The dominant C96Y mutation of one of the two murine insulin genes, Ins2 , causes diabetes mellitus in 'Akita' mice. Here we established pancreatic islet β β β β cell lines from heterozygous mice ( Ins2

The crystal structure of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) has been determined by x-ray diffraction methods at 2.8-Å resolution by using Escherichia coli PEPC complexed with L-aspartate, an allosteric inhibitor of all known PEPCs. The four subunits are arranged in a ''dimer-of-dimers'' form with respect to subunit contact, resulting in an overall square arrangement. The contents of ␣-helices and ␤-strands are 65% and 5%, respectively. All of the eight ␤-strands, which are widely dispersed in the primary structure, participate in the formation of a single ␤-barrel. Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers. The location of the catalytic site is likely to be near the C-terminal side of the ␤-barrel. The binding site for L-aspartate is located about 20 Å away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector binding. The participation of Arg-587 is unexpected, because it is known to be catalytically essential. Because this residue is in a highly conserved glycine-rich loop, which is characteristic of PEPC, L-aspartate seemingly causes inhibition by removing this glycine-rich loop from the catalytic site. There is another mobile loop from Lys-702 to Gly-708 that is missing in the crystal structure. The importance of this loop in catalytic activity was also shown. Thus, the crystal-structure determination of PEPC revealed two mobile loops bearing the enzymatic functions and accompanying allosteric inhibition by L-aspartate.

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the first step in the fixation of atmospheric CO(2) during C(4) photosynthesis. The crystal structure of C(4) form maize PEPC (ZmPEPC), the first structure of the plant PEPCs, has been determined at 3.0 A resolution. The structure includes a sulfate ion at the plausible binding site of an allosteric activator, glucose 6-phosphate. The crystal structure of E. coli PEPC (EcPEPC) complexed with Mn(2+), phosphoenolpyruvate analog (3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate), and an allosteric inhibitor, aspartate, has also been determined at 2.35 A resolution. Dynamic movements were found in the ZmPEPC structure, compared with the EcPEPC structure, around two loops near the active site. On the basis of these molecular structures, the mechanisms for the carboxylation reaction and for the allosteric regulation of PEPC are proposed.

Objective: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein.Homozygous WFS1 gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic b-cells are selectively lost from the patient's islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses. Design and methods: Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic b-cell-derived MIN6 cells, and Akita mouse-derived Ins2 96Y/Y insulinoma cells. The human WFS1 gene promoter-luciferase reporter analysis was also conducted. Result: Islet b-cells were the major site of Wfs1 expression. This expression was also found in d-cells, but not in a-cells. WFS1 expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increased WFS1 mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells. WFS1 gene expression was also increased in Ins2 96Y/Y insulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. The WFS1 gene promoter-luciferase reporter system revealed that the human WFS1 promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active in Ins2 96Y/Y cells than Ins2 wild/wild cells. Conclusion: Wfs1 expression, which is localized to b-and d-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link between Wfs1 and ER stress.