Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein

Sphingosine kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms. However, the molecular mechanism of the intracellular actions of SPP remains unclear. Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening. RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family. The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies. RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins. Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate. These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell.     

Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5′ cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species.     

Identification of ERK2-binding domain of EBITEIN1, a novel ERK2-binding protein

We recently cloned a cDNA encoding a novel extracellular signal-regulated kinase 2 (ERK2) binding protein, EBITEIN1, by yeast two-hybrid screening. In this study, we further characterized EBITEIN1. Binding experiments using various deletion mutants identified a 40-amino acid minimal sequence for binding ERK2. Binding experiments using substitution mutants indicated the crucial role of arginine residues in this sequence. Based on empirical and bioinformatic analyses, we propose two domains in EBITEIN1. One is the minimal sequence for binding ERK2 (EB domain) and the other is the EBITEIN1 C-terminal domain (ECT domain). These results might pave the way for further empirical and bioinformatic analyses of EBITEIN1- and ERK2-mediated events.     

Differential regulation of TIMP-1, -2, and -3 mRNA and protein expressions during mouse incisor development

Tissue inhibitors of metalloproteinases (TIMPs) possess multiple functions, in addition to their matrix metalloproteinase (MMP) inhibitory activity. The continuously growing incisor of mouse possesses a stem cell compartment at the apical end of the epithelium (the apical loop) and thus provides an excellent tool to analyze the mechanisms of organogenesis and cytodifferentiation. To understand the functions of TIMPs in tooth development, we have analyzed the gene expression and protein localization of TIMP-1, -2, and -3 during mouse incisor development, from embryonic day 13 (E13) to postnatal day 3 (P3). TIMP-1 was present on the basement membrane during early developmental stages. At P2, TIMP-1 was strongly detected along the apical loop, transiently disappeared from the basement membrane in the cytodifferentiation zone, and later reappeared at the distal end of functional ameloblasts. Expression of TIMP-2 protein was restricted to the outer part of the apical loop throughout the examined stages. At P2, TIMP-2 was present on the basement membrane at the outer part of the apical loop. The dental follicle also expressed Timp-2, and the corresponding protein was abundant within the extracellular matrix. Timp-3 mRNA was highly expressed in the mesenchyme surrounding the apical loop. During matrix formation, Timp-3 was expressed by subodontoblasts, and the protein was detected in this layer and between odontoblasts. Distinct temporal and spatial expression patterns of TIMPs suggest divergent functions of these factors in incisor organogenesis. This work was supported by INSERM, CNRS, ARC, French Ministry of Research (ACI), Japanese Ministry of Education, Culture, Sports, Science, and Technology, and Niigata University Research Projects.     

Identification and functional characterization of a PP1-binding site in BRCA1

The phosphorylation state of the tumor suppressor protein BRCA1 is tightly associated with its functions including cell cycle control and DNA repair. Protein kinases involved in the DNA damage checkpoint control, such as ATM, ATR, and hCds1/Chk2, have been shown to phosphorylate and activate BRCA1 upon DNA damage. We reported previously that protein phosphatase 1alpha (PP1alpha) interacts with and dephosphorylates hCds1/Chk2-phosphorylated BRCA1. This study demonstrates the identification of a PP1-binding motif 898KVTF901 in BRCA1. Mutation or deletion of critical residues in this PP1-binding motif substantially reduces the interaction between BRCA1 and PP1alpha. PP1alpha can also dephosphorylate ATM and ATR phosphorylation sites in BRCA1 and may serve as a general regulator for BRCA1 phosphorylation. Unlike wild-type BRCA1, expression of the PP1 non-binding mutant BRCA1 protein in BRCA1-deficient cells failed to enhance survival after DNA damage. Taken together, these results suggest that interaction with PP1alpha is important for BRCA1 function.     

