Huang DS et al. (2014) Curr Protein Pept Sci "Prediction of protein-protein interactions based on protein-protein correlation using least squares regression."

Zhang H, Han K, Huang DS, Zhang L, Deng S, Yang K

[Curr Protein Pept Sci, 2014]

In order to transform protein sequences into the feature vectors, several works have been done, such as computing auto covariance (AC), conjoint triad (CT), local descriptor (LD), moran autocorrelation (MA), normalized moreaubroto autocorrelation (NMB) and so on. In this paper, we shall adopt these transformation methods to encode the proteins, respectively, where AC, CT, LD, MA and NMB are all represented by '+' in a unified manner. A new method, i.e. the combination of least squares regression with '+' (abbreviated as LSR(+)), will be introduced for encoding a protein-protein correlation-based feature representation and an interacting protein pair. Thus there are totally five different combinations for LSR(+), i.e. LSRAC, LSRCT, LSRLD, LSRMA and LSRNMB. As a result, we combined a support vector machine (SVM) approach with LSR(+) to predict protein-protein interactions (PPI) and PPI networks. The proposed method has been applied on four datasets, i.e. Saaccharomyces cerevisiae, Escherichia coli, Homo sapiens and Caenorhabditis elegans. The experimental results demonstrate that all LSR(+) methods outperform many existing representative algorithms. Therefore, LSR(+) is a powerful tool to characterize the protein-protein correlations and to infer PPI, whilst keeping high performance on prediction of PPI networks.

Kotelnikova E et al. (2007) Evol Bioinform Online "Prediction of protein-protein interactions on the basis of evolutionary conservation of protein functions."

Kotelnikova E, Yuryev A, Kalinin A, Maslov S

[Evol Bioinform Online, 2007]

MOTIVATION: Although a great deal of progress is being made in the development of fast and reliable experimental techniques to extract genome-wide networks of protein-protein and protein-DNA interactions, the sequencing of new genomes proceeds at an even faster rate. That is why there is a considerable need for reliable methods of in-silico prediction of protein interaction based solely on sequence similarity information and known interactions from well-studied organisms. This problem can be solved if a dependency exists between sequence similarity and the conservation of the proteins' functions. RESULTS: In this paper, we introduce a novel probabilistic method for prediction of protein-protein interactions using a new empirical probabilistic formula describing the loss of interactions between homologous proteins during the course of evolution. This formula describes an evolutional process quite similar to the process of the Earth's population growth. In addition, our method favors predictions confirmed by several interacting pairs over predictions coming from a single interacting pair. Our approach is useful in working with "noisy" data such as those coming from high-throughput experiments. We have generated predictions for five "model" organisms: H. sapiens, D. melanogaster, C. elegans, A. thaliana, and S. cerevisiae and evaluated the quality of these predictions.

Yao Z et al. (2020) Nat Commun "Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition."

Aboualizadeh F, Boxem M, Kotlyar M, Lyakisheva A, Tang P, Stagljar I, Jurisica I, Akula I, Kroll J, Yao Z, Snider J

[Nat Commun, 2020]

Here, to overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. The bait/prey binding reconstitutes the intein, which splices the bait and prey peptides into a single intact protein that can be detected by regular protein detection methods such as Western blot analysis and ELISA, serving as readouts of PPIs. The method is robust and can be applied not only in mammalian cell lines but in animal models such as C. elegans. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. Additionally, we establish a SIMPL ELISA platform that enables high-throughput screening of PPIs and their inhibitors.

U Vivegananthan et al. (1999) International C. elegans Meeting "Protein-Protein Interactions Involving the Sex Determining Protein, FEM-3"

U Vivegananthan, AM Spence

[International C. elegans Meeting, 1999]

Male development in C. elegans depends on the activities of the three fem genes. Conversely, female development requires that fem activity be negatively regulated. We are interested in how the sex determining protein FEM-3 functions and how its activity is regulated. We believe that a physical interaction between FEM-3 and FEM-2 is required to promote the adoption of male cell fates (Chin-Sang and Spence, 1996. Genes and Dev. 10 : 2314-25). We also have evidence that an interaction between FEM-3 and the C-terminus of TRA-2A is required for the negative regulation of fem activity in XX somatic tissues (Mehra, A., Gaudet, J., Heck, L., Kuwabara, P. and Spence, A. submitted). We want to use genetic assays to test our hypotheses about the biological significance of the two known FEM-3 interactions. We screened in vitro mutagenized fem-3 cDNAS, in a modified version of the two-hybrid system, to look for FEM-3 mutants that are able to bind to FEM-2, or to TRA-2A, but not both. We have identified two mutations that selectively disrupt the FEM-3/FEM-2 interaction and five mutations that selectively disrupt the FEM-3/TRA-2A interaction. We are currently testing the effects of these mutants on sex determination in vivo . We predict that FEM-2 non-binding mutants of FEM-3 will be incapable of supporting male development. In contrast, we predict that the TRA-2A non-binding mutants of FEM-3 will dominantly masculinize the XX soma, irrespective of the level of TRA-2A activity.

