1999 — 2008 |
Lew, John |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism of Regulation in the Cyclin-Dependent Kinases @ University of California Santa Barbara
Mammalian cell division is controlled by a family of protein kinases known as the cyclin-dependent kinases (cdks). The goal of this project is to characterize the structural and mechanistic basis for catalysis, substrate specificity, and regulation by phosphorylation of two distinct cdk family members: cdk2/cyclinA and cdk5/p25. While cdk2/cyclinA is a classic cell division control enzyme, cdk5/p25 plays a unique role in the development of neurons - cells which classically do not divide. Activation of cdk2 typically depends on cyclin binding followed by phosphorylation. In contrast, cdk5 activation depends only on its interaction with p25, a neuronal-specific, non-cyclin-like protein. While several X-ray crystal structures of cdk2/cyclinA have been solved, there is little mechanistic information linking the abundant structural data with knowledge of this enzyme's unique substrate specificity and regulatory properties. We will describe the kinetic basis for activation of cdk2/cyclinA by phosphorylation at residue Thr160, and its inhibition by phosphorylation at Tyr15. We will describe the key structural determinants which are crucial for catalytic regulation and substrate recognition. We will compare the properties of cdk2 with those of cdk5 with an aim to understand the structural and kinetic basis for the unique regulatory properties of the cdk5/p25 complex. To achieve these goals, we will apply a variety of kinetic and biophysical techniques to isolate the individual steps of catalysis and their inherent rate constants, in both cdk2/cyclinA and cdk5/p25. We will determine which steps - substrate binding, phosphoryl group transfer, or product release - are controlled by phosphorylation and specific enzyme and substrate structural determinants. Our studies on cdk2/cyclinA and cdk5/p25 will provide the first description of how their chemical structures, catalytic mechanisms and biochemical properties are integrated, with a view to understanding the structure/function relationships of protein kinases in general.
|
1 |
2016 — 2017 |
Lew, John Reese, Benjamin E (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Procyanindin a-Type Dimer: a Novel Inhibitor of Tau Aggregation in Vivo @ University of California Santa Barbara
Project Summary Basic research has identified ?-amyloid plaque formation and tau aggregation as the primary biomarkers of Alzheimer?s Disease (AD), which are hypothesized to be causally involved in AD progression. Molecular interventions of primarily ?-amyloid aggregation have over the last decade, however, been met with overt failure. Thus, alternative targets are now essential. Several inhibitors of tau aggregation in vitro have been identified, but only one, methylene blue, is currently being assessed for efficacy in vivo. Most small molecule chemical inhibitors fail to enter the drug pipeline due to insolubility as well as inherent toxicity issues. Consequently, our laboratory has considered naturally occurring molecules present in edible plants as possible alternatives. Plant polyphenols have long been associated with various health beneficial effects owing classically to their anti-oxidant properties. However, distinct bio-activities of certain polyphenolic compounds are now believed to be responsible for a significant reduction in age-related diseases, cancer, cardiovascular diseases, and processes associated with neurodegeneration. We previously demonstrated that an extract of cinnamon containing a procyanidin A-linked trimer molecule (865 Da) was non-toxic and capable of inhibiting tau aggregation in vitro (Peterson et al., J. Alz. Dis., 2009, 17: 585-97). We now show that an aqueous extract of peanut skin (PSE) similarly inhibits tau aggregation. The bio-active component was isolated, and the purified compound contained intrinsic inhibitory activity. The compound was identified as a procyanidin A-linked dimer (576 Da). The corresponding, highly similar B-linked dimer did not inhibit, suggesting a specific interaction between tau and procyanidin oligomers of A-type linkage. Monomeric epicatechin, the molecule from which procyanidin oligomers are derived, do not inhibit, suggesting that the dimer is the smallest procyanidin molecule that retains tau aggregation inhibitory activity. Finally, peanut skin extract was orally administered to a mouse model of Alzheimer?s disease, and preliminary data suggests the potential of the extract to inhibit tau pathology in vivo. In this grant application, we propose to test if purified procyanidin A-linked dimer administered orally to Alzheimer?s disease mice displays efficacy to inhibit tau pathology and the behavioral symptoms associated with AD. We hypothesize that procyanidin A-linked dimer present in a common and inexpensive food source may offer potential as a novel and effective intervention of tau aggregation in humans.
|
1 |