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Ehud Gazit

Publications and source records attributed to Ehud Gazit.

At least 37 records · Page 2Linked to original sources
PubMed2005

Molecular dynamics simulation of the aggregation of the core-recognition motif of the islet amyloid polypeptide in explicit water.

The formation of amyloid fibrils is associated with major human diseases. Nevertheless, the molecular mechanism that directs the nucleation of these fibrils is not fully understood. Here, we used molecular dynamics simulations to study the initial self-assembly stages of the NH2-NFGAIL-COOH peptide, the core-recognition motif of the type II diabetes associated islet amyloid polypeptide. The simulations were performed using multiple replicas of the monomers in explicit water, in a confined box starting from a random distribution of the peptides at T = 300 K and T = 340 K. At both temperatures the formation of unique clusters was observed after a few nanoseconds. Structural analysis of the clusters clearly suggested the formation of "flat" ellipsoid-shaped clusters through a preferred locally parallel alignment of the peptides. The unique assembly is facilitated by a preference for an extended conformation of the peptides and by intermolecular aromatic interactions. Taken together, our results may provide a description of the molecular recognition determinants involved in fibril formation, in terms of the atomic detailed structure of nascent aggregates. These observations may yield information on new ways to control this process for either materials development or drug design.

Amyloid↗
PubMed2005

Novel electrochemical biosensing platform using self-assembled peptide nanotubes.

Here we describe a novel electrochemical biosensing platform based on biocompatible, well-ordered, self-assembled diphenylalanine peptide nanotubes. Voltammetric and time-based amperometric techniques were applied to demonstrate the ability of the peptide nanotubes to improve the electrochemical parameters of graphite electrodes. The findings clearly show that this novel class of peptide nanotubes provides an attractive component for future electroanalytical devices.

Biosensing Techniques↗
PubMed2005

Self-assembled peptide nanotubes are uniquely rigid bioinspired supramolecular structures.

We recently presented a novel class of self-assembled diphenylalanine-based peptide nanotubes. Here, for the first time, we present their mechanical properties, which we directly measured through indentation type experiments using atomic force microscopy. We find that the averaged point stiffness of the nanotubes is 160 N/m, and that they have a correspondingly high Young's modulus of approximately 19 GPa, as calculated by finite element analysis. This high value places these peptide nanotubes among the stiffest biological materials presently known, making them attractive building blocks for the design and assembly of biocompatible nanodevices.

Crystallization↗
PubMed2005

A model for the role of short self-assembled peptides in the very early stages of the origin of life.

The molecular basis of the origin of life is one of the most fundamental questions in modern biology. While the "RNA world" hypothesis offers a very sensible model for the evolvement of the current biochemical networks, there is a lack of knowledge about the early steps that led to the formation of the first RNA molecules. This issue is essential as it is practically impossible that complex molecules as functional RNA oligonucleotides had evolved spontaneously. It was recently demonstrated that peptide molecules as simple as dipeptides can self-assemble into well-ordered tubular, fibrilar, and closed-cage structures. Other studies have confirmed the ability of dipeptides to act as catalysts and the capability of other peptides, as short as tripeptides, to serve as a template for nucleotide binding and orientation. Unlike complex RNA molecules, the spontaneous formation of functional short peptides in the primordial earth conditions is very likely. We suggest a novel mechanism for the origin of life that is based on the ability of short peptides to form encapsulated structures, catalyst chemical reaction, and serve as highly ordered template for the assembly of nucleotides. This model may explain the early events that led to the formation of the current biochemical machinery that combines the elaborated and coordinated interaction between nucleic acids and proteins to allow the function of living systems.

Animals↗
PubMed2005

Mechanisms of amyloid fibril self-assembly and inhibition. Model short peptides as a key research tool.

