Identification of a spectrin-like protein in Sertoli cells

Sertoli cells prepared from rats ages 15 and 25 days were shown to contain a spectrin-like protein. Indirect immunofluorescence with monospecific antimouse erythrocyte immunoglobulin G (IgG) and with monospecific antimouse brain spectrin IgG revealed specific staining in Sertoli cells. Both antibodies precipitated two spectrin-like peptides of 240,000 and 235,000 daltons from cells solubilized with octyl glucoside. Proteins from Sertoli cell membranes were separated by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose membrane. Incubation of nitrocellulose membrane with either of the two antibodies, followed by horseradish peroxidase conjugated to second antibody, revealed only the larger, or alpha, spectrin subunit (Western blots). Both antibodies were used to provide immunoautoradiographic identification of the spectrin-like protein. In this procedure, spectrin and Sertoli cell membranes were shown to compete with [125I]-labeled spectrin from mouse erythrocytes for binding to antimouse erythrocyte spectrin IgG. Finally, two-dimensional proteolytic mapping of the 240,000- and 235,000-dalton peptides demonstrated limited spot homology with rat erythrocyte spectrin. However, subcellular fractions from Sertoli cells all contained a spectrin-like protein showing high homology from fraction to fraction. It is concluded that Sertoli cells contain a spectrin-like protein that is seen in cell fractions prepared by centrifugation, i.e., mitochondria, microsomes, nuclei, cytoplasm, and plasma membranes. Although homology with spectrin from erythrocytes or brain is not seen in peptide maps, the alpha subunit shares antigenic determinants with spectrin from erythrocytes. The beta subunit is believed to be precipitated by antispectrin as the result of binding to the alpha subunit, since the beta subunit shows no detectable antigenic homology with that of spectrin.     

Global geometric affinity for revealing high fidelity protein interaction network

Protein-protein interaction (PPI) network analysis presents an essential role in understanding the functional relationship among proteins in a living biological system. Despite the success of current approaches for understanding the PPI network, the large fraction of missing and spurious PPIs and a low coverage of complete PPI network are the sources of major concern. In this paper, based on the diffusion process, we propose a new concept of global geometric affinity and an accompanying computational scheme to filter the uncertain PPIs, namely, reduce the spurious PPIs and recover the missing PPIs in the network. The main concept defines a diffusion process in which all proteins simultaneously participate to define a similarity metric (global geometric affinity (GGA)) to robustly reflect the internal connectivity among proteins. The robustness of the GGA is attributed to propagating the local connectivity to a global representation of similarity among proteins in a diffusion process. The propagation process is extremely fast as only simple matrix products are required in this computation process and thus our method is geared toward applications in high-throughput PPI networks. Furthermore, we proposed two new approaches that determine the optimal geometric scale of the PPI network and the optimal threshold for assigning the PPI from the GGA matrix. Our approach is tested with three protein-protein interaction networks and performs well with significant random noises of deletions and insertions in true PPIs. Our approach has the potential to benefit biological experiments, to better characterize network data sets, and to drive new discoveries.     

Identification of a region of FtsA required for interaction with FtsZ

The assembly of the Z ring is the earliest step in bacterial cell division. In Escherichia coli this assembly requires either FtsA or ZipA which bind to a conserved, C-terminal 17 amino acid motif in FtsZ and to the membrane. The FtsZ-ZipA interaction is well characterized; however, nothing is known about the region of FtsA involved in the interaction with FtsZ even though the FtsA-FtsZ interaction is nearly ubiquitous in Eubacteria. FtsA is proposed to bind to the membrane through its conserved C-terminal amphiphatic helix before efficiently interacting with FtsZ. Based upon this model we designed a genetic screen to identify mutants specifically impaired for the FtsA-FtsZ interaction. The mutants obtained retain the ability to be targeted to the membrane but fail to be recruited to the Z ring or interact with FtsZ in the yeast two-hybrid system. These mutants do not complement an ftsA-depletion strain. Through this approach we have identified a region of FtsA containing some invariant residues which is required for binding to FtsZ. The results support our model that FtsA is targeted to the membrane before it interacts with FtsZ and demonstrates that this interaction plays an essential role in E. coli cell division.     

