The misfolding and aggregation of essential cellular proteins is a fundamental problem for all living organisms. Aggregation of even non-essential proteins can lead to debilitating diseases like type II diabetes, Alzheimer's, Huntington's and Parkinson's diseases. Importantly, protein folding and aggregation are heavily influenced by the cellular protein quality control machinery, involving networks of molecular chaperones. Precisely how different chaperone systems cooperate to dismantle and reactivate aggregated proteins, and how molecular chaperone action affects disease progression, is not well understood. This proposal will address three fundamental questions that impact this problem: First, what is the most accurate physical description of protein aggregate disassembly by molecular chaperones? Second, in what way do the structural properties of an aggregate nanoparticle impact how an they are taken apart? Third, how does the critical small heat shock (sHsp) class of molecular chaperones enhance protein aggregate disassembly? Addressing these questions in a detailed and quantitative manner is exceedingly difficult using standard approaches, because the complex and heterogeneous nature of protein aggregates can obscure key intermediates and transitions. Single particle analysis, in particular a fluorescence technique known as Burst Analysis Spectroscopy (BAS), is ideally suited to overcome this problem. BAS can quantify complex nanoparticle distributions in free solution, allowing for the detection of dynamically populated intermediates and sub-populations. This project will employ BAS to study the disassembly of protein aggregates by two model disaggregase systems, one from bacteria and one from yeast, at a level of detail unreachable by other approaches. The overall goal is to develop a mechanistic understanding of how different molecular chaperone networks recognize and dismantle protein aggregates that possess distinct physical properties. In service of this goal, this project will extend the capabilities of BAS to incorporate multi-color and Frster resonance energy transfer measurements. This project will also develop a set of new approaches that are complementary to BAS and permit more detailed analysis of the hydrodynamic and structural properties of aggregate nanoparticles by using (1) horizontal light sheet excitation and particle tracking in microfluidic flow and (2) Tip-Enhanced Raman spectroscopy (TERS). It is anticipated that the combination of these techniques will provide uniquely powerful approach to understanding protein disaggregation. Additionally, because the core components of the chaperone networks examined in this work are conserved, it is further expected that the discoveries made in these studies will contribute to a fundamentally better understanding of how molecular chaperones recognize and process protein aggregates in human cells impacted by protein misfolding diseases.
Human diseases as diverse as amyotrophic lateral sclerosis, cystic fibrosis, thalassemias, alpha1-antitrypsin deficiency, Type II diabetes and a variety of amyloid neuropathies such as Alzheimer's, Huntington's and Parkinson's disease share in common a link to the uncontrolled and pathological self-association of cellular proteins. How this process, known as protein aggregation, leads to disease is not well understood, though it is profoundly influenced by a specialized set of cellular proteins known as molecular chaperones. Our goal is to develop a clear understanding of how protein aggregates are recognized and dismantled by networks of molecular chaperones in order to help develop strategies for mitigating diseases of protein misfolding and aggregation.
