Author

Wen LiFollow

Date of Award

12-18-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Parjit Kaur

Second Advisor

Phang C.Tai

Third Advisor

Casonya Johnson

Abstract

The soil bacterium Streptomyces peucetius produces two widely used anticancer antibiotics doxorubicin and daunorubicin. Present within the biosynthesis gene cluster in S. peucetius is the drrAB operon which codes for a dedicated ATP-binding cassette type transporter for the export of these two closely related antibiotics. DrrAB system was believed to be the single-drug transporter due to its dedicated nature; however, our study demonstrated under both in vivo and in vitro conditions that DrrAB system can transport not only doxorubicin but also Hoechst 33342 and ethidium bromide. Moreover, many other well-studied multi-drug resistance proteins substrates (including verapamil, vinblastine and rifampicin) inhibit DrrAB-mediated doxorubicin transport, indicating they are also substrates of DrrAB pump. Kinetic studies show competitive inhibition of doxorubicin transport by Hoechst 33342 and rifampicin and non-competitive inhibition by verapamil, suggesting the possibility of more than one drug binding site in the DrrAB system. This is the first in-depth study of a drug resistance system from a producer organism, and it shows that a dedicated efflux system like DrrAB contains specificity for multiple drugs.

Our study also provides the first direct evidence for the dual role of the metalloprotease FtsH in the biogenesis of membrane proteins. We found that FtsH is not only responsible for proteolysis of unassembled DrrB protein but it also plays a much broader role in biogenesis of the DrrAB complex. DrrA and DrrB proteins expressed together in a temperature sensitive ftsH mutant strain were found to be non-functional due to their incorrect assembly. Simultaneous expression of wild-type FtsH in trans resulted in normal doxorubicin efflux. Strikingly, doxorubicin efflux could be restored in mutant cells irrespective of whether FtsH was expressed simultaneously with DrrAB or expressed after these proteins had already accumulated in an inactive conformation, thus providing crucial evidence for the ability of FtsH to refold the misassembled proteins. Complementation experiments also showed that the catalytic ATP binding domain of FtsH contains a chaperone-like activity, however both the catalytic and the proteolytic domains of FtsH are required to be present and work coordinately to participate in biogenesis of DrrAB complex in the membrane.

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