Why this research?

2. Why this research? • 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 • 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 • Transiently and permanently transfected construct into HeLa cells for in vitro work • 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 • “cancer in a test tube” would create a cure • First cells to survive indefinitely outside the body – Gey calls them “immortal” • 1st use was to develop vaccine that wiped out polio • 1974 “nasty rumor” of Walter Nelson-Rees; only one solution • Cells combined weight far exceeds that of her original body • Have been in space • www.jhu.edu/~jhumag/0400web/01.html

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

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

8. Figure 4

9. Figure 5

10. Future Directions and Questions • In vitro high-throughput screening and in vivo testing of promising leads • Unanswered questions: • 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 quickly does bortezomib work to inhibit the proteasome and what does this mean about its mechanism of action?