Neurochemistry international ( IF 4.2 ) Pub Date : 2020-08-03 , DOI: 10.1016/j.neuint.2020.104812 Marzia Soligo 1 , Antonio Chiaretti 2 , Eleonora Leotta 1 , Elena Lardone 1 , Chiara Boschelle 1 , Elide Mantuano 1 , Liana Veneziano 1 , Luigi Manni 1
The precursor of Nerve Growth Factor (proNGF) is the predominant form of NGF in the brain, where its tissue levels are increased in neurodegenerative diseases. proNGF exists in two main splicing variants, the long proNGF-A and the short proNGF-B. We demonstrated that proNGF-B is selectively increased in the hippocampus of rats affected by early diabetic encephalopathy and that native, purified proNGFs elicit different responses when used to stimulate PC12 cells. Therefore, the evaluation of the proNGF-B/proNGF-A ratio may be of important diagnostic and prognostic value in pathologies characterized by dysfunctions of NGF system. To date there is not clear pharmacological characterization of the different proNGFs variants, due to the lack of a proper recombinant proNGF-A.
Using a bioinformatics approach, we predicted aminoacid sites involved in proNGF-A intracellular cleavage/conversion into proNGF-B, we cloned and expressed non-cleavable proNGF-A in HeLa cells and pursued a first characterization of their secretion modalities. Finally, we studied the biological effects of different proNGF-A mutants, stimulating PC12 cells with conditioned media from transfected HeLa cells. Based on our results, we propose the A73Y mutation as essential to obtaining an intact proNGF-A, limiting its conversion to proNGF-B. proNGF-A A73Y is probably released in an activity dependent manner and, when supplied to PC12 cells, shows a moderate differentiative capacity opposed to high neuroprotective potential. This preliminary study lays the foundation for future research aimed at uncovering the selective biological activities of proNGF-A and proNGF-B, and at developing pharmacological treatments that target the unbalance of proNGF system, induced by neurodegeneration.
中文翻译:
人重组proNGF-A变体的构建和初步表征。
神经生长因子的前体 (proNGF) 是大脑中 NGF 的主要形式,其组织水平在神经退行性疾病中增加。proNGF 存在于两种主要的剪接变体中,长的 proNGF-A 和短的 proNGF-B。我们证明 proNGF-B 在受早期糖尿病脑病影响的大鼠的海马中选择性增加,并且当用于刺激 PC12 细胞时,天然纯化的 proNGF 会引起不同的反应。因此,proNGF-B/proNGF-A 比率的评估在以 NGF 系统功能障碍为特征的病理中可能具有重要的诊断和预后价值。迄今为止,由于缺乏合适的重组 proNGF-A,不同 proNGF 变体的药理学特征尚不清楚。
使用生物信息学方法,我们预测了参与 proNGF-A 细胞内裂解/转化为 proNGF-B 的氨基酸位点,我们在 HeLa 细胞中克隆并表达了不可裂解的 proNGF-A,并对其分泌方式进行了首次表征。最后,我们研究了不同 proNGF-A 突变体的生物学效应,用来自转染 HeLa 细胞的条件培养基刺激 PC12 细胞。根据我们的结果,我们提出 A73Y 突变对于获得完整的 proNGF-A 至关重要,从而限制了其向 proNGF-B 的转化。proNGF-A A73Y 可能以活性依赖性方式释放,并且当提供给 PC12 细胞时,显示出与高神经保护潜力相反的中等分化能力。