The Technical Tips handouts in the table below describe in greater detail the experimental methods and technologies used in the assigned literature listed in the Readings column.

1 Introduction We will introduce ourselves and briefly explain our backgrounds and interests in biological sciences. Every student will also briefly say why he/she has decided to take this course, and what his/her interests and expectations are.

We will discuss general aspects of the class syllabus, schedule and assignments. I will provide some tips about how to critically read a scientific paper, how to interpret data and methodologies and how to hypothesize models based on data.

I will introduce general aspects of the ubiquitin-proteasome system (UPS): its function, components and possible outcomes when any of its functions are damaged.

Papers and materials for the next session will be briefly introduced and provided.
Recommended Review Papers

Ciechanover, A., A. Orian, and A. L. Schwartz. "Ubiquitin-mediated proteolysis: biological regulation via destruction." Bioessays 22 (2000): 442-451.
2 Discovery of the Ubiquitin Conjugation System

The first session will lead us through the pioneer work and discovery of this essential system.

Required Review Papers

Hershko, A., A. Ciechanover, H. Heller, A. L. Haas, and I. A. Rose. "Proposed role of ATP in protein breakdown: Conjugation of proteins with multiple chains of the polypeptide of ATP-dependent proteolysis." Proc. Natl. Acad. Sci. USA 77 (1980): 1783-1786.

Ciechanover, A., D. Finley, and A. Varshavsky. "Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85." Cell 37 (1984): 57-66.

Technical Tips (PDF - 1.0 MB)

Recommended Review Papers

Hershko A., A. Ciechanover, and A. Varshavsky. "The ubiquitin system." Nature Medicine 6 (2000): 1073-1081.
3 Protein Degradation in Trafficking Membranes I: Endoplasmic Reticulum Associated Degradation (ERAD) Pathway

Proteins sorted to the secretory pathway such as membrane proteins enter this route through the ER. However, misfolded proteins can be retrotranslocated back to the cytosol by a quality control system present in this compartment. Retrotranslocated proteins are recognized by specific ER-associated E3 ubiquitin ligases that ubiquitinate the emerging protein for its proteasomal degradation. Interestingly, as we will see in the first paper, this pathway is not always used for protein removal but sometimes is instead for proteolytic activation. In this session we will learn how this system works and its importance for functions such as antigen presentation. We will also study in the second manuscript an example of how pathogenic agents can take over such cellular mechanism for their own benefit.

Required Review Papers

Hoppe, T., K. Matuschewski, M. Rape, S. Schlenker, H. D. Ulrich, and S. Jentsch.  "Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing." Cell 102 (2000): 577-586.

Margottin, F., S. P. Bour, H. Durand, L. Selig, S. Benichou, V. Richard, D. Thomas, K. Strebel, and R. Benarous. "A novel human WD protein, h- TrCP, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif." Mol. Cell 1 (1998): 565-574.

Technical Tips (PDF)

Recommended Review Papers

Hampton, R. Y. "ER-associated degradation in protein quality control and cellular regulation." Curr. Opin. Cell Biol. 14 (2002): 476-482.
4 Protein Degradation in Trafficking Membranes II: Endocytosis and lysosomal Degradation

Ubiquitin-dependent degradation of proteins in the secretory pathway does not occur only in the cytosol, mediated by the proteasome, but can also happen in a specialized compartment, the lysosome/vacuole. In this session we will study the importance of this alternative pathway for downregulation of the activities of plasma membrane proteins, such as receptors that act in signal transduction pathways. Again, we will have the opportunity to discuss how a virus can utilize components of a pathway for its own purposes.

Required Review Papers

Mosesson, Y., K. Shtiegman, M. Katz, Y. Zwang, G. Vereb, J. Szollosi, and Y. Yarden. "Endocytosis of receptor tyrosine kinases is driven by monoubiquitylation, not polyubiquitylation." J. Biol. Chem. 278 (2003): 21323-21326.

Pornillos, O., D. S. Higginson, K. M. Stray, R. D. Fisher, J. E. Garrus, M. Payne, G. P. He, H. E. Wang, S. G. Morham, and W. I. Sundquist. "HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein." J. Cell Biol. 162 (2003): 425-434.

