Biochemical and computational analysis of LNX1 interacting proteins

PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.     

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti‐aging and metabolic effects of sirtuins

Sirtuins are a family of NAD⁺‐dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti‐aging effects of human homologues of sirtuins, SIRT1‐7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti‐aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI‐TOF/TOF‐MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co‐immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti‐aging and metabolic regulatory activities of sirtuins.     

Novel cytosolic binding partners of the neural cell adhesion molecule: mapping the binding domains of PLC gamma, LANP, TOAD-64, syndapin, PP1, and PP2A

The neural cell adhesion molecule (NCAM) is implicated in important functions during development and maintenance of the nervous system. Two of the three major isoforms, NCAM 140 and NCAM 180, are transmembrane glycoproteins with large cytoplasmic domains of different length. The purpose of this study was to identify novel intracellular binding partners of NCAM 140 and NCAM 180. We expressed both cytoplasmic domains, as well as cytoplasmic fragments of NCAM, as fusion proteins in Escherichia coli and used them for ligand affinity chromatography or glutathione S-transferase (GST) pull-down assays. By peptide mass fingerprinting Western blot analysis, or both, we identified PLCgamma, LANP, syndapin, PP1, and PP2A as binding partners for both NCAM 140 and NCAM 180, whereas TOAD-64 was identified as a NCAM 180-specific interacting protein. Furthermore, we were able to show that binding of these novel binding proteins, as well as the previously described interaction partners ROK alpha (rho A binding kinase alpha) and alpha- and beta-tubulin, bind to specific cytosolic sequences of NCAM. For this purpose, we performed GST pull-down experiments using cytosolic fragments of NCAM as GST-fusion proteins and cytosolic- or cytoskeleton-enriched protein fractions of rat brain.     

Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

Identification and characterization of NIF3L1 BP1, a novel cytoplasmic interaction partner of the NIF3L1 protein

The NIF3L1 protein is strongly conserved during evolution from bacteria to mammals and recently its function in neuronal differentiation has been demonstrated. In the present study we identified novel binding partners of human NIF3L1 by screening a HeLa cDNA-library using the yeast two-hybrid system. We could show that the NIF3L1 protein is interacting with itself and with the NIF3L1 binding protein 1 (NIF3L1 BP1), a novel protein of 23.67kDa bearing a putative leucine zipper domain. Furthermore, both interactions were confirmed using the mammalian two-hybrid system. Deletion analyses clearly demonstrated that a C-terminal region of 100 amino acids of the NIF3L1 BP1 is sufficient for the interaction with NIF3L1. The NIF3L1 BP1 is ubiquitously expressed and cotransfection experiments revealed that NIF3L1 and NIF3L1 BP1 interact in the cytoplasm of human LNCaP cells. This study provides novel insights into the cellular function of the NIF3L1 protein.     

Dock7: a GEF for Rho-family GTPases and a novel myosin VI-binding partner in neuronal PC12 cells

Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI-Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization.     

Proteome-wide detection of Abl1 SH3-binding peptides by integrating computational prediction and peptide microarray

Protein-protein interactions are essential for regulating almost all aspects of cellular functions. Many of these interactions are mediated by weak and transient protein domain-peptide binding, but they are often under-represented in high throughput screening of protein-protein interactions using techniques such as yeast two-hybrid and mass spectrometry. On the other hand, computational predictions and in vitro binding assays are valuable in providing clues of in vivo interactions. We present here a systematic approach that integrates computer modeling and a peptide microarray technology to identify binding peptides of the SH3 domain of the tyrosine kinase Abl1 in the human proteome. Our study provides a comprehensive list of candidate interacting partners for the Abl1 protein, among which the presence of numerous methyltransferases and RNA splicing proteins may suggest a novel function of Abl1 in chromatin remodeling and RNA processing. This study illustrates a powerful approach for integrating computational and experimental methods to detect protein interactions mediated by domain-peptide recognition.     

Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

An original experimental method of direct molecular fishing has been developed for identification of potential partners of protein–protein and protein–peptide interactions. It is based on combination of surface plasmon resonance technology (SPR), size exclusion and affinity chromatography and mass spectrometric identification of proteins (LC-MS/MS). Previously, we demonstrated applicability of this method for protein interactomics using experimental model system, as well as in the pilot study in the frame of the Human Proteome Project (HPP). In the present paper, this method was successfully applied to identify possible molecular partners of 7 target proteins encoded by genes of 18 chromosome (also in the frame of the HPP). Fishing on the affinity sorbents with immobilized target proteins as ligands was carried out using total lysate of human liver tissue as well as pooled sets of fractions (individual for each bait-protein) obtained by means of a combination of size exclusion chromatography and SPR analysis for the presence of potential prey-proteins in each fraction. As a result we obtained lists of possible molecular partners of all 7 proteins and performed a comparative evaluation of direct fishing specificity for these target proteins. Direct molecular fishing was also successfully used for search of potential protein partners interacting with different isoforms of amyloid-beta peptide, playing a key role in the development of Alzheimer’s disease. The synthetic peptides that are analogues of the metal-binding domain isoforms of beta-amyloid were used as molecular baits and the fishing was performed in various fractions of immortalized human neural cells. As a result, 13 potential partner proteins were identified in the cytosol fraction of the cells by fishing on amyloid-beta peptide (1-16).     

Identification of novel SH3 domain ligands for the Src family kinase Hck. Wiskott-Aldrich syndrome protein (WASP), WASP-interacting protein (WIP), and ELMO1

The importance of the SH3 domain of Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, we used mass spectrometry to identify approximately 25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). We also focused on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegans gene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, we confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck. We also show that Hck and ELMO1 interact in intact cells and that ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck. The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck. Our results suggest that these proteins may be novel activators/effectors of Hck.     

Mapping protein receptor-ligand interactions via in vivo chemical crosslinking, affinity purification, and differential mass spectrometry

Protein receptor-ligand interactions play important roles in mediating enzyme catalysis, signal transduction, and other protein functions. Immunoaffinity purification followed by mass spectrometry analysis is a common method for identifying protein receptor-ligand complexes. However, it is difficult to distinguish between specific protein binding partners and non-specifically bound proteins that co-purify with the complex. In addition, weakly interacting binding partners may dissociate from the protein receptor-ligand complexes during immunoaffinity purification. The combination of chemical crosslinking, affinity purification, and differential mass spectrometry analysis provides a direct method for capturing stable, weak, and transient protein interactions that occur in vivo and in vitro. This approach enables the identification of functional receptor-ligand binding partners with high confidence. Herein, we describe a differential mass spectrometry approach coupled with in situ chemical crosslinking and immunoaffinity purification for identifying receptor-ligand binding partners. In particular, we identified a functional, counter-ligand structure of the natural killer cell p30-related protein.     

Defining the specificity space of the human SRC homology 2 domain

Src homology 2 (SH2) domains are the largest family of interaction modules encoded by the human genome to recognize tyrosine-phosphorylated sequences and thereby play pivotal roles in transducing and controlling cellular signals emanating from protein-tyrosine kinases. Different SH2 domains select for distinct phosphopeptides, and the function of a given SH2 domain is often dictated by the specific motifs that it recognizes. Therefore, deciphering the phosphotyrosyl peptide motif recognized by an SH2 domain is the key to understanding its cellular function. Here we cloned all 120 SH2 domains identified in the human genome and determined the phosphotyrosyl peptide binding properties of 76 SH2 domains by screening an oriented peptide array library. Of these 76, we defined the selectivity for 43 SH2 domains and refined the binding motifs for another 33 SH2 domains. We identified a number of novel binding motifs, which are exemplified by the BRDG1 SH2 domain that selects specifically for a bulky, hydrophobic residue at P + 4 relative to the Tyr(P) residue. Based on the oriented peptide array library data, we developed scoring matrix-assisted ligand identification (or SMALI), a Web-based program for predicting binding partners for SH2-containing proteins. When applied to SH2D1A/SAP (SLAM-associated protein), a protein whose mutation or deletion underlies the X-linked lymphoproliferative syndrome, SMALI not only recapitulated known interactions but also identified a number of novel interacting proteins for this disease-associated protein. SMALI also identified a number of potential interactors for BRDG1, a protein whose function is largely unknown. Peptide in-solution binding analysis demonstrated that a SMALI score correlates well with the binding energy of a peptide to a given SH2 domain. The definition of the specificity space of the human SH2 domain provides both the necessary molecular basis and a platform for future exploration of the functions for SH2-containing proteins in cells.     

