Sharon Gilead

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).

Self-organization of Short Peptide Fragments: From Amyloid Fibrils to Nanoscale Supramolecular Assemblies

Numerous supramolecular protein assemblies had been demonstrated to have either physiological or pathological activities. The most significant case of disease-associated self-organized structures is that of amyloid fibrils. The formation of these fibrils is the hallmark of major human disorders, including Alzheimers disease and type II diabetes. In this review we illustrate the molecular properties of the amyloid fibrils as supramolecular assemblies in the nanometric scale. We present recent advances in the elucidation of the molecular recognition and self-assembly processes that lead to the formation of these toxic structures, and we describe how the mechanistic study of amyloid formation process has led to unexpected discoveries of peptide-based nanostructures.

Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies

Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy.

Photoacoustic Doppler measurement of flow using tone burst excitation.

In this paper a novel technique for flow measurement which is based on the photoacoustic (PA) Doppler effect is described. A significant feature of the proposed approach is that it can be implemented using tone burst optical excitation thus enabling simultaneous measurement of both velocity and position. The technique, which is based on external modulation and heterodyne detection, was experimentally demonstrated by measurement of the flow of a suspension of carbon particles in a silicon tube and successfully determined the particles mean velocity up to values of 130 mm/sec, which is about 10 times higher than previously reported PA Doppler set-ups. In the theoretical part a rigorous derivation of the PA response of a flowing medium is described and some important simplifying approximations are highlighted.

Simultaneous spatial and spectral mapping of flow using photoacoustic Doppler measurement

We demonstrate the use of tone-burst excitation and time-gated spectral analysis for photoacoustic Doppler mapping of flow in an unperturbed vessel phantom and in a vessel with a spatially varying lumen. The method, which mimics pulsed Doppler ultrasound, enables simultaneous measurement of axial position and flow as well as complete characterization of the Doppler spectrum over a wide range of mean velocities (3.5 to 200 mm∕s). To generate the required optical excitation, a continuous cw laser source followed by an external electro-optic modulator is used. Stenoses at various levels are emulated in a C-flex tube with a flowing suspension of micrometer-scale carbon particles. Two-dimensional maps of spectral content versus axial position at different points along the vessel and for various levels of perturbations demonstrate the potential use of the method for characterization of flow irregularities.

A Bacteriophage Capsid Protein Provides a General Amyloid Interaction Motif (GAIM) That Binds and Remodels Misfolded Protein Assemblies

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (KD=9.4nM); (2); blocks fAβ assembly (IC50~50nM) and (3) dissociates fAβ (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.

Time-resolved photoacoustic Doppler characterization of flow using pulsed excitation

A new approach for implementing pulsed excitation enables time-resolved characterization of flow, using the photoacoustic Doppler effect. The method yields two-dimensional maps of the Doppler shift vs. axial position of flowing absorbing particles. It takes advantage of the unique flexibility and accuracy of external modulation which offers excellent control over the parameters of the pulsed optical excitation. The experimental setup comprised a CW tunable laser source operating in the fiber optic communications band (1510-1620nm) followed by an electro-optic modulator, electronically driven by an arbitrary waveform generator. Using the technique the flow of a suspension of carbon particles in a C-flex tube was measured over a wide range of velocities from 18 mm/sec up to 200mm/sec.

The use of pulse synthesis for optimization of photoacoustic measurements

In this paper the use of pulse shaping in photoacoustic (PA) measurements is presented. The benefits of this approach are demonstrated by utilizing it for optimization of either the responsivity or the sensitivity of PA measurements. The optimization is based on the observation that the temporal properties of the PA effect can be represented as a linear system which can be fully characterized by its impulse response. Accordingly, the response of the PA system to an input optical pulse, whose instantaneous power is arbitrarily shaped, can be analytically predicted via a convolution between the pulse envelope and the PA impulse response. Additionally, the same formalism can be used to show that the response of the PA system to a pulse whose instantaneous power is a reversed version of the impulse response, i.e. a matched pulse, would exhibit optimal peak amplitude when compared with all other pulses with the same energy. Pulses can also be designed to optimize the sensitivity of the measurement to a variation in a specific system parameter. The use of the matched pulses can improve SNR and enable a reduction in the total optical energy required for obtaining a detectable signal. This may be important for applications where the optical power is restricted or for dynamical measurements where long integration times are prohibited. To implement this new approach, a novel PA optical setup which enabled synthesis of excitation waveforms with arbitrary temporal envelopes was constructed. The setup was based on a tunable laser source, operating in the near-IR range, and an external electro-optic modulator. Using this setup, our approach for system characterization and response prediction was tested and the superiority of the matched pulses over other common types of pulses of equal energy was demonstrated.

NPT088 reduces both amyloid-β and tau pathologies in transgenic mice

Alzheimers disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid‐β (Aβ) and hyper‐phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein (“NPT088”) consisting of the active fragment of g3p and human‐IgG1‐Fc.

Coded photoacoustic Doppler excitation with near-optimal utilization of the time and frequency domains

We propose and experimentally demonstrate a new photoacoustic (PA) excitation and analysis method which achieves an almost complete utilization of the available time and frequency windows. The method, which enables spectral and spatial characterization of flow, is based on interleaving tens of tone-burst sequences at equally spaced frequencies. Depending on the application, the interleaved signals can be generated by a single optical source or by multiple sources, possibly at different wavelengths. Upon reception, the responses corresponding to the different tone-burst sequences are spectrally de-multiplexed. As demonstrated in the current work, this method can be used to improve the SNR of PA systems based on optical sources with limited peak power. Alternatively, if the interleaved excitation signals are at different wavelengths, the PA responses can be used for multispectral characterization of the medium.