Presentation Title

Incorporation of Antioxidant Luteolin into ApoE3NT and ApoAI containing HDL Nanodiscs

Faculty Mentor

Vasanthy Narayanaswami

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:00 AM

Location

Markstein 102

Session

oral 1

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

High-density lipoprotein (HDL) is a large spherical protein-lipid complex that is composed of proteins, such as apolipoprotein (apo) AI and apoE3, several molecules of lipids, and cholesterol. ApoE3 can bind to low-density lipoprotein receptors that facilitate cellular uptake of lipoproteins via receptor-mediated endocytosis. Other ways of entry for lipoproteins exist, such as via the selective influx pathway that involves scavenger receptor B1 (SR-B1). We propose to use these features of apolipoproteins to deliver the antioxidant luteolin (3′,4′,5,7-tetrahydroxyflavone) into breast cancer cells (MDA-MB-231). The overall goal is to incorporate luteolin into reconstituted high-density lipoproteins (rHDL). The objectives of the current study are to: (i) overexpress and purify recombinant human apoE3 encompassing residues 1-191 of the N-terminal domain (apoE3NT) and apoAI, bearing a His-tag; (ii) reconstitute HDL in the presence of phospholipids, protein, and luteolin; (iii) perform biophysical and biochemical characterization of rHDL. Overexpression of proteins was accomplished using E. coli BL21-Gold (DE3) pLysS cells and proteins purified by affinity chromatography using a Nickel Hi-Trap chelating column. SDS-PAGE analysis revealed ~95% pure proteins. They were mixed with one of the following phospholipids: POPC, DMPC, DMPE, or DMPG in the absence or presence of luteolin and sodium deoxycholate and dialyzed to obtain rHDL. The samples were ultracentrifuged and fractionated; protein-containing fractions pooled. Non-denaturing polyacrylamide gel electrophoresis (PAGE) confirmed formation of high molecular mass complexes (~600 kDa). Future studies will include utilizing HPLC to determine the efficiency of incorporation of luteolin into rHDL and assessing the effect of phospholipid head group variation on the incorporation. The effect of the presence of luteolin on cellular uptake of rHDL and its antioxidant activity against oxidative stress will be assessed in MDA-MB-231 cells. Successful completion of this study will aid in developing nanodiscs as a viable drug delivery vehicle for other hydrophobic drugs such as chemotherapeutic agents.

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Nov 23rd, 8:45 AM Nov 23rd, 9:00 AM

Incorporation of Antioxidant Luteolin into ApoE3NT and ApoAI containing HDL Nanodiscs

Markstein 102

High-density lipoprotein (HDL) is a large spherical protein-lipid complex that is composed of proteins, such as apolipoprotein (apo) AI and apoE3, several molecules of lipids, and cholesterol. ApoE3 can bind to low-density lipoprotein receptors that facilitate cellular uptake of lipoproteins via receptor-mediated endocytosis. Other ways of entry for lipoproteins exist, such as via the selective influx pathway that involves scavenger receptor B1 (SR-B1). We propose to use these features of apolipoproteins to deliver the antioxidant luteolin (3′,4′,5,7-tetrahydroxyflavone) into breast cancer cells (MDA-MB-231). The overall goal is to incorporate luteolin into reconstituted high-density lipoproteins (rHDL). The objectives of the current study are to: (i) overexpress and purify recombinant human apoE3 encompassing residues 1-191 of the N-terminal domain (apoE3NT) and apoAI, bearing a His-tag; (ii) reconstitute HDL in the presence of phospholipids, protein, and luteolin; (iii) perform biophysical and biochemical characterization of rHDL. Overexpression of proteins was accomplished using E. coli BL21-Gold (DE3) pLysS cells and proteins purified by affinity chromatography using a Nickel Hi-Trap chelating column. SDS-PAGE analysis revealed ~95% pure proteins. They were mixed with one of the following phospholipids: POPC, DMPC, DMPE, or DMPG in the absence or presence of luteolin and sodium deoxycholate and dialyzed to obtain rHDL. The samples were ultracentrifuged and fractionated; protein-containing fractions pooled. Non-denaturing polyacrylamide gel electrophoresis (PAGE) confirmed formation of high molecular mass complexes (~600 kDa). Future studies will include utilizing HPLC to determine the efficiency of incorporation of luteolin into rHDL and assessing the effect of phospholipid head group variation on the incorporation. The effect of the presence of luteolin on cellular uptake of rHDL and its antioxidant activity against oxidative stress will be assessed in MDA-MB-231 cells. Successful completion of this study will aid in developing nanodiscs as a viable drug delivery vehicle for other hydrophobic drugs such as chemotherapeutic agents.