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Two-hybrid Screening (two-hybrid + screening)
Kinds of Two-hybrid Screening Selected AbstractsIdentification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2007Masamichi Doiguchi Abstract Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insultsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2003Takako Niikura Abstract Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S -transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways. [source] Host factor Ebp1: Selective inhibitor of influenza virus transcriptaseGENES TO CELLS, Issue 2 2007Ayae Honda Influenza virus RNA polymerase is composed of three virus-coded proteins, and is involved in both transcription and replication of the negative-strand genome RNA. Subunit PB1 plays key roles in both the RNA polymerase assembly and the catalytic function of RNA polymerization. Using yeast two-hybrid screening, a HeLa cell protein with the molecular mass of 45 kDa was identified. After cloning and sequencing, this protein was identified to be Ebp1, ErbB3-binding protein. Epb1 specifically interacts with PB1 both in vitro and in vivo, and Epb1 contact site on PB1 was mapped at its binding site of transcription primers. Ebp1 was found to interfere with in vitro RNA synthesis by influenza virus RNA polymerase (3P complex), but no inhibition was observed for capped RNA endonuclease and RNA-cap binding, the intrinsic activities of RNA polymerase. Since inhibition was not observed against other nucleic acid polymerases tested, we propose that Ebp1 is a selective inhibitor of influenza viral RNA polymerase. Accordingly over-expression of Ebp1 interfered with virus production. The PB1-contact site on Ebp1 overlaps with the interaction site with ErbB3 (epidermal receptor tyrosine kinase), androgen receptor (AR) and retinoblastoma gene product (Rb), which are involved in controlling cell proliferation and differentiation. [source] Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activityGENES TO CELLS, Issue 9 2006Takashi Itoh It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase-activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab-GAP activity. In this study we established panels of the GTP-fixed form of 60 different Rabs constructed in pGAD-C1, a yeast two-hybrid bait vector. We also constructed a yeast two-hybrid prey vector (pGBDU-C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP-fixed Rabs and 40 TBC proteins by yeast two-hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5-like with Rab10). Measurement of in vitro Rab-GAP activity revealed that OATL1 and Evi5-like actually possess significant Rab2A- and Rab10-GAP activity, respectively, but that FLJ12085 do not display Rab5A-GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab-GAP domains, but that there is little correlation between the Rab-binding activity and Rab-GAP activity of other TBC proteins. [source] BIP, a BRAM-interacting protein involved in TGF-, signalling, regulates body length in Caenorhabditis elegansGENES TO CELLS, Issue 7 2001Katsura Sugawara Background The TGF-, superfamily has diverse biological activities and is involved in the early development of animals. We previously identified a novel family member, BMP receptor associated molecule (BRAM), which binds to the intracellular domain of BMP type IA receptor and is involved in the BMP signalling pathway. Results To identify novel molecules involved in TGF-, signalling pathways, we performed yeast two-hybrid screening using BRAM as bait. From a Xenopus cDNA library, we cloned a cDNA encoding 693 amino acids and containing the motif for an oxysterol binding protein (OSBP), which we designated BRAM interacting protein (BIP). We then isolated a BIP homologue from the Caenorhabditis elegans that encodes 733 amino acids and also contains the OSBP-like motif. Immunoprecipitation and Western blotting studies revealed that C. elegans BIP could interact with the C. elegans BRAM homologues BRA-1 and BRA-2. C. elegans BIP was expressed in pharyngeal muscle, hypodermis and several neuronal cells, an expression pattern overlaps with those of BRA-1 and BRA-2. Finally, we found that inhibition of BIP expression in C. elegans by double stranded RNA interference produces a Sma phenotype. Conclusions BIP was isolated using the yeast two-hybrid systems. BIP may function in the TGF-, pathway and regulate body length in C. elegans. [source] Germline mutations of the BRCA1-associated ring domain (BARD1) gene in breast and breast/ovarian families negative for