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The Generation of Antimicrobial Peptide Activity: A Trade-off between Charge and Aggregation?
Angewandte Chemie International Edition
Abstract
Action stations: Antimicrobial peptides (AMPs) can be derived from amyloid-prone regions by introduction of cationic residues at privileged positions (see picture). The design and testing of 24 de novo amyloid-derived AMPs are described, and the likelihood of evolution of amyloid-prone protein regions, devoid of antimicrobial activity, into potent antimicrobial domains is addressed.
... For example, the primary target for the Pr-AMP oncocin and its derivative is thought to be the ribosome exit channel (Roy et al., 2015;Seefeldt et al., 2015), but these peptides are also known to bind to and inhibit the bacterial Hsp70 homolog DnaK (Knappe et al., 2011), which will likely amplify the disruption of bacterial proteostasis of these peptides. Interestingly, many AMPs are known to form amyloid structure spontaneously (Zhao et al., 2006;Mahalka and Kinnunen, 2009;Torrent et al., 2011) and co-aggregation has been observed between AMPs and bacterial proteins (Code et al., 2009). Moreover, it was recently shown that some known amyloidogenic peptides are indeed toxic to bacterial cells (Last and Miranker, 2013), and it was even proposed that the Alzheimer β-peptide may itself be an overlooked AMP (Soscia et al., 2010). ...
... Moreover, it was recently shown that some known amyloidogenic peptides are indeed toxic to bacterial cells (Last and Miranker, 2013), and it was even proposed that the Alzheimer β-peptide may itself be an overlooked AMP (Soscia et al., 2010). Introduction of positively charged amino acids in amyloidogenic peptides lead to the identification of novel AMPs, highlighting the overlap between both types of sequences (Torrent et al., 2011). As it is further known that heterologous seeding of protein aggregation is significantly less efficient than homologous seeding (Morales et al., 2013), we speculated that aggregation-prone peptides whose sequences are derived from the aggregation-prone regions (APRs) of bacterial proteins could be used to induce toxic protein aggregation more efficiently in bacteria than in other organisms, potentially providing a new design paradigm for AMPs. ...
... The charged residues act as aggregation gatekeepers that slow down beta-aggregation and increase colloidal stability by charge repulsion (Rousseau et al., 2006b). As an additional benefit, the net positive charge of the peptides is expected to facilitate bacterial uptake (Hancock and Chapple, 1999;Torrent et al., 2011). Peptides were generated by parallel microscale solid-phase synthesis, which yields peptide preparations of > 80% purity for sequences below 20 amino acids without requiring highperformance liquid chromatography (HPLC) purification. ...
Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and haematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.
... The characteristics that usually define HDPs are their short amino acidic sequences (between 12 and 50 amino acids) [4], a net positive charge [5], a certain degree of hydrophobicity [9] and a wide range of broad-spectrum biological activities [10]. Among these activities, HDPs have microbicidal (effective against bacteria, virus, and fungi) [11][12][13], antibiofilm [14,15], and immunomodulatory activities [16][17][18]. ...
... Their well-known characteristics make them amenable to engineering [19][20][21] (Fig. 1A, B), peptide repurposing, such as engineered venoms that can be modified to become non-toxic HDPs [22] (Fig. 1A), development of multidomain proteins based on the combination of different HDPs (Fig. 1B), de novo designs [9,23] (Fig. 1C), or aid in the discovery of hidden peptides within larger protein structures [24] (Fig. 1D). All these new technologies yield an almost unlimited potential to modify known sequences or discover new peptides and modes of action. ...
The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides (HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for translational applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale development of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of HDPs in various cell factories.
... This behavior could be explained by considering that, differently from r(P)ApoB L Pro , a non-sigmoidal trend was obtained when r(P)ApoB L Ala was incubated with LPS in the presence of ThT. Indeed, sigmoidal trends have been described for several known amyloid peptides [17,26,27,63]. It has to be highlighted that the molecular mechanism at the basis of thioflavin-T binding to an aromatic-hydrophobic groove, spanning across four consecutive beta-strands, provides a generic mode of recognition for amyloid dyes [64], but this recognition mode might not be strictly related to the presence of mature fibrils. ...
... This behavior could be explained by considering that, differently from r(P)ApoBL Pro , a non-sigmoidal trend was obtained when r(P)ApoBL Ala was incubated with LPS in the presence of ThT. Indeed, sigmoidal trends have been described for several known amyloid peptides [17,26,27,63]. It has to be highlighted that the molecular mechanism at the basis of thioflavin-T binding to an aromatic-hydrophobic groove, spanning across four consecutive beta-strands, provides a generic mode of recognition for amyloid dyes [64], but this recognition mode might not be strictly related to the presence of mature fibrils. ...
Host defense peptides (HDPs) are gaining increasing interest, since they are endowed with multiple activities, are often effective on multidrug resistant bacteria and do not generally lead to the development of resistance phenotypes. Cryptic HDPs have been recently identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, with anti-biofilm, wound healing and immunomodulatory properties, and with the ability to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, a multidisciplinary approach was used, including time killing curves, differential scanning calorimetry, circular dichroism, ThT binding assays, and transmission electron microscopy analyses. The effects of a single point mutation (Pro → Ala in position 7) on the biological properties of ApoB-derived peptide r(P)ApoBLPro have been evaluated. Although the two versions of the peptide share similar antimicrobial and anti-biofilm properties, only r(P)ApoBLAla peptide was found to exert bactericidal effects. Interestingly, antimicrobial activity of both peptide versions appears to be dependent from their interaction with specific components of bacterial surfaces, such as LPS or LTA, which induce peptides to form β-sheet-rich amyloid-like structures. Altogether, obtained data indicate a correlation between ApoB-derived peptides self-assembling state and their antibacterial activity.
... 3,12,13 It has been reported previously that physicochemical properties of peptides and their antimicrobial activity are interrelated. 14,15 According to the structure-based classification of AMPs, they have four groups, namely, amphipathic α helices, β strand, loop, and extended structures. The β strand forming AMPs possess high β sheet content in their structure 16 which generally renders the peptide to be amyloidogenic. ...
... The peptide is expected to form two disulfide bonds (at the locations of C 6 -C 15 As it can be seen in Figure 1A, all the tools predicted PG-4 to be highly amyloidogenic. TANGO predicted the stretch 11 ...
Protegrin‐4 (PG‐4) is a member of the porcine leukocyte protegrins family of cysteine‐rich antimicrobial peptides (AMPs) isolated from Sus scrofa. It consists of 18 amino acid residues and works as a part of innate immune system. In this study, we examined the intrinsic aggregation propensity of this AMP using multiple computational algorithms, namely, TANGO, AGGRESCAN, FOLDAMYLOID, AMYLPRED, and ZYGGREGATOR, and found that the peptide is predicted to have a high propensity for the β sheet formation that disposes this peptide to be amyloidogenic. Under in vitro conditions, PG‐4 formed visible aggregates and displayed the hallmark properties of typical amyloids such as enhanced binding of Congo red, increased fluorescence with Thioflavin‐T, and fibrillar morphology under transmission electron microscopy. Then we examined its antimicrobial activity against Bacillus subtilis and found that the aggregated peptide retained its antimicrobial activity. Additionally, the aggregates remain non‐toxic to the HEK293 and Caco2 cells. Our study suggests that the inherent aggregation properties of AMP can rationally be explored as a potential source of peptide‐based antimicrobials with enhanced stability. Formation of amyloid by antimicrobial peptide protegrin‐4. The antimicrobial peptides (AMPs) are promising peptide‐based therapeutics, but their susceptibility towards proteases present in the biological fluids makes them lesser stable. We propose that AMPs having intrinsic aggregation potential can be converted into amyloid‐like structures. Having the exceptional stability of amyloids, the aggregated AMPs are expected to be proteolytically more stable and may remain effective for a longer duration, reducing the dose requirements and positively eradicating the toxicity problems of AMP‐based therapeutics.
... Interestingly, the increasing turbidity remarkably observed for the peptide C2 with LUVs (composed by POPC:POPG 50 mol%) is in accordance to lower MIC values for C2 compared to C1, in the absence of DTT, against the majority of strains evaluated here. Haney et al. [40] suggests that the trend of oligomerization presented by several peptides, would be a limitation for the antimicrobial activity, as also observed by Torrent et al. [41,42], who demonstrated the linear relationship between the tendency of peptide aggregation and a lower antimicrobial activity, although also demonstrated that antimicrobially active sequences could be generated from aggregation-prone regions of peptides and proteins [42]. In fact, it was demonstrated that the antimicrobial activity of beta-defensin-5 may be dependent on the peptide fold and quaternary structure [31]. ...
... Interestingly, the increasing turbidity remarkably observed for the peptide C2 with LUVs (composed by POPC:POPG 50 mol%) is in accordance to lower MIC values for C2 compared to C1, in the absence of DTT, against the majority of strains evaluated here. Haney et al. [40] suggests that the trend of oligomerization presented by several peptides, would be a limitation for the antimicrobial activity, as also observed by Torrent et al. [41,42], who demonstrated the linear relationship between the tendency of peptide aggregation and a lower antimicrobial activity, although also demonstrated that antimicrobially active sequences could be generated from aggregation-prone regions of peptides and proteins [42]. In fact, it was demonstrated that the antimicrobial activity of beta-defensin-5 may be dependent on the peptide fold and quaternary structure [31]. ...
Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence.
... The GAPR-1 structure bears some resemblance to amphipathic antimicrobial peptides in that they have a defined positively charged surface area and bind to negatively charged lipid membranes [9]. The lipid binding properties of these antimicrobial peptides involves conformational changes, aggregation, formation of oligomers and subsequently conversion to amyloid-type fibrils [10][11][12]. ...
... Membranes containing negatively charged lipids provide an environment promoting the local concentration of positively charged proteins or peptides at the membrane surface. The biophysical properties of the membrane subsequently elicit the conformational changes required for self-aggregation [9][10][11]24]. Here we provide evidence for induction of amyloid formation by GAPR-1 and CRISP2 by negatively charged lipid membranes. ...
Abstract Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) is a mammalian protein that is a member of the Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis related proteins group 1 (CAP) superfamily of proteins. A role for the common CAP domain in the function of the diverse superfamily members has not been described so far. Here, we show by a combination of independent techniques including electron microscopy, Thioflavin T fluorescence, and circular dichroism that GAPR-1 has the capability to form amyloid-like fibrils in the presence of liposomes containing negatively charged lipids. Surprisingly, GAPR-1 was also shown to bind the amyloid-oligomer specific antibody A11 in the absence of lipids, indicating that GAPR-1 has an intrinsic tendency to form oligomers. This behavior is characteristic for proteins that interfere with Aβ aggregation and indeed we found that GAPR-1 effectively inhibited aggregation of Aβ(1-40) peptide. Immuno-dot blot analysis revealed that GAPR-1 binds to prefibrillar oligomeric Aβ structures during the early stages of fibril formation. Another CAP domain-containing protein, CRISP2, was also capable of forming fibrils, indicating that oligomerization and fibril formation is a shared characteristic between CAP family members. We suggest that the CAP domain may regulate protein oligomerization in a large variety of proteins that define the CAP superfamily.
