Answer Key: UPR, proteasome, and recycling

BIOL230W Week 6 – What happens when things go wrong? UPR, proteasome, and recycling. We have made it all the way through gene expression! But what happens if there is a problem and what happens to all the stuff that the cell doesn’t need anymore? This week we will be looking at how the cell gets rid of unwanted materials. Day 1 Learning Objectives 1. Predict the result if a cell was unable to degrade misfolded proteins or remove damaged organelles. 2. Outline the three main processes that occur in the ER to properly fold proteins and the components that are required for each. 3. Describe three reasons for a misfolded protein (Mistake in transcription or translation/mutation/global problem with cellular conditions) 4. Compare and contrast the three different fates of misfolded proteins. 5. Compare and contrast the role of Sec61 (a translocon) in the UPR to its role in translation. Mini-Lecture: Overview of Degradation and Recycling in the Cell Look for the answers to these questions as we discuss: 1. What are the two main mechanisms of degradation in the cell? Proteasome (collection of proteins that degrades proteins), and lysosomes (sacs filled with enzymes) 2. What might be the result if misfolded proteins are not degraded? Exposed hydrophobic regions/amino acids, cause interactions with hydrophobic amino acids to cause aggregates, protein may gain a new function 3. Disulfide bonds typically form between cysteine side chains of two amino acids. Free cysteines (i.e., not covalently bonded with each other) would indicate a problem at which level of hierarchical structure? Tertiary Small Group Activity: Protein Glycosylation One of the processes that must be carried out for the proper folding of a protein is the addition of sugars to particular amino acids in the protein chain. This is known glycosylation. as A variety of sugars can be added in complex patterns. Glycosylation results in a molecule that is larger than the unglycosylated protein. Figure 1 shows the result of a technique known as immunoprecipitation. In short, this process allows the researcher to isolate a particular molecule from the other components of a cell. Here, the researchers have immunoprecipitated the CFTR protein from cells expressing the wild type CFTR gene (left) and from cells expressing ∆F508 mutant CFTR gene (right) at 24, 48 and 72 hours after expression was initiated. The proteins are then run on an electrophoresis gel (the proteins were run from the top to the bottom as the gel is shown on the image) and detected using radioactive labeling. Figure 1. Immunoprecipitation of CFTR protein from Wild-type CFTR (Left) and ∆F508 CFTR (right) expressing cells. 4. Using what you know about gel electrophoresis, which band (B or C) is the glycosylated form? Band C 1 BIOL230W Week 6 – What happens when things go wrong? UPR, proteasome, and recycling. 5. Why is the wildtype “B” band getting fainter over time? There is less of the non-glycosylated form as glycosylation occurs 6. In the wild-type sample, how does the diminishing “B” band relate to the “C” band that is increasing in intensity? The proteins are being glycosylated so there will be fewer non-glycosylated proteins and more glycosylated proteins as time goes on. Challenge Question: This will be answered later in the week, but if you have time think about it now… 7. Even though we see a diminishing “B” band over time in the ∆F508 expressing cells, we do not see a concomitant appearance of a “C” band. Why is there less protein at 72 hours in the ∆F508 cells? There is no glycosylation occurring because the protein is not being sent to the proteasome. Mini-Lecture: Three sequential processes respond to accumulation of misfolded proteins Step 1. Refolding 8. What might be the reason for one or two misfolded proteins? Mistake in transcription or translation 9. What might be the reason for a massive build-up of misfolded proteins? A mutation (either in gene or in the protein) 10. What is the first thing a cell will try when misfolded proteins are detected? To refold the protein 11. What would be the specific defect in a misfolded protein if a cell had a mutation in the gene that codes for PDI? Disulfide bonds will be unable to be reformed 12. Would the cell in Q11 have a defect in one specific protein or a broad range of different proteins? A broad range of different proteins 13. What characteristic of a misfolded protein is BiP looking for to identify a misfolded protein for refolding? Exposed hydrophobic domains Day 2 Learning Objectives 2 BIOL230W Week 6 – What happens when things go wrong? UPR, proteasome, and recycling. 1. Describe the purpose of ERAD. 2. Contrast the conditions that initiate ERAD to those of protein refolding. 3. Contrast the four steps of protein degradation via ERAD with regard to location and major components. 4. Describe how the structure of the proteasome relates to its three functions. 5. Describe structure and function of ubiquitin as it relates to ERAD. 6. Draw the path of a misfolded protein from the ER to the proteasome. Mini-Lecture: Three sequential processes respond to accumulation of misfolded proteins Step 2. Endoplasmic Reticulum Associated Degradation (ERAD) Accumulation of Misfolded Proteins 14. What characteristics would indicate that a protein should enter the ERAD pathway? If protein refolding is unsuccessful 15. A misfolded protein needs to move from Endoplasmic Reticulum to Cytoplasm in order to be degraded by the proteasome? (Think about compartments in the cell) 16. What molecule facilitates transport of the misfolded protein between the compartments described in Q15? Sec61 (translocon) Ubiquitination of Misfolded Proteins 17. What type of macromolecule is ubiquitin? Protein 18. In which cellular location is a ubiquitin added to a misfolded protein that has entered the ERAD pathway? In the cytoplasm Degradation of Misfolded Proteins 19. Which type of macromolecule does the proteasome degrade? Proteins 20. Which type of organelle is the proteasome? Proteinaceous 21. What type of bond does the proteasome break? Peptide bonds Mini-Lecture: The Proteasome-Structure and Function 3 BIOL230W Week 6 – What happens when things go wrong? UPR, proteasome, and recycling. 