Cancer-Derived Mutations in KEAP1 Impair NRF2 Degradation but not Ubiquitination

NRF2 is a transcription factor that mediates stress responses. Oncogenic mutations in NRF2 localize to one of its two binding interfaces with KEAP1, an E3 ubiquitin ligase that promotes proteasome-dependent degradation of NRF2. Somatic mutations in KEAP1 occur commonly in human cancer, where KEAP1 may function as a tumor suppressor. These mutations distribute throughout the KEAP1 protein but little is known about their functional impact. In this study, we characterized 18 KEAP1 mutations defined in a lung squamous cell carcinoma tumor set. Four mutations behaved as wild-type KEAP1, thus are likely passenger events. R554Q, W544C, N469fs, P318fs, and G333C mutations attenuated binding and suppression of NRF2 activity. The remaining mutations exhibited hypomorphic suppression of NRF2, binding both NRF2 and CUL3. Proteomic analysis revealed that the R320Q, R470C, G423V, D422N, G186R, S243C, and V155F mutations augmented the binding of KEAP1 and NRF2. Intriguingly, these 'super-binder' mutants exhibited reduced degradation of NRF2. Cell-based and in vitro biochemical analyses demonstrated that despite its inability to suppress NRF2 activity, the R320Q 'superbinder' mutant maintained the ability to ubiquitinate NRF2. These data strengthen the genetic interactions between KEAP1 and NRF2 in cancer and provide new insight into KEAP1 mechanics

Fracture Limits of Maxillary Fourth Premolar Teeth in Domestic Dogs Under Applied Forces

A cadaveric study was performed to investigate the external mechanical forces required to fracture maxillary fourth premolar teeth in domestic dogs and describe a clinically relevant model of chewing forces placed on functionally important teeth in which fracture patterns are consistent with those defined by the American Veterinary Dental College (AVDC). Twenty-four maxillary fourth premolar teeth were harvested from dog cadavers. Samples consisted of teeth with surrounding alveolar bone potted in polycarbonate cylinders filled with acrylic. The cylinders were held by an aluminum device at an angle of 60° with respect to the ground. An axial compression test was performed, creating a force upon the occluso-palatal aspects of the main cusps of the crowns of the teeth. The highest compressive force prior to failure was considered the maximum force sustained by the teeth. Results showed the mean maximum force (± SD) sustained by the tested teeth at the point of fracture was 1,281 N (± 403 N) at a mean impact angle (± SD) of 59.7° (± 5.2°). The most common fracture type that occurred among all samples was a complicated crown fracture (n = 12), followed by an uncomplicated crown fracture (n = 6), complicated crown-root fracture (n = 5), and uncomplicated crown-root fracture (n = 1). There was no statistically significant correlation between dog breed, age, weight, impact angle, crown height or crown diameter, and the maximum force applied at the point of fracture. The only independent variable that remained significantly associated with maximum force was the crown height to diameter ratio (p = 0.005), suggesting that a decreased ratio increases tooth fracture resistance. The methodology described herein has been successful in creating a pattern of fracture of maxillary fourth premolar teeth consistent with that defined by the AVDC under angled compression at forces within the maximum chewing capability of the average domestic dog