Cyclic ADP-ribose signal transduction : Molecular mechanism and pathophsiological significance

钙动员在多种细胞反应中起着核心作用，如胰腺β细胞中葡萄糖分泌胰岛素。我们最近发现环adp核糖（cADPR）是NAD^+的代谢物，是Ca^<2+>动员胰岛素分泌的新第二信使。在本项目中，我们已经阐明了哺乳动物细胞中cADPR信号转导的分子机制。我们通过定点诱变确定了NAD^+（底物）、cADPR（产物）和ATP（调节剂）与CD38（哺乳动物adp -核糖基环化酶/cADPR水解酶）的结合位点2。我们分离了人CD38基因并确定了其初级结构。该基因由8个外显子组成，在4号染色体p15带上延伸-100 kbp，为单拷贝基因。我们在日本NIDDM患者中发现了Arg140→Trp突变，突变蛋白表现出CD38形成cadpr的酶活性降低。我们在日本和高加索糖尿病患者中发现了抗CD38的自身抗体，该抗体可损害葡萄糖诱导的胰岛素分泌。我们证明了cADPR与大鼠胰岛红嘌呤受体（RyR）上的fk506结合蛋白12.6 （FKBP12.6）结合，并且cADPR与FKBP12.6的结合将RyR从FKBP12.6中释放出来，释放Ca^<2+>。此外，在心力衰竭中，ryanodine受体的fk506结合蛋白的化学计量减少。我们分离了人FKBP12.6基因并确定了其一级结构。该基因在2p21-23.5染色体上长约16kbp。我们发现CaM激酶II和a -激酶的激活大大增强了Ca^<2+>从微粒体的释放。我们通过同源重组产生了携带CD38基因零突变的敲除小鼠，发现CD38破坏会损害葡萄糖诱导的cADPR、细胞内Ca^<2+>浓度和胰岛素分泌的增加。使用CD38敲除小鼠，我们发现胰腺腺泡细胞中的毒蕈碱Ca^<2+>信号通路涉及一个CD38依赖性通路，负责两个cadpr依赖性Ca^<2+>释放机制，其中一个对ryanodine敏感，在重复Ca^<2+>尖尖的产生中起关键作用。我们确定了链球菌adp -核糖基环化酶/cADPR水解酶的一级结构。通过定点诱变，我们发现与人CD38的Lys-129对应的链球菌酶的Lys-162和/或Lys-163参与cADPR的结合，链球菌酶的Glu-307和人CD38的Glu-226是NAD^+结合所必需的。我们发现2型和3型ryanodine受体Ca^<2+>通道（RyR）在正常胰腺β-细胞中表达，而在糖尿病β-细胞中表达明显降低。我们已经在胰岛素产生β细胞中制造了缺乏两种类型的RyR基因的突变小鼠。少

Calcium mobilization plays a central role in a variety of cellular responses such as insulin secretion by glucose in pancreatic β-cells. We have recently found that cyclic ADP-ribose (cADPR), a metabolite of NAD^+, is a novel second messenger for Ca^<2+> mobilization for insulin secretion. In the project, we have elucidated the molecular mechanism of cADPR signal transduction in mammalian cells.1. We identified the binding sites for NAD^+ (substrate), cADPR (product) and ATP (regulator) for CD38 (mammalian ADP-ribosyl cyclase/cADPR hydrolase) by site-directed mutagenesis.2. We isolated the human CD38 gene and determined its primary structure. The gene consists of 8 exons that extend -100 kbp on band p15 of chromosome 4 as a single copy gene. We found an Arg140→Trp mutation in Japanese NIDDM patients and the mutated protein exhibited a decrease in the enzymic activity of CD38 to form cADPR.3. We found autoantibodies against CD38 that impair glucose-induced insulin secretion in Japanese … More and Caucasian subjects with diabetes.4. We demonstrated that cADPR binds to FK506-binding protein 12.6 (FKBP12.6) on rat islet ryanodine receptor (RyR) and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, releasing Ca^<2+>. In addition, in heart failure, the stoichiometry of FK506-binding protein per the ryanodine receptor was decreased. We isolated the human FKBP12.6 gene and determined the primary structure. The gene spans about 16 kbp on chromosome 2p21-23.5. We found that the Ca^<2+> release from microsomes was greatly enhanced by the activation of CaM kinase II and A-kinase.6. We produced knockout mice carrying a null mutation in the CD38 gene by homologous recombination and found that CD38 disruption impairs glucose-induced increases in cADPR, intracellular Ca^<2+> concentration, and insulin secretion.7. Using CD38 knockout mice, we found that muscarinic Ca^<2+> signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca^<2+> release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca^<2+> spikes.8. We determined the primary structure of streptococcal ADP-ribosyl cyclase/cADPR hydrolase. Using sitedirected mutagenesis, we found that Lys-162 and/or Lys-163 of the streptococcal enzyme, which correspond to Lys-129 of human CD38, participate in the cADPR binding and that Glu-307 of the streptococcal enzyme and Glu-226 of human CD38 are essential for the NAD^+ binding.9. We found type 2 and type 3 ryanodine receptor Ca^<2+> channel (RyR) is expressed in normal pancreatic β-cells and the expression is markedly reduced in diabetic β-cells. We have made mutant mice lacking the both types of RyR gene in insulin producing β-cells. Less