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Daniel Caput - One of the best experts on this subject based on the ideXlab platform.
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covalent modification of p73α by sumo 1 two hybrid Screening with p73 identifies novel sumo 1 interacting proteins and a sumo 1 interaction motif
Journal of Biological Chemistry, 2000Co-Authors: Adrian Minty, Xavier Dumont, Mourad Kaghad, Daniel CaputAbstract:Two-Hybrid Screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the Two-Hybrid system. We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.
Roberto Ravazzolo - One of the best experts on this subject based on the ideXlab platform.
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Confirmation of CLIM2/LMX1B interaction by yeast Two-Hybrid Screening and analysis of its involvement in nail-patella syndrome.
International journal of molecular medicine, 2003Co-Authors: Monica Marini, Ernie M.h.f. Bongers, Roberto Cusano, Marco Di Duca, Marco Seri, Nine V A M Knoers, Roberto RavazzoloAbstract:Nail-patella syndrome (NPS), an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns, and often associated with nephropathy and, less frequently, with open angle glaucoma, is caused by mutations in the LMX1B gene. Inter-familial and intra-familial phenotypic variability raises the question whether modifier genes can be identified to explain differences in the expression and severity of clinical features of NPS. Genes encoding proteins that interact with the LMX1B protein are good candidates and, therefore, methods to search for interactions can be used to this purpose. By the yeast Two-Hybrid Screening we detected the CLIM2 gene as a LMX1B interactor, confirming previous reports which described the same interaction by biochemical methods. Sequencing of the CLIM2 coding region in seven NPS cases in which no LMX1B mutation had been found, did not detect any molecular variant in these patients. Moreover, by genotyping a polymorphic dinucleotide repeat close to the CLIM2 gene in affected members of a large Dutch NPS family with high incidence of nephropathy, we were unable to find a correlation between the presence of a specific allele and the expression of nephropathy. In conclusion, although the results of this study could not provide any proof of CLIM2 involvement in the pathogenesis of NPS or in determination of the clinical phenotype, we suggest that the CLIM2 gene can be considered as a good candidate for further studies on normal and disturbed kidney development associated with NPS or other hereditary glomerulopathies.
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confirmation of clim2 lmx1b interaction by yeast two hybrid Screening and analysis of its involvement in nail patella syndrome
International Journal of Molecular Medicine, 2003Co-Authors: Monica Marini, Ernie M.h.f. Bongers, Roberto Cusano, Marco Di Duca, Marco Seri, Nine V A M Knoers, Roberto RavazzoloAbstract:Nail-patella syndrome (NPS), an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns, and often associated with nephropathy and, less frequently, with open angle glaucoma, is caused by mutations in the LMX1B gene. Inter-familial and intra-familial phenotypic variability raises the question whether modifier genes can be identified to explain differences in the expression and severity of clinical features of NPS. Genes encoding proteins that interact with the LMX1B protein are good candidates and, therefore, methods to search for interactions can be used to this purpose. By the yeast Two-Hybrid Screening we detected the CLIM2 gene as a LMX1B interactor, confirming previous reports which described the same interaction by biochemical methods. Sequencing of the CLIM2 coding region in seven NPS cases in which no LMX1B mutation had been found, did not detect any molecular variant in these patients. Moreover, by genotyping a polymorphic dinucleotide repeat close to the CLIM2 gene in affected members of a large Dutch NPS family with high incidence of nephropathy, we were unable to find a correlation between the presence of a specific allele and the expression of nephropathy. In conclusion, although the results of this study could not provide any proof of CLIM2 involvement in the pathogenesis of NPS or in determination of the clinical phenotype, we suggest that the CLIM2 gene can be considered as a good candidate for further studies on normal and disturbed kidney development associated with NPS or other hereditary glomerulopathies.
Manfred Koegl - One of the best experts on this subject based on the ideXlab platform.
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High-Throughput Yeast Two-Hybrid Screening of Complex cDNA Libraries
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Kerstin Mohr, Manfred KoeglAbstract:Yeast Two-Hybrid Screening can be used to find cDNAs encoding proteins which bind to a given bait protein in large, pooled cDNA libraries. Screening of complex, pooled libraries is slower and more laborious than Screening of arrayed collections of cDNAs, but has several advantages. First, the complexity of a pooled library can be orders of magnitude larger than the size of a typical arrayed library. Second, as long as available cDNA collections are incomplete and limited to full-length cDNAs, pooled cDNA libraries offer a more complete search space and are often the only way to do screens in organisms other than human and a few model organisms. We have streamlined and optimised the Screening of pooled libraries in a format which uses micro-titre plates and produces quantitative signals for the selection of hits. This format has the advantage that automation of the process is straightforward and allows a throughput of up to at least 1,000 screens per year per person.
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Improving yeast Two-Hybrid Screening systems.
Briefings in functional genomics & proteomics, 2007Co-Authors: Manfred Koegl, Peter UetzAbstract:Yeast Two-Hybrid (Y2H) Screening methods are an effective means for the detection of protein-protein interactions. Optimisation and automation has increased the throughput of the method to an extent that allows the systematic mapping of protein-protein interactions on a proteome-wide scale. Since Two-Hybrid screens fail to detect a great number of interactions, parallel high-throughput approaches are needed for proteome-wide interaction screens. In this review, we discuss and compare different approaches for adaptation of Y2H Screening to high-throughput, the limits of the method and possible alternative approaches to complement the mapping of organism-wide protein-protein interactions.
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Automated Yeast Two-Hybrid Screening for Nuclear Receptor-interacting Proteins
Molecular & cellular proteomics : MCP, 2004Co-Authors: Michael Albers, Harald Kranz, Ingo Kober, Carmen Kaiser, Martin Klink, Joerg Suckow, Rainer Kern, Manfred KoeglAbstract:High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast Two-Hybrid system, we completed a large interaction Screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast Two-Hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors.
Lutz Weber - One of the best experts on this subject based on the ideXlab platform.
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Development of a yeast Two-Hybrid screen for selection of human Ras-Raf protein interaction inhibitors.
Methods in molecular biology (Clifton N.J.), 2005Co-Authors: Vladimir Khazak, Erica A. Golemis, Lutz WeberAbstract:A yeast Two-Hybrid Screening system was developed to screen for small molecules that inhibit the interaction of the Ras and the Raf proteins. Hyperpermeable yeast strains useful for high-throughput Screening (HTS) for the Two-Hybrid system were created. Differential inhibition of the Ras-Raf vs the hsRPB4-hsRPB7 interaction allowed the identification of selective inhibitors.
Adrian Minty - One of the best experts on this subject based on the ideXlab platform.
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covalent modification of p73α by sumo 1 two hybrid Screening with p73 identifies novel sumo 1 interacting proteins and a sumo 1 interaction motif
Journal of Biological Chemistry, 2000Co-Authors: Adrian Minty, Xavier Dumont, Mourad Kaghad, Daniel CaputAbstract:Two-Hybrid Screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the Two-Hybrid system. We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.
