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Interacting Protein (interacting + protein)
Selected AbstractsChurchill and Sip1a repress fibroblast growth factor signaling during zebrafish somitogenesisDEVELOPMENTAL DYNAMICS, Issue 2 2010Fatma O. Kok Abstract Cell-type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild-type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity. Developmental Dynamics 239:548,558, 2010. © 2009 Wiley-Liss, Inc. [source] The embryonic expression patterns and the knockdown phenotypes of zebrafish ADP-ribosylation factor-like 6 interacting protein geneDEVELOPMENTAL DYNAMICS, Issue 1 2009Hsing-Yen Huang Abstract ADP-ribosylation factor-like 6 (Arl6) mutation is linked to human disease and Arl6 interacts with Arl6 interacting protein (Arl6ip). However, the expression pattern and function of Arl6ip during embryogenesis are unknown. To confirm whether abnormal Arl6ip function might result in embryonic defects in zebrafish, we examined the expression patterns of arl6ip during embryogenesis, and they were maternally expressed and exhibited in the brain, optic primordia, hypochord, spinal cord, myotome, heart, fin-bud, kidney, trunk, and retina. Knockdown of Arl6ip revealed the following phenotypic defects: microphthalmia, disorganized pigment pattern, flat head, defective tectum, deficient pectoral fins, abnormal pneumatic duct, pericardial edema, and deformed trunk. Particularly, histological dissection of the retinae of arl6ip -morphants revealed that neuronal differentiation is severely delayed, resulting in no formation of retinal layers. We further confirmed that opsins of arl6ip -morphants were not transcribed. Based on this evidence, Arl6ip may play important roles in zebrafish ocular, heart, and fin-bud development. Developmental Dynamics 238:232,240, 2009. © 2008 Wiley-Liss, Inc. [source] Identification of BOIP, a novel cDNA highly expressed during spermatogenesis that encodes a protein interacting with the orange domain of the hairy-related transcription factor HRT1/Hey1 in Xenopus and mouseDEVELOPMENTAL DYNAMICS, Issue 4 2003Reginald Van Wayenbergh Abstract Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity. Developmental Dynamics 228:716,725, 2003. © 2003 Wiley-Liss, Inc. [source] Somatodendritic localization of EFA6A, a guanine nucleotide exchange factor for ADP-ribosylation factor 6, and its possible interaction with ,-actinin in dendritic spinesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2007Hiroyuki Sakagami Abstract EFA6A is a member of the guanine nucleotide exchange factors that can specifically activate ADP ribosylation factor 6 (ARF6). In this study, we identified ,-actinin-1 as a possible interacting protein with EFA6A by the yeast two-hybrid screening with its C-terminal region as bait. The central region of ,-actinin-1 containing a part of spectrin repeat 1 and spectrin repeats 2,3 is responsible for this interaction. In the hippocampal formation, EFA6A immunoreactivity occurred at a high level as numerous fine puncta in the strata oriens, radiatum, lacunosum-moleculare of the hippocampal CA1,3 subfields and the dentate molecular layer, whereas the immunoreactivity was faint in the neuronal cell layers and the stratum lucidum, the mossy fiber-recipient layer of the CA3 subfield. Double-immunofluorescent analyses revealed a partial overlapping of EFA6A and ,-actinin at the dendritic spines of in vivo and cultured hippocampal neurons. Our present findings suggest that EFA6A may form a protein complex with ,-actinin and activate ARF6 in close proximity of the actin cytoskeleton and membrane proteins in the dendritic spines. [source] Human Werner helicase interacting protein 1 (WRNIP1) functions as a novel modulator for DNA polymerase ,GENES TO CELLS, Issue 1 2005Toshiki Tsurimoto Human WRNIP1, a Werner DNA helicase interacting protein 1, was expressed in insect cells and E. coli. The purified protein behaved as a homo-oligomeric complex with a native molecular mass indicative of an octamer, and the complex copurified with an ATPase activity that was stimulated by double-stranded DNA ends. As suggested by genetic studies of budding yeast WRNIP1/Mgs1, the purified human WRNIP1 complex interacted physically with human DNA polymerase , (pol ,), stimulating its DNA synthesis activity more than fivefold in the presence or absence of proliferating cell nuclear antigen. Analysis of reaction products demonstrated the stimulation to be partly due to an increased processivity of pol , but more importantly to an increase in its initiation