The formation of amyloid fibrils is associated with various human medical disorders of unrelated origin. Recent research indicates that self-assembled amyloid fibrils are also involved in physiological processes in several micro-organisms. Yet, the molecular basis for the recognition and self-assembly processes mediating the formation of such structures from their soluble protein precursors is not fully understood. Short peptide models have provided novel insight into the mechanistic issues of amyloid formation, revealing that very short peptides (as short as a tetrapeptide) contain all the necessary molecular information for forming typical amyloid fibrils. A careful analysis of short peptides has not only facilitated the identification of molecular recognition modules that promote the interaction and self-assembly of fibrils but also revealed that aromatic interactions are important in many cases of amyloid formation. The realization of the role of aromatic moieties in fibril formation is currently being used to develop novel inhibitors that can serve as therapeutic agents to treat amyloid-associated disorders.

Amino Acid Sequence↗
PubMed2004

Inhibition of islet amyloid polypeptide fibril formation: a potential role for heteroaromatic interactions.

The formation of amyloid fibril is associated with major human diseases, including Alzheimer's disease, prion diseases, and type 2 diabetes. Methods for efficient inhibition of amyloid fibril formation are therefore highly clinically important. A principal approach for the inhibition of amyloid formation is based on the use of modified molecular recognition elements. Here, we demonstrate efficient inhibition of amyloid formation of the type 2 diabetes-related human islet amyloid polypeptide (hIAPP) by a modified aromatic peptide fragment and a small aromatic polyphenol molecule. A molecular recognition assay using peptide array analysis suggested that molecular recognition between hIAPP and its core amyloidogenic module is mediated by aromatic rather than hydrophobic interactions. To study the possible effect of aromatic interactions on inhibition of hIAPP fibril formation, we have used peptide and small molecule inhibitors. The addition of a nonamyloidogenic peptide analogue of the core module NFGAILSS, in which phenylalanine was substituted with tyrosine (NYGAILSS), resulted in substantial inhibition of fibril formation by hIAPP. The inhibition was significantly stronger than the one achieved using a beta-sheet breaker-conjugated peptide NFGAILPP. On the basis of the molecular arrangement of the tyrosine-phenylalanine interaction, we suggest that the inhibition stems from the geometrical constrains of the heteroaromatic benzene-phenol interaction. In line with this notion, we demonstrate remarkable inhibition of hIAPP fibril formation and cytotoxicity toward pancreatic beta-cells by a small polyphenol molecule, the nontoxic phenol red compound. Taken together, our results provide further experimental support for the potential role of aromatic interactions in amyloid formation and establish a novel approach for its inhibition.

Amino Acid Substitution↗
PubMed2004

The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells.

The Bacillus thuringiensis delta-endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species. Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells. The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied. About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae. These results suggest differential interactions of the two toxins with BBMV encompassing all three domains. Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher Bmax and lower Kd than Cry1C domain II and further supported the existence of toxin multisite interactions. Competitive binding assays were used to estimate the sequence of interaction events. Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site. No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins. Taken together, all three domains specifically interact with the epithelial cell membrane. The folding of the three-domain toxin probably dictates the sequence of interaction events.

Amino Acid Sequence↗
PubMed2004

The von Hippel-Lindau tumor suppressor protein is a molten globule under native conditions: implications for its physiological activities.

The von-Hippel Lindau tumor suppressor protein (pVHL) is conserved throughout evolution, as its homologues are found in organisms ranging from mammals to the Drosophila melanogaster and Anopheles gambiae insects and the Caenorhabditis elegans nematode. Although the physiological role of pVHL is not fully understood, it has been shown to interact with a large number of unrelated proteins and was suggested to play a role in protein degradation as an E3 ubiquitin ligase component in the ubiquitin pathway. To gain insight into the molecular basis of pVHL activity, we analyzed its folding and stability in solution under physiologically relevant conditions. Dynamic light-scattering and gel filtration chromatography of the purified pVHL clearly indicated that the Stokes radius of the protein is larger than what would be expected from its crystal structure. However, under these conditions, the protein shows a clear secondary structure as determined by far-UV circular dichroism. Yet, the near-UV CD experiments show an absence of a tertiary structure. Upon the addition of urea, even at very low concentrations, the protein unfolds in a non-reversible manner, leading to the formation of amorphous aggregates. Furthermore, a large increase in fluorescence (>50-fold) is observed upon the addition of pVHL into a solution containing 8-anilino-1-naphthalene sulfonic acid. We therefore conclude that, under native conditions, the non-bound pVHL has a molten globule configuration with marginal stability. Although molten globular structures can be induced in many proteins under extreme conditions, this is one of the few reported cases of such a structure under the physiological conditions of pH, ionic strength, and temperature. The significance of the pVHL structural properties is being discussed in the context of its physiological activities.

