Benning Lauren N, Whisstock James C, Sun Jiuru, Bird Phillip I, Bottomley Stephen P
Department of Biochemistry and Molecular Biology, Monash University, P.O. Box 13D, Clayton, Victoria 3800, Australia.
Protein Sci. 2004 Jul;13(7):1859-64. doi: 10.1110/ps.04715304.
The metastable serpin architecture is perturbed by extremes of temperature, pH, or changes in primary sequence resulting in the formation of inactive, polymeric conformations. Polymerization of a number of human serpins in vivo leads to diseases such as emphysema, thrombosis, and dementia, and in these cases mutations are present within the gene encoding the aggregating protein. Here we show that aggregation of the human serpin, proteinase inhibitor-9 (PI-9), occurs under physiological conditions, and forms aggregates that are morphologically distinct from previously characterized serpin polymers. Incubation of monomeric PI-9 at 37 degrees C leads to the rapid formation of aggregated PI-9. Using a variety of spectroscopic methods we analyzed the nature of the structures formed after incubation at 37 degrees C. Electron microscopy showed that PI-9 forms ordered circular and elongated-type aggregates, which also bind the fluorescent dye Thioflavin T. Our data show that in vitro wild-type PI-9 forms aggregates at physiological temperatures. The biological implications of PI-9 aggregates at physiological temperatures are discussed.
亚稳态丝氨酸蛋白酶抑制剂结构会受到极端温度、pH值或一级序列变化的干扰，从而导致形成无活性的聚合构象。多种人类丝氨酸蛋白酶抑制剂在体内发生聚合会引发诸如肺气肿、血栓形成和痴呆等疾病，在这些情况下，编码聚集蛋白的基因中存在突变。在此我们表明，人类丝氨酸蛋白酶抑制剂蛋白酶抑制剂-9（PI-9）在生理条件下会发生聚集，并形成形态上不同于先前已表征的丝氨酸蛋白酶抑制剂聚合物的聚集体。将单体PI-9在37℃孵育会导致聚集的PI-9迅速形成。我们使用多种光谱方法分析了在37℃孵育后形成的结构的性质。电子显微镜显示PI-9形成有序的圆形和细长型聚集体，这些聚集体也能结合荧光染料硫黄素T。我们的数据表明，体外野生型PI-9在生理温度下会形成聚集体。文中讨论了PI-9聚集体在生理温度下的生物学意义。