... Some AMPs/HDPs translocate through cell membranes before reaching intracellular targets (non-membranolytic AMPs) 28 . Their global cationic and amphipathic characteristics are essential to their electrostatic interactions and hydrogen bonding with bacterial and eukaryotic membranes and are inherently linked to their functional promiscuity 32,33 . Current ML models excel at identifying sequences of antimicrobial nature or activity, but they rarely comprehend their intricate mechanism(s) of action. ...
Machine learning models are revolutionizing our approaches to discovering and designing bioactive peptides. However, these models often need protein structure awareness, as they heavily rely on sequential data. The models excel at identifying sequences of a particular biological nature or activity, but they frequently fail to comprehend their intricate mechanism(s) of action. To solve two problems at once, we studied the mechanisms of action and structural landscape of antimicrobial peptides as (i) membrane-disrupting peptides, (ii) membrane-penetrating peptides, and (iii) protein-affine peptides. Our in-depth analysis revealed that our preliminary best-performing classifiers (86–88% accuracy) trained on datasets with an over-represented distribution of α-helical and coiled structures. Consequently, our models would predict the antimicrobial activity of these structure classes more accurately. We mitigated this structural bias by implementing two strategies: subset selection and data reduction. The former gave three structure-specific models predicting the mechanisms of action of peptide sequences likely to fold into α-helices, coils, or mixed structures. The latter depleted over-represented structures, leading to general structure-agnostic predictors.
... Further, the aggregation propensity is associated with the formation of aggregates, which can lead to various issues, especially in biopharmaceutical and biotechnological applications. Primary structure, physicochemical properties, environmental conditions, concentration, and mechanical stress can influence the aggregation propensity of peptides [68]. A negative aggregation propensity value suggests that the molecule has a reduced tendency to aggregate, as shown for BiP_Aa_4 and BiP_Aa_6. ...
The demand for novel antimicrobial compounds is rapidly growing due to the rising appearance of antibiotic resistance in bacteria; accordingly, alternative approaches are urgently needed. Antimicrobial peptides (AMPs) are promising, since they are a naturally occurring part of the innate immune system and display remarkable broad-spectrum activity and high selectivity against various microbes. Marine invertebrates are a primary resource of natural AMPs. Consequently, cDNA expression (EST) libraries from the Cnidarian moon jellyfish Aurelia aurita and the Ctenophore comb jelly Mnemiopsis leidyi were constructed in Escherichia coli. Cell-free size-fractionated cell extracts (
... Oncocin also binds to and inhibits the bacterial Hsp70 homolog DnaK (Knappe et al., 2011), one of the key chaperones in bacteria, which will likely amplify the disruption of bacterial proteostasis by this peptide. Interestingly, many AMPs form amyloid structures spontaneously (Zhao et al., 2006;Mahalka and Kinnunen, 2009;Torrent et al., 2011) and some AMPs coaggregate with bacterial proteins (Code et al., 2009). ...
Cells have evolved a complex molecular network, collectively called the protein homeostasis (proteostasis) network, to produce and maintain proteins in the appropriate conformation, concentration and subcellular localization. Loss of proteostasis leads to a reduction in cell viability, which occurs to some degree during healthy ageing, but is also the root cause of a group of diverse human pathologies. The accumulation of proteins in aberrant conformations and their aggregation into specific beta-rich assemblies are particularly detrimental to cell viability and challenging to the protein homeostasis network. This is especially true for bacteria; it can be argued that the need to adapt to their changing environments and their high protein turnover rates render bacteria particularly vulnerable to the disruption of protein homeostasis in general, as well as protein misfolding and aggregation. Targeting bacterial proteostasis could therefore be an attractive strategy for the development of novel antibacterial therapeutics. This review highlights advances with an antibacterial strategy that is based on deliberately inducing aggregation of target proteins in bacterial cells aiming to induce a lethal collapse of protein homeostasis. The approach exploits the intrinsic aggregation propensity of regions residing in the hydrophobic core regions of the polypeptide sequence of proteins, which are genetically conserved because of their essential role in protein folding and stability. Moreover, the molecules were designed to target multiple proteins, to slow down the build-up of resistance. Although more research is required, results thus far allow the hope that this strategy may one day contribute to the arsenal to combat multidrug-resistant bacterial infections.
... Furthermore, predictive models based on artificial neural networks indicate that peptide aggregation in solution indeed contributes to low antimicrobial activity [38]. Interestingly, the addition of cationic residues to peptides has been shown to inhibit aggregation in solution while improving antimicrobial potency at the same time [39]. ...
Antimicrobial peptides (AMPs) are anti-infectives that have the potential to be used as a novel and untapped class of biotherapeutics. Modes of action of antimicrobial peptides include interaction with the cell envelope (cell wall, outer- and inner-membrane). A comprehensive understanding of the peculiarities of interaction of antimicrobial peptides with the cell envelope is necessary to perform a rational design of new biotherapeutics, against which working out resistance is hard for microbes. In order to enable de novo design with low cost and high throughput, in silico predictive models have to be invoked. To develop an efficient predictive model, a comprehensive understanding of the sequence-to-function relationship is required. This knowledge will allow us to encode amino acid sequences expressively and to adequately choose the accurate AMP classifier. A shared protective layer of microbial cells is the inner, plasmatic membrane. The interaction of AMP with a biological membrane (native and/or artificial) has been comprehensively studied. We provide a review of mechanisms and results of interactions of AMP with the cell membrane, relying on the survey of physicochemical, aggregative, and structural features of AMPs. The potency and mechanism of AMP action are presented in terms of amino acid compositions and distributions of the polar and apolar residues along the chain, that is, in terms of the physicochemical features of peptides such as hydrophobicity, hydrophilicity, and amphiphilicity. The survey of current data highlights topics that should be taken into account to come up with a comprehensive explanation of the mechanisms of action of AMP and to uncover the physicochemical faces of peptides, essential to perform their function. Many different approaches have been used to classify AMPs, including machine learning. The survey of knowledge on sequences, structures, and modes of actions of AMP allows concluding that only possessing comprehensive information on physicochemical features of AMPs enables us to develop accurate classifiers and create effective methods of prediction. Consequently, this knowledge is necessary for the development of design tools for peptide-based antibiotics.
... Developing predictive models by the artificial neural network it has been found that peptide aggregation in solution indeed contributes to low antimicrobial activity (Torrent et al. 2011a). Interestingly, the addition of cationic residues to peptides has been shown to inhibit aggregation in solution while improving the antimicrobial potency at the same time (Torrent et al. 2011b). ...
... Developing predictive models by the artificial neural network it has been found that peptide aggregation in solution indeed contributes to low antimicrobial activity (Torrent et al. 2011a). Interestingly, the addition of cationic residues to peptides has been shown to inhibit aggregation in solution while improving the antimicrobial potency at the same time (Torrent et al. 2011b). ...
Antimicrobial peptides (AMPs) are anti-infectives that have potential as a novel and untapped class of biotherapeutics. Modes of action of antimicrobial peptides imply interaction with cell envelope. Comprehensive understanding of peculiarities of interactions of antimicrobial peptides with cell envelope is necessary to perform the task-oriented design of new biotherapeutics, against which for microbes it is hard to work out resistance. In order to enable a de novo design with low costs and in high throughput, in silico predictive models have to be required. To develop the performant predictive model, comprehensive knowledge on mechanisms of action of AMPs has to be possessed. The last knowledge will allow us to encode amino acid sequences expressively and to get success to the choosing of the accurate classifier of AMPs. A shared protective layer of microbial cells is inner, plasmatic membrane. The interaction of AMP with a biological membrane (native and/or artificial) is the most comprehensively studied. We provide a review of mechanisms and results of interaction of AMP with the cell membrane, relying on the survey of physicochemical, aggregative and structural features of AMPs. Potency and mechanism of action of AMP have presented in the terms of amino acid compositions and distributions of the polar and apolar residues along the chain, that is in such physicochemical features of peptides as the hydrophobicity, hydrophilicity, and amphiphilicity. Many different approaches were used to classify AMPs. The survey of the knowledge on sequences, structures, and modes of actions of AMP, allows concluding that, only the physicochemical features of AMPs give the capability to perform the unambiguous classification. Comprehensive knowledge of physicochemical features of AMP is necessary to develop task-oriented methods of design of peptide-based antibiotics de novo.
... Other peptides, such as bactenecins, can also work at sublethal concentrations by inducing the depolarization of the cell membrane and consequent cell metabolic imbalance [40]. We can predict the peptide action on cell membranes based on their structural properties [41,42]. Many technologies can be used to study the membrane-perturbation mechanism [43]. ...
Background
Antimicrobial resistance (AMR) to conventional antibiotics is becoming one of the main global health threats and novel alternative strategies are urging. Antimicrobial peptides (AMPs), once forgotten, are coming back into scene as promising tools to overcome bacterial resistance. Recent findings have attracted attention to the potentiality of AMPs to work as antibiotic adjuvants.
Method
In this review, we have tried to collect the current available information on the mechanism of action of AMPs in synergy with other antimicrobial agents. In particular, we have focused on the mechanisms of action that mediate the inhibition of emergence of bacterial resistance by AMPs.
Results and conclusion
We find in the literature many examples where AMPs can significantly reduce the antibiotic effective concentration. Mainly, the peptides work at the bacterial cell wall and thereby facilitate the drug access to its intracellular target. Complementarily, AMPs can also contribute to permeate the exopolysaccharide layer of biofilm communities, or even prevent bacterial adhesion and biofilm growth. Secondly, we find other peptides that can directly block the emergence of bacterial resistance mechanisms or interfere with the community quorum sensing systems. Interestingly, the effective peptide concentrations for adjuvant activity and inhibition of bacterial resistance are much lower than the required for a direct antimicrobial action. Finally, many AMPs expressed by innate immune cells are endowed with immunomodulatory properties and can participate in the host response against infection. Recent studies in animal models confirm that AMPs work as adjuvants at non-toxic concentrations and can be safely administrated for novel combined chemotherapies.
... Du point de vue évolutif, cette tendance à la synergie entre PAM pourrait découler de mutations de peptides au caractère amyloïde vers un caractère antimicrobien, du fait de l'apparition de charges positives. 243 Malgré tout, la majorité des études portant sur ces mécanismes sont réalisées in vitro, il reste encore une zone d'ombre sur les mécanismes précis conduisant à l'action des PAM in vivo. 244 Un autre grand pan des interactions membranaires des PAM repose sur leur capacité à cibler des éléments spécifiques à la surface des bactéries. ...