Technical Tips (PDF)

Recommended Review Papers

Haglund, K., P. P. Di Fiore, and I. Dikic. "Distinct monoubiquitin signals in receptor endocytosis." Trends Biochem. Sci. 28 (2003): 598-604.

Amara, A.,  and D. R. Littman. "After Hrs with HIV." J. Cell Biol. 162 (2003): 371-375.
5 Role of Ubiquitination in Transcriptional Regulation Ubiquitination acts at multiple levels to regulate gene expression, mainly by "regulating the regulators", from targeting them for degradation, causing their proteolytic activation to controlling their intracellular localization. Moreover, in this class we will analyze how components of the proteasome themselves are physically recruited to the genes to regulate their expression. We will also study the importance of the ubiquitination of components of the transcriptional machinery to avoid the expression of damaged DNA, as an essential step for further action of DNA repair responses. Required Review Papers

Gonzalez, F., A. Delahodde, T. Kodadek, and S. A. Johnston. "Recruitment of a 19S proteasome subcomplex to an activated promoter." Science 296 (2002): 548-550.

Lee, K-B., D. Wang, S. J. Lippard, and P. A. Sharp. "Transcription-coupled and   DNA damage-dependent ubiquitination of RNA polymerase II." in vitro. Proc. Natl. Acad. Sci. USA 99 (2002): 4239-4244.

Technical Tips (PDF)

Recommended Review Papers

Muratani, M., and W. P. Tansey. "How the ubiquitin-proteasome system controls transcription." Nature Rev. Mol. Cell Biol. 4 (2003): 192-201.
6 Role of Ubiquitination in Cell Cycle Control and Programmed Cell Death The substantial importance of the ubiquitin-proteolytic system for cell's life will be evident in this session through the study of two fundamental cell pathways. In the first paper we will analyze the importance of ubiquitination for the progression of the cell division cycle. The second paper will allow us to discuss one of the multiple levels of ubiquitin-dependent regulation of the apoptotic (programmed cell death) pathway and its importance in tumorigenesis. Required Review Papers

Busino, L., M. Donzelli, M. Chiesa, D. Guardavaccaro, D. Ganoth, N. V. Dorrello, A. Hershko, M. Pagano, and G. F. Draetta. "Degradation of Cdc25A by  -TrCP during S phase and in response to DNA damage." Nature 426 (2003): 87-91.

Li, B., and Q. P. Dou. "Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression." Proc. Natl. Acad. Sci. USA 97 (2000): 3850-3855.

Technical Tips (PDF)

Recommended Review Papers

Reed, S. I. "Ratchets and clocks: The cell cycle, ubiquitylation and protein turnover." Nat. Rev. Mol. Cell Biol. 4 (2003): 855-864.

Yang, Y., and X. Yu. "Regulation of apoptosis: the ubiquitous way." FASEB J. 17 (2003): 790-799.
7 Ubiquitin-like Proteins Ubiquitin is not the only protein utilized to tag other proteins. In the past several years other ubiquitin-like modifiers with similar mechanisms of substrate conjugation have been discovered. In this session we will overview them and study their particular nonproteolytic functions. We will also speculate about the possible origins of the 'ubiquitin system' by analyzing its connection to other, more ancient, biosynthetic pathways present in bacteria. Required Review Papers

Li, T., E. Evdokimov, R-F. Shen, C-C. Chao, E. Tekle, T. Wang, E. R. Stadtman, D. C. H. Yang, and P. B. Chock. "Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis." Proc. Natl. Acad. Sci. USA 101 (2004): 8551-8556.

Furukawa, K., N. Mizushima, T. Noda, and Y. Ohsumi. "A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes." J. Biol. Chem. 275 (2000): 7462-7465.

Technical Tips (PDF)
8 Functions of the Ubiquitin-Proteasome System in the Immune System Although some aspects of ubiquitination controlling the immune response have been analyzed in session 3, we will now discuss an exceptional example of how viruses can deceive the protective mechanisms of antigen presentation. The second paper will introduce the role of ubiquitination in the control of another essential pathway that induces the inflammatory response and inhibits apoptosis. We will study a new case of a microbial system adapted against this system. Required Review Papers

Levitskaya, J., A. Sharipo, A. Leonchiks, A. Ciechanover, and M. G. Masucci. "Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1." Proc. Natl. Acad. Sci. USA 94 (1997): 12616-12621.

