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Biogenesis and degradation of membrane proteins, cellular quality control mechanisms, pathogenesis of cystic fibrosis

The post genomic era has provided massive information on the genetic basis of human disease, and attention is now increasingly focused on understanding how specific mutations disrupt protein function. A surprising finding in this area is that the endoplasmic reticulum (ER), the major intracellular organelle responsible for protein synthesis, plays a central role in diverse clinical disorders that share a common cellular defect. Subtle point mutations in a large number of key proteins disrupt protein folding, alert ER quality control machinery, and disrupt protein movement through the secretory pathway. Our laboratory is investigating two issues relevant to this problem. First, how does ER translocation machinery cellular together with chaperones facilitate protein folding in the ER? And second, how are misfolded proteins recognized and directed into degradation pathways? Specific projects in the Skach laboratory are investigating the molecular basis of cystic fibrosis by examining biogenesis and degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). Additional areas of interest include biogenesis pathways of P-glycoprotein (a major cause of drug resistance in cancer) and aquaporin water channels which play an important roles in kidney function and renal disease. Using a combination of biochemical, biophysical, molecular, and cell biological techniques, studies from our laboratory have demonstrated that these complex membrane proteins utilize a repertoire of stepwise folding pathways in which regions of protein are translocated across and integrated into the ER membrane. One goal is to understand how these steps are orchestrated by the Sec61 ER translocation machinery. These studies take advantage of in vitro and oocyte expression systems that enable us to incorporate photoactive crosslinkers and fluorescent probes for mapping protein-protein interactions that occur during and immediately following protein synthesis. Related studies are focused on disease-causing mutations found in cystic fibrosis patients that disrupt normal CFTR folding pathways. In this latter case, the misfolded protein is ubiquitinated, extracted from the membrane (a process termed retrotranslocation), and degraded by the 26S proteasome. Very little is known about misfolded CFTR is identified in cells and the molecular mediators of these events. We believe that understanding how cellular machinery facilitates biogenesis and quality control processes will have significant implications for medicine. It will enable us to better define disease pathology and provide insight into basic mechanisms of disease. Ultimately, our goal is to use knowledge to identify novel therapeutic targets for treating protein folding disorders.

Recent Publications:

1. Buck, T. Skach, W. Differential Stability of Biogenesis Intermediates Reveals a Common Pathway for Aquaporin-1 Topological Maturation, J. Biol. Chem. 280:2261-269, 2005.
   
   
2. Shibatani, T., David, L., McCormack, A., Frueh, K., Skach, W. Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain Sec61, TRAP and two potential new subunits. Biochem. 44:5982-5992, 2005.
   
   
3. Sadlish, H. Pitonzo, D., Johnson, A., Skach, W. Sequential triage of transmembrane segments by Sec61a during biogenesis of a native multispanning membrane protein. Nature Struct. Mol. Biol., 12:970-878, 2005
   
   
4. Oberdorf, J., Pitonzo, D., Skach, W. An energy dependent maturation step required for release of the cystic fibrosis transmembrane conductance Regulator from early biosynthetic machinery. J. Biol. Chem., 280:38193-38202, 2005.
   
   
5. Oberdorf, J., Carlson, E., Skach, W. Uncoupling proteasome peptidase and ATPase activities results in cytosolic release of an ER polytopic protein. J. Cell. Sci. 119:303-313, 2006
   
   
6. Pitonzo, D., Skach W. Molecular Mechanisms of Aquaporin Biogenesis by the Endoplasmic Reticulum Sec61 translocon. Bioch. Biophys. Acta. Biomembranes. In Press. 2006