Over 700 unique mutations have been found in the cystic fibrosis gene, and they can be classified in five general categories with respect to the cystic fibrosis transmembrane conductance regulator protein (CFTR). Therapy can now be addressed in a genotype-specific manner, and a new term has been coined to describe the novel concept- """"""""protein-repair therapy."""""""" The goal of this project this project is to develop protein- therapy for CF using a unique class of short chain fatty acid compounds that regulate gene expression. The delta508 mutation is present in over 70% of CF chromosomes and is a prototype Class II mutation, a so-called trafficking mutation. Because delta508 is also a partial conduction mutation, correction of the trafficking arrest should lead to improvement in epithelial chloride transport because the mutant channel conducts chloride. The hypothesis of this grant is that the butyrate compounds, specifically 4-phenylbutyrate (4-PBA), will correct the biosynthetic arrest in delta508 expression by regulation of endogenous chaperone molecules in the endoplasmic. Preliminary data will be presented, supporting a role for 4-PBA in regulating an important chaperone protein gene (Hsc70), thereby significantly increasing trafficking of delta508 protein in vitro. Part I of this grant explores the physiologic events surrounding the transfer of the mutant protein from the protein synthetic to degradative pathway.
Aim 1 is to study the interaction of 4-PBA and related butyrates with the biosynthesis and processing of delta508 in vitro in CF airway epithelial cell lines. Quantitative measurement of protein production, protein degradation (ubiquitinated ATP-dependent and independent degradation) and post- translation (molecular chaperoning) processing will be studied in the context of chaperone proteins. Part II examines the butyrate regulation of events at the level of gene regulation and mRNA production;
Aim 2 is to study the molecular biology of butyrate regulation of trafficking. Butyrate-sensitive sequences in the human CFTR and Hsc 70 promoters will be identified by reporter gene assays, electrophoretic mobility shift assays and DNAS footprinting. Butyrate-sensitive inducers and inhibitors and histone acetylation will be examined in detail.
Showing the most recent 10 out of 123 publications
