Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.     

High-avidity monoclonal antibodies against the human scavenger class B type I receptor efficiently block hepatitis C virus infection in the presence of high-density lipoprotein

The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformation-dependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.     

Inactivation of a plastid evolutionary conserved gene affects PSII electron transport, life span and fitness of tobacco plants

Chloroplasts contain a plastoquinone-NADH-oxidoreductase (Ndh) complex involved in protection against stress and the maintenance of cyclic electron flow. Inactivation of the Ndh complex delays the development of leaf senescence symptoms. Chlorophyll a fluorescence measurements, blue native gel electrophoresis, immunodetection and other techniques were employed to study tobacco (Nicotiana tabacum) Ndh-defective mutants (DeltandhF). The DeltandhF mutants compared with wild-type plants presented: (i) higher photosystem II : photosystem I (PSII : PSI) ratios; (ii) similar or higher levels of ascorbate, carotenoids, thylakoid peroxidase and superoxide dismutase, yield (Phi(PSII)) and maximal photochemical efficiency of PSII levels (F(v)/F(m)) than wild-type plant leaves of the same age; (iii) lower values of nonphotochemical quenching yield (Phi(NPQ)), but not at very high light intensities or during induced leaf senescence; (iv) a similar decrease of antioxidants during senescence; (v) no significant differences in the total foliar area and apical growth rate; and (vi) a production of viable seeds significantly higher than wild-type plants. These results suggest that the Ndh complex is involved in one of the redundant mechanisms that play a safety role in photosynthesis under stress, which has been conserved during evolution, but that its deletion increases fitness when plants are grown under favourable controlled conditions.     

Glycogen synthase binds to sarcoplasmic reticulum and is phosphorylated by CaMKII in fast-twitch skeletal muscle

We investigated the subcellular localization of glycogen synthase (GS) in the adductor muscle of anesthetized rabbits injected intravenously with propranolol. Under these experimental conditions, glycogen content was about 10 mmol/kg of fresh tissue. Immunofluorescent and fractionation studies showed that GS associated with sarcoplasmic reticulum (SR) membranes. Glycogen and GS always co-sedimented, suggesting a predominant role of glycogen in targeting of GS to SR. SR-associated GS was phosphorylated in vitro by SR-bound Ca2+-calmodulin dependent protein kinase (CaMKII) and dephosphorylated by endogenous protein phosphatase 1 (PP1c). Based on measurements of GS activity ratio, in vitro phosphorylation of GS by CaMKII did not significantly affect GS activity per se. However, GS activity ratio was slightly reduced, when SR membranes were further incubated with ATP after prior phosphorylation by CaMKII, suggesting that CaMKII might act sinergistically with other protein kinases. We propose that SR-bound CaMKII plays a role in regulation of glycogen metabolism in skeletal muscle, when intracellular Ca2+ is raised.     

Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice

Human autosomal recessive ichthyosis with hypotrichosis (ARIH) is an inherited disorder recently linked to homozygosity for a point mutation in the ST14 gene that causes a G827R mutation in the matriptase serine protease domain (G216 in chymotrypsin numbering). Here we show that human G827R matriptase has strongly reduced proteolytic activity toward small molecule substrates, as well as toward its candidate epidermal target, prostasin. To further investigate the possible contribution of low matriptase activity to ARIH, we generated an ST14 hypomorphic mouse strain that displays a 100-fold reduction in epidermal matriptase mRNA levels. Interestingly, unlike ST14 null mice, ST14 hypomorphic mice were viable and fertile but displayed a spectrum of abnormalities that strikingly resembled ARIH. Thus, ST14 hypomorphic mice developed hyperproliferative and retention ichthyosis with impaired desquamation, hypotrichosis with brittle, thin, uneven, and sparse hair, and tooth defects. Biochemical analysis of ST14 hypomorphic epidermis revealed reduced prostasin proteolytic activation and profilaggrin proteolytic processing, compatible with a primary role of matriptase in this process. This work strongly indicates that reduced activity of a matriptase-prostasin proteolytic cascade is the etiological origin of human ARIH and provides an important mouse model for the exploration of matriptase function in ARIH, as well as multiple other physiological and pathological processes.     

Characterization of the gene <Emphasis Type="Italic">Mre11</Emphasis> and evidence of silencing after polyploidization in <Emphasis Type="Italic">Triticum</Emphasis>

The MRE11 protein is a component of the highly conserved MRN complex, along with RAD50 and NBS1. This complex is crucial in the repair of breaks in double stranded DNA, and is involved in many other cell processes. The present paper reports the molecular characterization of Mre11 gene in all three genomes of wheat, making use of the diploid species Triticum monococcum (genome A) and Aegilops Tauschii (genome D), the tetraploid T. turgidum (genomes A and B), and the hexaploid T. aestivum (genomes A, B and D). The genomic sequences characterized ranged from 4,662 to 4,766 bp in length; the cDNA corresponding to the processed mRNA was 2,440–2,510 bp long. In all cases, Mre11 coded for a highly conserved protein of 699 amino acids with a structure involving 22 exons. Mre11 expression was determined by real-time PCR in all the species analysed. The tetraploid species showed an expression similar to that of the diploid Ae. tauschii and lower than that of T. monococcum. Stronger expression was detected in the hexaploid T. aestivum. The SSCP technique was modified by introducing fluorescent labelling to the procedure in order to analyse the expression of the different Mre11 genes (i.e., those belonging to the different genomes) in the polyploid species. In both polyploids, the Mre11 gene belonging to the B genome was the least expressed. This probably reflects a first step in the process of silencing duplicate genes after polyploidization.     

Growth rate of a non-fermentative <Emphasis Type="Italic">Escherichia coli</Emphasis> strain is influenced by NAD<Superscript>+</Superscript> regeneration

By complementing a non-fermentative Escherichia coli (ldhA ⁻ pflB ⁻) strain with the recombinant Zymomonas mobilis ethanol pathway (pdc, adhB), we evaluated the effect of different levels of enzymatic activity on growth rate demonstrating that there is a direct relationship between anaerobic growth rate and the total specific activity of pyruvate decarboxylase, which is the limiting enzyme of this specific fermentative NAD⁺ regenerating pathway. This relationship was proved to be useful to establish a selection strategy based on growth rate for the analysis of lctE libraries, which encode lactate dehydrogenase from Bacillus subtilis.     

Peripheral ghrelin interacts with orexin neurons in glucostatic signalling

Ghrelin interactions with glycemia in appetite control as well as the potential mechanisms involving the orexin and melanin-concentrating hormone (MCH) neurons in the orexigenic ghrelin signals were investigated by using a specific anti-ghrelin antibody (AGA). Our results confirm that peripheral ghrelin is an important signal in meal initiation and appetite. Employing immunohistochemistry techniques, we found that c-fos positive neurons in the lateral hypothalamus (LH) and perifornical area (PFA) increased after insulin or 2-deoxyglucose administration. Moreover, we have also demonstrated that peripheral ghrelin blockade by the AGA, reduces the orexigenic signal induced by insulin and 2-DG administration probably partly producing a decrease of c-fos immunoreactivity in the LH and PFA as well as a lower activation of orexin neurons. In contrast, the c-fos positive MCH neurons were not apparently affected. In summary, our findings suggest that peripheral ghrelin plays an important role in regulatory "glucostatic" feeding mechanisms by means of its role as a "hunger" signal affecting the LH and PFA areas, which may contribute to energy homeostasis through orexin neurons.