only a couple of affinity purification steps. We show that the lambda display system has a unique ability to display, at high density, proteins ranging in size from a few to at least 300 amino acid residues. This characteristic permits attenuation of the size bias in the selection procedure and, at the same time, offers a sensitive plaque assay that permits us to do away with the ligand background without unduly increasing the number of selection cycles. By using a proline-rich fragment of the synaptojanin 1 protein as a bait, we have identified, in a brain cDNA display library, seven ligands all containing either SH3 or WW domains. Four of these correspond to proteins that have already been validated as physiological partners, while the remaining three are new partners, whose physiological relevance remains to be established. Two different proline-rich regions of the p21-activated protein kinase 1 (Pak1) and WAVE/SCAR2 protein retrieve from the library different proteins containing SH3 or WW domains.

The hydrophobic core packing in four-alpha-helical bundles appears to be crucial for stabilizing the protein structure. To examine the structural basis of hydrophobic stabilization, the crystal structures of the Leu--Val (L41V) and Leu--Ala (L41A) substitutions of the core residue Leu41 of the ROP protein have been determined. Both substitutions are destabilizing and lead to formation of cavities. The main responses to mutations are the collapse of the central part of the alpha-helix containing the site of mutation, shifts of internal water molecules, and in L41A, the trapping of a water molecule in a cavity engineered by the mutation. For both mutants, these effects limit the increase in cavity size to less than 10 A3, while an increase of 37 A3 and 100 A3 is expected for L41V and L41A, respectively, in the absence of any cavity size reducing effects. The mobility of internal side-chains is increased and in L41A, it reaches values typical for exposed residues. A parameter (Deltanh) is introduced as a measure of the number of van der Waals contacts lost. For ROP, barnase and T4 lysozyme mutants, there is a good correlation between Deltanh and the free energy of unfolding DeltaDeltaG relative to wild-type protein. The Deltanh value turns out to be more suitable for analysing structural and energetic responses to mutation than other parameter, such as cavity volumes and packing densities. Possible evolutionary implications of the DeltaDeltaG versus Deltanh relationship are discussed.

Tyrosine phosphorylation and protein recognition, mediated by phosphotyrosine containing peptides, play an important role in determining the specific response of a cell, when stimulated by external signals. We have used peptide repertoires displayed by filamentous phage as a tool to study the substrate specificity of the protein tyrosine kinase (PTK) p55(fyn) (Fyn). Peptide libraries were incubated for a short time in the presence of Fyn and phages displaying efficiently phosphorylated peptides were selected by panning over anti-phosphotyrosine antibodies. The characterization of the peptides enriched after three phosphorylation/selection rounds allowed us to define a canonical substrate sequence for the kinase Fyn, E-(phi/T)YGx phi, where phi represents any hydrophobic residue. A peptide conforming to this sequence is a better substrate than a second peptide designed to be in accord with the consensus sequence recognised by the Fyn SH2 domain. When the library phosphorylation reaction is carried out in saturation conditions, practically all the tyrosine containing peptides are phosphorylated, irrespective of their context. These "fully modified" peptide libraries are a valuable tool to study the specificity of phosphotyrosine mediated protein recognition. We have used this new tool to identify a family of peptides that bind the PTB domain of the adapter protein Shc. Comparison of the peptide sequences permits us to confirm the essential role of N at position -3, while P often found at position -2 in natural targets is not absolutely required. Furthermore, our approach permits us to reveal an "extended" consensus indicating that residues that do not seem to influence binding in natural peptides can make productive contacts, at least in linear peptides.

The filamentous virus fd is an important model for membrane protein assembly and is used extensively as a cloning vector and vehicle for peptide display. The threadlike virion (∼6 × 880 nm) comprises a single-stranded DNA genome sheathed by ∼2700 copies of a 50-residue α-helical subunit (pVIII). pVIII contains two tyrosines, Y21 and Y24. Tyrosine side-chain orientations in the virion have been examined by polarized Raman microspectroscopy of oriented fibers of wild type fd and of mutants in which either Y21 or Y24 is replaced by methionine. Polarized Raman spectra were collected for two fiber orientations in the laboratory frame of reference (abc, where c is parallel to the virion or fiber axis). In one orientation, designated cc, electric vectors of the exciting (514.5 nm) and scattered radiation are parallel to c; in the other (bb), these vectors are parallel to b. The intensity ratio, I cc /I bb , was determined for each Raman band.

