ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism.
ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism.
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DOI：
10.3390/biomedicines9111646
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发表时间：
2021-11-09
期刊：
影响因子：
4.7
通讯作者：
Curtis R
中科院分区：
文献类型：
--
作者：
Bye J;Murray K;Curtis R
增加聚集抗性的常见策略是通过合理诱变对蛋白质进行增压，这会导致高胶体稳定性，但通常会产生降低构象稳定性的不良影响。我们证明，可以通过使用小的多价聚磷酸盐离子、三磷酸腺苷（ATP）和三聚磷酸盐（TPP）作为赋形剂来克服这种权衡。通过动态和静态光散射监测，这些离子在抑制热应激时卵清蛋白和牛血清白蛋白 (BSA) 的聚集方面同样有效。单体损失动力学研究，结合天然状态蛋白质-蛋白质相互作用和 ζ 电位的测量，表明离子通过结合和过度充电蛋白质来增加蛋白质胶体稳定性，从而减少聚集体生长。在研究的另外三种蛋白质中，核糖核酸酶 A (RNaseA)、α-胰凝乳蛋白酶原 (α-Cgn) 和溶菌酶，我们仅观察到 RNaseA 聚集体生长的减少，尽管溶菌酶和 α-Cgn 仍然会发生多磷酸盐离子的过度充电。由于这些盐不会改变蛋白质构象稳定性，一旦更好地阐明了决定多价离子结合是否会增加胶体稳定性的蛋白质结构因素，使用它们作为赋形剂可能是稳定生物制药的一种有前途的策略。我们的发现也具有生物学意义。最近，有人提出，ATP 在维持细胞内生物凝聚物和防止密集细胞环境中蛋白质聚集方面也发挥着重要作用。我们预计静电相互作用是决定 ATP 在体内维持蛋白质非分散状态的稳定能力的重要因素。
A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to high colloidal stability, but often has the undesirable effect of lowering conformational stability. We show this trade-off can be overcome by using small multivalent polyphosphate ions, adenosine triphosphate (ATP) and tripolyphosphate (TPP) as excipients. These ions are equally effective at suppressing aggregation of ovalbumin and bovine serum albumin (BSA) upon thermal stress as monitored by dynamic and static light scattering. Monomer loss kinetic studies, combined with measurements of native state protein–protein interactions and ζ-potentials, indicate the ions reduce aggregate growth by increasing the protein colloidal stability through binding and overcharging the protein. Out of three additional proteins studied, ribonuclease A (RNaseA), α-chymotrypsinogen (α-Cgn), and lysozyme, we only observed a reduction in aggregate growth for RNaseA, although overcharging by the poly-phosphate ions still occurs for lysozyme and α-Cgn. Because the salts do not alter protein conformational stability, using them as excipients could be a promising strategy for stabilizing biopharmaceuticals once the protein structural factors that determine whether multivalent ion binding will increase colloidal stability are better elucidated. Our findings also have biological implications. Recently, it has been proposed that ATP also plays an important role in maintaining intracellular biological condensates and preventing protein aggregation in densely packed cellular environments. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo.
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