L’analyse microbiologique pour confirmer l’absence de bactéries dans des échantillons biologiques normalement sains, comme le sang, est une routine dans de nombreux laboratoires. En effet, la présence de bactéries dans le sang, appelée bactériémie, peut avoir des conséquences très graves, voire mortelles pour le patient. Le protocole standard pour la détection des bactériémies repose jusqu’ici sur l’enrichissement des échantillons sanguins prélevés sur les patients lors de l’hémoculture, afin d’obtenir une population suffisante pour analyse. La lenteur de ce procédé retarde ainsi de parfois plusieurs jours le diagnostic et donc l’adaptation du traitement antibiotique administré au patient. Ces dernières décennies, des techniques comme l’identification par spectrométrie de masse ou les analyses moléculaires, ont permis de diminuer le délai requis pour identifier les pathogènes en cause. Dans ce contexte, l’emploi de biocapteurs est également une alternative. Ce travail propose d’inclure des sondes à large spectre dans un capteur optique par imagerie SPR (résonance de plasmons de surface). Ce système est déjà développé pour la reconnaissance spécifique de pathogènes au cours de leur croissance dans le sang. Les nouveaux ligands proposés et évalués sont les peptides antimicrobiens (PAM). Ces courts peptides cationiques et amphiphiles, présentent l’avantage d’un large spectre d’interaction couplé à une haute stabilité (chimique, thermique et séchage) comparativement aux anticorps employés jusqu’ici. Leur immobilisation sur des prismes SPRI permet d’évaluer simultanément l’affinité de plusieurs PAM à la même souche bactérienne. Les biocapteurs ainsi préparés ont permis de détecter des souches pathogènes d’Escherichia coli et Staphylococcus aureus en milieu de culture simple, comme en plasma et en sang dilué au milieu d’hémoculture. Le système obtenu permet la détection des pathogènes présents à une concentration initiale de l’ordre de 1 UFC.ml-1, en moins de 24 heures et quel que soit le milieu. Enfin, la mise en place d’analyses statistiques multidimensionnelles a abouti à une classification cohérente des espèces ciblées en milieu simple, comme en sang. Ces résultats montrent le potentiel de ce système pour parvenir à développer un biocapteur à large spectre capable à la fois de détecter mais aussi d’identifier par affinité croisée des pathogènes bactériens.
... This is especially important because coil to helix transition significantly contributes to membrane binding free energy. The ability to aggregate is another functional feature of AMPs, which relates them to amyloid peptides (Torrent et al., 2011). We assume that some patterns may also encode aggregation-prone regions which stimulate peptide self-association on a membrane surface, ultimately leading to its disruption. ...
Motivation:
The rational design of antimicrobial peptides (AMPs) with increased therapeutic potential requires deep understanding of the determinants of their activities. Inspired by the computational linguistic approach, we hypothesized that sequence patterns may encode the functional features of AMPs.
Results:
We found that α-helical and β-sheet peptides have non-intersecting pattern sets and therefore constructed new sequence templates using only helical patterns. Designed peptides adopted an α-helical conformation upon binding to lipids, confirming that the method captures structural and biophysical properties. In the antimicrobial assay, 5 of 7 designed peptides exhibited activity against Gram(+) and Gram(-) bacteria, with most potent candidate comparable to best natural peptides. We thus conclude that sequence patterns comprise the structural and functional features of α-helical AMPs and guide their efficient design.
Supplementary information:
Supplementary data are available at Bioinformatics online.
... A combination of these parameters defines the peptide DFTamP1, which effectively kills Gram-positive MRSA USA300 (10), a major community-associated resistant pathogen. In addition, other peptide discovery approaches have been developed based on the data from the APD (11)(12)(13). ...
As bacterial resistance to traditional antibiotics continues to emerge, new alternatives are urgently needed. Antimicrobial peptides (AMPs) are important candidates. However, how AMPs are designed with in vivo efficacy is poorly understood. Our study was designed to understand structural moieties of cationic peptides that would lead to their successful use as antibacterial agents. In contrast to the common perception, serum binding and peptide stability were not the major reasons for in vivo failure in our studies. Rather, our systematic study of a series of peptides with varying lysines revealed the significance of low cationicity for systemic in vivo efficacy against Gram-positive pathogens. We propose that peptides with biased amino acid compositions are not favored to associate with multiple host factors and are more likely to show in vivo efficacy. Thus, our results uncover a useful design strategy for developing potent peptides against multidrug-resistant pathogens.
... Amyloidogenic peptides are toxic to bacterial cells 46 , and it was even proposed that the Alzheimer β-peptide may itself be an overlooked AMP, for which C. albicans was identified as the most sensitive microbial target 47 . Further, introduction of positively charged amino acids into other amyloidogenic peptides lead to the identification of novel potent AMPs with broad spectrum antimicrobial activity 48 , which would support the hypothesis that supramolecular structures of CIDAMPs may also have microbicidal properties. Formation of intracellular protein aggregates induces oxidative stress, including production of free radicals 49 . ...
Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.
... We speculate that this improved peptide activity might be related to the fact that this optimization process showed evolutionary steps similar to the natural evolution of antimicrobial peptides ( Figure S10). 33 Furthermore, we tested the efficacy of the best peptide against drug-resistant bacterial strains ( Figure S11). To assess the efficacy in a comparable manner, we used drug-resistant E. coli MG1655 strains that we obtained by serially culturing the WT strain (the same strain used for the in vitro assay above) under single or multiple antibiotic stress. ...
... Besides, AMPs are also prone to aggregate and show occasionally poor solubility (198,199). Luckily, there are currently different strategies and predictive software available to prevent aggregation and improve physicochemical properties (42,200,201). Complementarily, cleavage protection can be enhanced through secondary structure stabilization (37, 202). ...
Tuberculosis (TB) continues to be a devastating infectious disease and remerges as a global health emergency due to an alarming rise of antimicrobial resistance to its treatment. Despite of the serious effort that has been applied to develop effective anti-tubercular chemotherapies, the potential of antimicrobial peptides (AMPs) remains underexploited. A large amount of literature is now accessible on the AMP mechanisms of action against a diversity of pathogens; nevertheless, research on their activity on mycobacteria is still scarce. In particular, there is an urgent need to integrate all available interdisciplinary strategies to eradicate extensively drug resistant Mycobacterium tuberculosis strains. In this context, we should not underestimate our endogenous antimicrobial proteins and peptides as ancient players of the human host defence system. We are confident that novel antibiotics based on human AMPs displaying a rapid and multifaceted mechanism, with reduced toxicity, should significantly contribute to reverse the tide of anti-mycobacterial drug resistance. In this review, we have provided an up to date perspective of the current research on AMPs to be applied in the fight against TB. A better understanding on the mechanisms of action of human endogenous peptides should ensure the basis for the best guided design of novel anti-tubercular chemotherapeutics.
... Aggregation and cell surface binding by P1 impede critical intermolecular interactions needed for proper S. mutans biofilm architecture, negatively impacting biofilm stability. In silico analyses of antimicrobial peptide sequences suggest that aggregation-prone regions are hotspots for the evolution of antimicrobial activity, when cationization in these regions results in amphipathic helical peptides (Torrent et al., 2011). A compendium of biofilm-active antimicrobial peptides, with their effects on medically relevant species, can be found in the open-access "BaAMPs" database (Di Luca et al., 2015). ...
Streptococcus mutans is the primary agent of dental cavities, in large part due to its ability to adhere to teeth and create a molecular scaffold of glucan polysaccharides on the tooth surface. Disrupting the architecture of S. mutans biofilms could help undermine the establishment of biofilm communities that cause cavities and tooth decay. Here we present a synthetic peptide P1, derived from a tick antifreeze protein, which significantly reduces S. mutans biofilm formation. Incubating cells with this peptide decreased biofilm biomass by approximately 75% in both a crystal violet microplate assay and an in vitro tooth model using saliva-coated hydroxyapatite discs. Bacteria treated with peptide P1 formed irregular biofilms with disconnected aggregates of cells and exopolymeric matrix that readily detached from surfaces. Peptide P1 can bind directly to S. mutans cells but does not possess bactericidal activity. Anti-biofilm activity was correlated with peptide aggregation and β-sheet formation in solution, and alternative synthetic peptides of different lengths or charge distribution did not inhibit biofilms. This anti-biofilm peptide interferes with S. mutans biofilm formation and architecture, and may have future applications in preventing bacterial buildup on teeth.
... Protein aggregation also has the beneficial aspect; protein aggregates contribute to exceptional stability, compactness and forms of organization that could not be achieved by monomeric or oligomeric conformations [7]. Several earlier studies reported that protein aggregation in organisms is beneficial for the adaptation in the diverse environment [30][31][32]. Considering the entire phenomenon due to aggregation property of protein we have predicted the protein aggregation score for all the genes of both the gene sets using a statistical mechanics algorithm, TANGO. Relative Aggregation Propensity (RAP) was obtained using the aggregation score derived from the TANGO program. ...
Paenibacillus sp. 32O-W, which is attributed for biodesulfurization of petroleum, has 56.34% genomic G+C content. Correspondence analysis on Relative Synonymous Codon Usage (RSCU) of the Paenibacillus sp. 32O-W genome has revealed the two different trends of codon usage variation. Two sets of genes have been identified representing the two distinct pattern of codon usage in this bacterial genome. We have measured several codon usage indices to understand the influencing factors governing the differential pattern of codon usage variation in this bacterial genome. We also observed significant differences in many protein properties between the two gene sets (e.g., hydrophobicity, protein biosynthetic cost, protein aggregation propensity). The compositional difference between the two sets of genes and the difference in their potential gene expressivity are the driving force for the differences in protein biosynthetic cost and aggregation propensity. Based on our results we argue that codon usage variation in Paenibacillus sp. 32O-W genome is actually influenced by both mutational bias and translational selection.
... Although natural and synthetic nanocarrier systems (e.g., liposome or colloidal delivery systems), prodrug-based approaches (e.g., cell-penetrating peptide conjugates), and chemical modifications via site-specific polyethylene glycol (PEG)ylation, lipidation, and glycosylation have frequently been used in peptide drug development[11,12], adverse consequences associated with these excipients, such as reduced bioactivity, aggregation and/or decreased solubility/stability of the formulated peptides, and toxicity-related issues are of concern[13,14]. This becomes much more pronounced for the AMPs since there is a good correlation between active AMPs sequences and the content of amino acid residues in the aggregation-prone region of amyloid peptides leading to their consequent physiochemical properties[15]. Peptide structural stability has also been described to be important for the high activity of AMPs, especially, at physiological salt concentrations[16,17]. Critically, the AMP sequences contain high numbers of residues sensitive to digestive serine and aspartate proteases[18], e.g., Lys and Arg as trypsin-sensitive cationic residues as well as hydrophobic residues such as Trp, Phe, Leu, Ile, Tyr, Ala, and Val that are sensitive to digestion by chymotrypsin, pepsin, and elastase proteases (Table 1); in particular these residues are functionally important for their activities and mediate peptide electrostatic attraction and hydrophobic insertion into the cell membrane[21]. ...
The emergence of multiresistant bacteria worldwide together with the shortage of effective antibiotics in the market emphasizes the need for the design and development of the promising agents for the treatment of superbug-associated infections. Antimicrobial peptides (AMPs) have been considered as excellent candidates to tackle this issue, and thousands of peptides of different lengths, amino acid compositions, and mode of action have been discovered and prepared to date. Nevertheless, it is of great importance to develop innovative formulation strategies for delivering these AMPs and to improve their low bioavailability and metabolic stability, particularly against proteases, if these peptides are to find applications in the clinic and administered orally or parenterally or used as dietary supplements. The purpose of this chapter is to describe basic experimental principles, based on analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS), for the prospective design of orally bioavailable AMPs considering the structural characteristics of the peptides and the substrate specificity of proteases that abound in the body especially at sites of infection.