frequency. Addition of ATP to reactions partially suppressed stimulation by WRNIP1. Furthermore, a mutant WRNIP1 lacking ATPase activity could stimulate pol , normally but was insensitive to suppression by ATP. These results indicate that WRNIP1 functions as a modulator for initiation or restart events during pol ,-mediated DNA synthesis and that its ATPase activity is utilized to sense DNA ends and to regulate the extent of stimulation. [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] BCCIP associates with the receptor protein tyrosine phosphatase PTPµJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2008Polly J. Phillips-Mason Abstract The receptor protein tyrosine phosphatase PTPµ belongs to a family of adhesion molecules that contain cell,cell adhesion motifs in their extracellular segments and catalytic domains within their intracellular segments. The ability of PTPµ both to mediate adhesion and exhibit enzymatic activity makes PTPµ an excellent candidate to transduce signals in response to cell,cell adhesion. In an effort to identify downstream signaling partners of PTPµ, we performed a modified yeast two-hybrid screen using the first tyrosine phosphatase domain of PTPµ as bait. We isolated an interacting clone encoding BRCA2 and CDKN1A interacting protein (BCCIP) from a HeLa cell library. BCCIP is a p21 and BRCA2 interacting protein that has been shown to play roles in both cell cycle arrest and DNA repair. In this manuscript, we confirm the interaction between BCCIP and PTPµ identified in yeast using in vitro biochemical studies and characterize BCCIP as a PTPµ binding protein. We demonstrate that BCCIP is phosphorylated by the Src tyrosine kinase and dephosphorylated by the PTPµ tyrosine phosphatase in vitro. Furthermore, we show that BCCIP is required for both the permissive and repulsive functions of PTPµ in neurite outgrowth assays, suggesting BCCIP and PTPµ are in a common signal transduction pathway. J. Cell. Biochem. 105: 1059,1072, 2008. © 2008 Wiley-Liss, Inc. [source] Aurora-A kinase phosphorylation of Aurora-A kinase interacting protein (AIP) and stabilization of the enzyme-substrate complexJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2007Hiroshi Katayama Abstract Aurora-A is an oncogenic kinase that plays essential roles in mitosis as well as cell survival. Aurora-A interacting protein (AIP) was identified as a negative regulator of Aurora-A with its ectopic over expression inducing destabilization of Aurora-A protein. Here we present evidence that in human cells, contrary to the earlier report, AIP functions in stabilizing rather than destabilizing Aurora-A. Furthermore, AIP is phosphorylated on Serine 70 by Aurora-A but not Aurora-B and expression of phosphorylation mimic mutant of AIP results in prolonged protein stability compared to unphosphorylatable mutant. We observed that when co-expressed with AIP, protein levels of both Aurora-A and Aurora-B are markedly elevated regardless of their kinase activities and phosphorylation state of AIP. Interaction of Aurora kinases with AIP is necessary for this elevated stability. This phenomenon is commonly detected in several human cancer cell lines used in this study. Depletion of AIP by RNA interference decreased Aurora-A but not Aurora-B in two of the three cell lines analyzed, indicating that under physiological condition, AIP functions in stabilization of Aurora-A but not Aurora-B, though this regulation may be dependent on additional factors as well. Further, AIP siRNA induced cell cycle arrest at G2/M, which is consistent with anticipated loss of function of Aurora-A in these cells. Thus, our study provides the first evidence of a role for AIP in G2/M cell cycle progression by cooperatively regulating protein stabilization of its up-stream regulator, Aurora-A kinase through protein,protein interaction as well as protein phosphorylation. J. Cell. Biochem. 102: 1318,1331, 2007. © 2007 Wiley-Liss, Inc. [source] Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2007Robert J. Nadeau Abstract Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGF-dependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when co-expressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels. J. Cell. Biochem. 