Protein Folding and Human Disease

Projects

Oral Presentation Guidelines

The goal of the oral presentation is for you to teach the class (including your instructors) about your topic. This will depend on the class size, but oral presentations are typically 30 minutes with a few interruptions for questions and clarification, followed by 5–10 minutes for questions and discussion period. Three days before your presentation send the instructor’s assistant a one paragraph abstract to be shared with the class. Include a reference to a review article and a recent research paper or paper you consider to be seminal. Your instructors and fellow students will look these over before class so they can think about your topic in advance and formulate better questions for discussion.

Also before your presentation, preferably, 5 to 6 days, and at the very least 2 days before, you will practice your talks to one or more of the people scheduled to present that same day. You will write a brief description of your suggestions for improvement of the oral presentation of your team and this will be emailed to the instructor on the day of your group’s presentations. It might be as brief as: “Jenny is an accomplished public speaker and gave a very well organized presentation. I suggested that she define her terms, and explain some concepts in the initial part of the talk.” More likely it will include more specifics. For example:

“Josh has had little practice in public speaking. I suggested that he simplify his slides, and taught him how to use animation in Powerpoint to layer information more effectively. I suggested that he begin with a very general description of pathology, but he felt that would be too boring. He may be right. I also suggested that he provide a handout for medical terminology, for future reference. His slides on amyloid in the CNS and its correlation with disease were very difficult to see. I suggested he find better illustrations.”

For your talk, use Powerpoints or other visual aids to illustrate key points. The general format:

• first a broad overview of the problem and its importance,
• pose the questions,
• describe key experiments,
• critically analyze and interpret the results,
• present an eye to the future.

You can’t cover everything that will appear in your final paper. The class presentation should be specifically constructed and tuned for an oral presentation. As in a professional presentation, your talk will be rehearsed and timed beforehand, with input from a peer reviewer in the class.

Final Paper Guidelines

The final papers are due the last week of classes. The format of the final papers should be a mini review of the selected topic. At the point you have completed the paper, you should be an expert on the subject, with the additional good feeling of having made your understanding available to your classmates and instructors during your oral presentation. The listed references are intended only to be starting points. You are responsible for finding the latest relevant results, using the tools built in to modern journal online resources. Just as with a scientific publication, the paper should be a single coherent integrated text.

The text should begin with an Introduction that gives the background and general context of the work, including the societal and biomedical importance. This is to be followed by a Critical Results and Discussion section that reviews the findings that you consider most relevant and important. Try to follow the format of first posing the question; then describing the experiment designed to answer the question; finally analyzing the resulting data. Reproduce a few figures to illustrate aspects of the subject. The paper should consist of a title page with an Abstract (<250 words) of the most important results and conclusions. The text proper should be 12–16 double spaced pages, with 16 pages the absolute limit. References and figures are additional. The references should include the full titles of the papers. The target audience is the other students in the class and your instructors.

One full week before the paper is due you will exchange drafts with the other members of your team.You will provide editorial suggestions—not just copy editing but substantive feedback on things that are unclear, things that are missing, things that are given too much space for their import, etc. Make a photocopy of your comments on your colleague’s paper and hand them in with the final paper. They are an important part of your class participation.

Student Presentation Topics

The references listed are to get you started. They do not represent a complete or systematic sampling of the literature. Finding, reading and assimilating a subset of critical papers is your responsibility.