... When the pH value of the solvent reaches a value of pI of the protein, it tends to precipitate and loses its biological function [25]. The analysed peptides had a pI close to 10, which has been reflected in the literature [26]. This observation is combined with the analysis of the peptides' net charge, estimated at pH 7, and content of particular amino acids. ...
The aim of this study was to evaluate the antimicrobial activity of meat protein-derived peptides against selected Gram-positive and Gram-negative bacteria. The in silico and in vitro approach was combined to determine the potency of antimicrobial peptides derived from pig (Sus scrofa) and cow (Bos taurus) proteins. The in silico studies consisted of an analysis of the amino acid composition of peptides obtained from the CAMPR3 database, their molecular weight and other physicochemical properties (isoelectric point, molar extinction coefficient, instability index, aliphatic index, hydropathy index and net charge). The degree of similarity was estimated between the antimicrobial peptide sequences derived from the slaughtered animals and the main meat proteins. Antimicrobial activity of peptides isolated from dry-cured meat products was analysed (in vitro) against two strains of pathogenic bacteria using the disc diffusion method. There was no evidence of growth-inhibitory properties of peptides isolated from dry-cured meat products against Escherichia coli K12 ATCC 10798 and Staphylococcus aureus ATCC 25923.
... This mode of action endows them with a broad-spectrum bactericidal activity; however, this comes at the cost of selectivity, since they can also potentially interact with the host cellular membranes. Interestingly enough, the determinants for antimicrobial activity and those for amyloid formation seem to overlap significantly, in such a way that many AMPs have been shown to selfassemble into amyloids both in solution and at the bacterial membrane , whereas positively charging classical amyloidogenic peptides may also result in strong antimicrobial activity (Torrent et al., 2011). Indeed, the Ab peptide and the islet amyloid polypeptide, linked to Alzheimer and type II diabetes, respectively, have been shown to behave as bona fide AMPs (Soscia et al., 2010;Last and Miranker, 2013). ...
A growing number of human diseases seem to be associated with protein misfolding and deposition into aggregates. In this issue of Molecular Microbiology, Bednarska and colleagues exploit the cytotoxic nature of protein aggregates to target bacterial infections. Protein aggregation is at the same time generic and sequence dependent; this allowed the authors to develop novel aggregation-prone antimicrobial peptides that penetrate bacteria and induce a peptide specific proteostatic collapse that leads to fast bacterial death, without any observable effects on host cells. The applicability of this intriguing strategy was demonstrated by curing animal models from bacterial sepsis. Although the precise mechanisms underlying the bactericidal activity of the peptide aggregates are still not clear, there is no doubt that this approach offers an exciting therapeutic alternative to conventional antibiotics. This article is protected by copyright. All rights reserved.
... These molecules can interact with the cell membrane, promoting a structural rearrangement. Antimicrobial peptides, for instance, can redistribute cholesterol, reduce membrane fluidity and create pores, damaging the membrane [55][56][57]. ...
Systemic inflammation and infections are associated with neurodegenerative diseases. Unfortunately, the molecular bases of this link are still largely undiscovered. We, therefore, review how inflammatory processes can imbalance membrane homeostasis and theorize how this may have an effect on the aggregation behavior of the proteins implicated in such diseases. Specifically, we describe the processes that generate such imbalances at the molecular level, and try to understand how they affect protein folding and localization. Overall, current knowledge suggests that microglia pro-inflammatory mediators can generate membrane damage, which may have an impact in terms of triggering or accelerating disease manifestation.
... Induced amphipathic α-helical conformations were found to play an important role in the antimicrobial activity of these peptides [88]. Additionally, most of the AMPs tend to form amyloid-like structures to destabilize phospholipid bilayers, suggesting that these aggregation-prone structures may have served as templates from which AMPs were evolutionarily derived [89]. Based on these structural guidelines and computer-assisted technologies that relate primary sequence to peptide structure, more potent, cost-effective, broad-spectrum peptides are being identified as potential next-generation, anti-infective peptides [87]. ...
The natural extracellular matrix (ECM), with its multitude of evolved cell-instructive and cell-responsive properties, provides inspiration and guidelines for the design of engineered biomaterials. One strategy to create ECM-mimetic materials is the modular design of protein-based engineered ECM (eECM) scaffolds. This modular design strategy involves combining multiple protein domains with different functionalities into a single, modular polymer sequence, resulting in a multifunctional matrix with independent tunability of the individual domain functions. These eECMs often enable decoupled control over multiple material properties for fundamental studies of cell-matrix interactions. In addition, since the eECMs are frequently composed entirely of bioresorbable amino acids, these matrices have immense clinical potential for a variety of regenerative medicine applications. This brief review demonstrates how fundamental knowledge gained from structure-function studies of native proteins can be exploited in the design of novel protein-engineered biomaterials. While the field of protein-engineered biomaterials has existed for over 20 years, the community is only now beginning to fully explore the diversity of functional peptide modules that can be incorporated into these materials. We have chosen to highlight recent examples that either (1) demonstrate exemplary use as matrices with cell-instructive and cell-responsive properties or (2) demonstrate outstanding creativity in terms of novel molecular-level design and macro-level functionality.
... Recent artificial neural-network prediction models of antimicrobial peptides have found that peptide aggregation in solution indeed contributes to a low antimicrobial activity [89]. Interestingly, the addition of cationic residues to peptides has been shown to inhibit aggregation in solution while improving the antimicrobial potency at the same time [90]. Propensity to aggregation is determined by sequence and consequently structural features of CAP. ...
Development of the new antimicrobial agents against antibiotic resistance
pathogens is the nowadays challenge. Antimicrobial peptides (AMP) occur as
important defence agents in many organisms and offer a viable alternative to
conventional antibiotics. Therefore they have become increasingly recognized in
current research as templates for prospective antibiotic agents. The efficient
designing of the new antimicrobials on the basis of antimicrobial peptides
requires comprehensive knowledge on those general physical-chemical
characteristics which allow to differ antimicrobial peptides from non-active
against microbs ones. According to supposed mechanisms of action, AMP interact
with and physically disrupt the bacterial membranes. Consequently,
hydrophobicity, amphiphilicity and intrinsic aggregation propensities are
considered as such major characteristics of the peptide, which determine the
results of peptide-membrane interactions. For some kind of peptides such
characteristics as hydrophobicity, amphiphilicity and aggregation bias
determines their ability to compose transmembrane domain of the membrane
protein, whilst for others the same properties are respond for their
antimicrpobial activity, i.e. give them ability of membrane permeability and
its damage. In this review we analyze the data about hydrophobicity,
amphiphilicity and intrinsic aggregation propensities available in literature
in order to compare antimicrobial and transmembrane peptides and show what is
the common and what is the difference in this respect between them.
... membrane fluidity and curvature) were found critical for the peptide action [13,15,18– 26]. Furthermore, we have recently suggested that antimicrobial activity could have arisen through cationization of amyloid-prone regions [27]. In this light, some AMPs have been described to form amyloid structures in vitro [28,29] and some amyloid peptides have also been considered as putative AMPs [30,31]. ...
Author Summary
Microbial infections are reported among the worst human diseases and cause millions of deaths per year over the world. Antibiotics are used to treat infections and have saved more lives than any other drug in human history. However, due to extended use, many strains are becoming refractive to common antibiotics. In this light, new promising compounds, like antimicrobial proteins and peptides (AMPs) are being investigated. Some AMPs also show agglutinating activity; this is the ability to clump bacteria after treatment. This feature is particularly appealing because agglutinating peptides could be used to keep bacteria to the infection focus, helping microbe clearance by host immune cells. In this study, we propose a novel mechanism to explain agglutinating activity at a molecular level using Eosinophil Cationic Protein. We show that the agglutinating mechanism is driven by the protein amyloid-like aggregation at the bacteria cell surface. Accordingly, elimination of the amyloid behavior abolishes both the agglutinating and the antimicrobial activities. This study provides a new concept on how Nature could exploit amyloid-like aggregates to fight bacterial infections. Moreover, these results could also add new insights in understanding the relation between infection and inflammation with dementia and amyloid-related diseases like Alzheimer.
... The overall picture for natural AMPs is shaped through evolution. For example, the preference of arginines in the AMPs in higher organisms (Figure 1B) is proposed to be significant in the emergence of adaptive immune systems (Torrent et al., 2011) and probably also confers the regulatory and integrative role to natural AMPs in host defense. We demonstrated that arginines are more effective in targeting MRSA or HIV-1 (Wang et al., 2010Wang et al., , 2012). ...
Antimicrobial peptides (AMPs) are critical components of natural host defense systems against infectious pathogens (Zasloff, 2002; Boman, 2003; Hancock and Sahl, 2006). They are ubiquitous in nature and have been found in nearly all forms of life, ranging from single-celled bacteria to multicellular organisms such as plants and animals. AMPs are short peptides (5–100 amino acids) with an average net charge of +3 (Wang, 2010). They can display broad or narrow-spectrum antimicrobial activities. The fact that AMPs are effective against multidrug resistance pathogens, including suppression of biofilm formation, deserves our attention (Menousek et al., 2012). In addition to direct bacterial elimination, these peptides have regulatory effects on immune systems. Consequently, AMPs are also referred to as host defense peptides (Hancock and Sahl, 2006). To decode the key elements behind the functional diversity of AMPs, we have been taking time and efforts in constructing a comprehensive database that annotates such information. The first version of the Antimicrobial Peptide Database (APD; http://aps.unmc.edu/AP/main.html) was established in 2003 (Wang and Wang, 2004) and the database has since been further developed (Wang et al., 2009). The APD contained 1973 entries as of May 2012. To facilitate our bioinformatic analysis, we will register a peptide into the APD if it is (1) from natural sources; (2) with minimal inhibitory concentration (MIC) of less than 100 μM or 100 μg/mL; (3) less than 100 amino acid residues; and (4) with a characterized amino acid sequence (Wang, 2010). The APD allows users to extract important parameters (e.g., charge, hydrophobicity, motif, and structure) that determine peptide function. In particular, our database enables the generation of the amino acid composition for a select peptide or a family of AMPs with a common feature. This bioinformatic tool thus uncovers the amino acid use in natural AMPs from different sources, with different functions, or three-dimensional structures. This opinion article highlights the critical roles of the amino acid composition in naturally occurring AMPs in terms of evolutional, structural, and functional significance. Moreover, its application in designing and predicting new AMPs will also be discussed.
Peptides and proteins are exposed to a variety of interfaces in a physiological environment, such as cell membranes, protein nanoparticles or viruses. These interfaces have a significant impact on the interaction, self-assembly, and aggregation mechanisms of biomolecular systems. Peptide self-assembly, particularly amyloid fibril formation, is associated with a wide range of functions; however, there is a link with neurodegenerative diseases such as Alzheimer's disease. This review highlights how interfaces affect peptide structure and the kinetics of aggregation leading to fibril formation. In nature, many surfaces are nanostructures such as liposomes, viruses or synthetic nanoparticles. Once exposed to a biological medium, nanostructures are coated with a corona, which then determines their activity. Both accelerating and inhibiting effects on peptide self-assembly have been observed. When amyloid peptides adsorb to a surface, they typically concentrate locally, which promotes aggregation into insoluble fibrils. Starting from a combined experimental and theoretical approach, we introduce and review models that allow for a better understanding of peptide self-assembly near hard and soft matter interfaces. We present research results from our laboratories, obtained in the last few years, and propose relationships between biological interfaces such as membranes and viruses and amyloid fibril formation. This article is protected by copyright. All rights reserved.
