The autoactivation of human single-chain urokinase-type plasminogen activator (uPA).

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-20 DOI:10.1016/j.jbc.2023.105179
Constanza Torres-Paris, Yueyi Chen, Lufan Xiao, Harriet J Song, Pingyu Chen, Elizabeth A Komives
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Abstract

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal β-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.

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人单链尿激酶型纤溶酶原激活剂(uPA)的自动激活。
大多数丝氨酸蛋白酶是作为非活性酶原合成的,通过另一种蛋白酶的切割以严格调节的机制激活。尿激酶型纤溶酶原激活剂(uPA)和纤溶酶相互切割和激活,构成一个正反馈回路。这种相互激活循环是如何开始的一直是个谜。我们使用氢-氘交换质谱法来表征非活性单链uPA(scuPA)与其活性形式的双链uPA(tcpPA)之间的动态差异。结果表明,与scuPA相比,tcpPA的C末端β-桶和新N末端周围的区域显著降低了动力学。我们还表明,酶原scuPA是无活性的,但在储存时,在没有外部蛋白酶的情况下可以变得有活性。除了纤溶酶外,tcpPA还可以通过在K158处切割来激活scuPA,这一过程被称为自动激活。出乎意料的是,即使该位点发生突变,tcuPA也能在158位切割。TcuPA也可以在K135或K136之后在无序接头中切割scuPA,从而产生uPA的可溶性蛋白酶结构域。纤溶酶仅在K158之后以比tcpPA更快的速率切割scuPA。我们提出了uPA受体二聚化可以促进细胞表面scuPA自身活化的机制。这些结果解决了文献中围绕uPA激活机制的长期争议。
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