Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia.
Illawarra Health and Medical Research Institute, Wollongong, Australia.
Protein Sci. 2021 Nov;30(11):2272-2286. doi: 10.1002/pro.4189. Epub 2021 Oct 4.
Proteostasis, the balance of protein synthesis, folding and degradation, is essential to maintain cellular function and viability, and the many known intracellular chaperones are recognized as playing key roles in sustaining life. In contrast, the identity of constitutively secreted extracellular chaperones (ECs) and their physiological roles in extracellular proteostasis is less completely understood. We designed and implemented a novel strategy, based on the well-known propensity of chaperones to bind to regions of hydrophobicity exposed on misfolding proteins, to discover new ECs present in human blood. We used a destabilized protein that misfolds at 37°C as "bait" to bind to potential ECs in human serum and captured the complexes formed on magnetic beads. Proteins eluted from the beads were identified by mass spectrometry and a group of seven abundant serum proteins was selected for in vitro analysis of chaperone activity. Five of these proteins were shown to specifically inhibit protein aggregation. Vitronectin and plasminogen activator-3 inhibited both the in vitro aggregation of the Alzheimer's β peptide (Aβ ) to form fibrillar amyloid, and the aggregation of citrate synthase (CS) to form unstructured (amorphous) aggregates. In contrast, prothrombin, C1r, and C1s inhibited the aggregation of Aβ but did not inhibit CS aggregation. This study thus identified five novel and abundant putative ECs which may play important roles in the maintenance of extracellular proteostasis, and which apparently have differing abilities to inhibit the amorphous and amyloid-forming protein aggregation pathways.
蛋白质稳态，即蛋白质合成、折叠和降解的平衡，对于维持细胞功能和生存能力至关重要，许多已知的细胞内伴侣被认为在维持生命方面发挥着关键作用。相比之下，构成性分泌的细胞外伴侣（ECs）的身份及其在细胞外蛋白质稳态中的生理作用还不完全清楚。我们设计并实施了一种新策略，该策略基于伴侣蛋白与错误折叠蛋白质暴露的疏水区结合的已知倾向，以发现存在于人血液中的新的 ECs。我们使用在 37°C 下错误折叠的不稳定蛋白质作为“诱饵”，与人血清中的潜在 ECs 结合，并将形成的复合物捕获在磁珠上。从珠子上洗脱的蛋白质通过质谱法鉴定，选择了一组七种丰富的血清蛋白进行体外伴侣活性分析。其中五种蛋白质被证明具有特异性抑制蛋白质聚集的活性。纤连蛋白和纤溶酶原激活物-3 抑制了阿尔茨海默病β肽（Aβ）形成纤维状淀粉样纤维的体外聚集，以及柠檬酸合酶（CS）形成无定形（无定形）聚集的聚集。相比之下，凝血酶原、C1r 和 C1s 抑制 Aβ的聚集，但不抑制 CS 的聚集。因此，该研究鉴定了五种新的丰富的潜在 ECs，它们可能在维持细胞外蛋白质稳态中发挥重要作用，并且显然具有不同的抑制无定形和淀粉样蛋白形成蛋白质聚集途径的能力。