POSSIBLE TOPICS	DESCRIPTIONS
Protein Folding and Chaperones
The Refolding Pathway for Apomyoglobin	Hemoglobin is one of the paradigms for a protein whose conformation controls critical physiological processes, and whose malfunction leads to a variety of anemias. Hydrogen exchange and NMR have been used to identify intermediates in the refolding of apomyoglobin. This allows interpretation of a variety of human hemoglobin. Barrick, D., and R. L. Baldwin. “Three-state Analysis of Sperm Whale Apomyoglobin Folding.” Biochemistry 32, no. 14 (1993): 3790–96. Jamin, M., and R. L. Baldwin. “Refolding and Unfolding Kinetics of the Equilibrium Folding Intermediate of Apomyoglobin.” Nature Structural Biology 3, no. 7 (1996): 613–8. Kay, M. S., et al. “Specificty of Native-like Interhelical Hydrophobic Contacts in Theapomyoglobin Intermediate.” Proceedings of the National Academy of Sciences of the United States of America 96, no. 5 (1999): 2007–12. Cavagnero, et al. “Effect of H Helix Destabilizing Mutations on Thekinetic and Equilibrium Folding of Apomyoglobin.” Journal of Molecular Biology 285, no. 1 (1999): 269–82. Hughson, F. M., Wright, P. E., et al (1990).
Trigger Factor and DNA K; The Conformations of Nascent Chains Emerging the Ribosome	What is the conformation of the newly synthesized polypeptide chain as it exits the ribosome? Does the ribosome play a role in early stages of protein folding? What are the roles of Trigger Factor? Crooke, Elliott, and William Wickner. “Trigger Factor: A Soluble Protein that Folds pro-OmpA into a Membrane-assembly-competent Form.” Proceedings of the National Academy of Sciences of the United States of America 84, no. 15 (1987): 5216–20. Hesterkamp, T., S. Hauser, et al. “Escherichia Coli Trigger Factor is a Prolyl Isomerase that Associates with Nascent Polypeptide Chains.” Proceedings of the National Academy of Sciences of the United States of America 93, no. 9 (1996): 4437–41. Hesterkamp, T., E. Deuerling, et al. “The Amino-terminal 118 Amino Acids of Escherichia Coli Trigger Factor Constitute a Domain that is Necessary and Sufficient for Binding to Ribosomes.” The Journal of Biological Chemistry 272, no. 35 (1997): 21865–71. Ullers, et al. “Interplay of Signal Recognition Particle and Trigger Factor at L23 Near the Nascent Chain Exit Site on the Escherichia coli Ribosome.” The Journal of Cell Biology 161, no. 4 (2003): 679–84. Li, Z., C. Liu, et al. “The Chaperone Activity of Trigger Factor is Distinct from its Isomerase Activity during Co-expression with Adenylate Kinase in Escherichia Coli.” Federation of Europian Biochemical Societies 506, no. 2 (2001): 108–12. Scholz, C., G. Stoller, et al. “Cooperation of Enzymatic and Chaperone Functions of Trigger Factor in the Catalysis of Protein Folding.” The EMBO Journal 16, no. 1 (1997): 54–8. Lyon, William, and Michael Caparon. “Trigger Factor-mediated Prolyl Isomerization Influences Maturation of the Streptococcus Pyogenes Cysteine Protease.” The Journal of Bacteriology 185, no. 12 (2003): 3661–7.
The Mechanism of GroE Function in Protein Folding and Assembly	How does the chaperone actually function in ensuring productive protein folding? How does the Jack in the Box work, how is ATP hydrolysis coupled to chaperone function? What is the relationship of GroES binding and release to folding within the GroEL lumen? (Paul Sigler, Arthur Horwich, Helen Saibl; Ulrich Hartl, George Lorimer;)
Function of the DNA K (HSP70) Class of Chaperonins	These appear to interact with newly synthesized chains at an earlier stage in folding than the GroE class, and function as a complex of DNA K, DNA J, and Grp E. They do not have a lumen, but appear to bind an unfolded peptide in an elongated cleft. Frydman, J., E. Nimmesgern, et al. “Folding of Nascent Polypeptide Chains in a High Molecular Mass Assembly with Molecular Chaperones.” Nature 370, no. 6485 (1994): 111–7. Zhu, X., et al. “Structural Analysis of Substrate Binding by the Molelcular Chaperone DNAK.” Science 272, no. 5268 (1996): 1606–14. Langer, et al. “Successive Action of DNAK, DNAJ, and Gro EL Along the Pathway of Chaperone Assisted Lagell Folding.” Nature 356 (1992): 683–9. Szabo, et al. “A Zinc-finger Like Domain of the Molecular Chaperone DNAJ is Involved in Binding to Denatured Protein Substrates.” The EMBO Journal 15, no. 2 (1996): 408–17.
Function of the Lens Chaperone Alpha-crystallin	This is a member of the small heat shock chapeorone family. It is present at high concentrations in the lens and is thought to protect lens crystallins from radiative or oxidative damage leading to cataract. Horvitz, J. “Alpha-Crystallin Can Function as a Molecular Chaperone.” Proceedings of the National Academy of Sciences of the United States of America 89, no. 21 (1992): 10449–53. Litt, M., P. Kramer, et al. “Autosomal Dominant Congenital Cataract Associated with a Missense Mutation in Human Alpha Crystallin Gene CRYAA.” Human Molecular Genetics 7, no. 3 (1998): 471–4. Van Montfort, R. L. M., et al. “Crystal Structure and Assembly of a Eukaryotic Small Heat Shock Protein.” Nature Structural and Biology 8, no. 3 (2001): 1025–30. Tanksale, A., M. Ghatge, et al. “α-crystallin Binds to the Aggregation-prone Molten-globule of Alkaline Proteases: Implications for Preventing Irreversible Thermal Denaturation.” Protien Science 11, no. 7 (2002): 1720–28.
