Interactions of Human hMSH2 with hMSH3 and hMSH2 with hMSH6: Examination of Mutations Found in Hereditary Nonpolyposis Colorectal Cancer

Mutations in the human mismatch repair protein hMSH2 have been found to cosegregate with hereditary nonpolyposis colorectal cancer (HNPCC). Previous biochemical and physical studies have shown that hMSH2 forms specific mispair binding complexes with hMSH3 and hMSH6. We have further characterized these protein interactions by mapping the contact regions within the hMSH2-hMSH3 and the hMSH2-hMSH6 heterodimers. We demonstrate that there are at least two distinct interaction regions of hMSH2 with hMSH3 and hMSH2 with hMSH6. Interestingly, the interaction regions of hMSH2 with either hMSH3 or hMSH6 are identical and there is a coordinated linear orientation of these regions. We examined several missense alterations of hMSH2 found in HNPCC kindreds that are contained within the consensus interaction regions. None of these missense mutations displayed a defect in protein-protein interaction. These data support the notion that these HNPCC-associated mutations may affect some other function of the heterodimeric complexes than simply the static interaction of hMSH2 with hMSH3 or hMSH2 with hMSH6.     

NEMO binding domain of IKK-2 encompasses amino acids 735-745

NF-kappaB activation is mediated by the IKK signalsome. Though this signalsome is comprised of IKK-1, IKK-2, and NEMO/IKKgamma, it is the interaction between IKK-2 and NEMO that is critical to formation of a functional signalsome. More specifically, previous reports have indicated that this interaction involves the C-terminal LDWSWL residues of IKK-2 (called the Nemo Binding Domain (NBD)) and the N-terminus of NEMO. In an effort to characterize the IKK-2:NEMO interaction, we have investigated several NBD-containing peptides for their ability to bind NEMO and inhibit the critical IKK-2:NEMO interaction. The six residue NBD peptide, LDWSWL, showed modest binding to NEMO and little inhibition of the IKK-2:NEMO interaction, whereas peptides containing the NBD plus additional flanking amino acids (NBD-containing peptides) more effectively bound NEMO and inhibited the interaction. These longer NBD-containing peptides may be required to give the NBD an appropriate conformation for recognition by NEMO and/or to provide for additional interactions with NEMO.     

Intra- and intermolecular interactions of the catalytic domains of human CD45 protein tyrosine phosphatase

We have investigated protein-protein interaction between distinct domains of the human CD45 cytoplasmic region using a yeast two-hybrid system. Consequently, we have found that the spacer region between two tandem PTP domains specifically interacts with the membrane-distal PTP domain (D2). This interaction is mediated by a stretch of amino acid residues in the carboxyl-terminal half of the spacer region. Although the membrane proximal region does not directly interact with either of the two PTP domains, it appears to function in stabilizing the interaction between the spacer region and D2. We also demonstrate that the interaction between the spacer region and D2 might take place intramolecularly.     

Mechanism and evolution of human ACE2 binding by SARS-CoV-2 spike

Spike glycoprotein of SARS-CoV-2 mediates viral entry into host cells by facilitating virus attachment and membrane fusion. ACE2 is the main receptor of SARS-CoV-2 and its interaction with spike has shaped the virus’ emergence from an animal reservoir and subsequent evolution in the human host. Many structural studies on the spike:ACE2 interaction have provided insights into mechanisms driving viral evolution during the on-going pandemic. This review describes the molecular basis of spike binding to ACE2, outlines mechanisms that have optimised this interaction during viral evolution, and suggests directions for future research.     

Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.

The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. However, the biological relevance of these two-hybrid interactions to viral positive-strand RNA replication has not been demonstrated. The brome mosaic virus (BMV) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the BMV 1a helicase-like and 2a polymerase-like proteins. We have tested wild-type 1a and 18 insertion mutations of 1a and found a perfect correlation between the in planta phenotypes and their ability to interact with 2a in the two-hybrid system. This finding allowed further characterization of the interaction between and among the BMV viral proteins. Using the two-hybrid assay, we have found that the interaction between the helicase-like region of 1a and the N terminus of 2a is stabilized by the presence of the centrally conserved polymerase-like domain of 2a. We have also identified a novel interaction between the 1a helicase-like protein and itself. Additionally, we have found this interaction in two related tripartite RNA viruses, cowpea chlorotic mottle virus and cucumber mosaic virus. We have demonstrated that this protein-protein interaction is specific to homologous pairings of the protein.     

