CHARACTERIZATION OF BCL-2 INTERACTING PARTNERS AT THE ENDOPLASMIC RETICULUM

Abstract

<p>Cancer occurs when cells acquire a number of mutations that trigger uncontrolled cell growth. The normal cellular response to this dysregulation of growth is the activation of programmed cell death. While focus in cancer research has been mainly concentrated in the mechanism of programmed cell death at the mitochondria, endoplasmic reticulum is slowly emerging as an essential platform for this regulatory mechanism.</p> <p>Bcl-2 is the founding member of the Bcl-2 family of protein, which contributes to the regulation of cell death at the mitochondria and at the endoplasmic reticulum. Previously in our lab, we have shown using MCF-7 cells stably expressing Bcl-2 targeted to the endoplasmic reticulum; they were protected from estrogen deprivation induced cell death. Thus the regulatory mechanism of Bcl-2 at the endoplasmic reticulum represents an interesting avenue to improve current cancer therapeutics.</p> <p>Two approaches were utilized to identify and characterize Bcl-2 and its interacting partners at the endoplasmic reticulum. Using an affinity tag fused to Bcl-2 that has been engineered to target the endoplasmic reticulum, tandem affinity purification was utilized to identify novel Bcl-2 interacting partners when estrogen receptor positive cells are treated with estrogen deprivation. Using fluorescent protein fused to the proteins of interest, Fluorescent Lifetime Imaging Measurement (FLIM) was used to characterize the interactions of Bcl-2 and its known interacting partner at the endoplasmic reticulum. The findings of this thesis verify the applications of the two aforementioned methods in the study of Bcl-2 interacting proteins at the endoplasmic reticulum.</p>