Anilino Naphthalenesulfonates↗
PubMed2004

Peptide sequence and amyloid formation; molecular simulations and experimental study of a human islet amyloid polypeptide fragment and its analogs.

We present a combined experimental and theoretical investigation of the tendencies to form amyloid fibrils by a hexapeptide derivative of the human islet amyloid polypeptide, the NFGAIL (22-27) fragment and its mutants. We performed a complete alanine scan of this fragment and studied the capability of the wild-type and its mutant analogs to form ordered fibrils by ultrastructural and biophysical analyses. In parallel, we conducted a meticulous characterization of each sequence-complex at an atomistic level by performing nine independent molecular dynamics simulations for a total of 36 ns. These allowed us to rationalize the experimental observations and to establish the role of every residue in the fibrillogenesis. The main factor that determines the formation of regular fibrils is a coherent organization of the intersheet space. In particular, phenylalanine side chains cement the macromolecular assemblies due to their aromatic chemical character and restricted conformational flexibility when interacting with aliphatic residues.

Alanine↗
PubMed2004

Amyloidogenic hexapeptide fragment of medin: homology to functional islet amyloid polypeptide fragments.

Medin is the main constituent of aortic medial amyloid that occurs in virtually all individuals older than sixty. It is derived from a proteolytic fragment of lactadherin, a mammary epithelial cell expressed glyco-protein that is secreted as part of the milk fat globule membrane. It was previously demonstrated that an octapeptide fragment of medin (NH2-NFGSVQFV-COOH) forms typical well-ordered amyloid fibrils. To obtain further insights into the molecular determinants that mediate this process by such a short peptide fragment, we examined the amyloidogenic potential of its truncated forms and analogues. Our results clearly indicated that a truncated fragment of medin, the hexapeptide, NFGSVQ can form typical amyloid fibrils. A shorter pentapeptide fragment, NFGSV, self-assembled into a gel structure that exhibited a network of fibrous structures. The amyloid forming NFGSVQ hexapeptide is noticeably similar to the short amyloidogenic peptide fragments of the islet amyloid polypeptide (IAPP), NFGAIL and NFLVH. Moreover, the substitution of the phenylalanine residue with either alanine or isoleucine significantly reduced the amyloidogenic potential of the peptide fragment. Taken together, the results are consistent with the assumed role of stacking interactions in the self-assembly processes that lead to the formation of amyloid fibrils. The results are discussed in the context of models for the mechanism of fibril formation and ways to design inhibitors.

Aged↗
PubMed2003

The YefM antitoxin defines a family of natively unfolded proteins: implications as a novel antibacterial target.

Although natively unfolded proteins are being observed increasingly, their physiological role is not well understood. Here, we demonstrate that the Escherichia coli YefM protein is a natively unfolded antitoxin, lacking secondary structure even at low temperature or in the presence of a stabilizing agent. This conformation of the protein is suggested to have a key role in its physiological regulatory activity. Because of the unfolded state of the protein, a linear determinant rather than a conformational one is presumably being recognized by its toxin partner, YoeB. A peptide array technology allowed the identification and validation of such a determinant. This recognition element may provide a novel antibacterial target. Indeed, a pair-constrained bioinformatic analysis facilitated the definite determination of novel YefM-YoeB toxin-antitoxin systems in a large number of bacteria including major pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. Taken together, the YefM protein defines a new family of natively unfolded proteins. The existence of a large and conserved group of proteins with a clear physiologically relevant unfolded state serves as a paradigm to understand the structural basis of this state.

Amino Acid Sequence↗
PubMed2003

The human islet amyloid polypeptide forms transient membrane-active prefibrillar assemblies.

