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The kinetics of islet amyloid polypeptide phase-separated system and hydrogel formation are critically influenced by macromolecular crowding
Biochemical Journal ( IF 4.4 ) Pub Date : 2021-08-13 , DOI: 10.1042/bcj20210384
Lior Pytowski 1 , David Vaux 1 , Létitia Jean 1
Affiliation  

Many protein misfolding diseases (e.g. type II diabetes and Alzheimer's disease) are characterised by amyloid deposition. Human islet amyloid polypeptide (hIAPP, involved in type II diabetes) spontaneously undergoes liquid–liquid phase separation (LLPS) and a kinetically complex hydrogelation, both catalysed by hydrophobic–hydrophilic interfaces (e.g. air–water interface and/or phospholipids–water interfaces). Gelation of hIAPP phase-separated liquid droplets initiates amyloid aggregation and the formation of clusters of interconnected aggregates, which grow and fuse to eventually percolate the whole system. Droplet maturation into irreversible hydrogels via amyloid aggregation is thought to be behind the pathology of several diseases. Biological fluids contain a high volume fraction of macromolecules, leading to macromolecular crowding. Despite crowding agent addition in in vitro studies playing a significant role in changing protein phase diagrams, the mechanism underlying enhanced LLPS, and the effect(s) on stages beyond LLPS remain poorly or not characterised.We investigated the effect of macromolecular crowding and increased viscosity on the kinetics of hIAPP hydrogelation using rheology and the evolution of the system beyond LLPS by microscopy. We demonstrate that increased viscosity exacerbated the kinetic variability of hydrogelation and of the phase separated-aggregated system, whereas macromolecular crowding abolished heterogeneity. Increased viscosity also strengthened the gel meshwork and accelerated aggregate cluster fusion. In contrast, crowding either delayed cluster fusion onset (dextran) or promoted it (Ficoll). Our study highlights that an in vivo crowded environment would critically influence amyloid stages beyond LLPS and pathogenesis.

中文翻译:

胰岛淀粉样多肽相分离系统的动力学和水凝胶的形成受到大分子拥挤的严重影响

许多蛋白质错误折叠疾病(例如 II 型糖尿病和阿尔茨海默病)的特征是淀粉样蛋白沉积。人胰岛淀粉样多肽(hIAPP,参与 II 型糖尿病)自发地经历液-液相分离(LLPS)和动力学复杂的水凝胶化,两者均由疏水-亲水界面(例如空气-水界面和/或磷脂-水界面)催化. hIAPP 相分离液滴的凝胶化引发淀粉样蛋白聚集和相互连接的聚集体簇的形成,这些聚集体生长并融合以最终渗透整个系统。液滴通过淀粉样蛋白聚集成熟为不可逆的水凝胶被认为是几种疾病的病理学背后的原因。生物流体含有高体积分数的大分子,导致大分子拥挤。尽管体外研究中添加拥挤剂在改变蛋白质相图方面发挥了重要作用,但增强 LLPS 的机制以及对 LLPS 以外阶段的影响仍然很差或没有表征。 我们研究了大分子拥挤和粘度增加的影响使用流变学研究 hIAPP 水凝胶化的动力学,并通过显微镜观察系统在 LLPS 之外的演变。我们证明增加的粘度加剧了水凝胶化和相分离-聚集系统的动力学变异性,而大分子拥挤消除了异质性。增加的粘度也加强了凝胶网状结构并加速了聚集体簇融合。相比之下,拥挤要么延迟簇融合开始(葡聚糖)要么促进它(Ficoll)。
更新日期:2021-08-13
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