Neish, A. S., A. T. Gewirtz, H. Zeng, A. N. Young, M. E. Hobert, V. Karmali, A. S. Rao, and J. L. Madara. "Prokaryotic regulation of epithelial responses by inhibition of I B-  ubiquitination." Science 289 (2000): 1560-1563.

Technical Tips (PDF)

Recommended Review Papers

Sijts, A., D. Zaiss, and P. M. Kloetzel. "The role of the ubiquitin-proteasome pathway in MHC class I antigen processing: implications for vaccine design." Curr. Mol. Med. 1 (2001): 665-676.

Ben-Neriah, Y. "Regulatory functions of ubiquitination in the immune system." Nature Immunol. 3 (2002): 20-26.
9 Ubiquitin and Cancer Examples of ubiquitin's importance in the development of cancer have already appeared in previous sessions. Now, the first paper will give us the opportunity to analyze how ubiquitination controls one of the main regulators of apoptosis, the p53 kinase. We will see how human papilloma virus deals with this threat by disabling one of our main protective mechanisms against cancer. The second example will introduce us in a case of genetically inherited susceptibility to cancer involving a defective ubiquitination system, Fanconi anemia. Required Review Papers

Scheffner, M., J. M. Huibregtse, R. D. Vierstra, and P. M. Howley. "The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53." Cell 75 (1993): 495-505.

Meetei, A. R., J. P. de Winter, A. L. Medhurst, M. Wallisch, Q. Waisfisz, H. J. van de Vrugt, A. B. Oostra, Z. Yan, C. Ling, C. E. Bishop, M. E. Hoatlin, H. Joenje, and W. Wang. "A novel ubiquitin ligase is deficient in Fanconi anemia." Nature Genetics 35 (2003): 165-170.

Technical Tips (PDF)

Recommended Review Papers

Scheffner, M. "Ubiquitin, E6-AP, and their role in p53 inactivation." Pharmacol. Ther. 78 (1998): 129-139.

D'Andrea, A. D., and M. Grompe. "The Fanconi anaemia/BRCA pathway." Nature Rev. Cancer 3 (2002): 23-34.
10 Ubiquitin and Neurodegenerative Diseases: Alzheimer's and Parkinson's Diseases In the last few years increasing evidence has proved that abnormal accumulation of certain forms of different proteins can cause toxicity and loss of function in specific tissues. Particularly, certain kinds of neurons have shown a special sensitivity, leading to several different neurodegenerative disorders and diseases. An altered functioning of the ubiquitin-proteasome system is tightly interconnected to this phenomenon, although whether its misfunctioning is a cause or a consequence for this abnormal accumulation of proteins is still topic of discussion. We will analyze in this session different aspects involving the ubiquitin-proteasome system in Alzheimer and Parkinson's diseases. Required Review Papers

Lam, Y. A., C. M. Pickart, A. Alban, M. Landon, C. Jamieson, R. Ramage, R. J. Mayer, and R. Layfield. "Inhibition of the ubiquitin-proteasome system in Alzheimer's disease." Proc. Natl. Acad. Sci. USA 97 (2000): 9902-9906.

Shimura, H., M .G. Schlossmacher, N. Hattori, M. P. Frosch, A. Trockenbacher, R. Schneider, Y. Mizuno, K. S. Kosik, and D. J. Selkoe. "Ubiquitination of a new form of  -synuclein by parkin from human brain: implications for Parkinson's disease." Science 293 (2001): 263-269.

Recommended Review Papers

Ciechanover, A., and P. Brundin. "The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg." Neuron 40 (2003): 427-446.
11 More Diseases Involving Ubiquitin: Huntington's and Von Hippel-Lindau Disease Huntington's is the most studied of the polyglutamine diseases of the nervous system caused by a mutation that involves a CAG triplet repeat expansion. Mutant forms of Huntingtin misfold promoting aggregation and leading to cell toxicity. In the second paper we will review the VHL (von Hippel-Lindau) syndrome, an autosomal dominant familial cancer syndrome that predisposes affected individuals to several different kinds of tumors. The gene mutated encodes for VHL, a protein member of a SCF E3 ligase complex acting in the regulation of hypoxia-inducible genes. Required Review Papers

Kalchman, M. A., R. K. Graham, G. Xia, H. B. Koide, J. G. Hodgson, K. C. Graham, Y. P. Goldberg, R. D. Gietz, C. Pickart, and M. R. Hayden. "Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme." J. Biol. Chem. 271 (1996): 19385-19394.

