Structural and Functional Basis for
Membrane Traffic and Misfolding Disease

William E. Balch

D. Hutt, V. Gupta, A.V. Koulov, P. LaPointe,
J. Matteson, A. Murray, A. Nauli, L. Page, H. Plutner,
A. Pottekat, A. Razvi, K. Subramanian, P. Szajner, I. Yonemoto*

*Joint project with Jeffery Kelly: Isaac Yonemoto

A major challenge in human health is to understand and treat the many conformational diseases that affect protein homeostasis (proteostasis) during development and aging. These diseases arise as a consequence of an imbalance between the energetics of the protein fold and the properties of the local folding environment. Such diseases include, among others, cystic fibrosis, childhood emphysema, type 2 diabetes and amyloidosis. Many of these are broadly classified as membrane trafficking disease because the defect in folding during transit through the mammalian exocytic pathway leads to loss of normal function and/or a gain-of-toxic function. Key goals of the laboratory are to define the basic operation of these trafficking pathways, determine the cause of the underlying folding disorder, and learn how these events can be altered to rescue the ability of the protein to function in the cell.

STRUCTURAL AND FUNCTIONAL BASIS FOR MEMBRANE TRAFFIC

Eukaryotic cells are highly compartmentalized; each compartment of the exocytic and endocytic pathways provides a unique chemical and biological environment in which protein folding and function can be modulated to maintain cellular proteostasis. Movement between these compartments involves the activity of both anterograde and retrograde transport tubules and vesicles. During export from the first compartment of the exocytic pathway, the endoplasmic reticulum (ER), assembly of vesicle budding sites involves the assembly of the coatomer complex II (COPII) coat. In collaboration with C. Potter and B. Carragher (Cell Biology), we have solved the 2-dimensional cryo-electron microscopy (cryoEM) structure of the COPII cage- a self-assembling polymeric scaffold. To collect cargo, this scaffold interacts with an adaptor protein complex (APC) that binds nascent cargo. We have recently solved the structure of the intact COPII coat containing both the cage scaffold and the adaptor complexes using cryoEM (Figure 1). The structural organization of this striking COPII coat system has led us to a new model that now describes the basis for cargo capture and trafficking through the early secretory pathway.

COPII vesicles also recruit GTPases, tethers and fusion components to promote vesicle fusion with downstream compartments. The x-ray structure of a Rab GTPase interacting p115 tether complex, solved in collaboration with I.A. Wilson (Molecular Biology), has now revealed a superhelical platform that directs function. We have shown that the assembly of this superhelical platform with other components of the tether-fusion system is likely regulated by the activity of the Hsp90 family of chaperone/co-chaperone components. Using bioinformatics and systems biology to build interaction networks, we are beginning to define how such systems likely integrate the folding and trafficking component networks with the overall structure and function of membrane compartments.

BIOLOGICAL AND STRUCTURAL BASIS FOR MISFOLDING DISEASE

Many mutations disrupt cargo traffic from the ER by preventing proper protein folding during synthesis, resulting in loss of recognition by the COPII machinery. In collaboration with J. Kelly (Chemistry) and E. Powers (Chemistry) we are studying the underlying basis for these events. Using biophysical modeling approaches we have developed a rigorous quantitative framework to describe in a global way the adaptable role of proteostasis and the chaperone environment in membrane trafficking and disease. In addition, in collaboration with J. Yates (Chemical Physiology) we have used mass spectrometry to analyze the proteome that regulates the trafficking and function of the wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that when defective is responsible for the disease cystic fibrosis (CF). We have discovered a cohort chaperones involving the Hsp90 chaperone system that dictates the CFTR folding environment (referred to as the chaperome). In disease, the Hsp90 system becomes trapped in an intermediate folding complex, blocking export and triggering degradation. A system-wide analysis of the role of folding pathway components using small interfering RNA (siRNA) to alter the steady-state distribution of chaperones led to restoration of the function of the chloride channel at the cell surface. These results, in combination with recent studies on the role transcriptional regulatory circuits controlling the expression of CF interactome components revealed an extensive proteostatic network necessary for CFTR function in health and disease. This environment is likely responsive to numerous environmental factors, such as caloric intake, that also influence the onset of other conformational diseases such as type 2 diabetes and neurodegenerative amyloid diseases of aging that we are studying in collaboration with J. Kelly, J. Buxbaum (Experimental Medicine) and A. Dillin (Salk).

Through such a multidisciplinary approach, we hope to gain critical insight into the fundamental principles of protein folding and trafficking, and a new understanding of a variety of inherited diseases sensitive to the proteostasis environment controlling human health and aging. We anticipate that knowledge of the function proteostatic pathways will enable the development of general, corrective small-molecule proteostasis regulators and chemical modulators of specific protein folds that will provide increased stability and thereby enhance the delivery and function of misfolded proteins in downstream environments, leading to alleviation of disease.

PUBLICATIONS:

Balch, W.E., Braakman, I., Frizzell, R., Guggino, W., Lukacs, G., Penland, C., Pollard, H., Skach, W., Sorscher, E., Thomas, P. In: Protein Misfolding Diseases: Current and Emerging Principles and Therapies. The Folding Biology of Cystic Fibrosis: a Consortium-based Approach to Disease. Ramirez-Alvarado, M., Kelly, J., Dobson, C. (Eds.). John Wiley & Sons, Inc., Wiley-Blackwell Division, Hoboken, NJ., in press.

Balch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W. Adapting proteostasis for disease intervention. Science. 319:916, 2008. (PubMed).

Brown, W.J., Plutner, H., Drecktrah, D., Judson, B.L., Balch, W.E. The lysophospholipid acyltransferase antagonist CI-976 inhibits a late step in COPII vesicle budding. Traffic 9:786, 2008.(PubMed).

Gurkan, C., Koulov, A.V. Balch, W.E. An evolutionary perspective on eukaryotic membrane trafficking. Adv. Exp. Med. Biol. 607:73, 2007.(PubMed).

Hutt, D.M., Balch, W.E. Rab1b silencing using small interfering RNA for analysis of disease-specific function. Methods Enzymol. 438:1, 2008.(PubMed).

Segatori, L., Mu, T.W., Ong, D.S.T., Balch, W.E., Kelly, J.W. Proteostasis regulators and pharmacologic chaperones synergize to restore protein homeostasis in loss-of-function diseases. Nature Medicine. In principle in press.

Stagg, S.M., LaPointe, P., Razvi, A., Gurkan, C., Potter, C.S., Carragher, B., Balch, W.E. Structural basis for cargo regulation of COPII coat assembly. Cell. In principle in press.

Wang, X., Koulov, A.V., Kellner, W.A., Riordan, J.R., Balch, W.E. Chemical and biological folding contribute to DeltaF508 CFTR trafficking in Cystic Fibrosis. Mol. Biol. Cell. In principle in press.

Wiseman, R.L., Powers, E.T., Buxbaum, J.N., Kelly, J.W., Balch, W.E. An adaptable standard for protein export from the endoplasmic reticulum. Cell. 131:809, 2007. (PubMed).