Biology of 14-3 3 proteins regulating neuronal cell death

本研究旨在从生物学和临床医学两个方面阐明影响儿童神经系统疾病的神经元细胞死亡的生物学机制和分子相互作用。脑脊液14-3-3蛋白的检测是阐明神经系统疾病潜在机制的有力工具。14-3-3脑脊液蛋白检测在克雅氏病等神经系统疾病中已有有益的研究，但在小脑疾病中尚无相关研究。为了评价14-3-3蛋白在小脑疾病患者脑脊液中的检测效果，我们采用免疫印迹法检测了7例小脑疾病患者脑脊液中的14-3-3蛋白，即急性小脑炎(2)、急性小脑共济失调(2)和小脑萎缩(3)。同时，我们还通过同工异构体特异性抗体鉴定了14-3-3蛋白的同工异构体。除1例急性小脑性共济失调患者外，6例患者脑脊液中均检测到14-3-3蛋白。有趣的是，在整个14-3-3阳性患者中只检测到…More 14-3-3 c异构体。对1例小脑萎缩患者14-3-3脑脊液蛋白的纵向分析显示，随着小脑萎缩的逐渐进展，14-3-3条带密度成比例降低。我们认为脑脊液中的14-3-3蛋白是一个重要的小脑破坏标志物，也是其他脑部疾病的重要标志物，14-3-3 ε的独特检测可能表明小脑参与了大脑。由于14-3-3蛋白是凋亡信号调节激酶1 （ASK1）的亲密伙伴，我们使用重组ASK1和14-3-3蛋白进行了体外蛋白激酶检测。我们清楚地表明，ASK1可以磷酸化带有丝氨酸和苏氨酸残基的14-3-3蛋白。有趣的是，磷酸化的14-3-3蛋白减少了ASK1的自磷酸化，这表明ASK1-14-3-3蛋白的相互作用可能影响ASK1的动力学活性。由于14-3-3蛋白通过结合共识基序与ASK1结合，蛋白-蛋白相互作用被认为是ASK1和14-3-3蛋白的关键生物学功能。（305字）少

We aimed in this study to elucidate the biological mechanisms regulating neuronal cell death and molecular interactions affecting the neurological diseases in children on both aspects, biological and clinical medicines.Detection of 14-3-3 protein in the CSF is a powerful tool for elucidating the underlying mechanisms of neurological disorders. There have been useful studies on 14-3-3 CSF protein detection in Creutzfeldt-Jakob disease and other neurological disorders, but none on cerebellar diseases. To evaluate detection of 14-3-3 protein in the CSF of patients with cerebellar diseases, we examined 14-3-3 protein in the CSF by immunoblotting in seven patients with cerebellar diseases, i.e., acute cerebellitis (2), acute cerebellar ataxia (2), and cerebellar atrophy (3). 14-3-3 protein isoforms were also identified by means of isoform-specific antibodies. 14-3-3 protein was detected in the CSF of six patients, the exception being one with acute cerebellar ataxia. Interestingly, only the … More 14-3-3 c isoform was detected throughout in 14-3-3 positive patients. Moreover, longitudinal analysis of 14-3-3 CSF protein in one patient with cerebellar atrophy showed that the 14-3-3 band density proportionally decreased when the cerebellar atrophy gradually progressed. We concluded that 14-3-3 protein in the CSF is a significant cerebellar destructive marker as well as one in other brain diseases, and the unique detection of 14-3-3 ε may indicate cerebellar involvement in the brain.Since 14-3-3 proteins are an intimate partner of apoptosis signal-regulating kinase 1 (ASK1), we examined in vitro protein kinse assay using recombinant ASK1 and 14-3-3 proteins. We clearly showed that ASK1 can phosphorylate 14-3-3 proteins with several portions of serine and threonine residues. Interestingly, phosphorylated 14-3-3 proteins diminished ASK1 autophosphorylation, suggesting that ASK1-14-3-3 proteins interaction may influence the kinetic activity of ASK1. As 14-3-3 proteins bind to ASK1 using a binding consensus motif, protein-protein interaction is supposed to be critical biological function on both ASK1 and 14-3-3 proteins. (305 words) Less