Stapled peptides have the ability to mimic α-helices involved in protein binding and have proved to be effective pharmacological agents for disrupting protein-protein interactions. DNA-binding proteins such as transcription factors bind their cognate DNA sequences via an α-helix interacting with the major groove of DNA. We previously developed a stapled peptide based on the bacterial alternative sigma factor RpoN capable of binding the RpoN DNA promoter sequence and inhibiting RpoN-mediated expression in Escherichia coli. We have elucidated a structure-activity relationship for DNA binding by this stapled peptide, improving DNA binding affinity constants in the high nM range. Lead peptides were shown to have low toxicity as determined by their low hemolytic activity at 100 μM and were shown to have anti-virulence activity in a Galleria mellonella model of Pseudomonas aeruginosa infection. These findings support further preclinical development of stapled peptides as antivirulence agents targeting P. aeruginosa.
Secondary structure changes are an inherent part of antimicrobial (AMP) and amyloidogenic peptide activity, especially in close proximity to membranes, and impact the peptides’ function and dysfunction roles. The formation, and stability of α‐helical components are regarded as essential ‘intermediates’ for both these functions. To illuminate the conformational transitions leading to amyloid formation we use short cationic AMPs, from an Australian toadlet, Uperoleia mjobergii, (Uperin 3 family, U3) and assess the impact on secondary structural elements in the presence of a membrane mimetic surfactant, sodium dodecyl sulfate (SDS). Specifically, Uperin 3.x, where x=4, 5, 6 wild‐type peptides and position seven variants for each, R7A or K7A, were investigated using a combination of experimental and simulation approaches. In water, U3 peptides remain largely unstructured as random coils, with the addition of salts initiating structural transitions leading to assembly towards amyloid. Solution NMR data show that an unstructured U3.5 wt peptide transitions in the presence of SDS to a well‐defined α‐helical structure that spans nearly the entire sequence. Circular dichroism (CD) and ThT fluorescence studies show that all six U3 peptides aggregate in solution, albeit with vastly varying rates, and a dynamic equilibrium between soluble aggregates rich in either α‐helices or β‐sheets may exist in solution. However, the addition of SDS leads to a rapid disaggregation for all peptides and stabilisation of predominantly α‐helical content in all the U3 peptides. Molecular dynamics (MD) simulations show that the adsorption of U3.5 wt/R7A peptides onto the SDS micelle is driven by Coulombic attraction between peptide cationic residues and the negatively charged sulfate head‐groups on SDS. Simulating the interactions of various kinds of β‐sheet dimers (of both U3.5 wt and its variant U3.5 R7A) with SDS micelles confirmed β‐sheet content decreases in the dimers after their attachment to the SDS micelle. Adsorbed peptides interact favourably with the hydrophobic core of the micelle, promoting intramolecular hydrogen bonds leading to stabilisation of the α‐helical structure in peptides, and resulting in a corresponding decrease in intermolecular hydrogen bonds responsible for β‐sheets.
Peptides constitute an essential component of all organisms' protein homeostasis ranging from bacteria, plants, and animals. They have organically been evolved to perform a wide range of essential functions, including their role as neurotransmitters, antimicrobial peptides (AMPs), and hormones. AMPs are short peptides synthesized by almost all organisms, implicated in guarding the host from various microbial infections. Their inherent ability to differentiate the target microbes from the host confers them excellent prospects in fighting against microbial infections and affirming their robust therapeutic potential against numerous drug‐resistant microbes. Amyloidogenic peptides (AMYs) represent another class of short peptides armed with inherent aggregation propensity and form fibrillar aggregates rich in cross β‐sheet structure. They are often involved in various degenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and type‐2 diabetes. Although these two distinct classes of peptides (i.e., AMPs and AMYs) appear to be functionally divergent, recent studies suggest that they possess a significant degree of structural and functional reciprocity. Consistent with this, many AMPs display amphiphilic nature, and hence, they can facilitate membrane remodeling processes, such as pore formation and fusion, similar to AMYs. The mounting evidence suggests the inherent ability of AMPs to self‐assemble to form amyloid‐like structures. On the other hand, the demonstration of antimicrobial properties of AMYs in their monomeric conformation provides a hint about the existence of an evolutionary linkage between these two classes of peptides. The congregation of specific amino acids to form aggregation‐prone regions in a protein/peptide might have served as an evolutionary reservoir from which AMPs and AMYs were consecutively evolved. The current article reviews the fundamental features of the AMPs, AMYs, and their inter‐relatedness and emerging paradigm for their inter‐conversion. Antimicrobial peptides (AMPs) and amyloid‐forming peptides (AMYs) represent two essential categories of biomolecules with their distinct structural and functional identities. Recent investigations in this field outline the pieces of evidence for structural and functional swapping among each other. The review summarizes the underlying fundamental mechanisms responsible for their mutualistic swapping among each other and provides a window of opportunity for developing novel AMPs/AMPYs with diverse potential applications.
Research and new tools in biomaterials development by using peptides are currently growing, as more functional and versatile building blocks are used to design a host of functional biomaterials via chemical modifications for health care applications. It is a field that is attracting researchers from across soft matter science, molecular engineering and biomaterials science.
Covering the fundamental concepts of self-assembly, design and synthesis of peptides, this book will provide a solid introduction to the field for those interested in developing functional biomaterials by using peptide derivatives. The bioactive nature of the peptides and their physical properties are discussed in various applications in biomedicine. This book will help researchers and students working in biomaterials and biomedicine fields and help their understanding of modulating biological processes for disease diagnosis and treatments.
Magainin 2 and PGLa are antimicrobial peptides found together in frog skin secretions. When added as a mixture they show an order of magnitude increase in antibacterial activity and in model membrane permeation assays. Here we demonstrate that both peptides can form fibers with beta-sheet/turn signature in ATR-FTIR- and CD-spectroscopic analyses, but with different morphologies in EM images. Whereas, fiber formation results in acute reduction of the antimicrobial activity of the individual peptides, the synergistic enhancement of activity remains for the equimolar mixture of PGLa and magainin 2 also after fibril formation. The biological significance and potential applications of such supramolecular aggregates are discussed.
Severe infections associated with antibiotic-resistant bacteria and biofilms have attracted increasing interest as these diseases are difficult to treat with current antibiotics. Typical cationic antimicrobial peptides dermaseptins are considered to be the most promising next-generation antibiotics because of their broad-spectrum antimicrobial activities and minor side effects. Two new dermaseptin peptides, DMS-PS1 and DMS-PS2, have been identified by “shotgun” molecular cloning of encoding cDNAs in the crude skin secretions of the waxy monkey tree frog, Phyllomedusa sauvagei. The mature peptide sequences predicted from the cloned cDNAs were separated from crude skin secretions and confirmed by mass spectrometry. Chemically synthetic replicates were assessed for various biological activities. Both dermaseptins were potently effective against a broad spectrum of microorganisms including antibiotic-resistant bacteria and displayed significant potency against gram-positive and gram-negative bacterial biofilms with low toxicity towards mammalian red blood cells. Remarkably, DMS-PS2 was effective against infections in murine skin caused by methicillin-resistant Staphylococcus aureus as a result of an induced wound. The actions of DMS-PS2 were with a membrane permeabilization mode. Overall, the data provided convincing evidence for the development of anti-infectious agents and/or biomaterials as a new therapeutic approach against bacterial infections.
Statement of Significance
Bacterial adhesion to biomaterials remains a major problem. Antimicrobial peptides (AMPs) are well-known components of the innate immune system that can be applied to overcome biofilm-associated infections. Cationic dermaseptin peptides showed significant broad-spectrum antimicrobial activities and activities against bacterial biofilms of persistent infections in association with weak toxicity for mammalian red blood cells. The membrane permeabilizing ability of DMS-PS2 was confirmed, and importantly, it demonstrated potent efficiency of the treatment of MRSA infected murine skin model. Furthermore, beyond our expectation, DMS-PS2 showed a self-aggregating parameter, indicating a promising potential for the use of immobilized AMPs in clinical applications., which makes it also a promising suggestion for infection-proof biomaterial development.
The emergence of bacterial resistance to the most commonly used antibiotics encourages the design of novel antimicrobial drugs. Antimicrobial proteins and peptides (AMPs) are the key players in host innate immunity. They exert a rapid and multifaceted action that reduces the development of bacterial adaptation mechanisms. Human antimicrobial RNases belonging to the vertebrate specific RNase A superfamily participate in the maintenance of tissue and body fluid sterility. Among the eight human canonical RNases, RNase 3 stands out as the most cationic and effective bactericidal protein against Gram-negative species. Its enhanced ability to disrupt the bacterial cell wall has evolved in detriment of its catalytic activity. Based on structure-functional studies we have designed an RNase 3/1 hybrid construct that combines the high catalytic activity of RNase 1 with RNase 3 bactericidal properties. Next, we have explored the ability of this hybrid RNase to target the development of bacterial resistance on an Acinetobacter baumannii cell culture. Synergy assays were performed in combination with colistin, a standard antimicrobial peptide used as an antibiotic to treat severe infections. Positive synergism was observed between colistin and the RNase 3/1 hybrid protein. Subsequently, using an in vitro experimental evolution assay, by exposure of a bacterial culture to colistin at incremental doses, we demonstrated the ability of the RNase 3/1 construct to reduce the emergence of bacterial antimicrobial resistance. The results advance the potential applicability of RNase-based drugs as antibiotic adjuvants.
Fast detection of bacteria in samples presumed to be un-contaminated, such as blood, is of great importance. Indeed, rapid diagnosis allows the set-up of appropriate antibiotic treatment. Besides clinical issues, there are many other domains, such as food processing or drug manufacturing, where the strict absence of any bacteria has to be assessed. Because the bacterial load found in most contaminated samples is often below the limit of detection for currently validated assays, a preliminary enrichment step is required to allow bacterial multiplication before proceeding to the analysis step, whatever it might be - cultural, immunological or molecular methods. In this study, we describe the use of a biosensor for single-step bacteria detection. The whole analysis is performed in less than 20 h, during the growth phase of the micro-organisms, using an array of antimicrobial peptides (AMPs) coupled with a surface plasmon resonance imager (SPRI). A wide range of bacterial strains are assayed, showing differentiated affinity patterns with the immobilized peptides, which are confirmed by multivariate analysis. This work establishes the evidence that antimicrobial peptides, mostly used so far in the antibiotic drug industry, are suited for the wide-spectrum detection of unknown bacteria in samples, even at very low initial loads. Moreover, the small set of AMPs that were assayed provided a specific affinity profile for each pathogen, as confirmed by multivariate analyses. Furthermore, this work opens up the possibility of applying this method in more complex and relevant samples such as foodstuff, urine or blood.
