Hippocalcin acts as a possible suppressor of neuronal apoptosis via NAIP-caspase and MLK3-JNK cascades

Hippocalcin （HIP）是主要表达于海马体锥体细胞的神经元钙传感器家族的成员。与野生型（+/+）小鼠相比，原代培养的HIP缺陷（-/-）小鼠海马神经元的存活率较低。相反，在培养的胶质细胞存活率方面，-/-和+/+小鼠之间没有差异。谷氨酸、高钾和霉素诱导钙内流进入原代培养的神经元，并利用Fra2AM监测钙内流和外排过程。在-/-小鼠中，这两个过程都受损。相反，在HIP过表达的COS7细胞中，两者都加速了。这表明HIP不仅作为钙缓冲剂，而且作为钙通道和钙泵激活剂。谷氨酸和高钾诱导的-/-海马切片中JNK的激活远高于+/+切片，表明HIP可能是JNK级联的钙依赖性抑制因子。然后，利用酵母双杂交系统组成的小鼠脑cDNA文库对HIP靶蛋白进行筛选。在3x10^6个转化子中，获得76个阳性克隆。Β-galactosidase表达分析显示有12个克隆与HIP相互作用。这些克隆分为混合谱系激酶（MLK） 3（7个克隆）、蛋白1B；序列分析显示，该转录因子具有f-box/WD40结构域（3个克隆）、NAIP（神经元凋亡抑制蛋白）3（3个克隆）和络合蛋白2。MLK3是JNK的激活剂，表现出最强的相互作用。发现HIP与MLK3的c端Pro/Ser/ thr富区相互作用。整个HIP构象参与了相互作用。神经视蛋白样蛋白（Neural visinin-like protein， NVP） 1、2和3是与HIP结构相关的蛋白，未显示出与MLK3的结合活性，NAIP是一种抑制神经元凋亡的caspase级联抑制剂。这些表明，HIP可能通过NAIP-caspase和MLK3-JNK级联反应抑制神经元凋亡。

Hippocalcin (HIP) is a member of neuronal calcium sensors family predominantly expressed in the hippocampal pyramidal cells. Primary cultured hippocampal neuron from HIP deficient (-/-) mice showed a low survivability compared to that from wild type (+/+) mice. Contrary, in the survivability of cultured glia, there is no difference between -/- and +/+ mice. Calcium influx into primary cultured neurons was induced by glutamate, high potassium and inomycin, and the influx and efflux processes were monitored using Fra2AM. Both processes were impaired in -/- mice. Contrary, both were accelerated in HIP over expressed COS7 cells. These indicate that HIP acts not only as a calcium buffer but also as calcium channel and pump activators. Glutamate- and high potassium- induced JNK activations in -/- hippocampal slices were much higher than those in +/+ slices, indicating that HIP acts as a possible calcium-dependent suppressor of JNK cascade. Then, we screened to search for HIP target proteins by using yeast two-hybrid system composed of mouse brain cDNA library. Among 3x10^6 transformants, 76 positive clones were obtained. Β-galactosidase expression analysis revealed that 12 clones interact with HIP. These clones were classified into mixed lineage kinase (MLK) 3 (7 clones), protein 1B; a transcription factor having f-box/WD40 domain (3 clones), NAIP (neuronal apoptosis inhibitory protein) 3 (3 clones) and complexin 2 by sequence analysis. MLK3 is a JNK activator, displayed the strongest interaction. HIP was found to interact with C-terminal Pro/Ser/Thr-rich region of MLK3. Whole HIP conformation involved in the interaction. Neural visinin-like protein (NVP) 1, 2 and 3, structurally related proteins to HIP, did not show the binding activity to MLK3, NAIP is known to be an inhibitor of caspase cascade that suppress the neuronal apoptosis. These indicate that HIP acts as a possible suppressor of neuronal apoptosis via NAIP-caspase and MLK3-JNK cascades.