Evaluating the consequences of polynucleotide kinase/phosphatase (PNKP) mutations in neurological diseases
Creators: Jiang, Bingcheng
Publisher: name: University of Alberta Library
Year: 2022

<p>Genomic stability is extremely important for developing or maintaining normal neurological functions, as the nervous system is constantly suffering from endogenous DNA damage. To maintain such stability, living cells are protected by several different DNA repair pathways. Polynucleotide kinase/phosphatase (PNKP) is a bifunctional DNA repair enzyme that possesses both the DNA 3’-phosphatase and DNA 5’-kinase activities. It is involved in several different repair pathways including the single strand break repair and the non-homologous end joining pathways. Mutations in PNKP have been found to be responsible for different neurological diseases, including Microcephaly, seizures and developmental delay (MCSZ), and Ataxia-ocular motor apraxia 4 (AOA4). <br>
Our focus was directed towards three different PNKP mutations reported in clinical cases. The first two mutations were found in a 3-year-old male MCSZ patient with cerebellar glioblastoma. Genetic screening for mutations associated with his clinical features showed that he carried 2 germline point mutations in PNKP, which caused two different single amino acid alterations in the protein (P101L and T323M). This is the first report of human cancer found in a patient with MCSZ. While the latter mutation has recently been identified in an MCSZ patient, the P101L mutation has not been previously reported. We have investigated the consequences of these mutations at the biochemical and cellular levels. Biochemically, the P101L variant retains relatively robust DNA kinase and phosphatase activity, but the alteration at T323 significantly diminishes both enzymatic activities, especially the phosphatase activity. This is due in part to the reduced affinity for DNA substrates. Expressing the mutated proteins in HeLa PNKP-knockout cells revealed that the P101L PNKP variant localizes primarily to the cytoplasm rather than the nucleus. We established that this is the result of the creation of a novel nuclear export signal. An increase in cytoplasmic PNKP was also observed in tissue from the patient. Further analysis indicated that cells expressing P101L and T323M variants have a slower repair of radiation-induced DNA single strand breaks than cells reconstituted with the wild-type protein, and that the repair of radiation- induced double strand breaks is particularly slow in T323M-expressing cells. We also observed a significant increase in cellular transformation by the cells expressing the mutant proteins using the soft agar assay, which may reflect an increased propensity for oncogenic transformation. <br>
To expand the spectrum of PNKP mutations and understand the molecular basis of AOA4, we picked one of the most frequently identified PNKP mutations associated with AOA4, G1123T, which gives rise to a G375W change in the protein. In vitro kinase and phosphatase assays revealed that the G375W PNKP mutant lacks kinase but retains near normal phosphatase activity. Furthermore, our results indicate that the loss of kinase activity can be attributed to near elimination of ATP binding, while DNA binding affinity remained unchanged. Cellular studies showed that the G375W-PNKP has the same subcellular localization as the wild type PNKP. It partially rescues radiation sensitivity in mutant cells compared to the PNKP knockout cells. Interestingly, G375W-PNKP increases paraquat sensitivity more than PNKP knockout cells. Further study showed mitochondria in G375W cells are more sensitive to paraquat treatment, indicating mitochondria in the mutant cell lines could be the main target during the disease’s development. <br>
Together, our study explored the consequences of different PNKP mutations found in MCSZ and AOA4, and provided evidence for different mechanisms underlying the development of these diseases. Understanding these consequences could lead to more targeted treatment in the future.</p>