Self-assembled nanomaterials are composed with building blocks through non-covalent interaction and spontaneously arranged into well-ordered nanostructures with defined functions. The well-organized arrangement and the two/three-dimension nanostructure of the architectures endow the nanomaterials abundant excellent biofunctions for antimicrobial applications and bacterial detections. Beyond nature inspired sources, the hybrid artificial nanomaterials including inorganic nanoparticles, nanosized small synthetic molecules assemblies, self-assembled multi-layer polymers are reviewed. We highlight recent contributions to developing strategies for the construction of self-assembled nanomaterials applied in bacterial infection. In addition, the design concept, assembled driving forces, nanostructural effect, bacterial targeting sites, stimuli-responsive factors, antimicrobial mechanism are also discussed and summarized. Especially, the relationship between the self-assembly and their biofunctions is emphasized. As the frontier field in supramolecular chemistry, in vivo self-assembled nanomaterials with specific stimuli-responsiveness and surprised biofunctions are also included in this review. Finally, we briefly outline our perspectives on how to explore and fabricate fascinated self-assembled nanomaterials and what the challenges are facing in this field
Self-assembled nanomaterials are composed of building blocks through non-covalent interaction and spontaneously arranged into well-ordered nanostructures with defined functions. The well-organized arrangement and the two-/three-dimensional nanostructure of the architectures endow the nanomaterials abundant excellent biofunctions for bacterial infection detection and therapy applications. Beyond nature-inspired sources, the hybrid artificial nanomaterials including inorganic nanoparticles, nanosized small synthetic molecules assemblies, self-assembled multilayer polymers are reviewed in this chapter. In addition, the design concept, assembled driving forces, nanostructural effect, antimicrobial mechanism, detection methods are also discussed and summarized. As the promising field, in vivo self-assembled nanomaterials with specific stimuli-responsiveness and surprised biofunctions are also included in this chapter to explore and fabricate fascinated self-assembled nanomaterials.
There is increasing interest in developing cationic host defense peptides (HDPs) and their synthetic derivatives as antimicrobial, immunomodulatory, and anti-biofilm agents. These activities are often evaluated without considering biologically relevant concentrations of salts or serum; furthermore certain HDPs have been shown to aggregate in vitro. Here we examined the effect of aggregation on the immunomodulatory activity of a synthetic innate defense regulator peptide, 1018 (VRLIVAVRIWRR-NH2). A variety of salts and solutes were screened to determine their influence on 1018 aggregation, revealing that this peptide "salts out" of solution in an anion-specific and concentration-dependent manner. Furthermore, the immunomodulatory activity of 1018 was found to be inhibited under aggregation-promoting conditions. A series of 1018 derivatives were synthesized with the goal of disrupting this self-assembly process. Indeed, some derivatives exhibited reduced aggregation while maintaining certain immunomodulatory functions, demonstrating that it is possible to engineer optimized synthetic HDPs to avoid unwanted peptide aggregation.
The amphibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum amphibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.
Antimicrobial peptides (AMP) are important components of the host innate immune response, as they exert broad-spectrum antimicrobial
activities against pathogenic microbes. The AMP allow housefly larva (maggots) to live in harsh environments filled with pathogenic
bacteria. In this study, maggot AMP were induced by incubation with inactivated Salmonella pullorum and crudely extracted. The concentration and antimicrobial activity of the maggot AMP were then measured. In bird experiments,
chickens were artificially infected with S. pullorum, and the maggot AMP extracts were used to treat the infected chickens. The expression level of AMP was significantly enhanced
by S. pullorum stimulation, and the antibiotic activity of the S. pullorum-induced AMP was significantly stronger than that of the noninduced AMP, especially against S. pullorum. In the bird experiments, based on survival rate, blood indicators, and intestinal bacterial changes, maggot AMP and antibiotics
were successful in treating the S. pullorum-infected chickens. In conclusion, AMP have the potential for further development as a convenient, alternative antibiotic
strategy to reduce the use of antibiotics and disease resistance.
Five analogs of a natural peptide (BmKn1) found in the venom of scorpion Buthus martensii Karsh have been synthesized and tested to compare their antimicrobial and hemolytic activity with the wild type. Circular dichroism spectra show that these peptides form an alpha helix structure and its amino acid positions predict an amphipathic nature. Results show that increasing hydrophobicity by substituting successively positions 5 and 9 of the sequence (on the hydrophobic side of the helix) with alanine, valine and leucine enhances antimicrobial activity and hemolysis. When changes are done on positions 7 and 10 (on the hydrophilic side) by introducing more positive charges with addition of lysine, both activities also increase. However, when negative charges are introduced instead (with glutamic acids), antimicrobial activity is observed but hemolysis is reduced to zero under the concentrations studied. Although strong inhibitory activity begins at low concentrations (10μg/mL), some peptides level off inhibition and no change is observed as concentrations are increased.
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Antimicrobial peptides (AMPs) show remarkable selectivity toward lipid membranes and possess promising antibiotic potential. Their modes of action are diverse and not fully understood, and innovative peptide design strategies are needed to generate AMPs with improved properties. We present a de novo peptide design approach that resulted in new AMPs possessing low-nanomolar membranolytic activities. Thermal analysis revealed an entropy-driven mechanism of action. The study demonstrates sustained potential of advanced computational methods for designing peptides with the desired activity.
Indolicidin (IR13), a 13-residue antimicrobial peptide from the cathelicidin family, is known to exhibit a broad spectrum of antimicrobial activity against various microorganisms. This peptide inhibits bacterial DNA synthesis resulting in cell filamentation. However, the precise mechanism remains unclear and requires further investigation. The central PWWP motif of IR13 provides a unique structural element that can wrap around, and thus stabilize, duplex B-type DNA structures. Replacements of the central Trp-Trp pair with Ala-Ala, His-His, or Phe-Phe residues in the PxxP motif significantly affects the ability of the peptide to stabilize duplex DNA. Results of microscopy studies in conjunction with spectroscopic data confirm that the DNA duplex is stabilized by IR13, thereby inhibiting DNA replication and transcription. In this study we provide high-resolution structural information on the interaction between indolicidin and DNA, which will be beneficial for the design of novel therapeutic antibiotics based on peptide scaffolds.
The RNase A superfamily lineage includes distant members with antimicrobial properties suggesting a common ancestral host defense role. In an effort to identify the minimal requirements for the eosinophil cationic protein (ECP or RNase 3) antimicrobial properties we have applied site directed mutagenesis on its closest family homologue, the eosinophil derived neurotoxin (EDN or RNase 2). Both eosinophil secretion proteins are involved in human immune defense, and are reported as among the most rapidly evolving coding sequences in primates. Previous studies in our laboratory defined two regions at the N-terminus involved in the protein antimicrobial action, encompassing residues 8 to 16 and 34 to 36. Here we demonstrate that switching two single residues is enough to provide EDN with ECP antipathogen properties. That is, the EDN double mutant Q34R/R35W displays an enhanced bactericidal activity, particularly towards Gram-negative bacteria, and a significant increase of its affinity towards the bacteria outer membrane lipopolysaccharides (LPS). Moreover, we confirmed the direct contribution of W35 residue in the LPS binding, membrane interaction and permeabilization processes. Furthermore, the additional T13 to I substitution provides EDN with an exposed hydrophobic patch required for the protein self-aggregation and triggers the bacteria agglutination, thereby increasing the final antimicrobial activity up to 20 fold. Our results highlight how single selected mutations can reshape the entire protein function. The present work provides an example of how structure guided protein engineering can successfully reproduce an evolution selection process towards the emergence of new physiological roles. This article is protected by copyright. All rights reserved.
A number of physicochemical characteristics have been described which contribute to the biological activity of antimicrobial peptides. This information was used to design a novel antimicrobial peptide sequence by using an intrinsically inactive membrane-associated peptide derived from the HIV glycoprotein, gp41, as a starting scaffold. This peptide corresponds to the tryptophan-rich membrane-proximal region of gp41, which is known to interact at the interfacial region of the viral membrane and adopts a helical structure in the presence of lipids. Three synthetic peptides were designed to increase the net positive charge and amphipathicity of this 19-residue peptide. Ultimately, the peptide with the greatest degree of amphipathicity and largest positive charge proved to be the most potent antimicrobial, and this peptide could be further modified to improve the antimicrobial activity. However, the other two peptides were relatively ineffective antimicrobials and instead proved to be extremely hemolytic. This work demonstrates a novel approach for the design of unexplored antimicrobial peptide sequences but it also reveals that the biological and cytotoxic activities of these polypeptides depend on a number of interrelated factors.
The amyloid beta-protein (Abeta) is believed to be the key mediator of Alzheimer's disease (AD) pathology. Abeta is most often characterized as an incidental catabolic byproduct that lacks a normal physiological role. However, Abeta has been shown to be a specific ligand for a number of different receptors and other molecules, transported by complex trafficking pathways, modulated in response to a variety of environmental stressors, and able to induce pro-inflammatory activities.
Here, we provide data supporting an in vivo function for Abeta as an antimicrobial peptide (AMP). Experiments used established in vitro assays to compare antimicrobial activities of Abeta and LL-37, an archetypical human AMP. Findings reveal that Abeta exerts antimicrobial activity against eight common and clinically relevant microorganisms with a potency equivalent to, and in some cases greater than, LL-37. Furthermore, we show that AD whole brain homogenates have significantly higher antimicrobial activity than aged matched non-AD samples and that AMP action correlates with tissue Abeta levels. Consistent with Abeta-mediated activity, the increased antimicrobial action was ablated by immunodepletion of AD brain homogenates with anti-Abeta antibodies.
Our findings suggest Abeta is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Abeta-mediated pathology and has important implications for ongoing and future AD treatment strategies.
Antimicrobial peptides (AMPs) are gaining popularity as better substitute to antibiotics. These peptides are shown to be active against several bacteria, fungi, viruses, protozoa and cancerous cells. Understanding the role of primary structure of AMPs in their specificity and activity is essential for their rational design as drugs. Collection of Anti-Microbial Peptides (CAMP) is a free online database that has been developed for advancement of the present understanding on antimicrobial peptides. It is manually curated and currently holds 3782 antimicrobial sequences. These sequences are divided into experimentally validated (patents and non-patents: 2766) and predicted (1016) datasets based on their reference literature. Information like source organism, activity (MIC values), reference literature, target and non-target organisms of AMPs are captured in the database. The experimentally validated dataset has been further used to develop prediction tools for AMPs based on the machine learning algorithms like Random Forests (RF), Support Vector Machines (SVM) and Discriminant Analysis (DA). The prediction models gave accuracies of 93.2% (RF), 91.5% (SVM) and 87.5% (DA) on the test datasets. The prediction and sequence analysis tools, including BLAST, are integrated in the database. CAMP will be a useful database for study of sequence-activity and -specificity relationships in AMPs. CAMP is freely available at http://www.bicnirrh.res.in/antimicrobial.
Much effort goes into identifying new antimicrobial compounds able to evade the increasing resistance of microorganisms to antibiotics. One strategy relies on antimicrobial peptides, either derived from fragments released by proteolytic cleavage of proteins or designed from known antimicrobial protein regions.