BRCA1 and BRCA2 alterationsGENES, CHROMOSOMES AND CANCER, Issue 3 2002Chiara Ghimenti BARD1 (BRCA1-associated RING domain) was identified by yeast two-hybrid screening as a protein interacting with BRCA1. Somatic and germline mutations of BARD1 have been detected in sporadic breast, ovarian, and endometrial cancers. The present study represents the first description of BARD1 germline mutations in hereditary breast and breast/ovarian cancer patients. We analyzed the BARD1 gene in 40 families with hereditary breast and breast/ovarian cancer, tested negative for BRCA1 and BRCA2 mutations. A mutational analysis by PCR-SSCP on the coding region and the exon,intron splice boundaries of the BARD1 gene yielded four different germline mutations. A group of 20 patients diagnosed with sporadic breast cancer below the age of 40 was also examined and only one germline mutation was found. A study of loss of heterozygosity at the BARD1 locus in neoplastic tissues from patients with BARD1 germline mutations was carried out. In all cases, we were unable to find any evidence for allelic deletions. The involvement of BARD1 mutations in the susceptibility to hereditary breast and breast/ovarian cancer is discussed. [source] The moleskin gene product is essential for Caudal-mediated constitutive antifungal Drosomycin gene expression in Drosophila epitheliaINSECT MOLECULAR BIOLOGY, Issue 3 2004S.-H. Han Abstract The homeobox gene, Caudal, encodes the DNA-binding nuclear transcription factor that plays a crucial role during development and innate immune response. The Drosophila homologue of importin-7 (DIM-7), encoded by moleskin, was identified as a Caudal-interacting molecule during yeast two-hybrid screening. Both mutation of the minimal region of Caudal responsible for moleskin binding and RNA interference (RNAi) of moleskin dramatically inhibited the Caudal nuclear localization. Furthermore, Caudal-mediated constitutive expression of antifungal Drosomycin gene was severely affected in the moleskin- RNAi flies, showing a local Drosomycin expression pattern indistinguishable from that of the Caudal- RNAi flies. These in vivo data suggest that DIM-7 mediates Caudal nuclear localization, which is important for the proper Caudal function necessary for regulating innate immune genes in Drosophila. [source] ,4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)-binding proteinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010William J. McDonald Abstract Mammalian ,4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. ,4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using ,4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of ,4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with ,4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between ,4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. ,4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD,,4 immunocomplex. Furthermore, deletion mutants of ,4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of ,4, independent of the ,4-PP2Ac binding site. Therefore, in addition to PP2Ac, ,4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression. J. Cell. Biochem. 110: 1123,1129, 2010. Published 2010 Wiley-Liss, Inc. [source] Petunia Germinating Pollen S/D3 Interacts with S-RNases in Petunia hybrida Vilm.JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 5 2006Yan-Xia Guo Abstract Self-incompatibility (SI) is a genetic mechanism of self/non-self pollen recognition to prevent self-fertilization in many flowering plants and, in most cases, this is controlled by a multi-allelic S-locus. S-RNase and S-locus F box (SLF) proteins have been shown to be the female and male determinants of gametophytic self-incompatibility (GSI), respectively, in the Solanaceae, Scrophulariaceae and Rosaceae. Nevertheless, it is thought that additional factors are required for the SI response. Herein, we constructed a mature anther cDNA library from a self-incompatible Petunia hybrida Vilm. line of the S3S3 haplotype. Using AhS2-RNase from Antirrhinum hispanicum as a bait for yeast two-hybrid screening, we found that petunia germinating pollen (PGP) S/D3 was capable of interacting physically with the bait. However, the interaction lacked haplotype specificity. The PGPS/D3 gene is a single copy gene that is expressed in tissues such as the style, ovary, pollen, and leaf. The PGPS/D3::GFP (green fluorescence protein) construct was detected in both the membrane and cytoplasm. The implications of these findings in the operation of S-RNase-based SI are discussed. (Managing