100: 151,160, 2007. © 2006 Wiley-Liss, Inc. [source] Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditionsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2009Lorena Perrone Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF-A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over-expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK-NF-,B signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP-induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over-expression in EC abolishes H3K9 tri-methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF-,B-binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy. J. Cell. Physiol. 221: 262,272, 2009. © 2009 Wiley-Liss, Inc [source] The zinc-finger protein ZFR is critical for Staufen 2 isoform specific nucleocytoplasmic shuttling in neuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2006George Elvira Abstract In mammalian neurons, transport and translation of mRNA to individual potentiated synapses is believed to occur via a heterogeneous population of RNA granules. To identify components of Staufen2-containing granules, we used the yeast two-hybrid system. A mouse fetal cDNA library was screened with the N-terminal fragment of Staufen2 as bait. ZFR, a three zinc finger protein, was identified as an interacting protein. Confocal microscopy showed that ZFR, although mainly nuclear, was also found in the somatodendritic compartment of primary hippocampal neurons where it localized as granule-like structures. Co-localization with Staufen2 was observed in several granules. Biochemical analyses (immunoprecipitation, cell fractionation) further confirmed the ZFR/Staufen2 association. ZFR was shown to interact with at least the Staufen262 isoform, but not with Staufen1. ZFR also co-fractionated with ribosomes and Staufen259 and Staufen252 in a sucrose gradient. Interestingly, knockdown expression of ZFR through RNA interference in neurons relocated specifically the Staufen262, but not the Staufen259, isoform to the nucleus. Our results demonstrate that ZFR is a native component of Staufen2-containing granules and likely plays its role during early steps of RNA transport and localization. They also suggest that one of these roles may be linked to Staufen262 -containing RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling. [source] Identification of a novel SNAP25 interacting protein (SIP30)JOURNAL OF NEUROCHEMISTRY, Issue 6 2002Ho-Ki Lee Abstract Soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including synaptosome-associated proteins of 25 kDa (SNAP25), syntaxins, and vesicle-associated membrane proteins (VAMP), are essential for regulated exocytosis of synaptic vesicles in neurotransmission. We identified a cDNA coding for a novel protein of 266 amino acids that we have named SIP30 (SNAP25 interacting protein of 30 kDa). SIP30 is expressed abundantly in brain and slightly in testis and kidney. In brain, SIP30 is highly expressed in the inferior and superior colliculi, which contain important relay nuclei of the auditory and visual systems. GST,pull-down and immunoprecipitation assays showed direct binding of SIP30 to SNAP25. Although SIP30 does not directly interact with syntaxin based on pull-down assays, syntaxin does co-immunoprecipitate with SIP30 suggesting that syntaxin is indirectly associated with SIP30, perhaps through SNAP25. [source] Phosphorylation-dependent dimerization and subcellular localization of islet-brain 1/c-Jun N-terminal kinase-interacting protein 1JOURNAL OF NEUROSCIENCE RESEARCH, Issue 16 2007T. Borsello Abstract Islet-brain 1 [IB1; also termed c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1] is involved in the apoptotic signaling cascade of JNK and functions as a scaffold protein. It organizes several MAP kinases and the microtubule-transport motor protein kinesin and relates to other signal-transducing molecules such as the amyloid precursor protein. Here we have identified IB1/JIP-1 using different antibodies that reacted with either a monomeric or a dimeric form of IB1/JIP-1. By immunoelectron microscopy, differences in the subcellular localization were observed. The monomeric form was found in the cytoplasmic compartment and is associated with the cytoskeleton and with membranes, whereas the dimeric form was found in addition in nuclei. After treatment of mouse brain homogenates with alkaline phosphatase, the dimeric form disappeared and the monomeric form decreased its molecular weight, suggesting that an IB1/JIP-1 dimerization is phosphorylation dependent and that IB1 exists in several phospho- forms. N-methyl-D-aspartate receptor activation induced a dephosphorylation of IB1/JIP-1 in primary cultures of cortical neurons and reduced homodimerization. In conclusion, these data suggest that IB1/JIP-1 monomers and dimers may differ in compartmental localization and thus function as a scaffold protein of