HSP90 A&B	This chaperone is specialized for the maturation of metastable proteins that often play key roles in cell circuitry, such as transcription factors, kinas, phosphatases. It cooperates with a host of other co-chaperones. To recognize its targets, it is expressed at high levels in all eukaryotic cells and is induced by many types of stress. It is an exciting new target for cancer therapeutics. Useful websites: International Conference on the Hsp90 Chaperone Machine and Files from Professor Didier Picard’s lab at the University of Geneva. Sanchez, E. R., S. Meshinchi, et al. “Relationship of the 90-kDa Murine Heat Shock Protein to the Untransformed and Transformed States of the L Cell Glucocorticoid Receptor.” The Journal of Biological Chemistry 262, no. 15 (1987): 6986–91. Prodromou, C., S. M. Roe, et al. “Identification and Structural Characterization of the ATP / ADP-binding Site in the Hsp90 Molecular Chaperone.” Cell 90, no. 1 (1997): 65–75. Southworth, D. R., and D. A. Agard. “Species-dependent Ensembles of Conserved Conformational States Define the Hsp90 Chaperone ATPase Cycle.” Molecular Cell 32, no. 5 (2008): 631–40. Picard, D., B. Khursheed, et al. “Reduced Levels of Hsp90 Compromise Steroid Receptor Action in Vivo.” Nature 348, no. 6297 (1990): 166–8. Whitesell, L., E. Mimnaugh, et al. “Inhibition of Heat Shock Protein HSP90-pp60v-src Heteroprotein Complex Formation by Benzoquinone Ansamycins: Essential Role for Stress Proteins in Oncogenic Transformation.” Proceedings of the National Academy of Sciences of the United States of America 91, no. 18 (1994): 8324–8. Rutherford, S. L., and S. Lindquist. “Hsp90 as a Capacitor for Morphological Evolution.” Nature 396 (1998): 336–42. Gorre, M. E., K. Ellwood-Yen, et al. “BCR-ABL Point Mutants Isolated from Patients with Imatinib Mesylate-resistant Chronic Myeloid Leukemia Remain Sensitive to Inhibitors of the BCR-ABL Chaperone Heat Shock Protein 90.” Blood 100, no. 8 (2002): 3041–044. Cowen, L. E., and S. Lindquist. “Hsp90 Potentiates the Rapid Evolution of New Traits: Drug Resistance in Diverse Fungi.” Science 309, no. 5744 (2005): 2185–9. Banerji, U., A. Affolter, et al. “BRAF and NRAS Mutations in Melanoma: Potential Relationships to Clinical Response to HSP90 Inhibitors.” Molecular Cancer Therapeutics 7, no. 4 (2008): 737–9.
Organismal Biology, Evolution, and Medicine
HSP104 & ClpB	These proteins belong to the AAA+ class of ATPases. They are large macromoleclar machines and use the energy of ATP to dissolve aggregated proteins. They play important roles in stress tolerance and in diverse processes such as the inheritance of prions in yeast. Chernoff, Y. O., S. L. Lindquist, et al. “Role of the Chaperone Protein Hsp104 in Propagation of the Yeast Prion-like Factor [psi+]”. Science 268, no. 5212 (1995): 880–4. Glover, J. R., and S. Lindquist. “Hsp104, Hsp70, and Hsp40: A Novel Chaperone System that Rescues Previously Aggregated Proteins.” Cell 94, no. 1 (1998): 73–82. Queitsch, C., S. W. Hong, et al. “Heat Shock Protein 101 Plays a Crucial Role in Thermotolerance in Arabidopsis [see comments]. Plant Cell 12, no. 4 (2000): 479–92. (the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation); Sanchez, Y., and S. L. Lindquist. “HSP104 Required for Induced Thermotolerance.” Science 248, no. 4959 (1990): 1112–5. Shorter, J., and S. Lindquist. “Hsp104 Catalyzes Formation and Elimination of Self-replicating Sup35 Prion Conformers.” Science 304, no. 5678 (2004): 1793–7. Lee, S., M. E. Sowa, et al. “The Structure of ClpB: A Molecular Chaperone that Rescues Proteins from an Aggregated State.” Cell 115, no. 2 (2003): 229–40. Lum, R., J. M. Tkach, et al. “Evidence for an Unfolding / Threading Mechanism for Protein Disaggregation by Saccharomyces Cerevisiae Hsp104.” The Journal of Biological Chemistry 279, no. 28 (2004): 29139–46. Schlieker, C., I. Tews, et al. “Solubilization of Aggregated Proteins by ClpB / DnaK Relies on the Continuous Extraction of Unfolded Polypeptides.” FEBS Letters 578, no. 3 (2004): 351–6. Weibezahn, J., C. Schlieker, et al. “Novel Insights into the Mechanism of Chaperone-assisted Protein Disaggregation.” Biological Chemistry 386, no. 8 (2005): 739–44. Zolkiewski, M. “ClpB Cooperates with DnaK, DnaJ, and GrpE in Suppressing Protein Aggregation: A Novel Multi-chaperone System from Escherichia Coli.” The Journal of Biological Chemistry 274, no. 40 (1999): 28083–6. Kenniston J. A., Baker T. A., et al. “Linkage Between ATP Consumption and Mechanical Unfolding during the Protein Processing Reactions of an AAA+ Degradation Machine.” Cell 114, no. 4 (2003): 511–20. Levchenko, I., Smith C.K., et al. “PDZ-like Domains Mediate Binding Specificity in the Clp / Hsp100 Family of Chaperones and Protease Regulatory Subunits.” Cell 91, no. 7 (1997): 939–47. Mogk, A., Schlieker C., et al. “Roles of Individual Domains and Conserved Motifs of the AAA+ Chaperone ClpB in Oligomerization, ATP Hydrolysis, and Chaperone Activity.” The Journal of Biological Chemistry 278, no. 20 (2003): 17615–24.