Identification and characterization of the Cdc42-binding site of IQGAP1

IQGAP1 is a multi-domained protein that integrates signaling of the Rho family GTPase Cdc42 with regulation of the cytoskeleton. Using SPOT analysis and in vitro peptide competition assays we have identified a 24 amino acid region of IQGAP1 that is necessary for Cdc42 binding. Both in vitro and in vivo analyses reveal that deletion of this sequence abolishes binding of IQGAP1 to Cdc42. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. An IQGAP1 mutant lacking the Cdc42 binding site mislocalizes to the cell periphery. These observations specifically define a short sequence of IQGAP1 that is required for its interaction with Cdc42 and demonstrate that Cdc42 binding is necessary for the normal subcellular distribution of IQGAP1.     

Identification of Pin1-binding phosphorylated proteins in the mouse brain

To determine the role of Pin1 in the neurotransmission pathway, Pin1-binding proteins in mouse brain extract were identified. The Pin1-binding proteins were extracted from mouse brain homogenate, and the trypsin-digested peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins that involve the neurotransmission pathway, such as synapsin I, synapsin II, and calcium/calmodulin-dependent protein kinase type II (CaMKII), were identified in a Mascot search. Pull-down and immunoprecipitation assay indicated that Pin1 binds CaMKII in a phosphorylation-specific manner. It was assumed that Pin1 participates in the neurotransmission pathway involving the phosphorylation signal by CaMKII.     

Expression, purification, and characterization of a novel recombinant fusion protein, rhTPO/SCF, in Escherichia coli

Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic effect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and purification of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1-157 a.a.) plus a truncated human stem cell factor (1-145 a.a.), linked by a peptide (GGGGSPGGSGGGGSGG). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaperones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was purified to homogeneity using anion exchange followed by metal affinity chromatography. Western blot analysis confirmed the identity of the purified protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines.     

Identification and characterization of the mouse MutS homolog 5: Msh5

We have identified and characterized the complete cDNA and gene for the mouse MutS homolog 5 (Msh5), as a step toward understanding the molecular genetic mechanisms involved in the biological function of this new MutS homologous protein in mammals. The Msh5 cDNA contains a 2502-bp open reading frame (ORF) that encodes an 833-amino acid protein with a predicted molecular weight of 92.6 kDa, which shares 89.8% amino acid sequence identity with the human hMSH5 protein. Northern blot analysis demonstrated the presence of a Msh5 mRNA approximately 2.9-kb in length, most abundantly expressed in mouse testis. Yeast two-hybrid analysis indicated that the mouse Msh5 protein positively interacted with the human hMSH4 protein—suggesting that Msh5 shares common functional properties with its human counterpart. Sequence and structural analyses show that the mouse gene Msh5 spans approximately 18 kb and contains 24 exons that range in length from 36 bp for exon 7 to 392 bp for exon 1. Structural comparison with the human hMSH5 gene revealed that all of the Msh5 internal exons, but not introns, are conserved in length with the human hMSH5. The Msh5 gene is located on mouse Chromosome (Chr) 17 in a location that is syntenic to the region of human Chr 6 harboring the hMSH5 gene. The identification and characterization of Msh5 will facilitate studies of the potential functional roles of this new member of the MutS family. Received: 11 May 1999 / Accepted: 16 July 1999     

Identification of functional p53-binding motifs in the mouse wig-1 promoter

With selective excitation around BChl-B800 and BChl-B850 absorption bands, we observed the evolution of excited-state dynamics in LH2 from Rhodobacter sphaeroides 601. The dynamical traces demonstrate a dominant excited-state absorption (ESA) followed concomitantly by an ultrafast transmission increase and decay with pulse-width limited time scale at 818 nm and 828 nm excitation. The ESA occurring prior to excitonic thermalization or ground-state bleach was observed at 840 nm as well. These experimental results indicate the competition between the transition from excitonic states to higher-lying excited states and interexciton relaxation, which are of physical significance for understanding excitation transfer and related mechanisms in LH2.     