To identify these antimicrobial determinants, we developed a theoretical approach that predicts antimicrobial proteins from their amino acid sequence in addition to determining their antimicrobial regions. A bactericidal propensity index has been calculated for each amino acid, using the experimental data reported from a high-throughput screening assay as reference. Scanning profiles were performed for protein sequences and potentially active stretches were identified by the best selected threshold parameters. The method was corroborated against positive and negative datasets. This successful approach means that we can spot active sequences previously reported in the literature from experimental data for most of the antimicrobial proteins examined.
The method presented can correctly identify antimicrobial proteins with an accuracy of 85% and a sensitivity of 90%. The method can also predict their key active regions, making this a tool for the design of new antimicrobial drugs.
Temporins constitute a family of amphipathic alpha-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic alpha-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies 'conformational switches', domains with equal probabilities for adopting random coil, alpha-helical and beta-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid beta-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.
Formation of amyloid-like fibrils is involved in numerous human protein deposition diseases, but is also an intrinsic property of polypeptide chains in general. Progress achieved recently now allows the aggregation propensity of proteins to be analyzed over large scales. In this work we used a previously developed predictive algorithm to analyze the propensity of the 34,180 protein sequences of the human proteome to form amyloid-like fibrils. We show that long proteins have, on average, less intense aggregation peaks than short ones. Human proteins involved in protein deposition diseases do not differ extensively from the rest of the proteome, further demonstrating the generality of protein aggregation. We were also able to reproduce some of the results obtained with other algorithms, demonstrating that they do not depend on the type of computational tool employed. For example, proteins with different subcellular localizations were found to have different aggregation propensities, in relation to the various efficiencies of quality control mechanisms. Membrane proteins, intrinsically disordered proteins, and folded proteins were confirmed to have very different aggregation propensities, as a consequence of their different structures and cellular microenvironments. In addition, gatekeeper residues at strategic positions of the sequences were found to protect human proteins from aggregation. The results of these comparative analyses highlight the existence of intimate links between the propensity of proteins to form aggregates with beta-structure and their biology. In particular, they emphasize the existence of a negative selection pressure that finely modulates protein sequences in order to adapt their aggregation propensity to their biological context.
The accumulation of beta-amyloid peptides (Abeta) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated Abeta is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent Abeta toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of Abeta1-42 and the toxic fragment Abeta25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) reduction. Formation of the toxic beta-sheet conformation by Abeta peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of Abeta1-40 and Abeta25-35 to negatively charged lipid vesicles and Abeta induced cell toxicity. These results suggest that Abeta toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.
Protein misfolding has been shown to be the direct cause of a number of highly devastating diseases such as Alzheimer's disease, Parkinson's disease, and Creutzfeldt-Jacob syndrome, affecting the aging population globally. The deposition in tissues of amyloid fibrils is a characteristic of all these diseases, and the mechanisms by which these protein aggregates form continue to be intensively investigated. In only a fraction of cases is an underlying mutation responsible, and accordingly, what initiates amyloid formation in vivo is the major question that is addressed. In this study, we show that membranes containing phosphatidylserine (PS), a negatively charged phospholipid, induce a rapid formation of fibers by a variety of proteins, viz., lysozyme, insulin, glyceraldehyde-3-phosphate dehydrogenase, myoglobin, transthyretin, cytochrome c, histone H1, and alpha-lactalbumin. Congo red staining of these fibers yields the characteristic light green birefringence of amyloid, and fluorescent lipid tracers further reveal them to include phospholipids. Our results suggest that PS as well as other acidic phospholipids could provide the physiological low-pH environment on cellular membranes, enhancing protein fibril formation in vivo. Interestingly, all the proteins mentioned above either are cytotoxic or induce apoptosis. PS-protein interaction could be involved in the mechanism of cytotoxicity of the aggregated protein fibrils, perturbing membrane functions. Importantly, our results suggest that this process induced by acidic phospholipids may provide an unprecedented and generic connection between three current major areas of research: (i) mechanism(s) triggering amyloid formation, (ii) cytotoxicity of amyloidal protein aggregates, and (iii) mechanism(s) of action of cytotoxic proteins.
Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibril-forming peptides in a beta-sheet conformation. One of the most abundant components in amyloid aggregates is the beta-amyloid peptide 1-42 (Abeta 1-42). Membrane alterations may proceed to cell death by either an oxidative stress mechanism, caused by the peptide and synergized by transition metal ions, or through formation of ion channels by peptide interfacial self-aggregation. Here we demonstrate that Langmuir films of Abeta 1-42, either in pure form or mixed with lipids, develop stable monomolecular arrays with a high surface stability. By using micropipette aspiration technique and confocal microscopy we show that Abeta 1-42 induces a strong membrane destabilization in giant unilamellar vesicles composed of palmitoyloleoyl-phosphatidylcholine, sphingomyelin, and cholesterol, lowering the critical tension of vesicle rupture. Additionally, Abeta 1-42 triggers the induction of a sequential leakage of low- and high-molecular-weight markers trapped inside the giant unilamellar vesicles, but preserving the vesicle shape. Consequently, the Abeta 1-42 sequence confers particular molecular properties to the peptide that, in turn, influence supramolecular properties associated to membranes that may result in toxicity, including: 1), an ability of the peptide to strongly associate with the membrane; 2), a reduction of lateral membrane cohesive forces; and 3), a capacity to break the transbilayer gradient and puncture sealed vesicles.
Antimicrobial peptides are an abundant and diverse group of molecules that are produced by many tissues and cell types in a variety of invertebrate, plant and animal species. Their amino acid composition, amphipathicity, cationic charge and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Although these models are helpful for defining mechanisms of antimicrobial peptide activity, their relevance to how peptides damage and kill microorganisms still need to be clarified. Recently, there has been speculation that transmembrane pore formation is not the only mechanism of microbial killing. In fact several observations suggest that translocated peptides can alter cytoplasmic membrane septum formation, inhibit cell-wall synthesis, inhibit nucleic-acid synthesis, inhibit protein synthesis or inhibit enzymatic activity. In this review the different models of antimicrobial-peptide-induced pore formation and cell killing are presented.
Antimicrobial peptides (AMPs) are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenge. These small cationic peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces and have demonstrated direct antimicrobial activity against various bacteria, viruses, fungi, and parasites. This review summarizes their progress to date as commercial antimicrobial drugs for topical and systemic indications.
Literature review.
Despite numerous clinical trials, no modified AMP has obtained Food & Drug Administration approval yet for any topical or systemic medical indications.
While AMPs are recognized as essential components of natural host innate immunity against microbial challenge, their usefulness as a new class of antimicrobial drugs still remains to be proven.
Protein aggregation correlates with the development of several debilitating human disorders of growing incidence, such as Alzheimer's and Parkinson's diseases. On the biotechnological side, protein production is often hampered by the accumulation of recombinant proteins into aggregates. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. AGGRESCAN is a web-based software for the prediction of aggregation-prone segments in protein sequences, the analysis of the effect of mutations on protein aggregation propensities and the comparison of the aggregation properties of different proteins or protein sets.
AGGRESCAN is based on an aggregation-propensity scale for natural amino acids derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities. It also provides new insights into the differential aggregation properties displayed by globular proteins, natively unfolded polypeptides, amyloidogenic proteins and proteins found in bacterial inclusion bodies.
By identifying aggregation-prone segments in proteins, AGGRESCAN http://bioinf.uab.es/aggrescan/ shall facilitate (i) the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases and (ii) the anticipation of aggregation phenomena during storage or recombinant production of bioactive polypeptides or polypeptide sets.
Antibacterial peptides are important components of the innate immune system, used by the host to protect itself from different types of pathogenic bacteria. Over the last few decades, the search for new drugs and drug targets has prompted an interest in these antibacterial peptides. We analyzed 486 antibacterial peptides, obtained from antimicrobial peptide database APD, in order to understand the preference of amino acid residues at specific positions in these peptides.
It was observed that certain types of residues are preferred over others in antibacterial peptides, particularly at the N and C terminus. These observations encouraged us to develop a method for predicting antibacterial peptides in proteins from their amino acid sequence. First, the N-terminal residues were used for predicting antibacterial peptides using Artificial Neural Network (ANN), Quantitative Matrices (QM) and Support Vector Machine (SVM), which resulted in an accuracy of 83.63%, 84.78% and 87.85%, respectively. Then, the C-terminal residues were used for developing prediction methods, which resulted in an accuracy of 77.34%, 82.03% and 85.16% using ANN, QM and SVM, respectively. Finally, ANN, QM and SVM models were developed using N and C terminal residues, which achieved an accuracy of 88.17%, 90.37% and 92.11%, respectively. All the models developed in this study were evaluated using five-fold cross validation technique. These models were also tested on an independent or blind dataset.
Among antibacterial peptides, there is preference for certain residues at N and C termini, which helps to demarcate them from non-antibacterial peptides. Both the termini play a crucial role in imparting the antibacterial property to these peptides. Among the methods developed, SVM shows the best performance in predicting antibacterial peptides followed by QM and ANN, in that order. AntiBP (Antibacterial peptides) will help in discovering efficacious antibacterial peptides, which we hope will prove to be a boon to combat the dreadful antibiotic resistant bacteria. A user friendly web server has also been developed to help the biological community, which is accessible at http://www.imtech.res.in/raghava/antibp/.
Alzheimer's disease (AD) is associated with dementia, brain atrophy and the aggregation and accumulation of a cortical amyloid-beta peptide (Abeta). Chronic bacterial infections are frequently associated with amyloid deposition. It had been known from a century that the spirochete Treponema pallidum can cause dementia in the atrophic form of general paresis. It is noteworthy that the pathological hallmarks of this atrophic form are similar to those of AD. Recent observations showed that bacteria, including spirochetes contain amyloidogenic proteins and also that Abeta deposition and tau phosphorylation can be induced in or in vivo following exposure to bacteria or LPS. Bacteria or their poorly degradable debris are powerful inflammatory cytokine inducers, activate complement, affect vascular permeability, generate nitric oxide and free radicals, induce apoptosis and are amyloidogenic. All these processes are involved in the pathogenesis of AD. Old and new observations, reviewed here, indicate that to consider the possibility that bacteria, including several types of spirochetes highly prevalent in the population at large or their persisting debris may initiate cascade of events leading to chronic inflammation and amyloid deposition in AD is important, as appropriate antibacterial and antiinflammatory therapy would be available to prevent dementia.
Eosinophil cationic protein (ECP) is an antimicrobial protein belonging to the superfamily of RNase A. ECP exhibits a broad spectrum of action against bacteria and, at higher concentrations, displays cytotoxic activity to eukaryotic cells. Recently, a powerful aggregation activity for lipid vesicles and for the gram-negative E. coli specie has also been related to the protein toxicity. Here we present the amyloid-like aggregation capacity of ECP. This is the first report of amyloid aggregation in a native nonengineered ribonuclease. The ECP aggregates are able to bind the amyloid-diagnostic dyes Thioflavin T and Congo Red and display a protofibril morphology when observed under electronic microscopy. We have also identified an N-terminus hydrophobic patch (residues 8-16) that is required for the amyloid aggregation process. A single substitution, I13A, breaks the aggregation prone sequence and abolishes the amyloid aggregation ability. Moreover, the corresponding R1N19 peptide is able to reproduce the protein amyloid-like aggregation behavior. The results may provide new clues on the protein antimicrobial mechanism and its toxicity to the host tissues in inflammation processes.