The in Vitro Refolding of Collagen	Short tripeptides with collagen-like sequences have recently been crystallized and their structure solved by X-ray diffraction. Using 2-D NMR it has been possible to follow the actual kinetics of the chain folding and association reaction and the effects of certain glycine substitutions. Baum, J., and B. Brodsky. “Real-time NMR Investigations of Triple-Helix Folding and Collagen Folding Diseases.” Folding and Design 2, no. 4 (1997): R53–60. Bhate, M., X. Wang, et al. “Folding and Conformational Consequences of Glycine to Alanine Replacements at Different Positions in a Collagen Model Peptide.” Biochemistry 41, no. 20 (2002): 6539–47. Fan, P., M. Li, et al. “Backbone Dynamics of (Pro-Hyp-Gly) 10 and a Designed Collagen-like Triple-Helical Peptide by 15N NMR Relaxation and Hydrogen-Exchange Measurements.” Biochemistry 32, no. 48 (1993): 13299–309.
Functions of Prolyl Hydroxylases in Collagen Chain Folding and Maturation	Prolyl hydroxylase is responsible for the formation of hydroxyproline on newly synthesized chains and is thought to be involved in regulating triple helix formation. Underhydroxylation of prolines is the molecular defect in scurvy, vitamin C deficiency. Myllyharju, J., and K. I. Kivirikko. “Identification of a Novel Proline-rich Peptide Binding Domain in Prolyl-4-hydroxylase.” The EMBO Journal 18, no. 2 (1999): 306–12. Kivirikko, K. I., R. Myllyla, et al. “Protein Hydroxylation: Prolyl-4-hydroxylase an Enzyme with Four Cosubstrates and a Multifunctional Subunit.” The FASEB Journal 3, no. 5 (1989): 1609–17. Walmsely, A. R., M. R. Batten, et al. “Intracellular Retention of Procollagen within the Endoplasmic Reticulum is Mediated by Prolyl4-hydrozylase.” The Journal of Biological Chemistry 274, no. 21 (1999): 14884–92.
The Role of Prolyl Isomerase in Protein Folding	What is the role of proline isomerization and proline isomerase in the folding of newly synthesized polypeptide chains within cells, including procollagen? Prolyl isomerase, originally called cyclophilin is the target of the cyclosporin class of immunosuppressive drugs. It turns out to function in many unexpected cellular processes. Harding, M. W., A. Galat, et al. “A Receptor for the Immunosuppressant FK506 is a Cis-trans Peptidyl-prolyl Isomerase.” Nature 341, no. 6244 (1989): 758–60. Fisher, G., B. Wittman-Liebold, et al. “Cyclophlin and Peptidyl-prolyl Cis-trans Isomerase are Probablyidentical Proteins.” Nature 337, no. 6206 (1989): 476–8. Steoinman, B., P. Bruckner, et al. “Cyclosporin A Slows Collagen Triple Helix Formation in Vivo; Inidirect Evidence for a Physiologic Role of Peptidyl Prolyl Cis-trans Isomerase.” The Journal of Biological Chemistry 266, no. 2 (1991): 1299–303.
Membrane Proteins and Transported Proteins
The SecB Chaperonins in Proteins Destined for Export	A number of proteins destined for export (and perhaps folding) outside the cell must be maintained in a non-folded state after synthesis. Some of those are maintained in this state by the secB protein of E.coli, whose mechanism has been studied in considerable detail. (Linda Randall and coworkers).
Chaperonin Function in Bacterial Pilus Assembly	Bacteria use extra cellular flagella and pili for swimming, attaching to other cells and transporting DNA. The folding and assembly of the proteins for these organelles utilize specialized chaperonins, some of which function in the bacterial periplasm. These function both in chain folding and in polymerization of the extracellular pilus organelle. Kuehn, M. J., S. Normark, et al. “Immunoglobulin-like PapD Chaperone Caps and Uncaps Interactive Surfaces of Nascently Translocated Pilus Subunits.” Proceedings of the National Academy of Sciences of the United States of America 88, no. 23 (1991): 10586–90. Slonim, L. N., J. S. Pinkner, et al. “Interactive Surface in the PapD Chaperone Cleft is Conserved in Pilus Chaperone Superfamily and Essential in Subunit Recognition and Assembly.” The EMBO Journal 11, no. 13 (1992): 4747–56. Dodson, K., F. Jacob-Dubuisson, et al. “Outer Membrane PapC Usher Discriminately Recognizes Periplasmic Chaperone-pilus Subunit Complexes.” Proceedings of the National Academy of Sciences of the United States of America 90, no. 8 (1993): 3670–74. Jones, C. H., J. Pinkner, et al. “FimC is a Periplasmic PapD-like Chaperone Which Directs Assembly of Type Pili in Bacteria.” Proceedings of the National Academy of Sciences of the United States of America 90, no. 18 (1993): 8397–401. Kuehn, M. J., D. J. Ogg, et al. “Structural Basis of Pilus Subunit Recognition by the PapD Chaperone.” Science 262, no. 5137 (1993): 1234–41.
Folding and Insertion of Bacteriorhodopsin	One of the very few membrane proteins whose three dimensional structure has been solved is the rhodopsin of the visual system. The best defined experiments on how these transmembrane helices associate within the membrane have been done with the bacterial and mammalian opsin. Rhodopsin mutation or damage is associated with a variety of human retinal pathologies. Booth, Paula J. “Unraveling the Folding of Bacteriorhodopsin.” Biochimica et Biophysica Acta 1460, no. 1 (2000): 4–14. Lu, Hui, and Paula J. Booth. “The Final Stages of Folding of the Membrane Protein Bacteriorhodopsin Occur by Kinetically Indistinguishable Parallel Folding Paths that are Mediated by pH.” Journal of Molecular Biology 299, no. 1 (2000): 233–43. Huang, Kuo-Sen, Hagan Bayley, et al. “Refolding of an Integral Membrane Protein.” The Journal of Biological Chemistry 256, no. 8 (1981): 3802–09.