Ivan, M., K. Kondo, H. Yang, W. Kim, J. Valiando, M. Ohh, A. Salic, J. M. Asara, W. S. Lane, and W. G. Kaelin Jr. "HIF  targeted for VHL- mediated destruction by proline hydroxylation: implications for O2 sensing." Science 292 (2001): 464-468.

Technical Tips (PDF)

Recommended Review Papers

Tarlac, V., and E. Storey. "Role of proteolysis in polyglutamine disorders." J. Neurosci. Res. 74 (2003): 406-416.

Kaelin Jr., W. G. "Molecular basis of the VHL hereditary cancer syndrome." Nature Rev. Cancer 2 (Sep 2002): 673-82.

12 Too Much Degradation can be as bad as not Enough: Cystic Fibrosis and Liddle's Syndrome Cystic fibrosis is a recessive disorder characterized by severe bronchopulmonary infections and pancreatic insufficiency. Mutations in the chloride ion channel CFTR cause its increased degradation rate, not allowing enough to reach the cell surface. Liddle syndrome is an autosomal dominant form of hypertension that results from mutations that stabilize the otherwise short half-life channel ENaC. Required Review Papers

Ward, C. L., S. Omura, and R. R. Kopito. "Degradation of CFTR by the ubiquitin-proteasome system." Cell 83 (1995): 121-127.

Malik, B., L. Schlanger, O. Al-Khalili, H-F. Bao, G. Yue, S. R. Price, W. E. Mitch, and D. G. Eaton. "ENaC degradation in A6 cells by the ubiquitin- proteasome system." J. Biol. Chem. 276 (2001): 12903-12910.

Technical Tips (PDF)

Recommended Review Papers

Plemper, R. K., and D. H. Wolf. "Retrograde protein translocation: ERADication of secretory proteins in health and disease." TIBS 24 (1999): 266-270.

Gormley, K., Y. Dong, and G. A. Sagnella. "Regulation of the epithelial sodium channel by accessory proteins." Biochem. J. 371 (2003): 1-14.
13 Potential Therapeutic Strategies in Ubiquitin-Related Diseases Several approaches are starting to emerge to battle disease by tackling defects in the ubiquitin-proteasome system. This session will allow us to discuss and speculate about some of the evolving methodologies. Required Review Papers

Ma, M. H., H. H. Yang, K. Parker, S. Manyak, J. M. Friedman, C. Altamirano, Z. Q. Wu, M. J. Borad, M. Frantzen, E. Roussos, J. Neeser, A. Mikail, J. Adams, N. Sjak-Shie, R. A. Vescio, J. R. Berenson. "The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents." Clin. Cancer Res. 9 (2003): 1136-44.

Sakamoto, K. M., K. B. Kim, A. Kumagai, F. Mercurio, C. M. Crews, and R. J. Deshaies. "Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation." Proc. Natl. Acad. Sci. USA 98 (2001): 8554-8559.

Recommended Review Papers

Richardson, P. G., T. Hideshima, and K. C. Anderson. "Bortezomib (PS-341): a novel, first-in-class proteasome inhibitor for the treatment of multiple myeloma and other cancers." Cancer Control 10 (2003): 361-369.

Lee, D. H., and A. L. Goldberg. "Proteasome inhibitors: valuable new tools for cell biologists." Trends Cell Biol. 8 (1998): 397-403.

Wong, B. R., F. Parlati, K. Qu, S. Demo, T. Pray, J. Huang, D. G. Payan, and M. K. Bennett. "Drug discovery in the ubiquitin regulatory pathway." Drug Discov. Today 8 (2003): 746-754.
14 Oral Presentations

Final Comments and Course Evaluation