Shc proteins are targets of activated tyrosine kinases and have been implicated in the transmission of activation signals to Ras. Upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated proteins and with the Grb2 adaptor protein. Two Shc isoforms of 52 and 46 kDa have been characterized. They share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal phospho-tyrosine binding domain (PTB). We report her ethe initial characterization of two Shc related human cDNAs: ShcB and ShcC. The ShcB and ShcC cDNAs code for proteins that are highly similar and share the same modular organization as Shc. PTB and SH2 domains of ShcB and ShcC have similar binding specificities in vitro and bind to activated EGFR in a phosphotyrosine-dependent manner. Based on these findings we propose to rename Shc as ShcA. Anti-ShcB and anti-ShcC antibodies recognize specific polypeptides of 52, 47 kDa (ShcB) and 54 kDa (ShcC) in mammalian cells. Since these two genes are predominantly expressed in specific brain tissues, these Shc family members may be involved in cell type-specific signaling, in the nervous system.

Rop is the simplest and most regular member of a family of proteins characterized by a bundle of four antiparallel helices. Rop is dimeric, each monomer being formed by two helices connected by a sharp bend. In this work we have extensively mutagenized three residues that form the connection between the two alpha-helices to ask whether the bend region contains any important folding information. The characterization of a collection of random mutants indicated that this structure is rather insensitive to amino acid substitutions and that most amino acids are tolerated in these positions by the Rop native structure. In order to identify the rare amino acid sequences that would prevent Rop from folding and/or dimerizing, we exploited the observation that Rop can functionally substitute the dimerization domain of the lambda repressor. In fact plasmids expressing a hybrid protein formed by the amino-terminal domain of the lambda repressor covalently linked to Rop, confer immunity to lambda infection on their hosts. We have shown that this property depends on the ability of the Rop moiety to fold and dimerize. The analysis of 380 Rop mutants containing random amino acid sequences at positions 30, 31 and 32 allowed us to identify three mutant Rop proteins that are defective in dimerization, probably as a consequence of their inability to fold. In these mutants the tripeptides VED, VPD and YPD substitute the wild-type DAD at positions 30, 31 and 32. Other combinations of amino acids are found resulting in levels of immunity that are lower than the wild-type but still sufficient to prevent single plaque formation. This result suggests that a smaller proportion of the corresponding Rop protein reaches a thermodynamic and proteolytically stable dimeric state.

We have approached the problem of folding and assembly of the heavy (H) chain of human ferritin by isolating point mutations that affect this process. Apoferritin is an ideal model system to approach the problem of protein folding and assembly into multimeric structures. We have developed a recombinant hybrid molecule that allows us to select for ferritin mutants in which the folding-assembly process is altered or completely impaired. The selection procedure is based on a recombinant protein which consists of a fusion between the H chain of human ferritin and the alpha-peptide of beta-galactosidase. In the wild type situation, the alpha-peptide domain is segregated inside the apoferritin shell upon assembly and is unable to interact with the substrate and perform its enzymic function. We show that by selecting for mutations that restore beta-galactosidase activity we are able to identify ferritin mutations that affect the folding-assembly process. The selective procedure was applied to the analysis of the amino acid side chains that are important for the attainment of the correct conformation of the carboxy-terminal E helix in the 4-fold axis.

Using the Escherichia coli Rop gene, we demonstrate a strategy that could be applied generally to optimize the initiation of translation of recombinant genes in E. coli. This involves cloning of the gene encoding the protein of interest in a suitable expression vector between an "efficient" ribosome binding site and the gene for the alpha-peptide of beta-galactosidase. By oligonucleotide-directed deletion mutagenesis, the two coding sequences are then fused in the correct frame. A second oligonucleotide is then used to place the initiator AUG (or GUG) at the correct distance from the Shine-Dalgarno sequence. In this step, however, we use an oligonucleotide that has a degenerate sequence. That is, on the basis of the "efficient" ribosome binding site sequence, we introduce random substitutions at various positions, both upstream and downstream from the initiator ATG, to obtain, after the mutagenesis experiment, a collection of random ribosome binding sites fused to the coding sequence. The development of blue colonies on indicator plates permits selection of clones in which an efficient ribosome binding site has been created for the specific gene of interest. We discuss the results obtained by applying the method to the Rop gene.