Glucose and type 2A protein phosphatase regulate the interaction between catalytic and regulatory subunits of AMP-activated protein kinase

We have expressed in yeast the different subunits of AMP-activated protein kinase (AMPK) and, by using the two-hybrid system, we have found a glucose-regulated interaction between alpha 2 catalytic and gamma 1 regulatory subunits. This regulation was not affected by known regulators of the corresponding yeast orthologue, the SNF1 complex, such as Reg1 or Hxk2, but it was affected by deletion of regulatory subunits of yeast type 2A protein phosphatase (PP2A) complex. We have also found that Tpd3 and PR65 alpha, the corresponding yeast and mammalian A subunits of PP2A, interacted with AMPK alpha 2 both in yeast and mammals, respectively. This interaction occurred only through the regulatory domain of this subunit. These results suggested a direct involvement of PP2A complex in regulating the interaction between AMPK alpha 2 and gamma 1 in a glucose-dependent manner.     

On the mechanism of nucleosome unfolding.

We have studied the relative stabilities to urea denaturation of histone-histone binding interactions as they occur both in chromatin and in histone complexes free in solution. We have used the two zero-length contact-site cross-linking agents, tetranitromethane and UV light, to measure the relative degree of H2B-H4 and H2A-H2B association under various conditions. The two interactions were disrupted coordinately when nuclei were treated with increasing concentrations of urea. In contrast, when histone complex in 2 M NaCl were treated with urea, the H2B-H4 interaction was found to be much less stable than the H2A-H2B interaction. We have shown previously that nucleosomes unfold at low ionic strengths such that the H2B-H4 but not the H2A-H2B interaction is broken in the process. We speculate that the preferential rupture of the H2B-H4 contact is of physiological significance.     

The interaction of Rh(II) and Rh(III) with DNA

The interaction of Rh2(II)(acetate)4, cis-[Rh(III)(en)2Cl2] Cl (en = ethylenediamine) and [Rh(III) (NH3)5Cl]Cl2 with calf thymus DNA has been studied at various r values [formula; see text] and interaction times. Electronic spectra, melting and cooling curves and sedimentation data indicate no interaction of the acetate complex with DNA, except in the case of a high r value and long interaction time. The other two complexes have been found to interact with the phosphate groups, thus stabilizing the macromolecule.     

Physicochemical characteristics of the interaction of the peat polymeric matrix with solvents of different natures

The features of the interaction in a system biocomposite (peat)-solvent (H₂O, 0.1 M NaOH, C₂H₅OH) have been considered. The enthalpies of interaction have been calculated, and the kinetic dependencies of changes in enthalpy characteristics in processes accompanied by the extraction of peat components have been obtained.     

Stimulation of the erythrocyte Ca2+-ATPase and of bovine brain cyclic nucleotide phosphodiesterase by chemically modified calmodulin

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic-nucleotide phosphodiesterase. Modification of arginine residues by 1,2-cyclohexanedione had no effect of the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N-chlorosuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C-terminal half of calmodulin are more important for the interaction with the Ca2+-ATPase.     

S-Nitrosation of arginase 1 requires direct interaction with inducible nitric oxide synthase

Arginase constrains endothelial nitric oxide synthase activity by competing for the common substrate, L -Arginine. We have recently shown that inducible nitric oxide synthase (NOS2) S-nitrosates and activates arginase 1 (Arg1) leading to age-associated vascular dysfunction. Here, we demonstrate that a direct interaction of Arg1 with NOS2 is necessary for its S-nitrosation. The specific domain of NOS2 that mediates this interaction is identified. Disruption of this interaction in human aortic endothelial cells prevents Arg1 S-nitrosation and activation. Thus, disruption of NOS2-Arg1 interaction may represent a therapeutic strategy to attenuate age related vascular endothelial dysfunction.     

Interaction between very-KIND Ras guanine exchange factor and microtubule-associated protein 2, and its role in dendrite growth--structure and function of the second kinase noncatalytic C-lobe domain

The kinase noncatalytic C-lobe domain (KIND) is a putative protein-protein interaction module. Four KIND-containing proteins, Spir-2 (actin-nuclear factor), PTPN13 (protein tyrosine phosphatase), FRMPD2 (scaffold protein) and very-KIND (v-KIND) (brain-specific Ras guanine nucleotide exchange factor), have been identified to date. Uniquely, v-KIND has two KINDs (i.e. KIND1 and KIND2), whereas the other three proteins have only one. The functional role of KIND, however, remains unclear. We previously demonstrated that v-KIND interacts with the high-molecular weight microtubule-associated protein 2 (MAP2), a dendritic microtubule-associated protein, leading to negative regulation of neuronal dendrite growth. In the present study, we analyzed the structure-function relationships of the v-KIND-MAP2 interaction by generating a series of mutant constructs. The interaction with endogenous MAP2 in mouse cerebellar granule cells was specific to v-KIND KIND2, but not KIND1, and was not observed for the KINDs from other KIND-containing proteins. The binding core modules critical for the v-KIND-MAP2 interaction were defined within 32 residues of the mouse v-KIND KIND2 and 43 residues of the mouse MAP2 central domain. Three Leu residues at amino acid positions 461, 474 and 477 in the MAP2-binding core module of KIND2 contributed to the interaction. The MAP2-binding core module itself promoted dendrite branching as a dominant-negative regulator of v-KIND in hippocampal neurons. The results reported in the present study demonstrate the structural and functional determinant underlying the v-KIND-MAP2 interaction that controls dendrite arborization patterns.     

