Reversible and efficient activation of HIV-1 cell entry by a tyrosine-sulfated peptide dissects endocytic entry and inhibitor mechanisms.

UNLABELLED: HIV-1 membranes contain gp120-gp41 trimers. Binding of gp120 to CD4 and a coreceptor (CCR5 or CXCR4) reduces the constraint on metastable gp41, enabling a series of conformational changes that cause membrane fusion. An analytic difficulty occurs because these steps occur slowly and asynchronously within cohorts of adsorbed virions. We previously isolated HIV-1JRCSF variants that efficiently use CCR5 mutants severely damaged in the tyrosine-sulfated amino terminus or extracellular loop 2. Surprisingly, both independent adaptations included gp120 mutations S298N, F313L, and N403S, supporting other evidence that they function by weakening gp120's grip on gp41 rather than by altering gp120 binding to specific CCR5 sites. Although several natural HIV-1 isolates reportedly use CCR5(Δ18) (CCR5 with a deletion of 18 N-terminal amino acids, including the tyrosine-sulfated region) when the soluble tyrosine-sulfated peptide is present, we show that HIV-1JRCSF with the adaptive mutations [HIV-1JRCSF(Ad)] functions approximately 100 times more efficiently and that coreceptor activation is reversible, enabling synchronous efficient entry control under physiological conditions. This system revealed that three-stranded gp41 folding intermediates susceptible to the inhibitor enfuvirtide form slowly and asynchronously on cell surface virions but resolve rapidly, with virions generally forming only one target. Adsorbed virions asynchronously and transiently become competent for entry at 37°C but are inactivated if the CCR5 peptide is absent during their window of opportunity. This competency is conferred by endocytosis, which results in inactivation if the peptide is absent. For both wild-type and adapted HIV-1 isolates, early gp41 refolding steps obligatorily occur on cell surfaces, whereas the final step(s) is endosomal. This system powerfully dissects HIV-1 entry and inhibitor mechanisms. IMPORTANCE: We present a powerful means to reversibly and efficiently activate or terminate HIV-1 entry by adding or removing a tyrosine-sulfated CCR5 peptide from the culture medium. This system uses stable cell clones and a variant of HIV-1JRCSF with three adaptive mutations. It enabled us to show that CCR5 coreceptor activation is rapidly reversible and to dissect aspects of entry that had previously been relatively intractable. Our analyses elucidate enfuvirtide (T-20) function and suggest that HIV-1 virions form only one nonredundant membrane fusion complex on cell surfaces. Additionally, we obtained novel and conclusive evidence that HIV-1 entry occurs in an assembly line manner, with some steps obligatorily occurring on cell surfaces and with final membrane fusion occurring in endosomes. Our results were confirmed for wild-type HIV-1. Thus, our paper provides major methodological and mechanistic insights about HIV-1 infection.

Kinetic mechanism for HIV-1 neutralization by antibody 2G12 entails reversible glycan binding that slows cell entry.

Despite structural knowledge of broadly neutralizing monoclonal antibodies (NMAbs) complexed to HIV-1 gp120 and gp41 envelope glycoproteins, virus inactivation mechanisms have been difficult to prove, in part because neutralization assays are complex and were previously not understood. Concordant with recent evidence that HIV-1 titers are determined by a race between entry of cell-attached virions and competing inactivation processes, we show that NMAb 2G12, which binds to gp120 N-glycans with α (1, 2)-linked mannose termini and inhibits replication after passive transfer into patients, neutralizes by slowing entry of adsorbed virions. Accordingly, apparent neutralization is attenuated when a kinetically competing virus inactivation pathway is blocked. Moreover, removing 2G12 from media causes its dissociation from virions coupled to accelerated entry and restored infectivity, demonstrating the reversibility of neutralization. A difference between 2G12 dissociation and infectivity recovery rates implies that the inhibited complexes at virus-cell junctions contain several 2G12's that must dissociate before entry commences. Quantitative microscopy of 2G12 binding and dissociation from single virions and studies using a split CCR5 coreceptor suggest that 2G12 competitively inhibits interactions between gp120's V3 loop and the tyrosine sulfate-containing CCR5 amino terminus, thereby reducing assembly of complexes that catalyze entry. These results reveal a unique reversible kinetic mechanism for neutralization by an antibody that binds near a critical V3 region in the glycan shield of gp120.

OregonFluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues.