Channels for Protein Import and Export	Many newly synthesized proteins have to transit a membrane, for example, for import into mitochondria or for entry to the endoplasmic reticulum. In general, the polypeptide chains have to be maintained in an unfolded state. The proteins forming these channels have recently been identified in a number of organisms. Eisele, and Rosenbuch_. The Journal of Biological Chemistry_ 265 (1990): 10217-22. Kleinschmidt, et al. “Outer Membrane Protein A of Escherichia coli Inserts and Folds into Lipid Bilayers by a Concerted Mechanism.” Biochemistry 38, no. 16 (1999): 5006–16. Kleinschmidt, and Tamm. “Folding Intermediates of a β-Barrel Membrane Protein. Kinetic Evidence for a Multi-Step Membrane Insertion Mechanism.” Biochemistry 35, no. 40 (1996): 12993–3000. Klose, et al. “Membrane Assembly of the Outer Membrane Protein OmpA of Escherichia Coli.” The Journal of Biological Chemistry 268, no. 34 (1993): 25664–70. Eppens, et al. “Folding of a Bacterial Outer Membrane Protein during Passage Through the Periplasm.” The EMBO Journal 16, no. 14 (1997): 4295–301. Surrey, et al. “Folding and Membrane Insertion of the Trimeric β-Barrel Protein OmpF.” Biochemistry 35, no. 7 (1996): 2283–8.
In Vivo Folding and Assembly of the Influenza Hemagglutinin	The intracellular assembly and maturation of this trimeric viral coat protein is one of the better model systems in eukaryotic cells. Braakman, I., Hoover-Litty H., et al. “Folding of Influenza Hemagglutinin in the Endoplasmic Reticulum.” The Journal of Cell Biology 114, no. 3 (1991): 401–11. Chen, W., Helenius J., et al. “Cotranslational Folding and Calnexin Binding during Glycoprotein Synthesis.” Proceedings of the National Academy of Sciences of the United States of America 92, no. 14 (1995): 6226–33. Chen, W., and Helenius H. “Role of Ribosome and Translocon Complex during Folding of Influenza Hemagglutinin in the Endoplasmic Reticulum of Living Cells.” Molecular Biology of the Cell 11, no. 2 (2000): 765–672. Copeland, C. S., K. P. Zimmer, et al. “Folding, Trimerization, and Transport are Sequential Events in the Biogenesis of Influenza Virus Hemagglutinin.” Cell 53, no. 2 (1988): 197–209. Hebert, D. N., Zhang J. X., et al. “The Number and Location of Glycans on Influenza Hemagglutinin Determine Folding and Association with Calnexin and Calreticulin.” The Journal of Cell Biology 139, no. 3 (1997): 613–23. Segal, M. S., Bye J. M., et al. “Disulfide Bond Formation During the Folding of Influenza Virus Hemagglutinin.” The Journal of Cell Biology 118, no. 2 (1992): 227–44. Tatu, U., Braakman I., et al. “Membrane Glycoprotein Folding, Oligomerization and Intracellular Transport: Effects of Dithiothreitol in Living Cells.” The EMBO Journal 12, no. 5 (1993): 2151–7. Tatu, U., Hammond C., et al. “Folding and Oligomerization of Influenza Hemagglutinin in the ER and the Intermediate Compartment.” The EMBO Journal 14, no. 7 (1995): 1340–8.
ERAD	Endoplasmic reticulin associated degradation. For many years scientists searched for the protease within the ER that was responsible for getting rid of misfolded proteins, only to realize that it doesn’t exist. Proteins in the ER are targeted for degradation and exported to the cytoplasm to be destroyed by the proteasome or sent to the lysosome (or vacuole) for degradation in that organelle. (see Prof. Lindquist for references)
Human Disease Associated with Protein Misfolding or Aggregation
Light Chain Amyloidosis	Cancer patients with a form of leukemia called multiple myeloma often accumulate amyloid deposits composed of the overproduced light chains. Aspects of this aggregation reaction have been elucidated through in vitro experiments. Hurle, et al. “A Role for Destabilizing Amino Acid Replacements in Light-chain Amyloidosis.” Proceedings of the National Academy of Sciences of the United States of America 91, no. 12 (1994): 5446–50. Helms, and Wetzel. “Specificity of Abnormal Assembly in Immunoglobulin Light Chain Deposition Disease and Amyloidosis.” Journal of Molecular Biology 257, no. 1 (1996): 77–86. Soloman, et al. “Light Chain-associated Amyloid Deposits Comprised of a Novel K Constant Domain.” Proceedings of the National Academy of Sciences of the United States of America 95, no. 16 (1998): 9547–51.
Superoxide Dismutase Defect in ALS (Lou Gehrig’s Disease).	Recent evidence indicates that amylotrophic lateral sclerosis is associated with a defect in the function of the widely distributed protein Superoxide Dismutase. The associated amino acid substitution may affect folding or stability rather than metabolic function. Rakhit, R., J. P. Crow, et al. “Monomeric Cu, Zn-superoxide Dismutase is a Common Misfolding Intermediate in the Oxidation Models of Sporadic and Familial Amyotrophic Lateral Sclerosis.” The Journal of Biological Chemistry 279 (2004): 15499–504. REVIEW: Valentine, J. S., and P. J. Hart. “Misfolded CuZnSOD and Amyotrophic Lateral Sclerosis.” Proceedings of the National Academy of Sciences of the United States of America 100, no. 7 (2003): 3617–22. Stathopulos, P. B., J. A. Rumfeldt, et al. “Cu/Zn Superoxide Dismutase Mutants Associated with Amyotrophic Lateral Sclerosis Show Enhanced Formation of Aggregates in Vitro.” Proceedings of the National Academy of Sciences of the United States of America 100, no. 12 (2003): 7021–6.