Interaction of the adenovirus IVa2 protein with viral packaging sequences

We have demonstrated previously that the adenovirus L1 52/55-kDa protein binds to the viral IVa2 protein in infected cells. The significance of this interaction was unclear, however, based on the known functions of these two proteins: the 52/55-kDa protein is required for viral DNA packaging, while the IVa2 protein is a transactivator of the major late promoter (MLP). In this report, we have attempted to elucidate a role for each of the two proteins in the other's known function. There is no apparent effect of the 52/55-kDa protein on the interaction of the IVa2 protein with the MLP. Surprisingly, however, we found that the IVa2 protein can interact with the adenoviral packaging signal and that this interaction involves DNA sequences that have previously been demonstrated to be required for packaging.     

The carbohydrate recognition by cytokines modulates their physiological activities

A variety of cytokines have been reported to be able to recognize specific carbohydrate moieties. To date, the role of carbohydrate recognition in cytokine function has been analyzed for several cytokines, including fibroblast growth factor (FGF), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2. The FGF family and their receptors have been found to recognize a heparan sulfate proteoglycan, which generates rigid complexes that induce signal transduction. We have found that IL-2 recognizes a high-mannose type glycan on the alpha subunit of the IL-2 receptor as well as a peptide portion of this subunit. Blocking this carbohydrate-IL-2 interaction diminished IL-2-induced signaling and T-cell proliferation. We have also shown that TNF-alpha recognizes the second mannose 6-phosphate diester of the glycan portion of glycosylphosphatidylinositol (GPI)-anchored glycoproteins. Blocking this GPI-anchored glycan-TNF-alpha interaction abrogates TNF-alpha-induced apoptosis. We aim to increase the number of cytokines which modulate their functions through the unique carbohydrate recognition, and open the way to systematically elucidate the biological functions of cytokine-carbohydrate interaction in immune system.     

Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, a homophilic cell adhesion molecule, and afadin, an actin filament-binding protein, which connects nectin to the actin cytoskeleton. Here we studied a mechanism of cell-cell adhesion of the nectin-afadin system by use of a cadherin-deficient L cell line stably expressing the intact form of mouse nectin-2alpha, a truncated form of nectin-2alpha incapable of interacting with afadin (nectin-2alpha-DeltaC), or a point-mutated form of nectin-2alpha capable of interacting with afadin and a cadherin-expressing EL cell line, which transiently expressed the point-mutated form of nectin-2alpha. We found that the interaction of nectin-2alpha with afadin was necessary for their clustering at cell-cell contact sites. However, nectin-2alpha-DeltaC showed cis dimerization and trans interaction, both of which did not require the interaction of nectin-2alpha with afadin. We have previously shown in EL cells that the interaction of nectin-1 with afadin is necessary for its recruitment to adherens junctions. We found that the trans interaction of nectin-2alpha was furthermore necessary for this recruitment. On the basis of these observations, we propose a model for the mechanism of cell-cell adhesion of nectin and roles of afadin in this mechanism.     

5T4 Interacts with TIP-2/GIPC, a PDZ Protein, with Implications for Metastasis

Overexpression of the 5T4 transmembrane glycoprotein can have marked effects on both the actin cytoskeleton and cell migration. Using a yeast two-hybrid approach, we describe a novel interaction between 5T4 and TIP-2/GIPC, a cytoplasmic interacting protein containing a PDZ domain. The cytoplasmic tail of 5T4 contains a class I PDZ-binding motif (Ser-Asp-Val) and we demonstrate that this region, in particular the terminal valine, is required for 5T4 interaction with TIP-2/GIPC. HeLa cells expressing hemagglutinin-tagged TIP-2/GIPC (HA-TIP-2/GIPC) have an altered distribution of endogenous 5T4, which colocalizes with HA-TIP-2/GIPC, thus supporting an interaction. Furthermore, TIP-2/GIPC can be coimmunoprecipitated with 5T4 from HeLa cell lysates. Identification of the 5T4 and TIP-2/GIPC interaction provides the first link between 5T4 and the actin cytoskeleton. Since other proteins, like 5T4, associate with TIP-2/GIPC and are linked with cancer, we explore the possibility that TIP-2/GIPC may be a common factor involved in the cancer process.     

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.     

Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.