Yu HY et al. (2004) Genome Res "Annotation transfer between genomes: protein-protein interologs and protein-DNA regulogs."

Yu HY, Vidal M, Xia Y, Luscombe NM, Gerstein M, Han JDJ, Lu HX, Zhu XW, Bertin N, Chung S

[Genome Res, 2004]

Proteins function mainly through interactions, especially with DNA and other proteins. While some large-scale interaction networks are now available for a number of model organisms, their experimental generation remains difficult. Consequently, interolog mapping--the transfer of interaction annotation from one organism to another using comparative genomics--is of significant value. Here we quantitatively assess the degree to which interologs can be reliably transferred between species as a function of the sequence similarity of the corresponding interacting proteins. Using interaction information from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Helicobacter pylori, we find that protein-protein interactions can be transferred when a pair of proteins has a joint sequence identity >80% or a joint E-value <10(-70). (These "joint" quantities are the geometric means of the identities or E-values for the two pairs of interacting proteins.) We generalize our interolog analysis to protein-DNA binding, finding such interactions are conserved at specific thresholds between 30% and 60% sequence identity depending on the protein family. Furthermore, we introduce the concept of a "regulog"--a conserved regulatory relationship between proteins across different species. We map interologs and regulogs from yeast to a number of genomes with limited experimental annotation (e.g., Arabidopsis thaliana) and make these available through an online database at http://interolog.gersteinlab.org. Specifically, we are able to transfer approximately 90,000 potential protein-protein interactions to the worm. We test a number of these in two-hybrid experiments and are able to verify 45 overlaps, which we show to be statistically significant.

Chin-Sang ID et al. (1996) Midwest Worm Meeting "Protein-protein interaction between FEM-3 and FEM-2, a Masculinizing Protein Phosphatase"

Chin-Sang ID, Spence AM

[Midwest Worm Meeting, 1996]

Male development in C. elegans requires the activities of the fem genes, fem-1, fem-2, and fem-3. The prevailing model of sex determination suggests that in the somatic tissues of XO animals, the FEM proteins negatively regulate TRA-1A, a sequence-specific DNA-binding protein that would otherwise cause female development. In the germline of both XX and XO animals, the FEM proteins regulate other targets to promote spermatogenesis. Greater understanding of the mechanism of sex determination requires knowledge of the interactions in which the FEM proteins engage and the regulatory mechanisms that they employ. The sequence of FEM-3 is unique and therefore offers no clues as to its biochemical activity. To address the question of how FEM-3 fulfills its role in sex determination, we employed the yeast two-hybrid system to screen for clones encoding FEM-3 - interacting proteins. Here we report the isolation of fem-2 cDNA clones on the basis of an interaction between FEM-2 and FEM-3. We used two assays to demonstrate that FEM-2 and FEM-3 associate in vitro. In the first, we used a monoclonal antibody against an epitope tag in FEM-3 to coimmunoprecipitate both proteins from in vitro translation reactions. In the second, we showed that glutathione Sepharose beads coated with bacterially expressed GST-FEM-2 specifically bound radiolabelled FEM-3 produced by in vitro translation in reticulocyte lysates. GST-coated beads did not bind FEM-3. Deletion analysis of FEM-2 suggests that a region of about 190 amino acids contains sequences required for FEM-3 binding. As reported by D. Pilgrim and colleagues (1), FEM-2 is similar in sequence to protein serine/threonine phosphatases of Type 2C (PP2C). We showed that GST-FEM-2 indeed exhibits casein phosphatase activity in vitro, and that its activity is magnesium-dependent, as is typical of PP2C. Substitution of a highly conserved arginine residue abolishes the casein phosphatase activity of FEM-2 but does not affect its FEM-3 - binding activity, suggesting that the mutant protein is not grossly abnormal in structure. When expressed from the heat shock promoter in transgenic fem-2 m-z- XO animals, FEM-2 lacking casein phosphatase activity is much less active in rescuing male development than wild-type FEM-2. The mutant and wild-type proteins exhibit similar levels of expression and stability. Our observations suggest that FEM-2 must be an active protein phosphatase to fulfill its normal role in sex determination. We conclude that the mechanism of sex determination in C. elegans involves protein phosphorylation, and that FEM-2 antagonizes the feminizing effect of a kinase that remains to be identified. We propose that interaction between FEM-2 and FEM-3 is necessary for normal male development, and we are currently testing the requirement for the interaction. Several possibilities exist as to the functional consequences of the FEM-2-FEM-3 interaction. For example, FEM-3 might regulate the localization or substrate specificity of FEM-2. (1) Pilgrim, D., et al. (1995). Mol. Biol. Cell 6: 1159-1171.