The CFTR Defect in Cystic Fibrosis	Cystic fibrosis is due to a defect in the chloride transporter protein in the respiratory tract. Recent evidence indicates that the most common inherited form is due to a protein folding defect. This is the most developed model of the role of protein folding defects in human disease. Thomas, et al. “Cystic Fibrosis Transmembrane Conductance Regulator: Nucleotide Binding to a Synthetic Peptide.” Science 251, no. 4993 (1992): 555–7. Qu, and Thomas. “Alteration of the Cystic Fibrosis Transmembrane Conductance Regulator Folding Pathway.” The Journal of Biological Chemistry 271, no. 13 (1996): 7261–4. War, and Kopito_. The Journal of Biological Chemistry_ 269 (1994): 25710–18. Zhang, et al. “Limited Proteolysis as a Probe for Arrested Conformational Maturation of DeltaF508 CFTR.” Nature Structural and Molecular Biology 5 (1998): 180–83. Johnston, et al. “Aggresomes: A Cellular Response to Misfolded Proteins.” The Journal of Biological Chemistry 143, no. 7 (1998): 1883–98.
Trans-thyretin and Amyloid Disease	A rare but well-studied class of amyloid diseases are due to deposition of the carrier protein trans-thyretin which is a retinol carrier protein. Features of the in vitro unfolding/ refolding reaction correlate with the conditions that yield pathology. Lashuel, H., Lai Z., et al. “Characterization of the Transthyretin Acid Denaturation Pathways by Analytical Ultracentrifugation: Implications for Wild-type, V30M, and L55P Amyloid Fibril Formation.” Biochemistry 37, no. 51 (1998): 17851–64. Colon, W., and J. W. Kelly. “Partial Denaturation of Transthyretin is Sufficient for Amyloid Fibril Formation in Vitro.” Biochemistry 31, no. 36 (1992): 8654–60. Lai, Z., W. Colon, et al. “The Acid-mediated Denaturation of Transthyretin Proceeds Through an Intermediate that Partitions into Amyloid.” Biochemistry 35, no. 20 (1996): 6470–82. Reixach N., S. Deechongkit, et al. “Tissue Damage in the Amyloidoses: Transthyretin Monomers and Nonnative Oligomers are the Major Cytotoxic Species in Tissue Culture.” Proceedings of the National Academy of Sciences of the United States of America 101, no. 9 (2004): 2817–22.
The Anti-trypsin Defect in Familial Lung Disease	Increased susceptibility to lung damage from smoking and dusts is associated with certain alleles of the anti-elastase that functions in the lung. Recent evidence reveals that their major familial form is due to a defect in the folding of the protein. Ryu, S. E., H. J. Choi, et al. “The Native Strains in the Hydrophobic Core and Flexible Reactive Loop of a Serine Protease Inhibitor: Crystal Structure of an Uncleaved α1-antitrypsin at 2.7 Å.” Structure 4, no. 10 (1996): 1181–92. Kim, S.-J., J.-R. Woo, et al. “A 2.1 Å Resolution Structure of an Uncleaved α1-antitrypsin Shows Variability of the Reactive Center and other Loops.” Journal of Molecular Biology 306, no. 1 (2001): 109–19. Cabrita, L. D., W. Dai, et al. “Different Conformational Changes within the F-helix Occur during Serpin Folding, Polymerization, and Proteinase Inhibition.” Biochemistry 43, no. 30 (2004): 9834–9. Im, H., M. S. Woo, et al. “Interactions Causing the Kinetic Trap in Serpin Protein Folding.” The Journal of Biological Chemistry 277, no. 48 (2002): 46347–54. Devlin, G. L., M. K. Chow, et al. “Acid Denaturation of α1-antitrypsin: Characterization of a Novel Mechanism of Serpin Polymerization.” Journal of Molecular Biology 324, no. 4 (2002): 859–70. Carrell, R. W., and B. Gooptu. “Conformational Changes and Disease-serpins Prions and Alzheimer’s.” Current Opinion in Structural Biology 8, no. 6 (1998): 799–809. Carrell, R. W., J. Whisstock, et al. “Conformational Changes in the Serpins and the Mechanism of α-1 Antichymotrypsin Deficiency.” American Journal of Respiratory and Critical Care Medicine 150 (1994): 171–5.
Mutations in Tumor Suppressor Proteins	Loss of function of a number of cellular proteins, which control DNA replication and cell division, is associated with tumor formation. Particularly well-studied are p53 and p21. There is considerable evidence for the p16 ankyrin proteins that some of these mutations may represent protein folding defects.
Prions
Prions	Prions are a diverse group of unrelated proteins that have very unusual folding properties. They can exist stably in profoundly different conformations, one of which (the prion conformation) can template other proteins of the same type to change into the prion conformation. This creates a protein conformational chain reaction that can cause terrible diseases, or serve as an epigenetic mechanism for the inheritance of new traits. (Prusiner, Lansbury).
