Charles A. Miller - US grants

Aryl Hydrocarbon Receptor (AHR) is a ligand-activated component of a heterodimeric transcription factor called the Aryl Hydrocarbon Receptor Complex (AHRC). AHRC mediates transcription of many gene products that lay a role in carcinogenesis, teratogenesis, metabolism, endocrine regulation, and immune function. Many pollutants, drugs, and naturally occurring compounds with a planar aromatic structure bind to AHR and activate AHR- mediated transcription. Since considerable financial and human resources are directed towards protecting people and the environment from exposure to ligands of AHRC, understanding this receptor is central to the validity of determining the health risks of aromatic compounds. Yeast lack the genes encoding AHR and the AHR dimerization partner, ARNT, that comprise the AHRC. Expression of human AHR and ARNT in yeast allows the use of genetic approaches to study AHR regulation and structure. This proposal specifically addresses understanding how two groups of proteins, the cyclophilins and 90 kilodalton heat shock proteins, influence the initial stages of the AHR signal transduction. Yeast genetic methods will also be used to select for new HSP90 and AHR alleles with altered function. Characterization of these mutated derivatives will further elucidate the structural features of HSP90 and AHR. These studies will provide significant new information about the molecular biology of AHR-mediated signal transduction.

DESCRIPTION (provided by applicant): Hsp90 proteins are the central members of a multi-protein complex called the Hsp90 chaperosome. Several of these Hsp90-client protein complexes are targets for cancer therapies or represent potential targets for future research. The steroid hormone receptors are the best-characterized client proteins of the Hsp90 chaperosome. Drugs designed to target a particular Hsp90 complex or its client protein (oncogenes/kinases/receptors) often produce toxic effects due to non-specific actions on other client protein-Hsp90 complexes. Consequently, a system to assess this "collateral damage" based on drug interactions with Hsp90 and/or its client proteins would be valuable in initial screens designed to eliminate toxic compounds. As a hypothetical example, if a compound (such as a geldanamycin derivative) designed to affect ErbB2-Hsp90 complexes also affected glucocorticoid receptor-Hsp90 complexes with similar potency, the compound would be flagged for potential toxicity and possibly eliminated from further testing. We are developing a "humanized" yeast-based system to assess drugs that may act on the Hsp90 chaperosome and its client proteins. The yeast chaperone components functioning in this pathway are being replaced by their human counterparts to create a relevant model for toxicity studies. Our previous studies with human aryl hydrocarbon receptor, Hsp90 isoproteins, and co-chaperones in yeast provide "proof of concept" for this model toxicity system. The proposed new yeast strains will provide novel, inexpensive, high-throughput toxicity screens that are relevant for the prediction of human toxicity. The Specific Aims of this proposal are 1) to construct yeast strains with human homodimeric steroid hormone receptors that coexpress critical human Hsp90 proteins and co-chaperones and 2) to validate signaling responses in these strains using known Hsp90 inhibitors and steroid hormone agonists and antagonists.

A trend in cochlear implants is the use of faster per-channel pulse carrier rates, promoted on the basis of increased information capacity. However, it is not clear that higher rates achieve this goal in most or ill individuals. Clinical studies present mixed results and research promoting high (e.g., 5000 pps) rales raises important questions. For example, while beneficial, desynchronizing, effects have been reported in many auditory nerve fibers, comparable numbers become adapted to the point of being unresponsive. Furthermore, physiologic dala have only been obtained from intact fibers, not from degenerated neurons typical of chronically deaf ears. Finally, while much is known about neural adaptation to acoustic stimuli, relatively little is known about electrical adaptation, even though the latter typically produces much larger functional changes. We hypothesize that at least part of the variability in performance with higher-rate carriers is due to across-user differences in the auditory nerve's response to high-rate stimuli. This research plan seeks to fill these gaps in our knowledge of how the auditory nerve encodes information presented as modulated pulse trains. Three Aims are proposed. Aim 1 will assess signal encoding in fibers excited by modulated carriers at rates relevant to modern and proposed speech processors. Data will be collected from intact and degenerated nerves for greater applicability to clinical cases. Fiber tracing techniques will help link physiology with anatomical status. Aim 2 will use that data to help develop a computational model of the nerve that accounts for many fiber properties (e.g., integration, refractoriness, and adaptation). This model wsll be used to predict the electrically evoked compound action potential (EC'AP) so that we can tesi: the capacity of ECAP measures to assess fiber functionality, a clinically relevant issue. Finally, Aim 3 will explore the feasibility of applying specific transforms to modulated trains to compensate for adaptation and refractory effects that limit information carried by modulated pulse trains. We expect the results of this work will guide future designs of cochlear-implant speech processors so that modulated stimuli can be better tailored to the encoding capacity of the user's auditory nerve.