There is widespread acceptance that cationic antimicrobial peptides, apart from their membrane-permeabilizing/disrupting properties, also operate through interactions with intracellular targets, or disruption of key cellular processes. Examples of intracellular activity include inhibition of DNA and protein synthesis, inhibition of chaperone-assisted protein folding and enzymatic activity, and inhibition of cytoplasmic membrane septum formation and cell wall synthesis. The purpose of this minireview is to question some widely held views about intracellular-targeting antimicrobial peptides. In particular, I focus on the relative contributions of intracellular targeting and membrane disruption to the overall killing strategy of antimicrobial peptides, as well as on mechanisms whereby some peptides are able to translocate spontaneously across the plasma membrane. Currently, there are no more than three peptides that have been convincingly demonstrated to enter microbial cells without the involvement of stereospecific interactions with a receptor/docking molecule and, once in the cell, to interfere with cellular functions. From the limited data currently available, it seems unlikely that this property, which is isolated in particular peptide families, is also shared by the hundreds of naturally occurring antimicrobial peptides that differ in length, amino acid composition, sequence, hydrophobicity, amphipathicity, and membrane-bound conformation. Microbial cell entry and/or membrane damage associated with membrane phase/transient pore or long-lived transitions could be a feature common to intracellular-targeting antimicrobial peptides and mammalian cell-penetrating peptides that have an overrepresentation of one or two amino acids, i.e. Trp and Pro, His, or Arg. Differences in membrane lipid composition, as well as differential lipid recruitment by peptides, may provide a basis for microbial cell killing on one hand, and mammalian cell passage on the other.
An increasing amount of information on the action of antimicrobial peptides (AMPs) at the molecular level has not yet been translated into a comprehensive understanding of effects in bacteria. Although some biophysical attributes of AMPs have been correlated with macroscopic features, the physiological relevance of other properties has not yet been addressed. Pertinent and surprising conclusions have therefore been left unstated. Strong membrane-binding and micromolar therapeutic concentrations of AMPs indicate that membrane-bound concentrations may be reached that are higher than intuitively expected, triggering disruptive effects on bacteria.
Permeation of the cell membrane leading to cell death is a mechanism used by a large number of membrane-lytic peptides. Some are linear, mostly helical, and others contain one or more disulfide bonds forming beta-sheet or both beta-sheet and alpha-helix structures. They are all soluble in solution but when they reach the target membrane, conformational changes occur which let them associate with and lyse the membrane. Some lytic peptides are not cell-selective and lyse different microorganisms and normal mammalian cells, while others are specific to either type of cells. Despite extensive studies, the mode of action of membrane-lytic peptides is not fully understood and the basis for their selectivity towards specific target cells is not known. Many studies have shown that peptide-lipid interactions leading to membrane permeation play a major role in their activity. Membrane permeation by amphipathic alpha-helical peptides has been proposed to occur via one of two general mechanisms: (i) transmembrane pore formation via a 'barrel-stave' mechanism; and (ii) membrane destruction/solubilization via a 'carpet' mechanism. This review, which is focused on the different stages of membrane permeation induced by representatives of amphipathic alpha-helical antimicrobial and cell non-selective lytic peptides distinguishes between the 'carpet' mechanism, which holds for antimicrobial peptides versus the 'barrel-stave' mechanism, which holds for cell non-selective lytic peptides.
Animals defend themselves against invading pathogenic micro-organisms by utilizing cationic anti-microbial peptides, which rapidly kill various micro-organisms without exerting toxicity against the host. Physicochemical peptide-lipid interactions provide attractive mechanisms for innate immunity. Many of these peptides form amphipathic secondary structures (alpha-helices and beta-sheets) which can selectively interact with anionic bacterial membranes by electrostatic interaction. Rapid, peptide-induced membrane permeabilization is an effective mechanism of anti-microbial action. Magainin 2 from frog skin forms a dynamic peptide-lipid supramolecular-complex pore that allows mutually coupled transmembrane transport of ions and lipids. The peptide molecule is internalized upon the disintegration of the pore. Several anti-microbial peptides are known to work synergistically.
Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.5 kDa amyloidogenic fragment derived from lactadherin. Subsequent characterization revealed that this peptide forms amyloid fibrils at pH 7.4 when incubated at 37 degrees C. Furthermore, although full-length lactoferrin does not by itself form amyloid fibrils, the protein does bind to the peptide fibrils as revealed by an increase in thioflavin T fluorescence and the presence of enlarged fibrils by transmission electron microscopy and polarized light microscopy. The binding of lactoferrin is a selective interaction with the NAGDVAFV fibrils. Lactoferrin does not bind to insulin or lysozyme fibrils, and the NAGDVAFV fibrils do not bind to soluble insulin or lysozyme. The lactoferrin appears to coat the peptide fibril surface to form mixed peptide/protein fibrils, but again there is no evidence for the formation of lactoferrin-only fibrils. This interaction, therefore, seems to involve selective binding rather than conventional seeding of fibril formation. We suggest that such a process could be generally important in the formation of amyloid fibrils in vivo since the identification of both full-length protein and protein fragments is common in ex vivo amyloid deposits.
Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.
Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.
It is said that nature is the greatest innovator, yet molecular conservation can be equally powerful. One key requirement for the survival of any host is its ability to defend against infection, predation and competition. Recent discoveries, including the presence of a multidimensional structural signature, have revealed a previously unforeseen structural and functional congruence among host defence effector molecules spanning all kingdoms of life. Antimicrobial peptides, kinocidins, polypeptide venoms and other molecules that were once thought to be distinct in form and function now appear to be members of an ancient family of host defence effectors. These molecules probably descended from archetype predecessors that emerged during the beginning of life on earth. Understanding how nature has sustained these host defence molecules with a potent efficacy in the face of dynamic microbial evolution should provide new opportunities to prevent or treat life-threatening infections.
Previous studies indicate that binding of alpha-synuclein to membranes is critical for its physiological function and the development of Parkinson's disease (PD). Here, we have investigated the association of fluorescence-labeled alpha-synuclein variants with different types of giant unilamellar vesicles using confocal microscopy. We found that alpha-synuclein binds with high affinity to anionic phospholipids, when they are embedded in a liquid-disordered as opposed to a liquid-ordered environment. This indicates that not only electrostatic forces but also lipid packing and hydrophobic interactions are critical for the association of alpha-synuclein with membranes in vitro. When compared to wild-type alpha-synuclein, the disease-causing alpha-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that the natural association of alpha-synuclein with membranes is altered in the inherited forms of Parkinson's disease.
Amyloid fibrils are highly ordered protein assemblies known to contribute to the pathology of a variety of genetic and aging-associated diseases. More recently, these fibrils have been shown to be useful as structural scaffolds in both natural biological systems and nanotechnology applications. The intense interest in amyloid fibrils has led to the investigation of well-characterized proteins, such as hen egg white lysozyme (HEWL), as model systems to examine structural and mechanistic principles that may be generally applicable to all amyloid fibrils. The purpose of this review is to critically examine the fibril-formation literature of proteins in the lysozyme family with respect to the known structure and folding properties of these proteins. The goal is to identify similarities and differences within the family, examine general misfolding / aggregation principles, and identify key areas of importance for future work on the fibril formation of these proteins.
Sterols impart significant changes to the biophysical properties of lipid bilayers. In this regard the impact of cholesterol on membrane organization and dynamics is particularly well documented and serves for comparison with other sterols. However, the factors underlying the molecular evolution of cholesterol remain enigmatic. To this end, cholesterol attenuates membrane perturbation by the so-called antimicrobial peptides (AMPs), produced ubiquitously by eukaryotic cells to combat bacterial infections by compromising the permeability barrier function of the microbial target membranes. In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Interestingly, sterol concentration dependent effects on the membrane association of these peptides were observed. At X(Sterol)=0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol>lanosterol> or =ergosterol, and cholesterol>lanosterol>ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cholesterol may have derived from the ability to protect the AMP-secreting host cell from the membrane damaging action of the antimicrobial peptides.
- H Zhao
- R Sood
- A Jutila
- S Bose
- G Fimland
- J Nissenmeyer
- P K Kinnunen
H. Zhao, R. Sood, A. Jutila, S. Bose, G. Fimland, J. NissenMeyer, P. K. Kinnunen, Biochim. Biophys. Acta Biomembr.
2006, 1758, 1461-1474.
- M Torrent
- F Odorizzi
- M V Nogues
- E Boix
M. Torrent, F. Odorizzi, M. V. Nogues, E. Boix, Biomacromolecules 2010, 11, 1983-1990.
- C Hertel
- E Terzi
- N Hauser
- R Jakob-Rotne
- J Seelig
- J A Kemp
C. Hertel, E. Terzi, N. Hauser, R. Jakob-Rotne, J. Seelig, J. A.
Kemp, Proc. Natl. Acad. Sci. USA 1997, 94, 9412-9416; b) H.
Zhao, E. K. Tuominen, P. K. Kinnunen, Biochemistry 2004, 43,
10302-10307.
- M Stockl
- P Fischer
- E Wanker
- A Herrmann
M. Stockl, P. Fischer, E. Wanker, A. Herrmann, J. Mol. Biol.
2008, 375, 1394-1404.
- S Thomas
- S Karnik
- R S Barai
- V K Jayaraman
- S Idiculathomas
S. Thomas, S. Karnik, R. S. Barai, V. K. Jayaraman, S. IdiculaThomas, Nucleic Acids Res. 2010, 38, D774-D780.
- E Monsellier
- M Ramazzotti
- N Taddei
- F Chiti
E. Monsellier, M. Ramazzotti, N. Taddei, F. Chiti, PLoS
Comput. Biol. 2008, 4, e1000199.
- A J Trexler
- M R Nilsson
A. J. Trexler, M. R. Nilsson, Curr. Protein Pept. Sci. 2007, 8, 537557.
- M N Melo
- R Ferre
- M A Castanho
M. N. Melo, R. Ferre, M. A. Castanho, Nat. Rev. Microbiol. 2009,
7, 245-250.
- Y J Gordon
- E G Romanowski
- A M Mcdermott
Y. J. Gordon, E. G. Romanowski, A. M. McDermott, Curr. Eye
Res. 2005, 30, 505-515.
- A K Mahalka
- P K Kinnunen
A. K. Mahalka, P. K. Kinnunen, Biochim. Biophys. Acta Biomembr. 2009, 1788, 1600-1609.
- S J Soscia
- J E Kirby
- K J Washicosky
- S M Tucker
- M Ingelsson
- B Hyman
- M A Burton
- L E Goldstein
- S Duong
- R E Tanzi
- R D Moir
S. J. Soscia, J. E. Kirby, K. J. Washicosky, S. M. Tucker, M.
Ingelsson, B. Hyman, M. A. Burton, L. E. Goldstein, S. Duong,
R. E. Tanzi, R. D. Moir, PLoS One 2010, 5, e9505.
- M R Yeaman
- N Y Yount
M. R. Yeaman, N. Y. Yount, Nat. Rev. Microbiol. 2007, 5, 727740.
- R Sood
- P K Kinnunen
R. Sood, P. K. Kinnunen, Biochim. Biophys. Acta Biomembr.
2008, 1778, 1460-1466.