Identification and characterization of the human protein kinase-like gene NTKL: mitosis-specific centrosomal localization of an alternatively spliced isoform

Although the centrosome has an essential role in mitosis, its molecular components have not been fully elucidated. Here, we describe the molecular cloning and characterization of the human gene NTKL, which encodes an evolutionarily conserved kinase-like protein. NTKL mRNA is found ubiquitously in human tissues. NTKL is located on 11q13 and is mapped around chromosomal breakpoints found in several carcinomas, suggesting that NTKL dysfunction may be involved in carcinogenesis. Alternative splicing generates two variant forms of NTKL mRNA that encode protein isoforms with internal deletions. When fused to green fluorescent protein, the full-length product and one of the variant proteins are found in cytoplasm. The other variant product also exists in the cytoplasm during interphase, but is found in the centrosomes during mitosis. Endogenous NTKL protein is also localized to the centrosomes during mitosis. This cell-cycle-dependent centrosomal localization suggests that NTKL is involved in centrosome-related cellular functions.     

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two‐hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation using the two‐hybrid system, and by co‐immunoprecipitation with anti‐PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co‐immunoprecipitation suggested that RACK1‐PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over‐expression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by ³H‐diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.     

Recombinant Gaussia luciferase with a reactive cysteine residue for chemical conjugation: expression, purification and its application for bioluminescent immunoassays

The vast majority of physiological processes in living cells are mediated by protein–protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80–100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase l, β-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.     

Bacterial beta-lactamase fragmentation complementation strategy can be used as a method for identifying interacting protein pairs

We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.     

In vivo interaction between the human dehydrodolichyl diphosphate synthase and the Niemann-Pick C2 protein revealed by a yeast two-hybrid system

Dehydrodolichyl diphosphate (DedolPP) synthase catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize DedolPP, a biosynthetic precursor for dolichol which plays an important role as a sugar-carrier lipid in the biosynthesis of glycoprotein in eukaryotic cells. During certain pathological processes like Alzheimer's disease or some neurological disorders, dolichol has been shown to accumulate in human brain. In order to understand the regulatory mechanism of dolichol in eukaryotes, we performed a yeast two-hybrid screen using full length human DedolPP synthase gene [Endo et al. BBA 1625 (2003) 291] as a bait to find some proteins specifically interacting with the enzyme. We identified Niemann-Pick Type C2 protein (NPC2) to show a specific interaction with human DedolPP synthase. This interaction was further confirmed by in vitro co-immunoprecipitation experiment, indicating the possible physiological interaction between NPC2 and DedolPP synthase proteins in human.     

Dyslexia-Associated Kiaa0319-Like Protein Interacts with Axon Guidance Receptor Nogo Receptor 1

To identify the putative interacting partners for Kiaa0319-like protein. KIAA0319-like, located near the dyslexia susceptibility locus, DYX8 in chromosome 1p34.3, has been suggested as a positional candidate for developmental dyslexia due to its homology with another gene, KIAA0319 which has been strongly established as a candidate gene for developmental dyslexia. Previous research has shown that a single marker, rs7523017 (P = 0.042) has been associated with developmental dyslexia by a Canadian group. There is little functional information about this gene and protein. In this article, we put forward further evidence that support Kiaa0319-like is a candidate for this disorder. A yeast-2-hybrid screen and co-immunopreciptiation assays were performed to find protein interacting partners of KIAA0319L. A human cortex immunohistochemistry assay was performed to show the colocalization of Kiaa0319-like and its specific interacting partner in cells. Nogo Receptor 1 (NgR1), an axon guidance receptor, was identified to have physical interactions with Kiaa0319-like protein. These two proteins interact predominantly in the cytoplasmic granules of cortical neurons in the human brain cortex. Based on this data, it can be concluded that Kiaa0319-like protein interacts with Nogo Receptor 1, supporting the idea that Kiaa0319-like protein participates in axon guidance.     

Functional analysis of a novel gene, DD3-3, from Dictyostelium discoideum

Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG.     

棉花锌指蛋白GhZFP1相互作用蛋白的酵母双杂交筛选

GhZFP1蛋白是从盐胁迫棉花幼苗cDNA文库中分离的一种CCCH型锌指蛋白.初步的生物学功能研究表明,过量表达该基因的转基因烟草耐盐性和抗病性显著提高.为深入研究GhZFP1蛋白的作用机制,构建pGBKT7-m1诱饵表达载体,利用酵母双杂交系统从盐胁迫诱导棉花cDNA文库中筛选与其相互作用的蛋白.通过阳性克隆的表型确定、PCR和限制性内切酶检测以及测序和生物信息学分析,获得9个与诱饵蛋白相互作用的靶蛋白.双分子荧光互补实验证明,GhZFP1与GZIRD19A确实存在互作关系.通过分析这些靶蛋白的已知功能,为研究GhZFP1锌指蛋白的未知生物学功能提供重要信息.     

