The highly complex process of DNA replication is susceptible to a large number of stressors that can lead to stalling, and ultimately collapse, of the replication fork. The cell has multiple pathways to repair stalled forks, enabling DNA synthesis to proceed, but failure to achieve this goal leads to genomic damage and potentially cell death. Thus, gaining an understanding of the mechanisms of DNA damage repair and replication fork stabilization is critically important. Recently, Vanderbilt Basic Sciences investigator David Cortez and his laboratory conducted a screen designed to discover proteins that are enriched at stalled replication forks. That screen identified 72 proteins, many, but not all of which were known to play a role in the DNA damage response. One previously uncharacterized protein, ETAA1, caught the investigator’s interest, leading them to work in collaboration with Walter Chazin and his laboratory to explore its function. Their research revealed that ETAA1 interacts with replication protein A (RPA), a protein known to bind to single-stranded DNA that is exposed during replication stress. They also showed that ETAA1 directly activates ATR, a kinase that participates in the DNA damage response. Their findings further show that ETAA1-mediated ATR activation occurs through a pathway that is distinct from the previously described ATR activation pathway, and that it results in phosphorylation of targets that are different from those previously identified. The findings open a new door to the understanding of the response to replication stress and, given the known association of ETAA1 with Ewing’s sarcoma, the research may also provide new insights into the role of ATR activation in the pathogenesis of some kinds of cancer. The work is published in the journal Nature Cell Biology [T. E. Bass, et al, (2016) Nat. Cell Biol., published online October 10, doi:10.1038/ncb3415].
Type 1 diabetes occurs when an abnormal autoimmune response destroys the β cells, a specialized group of insulin-producing cells, in the pancreas. Insulin is required to promote proper uptake and utilization of glucose, and its absence in Type 1 diabetes leads to the characteristic high blood sugar along with a range of metabolic derangements and eventually tissue damage. Current treatment of Type 1 diabetes focuses on replacing insulin by injection, but a much more satisfactory result would be achieved if it were possible to replace the missing β cells. Now, Vanderbilt Basic Sciences researchers Mark Magnuson and Guoqiang Gu and their laboratories report progress toward that goal. They created genetically altered mice that express three transcription factors known to promote β cell differentiation in the pancreas. They discovered that high expression of these three factors led to inflammation, and although many cells exhibited altered morphology and protein expression, no insulin-producing cells developed. However, when the researchers took care to prevent the inflammation, either by reducing the expression of the transcription factors or treating the mice to reduce the number of inflammatory macrophages, new cells with the characteristics of β cells developed. In addition, diabetic mice treated this way displayed a temporary improvement in their condition for as long as the transcription factors were expressed. The findings provide important insights into parameters that affect β cell reprogramming in the pancreas and help to lay the groundwork for better approaches to treatment of Type 1 diabetes in the future. The work is published in the journal Cell Reports [H. W. Clayton, et al. (2016) Cell Rep., 17, 2028].
Cystic fibrosis is a genetic disorder associated with recurrent lung infections, poor digestion, stunted growth, and a shortened life expectancy. It is caused by mutation of the gene that encodes the cystic fibrosis transmembrane conductance regulator protein (CFTR), which transports chloride ion across the apical membranes of epithelial cells, particularly in the respiratory and digestive tracts. A well-established but poorly understood clinical observation about cystic fibrosis is that female patients often suffer more severe disease than males. To better understand this phenomenon, Vanderbilt Basic Sciences investigator Chuck Sandersand his collaborators Carlos Vanoye (Northwestern University) and Wade Van Horn (Arizona State University) investigated the structure and function of KCNE3, a potassium channel modulating protein that is required for normal chloride ion transport. KCNE3 binds to the KCNQ1 potassium channel protein to form a dimeric channel that is permanently open. This enables a constant slow leak of potassium ions out of the cell that are then transported back into the with chloride ions. The investigators’ work revealed the three-dimensional structure of KCNE3, allowing them to predict precisely how it interacts with KCNQ1 to promote channel opening. The results also revealed how an estrogen-dependent phosphorylation of a key KCNE3 residue disrupts its interaction with KCNQ1. The latter finding helps to explain why cystic fibrosis is frequently more severe in female patients. Estrogen promotes a post-translational modification of KCNE3 that prevents its ability to maintain an open state of KCNQ1. This effect apparently does not cause problems in healthy individuals, but in cystic fibrosis patients who already suffer from impaired chloride transport, a reduction in the necessary potassium ion-based support mechanism can cause a significant exacerbation of the pathology. The findings provide important fundamental information concerning the function of key ion transport proteins and may lead to new approaches for the treatment of cystic fibrosis. The work is published in the journal Science Advances [B. M. Kroncke, et al. (2016), Sci. Adv., 2, e1501228].