Study of the molecular mechanism used by the peptidic vector pep-1 to introduce proteins inside cells

Tese de doutoramento em Bioquímica (Biofísica Molecular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2008The introduction of genetic material or proteins to originate a defined biochemical effect inside the cell has a remarkable potential for the treatment of Human diseases, however this is hampered by the cell membrane barrier for the entry of hydrophilic macromolecules. A possible strategy to overcome the membrane barrier was proposed after the discovery of basic peptidic sequences with ability to pass trough the membrane in a non-toxic and non-immunogenic manner. These peptides are commonly designated as cell-penetrating peptides (CPPs). Pep-1 is a CPP and has been successfully used to introduce several macromolecules biologically active inside cultured cells. The main goal of this thesis is to clarify the mechanism used by this peptide to pass through the membrane and to confirm if its efficiency as a carrier to introduce proteins inside cells. The interaction with membranes was followed in vitro with model membranes: large unilamellar vesicles; planar lipid membranes, giant unilamellar vesicles and supported bilayers. HeLa cells were used to follow the translocation of pep-1 associated with a protein. Fluorescence and UV-Vis spectroscopy methodologies, CD and ATR-FTIR spectroscopy, electrophysiological measurements and fluorescence microscopy were used to carry on the experimental work. It was shown that pep-1 is able to interact with and to destabilize the lipidic bilayers without evidence for pore formation at variance with other CPPs that use an endosomal pathway. Although all the evidences show that pep-1 translocates by a physically-mediated mechanism promoted by transmembrane potential with no evidences for the use of endosomal routes as an alternative pathway. Differences between this particular pep-1 and other CPPs can be related with the affinity for membrane. Peptides with higher affinity for membrane have more propensities to be internalized by a non-endocytic mechanism. Lower affinity for membranes favours endocytic uptake.Resumo alargado em português disponível no document

Bacteria may cope differently from similar membrane damage caused by the Australian tree frog antimicrobial peptide maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 mu M) than against E. coli (64 mu M) and an intermediate cytotoxicity against hRBC (30 mu M). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at similar to 20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes

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Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake

Chlorotoxin (CTX), a disulfide-rich peptide from the scorpion Leiurus qu'mquestriatus, has several promising biopharmaceutical properties, including preferential affinity for certain cancer cells, high serum stability, and cell penetration. These properties underpin its potential for use as a drug design scaffold, especially for the treatment of cancer; indeed, several analogs of CTX have reached clinical trials. Here, we focus on its ability to internalize into cells-a trait associated with a privileged subclass of peptides called cell-penetrating peptides-and whether it can be improved through conservative substitutions. Mutants of CTX were made using solid-phase peptide synthesis and internalization into human cervical carcinoma (HeLa) cells was monitored by fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 (ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in uptake compared to CTX. We further showed that these mutants internalize into HeLa cells largely via an energy-dependent mechanism. Importantly, the mutants have high stability, remaining intact in serum for over 24 h; thus, retaining the characteristic stability of their parent peptide. Overall, we have shown that simple conservative substitutions can enhance the cellular uptake of CTX, suggesting that such type of mutations might be useful for improving uptake of other peptide toxins

Mode-of-Action of Antimicrobial Peptides: Membrane Disruption vs. Intracellular Mechanisms

Antimicrobial peptides are an attractive alternative to traditional antibiotics, due to their physicochemical properties, activity toward a broad spectrum of bacteria, and mode-of-actions distinct from those used by current antibiotics. In general, antimicrobial peptides kill bacteria by either disrupting their membrane, or by entering inside bacterial cells to interact with intracellular components. Characterization of their mode-of-action is essential to improve their activity, avoid resistance in bacterial pathogens, and accelerate their use as therapeutics. Here we review experimental biophysical tools that can be employed with model membranes and bacterial cells to characterize the mode-of-action of antimicrobial peptides

Cyclotide structure and function: the role of membrane binding and permeation

There is growing interest in the use of peptides as therapeutic drugs and, in particular, in the potential of cyclotides, a family of cyclic peptides with remarkable stability and amenability to sequence engineering, as scaffolds in drug design. As well as having an ultrastable structure, many natural cyclotides have intrinsic biological activities with potential pharmaceutical or agricultural applications. Some cyclotides also have the ability to cross membrane barriers and to enter into cells; in particular, cyclotides that belong to the Möbius and bracelet subfamilies have been found to harbor lipid-binding domains, which allow for the specific recognition of phosphatidylethanolamine phospholipids in biological membranes. This lipid selectivity is intimately correlated with the highly conserved three-dimensional structures of cyclotides and is important for their reported biological properties and cell penetration ability. The membrane binding features of Möbius and bracelet cyclotides contrast with the lack of membrane binding of trypsin inhibitor cyclotides, which have physicochemical properties and bioactivities different from those of the other two subfamilies of cyclotides but are also able to enter cells. This review discusses the structures of cyclotides with regard to their myriad of biological activities and describes the role of membrane binding in their functions and ability to enter cells

Consequences of nonlytic membrane perturbation to the translocation of the cell penetrating peptide ppep-1 in lipidic vesicles

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 Forster 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

Is PrP(106-126) Fragment Involved in the Membrane Activity of the Prion Protein?

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)) becomes 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 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