George B. Benedek - US grants

Our long-term objective is to identify the molecular factors responsible for light scattering and opacification of the lens. Light scattering can be produced in the lens by the formation of protein aggregates, the condensation of proteins into coexisting protein-rich and protein-poor liquid phases and the condensation of proteins into crystalline solid phases. In previous years, we have identified various molecular mechanisms that produce such condensates, and thereby result in light scattering and opacification in gamma crystallin solutions in vitro. Some of these mechanisms are now known to be responsible for several human genetic cataracts. We now wish to extend our work by conducting investigations of the condensates formed by the beta crystallins. Moreover, with the extensive knowledge base of gamma crystallins, we also want to explore the thermodynamic and kinetic behavior of the mixtures of beta and gamma crystallins, in an effort to emulate more realistically the situation present in the lens cytoplasm. To achieve these objectives, we propose the following Specific Aims: 1. To determine the phase diagrams, and the kinetics of aggregation of aqueous solutions of recombinant human beta crystallins, and their naturally occurring mutant and truncated forms. 2. To explore the interactions between beta and gamma crystallins, by studying the phase-diagrams and kinetics of aggregation of solutions of beta and gamma crystallin mixtures. 3. To develop a theoretical description which can predict the phase diagrams, and the kinetics of aggregation for solutions of pure beta and gamma crystallins as well as their mixtures.

This project is an experimental and theoretical investigation of phase separations of ternary aqueous protein solutions. The solutions will be studied by static and dynamic light scattering spectroscopy as well as biochemical chromatographic and centrifugation methods. The theoretical studies will use the Carnahan-Starling mixing entropy and a mean field model for the interaction energies in order to construct a Gibbs free energy for the system. This will potentially provide a route to interpretation and prediction of phase boundaries and compositions of coexisting phases. The results will be of significance for the general area of biomolecular materials purification and processing.

We hypothesize that amyloid beta-protein (A-beta) assembly into oligomers and polymers is a seminal[unreadable] neuropathogenetic process in Alzheimer's disease (AD) and in cerebral amyloid angiopathy (CAA).[unreadable] Inhibiting formation of, or disrupting, A-beta assemblies thus could be of benefit in the treatment of these[unreadable] disorders. To test this hypothesis, a detailed mechanistic knowledge of the assembly process is necessary.[unreadable] Our previous work towards this goal revealed the existence of protofibrillar fibrillogenesis intermediates.[unreadable] These have been found to be neurotoxic in vitro. Recently, in collaborative studies with the Lannfelt group,[unreadable] we reported that protofibrils may be the causative agents in an "Arctic" form of AD. Thus, a concordance of[unreadable] results from fundamental studies of A-beta assembly in vitro with those from clinical investigations in[unreadable] humans supports the involvement of prefibrillar assemblies in AD pathogenesis. In order to understand the[unreadable] structural and thermodynamic principles underlying the formation of these toxic A-beta assemblies, rigorous[unreadable] in vitro studies are necessary. This elucidation process should facilitate later rational design and testing of[unreadable] therapeutic agents. Here, we seek to delve deeply into the thermodynamics and structural biology of early[unreadable] A-beta assembly reactions in order to understand the fundamental factors controlling these processes and[unreadable] to identify and characterize the structures formed. These structures include small prenuclear oligomers,[unreadable] larger micelle-like oligomers, fibril nuclei, and protofibrils. Importantly, knowledge gained regarding the[unreadable] kinetics of formation and the stability of these assemblies will be used to enable functional assays of the[unreadable] biological effects of the assemblies. We propose to accomplish these goals in the context of the following[unreadable] four interconnected aims.[unreadable] Aim 1. To elucidate the thermodynamics of A-beta fibril formation at neutral pH.[unreadable] Aim 2. To determine the structural features of early A-beta assemblies.[unreadable] Aim 3. To determine the mechanism of transformation of early A-beta assemblies into fibril nuclei.[unreadable] Aim 4. To determine the biological activity of early intermediates.