SUMMARY Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca2+ levels and activates the CamKKβ-AMPK pathway via phospholipase C and the ryanodine receptor Ca2+-release channel. As a consequence, resveratrol increases NAD+ and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.

Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants.

SUMMARY Autophagy is an important intracellular catabolic mechanism that mediates the degradation of cytoplasmic proteins and organelles. We report a potent small molecule inhibitor of autophagy named “spautin-1” for specific and potent autophagy inhibitor-1. Spautin-1 promotes the degradation of Vps34 PI3 kinase complexes by inhibiting two ubiquitin-specific peptidases, USP10 and USP13, that target the Beclin1 subunit of Vps34 complexes. Beclin1 is a tumor suppressor and frequently monoallelically lost in human cancers. Interestingly, Beclin1 also controls the protein stabilities of USP10 and USP13 by regulating their deubiquitinating activities. Since USP10 mediates the deubiquitination of p53, regulating deubiquitination activity of USP10 and USP13 by Beclin1 provides a mechanism for Beclin1 to control the levels of p53. Our study provides a molecular mechanism involving protein deubiquitination that connects two important tumor suppressors, p53 and Beclin1, and a potent small molecule inhibitor of autophagy as a possible lead compound for developing anticancer drugs.

Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicleenclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte, where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from ~2 μM to ~80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from ~1 μM to ~40 nM) relieves the inhibition of PDE3A, increasing its activity by ~5-fold. This causes a decrease in oocyte cAMP (from ~700 nM to ~140 nM), leading to the resumption of meiosis.

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.

Mutations in DJ-1, a protein of unknown function, were recently identified as the cause for an autosomal recessive, early onset form of familial Parkinson's disease. Here we report that DJ-1 is a dimeric protein that exhibits protease activity but no chaperone activity. The protease activity was abolished by mutation of Cys-106 to Ala, suggesting that DJ-1 functions as a cysteine protease. Our studies revealed that the Parkinson's disease-linked L166P mutation impaired the intrinsic folding propensity of DJ-1 protein, resulting in a spontaneously unfolded structure that was incapable of forming a homodimer with itself or a heterodimer with wild-type DJ-1. Correlating with the disruption of DJ-1 structure, the L166P mutation abolished the catalytic function of DJ-1. Furthermore, as a result of protein misfolding, the L166P mutant DJ-1 was selectively polyubiquitinated and rapidly degraded by the proteasome. Together these findings provide insights into the molecular mechanism by which loss-of-function mutations in DJ-1 lead to Parkinson's disease.

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We have solved the structure of a chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5), the 1.9-crstal structure of the monofunctional enzyme from BaciUls sublis. The structure determination process was an unusual one, involving 12 monomers of the enzyme in the asymmetric unit. This structure was solved by the multiple isomorphous replacement method with partal structure phase combination and mo lar averaging. The final model, which includes 1380 residues and 522 water molecules in an asymmetric unit, has been refind at 1.9 A and the current crystallographic R value b 0.201. The B. subtiUs chorismate mutase is a homotrimer, with -sheets from each monomer packdng to form the core of a pseudo-a1-barrel with helices on the outside ofthe trimer. In addition, the active sites have been located by using data from a complex with an endo-oxabicydic inhibitor that mimics the transition state of the reaction. The structure of this complex has been reined to 2.2 A with a current R value of 0.182 for a model that clude 1388 residues, 12 inhibitor moleculs, and 530 water molecules in the asymmetric unit. In each trimer, three equivalent active sites are located at the interfaces of two adjacent subunits.