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Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed ‘clamp-like’ mechanism of binding and inhibition
BMC Molecular and Cell Biology ( IF 2.4 ) Pub Date : 2020-12-07 , DOI: 10.1186/s12860-020-00335-5
Izanara C Pritsch 1, 2 , Irina G Tikhonova 3 , Heather L Jewhurst 2, 4 , Orla Drysdale 2 , Krystyna Cwiklinski 2, 4 , Marcelo B Molento 1, 5 , John P Dalton 2, 4 , Carolina De M Verissimo 2, 4
Affiliation  

The zoonotic worm parasite Fasciola hepatica secretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues. Immunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinant F. hepatica FhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (Ki) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and < 0.002 nM, respectively. These values are at least 1000-fold lower than those Ki obtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (Tyr46Lys47) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu66 and Glu68) that allow the propeptide to block the active site. The FhCL3 peptidase involved in host invasion by F. hepatica is produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a “clamp-like” mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.

中文翻译:

通过其前肽对Fasciola hepatica新近被囊化的幼组织蛋白酶L3(FhCL3)的调节:拟议的“钳样”结合和抑制机制

人畜共患病的蠕虫寄生虫Fasciola hepatica分泌大量组织蛋白酶L肽酶,这些酶与毒力,侵袭性,摄食和迁移有关。肽酶以无活性酶原的形式产生,可通过自动催化去除其N端前结构域或前肽在低pH下激活。前肽以高特异性结合其同源酶。然而,鲜为人知的是,感染性新近被囊泡的幼虫(NEJs)分泌的组织蛋白酶L肽酶FhCL3的前肽在成熟酶被分泌到宿主组织之前调节其抑制和激活的机制。免疫定位/免疫印迹研究表明,FhCL3酶原是由NEJs肠胃的胃真皮细胞产生和分泌的。FhCL3的重组前肽(ppFhCL3)被证明是天然和重组F.hepatica FhCL3肽酶以及组织蛋白酶L家族其他成员的高效抑制剂。FhCL1,FhCL2和FhCL3获得的抑制常数(Ki)值分别为0.04 nM,0.004 nM和<0.002 nM。这些值比人组织蛋白酶L(HsCL)和人组织蛋白酶K(HsCK)获得的Ki至少低1000倍,证明了ppFhCL3对寄生虫组织蛋白酶L的选择性。通过利用3-D结构数据,我们确定了关键的分子相互作用ppFhCL3和FhCL3成熟域之间的特异性结合。使用ppFhCL3的重组变体,我们证明了一对前肽残基(Tyr46Lys47)对于与成熟酶的前肽结合环(PBL)和其他残基(Leu66和Glu68)的相互作用至关重要,这些残基允许前肽阻断活性现场。参与肝炎链球菌侵袭的FhCL3肽酶在NEJs肠中作为酶原产生。对其激活的调节涉及前肽内的特定结合位点,这些位点相互依存并充当抑制的“钳样”机制。这些相互作用被NEJs肠的低pH破坏,从而引发自催化活化。我们的酶动力学数据表明ppFhCL3对其同源FhCL3酶具有很高的效力和选择性,这些信息可用于设计寄生虫组织蛋白酶L肽酶的抑制剂。
更新日期:2020-12-07
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