TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid-protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.

The planar cell polarity (PCP) pathway is required for fetal tissue morphogenesis as well as for maintenance of adult tissues in animals as diverse as fruit flies and mice. One of the key members of this pathway is Prickle (Pk), a protein that regulates cell movement through its association with the Dishevelled (Dsh) protein. Pk presents three LIM domains and a PET domain of unknown structure and function. Both the PET and LIM domains control membrane targeting of Dsh, which is necessary for Dsh function in the PCP pathway. Here, we show that the PET domain is monomeric and presents a nonglobular conformation with some properties of intrinsically disordered proteins. The PET domain adopts a helical conformation in the presence of 2,2,2-trifluoroethanol (TFE), a solvent known to stabilize hydrogen bonds within the polypeptide backbone, as analyzed by circular dichroism (CD) and NMR spectroscopy. Furthermore, we found that the conserved and single tryptophan residue in PET, Trp 536, moves to a more hydrophobic environment when accompanied with membrane penetration and that the protein becomes more helical in the presence of lipid micelles. The presence of LIM domains, downstream of PET, increases protein folding, thermostability, and tolerance to limited proteolysis. In addition, pull-down and tryptophan fluorescence analyses suggest that the LIM domains physically interact to regulate membrane penetration of the PET domain. The findings reported here favor a model where the PET domain is engaged in Pk membrane insertion, whereas the LIM domains modulate this function.

Introduction. Motor vehicle crashes remain the leading cause of fatalities among teens in the U.S. (National Center for Injury Prevention and Control, 2013). Prior research suggests that real-time and post hoc feedback can improve teen driver behavior. The Driver Coach Study (DCS) aimed to improve teens’ safe driving habits by providing them real-time feedback and post hoc feedback to a broader range of risky driving behaviors that have never been used in previous studies. Exposure data were also collected so that rates of risky driving behaviors over time could be assessed. Post hoc feedback, which included an electronic report card of risky driving behavior as well as video clips, was provided to both teens and parents via email and secure website link. Method. Ninety-two teen/parent dyads were recruited in southwest Virginia to have a data acquisition system (DAS) installed in their vehicles within two weeks of receiving their learner’s permit. Data were collected through the nine-month (minimum) learner’s permit phase plus seven months of provisional licensure. Feedback was only provided for the first six months of post licensure, then turned off to assess whether teenagers returned to unsafe driving behavior. Trained data coders reviewed 15 seconds of video surrounding each risky driving maneuver, and recorded driver errors such as poor vehicle control, poor speed selection, drowsiness, etc., for each event. Results. In this paper, the relationship between driver coaching and driver errors will be examined across the six-month feedback phase and also compared to the seventh month when feedback was turned off. Conclusions. This study has implications for the design of future monitoring and feedback systems, as it is currently unknown whether these devices can improve novice drivers’ crash rates.

No abstract

No abstract