Scientific Report 2008

Cell Biology

Structural and Functional Basis for Membrane Traffic and Misfolding Diseases

W.E. Balch, S. Becker, J. Coppinger, V. Gupta, J. Hulleman, D. Hutt, A.V. Koulov, P. LaPointe, J. Matteson, A. Murray, A. Nauli, L. Page, S. Pankow, H. Plutner, A. Pottekat, A. Razvi, L. Ryno, K. Subramanian, P. Szajner, I. 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 are due to an imbalance between the energetics of the protein fold and the properties of the local folding environment. Such diseases include cystic fibrosis, childhood emphysema, type 2 diabetes, and amyloidosis. Many of these are broadly classified as membrane-trafficking diseases because the defect in protein folding during transit through the mammalian exocytic pathway leads to loss of normal function and/or a gain of toxic function. Our key goals are to define the basic operation of these trafficking pathways, determine the cause of the underlying folding disorders, and learn how these events can be altered to rescue the ability of a protein to function in a 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, assembly of vesicle-budding sites involves assembly of the coatomer complex II (COPII) coat. In collaboration with C. Potter and B. Carragher, Department of Cell Biology, we have solved the 2-dimensional electron cryomicroscopy structure of the COPII cage, a self-assembling polymeric scaffold. To collect cargo, this scaffold interacts with an adaptor protein complex that binds nascent cargo. Using electron cryomicroscopy, we recently solved the structure of the intact COPII coat containing both the cage scaffold and the adaptor complexes (Fig. 1). The structural organization of this striking COPII coat system led 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 complex composed of Rab GTPase interacting with the p115 tether, solved in collaboration with I.A. Wilson, Department of Molecular Biology, 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-cochaperone 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

Fig.1 A 3-dimensional view looking through the coat from the pentagonal cage of the intact COPII coat directing export of cargo from the endoplasmic reticulum. The self-assembling nanoparticle contains the outer layer (green-yellow) scaffold composed of Sec13-31 (the cage) and the inner layer adaptor protein complex (red) consisting of Sec23-24. The COPII coat structure was solved by using electron cryomicroscopy. Reprinted from Mu, T.W., Ong, D.S., Wang, Y.J., Balch, W.E., Yates, J.R. III, Segatori, L., Kelly, J.W. Chemical and biological approaches synergize to ameliorate protein-folding diseases. Cell 134:769, 2008.

Biological And Structural Basis For Misfolding Disease

Many mutations disrupt cargo traffic from the endoplasmic reticulum by preventing proper protein folding during synthesis, resulting in loss of recognition by the COPII machinery. In collaboration with J.W. Kelly and E. Powers, Department of 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.R. Yates, Department of Chemical Physiology, we 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. We have discovered a cohort of 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. In a system-wide analysis of the role of folding pathway components, using small interfering RNA 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 those of recent studies on the role of transcriptional regulatory circuits that control the expression of the components of the entire set of molecular interactions in cells in cystic fibrosis, revealed an extensive proteostatic network necessary for CFTR function in health and disease (referred to as the CFTR “interactome”). 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, which we are studying in collaboration with Dr. Kelly, J. Buxbaum, Department of Experimental Medicine, and A. Dillin, Salk Institute for Biological Studies, La Jolla, California, and R. Morimoto, Northwestern University, Evanston, Illinois.

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 that controls human health and aging. We anticipate that knowledge of the function of 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. The folding biology of cystic fibrosis: a consortium-based approach to disease. In: Protein Misfolding Diseases: Current and Emerging Principles and Therapies. Ramirez-Alvarado, M., Kelly, J.W., Dobson, C.M. (Eds.). Wiley & Sons, Hoboken, NJ, in press.

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

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.

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

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

Mu, T.W., Ong, D.S., Wang, Y.J., Balch, W.E., Yates, J.R. III, Segatori, L., Kelly, J.W. Chemical and biological approaches synergize to ameliorate protein-folding diseases. Cell 134:769, 2008.

Powers, E.T., Balch, W.E. Costly mistakes: translational infidelity and protein homeostasis. Cell 134:204, 2008.

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 134:474, 2008.

Subramanian, K., Balch, W.E. NPC1/NPC2 function as a tag team duo to mobilize cholesterol. Proc. Natl. Acad. Sci. U. S. A. 105:15223, 2008.

Wang, X., Koulov, A.V., Kellner, W.A., Riordan, J.R., Balch, W.E. Chemical and biological folding contribute to temperature-sensitive CFTR trafficking. Traffic 9:1878, 2008.

Yonemoto, I.T., Kroon, G.J., Dyson, H.J., Balch, W.E., Kelly, J.W. Amylin proprotein processing generates progressively more amyloidogenic peptides that initially sample the helical state. Biochemistry 47:9900, 2008.