22. Why is it important for the 19S subunit to unfold the protein? To allow it to feed through the proteasome (regulate entry to proteasome) 23. Which subunit is required for enzymatic cleavage of misfolded proteins? 20S (beta subunits) 24. Does the proteasome exhibit quaternary structure? Yes 25. Why when you add a 19S and a 20S subunit is the resulting protein complex only 26S? Study Activity: Draw the path a misfolded protein takes from the ER to the proteasome where it will be degraded.Draw and label the following components. ER membrane, ER lumen, Cytoplasm, Sec61, misfolded protein, Ubiquitin For at home review: Three sequential processes respond to misfolded proteins in the ER Look for the answers to these questions as you listen. It may help to refer to figure 2 as well. Figure 2. (A) Proteins of the endomembrane system are translocated to the ER co-translationally through ER channels called translocons (composed of sec61, signal-recognition particle (SRP) and other proteins). (B) In the oxidizing environment of the ER lumen, chaperones like BiP, calnexin and protein disulphide isomerase (PDI) facilitate the proper folding of the protein by preventing protein aggregation, monitoring glycosylation (glycans) and disulphide bond formation. (C) Once correctly folded and modified, the protein will exit the ER through transport vesicles.(D) If the ER quality-control system cannot repair a misfolded protein, it will be retrotranslocated to the cytosol and degraded by the proteasome. (E) Changes in the ER environment shift the balance from normal to improper folding (thicker arrow), leading to the accumulation of unfolded proteins. This 4 BIOL230W Week 6 – What happens when things go wrong? UPR, proteasome, and recycling. activates the unfolded protein response (UPR) via UPR transducers (ER integral membrane proteins that recognize an accumulation of misfolded proteins and initiate a signal to the nucleus). 26. What general events are happening between step “a” and “b”? 27. If glycosylation activity is reduced what will not be added to the mature protein? 28. Why is there a bi-directional arrow between “folded” and “malfolded (misfolded)” protein? 29. This diagram indicates that misfolded proteins have three possible fates, what are they? Day 3 Learning Objectives: 1. Describe the cellular conditions that result in the initiation of the UPR 2. Compare the three major cellular processes that can be modified by the Unfolded Protein Response (UPR) 3. Compare and contrast autophagy and proteasome mediated degradation (including initiation, targets, and location in the cell). 4. Analyze how the dynamic nature of mitochondria and autophagy helps protect a cell from damage. Mini lecture: If ERAD is not enough. The unfolded protein response (UPR) is the third and final cellular response to misfolded proteins in the ER. It is initiated by an accumulation of misfolded proteins in the ER and results two outcomes in based on cell signaling. Initiation of the UPR 30. Compare and contrast the two functions of BiP (What is similar about the two functions and what is different?). Binds to unfolded proteins, binds to transmembrane protein which turns on unfolded protein response. In one location it turns a UPR off, and in another one encourages the breakdown of a misfolded protein. 31. What type of problem might result in the activation of the UPR? When protein levels get very high, and draw BiP off of the membrane, which stops it from turning off UPR. Cellular responses 32. What are the three outcomes of the UPR?? Increase refolding, increase ERAD, and decrease the synthesis of unnecessary proteins 5 BIOL230W Week 6 – What happens when things go wrong? UPR, proteasome, and recycling. 33. What is the common goal of the three outcomes (from Q32)? Decrease the amount of unnecessary/misfolded proteins Mini-Lecture: Autophagy Small group discussion: Autophagy Components of a cell have a limited lifespan. Once proteins, organelles and other cellular molecules have served their purpose or the needs of the cell change, the large molecules are broken down and their parts are used to make more of what the cell needs. 34. Which degradative process (lysosomal or proteasome mediated) would recycle the following materials? a. Misfolded CFTR protein: Proteasome b. Damaged mitochondria: Lysosomal 35. Would a researcher expect to detect autophagy occurring in a normally functioning cell? No 36. Would a researcher expect to see more autophagy or less autophagy in a cell lacking a major energy source such as glucose? More autophagy 37. Why would increasing membrane production be an important part of inducing Autophagy? To provide the materials for a membrane to form around the damaged object End of Week 3 Summary A great technique to achieve deeper learning is to make connections between topics that may seem unrelated at first. Concept maps and flow charts are diagrams that you can make that help to accomplish this goal. Step 1. Think of a list of terms or concepts that make up a subject that you are interested in. We have already done this for you (see below). Step 2. Organize the words into small groups to which you think they would belong based on your understanding of the concepts or terms. Step 3. Draw lines or arrows that connect the groups or the individual terms or concepts when you think of a way that they are connected. Try labeling the connecting lines with what you see think connects them. Draw a concept map or a flow chart connecting the following terms. This could be a house point submission! 6 BIOL230W Week 6 – What happens when things go wrong? UPR, proteasome, and recycling. Proteasome, mutation, mistakes in transcription, disulfide bonds, starvation, translational inhibition, transcription factors, UPR, ER, BiP, protein, mitochondria, Sec61, miRNA, protein misfolding, ERAD, PDI, changes in gene expression, sugars. 7