ORCID Profile
0000-0001-7891-7562
Current Organisations
Universidade de Lisboa Faculdade de Medicina
,
Instituto de Medicina Molecular
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Publisher: Wiley
Date: 08-01-2008
DOI: 10.1002/PSC.1003
Abstract: The Cell membrane is impermeable for most peptides, proteins, and oligonucleotides. Moreover, some cationic peptides, the so-called cell-penetrating peptides (CPPs), are able to translocate across the membrane. This observation has attracted much attention because these peptides can be covalently coupled to different macromolecules, which are efficiently delivered inside the cell. The mechanism used by these peptides to pass across the membrane is a controversial matter of debate. It has been suggested that endocytosis is the main mechanism of internalization and this was confirmed by several studies for different peptides. Pep-1 is an exception worthy of attention for its ability to translocate cargo macromolecules without the need to be covalently attached to them. A preferential internalization by an endocytosis-independent mechanism was demonstrated both in vitro and in vivo. Pep-1 has a high affinity to lipidic membranes, it is able to insert and induce local destabilization in the lipidic bilayer, although without pore formation. No cytotoxic effects were found for pep-1 concentrations where translocation is fully operative. At much higher concentrations, membrane disintegration takes place by a detergent-like mechanism that resembles anti-microbial peptide activity. In this review, the ability of pep-1 to transverse the membrane by an endocytosis-independent mechanism, not mediated by pores as well as an ability to induce membrane disintegration at high peptide concentration, is demonstrated.
Publisher: Wiley
Date: 26-07-2005
DOI: 10.1016/J.FEBSLET.2005.06.085
Abstract: Cell-penetrating peptides (CPPs) are able to translocate across biological membranes and deliver bioactive proteins. Cellular uptake and intracellular distribution of CPPs is commonly evaluated with fluorescent labels, which can alter peptide properties. The effect of carboxyfluorescein label in the Lys-rich domain of the hipathic CPP pep-1, was evaluated and compared with non-labelled pep-1 in vitro and in vivo. A reduced membrane affinity and an endosomal-dependent translocation mechanism, at variance with non-labelled pep-1, were detected. Therefore, the charged domain is not a mere enabler of peptide adsorption but has a crucial role in the translocation pathway of non-labelled pep-1.
Publisher: American Chemical Society (ACS)
Date: 07-07-2005
DOI: 10.1021/BI0502644
Abstract: The cell-penetrating peptide (CPP) pep-1 is capable of introducing large proteins into different cell lines, maintaining their biological activity. Two possible mechanisms have been proposed to explain the entrance of other CPPs in cells, endosomal-dependent and independent types. In this work, we evaluated the molecular mechanisms of pep-1-mediated cellular uptake of beta-galactosidase (beta-Gal) from Escherichia coli in large unilamellar vesicles (LUV) and HeLa cells. Fluorescence spectroscopy was used to evaluate the translocation process in model systems (LUV). Immunofluorescence microscopy was used to study the translocation in HeLa cells. Enzymatic activity detection enabled us to monitor the internalization of beta-Gal into LUV and the functionality of the protein in the interior of HeLa cells. Beta-Gal translocated into LUV in a transmembrane potential-dependent manner. Likewise, the extent of beta-Gal incorporation was extensively decreased in depolarized cells. Furthermore, beta-Gal uptake efficiency and kinetics were temperature-independent, and beta-Gal did not colocalize with endosomes, lysosomes, or caveosomes. Therefore, beta-Gal translocation was not associated with the endosomal pathway. Although an excess of pep-1 was mandatory for beta-Gal translocation in vivo, transmembrane pores were not formed as concluded from the trypan blue exclusion method. These results altogether indicated that protein uptake both in vitro with LUV and in vivo with HeLa cells was mainly, if not solely, dependent on negative transmembrane potential across the bilayer, which suggests a physical mechanism governed by electrostatic interactions between pep-1 (positively charged) and membranes (negatively charged).
