Assessing Cysteine Residue Thiol Status of t‐DARPP, A Protein Involved in Chemoresistance

Abstract

The gene PPP1R1B expresses t‐Darpp, a protein that activates protein kinase A and the AKT pathway in breast cancer cells. t‐Darpp, when overexpressed in breast cancer cells, also confers resistance to trastuzumab, a therapeutic that binds to the Her2 receptor. Although t‐Darpp expression is relatively high in gastric, breast and colon cancers, its structure is unknown. Human t‐Darpp has two cysteine residues that can be exploited to explore t‐Darpp structure, Cys36 and Cys119. Methoxypolyethylene glycol‐maleimide (Mal‐PEG) was used to determine whether recombinant t‐Darpp cysteine thiols are on the surface of the protein and to find out if they are reversibly oxidized. Later, a quantitative analysis was conducted using Ellman's test. 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (Ellman's reagent) was used to determine whether recombinant t‐Darpp cysteine thiols are free or not. Both studies suggest that the two cysteine thiols are either buried or oxidized. Further work will be conducted to distinguish between these two possibilities.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.