弗林,一种专门的内切蛋白酶,将前蛋白转化为生物活性蛋白。弗林蛋白酶的功能对于正常细胞以及包括恶性肿瘤和炭疽在内的多种病理是重要的。弗林蛋白酶被认为在高尔基区室和细胞表面之间循环。迄今为止,细胞对炭疽保护性抗原83(PA83)的加工被认为是存在大量细胞表面弗林蛋白酶的证据。为了监测弗林,我们设计了一种裂解激活的FRET生物传感器,其中增强的青色和黄色荧光蛋白通过源自炭疽PA83的肽序列SNSRKKR/STSAGP连接。由于炭疽序列对弗林蛋白酶蛋白水解和基于FRET的检测的敏感性和选择性,生物传感器记录了体外反应和基于细胞的测定中弗林蛋白酶的毫摩尔水平。使用由于其大小以及其他相关方法而无法渗透细胞的生物传感器,我们确定了非常低的水平,如果有的话,细胞表面的弗林蛋白酶存在于完整细胞和强制弗林蛋白酶过表达的细胞中。这一观察结果与现有的关于弗林蛋白酶在细胞表面和炭疽疾病机制的概念形成鲜明对比。接下来,我们使用基于细胞的测试证明,PA83实际上,在细胞外环境中被弗林蛋白酶处理,然后才产生的PA63结合炭疽毒素细胞表面受体。我们还确定生物传感器,但不是传统的肽底物,允许连续监测癌细胞提取物中的弗林蛋白酶活性。我们的结果表明,没有生理相关水平的细胞表面弗林蛋白酶,因此,炭疽病的机制应该重新调查.此外,生物传感器的可用性是对癌细胞中弗林蛋白酶活性进行非侵入性监测的基础。概念上,我们开发的生物传感器可以作为其他蛋白酶激活生物传感器的原型。
Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins.
Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax.
Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR / STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to
furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced
furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors.