RyR2 and calpain-10 delineate a novel apoptosis pathway in pancreatic islets

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic beta-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism. Increased calpain activity and calpain gene expression suggested a role for a calpain-dependent death pathway. Using a combination of pharmacological and genetic approaches, we demonstrated that the calpain-10 isoform mediated ryanodine-induced apoptosis. Apoptosis induced by the fatty acid palmitate and by low glucose also required calpain-10. Ryanodine-induced calpain activation and apoptosis were reversed by glucagon-like peptide or short-term exposure to high glucose. Thus RyR2 activity seems to play an essential role in beta-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.     

Identification and characterization of GCP16, a novel acylated Golgi protein that interacts with GCP170

GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH2-terminal region (positions 137-237). Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed. Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [3H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys69 and Cys72, accounting for its tight association with the membrane. A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface.     

Further insight into the mechanism of stereoselective proton abstraction by bacterial copper amine oxidase

During the catalytic reaction of copper amine oxidase, one of the two prochiral hydrogen atoms at the C1 position of substrate amine is stereoselectively abstracted by a conserved Asp residue serving as a general base. Using stereospecifically deuterium-labeled enantiomers of 2-phenylethylamine, we previously showed that the pro-S alpha-proton is abstracted by the enzyme from Arthrobacter globiformis (AGAO) [Uchida, M., et al. (2003) Biosci. Biotechnol. Biochem. 67, 2664-2667]. More recently, we have also demonstrated that the pro-S selectivity of alpha-proton abstraction is fully retained even in the reaction of a mutant AGAO lacking the catalytic base [Chiu, Y.-C., et al. (2006) Biochemistry 45, 4105-4120]. On the basis of these findings, we have proposed that the stereoselectivity of alpha-proton abstraction is primarily determined by the conformation of the Schiff base intermediate formed between the substrate and the topa quinone cofactor (TPQ), stabilized by the binding of the distal part of the substrate to a hydrophobic pocket of the enzyme. In this conformation, the pro-S hydrogen atom to be abstracted is nearly perpendicular to the plane of the Schiff base-TPQ conjugate system, achieving the maximum overlap of sigma- and pi-orbitals. To further elucidate the stereochemical details, we have synthesized stereospecifically deuterium-labeled enantiomers of ethylamine, a very poor substrate for AGAO, in addition to those structurally related to the preferred substrate, 2-phenylethylamine. In marked contrast to the nearly complete pro-S selectivity of alpha-proton abstraction for most substrates that have been examined, the stereoselectivity for ethylamine decreased significantly to as little as 88%. The crystal structure of AGAO soaked with ethylamine showed very poor electron densities for the substrate Schiff base intermediate, showing that its conformation is not defined uniquely. Thus, the stereoselectivity of alpha-proton abstraction during the copper amine oxidase reaction is closely associated with the conformational flexibility of the substrate Schiff base intermediate.     

Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-β receptor and inhibits TGF-β1 binding to cell surface receptors, thereby inhibiting TGF-β1 signaling. Klotho suppresses TGF-β1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-β1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.