Identification and characterization of ErbB-3-binding protein-1 as a target for immunotherapy

Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients' sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1). Immunohistochemical staining of colorectal tumors and neighboring normal colon tissue indicated the observed cancer-related immunogenicity of Ebp1 to be related to overexpression. Via reverse immunology, five potential HLA-A2-restricted T cell epitopes were identified, of which two (Ebp1(45-54) and Ebp1(59-67)) bound HLA-A2 with intermediate and high affinity, respectively. Analysis of their immunogenicity in vitro indicated that only the high-affinity Ebp1(59) epitope gave rise to CD8(+) T cells capable of recognizing both exogenously loaded Ebp1 peptide and endogenously expressed Ebp1 on target cells. In addition, in vivo CD8(+) T cell responsiveness against the Ebp1(59) epitope could be detected in two of nine and three of six cancer patients PBMC and tumor draining lymph nodes, respectively, but not in nine of nine healthy donors tested. These data confirm that Ebp1 is an immunogenic protein, capable of eliciting CD8-mediated responses in vivo and in vitro, providing a rationale for further exploration of Ebp1 as a possible target for anticancer immunotherapy.     

Expression, purification, and characterization of Tara, a novel telomere repeat-binding factor 1 (TRF1)-binding protein

Tara was originally identified as a binding protein of guanine nucleotide exchange factor Trio. Although Tara may be involved in many fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation, aging, and cancer, the exact molecular mechanisms are poorly understood. We expressed recombinant Tara in Escherichia coli and purified the protein to approximately 99% purity using affinity chromatography and gel-filtration chromatography. The identity of the purified protein was confirmed by mass spectrometry. Non-denaturing polyacrylamide gel electrophoresis and gel-filtration chromatography showed that Tara forms multimer in vitro. The purified Tara was used to generate polyclonal antibody, which could specifically recognize both the recombinant and endogenous Tara. Using the pull-down assay, we showed that the purified Tara interacted with TRF1, suggesting that the purified protein is functional and biologically active. The availability of purified Tara and anti-Tara antibody provides critical reagents for elucidating Tara's cellular function and its molecular mechanism.     

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.     

Identification and characterization of the surface-layer protein of Clostridium tetani

Many bacterial species produce a paracrystalline layer, the surface layer, which completely surrounds the exterior of the cell. In some bacteria, the surface layer is implicated in pathogenesis. Two proteins present in cell wall extracts from Clostridium tetani have been investigated and identified one of these has been unambiguously as the surface-layer protein (SLP). The gene, slpA, has been located in the genome of C. tetani E88 that encodes the SLP. The molecular mass of the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is considerably larger than that predicted from the gene; however the protein does not appear to be glycosylated. Furthermore, analysis of five C. tetani strains, including three recent clinical isolates, shows considerable variation in the sizes of the SLP.     

Identification and characterization of merozoite surface protein 1 epitope

Malaria is an important tropical infection which urgently requires intervention of an effective vaccine. Antigenic variations of the parasite and allelic diversity of the host are main problems in the development of an effective malaria vaccine. Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum­derived antigens are shown to play an important role for the protection against malaria. The merozoite surface protein 1 (MSP1) is expressed in all the four life-cycle stages of Plasmodium falciparum and did not find any sequence similarity to human and mouse reference proteins. MSP1 is a known target of the immune response and a single CTL epitope binding to the HLA­A*0201 is available for merozoite form. Here, we report the results from the computational characterization of MSP1, precursor (1720 residue) and screening of highest scoring potential CTL epitopes for 1712 overlapping peptides binding to thirty four HLA class­I alleles and twelve HLA class­I supertypes (5 HLA­A and 7 HLA­B) using bioinformatics tools. Supertypes are the clustered groups of HLA class­I molecules, representing a sets of molecules that share largely overlapping peptide binding specificity. The prediction results for MSP1 as adhesin and adhesin-like in terms of probability is 1.0. Results also show that MSP1 has orthologs to other related species as well as having non allergenicity and single transmembrane properties demonstrating its suitability as a vaccine candidate. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.