Yaron Bram

Cognitive-Performance Recovery of Alzheimer's Disease Model Mice by Modulation of Early Soluble Amyloidal Assemblies†

A rationally designed oligomerization inhibitor interacts with early intermediate assemblies of amyloid-beta polypeptide (Abeta) through the aromatic elements and inhibits their assembly into the toxic oligomers that cause Alzheimers disease by a unique C(alpha)-methylation beta-breakage strategy. The electrostatic potential of the low-energy conformation of the dipeptide inhibitor bound to Abeta is shown.

Ostwald’s rule of stages governs structural transitions and morphology of dipeptide supramolecular polymers

The self-assembly of molecular building blocks into nano- and micro-scale supramolecular architectures has opened up new frontiers in polymer science. Such supramolecular species not only possess a rich set of dynamic features as a consequence of the non-covalent nature of their core interactions, but also afford unique structural characteristics. Although much is now known about the manner in which such structures adopt their morphologies and size distributions in response to external stimuli, the kinetic and thermodynamic driving forces that lead to their transformation from soluble monomeric species into ordered supramolecular entities have remained elusive. Here we focus on Boc-diphenylalanine, an archetypical example of a peptide with a high propensity towards supramolecular self-organization, and describe the pathway through which it forms a range of nano-assemblies with different structural characteristics. Our results reveal that the nucleation process is multi-step in nature and proceeds by Ostwalds step rule through which coalescence of soluble monomers leads to the formation of nanospheres, which then undergo ripening and structural conversions to form the final supramolecular assemblies. We characterize the structures and thermodynamics of the different phases involved in this process and reveal the intricate nature of the transitions that can occur between discrete structural states of this class of supramolecular polymers.

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.

Monitoring and Targeting the Initial Dimerization Stage of Amyloid Self‐Assembly

Amyloid deposits are pathological hallmark of a large group of human degenerative disorders of unrelated etiologies. While accumulating evidence suggests that early oligomers may account for tissue degeneration, most detection tools do not allow the monitoring of early association events. Here we exploit bimolecular fluorescence complementation analysis to detect and quantify the dimerization of three major amyloidogenic polypeptides; islet amyloid polypeptide, β-amyloid and α-synuclein. The constructed systems provided direct visualization of protein-protein interactions in which only assembled dimers display strong fluorescent signal. Potential inhibitors that interfere with the initial intermolecular interactions of islet amyloid polypeptide were further identified using this system. Moreover, the identified compounds were able to inhibit the aggregation and cytotoxicity of islet amyloid polypeptide, demonstrating the importance of targeting amyloid dimer formation for future drug development.

The Effect of Chemical Chaperones on the Assembly and Stability of HIV-1 Capsid Protein

Chemical chaperones are small organic molecules which accumulate in a broad range of organisms in various tissues under different stress conditions and assist in the maintenance of a correct proteostasis under denaturating environments. The effect of chemical chaperones on protein folding and aggregation has been extensively studied and is generally considered to be mediated through non-specific interactions. However, the precise mechanism of action remains elusive. Protein self-assembly is a key event in both native and pathological states, ranging from microtubules and actin filaments formation to toxic amyloids appearance in degenerative disorders, such as Alzheimers and Parkinsons diseases. Another pathological event, in which protein assembly cascade is a fundamental process, is the formation of virus particles. In the late stage of the virus life cycle, capsid proteins self-assemble into highly-ordered cores, which encapsulate the viral genome, consequently protect genome integrity and mediate infectivity. In this study, we examined the effect of different groups of chemical chaperones on viral capsid assembly in vitro, focusing on HIV-1 capsid protein as a system model. We found that while polyols and sugars markedly inhibited capsid assembly, methylamines dramatically enhanced the assembly rate. Moreover, chemical chaperones that inhibited capsid core formation, also stabilized capsid structure under thermal denaturation. Correspondingly, trimethylamine N-oxide, which facilitated formation of high-order assemblies, clearly destabilized capsid structure under similar conditions. In contrast to the prevailing hypothesis suggesting that chemical chaperones affect proteins through preferential exclusion, the observed dual effects imply that different chaperones modify capsid assembly and stability through different mechanisms. Furthermore, our results indicate a correlation between the folding state of capsid to its tendency to assemble into highly-ordered structures.

Targeting the Early Step of Building Block Organization in Viral Capsid Assembly

Viral assembly, similar to other self-organizing protein systems, relies upon early building blocks, which associate into the late supramolecular structures. An initial and crucial event during HIV-1 core assembly is the dimerization of the capsid protein C-terminal domain, which stabilizes the viral capsid lattice. Thus, monitoring and manipulating this stage is desirable both from mechanistic as well as clinical perspectives. Here, we developed a fluorescent-based method for the detection and visualization of these early capsid interactions. We detected strong dimeric interactions, which were influenced by mutations in the capsid protein. We utilized this assay for potential assembly inhibitors screening, which resulted in the identification of a leading compound that hinders the assembly of capsid protein in vitro. Moreover, a derivative of the compound impaired virus production and infectivity in cell cultures. These findings demonstrate that the described assay efficiently detects the very first association events in HIV-1 capsid formation and emphasize the significance of targeting early intermolecular interactions.

Formation of multimeric antibodies for self-delivery of active monomers

Abstract Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.