【作者】 孔凡楼；

【导师】 梁毅；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 透析相关性淀粉样疾病是经过长期血液透析后,出现的一种以渐进性丧失行动能力为临床特征、关节和骨骼及内脏出现由β2微球蛋白(β2M)形成的淀粉样纤维为病理特征的非神经性疾病。国内外已对β2M形成纤维做了很多实验研究,但它在体内形成淀粉样纤维的机制及其在透析相关性疾病中的病理学作用至今仍不清楚。透析相关性疾病还涉及到一些其它因素的存在,目前从透析相关性疾病病变组织中的淀粉样纤维分离得到的p2M主要是经晚期糖基化修饰过的β2M,然而糖基化修饰在β2M的错误折叠和聚集中起到的作用并不清晰。我们研究了晚期糖基化修饰诱导β2M发生聚集的分子机制。SDS-PAGE实验结果表明,糖基化试剂D-核糖很快与β2M发生反应,并对其晚期糖基化修饰,进而生成二聚体、三聚体及更高分子量的聚集体。MALD-TOF质谱实验结果显示,糖基化3天的β2M分子量与未被修饰的β2M分子量相比增加了803Da,表明此时约有6个核糖分子因修饰而连接到一个β2M分子上了,同时糖基化过程中有大量的二聚体合三聚体生成。荧光和Western bolt实验证实了在糖基化过程中生成了晚期糖基化终产物。总之,我们发现D-核糖能快速晚期糖基化修饰的β2M,晚期糖基化修饰诱导β2M发生聚集,生成具有ThT结合特性的聚集体。该聚集体具有明显的蛋白酶解抗性。接着我们对糖基化修饰诱导p2M生成的聚集体的细胞毒性进行了研究。该聚集体对人神经瘤母细胞SH-SY5Y及正常人包皮成纤细胞FS-2具有很高的细胞毒性,而且这种细胞毒性具有浓度依赖性：随着糖基化孵育时间的增加,糖基化β2M聚集体生成得越多,细胞毒性也越强。Annexin-V FITC和PI双染流式细胞技术检测到该聚集体诱导的凋亡和坏死的细胞比例远远高于对照及正常细胞组中的凋亡和坏死细胞的比例,Hoechst33342荧光染色结果也证实了糖基化p2M聚集体能诱导SH-SY5Y细胞凋亡。1.0mM抗氧化剂N-乙酰半胱氨酸处理能清除胞内活性氧簇(ROS),从而阻止该聚集体诱导的SH-SY5Y及FS-2细胞的凋亡,显示糖基化β2M聚集体发挥细胞毒性遵循胞内ROS介导的机制。总之,我们发现糖基化修饰诱导β2M生成的聚集体具有蛋白酶解抗性和很高的细胞毒性,诱导细胞生成胞内ROS,进而诱导细胞发生凋亡。由于生成的聚集体具有蛋白酶解抗性,一旦其在体内形成,就会存在很长时间,同时该聚集体具有很高的细胞毒性,会对人体所造成较大的伤害,因此晚期糖基化修饰的p2M生成的聚集体可能是透析相关性淀粉样纤维疾病发病的因素之一。我们使用大分子拥挤试剂Ficoll70和dextran70来模拟生理拥挤环境,研究了大分子拥挤对β2M错误折叠的影响。我们的实验结果表明,大分子拥挤显著促进了β2M形成淀粉样纤维的成核步骤和延伸步骤,分别表现为成核时间的极大缩短和延伸速率的明显增加。大分子拥挤也明显抑制了成熟β2M淀粉样纤维的解聚,而且这种抑制效果对大分子拥挤试剂具有浓度依赖性,相同浓度的Ficoll70和dextran70的抑制效果则几乎相同。这表明生理拥挤环境中,p2M更容易发生错误折叠,而且生成的淀粉样纤维结构更加稳定更不易解聚,因此p2M所处的生理拥挤环境(如血清和骨滑液)在β2M形成淀粉样纤维的进程中起到重要的作用,因此我们在研究透析相关淀粉样疾病发病机理时也需要考虑生理拥挤环境这一重要因素。更多还原

【Abstract】 β2-Microglobulin (β2M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). Although glycated P2M is found as a major component of the amyloid deposits in HAA, the effect of glycation on the misfolding and aggregation of β2M has not been studied so far. D-ribose, present in all living cells and blood, is not only an efficient glycating agent, but also an essential component for energy production in the human body. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β2M in vitro. We find that glycating agent D-ribose interacts with human β2M to generate AGEs which form aggregates in a time-dependent manner. The molecular mass of ribosylated β2M monomer incubated for3days increased up to12,694Da. The extra803Da indicated that about6ribose groups were bound to β2M on average, compared with that of the native P2M. Ribosylated β2M molecules are highly oligomerized compared with unglycated β2M, and have a granular morphology. Furthermore, we demonstrated that ribosylated β2M aggregates could not be digested by trypsin and showed increased resistance to PK. Therefore, once glycated β2M aggregates have formed, they are difficult to be degraded by proteases and can persist in human tissues for a long period.Next, we employed MTT reduction assay to measure the cytotoxicity of ribosylated β2M to human SH-SY5Y neuroblastoma cells and human foreskin fibroblast FS2cells. Because glycated β2M is the major component of the amyloid deposits in HAA, we tried to define the cytotoxic role of ribosylated β2M aggregates. Our data demonstrate that ribosylated P2M aggregates are highly toxic to both SH-SY5Y cells and FS2cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2cells, indicating that the cytotoxicity of ribosylated β2M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, D-ribose reacts with β2M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. In the light of the higher cytotoxicity of ribosylated β2M aggregates than unglycated β2M aggregates, once glycated β2M aggregates have formed, they can persist in human tissues for a long time period and can be more harmful to cells than unglycated β2M aggregates, emphasizing the need for lowering circulating glycated β2M levels in dialysis patients. These findings suggest that ribosylated β2M aggregates could contribute to the dysfunction and death of cells and could play an important role in pathogenesis of β2M-associated diseases such as HAA.Finally, we investigated the effects of macromolecular crowding agents, Ficoll70and dextran70, on the depolymerization of amyloid fibrils at pH7.5, as well as on fibril formation by P2M at an acidic pH, using thioflavin T binding assays, transmission electron microscopy and circular dichroism. On the one hand, depolymerization of β2M amyloid fibrils formed by β2M was inhibited by Ficoll70and dextran70in a dose-dependent way. On the other hand, macromolecular crowding dramatically accelerated amyloid formation by monomeric β2M at an acidic pH. A sigmoidal equation has been used to fit these kinetic data, yielding lag times and apparent rate constants for the growth of fibrils of β2M. These biochemical data indicate that macromolecular crowding significantly accelerated the nucleation step of P2M fibril formation. The above results suggest that crowded physiological environment could enhance the deposition of β2M amyloid fibrils in vivo, possibly by accelerating the formation of β2M amyloid fibrils and inhibiting depolymerization of β2M amyloid fibrils at the same time.更多还原