2. Why this research? Proteasome activity before now measured with isolated 20S subunit – no 19S involvement Proteasome activity before now measured with isolated 20S subunit – no 19S involvement May not accurately determine activity of intact 26S proteasome, especially inside a tumor or target tissue May not accurately determine activity of intact 26S proteasome, especially inside a tumor or target tissue A mutant multi-ubiquitin tagging system had already been developed (tagged to B- lactamase) A mutant multi-ubiquitin tagging system had already been developed (tagged to B- lactamase)

3. Methodology Created construct of luciferase gene and 4 mutant ubiquitins all with a cytomegalovirus promoter Created construct of luciferase gene and 4 mutant ubiquitins all with a cytomegalovirus promoter Transiently and permanently transfected construct into HeLa cells for in vitro work Transiently and permanently transfected construct into HeLa cells for in vitro work In vivo tumors created by injecting these transfectants subcutaneously in nude mice In vivo tumors created by injecting these transfectants subcutaneously in nude mice

4. Luciferase

5. HeLa cells = HeLa cool Cervical cancer cells taken from Henrietta Lacks fifty years ago at Hopkins Cervical cancer cells taken from Henrietta Lacks fifty years ago at Hopkins “cancer in a test tube” would create a cure “cancer in a test tube” would create a cure First cells to survive indefinitely outside the body – Gey calls them “immortal” First cells to survive indefinitely outside the body – Gey calls them “immortal” 1 st use was to develop vaccine that wiped out polio 1 st use was to develop vaccine that wiped out polio 1974 “nasty rumor” of Walter Nelson-Rees; only one solution 1974 “nasty rumor” of Walter Nelson-Rees; only one solution Cells combined weight far exceeds that of her original body Cells combined weight far exceeds that of her original body Have been in space Have been in space www.jhu.edu/~jhumag/0400web/01.html www.jhu.edu/~jhumag/0400web/01.html

6. Figure 1 MG132 has no effect on FL activity; large effect on Ub-FL MG132 has no effect on FL activity; large effect on Ub-FL Time delay (increase at 6h, equiv at 8h) Time delay (increase at 6h, equiv at 8h) Transient transfection (a); stable (b) Transient transfection (a); stable (b) Cyclohexamide added in b to inhibit protein synthesis (Ub-FL gets quickly degraded) Cyclohexamide added in b to inhibit protein synthesis (Ub-FL gets quickly degraded)

7. MG132lactacystin bortezomib MG132 lactacystin bortezomib [ ] Dependence Time Dependence

8. Figure 4

9. Figure 5

10. Future Directions and Questions In vitro high-throughput screening and in vivo testing of promising leads In vitro high-throughput screening and in vivo testing of promising leads Unanswered questions: Unanswered questions: Why is proteasome inhibition by this compound so slow, and why does the Ub-FL trail it so significantly? Why is proteasome inhibition by this compound so slow, and why does the Ub-FL trail it so significantly? How does proteasome inhibition affect translation? How does proteasome inhibition affect translation? How quickly does bortezomib work to inhibit the proteasome and what does this mean about its mechanism of action? How quickly does bortezomib work to inhibit the proteasome and what does this mean about its mechanism of action?