| 1 |
Introduction and Course Overview | The first meeting will consist of introductions, both of the instructor and the students. The instructor will present a general overview of the course, discuss the format, and explain the preparation that will be expected of each student each week. The instructor will also provide an overview of the course material with an introduction to the historical aspect and concepts that have emerged to explain why and how protein misfolding and aggregation occurs and why degradation is a crucial cellular response. |
| 2 |
ER Quality Control and Degradation | Protein quality control involves an elaborate machinery that recognizes and retains newly synthesized misassembled proteins and targets them for degradation. ER quality control involves a multi-step pathway, which includes transport of misfolded substrates from the ER to the cytosol, where they are tagged for disposal by the addition of ubiquitin chains and degraded at the proteasome. In this session, we will discuss what is known about this pathway - how this multi-step process involves substrate recognition, targeting for dislocation, removal from the ER membrane, deglycosylation, ubiquitination and finally proteolysis. |
| 3 |
Discovery of Ubiquitin as a Tag for Proteasomal Degradation | In the early 1980s ubiquitin was discovered in an attempt to characterize and purify a non-lysosomal proteolytic system that was responsible for turnover of short-lived proteins. The discovery of the ubiquitin system was a paradigm and provided a way to understand how a protease and substrate can reside in the same compartment. Unless a protein is tagged by ubiquitin (a 76 amino acid protein), it is not recognized by the proteasome and consequently not degraded. We will cover the experimental design and rationale that led to the initial discovery of ubiquitination as a method of tagging polypeptides destined for destruction. |
| 4 |
The Ubiquitin Conjugation Cascade | Two monumental papers published in 1984 by the Varshavsky lab revealed that the ubiquitin/proteasome pathway is the principle method of degradation of short-lived proteins in mammalian cells, setting the stage for future studies of this pathway's multiple regulatory roles. We will discuss the impact of these papers and some of the subsequent insights that have led to our current understanding and appreciation of the breadth of ubiquitin-mediated cell-signaling. |
| 5 |
Biology Seminar | We will attend a research lecture at Harvard Medical School, Boston, about protein aggregation and its impact on human physiology. The speaker, Dr. F. Ulrich Hartl from Max Planck Institute of Biochemistry, Germany, will be presenting his lecture on "Proteotoxicity of β-aggregation: Mechanisms and defense by molecular chaperones." This lecture (experimental approach and key results) will be discussed in the next session. |
| 6 |
Biochemical Approaches to Measure Protein Dislocation | An approach that has been historically used to gain insight into the molecular mechanisms underlying protein dislocation and degradation – also referred to as ER-associated degradation (ERAD) - is to biochemically monitor the fate of model substrates, typically either radioactively or fluorescently labelled. Blocking proteasomal proteolysis either by chemical or enzymatic means provides an opportunity to follow the trajectory and the intermediate stages of these model substrates from the ER lumen to the cytoplasm. This week we will become familiar with routine biochemical techniques that are typically used to study this pathway. |
| 7 |
The Putative Dislocon: Lessons from Yeast and Mammalian Systems | Export of proteins across the ER bilayer presumably occurs through an aqueous channel that allows passage of polypeptides through the highly hydrophobic membrane environment. We will discuss available data from studies of yeast and mammalian cells concerning the most promising candidates that have been proposed to constitute such a channel. |
| 8 |
Substrate Recognition: The Glycan Destruction Signal for ERAD | Owing to the extreme diversity that exists among proteins that must be examined by the ER quality control machinery, an interesting question is how recognition of misfolded substrates occurs. For glycoproteins, a possible mechanism is through calnexin/calreticulin lectin-type chaperones, which retain glycoproteins in the ER until productive folding takes place. |
| 9 |
Cytosolic Regulation of Misfolded/Mislocalized Protein Degradation | The ER quality control pathway employs membrane-bound ubiquitin ligases and the ATPase p97 to dislocate misfolded proteins for proteasomal degradation. How such dislocated polypeptides bearing hydrophobic transmembrane segments remain soluble in the cytosol escaping premature aggregation has been an important line of research. Emerging data suggest a tight regulation that exists through cytosolic chaperone complexes guarantee delivery of these misfolded proteins for proteasomal proteolysis. |
| 10 |
Viral Avoidance and Exploitation of the Ubiquitin Proteasome System | Intracellular pathogens exploit the ubiquitin proteasome system mostly to destroy or avoid destruction of specific cellular proteins. These activities create a more hospitable environment for the pathogens in the host cells by escaping detection by the immune system. In this session we will focus on a few examples of viral interference with the Ubproteasome system that illustrate how this system is crucial for aspects of pathogen life cycles. |
| 11 |
Toxin Invasion through the ERAD Machinery | A number of toxins, such as cholera, ricin and Pseudomonas aeruginosa exotoxin A, exploit membrane trafficking pathways of the host cell machinery to enter and intoxicate. Once in the ER, the enzymatically active subunit of these protein toxins can be translocated to the cytosol. How does this occur? Such toxins are thought to masquerade as misfolded proteins to hijack the ER quality control system that normally targets misfolded ER proteins to the cytosol for proteasomal degradation. Taking cholera as an example, we will discuss how host-pathogen interactions might be occurring through the ER quality control pathway. |
| 12 |
Student Oral Presentations | See the Assignments section for a description of what is expected. The students will receive written evaluations of their presentations from the instructor. |
| 13 |
ER-associated Degradation in Disease | Emerging data about ER-associated degradation have revealed that a malfunction of this pathway caused by genetic mutations and environmental factors can result in various diseases, such as diabetes, inflammation and neurodegenerative disorders, including Alzheimer's, Parkinson's and Huntington's diseases. Mutations in the human cystic fibrosis transmembrane conductance regulator (CFTR) gene result in cystic fibrosis (CF) due to a failure to hydrate exocrine secretions adequately. The most common mutation, ΔF508, has the primary consequence of preventing biosynthetic maturation and transport beyond the ER. In this session we will discuss the molecular basis of CF and the role of ER-associated degradation in this disease. |