the JNK signaling cascade in the cytoplasm or as a transcription factor in nuclei. © 2007 Wiley-Liss, Inc. [source] A network analysis of the single nucleotide polymorphisms in acute allergic diseasesALLERGY, Issue 1 2010J. Renkonen Abstract Background:, Genetics of acute allergies has focused on identifying single nucleotide polymorphisms (SNPs) within genes relevant in the pathogenesis. In this study, we begin a systems biology analysis of the interconnectivity and biological functions of these genes, their transcripts and their corresponding proteins. Methods:, The literature (Pubmed) was searched for SNPs within genes relevant in acute allergic diseases. The SNP-modified genes were converted to corresponding proteins and their protein,protein interactions were searched from six different databases. This interaction network was analysed with annotated vocabularies (ontologies), such as Gene Ontology, Reactome and Nature pathway interaction database. Time-series transcriptomics was performed with nasal epithelial cells obtained from allergic patients and their healthy control subjects. Results:, A total of 39 genes with SNPs related to acute allergic diseases were found from a literature search. The corresponding proteins were then hooked into a large protein,protein interaction network with the help of various databases. Twenty-five SNP-related proteins had more than one interacting protein and a network contained 95 proteins, and 182 connections could be generated. This network was 10-fold enriched with protein kinases and proteins involved in the host,virus interaction compared with background human proteome. Finally, eight of the 95 nodes on our network displayed nasal epithelial transcriptomal regulation in a time-series analysis collected from birch allergic patients during the spring pollen season. Conclusions:, Signal transduction with special reference to host,virus interactions dominated in the allergy-related protein interaction network. Systems level analysis of allergy-related mutation can provide new insights into pathogenetic mechanisms of the diseases. [source] Structural characterization of Lyn-SH3 domain in complex with a herpesviral protein reveals an extended recognition motif that enhances binding affinityPROTEIN SCIENCE, Issue 10 2005Finn Bauer Abstract The Src homology 3 (SH3) domain of the Src family kinase Lyn binds to the herpesviral tyrosine kinase interacting protein (Tip) more than one order of magnitude stronger than other closely related members of the Src family. In order to identify the molecular basis for high-affinity binding, the structure of free and Tip-bound Lyn-SH3 was determined by NMR spectroscopy. Tip forms additional contacts outside its classical proline-rich recognition motif and, in particular, a strictly conserved leucine (L186) of the C-terminally adjacent sequence stretch packs into a hydrophobic pocket on the Lyn surface. Although the existence of this pocket is no unique property of Lyn-SH3, Lyn is the only Src family kinase that contains an additional aromatic residue (H41) in the n-Src loop as part of this pocket. H41 covers L186 of Tip by forming tight hydrophobic contacts, and model calculations suggest that the increase in binding affinity compared with other SH3 domains can mainly be attributed to these additional interactions. These findings indicate that this pocket can mediate specificity even between otherwise closely related SH3 domains. [source] Effect of Polymorphisms in Four Candidate Genes for Fertility on Litter Size in a German Pig LineREPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2010A Spötter Contents We carried out an SNP discovery project in pigs for candidate genes playing potentially important roles in embryonic development. Using eight pigs one each from eight breeds (Meishan, Mangalitza, Duroc, Pietrain, German Landrace, Hampshire, Husum Red Pied, German Large White), 36 SNPs were identified in intronic sequences of 21 porcine candidate genes based on sequencing of PCR products. The primer pairs were designed using porcine EST sequences allowing amplification of introns. These SNPs were tested for their association with the number of piglets born alive in German Large White sows using a discordant approach. Significant effects (p < 0.001 and p < 0.05, respectively) of intronic SNPs on litter size were found for four genes: mitogen-activated protein kinase kinase kinase 3 (MAP3K3), vascular endothelial growth factor receptor (KDR), erbb2 interacting protein (ERBB2IP) and peroxisome proliferator-activated receptor