Antiamyloidogenic and proamyloidogenic chaperone effects of C-reactive protein and serum amyloid P component.
Antiamyloidogenic and proamyloidogenic chaperone effects of C-reactive protein and serum amyloid P component.
复制标题
C 反应蛋白和血清淀粉样蛋白 P 成分的抗淀粉样蛋白生成和促淀粉样蛋白生成伴侣作用。
DOI：
10.1080/13506129.2017.1295943
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发表时间：
2017
期刊：
影响因子：
5.5
通讯作者：
Naiki H.
中科院分区：
文献类型：
--
作者：
Ozawa D;Nomura R;Mangione PP;Hasegawa K;Okoshi T;Porcari R;Bellotti V;Naiki H.
研究背景C反应蛋白（CRP）和血清淀粉样蛋白P成分（SAP）是人体内两种主要的五聚蛋白，是调节先天免疫系统的可溶性模式识别分子。它们具有独特的五聚体结构，并通过它们的B面与它们的配体钙依赖性地结合。在体外酸性pH条件下，五聚体CRP与各种蛋白质（包括淀粉样蛋白β（Aβ））的结合不依赖于钙离子[引文1]。据推测，五聚体CRP保护免受酸性炎症环境中蛋白质错误折叠和聚集引起的毒性病症，但CRP的伴侣活性仍然知之甚少。SAP普遍存在于所有细胞外淀粉样蛋白沉积物中[引文2]。其在淀粉样蛋白生成中的主要作用被认为是通过钙依赖性地与淀粉样蛋白原纤维表面的B面结合来增强淀粉样蛋白原纤维的形成和沉积。另一方面，SAP抑制Aβ的淀粉样纤维形成[引文3]，并在体外提高变性乳酸脱氢酶的重折叠产率[引文4]。迄今为止，还没有令人信服的数据或模型来解释SAP的促淀粉样蛋白生成活性和抗淀粉样蛋白生成活性之间的差异。在这项研究中，我们研究了CRP和SAP对体外淀粉样纤维形成和无定形蛋白聚集的影响[引文5]。材料和方法人CRP和SAP是Mark B教授的善意礼物。佩皮斯，伦敦大学学院。淀粉样原纤维的形成通过硫磺素T测定和电子显微镜监测。通过浊度测定监测无定形蛋白聚集。蛋白质-蛋白质相互作用进行了评估，通过酶联免疫吸附试验和蛋白质印迹analysis.ResultsWe第一次评估的组装状态的CRP和SAP在各种缓冲液条件。在无Ca 2+和有Ca 2+时，CRP均以五聚体形式存在.另一方面，SAP在无Ca 2+的Tris-EDTA缓冲液中组装成稳定的十聚体。在由50 mMMES-NaOH（pH7.0）、500 mMNaCl和2 mMCaCl 2组成的MES-Ca缓冲液中，SAP甚至在37 ℃孵育72小时后仍保持五聚体。我们接下来检查了CRP和SAP在不同缓冲液条件下对淀粉样蛋白原纤维形成的影响。CRP和SAP以剂量依赖性和亚化学计量方式抑制Aβ（1-40）和D 76 N β 2-微球蛋白（β2-m）纤维形成，且不依赖于Ca 2+。由于无钙SAP具有B面对B面的十聚体，这些数据可能表明SAP的A面可能以不依赖于Ca 2+的方式具有抗淀粉样蛋白生成活性。有趣的是，在Ca 2+存在下，SAP首先抑制，然后显著加速D 76 N β2-m原纤维的形成（图1）。电子显微镜下，D 76 N β2-m原纤维的表面被五聚体SAP包被。这些数据表明SAP首先可能通过A面表现出抗淀粉样蛋白生成活性，然后通过B面表现出促淀粉样蛋白生成活性。我们接下来进行了酶联免疫吸附试验，以评估CRP和SAP与新鲜和聚集的Aβ（1-40）和D 76 N β2-m的相互作用。CRP和SAP与新鲜和预聚集的Aβ（1-40）和D 76 N β2-m在纤维形成途径上相互作用。最后，SAP不仅抑制淀粉样纤维的形成，而且抑制热诱导的人谷胱甘肽S-转移酶的无定形聚集。
BackgroundC-reactive protein (CRP) and serum amyloid P component (SAP), two major classical pentraxins in humans, are soluble pattern recognition molecules that regulate the innate immune system. They have a unique pentameric structure and bind to their ligands calcium-dependently with their B faces. Pentameric CRP binds calcium-independently to various proteins, including amyloid β (Aβ), at acidic pH in vitro [Citation 1]. It is hypothesized that pentameric CRP protects against toxic conditions caused by protein misfolding and aggregation in acidic inflammatory environments, but the chaperone activity of CRP remain poorly understood. SAP is present universally in all extracellular amyloid deposits [Citation 2]. Its primary role in amyloidogenesis is thought to enhance the formation and deposition of amyloid fibrils by binding to the surface of amyloid fibrils calcium-dependently with the B face. On the other hand, SAP inhibits the amyloid fibril formation of Aβ [Citation 3] and enhances the refolding yield of denatured lactate dehydrogenase in vitro [Citation 4]. No convincing data or models have been published thus far to explain the discrepancy between the pro-and anti-amyloidogenic activities of SAP. In this study, we investigated the effects of CRP and SAP on amyloid fibril formation and amorphous protein aggregation in vitro [Citation 5].Materials and methodsHuman CRP and SAP were kind gifts from Professor Mark B. Pepys, UCL. Amyloid fibril formation was monitored by thioflavin T assay and electron microscopy. Amorphous protein aggregation was monitored by turbidity assay. Protein–protein interaction was assessed by enzyme-linked immunosorbent assay and western blotting analysis.ResultsWe first evaluated the assembly states of CRP and SAP in various buffer conditions. In the absence and presence of Ca 2+, CRP existed as pentamers. On the other hand, SAP assembled into the stable decamers in Ca 2+-free Tris-EDTA buffer. In MES-Ca buffer, comprised of 50 mM MES-NaOH (pH 7.0), 500 mM NaCl and 2 mM CaCl 2, SAP remained pentameric even after 72-h incubation at 37 C. We next examined the effects of CRP and SAP on amyloid fibril formation in different buffer conditions. CRP and SAP dose-dependently and substoichiometrically inhibited both Aβ (1–40) and D76N β 2-microglobulin (β2-m) fibril formation in a Ca 2+-independent manner. As calcium-free SAP exhibits a B-face-to-B-face decamer, these data may indicate that the A-face of SAP may have anti-amyloidogenic activity in a Ca 2+-independent manner. Interestingly, in the presence of Ca 2+, SAP first inhibited, then significantly accelerated D76N β2-m fibril formation (Figure 1). Electron microscopically, the surface of the D76N β2-m fibril was coated with pentameric SAP. These data suggest that SAP first exhibits antiamyloidogenic activity possibly via A face, followed by proamyloidogenic activity via B face. We next performed an enzyme-linked immunosorbent assay to assess the interaction of CRP and SAP with fresh and aggregated Aβ (1–40) and D76N β2-m. CRP and SAP interacted with fresh and preaggregated Aβ (1–40) and D76N β2-m on the fibril-forming pathway. Finally, SAP inhibited not only amyloid fibril formation but also the heat-induced amorphous aggregation of human glutathione S-transferase.