Understanding protein-protein interaction network: a case study with glutaminase interacting protein

Joint Annual Meeting of the ASPET/BPS at Experimental Biology (EB) -- APR 20-24, 2013 -- Boston, MAWOS: 000319883500016ASPET, British Pharmacol Soc (BPS

Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

WOS: 000294317300024PubMed ID: 21787750The 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-I, 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, beta-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kit 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. (C) 2011 Elsevier Inc. All rights reserved.USDA PECASE [2003-35302-12930]; NSFNational Science Foundation (NSF) [IBN-0628064]; USDAUnited States Department of Agriculture (USDA) [2011-65503-20030]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [DK082397]; NSERCNatural Sciences and Engineering Research Council of Canada [155268]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108T945]This research was financially supported by USDA PECASE Presidential Early Career Award for Scientists and Engineers award 2003-35302-12930, NSF Grant IBN-0628064, USDA Grant 2011-65503-20030 and NIH Grant DK082397 to Smita Mohanty, NSERC Grant 155268 to Melanie Dobson, and the Grant TUBITAK 108T945 to Zeki Topcu. We thank Dr. David L. Zoetewey for critical reading of this manuscript

New partner proteins containing novel internal recognition motif for human glutaminase interacting protein (hGIP)

WOS: 000316038500003PubMed ID: 23395680Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein-protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human glutaminase interacting protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. (C) 2013 Elsevier Inc. All rights reserved.United States Department of AgricultureUnited States Department of Agriculture (USDA) [2003-35302-12930, 2011-65503-20030]; National Science FoundationNational Science Foundation (NSF) [IOS-0628064]; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [DK082397]; NSERCNatural Sciences and Engineering Research Council of Canada [155268]; [TUBITAK 108T945]This research was financially supported by United States Department of Agriculture PECASE award 2003-35302-12930, and AFRI award 2011-65503-20030, National Science Foundation Grant IOS-0628064, and National Institutes of Health Grant DK082397 to Smita Mohanty, NSERC Grant 155268 to Melanie Dobson, and the Grant TUBITAK 108T945 to Zeki Topcu

Specificity and Promiscuity in Human Glutaminase Interacting Protein Recognition: Insight from the Binding of the Internal and C‑Terminal Motif

A large number of cellular processes are mediated by protein–protein interactions, often specified by particular protein binding modules. PDZ domains make up an important class of protein–protein interaction modules that typically bind to the C-terminus of target proteins. These domains act as a scaffold where signaling molecules are linked to a multiprotein complex. Human glutaminase interacting protein (GIP), also known as tax interacting protein 1, is unique among PDZ domain-containing proteins because it is composed almost exclusively of a single PDZ domain rather than one of many domains as part of a larger protein. GIP plays pivotal roles in cellular signaling, protein scaffolding, and cancer pathways via its interaction with the C-terminus of a growing list of partner proteins. We have identified novel internal motifs that are recognized by GIP through combinatorial phage library screening. Leu and Asp residues in the consensus sequence were identified to be critical for binding to GIP through site-directed mutagenesis studies. Structure-based models of GIP bound to two different surrogate peptides determined from nuclear magnetic resonance constraints revealed that the binding pocket is flexible enough to accommodate either the smaller carboxylate (COO^–) group of a C-terminal recognition motif or the bulkier aspartate side chain (CH₂COO^–) of an internal motif. The noncanonical ILGF loop in GIP moves in for the C-terminal motif but moves out for the internal recognition motifs, allowing binding to different partner proteins. One of the peptides colocalizes with GIP within human glioma cells, indicating that GIP might be a potential target for anticancer therapeutics

Modulation of Xenobiotic Receptors by Steroids

Nuclear receptors (NRs) are ligand-activated transcription factors that regulate the expression of their target genes. NRs play important roles in many human diseases, including metabolic diseases and cancer, and are therefore a key class of therapeutic targets. Steroids play important roles in regulating nuclear receptors; in addition to being ligands of steroid receptors, steroids (and their metabolites) also regulate other NRs, such as the pregnane X receptor and constitutive androstane receptor (termed xenobiotic receptors), which participate in steroid metabolism. Xenobiotic receptors have promiscuous ligand-binding properties, and their structurally diverse ligands include steroids and their metabolites. Therefore, steroids, their metabolism and metabolites, xenobiotic receptors, steroid receptors, and the respective signaling pathways they regulate have functional interactions. This review discusses these functional interactions and their implications for activities mediated by steroid receptors and xenobiotic receptors, focusing on steroids that modulate pathways involving the pregnane X receptor and constitutive androstane receptor. The emphasis of the review is on structure-function studies of xenobiotic receptors bound to steroid ligands