The copy number of E. coli plasmids of the ColE1 family is regulated at the level of the formation of an RNA molecule that serves as primer for the enzyme DNA polymerase I. Negative regulation is obtained by the interaction between teh precursor of the primer and RNA I, a plasmid-encoded small RNA. The RNA-RNA interaction between the two regulatory molecules is modulated by the protein Rop.

We characterize two dominant copy number mutants of a derivative of plasmid pMB1. One of the two mutations maps in the -35 region of the primer promoter and results in increased promoter activity. The analysis of the secondary structure in the proximity of the mutant sequence suggests a possible mechanism which could be the basis of the promoter-up phenotype. By comparing the properties of the mutant and the wild type plasmid in an in vitro system, we confirm that the primer and not its coding sequence is the target of RNA I inhibition. The second mutation affects the sequence of the primer so that it is less sensitive to inhibition by RNA I. We propose that this mutation stabilizes a secondary structure necessary for primer formation.

We show that a protein of 63 amino acids is the product of the rop gene, a gene which negatively regulates the copy number of plasmids of the ColE1 family. Rop protein purified to homogeneity inhibits ColE1 plasmid replication in a bacterial extract. Furthermore, we show that Rop inhibition requires RNA I. In a purified in vitro system that can support primer transcription and processing, Rop affects primer formation in two ways: first, it elicits transcription termination oat nucleotide 220, and second, it increases the ability of RNA I to inhibit RNAase H processing of the primer. The analysis of these data and the comparison with the results obtained in vivo with transcription fusion experiments allow us to propose a tentative model of the molecular mechanism underlying Rop-RNA I inhibition.

The analysis of a large number of independent mutants in the target of one of the inhibitors of pMB1 replication suggests that RNA1 regulates primer formation by base-pairing with the complementary sequence in the primer precursor. We conclude that the number of bases that are involved in the hydrogen bonding responsible for the specificity of the mechanism that controls plasmid replication and incompatibility properties is not much larger than seven. Five of these bases are located in the central loop and two in loop I of the RNA primer cloverleaf structure. Twenty-two single, double or triple mutants, with different nucleotide sequences in these seven bases, maintain an active mechanism of control, though with altered specificity. The efficiency of the inhibition mechanism correlates with the ΔG value of the hydrogen bonds between the nucleotides of the two heptamers postulated to be involved in the interaction. The implications of these findings are discussed, and a molecular model of the interaction between RNA1 and the primer precursor is presented.

Four single base pair mutations in the region that codes for RNA I create new incompatibility groups while preserving the mechanism of control of initiation of DNA replication in a ColE1-type plasmid. Sequence analysis of these base substitutions points to the primary importance of the central loop of the cloverleaf structure of RNA I in this control mechanism.

The analysis of a large number of independent mutants in the target of one of the inhibitors of pMB1 replication suggests that RNA1 regulates primer formation by base-pairing with the complementary sequence in the primer precursor. We conclude that the number of bases that are involved in the hydrogen bonding responsible for the specificity of the mechanism that controls plasmid replication and incompatibility properties is not much larger than seven. Five of these bases are located in the central loop and two in loop I of the RNA primer cloverleaf structure. Twenty-two single, double or triple mutants, with different nucleotide sequences in these seven bases, maintain an active mechanism of control, though with altered specificity. The efficiency of the inhibition mechanism correlates with the delta G value of the hydrogen bonds between the nucleotides of the two heptamers postulated to be involved in the interaction. The implications of these findings are discussed, and a molecular model of the interaction between RNA1 and the primer precursor is presented.

