Why this research?Why this research?

Proteasome activity before now measured with Proteasome activity before now measured with isolated 20S subunit – no 19S involvementisolated 20S subunit – no 19S involvement

May not accurately determine activity of intact May not accurately determine activity of intact 26S proteasome, especially inside a tumor or 26S proteasome, especially inside a tumor or target tissuetarget tissue

A mutant multi-ubiquitin tagging system had A mutant multi-ubiquitin tagging system had already been developed (tagged to B-already been developed (tagged to B-lactamase)lactamase)

MethodologyMethodology

Created construct of luciferase gene and 4 Created construct of luciferase gene and 4 mutant ubiquitins all with a cytomegalovirus mutant ubiquitins all with a cytomegalovirus promoterpromoter

Transiently and permanently transfected Transiently and permanently transfected construct into HeLa cells for in vitro workconstruct into HeLa cells for in vitro work

In vivo tumors created by injecting these In vivo tumors created by injecting these transfectants subcutaneously in nude micetransfectants subcutaneously in nude mice

LuciferaseLuciferase

HeLa cells = HeLa coolHeLa cells = HeLa cool Cervical cancer cells taken from Henrietta Lacks fifty years Cervical cancer cells taken from Henrietta Lacks fifty years

ago at Hopkinsago at Hopkins ““cancer in a test tube” would create a curecancer in a test tube” would create a cure First cells to survive indefinitely outside the body – Gey calls First cells to survive indefinitely outside the body – Gey calls

them “immortal”them “immortal” 11stst use was to develop vaccine that wiped out polio use was to develop vaccine that wiped out polio 1974 “nasty rumor” of Walter Nelson-Rees; only one solution1974 “nasty rumor” of Walter Nelson-Rees; only one solution Cells combined weight far exceeds that of her original bodyCells combined weight far exceeds that of her original body Have been in spaceHave been in space www.jhu.edu/~jhumag/0400web/01.htmlwww.jhu.edu/~jhumag/0400web/01.html

Figure 1Figure 1

MG132 has no effect on FL activity; large effect on Ub-FLMG132 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 Cyclohexamide added in b to inhibit protein synthesis (Ub-FL

gets quickly degraded)gets quickly degraded)

MG132 lactacystin

bortezomib

MG132 lactacystin

bortezomib

[ ] Dependence

Time Dependence

Figure 4Figure 4

Figure 5Figure 5

Future Directions and QuestionsFuture Directions and Questions

In vitro high-throughput screening and in vivo In vitro high-throughput screening and in vivo testing of promising leadstesting of promising leads

Unanswered questions:Unanswered questions: Why is proteasome inhibition by this compound so Why is proteasome inhibition by this compound so

slow, and why does the Ub-FL trail it so slow, and why does the Ub-FL trail it so significantly?significantly?

How does proteasome inhibition affect translation?How does proteasome inhibition affect translation? How quickly does bortezomib work to inhibit the How quickly does bortezomib work to inhibit the

proteasome and what does this mean about its proteasome and what does this mean about its mechanism of action? mechanism of action?