Conformational Isomerization Involving Conserved Proline Residues Modulates Oligomerization of the NS1 Interferon Response Inhibitor from the Syncytial Respiratory Virus

Interferon response suppression by the respiratory syncytial virus relies on two unique nonstructural proteins, NS1 and NS2, that interact with cellular partners through high-order complexes. We hypothesized that two conserved proline residues, P81 and P67, participate in the conformational change leading to oligomerization. We found that the molecular dynamics of NS1 show a highly mobile C-terminal helix, which becomes rigid upon in silico replacement of P81. A soluble oligomerization pathway into regular spherical structures at low ionic strengths competes with an aggregation pathway at high ionic strengths with an increase in temperature. P81A requires higher temperatures to oligomerize and has a small positive effect on aggregation, while P67A is largely prone to aggregation. Chemical denaturation shows a first transition, involving a high fluorescence and ellipticity change corresponding to both a conformational change and substantial effects on the environment of its single tryptophan, that is strongly destabilized by P67A but stabilized by P81A. The subsequent global cooperative unfolding corresponding to the main β-sheet core is not affected by the proline mutations. Thus, a clear link exists between the effect of P81 and P67 on the stability of the first transition and oligomerization/aggregation. Interestingly, both P67 and P81 are located far away in space and sequence from the C-terminal helix, indicating a marked global structural dynamics. This provides a mechanism for modulating the oligomerization of NS1 by unfolding of a weak helix that exposes hydrophobic surfaces, linked to the participation of NS1 in multiprotein complexes.Fil: Conci, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Álvarez Paggi, Damián Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: de Oliveira, Guilherme A. P.. Universidade Federal do Rio de Janeiro; BrasilFil: Pagani, Talita Duarte. Universidade Federal do Rio de Janeiro; BrasilFil: Esperante, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Borkosky, Silvina Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Aran, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Alonso, Leonardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mohana Borges, Ronaldo. Universidade Federal do Rio de Janeiro; BrasilFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Characterisation of an ABC transporter of a resistant Candida glabrata clinical isolate

BACKGROUND Candida glabrata ranks second in epidemiological surveillance studies, and is considered one of the main human yeast pathogens. Treatment of Candida infections represents a contemporary public health problem due to the limited availability of an antifungal arsenal, toxicity effects and increasing cases of resistance. C. glabrata presents intrinsic fluconazole resistance and is a significant concern in clinical practice and in hospital environments. OBJECTIVE The aim of this study was to characterise the azole resistance mechanism presented by a C. glabrata clinical isolate from a Brazilian university hospital. METHODS Azole susceptibility assays, chemosensitisation, flow cytometry and mass spectrometry were performed. FINDINGS Our study demonstrated extremely high resistance to all azoles tested: fluconazole, voriconazole, posaconazole and itraconazole. This isolate was chemosensitised by FK506, a classical inhibitor of ABC transporters related to azole resistance, and Rhodamine 6G extrusion was observed. A mass spectrometry assay confirmed the ABC protein identification suggesting the probable role of efflux pumps in this resistance phenotype. MAIN CONCLUSIONS This study emphasizes the importance of ABC proteins and their relation to the resistance mechanism in hospital environments and they may be an important target for the development of compounds able to unsettle drug extrusion.

Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein

© 2013 Freire et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.This work was supported by Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) [PTDC/QUI-BIQ/112929/2009], by the European Union [projects FP7-PEOPLE IRSES (MEMPEPACROSS) and FP7-HEALTH-F3-2008-223414 (LEISHDRUG)], by the Spanish Ministry of Economy and Competitiveness (SAF2011-24899), the Generalitat de Catalunya (2009 SGR 492), by the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnoloógico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the National Institute of Science and Technology in Dengue (INCT-Dengue). JMF also acknowledges FCT-MEC for Ph.D. fellowship SFRH/BD/70423/2010

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

<p>Abstract</p> <p>Background</p> <p>The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction.</p> <p>Results</p> <p>The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr.</p> <p>Conclusion</p> <p>Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.</p

Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines

The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection

Uso das Pironofatoquinona como antiviral; composição farmacêutica contendo as Piranonaftoquinonas; medicamento contendo as Piranonaftoquinonas para tratamento de infecções causadas por virus da dengue

DepositadaDescreve o uso das Piranonaftoquinonas, assim como seus derivados, seus isômeros e seus sais como um agente antiviral, por inibir as enzimas ATPases viral. Além disso, também, faz parte desta invenção uma composição farmacêutica contendo as Piranonaftoquinonas descritas

Compostos peptídeos miméticos derivados do ácido tartárico potencialmente ativos contra vírus da Hepatite C e composição farmacêutica contendo tais compostos

DepositadaA presente invenção se refere a compostos peptídeo mimético derivados do ácido tartárico potencialmente ativos contra vírus da Hepatite C e composição farmacêutica contendo tais compostos, desenhados como inibidores de polimerases e serina protease do vírus da Hepatite C (HCV), sintéticos, caracterizados por possuir uma estrutura do tipo 1,4:3,6-dianidromanitol e um cerne tipo ácido tartárico. As porções laterais são caracterizadas por possuir ligações peptídeos miméticas provenientes do acoplamento com diversos aminoésteres

Characterisation of an ABC transporter of a resistant Candida glabrata clinical isolate

BACKGROUND Candida glabrata ranks second in epidemiological surveillance studies, and is considered one of the main human yeast pathogens. Treatment of Candida infections represents a contemporary public health problem due to the limited availability of an antifungal arsenal, toxicity effects and increasing cases of resistance. C. glabrata presents intrinsic fluconazole resistance and is a significant concern in clinical practice and in hospital environments. OBJECTIVE The aim of this study was to characterise the azole resistance mechanism presented by a C. glabrata clinical isolate from a Brazilian university hospital. METHODS Azole susceptibility assays, chemosensitisation, flow cytometry and mass spectrometry were performed. FINDINGS Our study demonstrated extremely high resistance to all azoles tested: fluconazole, voriconazole, posaconazole and itraconazole. This isolate was chemosensitised by FK506, a classical inhibitor of ABC transporters related to azole resistance, and Rhodamine 6G extrusion was observed. A mass spectrometry assay confirmed the ABC protein identification suggesting the probable role of efflux pumps in this resistance phenotype. MAIN CONCLUSIONS This study emphasizes the importance of ABC proteins and their relation to the resistance mechanism in hospital environments and they may be an important target for the development of compounds able to unsettle drug extrusion

Diabetogenic viruses: linking viruses to diabetes mellitus

Diabetes Mellitus (DM) is a group of chronic metabolic diseases distinguished by elevated glycemia due to the alterations in insulin metabolism. DM is one of the most relevant diseases of the modern world, with high incidence and prevalence worldwide, associated with severe systemic complications and increased morbidity and mortality rates. Although genetic factors and lifestyle habits are two of the main factors involved in DM onset, viral infections, such as enteroviruses, cytomegalovirus, hepatitis C virus, human immunodeficiency virus, severe acute respiratory syndrome coronavirus 2, among others, have been linked as triggers of type 1 (T1DM) and type 2 (T2DM) diabetes. Over the years, various groups identified different mechanisms as to how viruses can promote these metabolic syndromes. However, this field is still poorly explored and needs further research, as millions of people live with these pathologies. Thus, this review aims to ex-plore the different processes of how viruses can induce DM and their contribution to the prevalence and incidence of DM worldwide