Molecular chaperones are abundant and conserved proteins in the cytosol, mitochondria, nucleus and endoplasmic reticulum that help protein clients fold to their active conformation known as the native state. The structure and mechanism of these chaperones holds significance because of their association with cancer and metabolic disease. The heat shock protein 70 (Hsp70) family of chaperones includes the ATP-dependent BiP (Grp78) located in the endoplasmic reticulum. Its protein-folding activity is enhanced by cochaperones known as J proteins of the Hsp40 family. A J protein known as JB11 promotes protein folding by bringing clients to the substrate-binding domain of BiP and stimulating its ATP hydrolysis activity. We investigated the ATP hydrolysis activity of BiP and its stimulation by JB11 by constructing an ATP-binding (T229A) mutant of BiP and a BiP-binding (H53Q) mutant of JB11. After purifying wild-type and mutant BiP and JB11, we determine the rates of ATP hydrolysis of the chaperones using an enzyme-coupled ATPase assay. We found that the BiP mutant had a decreased rate of ATP hydrolysis compared to the wild-type. It remained defective with the enhancement of a client protein, L2, and nucleotide exchange factor, Grp170, demonstrating the importance of this N-terminal domain residue in ATP binding of BiP. The wild-type JB11 enhanced the ATP hydrolysis rate of BiP alone and in its chaperone system; the BiP-binding JB11 mutant was defective in its enhancement since it was not able to bind BiP. An understanding of the activities of these molecular chaperones supports research on pharmaceuticals that inhibit their activity in diseased cells, especially because of their upregulation and role as a biomarker in many cancers. This project is significant to my future career in medicine because it gives me the opportunity to understand the research behind therapeutics used to treat patients with serious illnesses such as cancer.
Author: Yasmeen Fleifil
Advisor: Andrea Kravats, Chemistry and Biochemistry
Graduate Advisor: Yaa Amankwah Sarfowah, Chemistry and Biochemistry
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