Food grade expression system using L. plantarumAntibody Engineering
Molecular and Cellular Mechanisms of Neutraceuticals
Development of Biologics Production Platforms
Generation and Manufacturing of Bioactive oligosaccharides
Molecular biology
Phage display technolgy
Protein, Expression Engineering
Current Research
Food grade expression system using L. plantarumAntibody Engineering
Molecular and Cellular Mechanisms of Neutraceuticals
Development of Biologics Production Platforms
Generation and Manufacturing of Bioactive oligosaccharides
Molecular biology
Phage display technolgy
Protein, Expression Engineering
Year
Month
Title
Journal
Information
2000
Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways Adams A., Thorn J.M., Yamabhai M., Kay B.K., O'Bryan J.P.
Journal of Biological Chemistry
Abstract: Intersectin is a member of a growing family of adaptor proteins that possess conserved Eps15 homology (EH) domains as well as additional protein recognition motifs. In general, EH domain-containing proteins play an integral role in clathrin-mediated endocytosis. Indeed, intersectin functions in the intermediate stages of clathrin-coated vesicle assembly. However, recent evidence suggests that components of the endocytic machinery also regulate mitogenic signaling pathways. In this report, we provide several lines of evidence that intersectin has the capacity to activate mitogenic signaling pathways. First, intersectin overexpression activated the Elk-1 transcription factor in an MAPK-independent manner. This ability resides within the EH domains, as expression of the tandem EH domains was sufficient to activate Elk-1. Second, intersectin cooperated with epidermal growth factor to potentiate Elk-1 activation; however, a similar level of Elk-1 activation was obtained by expression of the tandem EH domains suggesting that the coiled-coil region and SH3 domains act to regulate the EH domains. Third, intersectin expression was sufficient to induce oncogenic transformation of rodent fibroblasts. And finally, intersectin cooperated with progesterone to accelerate maturation of Xenopus laevis oocytes. Together, these data suggest that intersectin links endocytosis with regulation of pathways important for cell growth and differentiation.
The EH network Santolini E., Salcini A.E., Kay B.K., Yamabhai M., Di Fiore P.P.
Experimental Cell Research
Abstract: The EH domain is an evolutionary conserved protein-protein interaction domain present in a growing number of proteins from yeast to mammals. Even though the domain was discovered just 5 years ago, a great deal has been learned regarding its three-dimensional structure and binding specificities. Moreover, a number of cellular ligands of the domain have been identified and demonstrated to define a complex network of protein-protein interactions in the eukaryotic cell. Interestingly, many of the EH-containing and EH-binding proteins display characteristics of endocytic 'accessory' proteins, suggesting that the principal function of the EH network is to regulate various steps in endocytosis. In addition, recent evidence suggests that the EH network might work as an 'integrator' of signals controlling cellular pathways as diverse as endocytosis, nucleocytosolic export, and ultimately cell proliferation.
Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells Hussain N.K., Yamabhai M., Ramjaun A.R., Guy A.M., Baranes D., O'Bryan J.P., Der C.J., Kay B.K., McPherson P.S.
Journal of Biological Chemistry
Abstract: We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C- terminal extension containing DbI homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is colocalized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.
Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery Kay B.K., Yamabhai M., Wendland B., Emr S.D.
Protein Science
Abstract: We have identified a ~ 140 amino acid domain that is shared by a variety of proteins in budding and fission yeast, nematode, rat, mouse, frog, oat, and man. Typically, this domain is located within 20 residues of the N- terminus of the various proteins. The percent identity among the domains in the 12 proteins ranges from 42 to 93%, with 16 absolutely conserved residues: N-x11-13-V-x2-A-T-x34-36-R-x7-8-W-R-x3-K-x12-G-x-E-x15-L- X11-12-D-x-G-R-x11-D-x7-R. Even though these proteins share little beyond their segment of homology, data are emerging that several of the proteins are involved in endocytosis and or regulation of cytoskeletal organization. We have named this protein segment the ENTH domain, for Epsin N-terminal Homology domain, and hypothesize that it is a candidate for binding specific ligands and/or enzymatic activity in the cell.
Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries Kay B.K., Yamabhai M., Kasanov J., Jvoiirakine A.
FASEB Journal
Abstract: We have const rucied recombinant barteriophage M13 libraries expressing billions of different combinatorial peptides attached to the N-termiiius of the cap si(i protein, pill. We have screened the libraries by affinity selection for members which interact with a variety of targets and have isolated p h age that bind specifically 1o antibodies (monoclonal and polyclonai), cytoskeletal proteins, signal transduction proteins, enzymes, and various protein interaction modules (i.e.. SH3, WW, EH domains). Three important lessons have been learned from this work: 1) isolated phage bind at bone fide binding sites of target proteins, 2) the phage-displayed pe.pt.ide often resembles, in primary structure, known proteins that interact with the target protein, and 3} peptides synthesized corresponding to the phage-diispiayed sequence can act as agonists or antagonists in vitro and in vivo.
Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains Yamabhai M., Hoffman N.G., Hardison N.L., McPherson P.S., Castagnoli L., Cesareni G., Kay B.K.
Journal of Biological Chemistry
Abstract: We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid- long protein containing two Eps15 homology (EH) domains, a central coiled- coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX(1- 2)NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin- binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.
Examining the specificity of Src homology 3 domain-ligand interactions with alkaline phosphatase fusion proteins Yamabhai M., Kay B.K.
Analytical Biochemistry
Abstract: Sixteen-amino-acid-long peptides, corresponding to the optimal ligand preferences of the Src homology 3 (SH3) domains of Abl, Cortactin, Crk, p53BP2, and Src, were fused to the N-terminus of Escherichia coli alkaline phosphatase (AP). These secreted fusion proteins have been used as one-step detection probes of peptide ligand-SH3 domain interactions on microtiter plates and membranes. The binding of both the class I and II SH3 ligand-AP fusion proteins to their targets is robust and specific in comparison to chemically synthesized biotinylated peptides, used either in monovalent or tetravalent formats. p53BP2 and Cortactin SH3 ligand-AP fusions have been used to screen a mouse embryo λ cDNA expression library and resulted in the cloning of p53BP2 and several known proteins with SH3 domains similar to that of Cortactin, respectively. In addition, the ~60-amino-acid-long SH3 domains of Src and Abl were fused to AP and the resulting fusion proteins were found to bind specifically to their respective peptide ligands in microtiter plates and proteins containing praline-rich regions in screens of a λ cDNA expression library. Thus, SH3 peptide ligand- and SH3 domain-AP fusion proteins are convenient and sensitive reagents for examining the specificity of SH3 domain-ligand interactions, identifying potentially interacting proteins, and establishing high-throughput screens of combinatorial chemical libraries.