Identification of proteins with high affinity for refolded and native PrPC

PrPC, the cellular prion protein, is widely expressed in most tissues, including brain, muscle and the gastrointestinal tract, but its physiological role remains unclear. During propagation of transmissible spongiform encephalopathies (TSEs), prion protein is converted to the pathological isoform, PrPSc, in a process believed to be mediated by as-yet-unknown host factors. The identification of proteins associated with PrP may provide information about the biology of prions and the pathogenesis of TSEs. In the present work, we report proteins identified from brain tissue based on their ability to bind to recombinant PrP (recPrP) or form multimolecular complexes with native PrPC in the presence of cross-linkers. Immobilized his-tagged recPrP was used as an affinity matrix to isolate PrP-interacting proteins from brain homogenates of normal individuals. In parallel, PrPC-associated proteins were characterized by cross-linking and co-immunoprecipitation assays. The unknown molecules were identified by MS and the results of LC-MS/MS analysis were subsequently verified by Western blot. Both techniques resulted in identification of proteins participating in the formation of cytoskeleton and signal transduction, further supporting the hypothesis that PrP is involved in the organization and function of receptors throughout the nervous system.     

The tandem beta-zipper model defines high affinity fibronectin-binding repeats within Staphylococcus aureus FnBPA

Binding of the fibronectin-binding protein FnBPA from Staphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.     

Alpha-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids

Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to alpha-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of alpha-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in alpha-helix content, but the helix content was somewhat less than in reconstituted discoidal PC.apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of alpha-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that alpha-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.     

Identification of residues required for the interaction of BARD1 with BRCA1.

The breast and ovarian cancer predisposition gene product BRCA1, binds to BARD1 at its N terminus. In cells BRCA1 is found as a heterodimer with BARD1 and may represent the functionally active form of BRCA1. Using yeast two-hybrid and split-hybrid screens we have identified 16 independent missense mutations which prevent the ability of the BARD1 N terminus to heterodimerize with BRCA1. With reference to the recent structure of the BARD1center dotBRCA1 RING complex (Brzovic, P. S., Rajagopal, P., Hoyt, D. W., King, M-C., and Klevit, R. E. (2001) Nat. Struct. Biol. 8, 833--837) we note two classes of mutation; those that map to the hydrophobic core forming the BARD1:BRCA1 interface and are substitutions of leucine, and those that map to residues forming intramolecular contacts either in helical packing, or in the conserved zinc chelating cysteine residues of the RING itself. The directed mutation of charged residues predicted to play a role in the interaction could not alone prevent heterodimer formation suggesting that, while polar interactions may participate in the specificity of the interaction, they are not crucial. Together these data provide functional evidence for the requirement of a hydrophobic interface and illustrate that disruption of the tertiary structure by mutations away from the interface itself are able to prevent formation of the heterodimer.     

The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis

Rhizopus oryzae is the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first‐line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron‐depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidosis (DKA) mice. In addition, we disrupted FTR1 by double cross‐over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti‐Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus, FTR1 is a virulence factor for R. oryzae, and anti‐Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis.     

Identification of a 33-kilodalton cytoskeletal protein with high affinity for the sodium channel

The voltage-sensitive sodium channel is an intrinsic membrane protein that is nonrandomly distributed in neurons, suggesting a possible interaction with other cellular constituents. In this study, we have directly tested the hypothesis that components of the cytoskeleton interact with sodium channels. Utilizing the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blot overlay, we have identified a 33-kilodalton cytoskeletal protein (p33) that binds 32P-labeled sodium channel purified from rat brain. This binding is a high-affinity (KD less than 1 nM) protein-protein interaction that is blocked by low concentrations of unlabeled sodium channels but is not blocked by monosaccharides, the complex glycoprotein fetuin, the transmembrane protein Na+-K+-ATPase, or bovine serum albumin. Levels of p33 are highest in lung and spleen while lower levels are found in brain, peripheral nerve, skeletal muscle, liver, and testes. This tissue distribution implies that the sodium channel may not be the only ligand for p33.     

Identification of molecular determinants required for interaction of ubiquitin-conjugating enzymes and RING finger proteins.