delta (PPARD). These SNPs can be further tested in upcoming association studies for their influence on litter size in different breeds using larger sample sizes. [source] The Arabidopsis ATRIP ortholog is required for a programmed response to replication inhibitorsTHE PLANT JOURNAL, Issue 3 2009Paul R. Sweeney Summary The programmed response to replication inhibitors in eukaryotic cells requires the protein kinase ATR (ataxia telangiectasia mutated and rad3-related), which is activated primarily through the persistence of replication protein A (RPA)-bound single-stranded DNA at stalled replication forks and sites of DNA damage undergoing excision repair. Once activated, ATR initiates a cascade of events, including cell-cycle arrest and induction of DNA repair, to mitigate the mutagenic effects of DNA replication in the presence of damage and/or blockage. While many of the molecular regulators of ATR have been determined in yeast and animal cells, little is known about ATR regulation in plants. To genetically define ATR regulatory pathways in Arabidopsis, we describe here a genetic screen for identifying mutants that display a characteristic phenotype of Arabidopsis atr null mutants , hypersensitivity to the replication blocking agent hydroxyurea (HU). Employing this screen, we isolated a novel mutant, termed hus2 (hydroxyurea-sensitive), that displays hypersensitivity to HU, aphidicolin and ionizing radiation, similar to atr mutants. In addition, cell-cycle progression in response to replication blocks and ionizing radiation is defective in hus2, displaying a nearly identical phenotype to atr mutants. Positional cloning of hus2 reveals a gene sequence similar to yeast Rad26/Ddc2 and ATRIP (ATR interacting protein), suggesting that hus2 encodes an Arabidopsis ATRIP ortholog. [source] A mutation in NF,B interacting protein 1 causes cardiomyopathy and woolly haircoat syndrome of Poll Hereford cattleANIMAL GENETICS, Issue 1 2009M. A. Simpson Summary Cardiomyopathy and woolly haircoat syndrome (CWH) of Poll Hereford cattle is a lethal, autosomal recessive disorder. Cardiac and haircoat changes are congenital, neonatal ocular keratitis develops in some cases and death usually occurs within the first 12 weeks of life. We undertook a homozygosity mapping approach to identify the chromosomal location of the causative gene. Seven candidate genes were examined for homozygosity in affected animals: desmoplakin and junction plakoglobin (both previously implicated in human cardiocutaneous syndromes), desmocollin 2, desmoglein 2, plakophilin 2, nuclear factor kappa B (NFKB1) and NF,B interacting protein 1 (PPP1R13L, also known as NKIP1). Homozygosity in 13 affected animals was observed at the PPP1R13L locus, located on bovine chromosome 18. Subsequent sequence analysis revealed a 7-bp duplication (c.956_962dup7) in exon 6 of this 13-exon gene. This frameshift variant is predicted to result in the substitution of three amino acids and the introduction of a premature stop codon at position 325 of the protein product (p.Ser322GlnfsX4). PPP1R13L interacts with NF,B, a family of structurally related transcription factors that regulate genes controlling inflammation, immune responses and cell proliferation and survival. CWH represents a large-animal model for cardiocutaneous disorders caused by a mutation in the PPP1R13L gene. The identification of this bovine mutation also indicates that PPP1R13L and other genes affecting NF,B activity may be candidate genes in the study of human cardiovascular disease. [source] Crystallization and preliminary X-ray crystallographic studies of the sixth PDZ domain of glutamate-receptor interacting protein 1 (GRIP1) from Rattus norvegicusACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002Seong Ho Park The sixth PDZ domain from GRIP1 and its complex with the octapeptide of the liprin-,1 C-terminus were crystallized at 294,K by the hanging-drop vapour-diffusion method. The native crystal belongs to space group P6122 (or P6522), with unit-cell parameters a = b = 40.3, c = 222.9,Å. The complex crystal belongs to space group R32, with unit-cell parameters a = b = 117.8, c = 102.0,Å. Native and peptide-complex diffraction data were collected to resolutions of 1.5 and 1.8,Å, respectively, using synchrotron X-rays. [source] Structure of full-length ubiquitin-conjugating enzyme E2-25K (huntingtin-interacting protein 2)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Randall C. Wilson The ubiquitin-conjugating enzyme E2-25K has been identified as a huntingtin (the key protein in Huntington's