The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.
The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.
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DOI：
10.7554/elife.08887
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发表时间：
2015-12-17
期刊：
影响因子：
7.7
通讯作者：
Rampoldi L
中科院分区：
文献类型：
--
作者：
Brunati M;Perucca S;Han L;Cattaneo A;Consolato F;Andolfo A;Schaeffer C;Olinger E;Peng J;Santambrogio S;Perrier R;Li S;Bokhove M;Bachi A;Hummler E;Devuyst O;Wu Q;Jovine L;Rampoldi L
尿调蛋白是尿液中含量最丰富的蛋白质。它仅由肾上皮细胞产生，在肾功能和疾病中起关键作用。尿调蛋白主要发挥其作为细胞外基质的功能，其组装依赖于保守的特异性蛋白水解裂解，导致透明带（ZP）聚合结构域的构象活化。通过一个全面的方法，包括广泛的表征细胞模型和特定的基因敲除小鼠中的尿调节素加工，我们证明了膜结合丝氨酸蛋白酶hepsin是负责生理切割的尿调节素的酶。我们的研究结果定义了尿调节蛋白生物学的一个关键方面，并确定了第一个体内肝蛋白酶底物。hepsin作为参与ZP结构域蛋白释放的第一种蛋白酶的鉴定可能与该蛋白家族的其他成员相关，包括几种细胞外蛋白，如卵壳蛋白和内耳tectorins。http://dx.doi.org/10.7554/eLife.08887.001人类和其他动物体内的几种蛋白质含有一个称为“透明蛋白结构域”的区域。该结构域使这些蛋白质能够彼此结合并形成长纤维。尿调蛋白是五十多年前首次发现的一种蛋白质。已知这种蛋白质在高血压和肾衰竭等人类疾病中发挥作用，但尿调节素的生物学目的仍然难以捉摸。尿调蛋白仅在肾脏中产生，并且是健康个体尿液中最丰富的蛋白质。尿调蛋白还含有所谓的“外部疏水补丁”，必须在透明质酸结构域开始形成细丝之前去除。当尿调节素被一种未知的酶切割时，这种疏水性的补丁被去除;这种切割将尿调节素蛋白质的其余部分从肾脏细胞的表面释放到尿液中。Brunati等人现在已经测试了一组候选酶，并确定一种称为hepsin的酶能够切割尿调蛋白。Hepsin嵌入在肾脏细胞的细胞膜中。当在实验室培养的细胞中人为降低hepsin的水平时，尿调素仍然锚定在细胞表面，其加工过程发生改变，并且不形成细丝。Brunati等人接下来分析了编码hepsin的基因被删除的小鼠。虽然这些动物的内脏器官没有任何重大缺陷，但它们尿液中的尿调节素水平要低得多。此外，这种残留的尿蛋白没有被适当地切割，并且它没有组装成细丝。因此，这些发现揭示了hepsin是负责在尿液中释放尿调素的酶。这一发现可以用来改变尿调节素的释放水平，而进一步使用缺乏hepsin的小鼠进行研究也可能有助于理解尿调节素的生物学作用。最后，重要的是要了解hepsin或类似的酶是否也负责释放其他含有透明质酸结构域的蛋白质。DOI：http://dx.doi.org/10.7554/eLife.08887.002网站
Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins. DOI: http://dx.doi.org/10.7554/eLife.08887.001 Several proteins in humans and other animals contain a region called a 'zona pellucida domain'. This domain enables these proteins to associate with each other and form long filaments. Uromodulin is one such protein that was first identified more than fifty years ago. This protein is known to play a role in human diseases such as hypertension and kidney failure, but uromodulin’s biological purpose still remains elusive. Uromodulin is only made in the kidney and it is the most abundant protein in the urine of healthy individuals. Uromodulin also contains a so-called 'external hydrophobic patch' that must be removed before the zona pellucida domain can start to form filaments. This hydrophobic patch is removed when uromodulin is cut by an unknown enzyme; this cutting releases the rest of the uromodulin protein from the surface of the cells that line the kidney into the urine. Brunati et al. have now tested a panel of candidate enzymes and identified that one called hepsin is able to cut uromodulin. Hepsin is embedded in the cell membrane of the cells that line the kidney. When the level of hepsin was artificially reduced in cells grown in the laboratory, uromodulin remained anchored to the cell surface, its processing was altered and it did not form filaments. Brunati et al. next analysed mice in which the gene encoding hepsin had been deleted. While these animals did not have any major defects in their internal organs, they had much lower levels of uromodulin in their urine. Furthermore, this residual urinary protein was not cut properly and it did not assemble into filaments. Thus, these findings reveal that hepsin is the enzyme that is responsible for releasing uromodulin in the urine. This discovery could be exploited to alter the levels of uromodulin release, and further studies using mice lacking hepsin may also help to understand uromodulin’s biological role. Finally, it will be important to understand if hepsin, or a similar enzyme, is also responsible for the release of other proteins containing the zona pellucida domain. DOI: http://dx.doi.org/10.7554/eLife.08887.002