Publisher: Elsevier BV
Date: 02-2018
Publisher: Elsevier BV
Date: 08-2008
Publisher: MDPI AG
Date: 29-03-2022
Publisher: Elsevier BV
Date: 05-2005
DOI: 10.1016/J.BBAMEM.2004.11.017
Abstract: Pep-1 is a cell penetrating peptide (CPP) derived from the nuclear localization sequence of Simian Virus 40 large antigen T and from reverse transcriptase of Human Immunodeficiency Virus. Although it has been successfully used to transport proteins into cells, its action at the molecular level is not yet clear, mainly the local environmental factors that condition partition and translocation. Characterization in aqueous medium and quantification of partition into bilayers were carried out. Dynamic light scattering studies show that pep-1 self-associates in aqueous medium. The role of the bilayer phase, anionic lipids, ionic strength of the medium, reducing agents and pep-1 concentration on the extent and kinetics of partition were studied. Unlike others cationic CPP (e.g. penetratin) pep-1 has a high affinity to neutral vesicles (Kp = 2.8 x 10(3)), which is enhanced by anionic lipids. In a reduction environment partition is strongly inhibited (Kp = 2.2 x 10(2)), which might be a key-feature in the biological action of pep-1. Peptide incorporation takes place in the millisecond time-range to the lipidic interfaces. These environmental factors are systematized to enlighten how they help cellular uptake.
Publisher: American Chemical Society (ACS)
Date: 07-08-2019
DOI: 10.1021/ACSCHEMBIO.9B00593
Abstract: The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using
Publisher: Frontiers Media SA
Date: 12-06-2019
Publisher: Informa UK Limited
Date: 2007
DOI: 10.1080/09687860601142936
Abstract: Pep-1 is a cell-penetrating peptide (CPP) with the ability to translocate across biological membranes and introduce active proteins inside cells. The uptake mechanism used by this CPP is, as yet, unknown in detail. Previous results show that such a mechanism is endocytosis-independent and suggests that physical-chemical interactions between the peptide and lipid bilayers govern the translocation mechanism. Formation of a transmembrane pore has been proposed but this issue has always remained controversial. In this work the secondary structure of pep-1 in the absence resence of lipidic bilayers was determined by CD and ATR-FTIR spectroscopies and the occurrence of pore formation was evaluated through electrophysiological measurements with planar lipid membranes and by confocal microscopy using giant unilamellar vesicles. Despite pep-1 hydrophobic domain tendency for hipathic alpha-helix conformation in the presence of lipidic bilayers, there was no evidence for membrane pores in the presence of pep-1. Furthermore, alterations in membrane permeability only occurred for high peptide/lipid ratios, which induced the complete membrane disintegration. Such observations indicate that electrostatic interactions are of first importance in the pep-1-membrane interactions and show that pores are not formed. A peptide-lipid structure is probably formed during peptide partition, which favours peptide translocation.
Publisher: Informa UK Limited
Date: 2007
DOI: 10.1080/09687860601102476
Abstract: Membrane translocation is a crucial issue when addressing the activity of both cell-penetrating and antimicrobial peptides. Translocation is responsible for the therapeutic potential of cell-penetrating peptides as drug carriers and can dictate the killing mechanisms, selectivity and efficiency of antimicrobial peptides. It is essential to evaluate if the internalization of cell-penetrating peptides is mediated by endocytosis and if it is able to internalize attached cargoes. The mode of action of an antimicrobial peptide cannot be fully understood if it is not known whether the peptide acts exclusively at the membrane level or also at the cytoplasm. Therefore, experimental methods to evaluate and quantify translocation processes are of first importance. In this work, over 20 methods described in the literature for the assessment of peptide translocation in vivo and in vitro, with and without attached macromolecular cargoes, are discussed and their applicability, advantages and disadvantages reviewed. In addition, a classification of these methods is proposed, based on common approaches to detect translocation.
Publisher: American Chemical Society (ACS)
Date: 07-07-2004
DOI: 10.1021/BI036325K
Abstract: The action of the cell penetrating pep-1 at the molecular level is not clearly understood. The ability of the peptide to induce (1) vesicle aggregation, (2) lipidic fusion, (3) anionic lipid segregation, (4) pore or other lytic structure formation, (5) asymmetric lipidic flip-flop, and (6) peptide translocation across the bilayers in large unilamellar vesicles was studied using photophysical methodologies mainly related to fluorescence spectroscopy. Neflometry and turbidimetry techniques show that clustering of vesicles occurs in the presence of the peptide in a concentration- and anionic lipid content-dependent manner. Results from Forstër resonance energy transfer-based methodologies prove lipidic fusion and anionic lipid segregation, but no evidence for pores or other lytic structures was found. Asymmetric lipid flip-flop was not detected either. A specific method related to the quenching of the rhodamine-labeled lipids by pep-1 was developed to study the eventual translocation of the peptide. Translocation does not occur in symmetrical neutral and negatively charged vesicles, except when a valinomycin-induced transmembrane potential exists. Our work strongly suggests that the main driving force for peptide translocation is charge asymmetry between the outer and inner leaflet of biological membranes and reveals that pep-1 is able to perturb membranes without being cytotoxic. This nonlytic perturbation is probably mandatory for translocation to occur.