editor: Li-Hui Zhao) [source] Cytoskeleton-associated, carbohydrate-metabolizing enzymes in maize identified by yeast two-hybrid screeningPHYSIOLOGIA PLANTARUM, Issue 2 2005Daniela Holtgräwe We have used yeast two-hybrid screens and biochemical methods to identify glycolytic enzymes that interact with subcellular structures in hypoxic maize seedlings. As binding domain-bait fusion constructs, we have cloned actin, cytosolic aldolase, the three sucrose synthase (SUS) isoforms SUS1, SUS3, and SH1 as well as the SNF1-related protein kinase into yeast and identified cytosolic isoforms of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase, tubulin, and mitochondrial porin voltage-dependent anion channel protein (VDAC) as well as protein kinases and proteins involved in ubiquitinylation and proteasome-linked degradation as interacting activation domain-prey clones. The results were further confirmed using overlay blots (VDAC) as well as co-polymerization and co-precipitation assays (tubulin and actin). Some results were obtained that support the idea of metabolite and modification effects on the association, namely guanosine triphosphate (GTP)/MgCl2 was necessary for the binding of enolase to actin. GAPDH is inactivated upon association with tubulin but then serves to stabilize the microtubules. The findings support the idea of the dynamic formation of locally associated complexes of enzymes involved in sucrose breakdown and glycolysis in plant cells depending on their metabolic state. [source] Interaction of the plant glycine-rich RNA-binding protein MA16 with a novel nucleolar DEAD box RNA helicase protein from Zea maysTHE PLANT JOURNAL, Issue 6 2004Elisenda Gendra Summary The maize RNA-binding MA16 protein is a developmentally and environmentally regulated nucleolar protein that interacts with RNAs through complex association with several proteins. By using yeast two-hybrid screening, we identified a DEAD box RNA helicase protein from Zea mays that interacted with MA16, which we named Z. maysDEAD box RNA helicase 1 (ZmDRH1). The sequence of ZmDRH1 includes the eight RNA helicase motifs and two glycine-rich regions with arginine,glycine-rich (RGG) boxes at the amino (N)- and carboxy (C)-termini of the protein. Both MA16 and ZmDRH1 were located in the nucleus and nucleolus, and analysis of the sequence determinants for their cellular localization revealed that the region containing the RGG motifs in both proteins was necessary for nuclear/nucleolar localization The two domains of MA16, the RNA recognition motif (RRM) and the RGG, were tested for molecular interaction with ZmDRH1. MA16 specifically interacted with ZmDRH1 through the RRM domain. A number of plant proteins and vertebrate p68/p72 RNA helicases showed evolutionary proximity to ZmDRH1. In addition, like p68, ZmDRH1 was able to interact with fibrillarin. Our data suggest that MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA (rRNA) metabolism. [source] TUSC4/NPRL2, a novel PDK1-interacting protein, inhibits PDK1 tyrosine phosphorylation and its downstream signalingCANCER SCIENCE, Issue 9 2008Atsuo Kurata 3-Phosphoinositide,dependent protein kinase-1 (PDK1) is a key regulator of cell proliferation and survival signal transduction. PDK1 is known to be constitutively active and is further activated by Src-mediated phosphorylation at the tyrosine-9, -373, and -376 residues. To identify novel regulators of PDK1, we performed E. coli -based two-hybrid screening and revealed that tumor suppressor candidate 4 (TUSC4), also known as nitrogen permease regulator-like 2 (NPRL2), formed a complex with PDK1 and suppressed Src-dependent tyrosine phosphorylation and activation of PDK1 in vitro and in cells. The NH2 -terminal 133 amino acid residues of TUSC4 were involved in binding to PDK1. The deletion mutant of TUSC4 that lacked the NH2 -terminal domain showed no inhibitory effects on PDK1 tyrosine phosphorylation or activation. Thus, complex formation is indispensable for TUSC4-mediated PDK1 inactivation. The siRNA-mediated down-regulation of TUSC4 induced cell proliferation, while ectopic TUSC4 expression inactivated the PDK1 downstream signaling pathway, including Akt and p70 ribosomal protein S6 kinase, and increased cancer cell sensitivity to several anticancer drugs. Our results suggest that TUSC4/NPRL2, a novel PDK1-interacting protein, plays a role in regulating the Src/PDK1 signaling pathway and cell sensitivity to multiple cancer chemotherapeutic drugs. (Cancer Sci 2008; 99: 1827,1834) [source] | |||||||||||||||||||||||||