Non-destructive fluorophore diffusion across cell membranes to provide an unbiased fluorescence intensity readout is critical for quantitative imaging applications in live cells and tissues. Commercially available small-molecule fluorophores have been engineered for biological compatibility, imparting high water solubility by modifying rhodamine and cyanine dye scaffolds with multiple sulfonate groups. The resulting net negative charge, however, often renders these fluorophores cell-membrane-impermeant. Here we report the design and development of our biologically compatible, water-soluble and cell-membrane-permeable fluorophores, termed OregonFluor (ORFluor). By adapting previously established ratiometric imaging methodology using bio-affinity agents, it is now possible to use small-molecule ORFluor-labelled therapeutic inhibitors to quantitatively visualize their intracellular distribution and protein target-specific binding, providing a chemical toolkit for quantifying drug target availability in live cells and tissues.

Computational Modeling of TP63-TP53 Interaction and Rational Design of Inhibitors: Implications for Therapeutics.

Tumor protein p63 (TP63) is a member of the TP53 protein family that are important for development and in tumor suppression. Unlike TP53, TP63 is rarely mutated in cancer, but instead different TP63 isoforms regulate its activity. TA isoforms (TAp63) act as tumor suppressors, whereas ΔN isoforms are strong drivers of squamous or squamous-like cancers. Many of these tumors become addicted to ΔN isoforms and removal of ΔN isoforms result in cancer cell death. Furthermore, some TP53 conformational mutants (TP53CM) gain the ability to interact with TAp63 isoforms and inhibit their antitumorigenic function, while indirectly promoting tumorigenic function of ΔN isoforms, but the exact mechanism of TP63-TP53CM interaction is unclear. The changes in the balance of TP63 isoform activity are crucial to understanding the transition between normal and tumor cells. Here, we modeled TP63-TP53CM complex using computational approaches. We then used our models to design peptides to disrupt the TP63-TP53CM interaction and restore antitumorigenic TAp63 function. In addition, we studied ΔN isoform oligomerization and designed peptides to inhibit its oligomerization and reduce their tumorigenic activity. We show that some of our peptides promoted cell death in a TP63 highly expressed cancer cell line, but not in a TP63 lowly expressed cancer cell line. Furthermore, we performed kinetic-binding assays to validate binding of our peptides to their targets. Our computational and experimental analyses present a detailed model for the TP63-TP53CM interaction and provide a framework for potential therapeutic peptides for the elimination of TP53CM cancer cells.

Turning antibodies off and on again using a covalently tethered blocking peptide.

In their natural form, antibodies are always in an "on-state" and are capable of binding to their targets. This leads to undesirable interactions in a wide range of therapeutic, analytical, and synthetic applications. Modulating binding kinetics of antibodies to turn them from an "off-state" to an "on-state" with temporal and spatial control can address this. Here we demonstrate a method to modulate binding activity of antibodies in a predictable and reproducible way. We designed a blocking construct that uses both covalent and non-covalent interactions with the antibody. The construct consisted of a Protein L protein attached to a flexible linker ending in a blocking-peptide designed to interact with the antibody binding site. A mutant Protein L was developed to enable photo-triggered covalent crosslinking to the antibody at a specific location. The covalent bond anchored the linker and blocking peptide to the antibody light chain keeping the blocking peptide close to the antibody binding site. This effectively put the antibody into an "off-state". We demonstrate that protease-cleavable and photocleavable moieties in the tether enable controlled antibody activation to the "on-state" for anti-FLAG and cetuximab antibodies. Protein L can bind a range of antibodies used therapeutically and in research for wide applicability.

Molecular modelling of the FOXO4-TP53 interaction to design senolytic peptides for the elimination of senescent cancer cells.

BACKGROUND: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. METHODS: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. FINDINGS: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. INTERPRETATION: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. FUNDING: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University.

Recognition of galactose-deficient O-glycans in the hinge region of IgA1 by N-acetylgalactosamine-specific snail lectins: a comparative binding study.

Aberrancies in IgA1 glycosylation have been linked to the pathogenesis of IgA nephropathy (IgAN), a kidney disease characterized by deposits of IgA1-containing immune complexes in the glomerular mesangium. IgA1 from IgAN patients is characterized by the presence of galactose (Gal)-deficient O-glycans in the hinge region that can act as epitopes for anti-glycan IgG or IgA1 antibodies. The resulting circulating immune complexes are trapped in the glomerular mesangium of the kidney where they trigger localized inflammatory responses by activating mesangial cells. Certain lectins recognize the terminal N-acetylgalactosamine (GalNAc)-containing O-glycans on Gal-deficient IgA1 and can be potentially used as diagnostic tools. To improve our understanding of GalNAc recognition by these lectins, we have conducted binding studies to assess the interaction of Helix aspersa agglutinin (HAA) and Helix pomatia agglutinin (HPA) with Gal-deficient IgA1. Surface plasmon resonance spectroscopy revealed that both HAA and HPA bind to a Gal-deficient synthetic hinge region glycopeptide (HR-GalNAc) as well as various aberrantly glycosylated IgA1 myeloma proteins. Despite having six binding sites, both HAA and HPA bind IgA1 in a functionally bivalent manner, with the apparent affinity for IgA1 related to the number of exposed GalNAc groups in the IgA1 hinge. Finally, HAA and HPA were shown to discriminate very effectively between the IgA1 secreted by cell lines derived from peripheral blood cells of patients with IgAN and that from cells of healthy controls. These studies provide insight into lectin recognition of the Gal-deficient IgA1 hinge region and lay the groundwork for the development of reliable diagnostic tools for IgAN.

