Details

Autor(en) / Beteiligte

• Farokhnia, Roxana
• Olivera, Maisa C.
• Okwuone, Dakota D.D.
• Jensen, Madeline K.
• Edgar, Jennifer M.
• Wise, John G.
• Vogel, Pia D.

Titel

Reconstituting human P‐glycoprotein (MDR1) into membrane nanodiscs

Ist Teil von

• The FASEB journal, 2019-04, Vol.33 (S1), p.782.9-782.9

Ort / Verlag

The Federation of American Societies for Experimental Biology

Erscheinungsjahr

2019

Quelle

Access via Wiley Online Library

Beschreibungen/Notizen

• P‐glycoprotein (P‐gp) is a multidrug resistance protein found in the membrane of many cells. As an ATP powered pump, P‐gp exports toxins and other hydrophobic compounds outward across the plasma membrane. This trait becomes problematic when overexpression of P‐gp occurs in cancer cells and causes chemotherapeutics to be pumped out of the cancer cells, rendering therapy ineffective. For biochemical and/or biophysical assessment of P‐glycoprotein, the protein is removed from the membrane by detergents and purified into detergent micelles using affinity chromatography. Although P‐gp is active when dissolved into micelles, the detergent environment has been shown to affect its biological activity. To avoid this issue, native‐like membrane nanodiscs have been introduced and used successfully over the last several years. Nanodiscs typically contain the P‐glycoprotein surrounded by lipids and enclosed within membrane scaffold proteins (MSP) which create an amphipathic belt around the membrane disc for water solubility. This more native‐like environment within the nanodiscs allows access to both the nucleotide and drug binding domains of the inserted proteins. In order to effectively assemble proteins into nanodiscs, specific ratios of protein to MSP to lipid are needed. In our lab, different ratios of these components have been used to optimize the assembly of the mouse cysteine‐less variant of P‐gp (MDR3CL) into nanodiscs. For the human P‐gp (MDR1) preliminary assembly successes were achieved at a 1:10:500 ratio (P‐gp: MSP: phosphatidylcholine). However, the yield of P‐gp assembled into nanodiscs was very low (about 30%). Here we report our efforts to optimize the nanodisc assembly with the goal of increasing P‐gp reconstitution yield and specific ATP hydrolysis activity. We will use the nanodisc reconstituted P‐glycoprotein in our efforts to investigate efficacy and mechanism of inhibition of novel P‐glycoprotein inhibitors discovered in our lab. Support or Funding Information This work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work. This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.