Deciphering the “chaperone code”
Hsp70 is a universally conserved molecular chaperone that performs a wide variety of functions in the cell including folding of both newly synthesized and denatured protein “clients”, protein transport and disaggregation of oligomerized proteins. Although transcriptional regulation of Hsp70 has been highly studied, little is known about post-translational modifications on Hsp70 (known as the “Chaperone Code”) and their effects on in vivo function.
Our central hypothesis is that Hsp70 phosphorylation regulates co-chaperone and client protein interactions and may be altered in cancer cells resulting in increased oncoprotein stability.
1. Understanding the role of Hsp70 phosphorylation in cancer
Eukaryotes coordinate cell cycle progression with environmental cues to maintain proper growth and proliferation, and misregulation of this process is associated with the emergence of cancer. The G1 cyclin, Cyclin D1 forms a complex with CDK4 to phosphorylate important cell cycle proteins such as Rb to drive G1/S progression. As such, down regulation of Cyclin D1 function may offer a way to curb uncontrolled cell cycle progression seen in cancer.
We have previously demonstrated that CDK-dependent phosphorylation of Hsp70 impacts cell cycle progression by altering binding and stability of Cyclin D1. Although several studies have identified key proteins involved in G1 cyclin stability, the exact mechanism that controls Cyclin D1 destruction in response to Hsp70 phosphorylation has yet to be determined. This work will provide a pathway to a novel form of anticancer therapeutic strategy whereby Hsp70 phosphorylation is altered to destabilize specific oncoproteins.
2. Regulation of ribonucleotide reductase by chaperones
Ribonucleotide reductase (RNR) is a key enzyme in dNTP synthesis. Inhibition of RNR results in stalled DNA replication and repair, making it a promising target for cancer intervention. Our results show a dependence of RNR activity on Hsp70 and Hsp90, with pre-treatment of cancer cells with chaperone inhibitors sensitizing breast cancer cells to RNR inhibitors. This offers the potential of a novel cancer therapeutic strategy that exploits the synergy of chaperone and ribonucleotide reductase inhibitors. We are currently interested in the co-chaperones involved in RNR regulation, the exact nature of the RNR-chaperone interaction and isolation of novel molecules that may disrupt this interaction for anticancer purposes.
3. Using CRISPR-CAS9 to study molecular chaperone function
The CRISPR/Cas system is a novel genome engineering technology originally repurposed from the bacterial Type II CRISPR system. Using CRISPR/Cas in mammalian cells, genes may be deleted, mutated or epitope tagged with relative ease. CRISPR-mediated DNA insertion into the genome is permanent and highly specific, minimizing the cell heterozygosity observed in traditional stable expression technologies that rely on random chromosomal integration.
In the Truman Lab we are currently using CRISPR to:
- manipulate chaperone/co-chaperone function
- epitope tag chaperones for proteomic studies
- randomly mutate chaperones in combination with anticancer drug screens.
If you are interested in collaborating with us on a CRISPR project, please email Dr. Andy Truman at: atruman1 at uncc.edu, we would be happy to help!
4. Using cross-linking mass spectrometry to understand Hsp70 interactions
Hsp70 interacts with a vast number of proteins in the cell. Standard techniques such as Co-immunoprecipitation and Yeast 2-hybrid cannot discriminate direct vs. indirect interactors. Cross-linking of Hsp70 complexes with an MS-cleavable linker allows not not only identification of novel Hsp70 interactors but is allowing us to determine the surface of interaction for all these proteins.