原癌基因LMO3相互作用蛋白的筛选及鉴定

目的 通过筛选LMO3的相互作用蛋白,进一步了解LMO3的作用及可能机制。方法酵母双杂交方法筛选LMO3相瓦作用蛋白,并通过酵母结合试验、免疫共沉淀及荧光共定位等进行验证。结果在初步获得相互作用蛋白：钙-整合素结合蛋白（Calcium—and integrin—binding protein,CIB）的基础上,在酵母中证实了LMO3与CIB的相互作用,并通过酵母结合试验确定了CIB与LMO3的相互作用位点,发现CIB可与LMO3的第一个LIM结构域（LIMI）及全长LMO3结合,免疫共沉淀试验确证了它们可以在真核细胞内结合,荧光共定位表明与CIB的相互作用可使LMO3在C8细胞中的定位由细胞核移到细胞质。结论LMO3可以与CIB在真核细胞中发生相互作用,提示LMO3可能通过与CIB的相互作用参与细胞相关功能的调节。     

Identification of a novel protein interacting with laforin,the EPM2a progressive myoclonus epilepsy gene product

We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy. The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs. Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated. The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed. The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3' untranslated regions, maps to human chromosome 3p22.1. The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs. Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found. The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.     

The physical and functional interaction of NDRG2 with MSP58 in cells

NDRG2, a member of N-Myc downstream regulated gene family, exerts the important functions in cell differentiation and tumor suppression. Although the ectopic expressed Ndrg2 inhibits the proliferation of tumor cells, its intracellular signal transduction pathway is hardly known. Here, we identified MSP58, a 58-kDa microspherule protein, as an interacting partner of human Ndrg2 by using yeast two-hybrid screening. The interaction was confirmed by glutathione S-transferase pull-down assay in vitro and by co-immune-precipitation assay in vivo. The forkhead associated domain of MSP58 is essential for its interaction with Ndrg2. Ndrg2 could co-localize with MSP58 in nuclear of HeLa cell during cell stress. Furthermore, the modulation of Ndrg2 level influences the cell cycle process together with MSP58. In conclusion, the findings offered a novel insight into the physiological roles of Ndrg2.     

The Nucleolar PICT-1/GLTSCR2 Protein Forms Homo-Oligomers

The human “protein interacting with carboxyl terminus 1” (PICT-1), also designated as the “glioma tumor suppressor candidate region 2 gene product”, GLTSCR2, is a nucleolar protein whose activity is, as yet, unknown. Contradictory results regarding the role of PICT-1 in cancer have been reported, and PICT-1 has been suggested to function either as a tumor suppressor protein or as an oncogene. In this study, we demonstrate self-association of PICT-1. Through yeast two-hybrid assay, we identified PICT-1 as its own interaction partner. We confirmed the interaction of PICT-1 with itself by direct yeast two-hybrid assay and also showed self-association of PICT-1 in mammalian cells by co-immunoprecipitation and fluorescence resonance energy transfer assays. Furthermore, we confirmed direct self-association of PICT-1 by using in vitro microfluidic affinity binding assays. The later assay also identified the carboxy-terminal domain as mediating self-interaction of PICT-1. Glutaraldehyde cross-linking and gel-filtration assays suggest that PICT-1 forms dimers, though it may form higher-order complexes as well. Our findings add another layer of complexity in understanding the different functions of PICT-1 and may help provide insights regarding the activities of this protein.     

酵母双杂交筛选与蛋白激酶CK2α′相互作用蛋白——泛素-52氨基酸融合蛋白的鉴定

蛋白激酶CK2是一种真核细胞中普遍存在的信使非依赖性丝/苏氨酸蛋白激酶. 为研究CK2α′亚基在精子发生中的作用机制,将构建于pACT2质粒的人睾丸cDNA文库和人蛋白激酶CK2α′为诱饵蛋白进行酵母双杂交实验. 以初步筛选与人蛋白激酶CK2α′相互作用蛋白的阳性候选克隆,筛选获得8个阳性克隆,其中1个与人泛素-52氨基酸融合蛋白基因（UBA52）的cDNA序列有高度同源性（100％）. GST pull-down实验在细胞外进一步证实了CK2α′与UBA52之间存在相互作用. 本实验证明,人泛素-52氨基酸（UBA52）融合蛋白是人CK2α′亚基的相互作用蛋白, 它们之间的相互作用对精子发生机制的影响尚不清楚,进一步分子机制研究正在进行中.     

Alix (ALG-2-interacting protein X), a protein involved in apoptosis,binds to endophilins and induces cytoplasmic vacuolization

ALG-2-interacting protein X (Alix), also known as AIP1, is a cytoplasmic protein ubiquitously expressed and concentrated in phagosomes and exosomes. Alix may regulate apoptosis since it binds apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein necessary for cell death, and also overexpression of its C-terminal half (Alix-CT) blocks death induced by several stimuli. This part of Alix contains a long proline-rich domain containing several potential SH3-binding sites. Using Alix as bait in a yeast two-hybrid system to screen a mouse brain library, we have found that SH3p4, SH3p8, and SH3p13, collectively known as endophilins, bind to Alix. Co-immunoprecipitations and overlay experiments allowed us to demonstrate that endophilins bind to Alix-CT through an SH3/proline-rich domain interaction. We have narrowed the region of Alix interacting with endophilins down to 14 amino acids containing a PXRPPPP consensus sequence, also present in synaptojanin and germinal center kinase-like kinase, allowing their interaction to endophilins. We further show that overexpression of Alix-CT, which blocks cell death, leads to cytoplasmic vacuolization into tubulo-vesicular structures delineated by Alix-CT. This vacuolization phenomenon is greatly enhanced upon co-expression with endophilins and may be part of the protecting mechanism afforded by Alix-CT.