XPD localizes in mitochondria and protects the mitochondrial genome from oxidative DNA damage.

Xeroderma pigmentosum group D (XPD/ERCC2) encodes an ATP-dependent helicase that plays essential roles in both transcription and nucleotide excision repair of nuclear DNA, however, whether or not XPD exerts similar functions in mitochondria remains elusive. In this study, we provide the first evidence that XPD is localized in the inner membrane of mitochondria, and cells under oxidative stress showed an enhanced recruitment of XPD into mitochondrial compartment. Furthermore, mitochondrial reactive oxygen species production and levels of oxidative stress-induced mitochondrial DNA (mtDNA) common deletion were significantly elevated, whereas capacity for oxidative damage repair of mtDNA was markedly reduced in both XPD-suppressed human osteosarcoma (U2OS) cells and XPD-deficient human fibroblasts. Immunoprecipitation-mass spectrometry analysis was used to identify interacting factor(s) with XPD and TUFM, a mitochondrial Tu translation elongation factor was detected to be physically interacted with XPD. Similar to the findings in XPD-deficient cells, mitochondrial common deletion and oxidative damage repair capacity in U2OS cells were found to be significantly altered after TUFM knock-down. Our findings clearly demonstrate that XPD plays crucial role(s) in protecting mitochondrial genome stability by facilitating an efficient repair of oxidative DNA damage in mitochondria.

Characterization of a chemical affinity probe targeting Akt kinases.

Protein kinases are key regulators of cellular processes, and aberrant function is often associated with human disease. Consequently, kinases represent an important class of therapeutic targets and about 20 kinase inhibitors (KIs) are in clinical use today. Detailed knowledge about the selectivity of KIs is important for the correct interpretation of their pharmacological and systems biological effects. Chemical proteomic approaches for systematic kinase inhibitor selectivity profiling have emerged as important molecular tools in this regard, but the coverage of the human kinome is still incomplete. Here, we describe a new affinity probe targeting Akt and many other members of the AGC kinase family that considerably extends the scope of KI profiling by chemical proteomics. In combination with the previously published kinobeads, the synthesized probe was applied to selectivity profiling of the Akt inhibitors GSK690693 and GSK2141795 in human cancer cells. The results confirmed the inhibition of all Akt isoforms and of a number of known as well as CDC42BPB as a novel putative target for GSK690693. This work also established, for the first time, the kinase selectivity profile of the clinical phase I drug GSK2141795 and identified PRKG1 as a low nanomolar kinase target as well as the ATP-dependent 5'-3' DNA helicase ERCC2 as a potential new non-kinase off-target.