We describe the construction and properties of pEMBLY plasmids. They belong to a new family of yeast shuttle vectors which are derived from plasmid vector pEMBL9 and offer the following improvement: relatively small size; large number of cloning sites; screening for insert-containing plasmids on indicator plates; different combinations of genes which complement auxotrophic deficiencies and sequences that support DNA replication in Saccharomyces cerevisiae; and ability to isolate the plasmid DNA in single-stranded (ss) form. The yeast S. cerevisiae can be efficiently transformed by these plasmids in both the ss and double-stranded (ds) forms. Finally, the presence of the phage f1 intergenic region allows one to obtain the cloned sequences in the ss form upon infection with the wild-type ss phage [Dotto et al., Virology 114 (1981) 463-473].

Plasmids carrying cloned lambda att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of lambda. The cross, referred to as "lifting" the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed "lifting" lambda phages. One phage donates a short left arm and the other donates a short right arm. These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria. However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids. We call these recombinants phasmids. They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components. These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments. Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.

Plasmids of the ColEl family provide a convenient model system for the investigation of the molecular mechanisms which regulate initiation of DNA replication in Escherichia coli. ColEl replicates in bacteria at a copy number of approximately 15. The observations that this copy number could be increased by mutagenesis and that some of these mutants are complemented in trans by plasmids of the same incompatibility group suggest that ColEl replication is negatively regulated (1–6). An attractive model of the processes of initiation of DNA replication was proposed by Tomizawa and coworkers who were able to reassemble from purified enzymes an in vitro system which can carry out the biochemical steps involved in primer formation and initial deoxyribonucleotide polymerization (3, 7). When origin sequences are incubated in the presence of RNA polymerase, two main transcripts can be identified (Figure 1). The longer one (RNA2) starts from a promoter 550 nucleotides upstream from the replication origin and is believed to form a hybrid with the DNA template in the origin region. This hybrid is processed by RNAseH to yield an RNA molecule of 550 nucleotides which can serve as primer for deoxyribonucleotide polymerization by DNA polymerase I.

The endogenous 2 mu plasmid of yeast encodes a site-specific recombinase FLP that can cause an amplification of plasmid copy number. Using strains overexpressing 2 mu plasmid proteins from chromosomal constructs to disrupt the normal balance of gene products, we show here that copy number is controlled by regulating the transcript level of FLP. Expression of FLP is negatively regulated over a 100-fold range by the joint action of the plasmid-encoded REP1 and REP2 proteins, which also have a role in plasmid partition. We also show that the product of the fourth plasmid open reading frame D increases FLP expression by relieving the repression caused by REP1 and REP2. This is the first demonstration of a function for this gene, which we call RAF. The transcription of RAF is also repressed by REP1 and REP2 acting together, but requires a higher level for complete inhibition than that required to repress FLP. Copy number is therefore negatively regulated by REP1-REP2 concentration both by direct repression of FLP and indirectly, by control of the positive element, the anti-repressor RAF. We propose that these antagonistic regulatory mechanisms amplify the signal produced by a small fall in copy number.

snR17, one of the most abundant capped small nuclear RNAs of Saccharomyces cerevisiae, is equivalent to U3 snRNA of other eukaryotes. It is 328 nucleotides in length, 1.5 times as long as other U3 RNAs, but shares significant homology both in nucleotide sequence and in predicted secondary structure. Human scleroderma antiserum specific to nucleolar U3 RNP can enrich snR17 from sonicated yeast nuclear extracts. Unlike other yeast snRNAs which are encoded by single copy genes, snR17 is encoded by two genetically unlinked genes: SNR17A and SNR17B. The RNA snR17A is more abundant than snR17B. Deleting one or other of the genes has no obvious phenotypic effect, except that the steady-state level of snR17B is increased in snr17a- strains. Haploid strains with both genes deleted are inviable, therefore yeast U3 is essential.

The rop gene participates in the control of plasmid copy number by interfering with transcripts originating from the primer promoter. Here we show that this inhibition mechanism requires RNA1 in trans. Mutations in the RNA1 coding sequence that result in plasmids with altered incompatibility properties do not affect the ability of the molecule to participate in the Rop inhibition mechanism. Furthermore we show that the target of the Rop-RNA1 inhibitory mechanism is located, at least in part, after the 52nd nucleotide of the sequence encoding the primer transcript.

To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H). As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction. The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e. at pH 7 and in presence of iron core. We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus. These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation. The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core. It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation.