Discovering the Secrets of a Cancer Drug Resistance Protein

March 21, 2017

One way that many forms of cancer evade the toxic effects of chemotherapeutic drugs is through expression of the P-glycoprotein. This membrane protein uses energy from the hydrolysis of ATP to transport over 200 structurally distinct compounds, including many drugs, out of the cell. Because of its widespread expression in many tissues of the body the P-glycoprotein also affects the distribution and excretion of a large number of other drugs and toxicants. Consequently, for over 40 years, researchers have attempted to understand the structure and function of the P-glycoprotein in the hope of discovering new ways to productively modulate its activity. Now, Vanderbilt Basic Sciences investigator Hassane Mchaourab, his collaborators Robert Nakamoto (University of Virginia) and Emad Tajkhorshid (University of Illinois at Urbana-Champaign), and their laboratories have learned how the P-glycoprotein harnesses the energy from ATP hydrolysis to propel a substrate molecule across the cell membrane. The Mchaourab laboratory uses a technique called double electron-electron resonance (DEER) to measure the distance between two previously labeled sites on a protein. DEER enables the researchers to monitor changes in this distances that occur as the protein changes from one functional state to another. They discovered that the biggest change in P-glycoprotein structure occurs right at the time of ATP hydrolysis, and that the two sites on the protein where ATP hydrolysis occurs function asymmetrically. These, along with other data, enabled them to use computational methods to construct a model of the protein’s structure just following substrate transport. They were also able to propose a mechanism that describes how ATP hydrolysis leads to a series of structural changes in the protein that result in substrate transport. This important discovery provides new insight into the functioning of P-glycoprotein that may lead to future success in preventing cancer drug resistance. The work is published in the journal Nature [B. Verhalen, R Dastvan, et al., (2017) Nature, published online March 16, 2017, DOI:10.1038/nature21414].

Electric Charge Transfer-Based Control of DNA Replication

March 7, 2017

DNA replication actually starts with the synthesis of a short stretch of RNA by the enzyme DNA primase. Extension of the RNA with a similarly short stretch of DNA by DNA polymerase α (pol α) follows before the more efficient and highly processive DNA polymerases take over to finish the job. The mechanisms that regulate the initiation and termination of the RNA primer are poorly understood, but new insight now comes from the work of Vanderbilt Basic Sciences investigator Walter Chazin, his collaborator Jacqueline Barton (California Institute of Techology), and their laboratories. The Chazin and Barton labs focused on an interesting property of DNA – its ability to transfer an electric charge for a long distance along the duplex. They noted that DNA primase, pol α, and a number of other DNA-processing enzymes contain a [4Fe4S] cluster that is readily oxidized and reduced. They hypothesized that charge transfers along DNA could serve as a mechanism for signaling between bound [4Fe4S] cluster-containing proteins, and that this signaling could regulate DNA processing activity. To test their hypothesis, they used a DNA-coated gold electrode to show that the [4Fe4S] cluster of DNA primase is susceptible to direct oxidation and reduction by charge transfer through the DNA duplex. They identified three tyrosine residues that form a charge transfer network between the DNA binding site and the [4Fe4S] cluster of DNA primase, and they showed that interruption of this network led to defects in primer initiation and termination. These findings support the hypothesis that DNA charge transfer, in conjunction with redox centers located within DNA processing enzymes, plays a key role in the regulation of enzyme activity. Further work will seek to define the scope of this interesting new mechanism of enzyme modulation. The work is published in the journal Science [E. O’Brien et al., (2017) Science, 355, eaag1789].

Unlocking the Mysteries of a Deadly Form of Lung Cancer

January 4, 2017

Small cell lung cancer (SCLC) is a highly aggressive, particularly deadly form of cancer noted for its early metastasis and resistance to therapy. One explanation for these traits is that SCLC is usually composed of heterogeneous cell populations, most often including cells with neuroendocrine (NE) traits and those with mesenchymal-like (ML) traits. These distinct populations are not associated with specific genetic mutations as are frequently seen in other forms of cancer, and their origin is not fully understood. Now, however, Vanderbilt Basic Sciences researchers Vito Quaranta and Jonathan Irish and their laboratories report the results of gene expression studies using data from 53 SCLC cell lines and 28 patient tumors. Their results confirmed that most tumors and cell lines comprised two major cell types, NE and ML, and they were able to identify a set of 33 transcription factors that were differentially activated in the two primary phenotypes. Careful examination of each of the cell lines and tumors revealed, however, that not all of them fit into the NE or ML classification. Single cell flow cytometry of these samples demonstrated that the cells were a hybrid between the NE and ML phenotypes. Further studies showed that treatment of SCLC with standard forms of chemotherapy resulted in a shift of the cells towards a hybrid phenotype. The researchers concluded that the heterogeneity observed in SCLC is the result of the level of activation or inactivation of 33 key transcription factors and that changes in the activity of those factors in response to stress, including therapy, can lead to adaptations that result in cell survival and drug resistance. These insights will be valuable in designing new approaches to SCLC treatment in the future. The work is published in the journal Cancer Research [A. R. Udyavar, et al. (2016) Cancer Res., published online December 8, DOI: 10.1158/0008-5472.CAN-16-1467].