Topic A: properties and transmissibility	Mammalian PrP Cellular properties of the rodent prion agent and its transmissibility. Prusiner, et al. “Prion Protein Biology.” Cell 93, no. 3 (1998): 337–48. Caughey, and Bruce Chesebro. “Prion Protein and the Transmissible Spongiform Encephalopathies.” Trends in Cell Biology 7, no. 2 (1997): 56–62. and work forward; Pillot, et al. “The 118–135 Peptide of the Human Prion Protein Forms Amyloid Fibrils and Induces Liposome Fusion.” Journal of Molecular Biology 274, no. 3 (1997): 381–93. Raymond, G., A. Bossers, et al. “Evidence of a Molecular Barrier Limiting Susceptibility of Humans, Cattle and Sheep to Chronic Wasting Disease.” The EMBO Journal 19, no. 17 (2000): 4425–30. Pertez, D., M. Scoot, et al. “Strain-specific Relative Conformational Stability of the Scrapie Prion Protein.” Protein Science 10, no. 4 (2001): 854–63.
Topic B: structural characterization of mammalian prion protein	Mammalian PrP Structural characterization of the isolated prion protein. Zhang, et al. “Physical Studies of Conformational Plasticity in a Recombinant Prion Protein.” Biochemistry 36, no. 12 (1997): 3543–53. Liemann, and Glockshuber. “Influence of Amino Acid Substitutions Related to Inherited Human Prion Diseases on the Thermodynamic Stability of the Cellular Prion Protein.” Biochemistry 38, no. 11 (1999): 3258–67. Jackson, et al. “Reversible Conversion of Monomeric Human Prion Protein between Native and Fibrilogenic Conformations.” Science 283, no. 5409 (1999): 1935–7. Peretz, et al. “A Conformational Transition at the N Terminus of the Prion Protein Features in Formation of the Scrapie Isoform.” Journal of Molecular Biology 273, no. 3 (1997): 614–22. Goverts, C., H. Wille, et al. “Evidence for the Assembly of Prions with Left-handed β Helices into Trimers.” Proceedings of the National Academy of Sciences of the United States of America 101, no. 22 (2004): 8342–7. Wille, H., M. D. Michelitsch, et al. “Structural Studies of the Scrapie Prion Protein by Electron Crystallography.” Proceedings of the National Academy of Sciences of the United States of America 99, no. 6 (2002): 3563–8.
Topic C: characterization of prion protein in yeast	Yeast Prions Characterization of the yeasts prion protein, genetics & cell biology. Sophisticated genetic analysis available in yeast has allowed a very penetrating analysis of the origin and pheotypes of the sup35 yeast prion protein. Uptain, S. M., and S. Lindquist. “Prions as Protein-based Genetic Elements.” Annual Review of Microbiology 56 (2002): 703–41. Krishnan, R., and S. L. Lindquist. “Structural Insights into a Yeast Prion Illuminate Nucleation and Strain Diversity.” Nature 435 (2005): 765–72. Serio, T. R., and S. L. Lindquist. “The Yeast Prion [PSI+]: Molecular Insights and Functional Consequences.” Advances in Protein Chemistry 59 (2001): 391–412. Scheibel, T., A. S. Kowal, et al. “Bidirectional Amyloid Fiber Growth for a Yeast Prion Determinant.” Current Biology 11, no. 5 (2001): 366–9. Scheibel, T., J. Blookm, et al. “The Elongation of Yeast Prion Fibers Involves Separable Steps of Association and Conversion.” Proceedings of the National Academy of Sciences of the United States of America 101, no. 8 (2004): 2287–92.
Alzheimer’s Disease
Amyloid Deposits in Alzheimers Disease	Alzheimers patients have insoluble protein deposits in a number of their tissues. A major class are characterized by a distinctive cross beta rod structure. Jarret, et al. “The Carboxy Terminus of the Beta Amyloid Protein is Critical for the Seeding of Amyloid Formation: Implications for the Pathogenesis of Alzheimer’s Disease.” Biochemistry 32, no. 18 (1993): 4693–7. Harper, J. D., C. M. Leber, et al. “Atomic Force Microscopic Imaging of Seeded Fibril Formation and Fibril Branching by the Alzheimer’s Disease Amyloid-β Protein.” Chemistry and Biology 4, no. 12 (1997): 951–9. Pike, et al. “Amino-terminal Deletions Enhance Aggregation of β-Amyloid Peptides in Vitro.” The Journal of Biological Chemistry 270 (1995): 23895–8.
Topic A: proteolytic cleavage	Studies on the generation of the Alzheimer’s peptide by proteolytic cleavage from its precursor. Strooper, B. De, P. Saftig, et al. “Deficiency of Presenilin-1 Inhibits Normal Cleavage of Amyloid Precursor Protein.” Nature 391 (1998): 387–90. Esch, F. S., P. S. Keim, et al. “Cleavage of Amyloid Beta-peptide during Constitutive Processing of its Precursor.” Science 248, no. 4959 (1990): 1122–4. Haass, C., and D. J. Selkoe. “Cellular Processing of β-Amyloid Precursor Protein and the Genesis of Amyloid Beta-Peptide.” Cell 75, no. 6 (1993): 1039–42. Haass, C., M. G. Schlossmacher, et al. “Amyloid β-peptide is Produced by Cultured Cells during Normal Metabolism.” Nature 359, no. 6393 (1992): 322–5. Kimberly, W. T., and M. S. Wolfe. “Identity and Function of γ-secretase.” Journal of Neuroscience Research 74, no. 3 (2003): 353–60. Mandelkow, E. “Alzheimer’s Disease: The Tangled Tale of Tau.” Nature 402 (1999): 588–9. Naslund, J., Haroutunian, V., et al. “Correlation Between Elevated Levels of Amyloid β-peptide in the Brain and Cognitive Decline.” Journal of the American Medical Association 283, no. 12 (2000): 1571–7.