Tan D et al. (2016) Elife "Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states."

Zhang P, Zhang MJ, Gao N, Tan D, Feng B, Wang HW, Ma C, Lei X, Ma S, He SM, Liu X, Yang B, Liu J, Li Q, Fan SB, Liu C, Ye K, Ding YH, Dong MQ, Tao L, Li X

[Elife, 2016]

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.

Kroll, Jason et al. (2021) International Worm Meeting "Light-induced protein clustering to study protein-protein interactions in C. elegans"

Kroll, Jason, Boxem, Mike, Remmelzwaal, Sanne

[International Worm Meeting, 2021]

Interactions among proteins are fundamental for life, and misregulation of them can lead to diseases such as cancer. We adapted the light-activated reversible inhibition by assembled trap (LARIAT) system and the light-induced co-clustering assay for use in C. elegans. This assay can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. In the LARIAT protein-protein interaction assay, cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) bind each other in a blue-light dependent manner, forming small but densely packed protein clusters within the cell. Fusion of an anti-GFP nanobody to the CRY2 protein traps any GFP-tagged protein in the clusters. Co-clustering of a mCherry/mScarlet fluorescent protein can be monitored for inclusion in the CRY2/GFP clusters, indicating an interaction between the two fluorescently tagged proteins. We tested the system in C. elegans with an array of positive and negative reference protein pairs, using endogenously tagged GFP and mCherry/mScarlet alleles. CRY2/CIB1 proteins were expressed from extrachromosomal arrays since genome integration is unnecessary for the assay. Light-induced clusters form rapidly within seconds in multiple tissue types. Assay performance was extremely robust with no false positives detected in the negative reference pairs, and no lethality associated with the clustering, even with proteins essential for development and cell polarity. We have generated a toolkit containing plasmids with different promoters, an anti-mCherry nanobody variant, and a plasmid with an empty promoter module to enable one-step cloning of any particular promoter using SapTrap assembly. The light induced co-clustering assay is a powerful and rapid technique that will be valuable for the C. elegans community, especially due to the fact that the assay uses a universal plasmid for every protein pair, and existing fluorescently tagged strains can be used without need for additional cloning or genetic modification of the genome.

Bazopoulou MD et al. (2004) Focus on Protein Research "Protein turnover and ageing."

Tavernarakis N, Troullinaki K, Bazopoulou MD

[Focus on Protein Research, 2004]

Smith HE et al. (1998) J Mol Biol "Identification of protein-protein interactions of the major sperm protein (MSP) of Caenorhabditis elegans."

Ward S, Smith HE

[J Mol Biol, 1998]

In nematodes, sperm are amoeboid cells that crawl via an extended pseudopod. Unlike those in other crawling cells, this pseudopod contains little or no actin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility occurs via the regulated assembly and disassembly of MSP filaments. Filaments composed of MSP dimers are thought to provide the motive force. We have employed the yeast two-hybrid system to investigate MSP-MSP interactions and provide insights into the process of MSP filament formation. Fusions of the Caenorhabditis elegans msp-142 gene to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) interact to drive expression of a lacZ reporter construct. A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones that fail to interact with LEXA-MSP. Single missense mutations were identified and mapped to the crystal structure of A. suum MSP. Two classes of mutations predicted from the structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface. Multiple additional mutations were obtained in the two carboxy-terminal beta strands, a region not predicted to be involved in protein folding or dimer formation. Size fractionation of bacterially expressed MSPs indicates that mutations in this region do not abolish dimer formation. A number of compensating mutations that restore the interaction also map to this region. The data suggest that the carboxy-terminal beta strands are directly involved in interactions required for MSP filament assembly.