disease) interacting protein and has been shown to play a role in mediating the toxicity of A,, the principal protein involved in Alzheimer's disease pathogenesis. E2-25K is a dual-domain protein with an ubiquitin-associated (UBA) domain as well as a conserved ubiquitin-conjugating (UBC) domain which catalyzes the formation of a covalent bond between the C-terminal glycine of an ubiquitin molecule and the ,-amine of a lysine residue on the acceptor protein as part of the ubiquitin-proteasome pathway. The crystal structures of E2-25K M172A mutant protein at pH 6.5 and pH 8.5 were determined to 1.9 and 2.2,Å resolution, respectively. Examination of the structures revealed domain,domain interactions between the UBC and UBA domains which have not previously been reported. [source] Human GCIP interacts with CT847, a novel Chlamydia trachomatis type III secretion substrate, and is degraded in a tissue-culture infection modelCELLULAR MICROBIOLOGY, Issue 10 2007Blandine Chellas-Géry Summary The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole and employs a type III secretion mechanism to translocate host-interactive proteins. These proteins most likely contribute to pathogenesis through modulation of host cell mechanisms crucial for the establishment and maintenance of a permissive intracellular environment. Using a surrogate Yersinia type III secretion system (T3SS), we have identified the conserved gene product CT847 as a chlamydial T3SS substrate. Yeast two-hybrid studies using CT847 as bait to screen a HeLa cell cDNA library identified an interaction with mammalian Grap2 cyclin D- interacting protein (GCIP). Immunoblot analyses of C. trachomatis -infected HeLa cells showed that GCIP levels begin to decrease (as compared with mock-infected HeLa cells) between 8 h and 12 h post infection. GCIP was virtually undetectable in 24 h time point material. This decrease was inhibited by proteasome inhibitors lactacystin and MG-132, and the T3SS inhibitor Compound 1. CT847 was detectible in purified reticulate body but not elementary body lysates, and reverse transcription polymerase chain reaction (RT-PCR) expression analyses indicate a mid-cycle expression pattern. Both of these findings are consistent with CT847 contributing to the observed effect on GCIP. Given the established roles of GCIP, we believe that we have discovered a novel C. trachomatis antihost protein whose activity is relevant to chlamydial pathogenesis. [source] Ki-1/57 interacts with PRMT1 and is a substrate for arginine methylationFEBS JOURNAL, Issue 17 2006Dario O. Passos The human 57 kDa Ki-1 antigen (Ki-1/57) is a cytoplasmic and nuclear protein, associated with Ser/Thr protein kinase activity, and phosphorylated at the serine and threonine residues upon cellular activation. We have shown that Ki-1/57 interacts with chromo-helicase DNA-binding domain protein 3 and with the adaptor/signaling protein receptor of activated kinase 1 in the nucleus. Among the identified proteins that interacted with Ki-1/57 in a yeast two-hybrid system was the protein arginine-methyltransferase-1 (PRMT1). Most interestingly, when PRMT1 was used as bait in a yeast two-hybrid system we were able to identify Ki-1/57 as prey among 14 other interacting proteins, the majority of which are involved in RNA metabolism or in the regulation of transcription. We found that Ki-1/57 and its putative paralog CGI-55 have two conserved Gly/Arg-rich motif clusters (RGG/RXR box, where X is any amino acid) that may be substrates for arginine-methylation by PRMT1. We observed that all Ki-1/57 protein fragments containing RGG/RXR box clusters interact with PRMT1 and are targets for methylation in vitro. Furthermore, we found that Ki-1/57 is a target for methylation in vivo. Using immunofluorescence experiments we observed that treatment of HeLa cells with an inhibitor of methylation, adenosine-2,,3,-dialdehyde (Adox), led to a reduction in the cytoplasmic immunostaining of Ki-1/57, whereas its paralog CGI-55 was partially redistributed from the nucleus to the cytoplasm upon Adox treatment. In summary, our data show that the yeast two-hybrid assay is an effective system for identifying novel PRMT arginine-methylation substrates and may be successfully applied to other members of the growing family of PRMTs. [source] Novel brain 14-3-3 interacting proteins involved in neurodegenerative diseaseFEBS JOURNAL, Issue 16 2005Shaun Mackie We isolated two novel 14-3-3 binding proteins using 14-3-3 , as bait in a yeast two-hybrid screen of a human brain cDNA library. One of these