The formation of amyloid fibrils by the human islet amyloid polypeptide is associated with type II diabetes. While it was previously suggested that the formed fibrils are toxic to pancreatic beta-cells due to membrane permeation activity, more recent studies suggested that protofibrillar assemblies have significantly higher potency in permeating lipid bilayers. Here, we specifically studied the membrane interaction activity of soluble and insoluble islet amyloid polypeptide assemblies at high temporal resolution. A colorimetric analysis using lipid/polydiacetylene (PDA) biomimetic vesicles clearly demonstrated the transient formation of soluble assemblies that strongly interact with the lipid vesicles. A peak in the level of membrane binding of the soluble fraction, as reflected by the colorimetric assay, was observed after incubation for approximately 1 h, followed by a decrease in the level of membrane interaction of the assemblies. The transient nature of the membrane-active assemblies was independently confirmed by a fluorescence quenching assay. Ultrastructural analysis using transmission electron microscopy provided morphological evidence of prefibrillar assemblies, supported the transient existence of membrane interacting soluble species, and facilitated observation of the non-membrane-active filaments in the solution. Taken together, our results provide experimental evidence for the formation of transient soluble prefibrillar assemblies which are highly membrane-active. The implications of these observations are discussed in light of designed fibrillization inhibitors.

Amyloid↗
PubMed2003

Casting metal nanowires within discrete self-assembled peptide nanotubes.

Tubular nanostructures are suggested to have a wide range of applications in nanotechnology. We report our observation of the self-assembly of a very short peptide, the Alzheimer's beta-amyloid diphenylalanine structural motif, into discrete and stiff nanotubes. Reduction of ionic silver within the nanotubes, followed by enzymatic degradation of the peptide backbone, resulted in the production of discrete nanowires with a long persistence length. The same dipeptide building block, made of D-phenylalanine, resulted in the production of enzymatically stable nanotubes.

Amino Acid Motifs↗
PubMed2002

Identification and characterization of a novel molecular-recognition and self-assembly domain within the islet amyloid polypeptide.

The islet amyloid polypeptide (hIAPP) is a 37 amino acid residue polypeptide that was found to accumulate as amyloid fibrils in the pancreas of individuals with type II diabetes. Previous studies identified various fragments of hIAPP that can form amyloid fibrils in vitro (e.g. hIAPP(8-20), hIAPP(23-27), and hIAPP(30-37)). However, no comparative and systematic information was available on the role of these structural domains (or others) in the process of molecular recognition that mediates fibrillization, in the context of the full-length polypeptide. To systematically map and compare potential recognition domains, we studied the ability of hIAPP to interact with an array of 28 membrane-spotted overlapping peptides that span the entire sequence of hIAPP (i.e. hIAPP(1-10), hIAPP(2-11...), hIAPP(28-37)). Our study clearly identified a major domain of molecular recognition within hIAPP, as the polypeptide was found to bind with high affinity to a defined linear group of peptides ranging from hIAPP(7-16) to hIAPP(12-21). The maximal binding of the full-length polypeptide was to the hIAPP(11-20) peptide fragment (with the sequence RLANFLVHSS). In order to define the minimal fragment, within this apparent recognition motif, that is capable of self-association and thus may serve as the core molecular recognition motif, we examined the ability of truncated analogs of the recognition sequence to self-assemble into amyloid fibrils. The shortest active fragments capable of self-assembly were found to be the pentapeptides FLVHS and NFLVH. The apparent role of this motif in the process of hIAPP self-assembly is consistent with the profile of the hIAAP-binding distribution to the peptide array. The identification of such short recognition motifs is extremely useful in the attempts to develop means to block amyloid fibril formation by hIAPP. It is worth mentioning that this is only the second time in which peptides as short as a pentapeptide were shown to form amyloid fibrils (the other pentapeptide is FGAIL).

Amino Acid Motifs↗
PubMed2002

Amyloid fibril formation by pentapeptide and tetrapeptide fragments of human calcitonin.

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH(2)-DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH(2)-DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH(2)-FNKF-COOH tetrapeptide, the NH(2)-DFN-COOH tripeptide, or the NH(2)-DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.

Amino Acid Sequence↗