Publisher: Portland Press Ltd.
Date: 2004
DOI: 10.1042/BJ20031350
Abstract: Partition of the intrinsically fluorescent HIV fusion inhibitor enfuvirtide into lipidic membranes is relatively high (ΔG=6.6 kcal·mol−1) and modulated by cholesterol. A shallow position in the lipidic matrix makes it readily available for interaction with gp41. No conformational energetic barrier prevents enfuvirtide from being active in both aqueous solution and lipidic membranes. Lipidic membranes may play a key role in the enfuvirtide biochemical mode of action.
Publisher: Bentham Science Publishers Ltd.
Date: 08-2010
DOI: 10.2174/138920310791330604
Abstract: Prion diseases are a class of fatal neurodegenerative disorders that affect mammals and are characterized by their unique transmissibility and the nature of the infectious agent. When the physiological prion protein (PrP(C)) become corrupted (PrP(Sc)) it accumulates in the brain, promoting infection and self-propagation via recruitment of PrP(C). Although with identical sequence, PrP(C) and PrP(Sc) differ in their physicochemical properties: PrP(C) is soluble, has an alpha-helical structure and is sensitive to enzymatic degradation, whereas PrP(Sc) is insoluble, forms beta-aggregates and is resistant to proteolysis. The fragment PrP(16-126) possess similar physicochemical and pathological properties to PrP(sc), and therefore is commonly used as a model to study pathogenic effects. Although the pathogenicity of prion diseases is still unclear, strong evidences suggest that the cell membrane is relevant not only in infection and propagation of the disease but also in the manifestation of the clinical symptoms. In particular, the fragment PrP(106-126) has been implicated in the perturbation of the membranes and in the manifestation of Prion diseases. However, this is controversial. This review will discuss the effect of PrP(106-126) on the cell membrane based on its effect on model phospholipid bilayers. Different conditions were studied, including membrane charge, viscosity, lipid composition, pH, and ionic strength, revealing that PrP(106-126) only interacts with lipid membranes at conditions with no physiological relevance. Such findings are here reviewed and correlated with the full-length protein effect.
Publisher: Elsevier BV
Date: 03-2013
DOI: 10.1016/J.BBAMEM.2012.12.002
Abstract: BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required.
Publisher: Elsevier BV
Date: 07-2011
Publisher: American Chemical Society (ACS)
Date: 06-08-2020
Publisher: Springer Science and Business Media LLC
Date: 04-07-2020
Publisher: Elsevier BV
Date: 09-2012
Publisher: Wiley
Date: 2010
DOI: 10.1002/BIP.21367
Abstract: The use of peptide carriers, termed "cell-penetrating peptides (CPPs)" has attracted much attention due to their potential for cellular delivery of hydrophilic molecules with pharmacological interest, overcoming the membrane barrier. These peptides are able to deliver attached cargos in a nontoxic manner, with the uptake mechanisms being either endosomally or physically driven. Pep-1 is a CPP of particular interest, not only due to outstanding delivery rates but also because its mechanism of membrane translocation is exclusively physically driven which appears to be dependent on a very high affinity for the phospholipid bilayer in the cell membrane. In this study, pep-1-lipid interactions were further explored by characterization of the pep-1-lipid association/dissociation by surface plasmon resonance. Although a high affinity of pep-1 for lipid bilayers was observed in all conditions tested, negatively charged phospholipids resulted in a larger peptide/lipid ratio. We also show that pep-1-membrane interaction is a fast process described by a multistep model initiated by peptide adsorption, primarily governed by electrostatic attractions, and followed by peptide insertion in the hydrophobic membrane core. In the context of a cell-based process, the translocation of pep-1 is a physical mechanism promoted by peptide primary hipathicity and asymmetric properties of the membrane. This explains the high efficiency rates of pep-1 when compared with other CPPs.
Publisher: Frontiers Media SA
Date: 04-05-2017
Publisher: Portland Press Ltd.
