subunits of hemoglobin in the presence and absence of heme. 1A. Describe the locations in the gradient of the mRNAs for the α and β subunits of hemoglobin in the presence and absence of heme. near the top of the gradient - corresponds to the location of free ribosomes and ribonucleoproteins. This RNA is not being translated in the middle and near the bottom of the gradient – corresponds to polyribosomes active in translation In the absence of heme, the majority of the globin mRNAs are at the top of the gradient in the untranslated fraction. In the presence of heme, the majority of the globin mRNAs are associated with the polyribosome peak, which is strong evidence they are being translated.   No Heme Heme RNAs

1B. Which step in protein synthesis is affected by the absence of heme, initiation or elongation? Initiation. When initiation is slow relative to elongation, the few ribosomes that initiate protein synthesis will have time to run off the message before new ribosomes can re-initiate protein synthesis. When elongation is slow relative to initiation, the message is able to accumulate the slower moving ribosomes. There are two explanations for the results. The absence of heme either increased elongation or decreased initiation. Since overall protein synthesis is inhibited, the second explanation must be correct.  

1C. Describe the likely mechanism by which the absence of heme leads to an inhibition of the rate of protein synthesis   Heme inhibits a protein kinase that phosphorylates eIF-2. In the absence of heme, eIF-2 becomes phosphorylated and forms a stable complex with eIF-2B. eIF-2B is a guanine nucleotide exchange factor that facilitates the activation of eIF-2 by exchanging the bound GDP with GTP. eIF-2B is limiting. When eIF-2B is sequestered in a stable complex with phosphorylated eIF-2, it is unavailable to recycle the remaining eIF-2, and translation initiation is blocked

2A. Describe the effect of the loss of one good copy of the CHIP gene on animals that express the mutant huntingtin (HD) gene Removing one good copy of CHIP appears to have no effect on the appearance of the normal mouse (wt in Panels B and C), but leads to an earlier onset of the disease in mice expressing abnormal huntingtin and significantly reduces their life span (Panel C).    

2B. Suggest a role for the proteasome and CHIP in Huntington disease . The proteasome and CHIP participate in the removal of the mutant huntingtin protein, thereby delaying the onset of HD. Factors that reduce the ability of the proteasome to lower the levels of mutant huntingtin (e.g. proteasome inhibitors) or that block the ability of CHIP to tag the mutant huntingtin with polyubiquitin, would decrease the length of the disease-free interval and cause an earlier age of death.    

3A. Briefly describe how the transcriptional regulatory activity of NF-κB is regulated - include a description of IκB and its regulation .    NF-κB is a transcription factor that is regulated by its inhibitor, IκB. IκB in turn is regulated by ubiquitination and proteasome degradation. Phosphorylation of IκB by a protein kinase linked to a growth factor receptor initiates the ubiquitination of IκB by the ubiquitination conjugation system. Inhibitors of IκB degradation in turn inhibit NF-κB, which in turn blocks transcription of pro-survival genes that contain an NF-κB response element.

3B. Describe the results in Figure 1 and provide an explanation of the effect of PS-341 on the levels of the phosphorylated and nonphosphorylated forms of IκB and ubiquitinated cellular proteins. What is the purpose of including vinculin in the analysis?   In Figure 1, the proteasome inhibitor, PS-341, blocks the degradation of IκBα, leading to the accumulation of both phosphorylated and nonphosphorylated forms of IκBα. Levels of ubiquitinated proteins also increase, since they cannot be degraded by the proteasome. The control protein vinculin, which is not regulated by ubiquitination and proteasome degradation, shows no change in response to the proteasome inhibitor. Vinculin is a negative control in this experiment.

3C. In an experiment not shown here, it was found that bortezomib decreased the amount of NF-κB in cells that were bound to regulatory (promoter) regions of genes. Please explain   Bortezomib blocked the degradation of IκB. As a result NF-κB remained Bound to IκB in the cytosol and was unavailable to activate gene expression in the nucleus.

3D. Describe the results in Figures 3A and 3B 3D. Describe the results in Figures 3A and 3B. Explain the ability of the bortezomib and PS-341 to inhibit tumor growth and sensitize tumor cells to an apoptotic inducer in mice transplanted with human ALT leukemic cells.   Figure 3A shows that PS-341 sensitizes the leukemia cells to killing by HAT presumably by decreasing the expression of pro-survival genes activated by NF-κB. Figure 3B shows that bortezomib inhibits the tumor growth in mice implanted, presumably by promoting apoptosis by preventing NF-κB mediated transcription of pro-survival genes. HAT is an antibody to the IL-2 receptor and induces apoptosis in leukemia cells. Figure 3A. Kaplan-Meier survival plot of MET-1 NOD/SCID mice. HAT, alone or combined with PS-341, significantly prolonged the life span of mice compared with control mice. Figure 3B. Antitumor efficacy of bortezomib against ATL-derived cell line xenografts in SCID mice.

3E. What additional studies might be needed to determine whether proteasome inhibitors should be used to treat human ATL?   Initial toxicity studies in humans (phase I clinical trial) to determine the maximal tolerable dose of drug that can be safely administered in humans. Studies of drug efficacy against human cancer that demonstrate proteasome inhibitors are significantly more effective than current drugs or that demonstrate proteasome inhibitors enhance the efficacy of other commonly used drugs when given together.

4A. Describe the results in Figure 3, and indicate how these results could be used to predict life expectancy for any new patients with HCC The results suggest HCC patient outcomes may be predicted based on which miRNA expression pattern their tumor displays – one like the tumors under the left blue triangle or one like the tumors under the right triangle. More than 2/3 of the informative miRNAs (those miRNAs whose expression differs between the two groups) are decreased in the high-risk group compared to the low risk group. As a result the expression of their target genes should be elevated. If some of the protein products of these genes can be identified, they could be targets for drug development.   Low expression High expression High risk Low risk

4B. Are oncogenes or tumor suppressor genes more likely to be the target of miRNAs over-expressed in tumors from patients with a poorer prognosis? Which category of genes is likely to be the target of miRNAs whose expression is reduced in the more aggressive tumors?   miRNAs targeting tumor suppressor proteins would be increased in tumors from patients with a poor prognosis. miRNAs targeting oncogenes would be decreased in tumors from patients with a poor prognosis.

4C. If only the seed sequence and not the majority of the miRNA show perfect complementarity to the target, what is the expected outcome?   Degradation of the mRNA would be relatively slow. Translation of the mRNA would be inhibited, they would accumulate in P-bodies and eventually be degraded 4D. Had the miRNAs shown much greater complementarity over the majority of its length toward the target mRNA (near perfect complementarity across its entire length), what would have been the expected outcome? Rapid degradation of the mRNA. Endonucleolytic cleavage of the target mRNA by the RISC complex, followed by a rapid degradation of the cleavage fragments by exonucleases acting on their unprotected ends.