DETECT PROTEIN AGGREG ON CELL SURFACE: CONCANAVALIN A OLIGOMERS FORM

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 许多神经退行性疾病涉及蛋白质聚集和淀粉样蛋白形成。最近出现的证据表明，小短暂的prefibrillar寡聚体作为主要的病原体。值得注意的是，在用错误折叠的非致病性蛋白质处理的培养物中的细胞的行为和在病理条件下的细胞的行为之间存在严格的相似性，这种情况连同观察到的低聚物和原纤维的特征在于共同的结构特征表明，细胞毒性的共同机制可能存在，并且必须在涉及聚集的共同相互作用中仔细研究。 本文报道了刀豆球蛋白A（ConA）聚集及其对细胞作用的实验研究。在体外，接近生理温度，这种蛋白质很容易形成原纤维，涉及二级结构的变化，导致b-聚集体结构。ConA对细胞培养物的影响进行了测试，并通过共聚焦荧光显微镜研究了这些样品中蛋白质聚集体的形成。我们使用N&B分析方法来监测活细胞中ConA聚集。N&B分析显示，即使在非常低的蛋白质浓度下，ConA寡聚体也在细胞膜上快速和渐进地形成;不幸的是，细胞的形态变化表明细胞的渐进压实和死亡。细胞表面可能为聚集提供了成核位点，高的局部浓度和大分子拥挤有利于聚集，小聚集体的形成可能刺激非特异性细胞反应，这是蛋白质结构的反应区域暴露和cross-b结构逐渐形成的结果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A number of neurodegenerative diseases involve protein aggregation and amyloid formation. Recently evidence has emerged indicating small-transient prefibrillar oligomers as the primary pathogenic agents. Noteworthy, strict analogies exist between the behaviour of cells in culture treated with misfolded non-pathogenic proteins and in pathologic conditions, this instance together with the observation that the oligomers and fibrils are characterised by common structural features suggest that common mechanisms for cytotoxicity could exists and have to be perused in common interactions involved in aggregation. We here report an experimental study on ConcanavalinA (ConA) aggregation and its effects on cells. In vitro, close to physiological temperature, this protein readily forms fibrils involving secondary structure changes leading to b-aggregate structures. The effect of a ConA on cell cultures was tested and the formation of protein aggregates in these samples was studied by confocal fluorescence microscopy. We used the N&B analysis method to monitor ConA aggregation in live cells. The N&B analysis shows a rapid and progressive formation of ConA oligomers on cell membrane, even at very low protein concentration; simultaneusly, the morphology of the cell changes indicating the progressive cell compaction and death. Cell surface probably provides nucleation sites for aggregation where high local concentration and macromolecular crowding favor aggregation.The formation of small aggregates may stimulate non-specific cellular response as a result of the exposure of reactive regions of protein structure and of the progressive formation of cross-b structures.