Identification and characterization of a novel tight junction-associated family of proteins that interacts with a WW domain of MAGI-1

The membrane-associated guanylate kinase protein, MAGI-1, has been shown to be a component of epithelial tight junctions in both Madin-Darby canine kidney cells and in intestinal epithelium. Because we have previously observed MAGI-1 expression in glomerular visceral epithelial cells (podocytes) of the kidney, we screened a glomerular cDNA library to identify the potential binding partners of MAGI-1 and isolated a partial cDNA encoding a novel protein. The partial cDNA exhibited a high degree of identity to an uncharacterized human cDNA clone, KIAA0989, which encodes a protein of 780 amino acids and contains a predicted coiled-coil domain in the middle of the protein. In vitro binding assays using the partial cDNA as a GST fusion protein confirm the binding to full-length MAGI-1 expressed in HEK293 cells, as well as endogenous MAGI-1, and also identified the first WW domain of MAGI-1 as the domain responsible for binding to this novel protein. Although a conventional PPxY binding motif for WW domains was not present in the partial cDNA clone, a variant WW binding motif was identified, LPxY, and found to be necessary for interacting with MAGI-1. When expressed in Madin-Darby canine kidney cells, the full-length novel protein was found to colocalize with MAGI-1 at the tight junction of these cells and the coiled-coil domain was found to be necessary for this localization. Because of its interaction with MAGI-1 and its localization to cell-cell junctions, this novel protein has been given the name MAGI-1-associated coiled-coil tight junction protein (MASCOT).     

Cellular Localization and Characterization of Cytosolic Binding Partners for Gla Domain-containing Proteins PRRG4 and PRRG2

The genes encoding a family of proteins termed proline-rich γ-carboxyglutamic acid (PRRG) proteins were identified and characterized more than a decade ago, but their functions remain unknown. These novel membrane proteins have an extracellular γ-carboxyglutamic acid (Gla) protein domain and cytosolic WW binding motifs. We screened WW domain arrays for cytosolic binding partners for PRRG4 and identified novel protein-protein interactions for the protein. We also uncovered a new WW binding motif in PRRG4 that is essential for these newly found protein-protein interactions. Several of the PRRG-interacting proteins we identified are essential for a variety of physiologic processes. Our findings indicate possible novel and previously unidentified functions for PRRG proteins.     

Identification of a potential regulator of the gap junction protein pannexin1

Recent studies have revealed a second class of gap-junction-forming proteins in vertebrates. These genes are termed pannexins, and it has been suggested that they perform similar functions as connexins. Pannexin1 is expressed in diverse tissues including the central nervous system and seems to form gap junction channels in the Xenopus oocyte expression system. Since protein interacting partners have frequently been described for connexins, the most prominent family of gap junction forming proteins, we thus started to search for candidate genes of pannexin interacting partners. Kvbeta3, a protein belonging to the family of regulatory beta-subunits of the voltage-dependent potassium channels, was identified as a binding partner of pannexin1 in an E. coli two-hybrid system. This result was verified by confocal laser scanning microscopy using double transfected Neuro2A cells. The colocalization of both proteins at the plasma membrane is suggestive of functional interaction.