Analysis of IgA1 N-glycosylation and its contribution to FcalphaRI binding.

The IgA isotype of human antibodies triggers inflammatory responses via the IgA-specific receptor FcalphaRI (CD89). Structural studies have suggested that IgA1 N-glycans could modulate the interaction with FcalphaRI. We have carried out detailed biophysical analyses of three IgA1 samples purified from human serum and recombinant IgA1-Fc and compared their binding to FcalphaRI. Analytical ultracentrifugation revealed wide variation in the distribution of polymeric species between IgA1 samples, and Fourier transform ion cyclotron resonance mass spectrometry showed overlapping but distinct populations of N-glycan species between IgA1 samples. Kinetic and equilibrium data from surface plasmon resonance experiments revealed that variation in the IgA1 C H2 N-glycans had no effect on the kinetics or affinity constants for binding to FcalphaRI. Indeed, complete enzymatic removal of the IgA1 N-glycans yielded superimposable binding curves. These findings have implications for renal diseases such as IgA nephropathy.

Ultrafiltered recombinant AAV8 vector can be safely administered in vivo and efficiently transduces liver.

Viral vectors are extensively purified for use in biomedical research, in order to separate biologically active virus particles and to eliminate production related impurities that are assumed to be detrimental to the host. For recombinant adeno-associated virus (rAAV) vectors this is typically accomplished using density gradient-based methods, which are tedious and require specialized ultracentrifugation equipment. In order to streamline the preparation of rAAV vectors for pilot and small animal studies, we recently devised a simple ultrafiltration approach that permits rapid virus concentration and partial removal of production-related impurities. Here we show that systemic administration of such rapidly prepared (RP) rAAV8 vectors in mice is safe and efficiently transduces the liver. Across a range of doses, delivery of RP rAAV8-CMV-eGFP vector induced enhanced green fluorescent protein (eGFP) expression in liver that was comparable to that obtained from a conventional iodixanol gradient-purified (IP) vector. Surprisingly, no liver inflammation or systemic cytokine induction was detected in RP rAAV injected animals, revealing that residual impurities in the viral vector preparation are not deleterious to the host. Together, these data demonstrate that partially purified rAAV vector can be safely and effectively administered in vivo. The speed and versatility of the RP method and lack of need for cumbersome density gradients or expensive ultracentrifuge equipment will enable more widespread use of RP prepared rAAV vectors, such as for pilot liver gene transfer studies.

A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders.

Multimeric scaffolds displaying the HIV-1 envelope MPER induce MPER-specific antibodies and cross-neutralizing antibodies when co-immunized with gp160 DNA.

Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.

HIV-1 envelope glycoprotein resistance to monoclonal antibody 2G12 is subject-specific and context-dependent in macaques and humans.

HIV-1 Envelope (Env) protein is the sole target of neutralizing antibodies (NAbs) that arise during infection to neutralize autologous variants. Under this immune pressure, HIV escape variants are continuously selected and over the course of infection Env becomes more neutralization resistant. Many common alterations are known to affect sensitivity to NAbs, including residues encoding potential N-linked glycosylation sites (PNGS). Knowledge of Env motifs associated with neutralization resistance is valuable for the design of an effective Env-based vaccine so we characterized Envs isolated longitudinally from a SHIV(SF162P4) infected macaque for sensitivity to neutralizing monoclonal antibodies (MAbs) B12, 2G12, 4E10 and 2F5. The early Env, isolated from plasma at day 56 after infection, was the most sensitive and the late Env, from day 670, was the most resistant to MAbs. We identified four PNGS in these Envs that accumulated over time at positions 130, 139, 160 and 397. We determined that removal of these PNGS significantly increased neutralization sensitivity to 2G12, and conversely, we identified mutations by in silico analyses that contributed resistance to 2G12 neutralization. In order to expand our understanding of these PNGS, we analyzed Envs from clade B HIV-infected human subjects and identified additional glycan and amino acid changes that could affect neutralization by 2G12 in a context-dependent manner. Taken together, these in vitro and in silico analyses of clade B Envs revealed that 2G12 resistance is achieved by previously unrecognized PNGS substitutions in a context-dependent manner and by subject-specific pathways.

Fluorescence resonance energy transfer analysis of merlin conformational changes.