Topic B: amyloid plaques and interactions	Formation of amyloid plaques in vivo, including interactions with other factors such as the ApoE lipprotein, chaperonins, etc. Esch, et al. “Cleavage of Amyloid Beta Peptide during Constitutive Processing of its Precursor.” Science 248, no. 4959 (1990): 1122–4. Haas, and Selkoe. “Cellular Processing of β-amyloid Precursor Protein and the Genesis of Amyloid Beta-peptide.” Cell 75, no. 6 (1993): 1039–42. Haas, et al. “Amyloid β-peptide is Produced by Cultured Cells during Normal Metabolism.” Nature 359 (1992): 322–5. Scheuner, et al. “Secreted Amyloid β-protein Similar to that in the Senile Plaques of Alzheimer’s Disease is Increased in Vivo by the Presenilin 1 and 2 and APP Mutations Linked to Familial Alzheimer’s Disease.” Nature Medicine 2, no. 8 (1996): 864–70. Wild-Bode, et al. “Intracellular Generation and Accumulation of Amyloid β-Peptide Terminating at Amino Acid 42.” The Journal of Biological Chemistry 272 (1997_)_: 16085–88. Allsop, D., C. W. Wong, et al. “Immunohistochemical Evidence for the Derivation of a Peptide Ligand from the Amyloid Beta-protein Precursor of Alzheimer Disease.” Proceedings of the National Academy of Sciences of the United States of America 85, no. 8 (1988): 2790–94.
Topic C: therapeutic approaches	Therapeutic Approaches to Retarding or Alleviating Alzheimers (refs. to follow) Therapeutic strategies for alleviating Alzheimers disease cover a vast landscape. They include, among others: attempts to reduce the production of ABeta by inhibiting a protease that cleaves APP, immunizing patients against ABeta, protecting mitochondrial function, providing nerve growth factors, and blocking amyloid assembly. Provide an overview, and then choose three strategies and report on them in detail. The rationale, evidence of success, reasons they might fail.
Parkinson’s Disease
Parkinson’s Disease and Alpha-synuclein Aggregation	Parkinson’s disease, due to damage to a very specific region of the brain, is associated with aggregation of a plentiful brain protein alpha-synuclein.
Topic A: alpha-synuclein aggregation and polymerization	In vitro characterization of alpha-synuclein aggregation and polymerization: Lashuel, H. A., B. M. Petre, et al. “α-synuclein, Especially the Parkinson’s Disease Associated Mutants, Form Pore-like Annular and Tubular Protofibrils.” Journal of Molecular Biology 322, no. 5 (2002): 1089–102. Park, J. Y., and P. T. Lansbury. “β-synuclein Inhibits Formation of α-synuclein Protofibrils: A Possible Therapeutic Strategy Against Parkinson’s Disease.” Biochemistry 42, no. 13 (2003): 3696–700. Zhu, M., J. Li, et al. “The Association of α-synuclein with Membranes Affects Bilayer Structure, Stability, and Fibril Formation.” The Journal of Biological Chemistry 278 (2003): 40186–97.
Topic B: Etiology of Parkinson’s	Etiology of Parkinson’s disease; MPTP, rotenone, paraquat as etiological agents; inherited mutations causing early onset PD. Polymeropoulos, et al. “Mutation in the α-synuclein Gene Identified in Families with Parkinson’s Disease.” Science 276, no. 5321 (1997): 2045–7. Mezey, et al. “Alpha Synuclein in Neurodegenerative Disorders: Murderer or Accomplice?” Nature Medicine 4 (1998): 755–7. Leroy, et al. “Deletions in the Parkin Gene and Genetic Heterogeneity in a Greek family with Early Onset Parkinson’s Disease.” Human Genetics 103, no. 4 (1998): 424–7. Li, J., V. N. Uversky, et al. “Effect of Familial Parkinson’s Disease Point Mutations A30P and A53T on the Structural Properties, Aggregation, and Fibrillation of Human α-synuclein.” Biochemistry 40, no. 38 (2001): 11604–13.
Huntington’s Disease
Protein Aggregation in Huntington’s Disease	The modified protein product associated with CCC expansions in Huntington’s disease has recently been identified. The presence of polyglutamine insertions and expansions appears to be a more general source of cellular pathology. An aggregated form of Huntington’s is found within the cell nucleus.
Topic A: polyglutamine aggregation	In vitro characterization of polyglutamine aggregation. Perutz, et al. “Glutamine Repeats as Polar Zippers: Their Possible Role in Inherited Neurodegenerative Diseases.” Proceedings of the National Academy of Sciences of the United States of America 91, no. 12 (1991): 5355–8. Perutz, et al. “Polar Zippers.” Current Biology 3, no. 5 (1993): 249–53. DeFiglia, et al. “Aggregation of Huntingtin in Neuronal Intranuclear Inclusions and Dystrophic Neurites in Brain.” Science 277, no. 5334 (1997): 1990–93. Scherzinger, et al. “Huntingtin-encoded Polyglutamine Expansions Form Amyloid-like Protein Aggregates In Vitro and In Vivo.” Cell 90, no. 3 (1997): 549–58. Scherzinger, et al. “Self-assembly of Polyglutamine-containing Huntingtin Fragments into Amyloid-like Fibrils: Implications for Huntington’s Disease Pathology.” Proceedings of the National Academy of Sciences of the United States of America 96, no. 8 (1998): 4604–09.
Topic B: polyglutamine aggregation in experimental organisms	Investigation of polyglutamine aggregation in experimental organisms; caenorhabditis elegans, Drosophila, or mice. Morley, J. F., H. R. Brignull, et al. “The Threshold for Polyglutamine-expansion Protein Aggregation and Cellular Toxicity is Dynamic and Influenced by Aging in Caenorhabditis Elegans.” Proceedings of the National Academy of Sciences of the United States of America 99, no. 16 (2002): 10417–22.