encoded the C-terminus of a neural specific armadillo-repeat protein, ,-catenin (neural plakophilin-related arm-repeat protein or neurojungin). ,-Catenin from brain lysates was retained on a 14-3-3 affinity column. Mutation of serine 1072 in the human protein and serine 1094 in the equivalent site in the mouse homologue (in a consensus binding motif for 14-3-3) abolished 14-3-3 binding to ,-catenin in vitro and in transfected cells. ,-catenin binds to presenilin-1, encoded by the gene most commonly mutated in familial Alzheimer's disease. The other clone was identified as the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). Human IRSp53 interacts with the gene product implicated in dentatorubral-pallidoluysian atrophy, an autosomal recessive disorder associated with glutamine repeat expansion of atrophin-1. [source] Uncoupling proteins 2 and 3 interact with members of the 14.3.3 familyFEBS JOURNAL, Issue 9 2000Benoit Pierrat Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms ,, ,, , were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the , isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 , could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria. [source] VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like mannerGENES TO CELLS, Issue 7 2003Tomoyuki Fukuda Background: Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus. Results: We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5, clb6, mutation reduces VDE-mediated DSBs. Conclusion: The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism. [source] Isolation and expression of a novel mitochondrial septin that interacts with CRMP/CRAM in the developing neuronesGENES TO CELLS, Issue 2 2003Shusuke Takahashi Background: Collapsin response mediator proteins (CRMPs) and CRAM belong to the unc-33 gene family which is implicated in axon guidance and outgrowth during neural development. However, their exact roles remain largely unknown. To understand the molecular basis of CRMP/CRAM function, we have undertaken to identify CRMP/CRAM interacting proteins. Results: We have identified a novel mitochondrial septin (M-septin) as one of the CRMP/CRAM interacting proteins from the developing rat brain. M-septin is a major, alternatively spliced variant of the H5 gene in developing mouse brain and its expression is up-regulated during the neuronal differentiation of embryonal carcinoma P19 cells. In COS-7 cells, M-septin is specifically localized to mitochondria whereas H5 is diffusely distributed to the perinuclear cytoplasm and plasma membranes. In contrast to H5, M-septin induces the mitochondrial translocation of CRAM but not CRMP2. Finally, M-Septin is found to be transiently translocated to mitochondria before the induction of the neurites and then dissociates from the mitochondria after neurite extension in P19 cells. Conclusions: Our results suggest that M-septin has a role which is distinct from H5, and together with CRMP/CRAM, may play an important role in the neuronal differentiation and axon guidance through the control of mitochondrial function. [source] Modulation of astrocyte P2Y1 receptors by the carboxyl terminal domain of the gap junction protein Cx43GLIA, Issue 2 2008Eliana Scemes Abstract Gap junction proteins, connexins, provide intercellular channels that allow ions and small signaling molecules to be transmitted to adjacent coupled cells. Besides this function, it is becoming apparent that connexins also exert channel-independent effects, which are likely mediated by processes involving protein,protein interactions. Although a number of connexin interacting proteins have been identified, only little is known about the functional consequences of such interactions. We have previously shown that deletion of the astrocytic gap junction protein, connexin43 (Cx43) causes a right-ward shift in the dose-response curve to P2Y1R agonists and decreased P2Y1R expression levels. To evaluate whether these changes were due to reduced gap junctional communication or to protein,protein interactions, Cx43-null astrocytes were transfected with full-length Cx43 and Cx43 domains, and P2Y1R function and expression levels evaluated. Results indicate that restoration of P2Y1R function is independent of gap junctional communication and that the Cx43 carboxyl terminus spanning the SH3 binding domain (260,280) participates in the rescue of P2Y1R pharmacological behavior (shifting to the left the P2Y1R dose-response curve) without affecting its expression levels. These results suggest that the Cx43 carboxyl-terminus domain