Structure-function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody

The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide binding to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the K(d) values of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses. Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH(2) could effectively inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting 2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have significantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative development of a vaccine based on the 4E10 epitope.     

Cortisol diurnal patterns,associations with depressive symptoms,and the impact of intervention in older adults: Results using modern robust methods aimed at dealing with low power due to violations of standard assumptions

Advances in salivary bioscience enable the widespread integration of biological measures into the behavioral and social sciences. While theoretical integration has progressed, much less attention has focused on analytical strategies and tactics. The statistical literature warns that common methods for comparing groups and studying associations can have relatively poor power compared to more modern robust techniques. Here we illustrate, in secondary data analyses using the USC Well Elderly II study (n = 460, age 60–95, 66% female), that modern robust methods make a substantial difference when analyzing relations between salivary analyte and behavioral data. Analyses that deal with the diurnal pattern of cortisol and the association of the cortisol awakening response with depressive symptoms and physical well-being are reported. Non-significant results become significant when using improved methods for dealing with skewed distributions and outliers. Analytical strategies and tactics that employ modern robust methods have the potential to reduce the probability of both Type I and Type II errors in studies that compare salivary analytes between groups, across time, or examine associations with salivary analyte levels.     

The Cellular Prion Protein Interacts with the Tissue Non-Specific Alkaline Phosphatase in Membrane Microdomains of Bioaminergic Neuronal Cells

Background

The cellular prion protein, PrP^C, is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP^C in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP^C acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP^C, we have described a neuronal specificity pointing to a role of PrP^C in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11^5-HT) or noradrenergic (1C11^NE) derivatives.

Methodology/Principal Findings

The neuronal specificity of PrP^C signaling prompted us to search for PrP^C partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP^C with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11^5-HT and 1C11^NE cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11^5-HT and 1C11^NE bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP.

Conclusion/Significance

The identification of a novel PrP^C partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP^C and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP^C-laminin interplay. The partnership between TNAP and PrP^C in neuronal cells may provide new clues as to the neurospecificity of PrP^C function.     

Recombinant Human Collagen XV Regulates Cell Adhesion and Migration

The C-terminal end of collagen XV, restin, has been the focus of several studies, but the functions of full-length collagen XV have remained unknown. We describe here studies on the production, purification, and function of collagen XV and the production of a monoclonal N-terminal antibody to it. Full-length human collagen XV was produced in insect cells using baculoviruses and purified from the cell culture medium. The yield was 15 mg/liter of cell culture medium. The collagen XV was shown to be trimeric, with disulfide bonds in the collagenous region. Rotary shadowing electron microscopy revealed rod-like molecules with a mean length of 241.8 nm and with a globular domain at one end. The globular domain was verified to be the N-terminal end by N-terminal antibody binding. The molecules show flexibility in their conformation, presumably due to the many interruptions in their collagenous domains. The ability of collagen XV to serve as a substrate for cells was tested in cell adhesion assays, and it was shown that cells did not bind to collagen XV-coated surfaces. When added to the culture medium of fibroblasts and fibrosarcoma cells, however, collagen XV rapidly bound to their fibronectin network. Solid phase assays showed that collagen XV binds to fibronectin, laminin, and vitronectin and that it binds to the collagen/gelatin-binding domain of fibronectin. No binding was detected to fibrillar collagens, fibril-associated collagens, or decorin. Interestingly, collagen XV was found to inhibit the adhesion and migration of fibrosarcoma cells when present in fibronectin-containing matrices.     

Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B₁), and pyridoxine (vitamin B₆) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.     

Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.     

Another look at the interaction between mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> and flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>

Yeast flavocytochrome b ₂ tranfers reducing equivalents from lactate to oxygen via cytochrome c and cytochrome c oxidase. The enzyme catalytic cycle includes FMN reduction by lactate and reoxidation by intramolecular electron transfer to heme b ₂. Each subunit of the soluble tetrameric enzyme consists of an N terminal b ₅-like heme-binding domain and a C terminal flavodehydrogenase. In the crystal structure, FMN and heme are face to face, and appear to be in a suitable orientation and at a suitable distance for exchanging electrons. But in one subunit out of two, the heme domain is disordered and invisible. This raises a central question: is this mobility required for interaction with the physiological acceptor cytochrome c, which only receives electrons from the heme and not from the FMN? The present review summarizes the results of the variety of methods used over the years that shed light on the interactions between the flavin and heme domains and between the enzyme and cytochrome c. The conclusion is that one should consider the interaction between the flavin and heme domains as a transient one, and that the cytochrome c and the flavin domain docking areas on the heme b ₂ domain must overlap at least in part. The heme domain mobility is an essential component of the flavocytochrome b ₂ functioning. In this respect, the enzyme bears similarity to a variety of redox enzyme systems, in particular those in which a cytochrome b ₅-like domain is fused to proteins carrying other redox functions.