Recent results from several laboratories suggest that the interaction of E2 ubiquitin-conjugating enzymes with the RING finger domain has a central role in mediating the transfer of ubiquitin to proteins. Here we present a mutational analysis of the interaction between the E2 enzyme UbcM4/UbcH7 and three different RING finger proteins, termed UIPs, which, like Parkin, contain a RING1-IBR-RING2 motif. The results show that the E2 enzyme binds to the RING1 domain but not to the other cysteine/histidine-rich domains of the RING1-IBR-RING2 motif. Three regions within the UbcM4 molecule are involved in this interaction: the H1 alpha helix, loop L1, connecting the third and fourth strand of the beta sheet, and loop L2, located between the fourth beta strand and the second alpha helix. Loop L2 plays an important role in determining the specificity of interaction. The effects of L2 mutations on UbcM4/UIP interaction are different for each UIP, indicating that RING finger domains can vary considerably in their structural requirements for binding to E2 enzymes. The result that single amino-acid changes can regulate binding of E2 enzymes to different RING finger proteins suggests a novel approach to experimentally manipulate proteolytic pathways mediated by RING finger proteins.     

Identification of high affinity receptors for human monocyte chemoattractant protein-1 on human monocytes

The binding of human monocyte chemoattractant protein-1 (MCP-1) to human monocytes was studied. MCP-1 was radioiodinated with Iodo-beads (Pierce Chemical Co., Rockford, IL) without significant loss of biologic activity. 125I-MCP-1 binding to PBMC occurred within 5 min at 0 degrees C and the binding was inhibited by unlabeled MCP-1 dose dependently but not by neutrophil attractant/activation protein-1 or FMLP. 125I-MCP-1 bound to monocytes; no significant binding to either neutrophils or lymphocytes was observed. Scatchard plot analysis indicated that monocytes had a minimum of 1700 +/- 600 binding sites per cell with a Kd of 1.9 +/- 0.2 x 10(-9) M. For analysis of binding by flow cytometry, MCP-1 was biotinylated. In contrast to radioiodination, biotinylation resulted in loss of activity; potency was 10-fold less, but the efficacy was retained. Detection by flow cytometry of bound biotinylated MCP-1 with avidin-FITC confirmed results obtained with 125I-MCP-1. Biotinylated MCP-1 bound to monocytes but not to lymphocytes; and the binding was inhibited by a 100-fold excess of unlabeled MCP-1.     

Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay

The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.     

Identification of a surface protein of the rabbit blood platelet with high affinity for collagen

Platelet membrane components adhering with high affinity to collagen fibers were studied by means of an affinity column in which fibrillar type I collagen was physically immobilized. Intact rabbit platelets in 1 mM EGTA adhered to the column but did not aggregate. Adhesion was dependent on the collagen concentration and on the number of platelets applied. Passage through the column without adhesion did not affect the potential for subsequent platelet binding. Surface-labelled whole platelets were passaged through this column, lysed in Triton and in SDS and labelled components adhering to the collagen were analysed on SDS-polyacrylamide gels. It was found that Triton lysis removed most of the major surface glycoproteins but left the cytoskeleton on the column. Subsequent SDS elution removed the cytoskeletal proteins along with the remaining major surface glycoproteins. The label left on the column could not be eluted with 8 M urea or up to 4 M NaCl. Collagenase digestion of the column collagen released a single surface glycoprotein of Mr 80,000. Limited chymotryptic digestion of the labelled platelets prior to their application to the column did not affect their binding. A radiolabelled band of the same molecular weight (MW) became bound to the collagen following passage of the chymotrypsin-treated platelets. This band was trypsin-sensitive following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These results, along with other published evidence, suggest that at least one platelet membrane component, expressed on the surface of the unstimulated platelet, binds with high affinity to fibrillar type I collagen and is probably involved in platelet collagen recognition.     

Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.     

Identification of novel endogenous cytochrome p450 arachidonate metabolites with high affinity for cannabinoid receptors

Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.     

Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction

Members of the hect domain protein family are characterized by sequence similarity of their C-terminal regions to the C terminus of E6-AP, an E3 ubiquitin-protein ligase. An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between E6-AP and ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSP5, form ubiquitin thioester complexes, indicating that hect domain proteins in general have E3 activity. We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RSP5. Of particular importance is a phenylalanine residue at position 62 of UbcH5 that is conserved among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions. The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with hect domain proteins for the cellular function of UBC4/UBC5 subfamily members.