Date: 13-09-2006
DOI: 10.1042/BJ20061100
Abstract: Some cationic peptides, referred to as CPPs (cell-penetrating peptides), have the ability to translocate across biological membranes in a non-disruptive way and to overcome the impermeable nature of the cell membrane. They have been successfully used for drug delivery into mammalian cells however, there is no consensus about the mechanism of cellular uptake. Both endocytic and non-endocytic pathways are supported by experimental evidence. The observation that some AMPs (antimicrobial peptides) can enter host cells without damaging their cytoplasmic membrane, as well as kill pathogenic agents, has also attracted attention. The capacity to translocate across the cell membrane has been reported for some of these AMPs. Like CPPs, AMPs are short and cationic sequences with a high affinity for membranes. Similarities between CPPs and AMPs prompted us to question if these two classes of peptides really belong to unrelated families. In this Review, a critical comparison of the mechanisms that underlie cellular uptake is undertaken. A reflection and a new perspective about CPPs and AMPs are presented.
Publisher: Springer Science and Business Media LLC
Date: 08-03-2013
DOI: 10.1007/S00726-013-1484-2
Abstract: The adverse side-effects associated with opioid administration restrain their use as analgesic drugs and call for new solutions to treat pain. Two kyotorphin derivatives, kyotorphin-amide (KTP-NH₂) and ibuprofen-KTP-NH₂ (IbKTP-NH₂) are promising alternatives to opioids: they trigger analgesia via an indirect opioid mechanism and are highly effective in several pain models following systemic delivery. In vivo side-effects of KTP-NH₂ and IbKTP-NH₂ are, however, unknown and were evaluated in the present study using male adult Wistar rats. For comparison purposes, morphine and tramadol, two clinically relevant opioids, were also studied. Results showed that KTP-derivatives do not cause constipation after systemic administration, in contrast to morphine. Also, no alterations were observed in blood pressure or in food and water intake, which were only affected by tramadol. A reduction in micturition was detected after KTP-NH₂ or tramadol administrations. A moderate locomotion decline was detected after IbKTP-NH₂-treatment. The side-effect profile of KTP-NH₂ and IbKTP-NH₂ support the existence of opioid-based mechanisms in their analgesic actions. The conjugation of a strong analgesic activity with the absence of the major side-effects associated to opioids highlights the potential of both KTP-NH₂ and IbKTP-NH₂ as advantageous alternatives over current opioids.
Publisher: Wiley
Date: 26-08-2013
Abstract: Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high-throughput screening with bactenecin as the template. Sub3 has potent activity against Gram-negative and Gram-positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ζ potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat.
Publisher: American Chemical Society (ACS)
Date: 08-04-2009
DOI: 10.1021/BI900009D
Abstract: Prion diseases result from a post-translational modification of the physiological prion protein (PrP(C)) into a scrapie isoform (PrP(Sc)). The PrP(106-126) fragment is conserved among various abnormal variants and shows PrP(Sc) pathogenic properties. It has been proposed that the PrP(106-126) fragment may exhibit its toxic effects through membrane pore formation. Our previous studies showed that PrP(106-126) does not interact with membranes under physiological conditions. In the present study, PrP(106-126) affinity for membranes was increased by modifying PrP(106-126) with a M112W substitution, and pore formation was further evaluated. However, while the peptide exhibited an increased local concentration in the membrane, this did not lead to the induction of membrane permeabilization, as verified by fluorescence methodologies and surface plasmon resonance. These results further support the idea that PrP(106-126) toxicity is not a consequence of peptide-membrane interaction and pore formation.
Publisher: Hindawi Limited
Date: 2012
DOI: 10.1155/2012/460702
Abstract: The increasing bacteria resistance to conventional antibiotics has led to the need for alternative therapies. Being part of the human innate defence system and with a broad spectrum of activity against bacteria, viruses, protozoa, and cancer cells, antimicrobial peptides (AMPs) are a very promising alternative. The mechanism of action of AMPs seems to broadly correlate with their ability to target the bacterial cell membrane. To understand and improve their effect, it is of major importance to unravel their mechanism of action and, in particular, to understand the peptide-membrane binding. Several biophysical techniques such as fluorescence spectroscopy, circular dichroism, zeta potential determination, and atomic force microscopy can be used to achieve this goal. Characteristics of AMPs-membranes interactions and the use of these biophysical techniques will be discussed.
Location: Portugal
No related grants have been discovered for Miguel Castanho.