Fernando Camacho

Control of Mammalian Circadian Rhythm by CKIε-Regulated Proteasome-Mediated PER2 Degradation

ABSTRACT The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIε (casein kinase Iε) has been postulated to prime PER2 for degradation. Supporting this idea, CKIε inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIε inhibition also slows the degradation of PER2 in cells. CKIε-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein β-TrCP to a specific site, and dominant negative β-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.

Phosphorylation and destabilization of human period I clock protein by human casein kinase Iε

Period (PER), a central component of the circadian clock in Drosophila, undergoes daily oscillation in abundance and phosphorylation state. Here we report that human casein kinase lε (hCKIε) can phosphorylate human PERI (hPERI). Purified recombinant hCKIε (but not a kinase negative mutant of hCKIε, hCKIε-K38R) phosphorylated hPERI in vitro. When co-transfected with wild-type hCKIε, in 293T cells, hPERI showed a significant increase in phosphorylation as evidenced by a shift in molecular mass. Furthermore, phosphorylation of hPERI by hCKIε caused a decrease in protein stability in hPERI. Whereas phosphorylated hPERI had a half-life of approximately 12 h, unphosphorylated hPERI remained stable in the cell for > 24 h. hPERI protein could also be co-immunoprecipitated with transfected hCKIε as well as endogenous hCKIε, indicating physical association between hPERI and hCKIε proteins in vivo.

Synthesis and preliminary structure-activity relationships of 1-[(3-fluoro-4-pyridinyl)amino]-3-methyl-1H-indol-5-yl methyl carbamate (P10358), a novel acetylcholinesterase inhibitor

Abstract A series of carbamate analogs of besipirdine (HP 749) was synthesized as potential agents with enhanced cholinomimetic properties for the treatment of Alzheimers disease. Compound 5a (P10358, 1-[3-fluoro-4-pyridinyl)amino]-3-methyl-1H-indol-5-yl methyl carbamate) emerged as a potent, reversible acetylcholinesterase inhibitor that significantly enhanced performance on oral or parenteral administration in learning and memory paradigms.

P11467: An Orally-Active Acetylcholinesterase Inhibitor and α 2 -Adrenoceptor Antagonist for Alzheimer’s Disease

Alzheimer’s disease (AD) is a complex and multifaceted neurodegenerative disease characterized by cognitive and behavioral abnormalities. The primary cognitive deficit has been correlated with extensive cholinergic dysfunction and the efficacy of cholinergic therapies in this disease validates and supports the cholinergic hypothesis of AD (Weinstock, 1995). To date, tacrine has shown clinical efficacy in 20 to 30% of AD patients and remains the only acetylcholinesterase inhibitor (AChEI) to receive FDA approval for symptomatic treatment (Knapp et al., 1994). However, the limited efficacy of pure cholinergic therapies suggests that other neurochemical deficiencies are involved.