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rBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures

Publication . Domingues, Marco M.; Bianconi, M. Lucia; Barbosa, Leandro R. S.; Santiago, Patrícia S.; Tabak, Marcel; Castanho, Miguel A. R. B.; Itri, Rosangela; Santos, Nuno C.

rBPI21 belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI21 at the membrane level. Using isothermal titration calorimetry, we observed that rBPI21 interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI21 is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI21 promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI21 action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI21 may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death.

2013Journal article

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Dengue virus capsid protein interacts specifically with very low-density lipoproteins

Publication . Faustino, André F.; Carvalho, Filomena A.; Martins, Ivo C.; Castanho, Miguel A. R. B.; Mohana-Borges, Ronaldo; Almeida, Fábio C. L.; Da Poian, Andrea T.; Santos, Nuno C.

Dengue affects millions of people worldwide. No specific treatment is currently available, in part due to an incomplete understanding of the viral components' interactions with host cellular structures. We tested dengue virus (DENV) capsid protein (C) interaction with low- and very low-density lipoproteins (LDL and VLDL, respectively) using atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR and computational analysis. Data reveal a specific DENV C interaction with VLDL, but not LDL. This binding is potassium-dependent and involves the DENV C N-terminal region, as previously observed for the DENV C-lipid droplets (LDs) interaction. A successful inhibition of DENV C-VLDL binding was achieved with a peptide drug lead. The similarities between LDs and VLDL, and between perilipin 3 (DENV C target on LDs) and ApoE, indicate ApoE as the molecular target on VLDL. We hypothesize that DENV may form lipoviroparticles, which would constitute a novel step on DENV life cycle.

2014Journal article

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Peptides as models for the structure and function of viral capsid proteins : insights on dengue virus capsid

Publication . Freire, João Miguel; Veiga, Ana Salomé; de la Torre, Beatriz G.; Santos, Nuno C.; Andreu, David; Da Poian, Andrea T.; Castanho, Miguel A. R. B.

The structural organization of viral particles is among the most astonishing examples of molecular self-assembly in nature, involving proteins, nucleic acids, and, sometimes, lipids. Proper assembly is essential to produce well structured infectious virions. A great variety of structural arrangements can be found in viral particles. Nucleocapsids, for instance, may display highly ordered geometric shapes or consist in macroscopically amorphous packs of the viral genome. Alphavirus and flavivirus are viral genera that exemplify these extreme cases, the former comprising viral particles structured with a T=4 icosahedral symmetry, whereas flavivirus capsids have no regular geometry. Dengue virus is a member of flavivirus genus and is used in this article to illustrate how viral protein-derived peptides can be used advantageously over full-length proteins to unravel the foundations of viral supramolecular assemblies. Membrane- and viral RNA-binding data of capsid protein-derived dengue virus peptides are used to explain the amorphous organization of the viral capsid. Our results combine bioinformatic and spectroscopic approaches using two- or three-component peptide and/or nucleic acid and/or lipid systems.

2013Journal article

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Nucleic acid delivery by cell penetrating peptides derived from dengue virus capsid protein : design and mechanism of action

Publication . Freire, João M.; Veiga, Ana Salomé; Figueiredo, Inês Rego de; Torre, Beatriz G. de la; Santos, Nuno C.; Andreu, David; Poian, Andrea T. Da; Castanho, Miguel A. R. B.

Cell penetrating peptides (CPPs) can be used as drug delivery systems for different therapeutic molecules. In this work two novel CPPs, pepR and pepM, designed from two domains of the dengue virus (DENV) capsid protein, were studied for their ability to deliver nucleic acids into cells as non-covalently bound cargo. Translocation studies were performed by confocal microscopy in HepG2, BHK and HEK cell lineages, astrocytes and peripheral blood mononuclear cells. Combined studies in HepG2 cells, astrocytes and BHK cells, at 4 and 37 °C or using specific endocytosis inhibitors, revealed that pepR and pepM use distinct internalization routes: pepM translocates lipid membranes directly, while pepR uses an endocytic pathway. To confirm these results, a methodology was developed to monitor the translocation kinetics of both peptides by real-time flow cytometry. Kinetic constants were determined, and the amount of nucleic acids delivered was estimated. Additional studies were performed in order to understand the molecular bases of the peptide-mediated translocation. Peptide– nucleic acid and peptide–lipid membrane interactions were studied quantitatively based on the intrinsic fluorescence of the peptides. pepR and pepM bound ssDNA to the same extent. Partition studies revealed that both peptides bind preferentially to anionic lipid membranes, adopting an α-helical conformation. However, fluorescence quenching studies suggest that pepM is deeply inserted into the lipid bilayer, in contrast with pepR. Moreover, only pepM is able to promote the fusion and aggregation of vesicles composed of zwitterionic lipids. Altogether, the results show that DENV capsid protein derived peptides serve as good templates for novel CPP-based nucleic acid delivery strategies, defining different routes for cell entry.

2013Journal article

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Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein

Publication . Freire, João Miguel; Veiga, Ana Salomé; Conceição, Thaís M.; Kowalczyk, Wioleta; Mohana-Borges, Ronaldo; Andreu, David; Santos, Nuno C.; Poian, Andrea T. da; Castanho, Miguel A. R. B.

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.

2013Journal article

Open access