Dante Davide, Jangra Jatin, Baidya Anurag T K, Kumar Rajnish, Darreh-Shori Taher
Division of Clinical Geriatrics, Centre for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, 141 57 Stockholm, Sweden.
Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (B.H.U.), Varanasi 221005, India.
Int J Mol Sci. 2024 Dec 19;25(24):13602. doi: 10.3390/ijms252413602.
Choline-acetyltransferase (ChAT) is the key cholinergic enzyme responsible for the biosynthesis of acetylcholine (ACh), a crucial signaling molecule with both canonical neurotransmitter function and auto- and paracrine signaling activity in non-neuronal cells, such as lymphocytes and astroglia. Cholinergic dysfunction is linked to both neurodegenerative and inflammatory diseases. In this study, we investigated a serendipitous observation, namely that the catalytic rate of human recombinant ChAT (rhChAT) protein greatly differed in buffered solution in the presence and absence of Triton X-100 (TX100). At a single concentration of 0.05% (/), TX100 boosted the specific activity of rhChAT by 4-fold. Dose-response analysis within a TX100 concentration range of 0.8% to 0.008% (accounting for 13.7 mM to 0.013 mM) resulted in an S-shaped response curve, indicative of an over 10-fold boost in the catalytic rate of rhChAT. This dramatic boost was unlikely due to a mere structural stabilization since it remained even after the addition of 1.0 mg/mL gelatin to the ChAT solution as a protein stabilizer. Furthermore, we found that the catalytic function of the ACh-degrading enzyme, AChE, was unaffected by TX100, underscoring the specificity of the effect for ChAT. Examination of the dose-response curve in relation to the critical micelle concentration (CMC) of TX100 revealed that a boost in ChAT activity occurred when the TX100 concentration passed its CMC, indicating that formation of micelle-ChAT complexes was crucial. We challenged this hypothesis by repeating the experiment on Tween 20 (TW20), another non-ionic surfactant with ~3-fold lower CMC compared to TX100 (0.06 vs. 0.2 mM). The analysis confirmed that micelle formation is crucial for ultra-boosting the activity of ChAT. In silico molecular dynamic simulation supported the notion of ChAT-micelle complex formation. We hypothesize that TX100 or TW20 micelles, by mimicking cell-membrane microenvironments, facilitate ChAT in accessing its full catalytic potential by fine-tuning its structural stabilization and/or enhancing its substrate accessibility. These insights are expected to facilitate research toward the development of new cholinergic-enhancing therapeutics through the formulation of micelle-embedded ChAT nanoparticles.
胆碱乙酰转移酶（ChAT）是负责乙酰胆碱（ACh）生物合成的关键胆碱能酶，乙酰胆碱是一种重要的信号分子，在非神经元细胞（如淋巴细胞和星形胶质细胞）中具有典型的神经递质功能以及自分泌和旁分泌信号活性。胆碱能功能障碍与神经退行性疾病和炎症性疾病都有关联。在本研究中，我们调查了一个意外发现，即人重组ChAT（rhChAT）蛋白在有和没有Triton X-100（TX100）的缓冲溶液中的催化速率有很大差异。在0.05%（/）的单一浓度下，TX100使rhChAT的比活性提高了4倍。在0.8%至0.008%（相当于13.7 mM至0.013 mM）的TX100浓度范围内进行剂量反应分析，得到一条S形反应曲线，表明rhChAT的催化速率提高了10倍以上。这种显著的提高不太可能仅仅是由于结构稳定，因为在向ChAT溶液中添加1.0 mg/mL明胶作为蛋白质稳定剂后，这种提高仍然存在。此外，我们发现乙酰胆碱降解酶AChE的催化功能不受TX100影响，这突出了该效应对ChAT的特异性。对与TX100的临界胶束浓度（CMC）相关的剂量反应曲线进行检查发现，当TX100浓度超过其CMC时，ChAT活性会提高，这表明胶束 - ChAT复合物的形成至关重要。我们通过对吐温20（TW20）重复该实验来验证这一假设，吐温20是另一种非离子表面活性剂，其CMC比TX100低约3倍（0.06对0.2 mM）。分析证实胶束形成对于超增强ChAT的活性至关重要。计算机模拟分子动力学模拟支持了ChAT - 胶束复合物形成的观点。我们假设TX100或TW20胶束通过模拟细胞膜微环境，通过微调其结构稳定性和/或增强其底物可及性，促进ChAT发挥其全部催化潜力。这些见解有望通过制备包埋胶束的ChAT纳米颗粒来促进新型胆碱能增强疗法的研发。
