Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) ε or δ can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1ε and CK1δ using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1δ is the principal regulator of the clock period: pharmacological inhibition of CK1δ, but not CK1ε, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1δ inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1δ inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2 −/− mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2 −/− mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1δ offers an avenue for therapeutic modulation of perturbed circadian behavior.circadian clock | suprachiasmatic nucleus | period protein | Tau mutation | pacemaking

AMP-activated protein kinase (AMPK) is a principal metabolic regulator affecting growth and response to cellular stress. Comprised of catalytic and regulatory subunits, each present in multiple forms, AMPK is best described as a family of related enzymes. In recent years, AMPK has emerged as a desirable target for modulation of numerous diseases, yet clinical therapies remain elusive. Challenges result, in part, from an incomplete understanding of the structure and function of full-length heterotrimeric complexes. In this work, we provide the full-length structure of the widely expressed α1β1γ1 isoform of mammalian AMPK, along with detailed kinetic and biophysical characterization. We characterize binding of the broadly studied synthetic activator A769662 and its analogs. Our studies follow on the heels of the recent disclosure of the α2β1γ1 structure and provide insight into the distinct molecular mechanisms of AMPK regulation by AMP and A769662.

The circadian clock links our daily cycles of sleep and activity to the external environment. Deregulation of the clock is implicated in a number of human disorders, including depression, seasonal affective disorder, and metabolic disorders. Casein kinase 1 epsilon (CK1) and casein kinase 1 delta (CK1␦) are closely related Ser-Thr protein kinases that serve as key clock regulators as demonstrated by mammalian mutations in each that dramatically alter the circadian period. Therefore, inhibitors of CK1␦/ may have utility in treating circadian disorders. Although we previously demonstrated that a pan-CK1␦/ inhibitor, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), causes a significant phase delay in animal models of circadian rhythm, it remains unclear whether one of the kinases has a predominant role in regulating the circadian clock. To test this, we have characterized 3-(3-, a novel and potent inhibitor of CK1 (IC 50 ϭ 32 nM) with greater than 20-fold selectivity over CK1␦. PF-4800567 completely blocks CK1-mediated PER3 nuclear localization and PER2 degradation. In cycling Rat1 fibroblasts and a mouse model of circadian rhythm, however, PF-4800567 has only a minimal effect on the circadian clock at concentrations substantially over its CK1 IC 50 . This is in contrast to the pan-CK1␦/ inhibitor PF-670462 that robustly alters the circadian clock under similar conditions. These data indicate that CK1 is not the predominant mediator of circadian timing relative to CK1␦. PF-4800567 should prove useful in probing unique roles between these two kinases in multiple signaling pathways.All living things, from fungi to humans, have regular cycles aligning them with the daily events in their environment. These cycles, known as circadian rhythms, are controlled in mammals by the master clock located in the suprachiasmatic nucleus of the hypothalamus (Antle and Silver, 2005;Gallego and Virshup, 2007). At the cellular level, the molecular events behind clock cycling are described by the regular increase and decrease in mRNAs and proteins that define feedback loops, resulting in approximately 24-h cycles. The suprachiasmatic nucleus is primarily regulated, or entrained, directly by light via the retinohypothalamic tract. The cycling outputs of the suprachiasmatic nucleus, not fully identified, regulate multiple downstream rhythms, such as those in sleep and awakening, body temperature, and hormone secretion (Schibler et al., 2003;Ko and Takahashi, 2006). As anyone who has experienced jet lag knows, misalignment of the internal clock with the external environment profoundly affects well being. Furthermore, diseases, such as depression, seasonal affective disorder, and metaArticle, publication date, and citation information can be found at

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb ␦-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced ␦-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210-and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CryIAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.

, and I375A had reduced toxicity to H. virescens. In contrast to our findings with M. sexta, the reduction in toxicity of these mutants was correlated directly with loss of initial binding to H. virescens BBMV, indicating that these residues perform functionally distinct roles in binding and toxicity to different insects. In ligand blots, CryIAb recognized a major 210-kDa peptide in M. sexta BBMV and a 170-kDa peptide in H. virescens BBMV.

The elevation of serum alanine aminotransferase (ALT) is regarded as an indicator of liver damage based on the presumption that ALT protein is specifically and abundantly expressed in the liver. However, ALT elevation is also observed in non-liver injury conditions (for example, muscle injury) and in apparently healthy people. Conversely, serum ALT activity is normal in many patients with confirmed liver diseases (for example, cirrhosis and hepatitis C infection). To improve the diagnostic value of the ALT assay and to understand the molecular basis for serum ALT changes in various pathophysiological conditions, we have cloned rat ALT isoenzyme ALT1 and ALT2 complementary DNAs (cDNAs), examined their tissue expressions at the messenger RNA and protein levels, and determined ALT1 and ALT 2 serum levels in response to liver damage in rodents. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis shows that ALT1 messenger RNA is widely distributed and mainly expressed in intestine, liver, fat tissues, colon, muscle, and heart, in the order of high to low expression level, whereas ALT2 gene expression is more restricted, mainly in liver, muscle, brain, and white adipose tissue. The tissue distribution pattern of ALT1 and ALT2 proteins largely agrees with their messenger RNA expression. Interestingly, hepatic ALT2 protein is approximately four times higher in male rats than in female rats. In addition, ALT isoenzymes distribute differentially at the subcellular level in that ALT1 is a cytoplasmic protein and ALT2 a mitochondrial protein, supporting bioinformatic prediction of mitochondrial localization of ALT2. Conclusion: Using animal models of hepatoxicity induced by carbon tetrachloride and acetaminophen, we found that both serum ALT1 and ALT2 protein levels were significantly elevated and correlated with ALT activity, providing, for the first time, the molecular basis for the elevated total serum ALT activity. (HEPATOLOGY 2009;49:598-607.)

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis ␦-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) Ϸ4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering ␦-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.