provides a binding site for an intracellular molecule, most likely a member of the c-Src tyrosine kinase family, which affects P2Y1R-induced calcium mobilization. It is here proposed that a nonchannel function of Cx43 is to serve as a decoy for such kinases. Such modulation of P2Y1R is expected to influence several neural cell functions, especially under inflammation and neurodegenerative disorders where expression levels of Cx43 are decreased. © 2007 Wiley-Liss, Inc. [source] Orthogonal Chemical Genetic Approaches for Unraveling Signaling PathwaysIUBMB LIFE, Issue 6 2005Kavita Shah Abstract While chemical genetic approach uses small molecules to probe protein functions in cells or organisms, orthogonal chemical genetics refers to strategies that utilize reengineered protein-small molecule interfaces, to alter specificities, in order to probe their functions. The advantage of orthogonal chemical genetics is that the changes at the interfaces are generally so minute that it goes undetected by natural processes, and thus depicts a true physiological picture of biological phenomenon. This review highlights the recent advances in the area of orthogonal chemical genetics, especially those designed to probe signaling processes. Dynamic protein-protein and enzyme-substrate interactions following stimuli form the foundation of signal transduction. These processes not only break spatial and temporal boundaries between interacting proteins, but also impart distinct regulatory properties by creating functional diversity at the interfaces. Functional and temporal modulation of these dynamic interactions by specific chemical probes provides extremely powerful tools to initiate, ablate, decouple and deconvolute different components of a signaling pathway at multiple stages. Not surprisingly, multiple receptor-ligand reengineering approaches have been developed in the last decade to selectively manipulate these transient interactions with the aim of unraveling signaling events. However, given the diversity of protein-protein interactions and novel chemical genetic probes developed to perturb these processes, a short review cannot do adequate justice to all aspects of signaling. For this reason, this review focuses on some orthogonal chemical-genetic strategies that are developed to study signaling processes involving enzyme-substrate interactions. IUBMB Life, 57: 397-405, 2005 [source] Syntaxin 16: Unraveling cellular physiology through a ubiquitous SNARE moleculeJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Yanan Chen Syntaxin 16 (Syx16) is member of the soluble N -ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of molecules that functions in membrane fusion in eukaryotic cells. A rather ubiquitously expressed, tail-anchored membrane protein localized mainly at the trans-Golgi network (TGN), it mediates primarily retrograde endosomal-TGN transport. In spite of its ubiquitous expression, Syx16 has specific and interesting roles in the physiology of specialized cells, including Glut4 dynamics, dendritic outgrowth-related membrane traffic, and cytokinesis. We discussed these physiological functions of Syx16 in the light of what is known of its subcellular localization, vesicular trafficking pathways involved, cognate SNARE partners and other interacting proteins. Further, we speculate on some possible pathophysiological roles of Syx16. J. Cell. Physiol. 225: 326,332, 2010. © 2010 Wiley-Liss, Inc. [source] Emerging functions of p21-activated kinases in human cancer cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002Rakesh Kumar The p21 activated kinases (Paks), an evolutionarily conserved family of serine/threonine kinases, are important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, and angiogenesis. Paks are widely expressed in numerous tissues and are activated by growth factors and extracellular signals through GTPase-dependent and -independent mechanisms. Overexpression of Paks in epithelial cancer cells has been shown to increase migration potential, increase anchorage independent growth, and cause abnormalities in mitosis. Dysregulation of Paks has been reported in several human tumors and neurodegenerative diseases. A growing list of novel Pak interacting proteins has opened up exciting avenues of investigation by which to understand the functions of Paks in tumorigenesis. In this review, we will summarize the current knowledge of the Paks family with respect to emerging cellular functions and possible contributions to cancer. © 2002 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||