Neurofibromatosis type 2 is an inherited autosomal disorder caused by biallelic inactivation of the NF2 tumor suppressor gene. The NF2 gene encodes a 70-kDa protein, merlin, which is a member of the ezrin-radixin-moesin (ERM) family. ERM proteins are believed to be regulated by a transition between a closed conformation, formed by binding of their N-terminal FERM domain and C-terminal tail domain (CTD), and an open conformation, in which the two domains do not interact. Previous work suggests that the tumor suppressor function of merlin is similarly regulated and that only the closed form is active. Therefore, understanding the mechanisms that control its conformation is crucial. We have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer, both as purified protein and in live cells. Using these tools, we find that merlin exists predominately as a monomer in a stable, closed conformation that is mediated by the central alpha-helical domain. The contribution from the FERM-CTD interaction to the closed conformation appears to be less important. Upon phosphorylation or interaction with an effector protein, merlin undergoes a subtle conformational change, suggesting a novel mechanism that modulates the interaction between the FERM domain and the CTD.

Structural basis of nucleotide exchange and client binding by the Hsp70 cochaperone Bag2.

Cochaperones are essential for Hsp70- and Hsc70-mediated folding of proteins and include nucleotide-exchange factors (NEFs) that assist protein folding by accelerating ADP-ATP exchange on Hsp70. The cochaperone Bag2 binds misfolded Hsp70 clients and also acts as an NEF, but the molecular basis for its function is unclear. We show that, rather than being a member of the Bag domain family, Bag2 contains a new type of Hsp70 NEF domain, which we call the 'brand new bag' (BNB) domain. Free and Hsc70-bound crystal structures of Bag2-BNB show its dimeric structure, in which a flanking linker helix and loop bind to Hsc70 to promote nucleotide exchange. NMR analysis demonstrates that the client binding sites and Hsc70-interaction sites of the Bag2-BNB overlap, and that Hsc70 can displace clients from Bag2-BNB, indicating a distinct mechanism for the regulation of Hsp70-mediated protein folding by Bag2.

Representações sociais de paternidade na revista Pais & Filhos (1969-2008) / Social representations of fatherhood in Pais & filhos magazine (1969-2008)

O objetivo dessa investigação foi identificar e analisar as representações sociais de paternidade veiculadas pela revista Pais & Filhos entre 1969 e 2008. Foram selecionados, para a análise, 40 exemplares dessa revista (um exemplar por ano; seleção aleatória do mês). Desse conjunto, foram transcritos todos os 190 itens (reportagens, entrevistas, colunas de especialistas, anúncios publicitários) que apresentavam os vocábulos “pais”, “pai”, “paternidade”, “paternal” e variantes. O corpus formado foi submetido à análise lexical realizada pelo software ALCESTE. Foram identificadas as seguintes classes de formas reduzidas: “nós, as crianças e nossas histórias”, “relacionando-se com crianças e adolescentes”, “terapia”, “as doenças dos bebês” e “pai jurídico”. Em seu conjunto, os resultados indicam que, no corpus analisado, a representaçãosocial de paternidade é composta por elementos que concedem ao pai o lugar de coadjuvante ou, mais recorrentemente, o identificam como uma referência problemática (causador de problemas ou como eventual possibilidade de solução).

This research aimed to identify and analyze the social representations of fatherhood conveyed by the Pais & Filhos magazine from 1969 to 2008. Forty issues of the magazine were selected for analysis (one piece per year, at random selection of the month). From this group, all 190 items (reports, interviews, expert columns and advertisements) that showed the words “parents”, “father”, “paternity”, parental” and variants were transcribed. The corpus formed was subjected to lexical analysis in the software ALCESTE. We identified the following classes of reduced form: "we, the children and our stories," “interacting with children and adolescents", "therapy", "babies’ diseases” and " “legal father". Taken together, these results indicate that in the corpus examined, the social representation of fatherhood is made of elements that give the father the supporting role, or more frequently, identify the father as a problematic reference (troublemaker or as an eventual possibility of solution).

IgA and IgA-specific receptors in human disease: structural and functional insights into pathogenesis and therapeutic potential.

IgA antibodies play an important role in humoral immunity. IgA is the predominant antibody in mucosal secretions and the second most prevalent in the serum. It occupies a unique position among human antibodies in that it can both trigger and suppress inflammatory responses, depending on the situation. Recent structural and functional studies have revealed details of the structure of IgA and its interaction with key cell-surface receptors. We look at the role IgA and IgA receptors (particularly FcalphaRI) play in the pathogenesis of diseases such as IgA nephropathy and other autoimmune conditions. Finally, we address the potential of IgA as a therapeutic tool to either trigger specific inflammatory responses to destroy target cells or suppress inflammatory responses in the case of autoimmune diseases, and the promise of mucosal vaccines for eliciting specific IgA responses to pathogens in mucosal environments.