Clancy Danielle M, Henry Conor M, Davidovich Pavel B, Sullivan Graeme P, Belotcerkovskaya Ekaterina, Martin Seamus J
Molecular Cell Biology Laboratory Department of Genetics The Smurfit Institute Trinity College Dublin 2 Ireland.
Cellular Biotechnology Laboratory Saint-Petersburg State Institute of Technology Russian Federation.
FEBS Open Bio. 2016 Mar 12;6(4):338-48. doi: 10.1002/2211-5463.12044. eCollection 2016 Apr.
Recent evidence has strongly implicated IL-36 cytokines as key initiators of inflammation in the skin barrier. IL-36 cytokines belong to the extended IL-1 family and, similar to most members of this family, are expressed as inactive precursors that require proteolytic processing for activation. Because the proteases responsible for activation of members of the IL-36 subfamily have not been reported, we have developed a method for the production of biologically active IL-36 through introduction of a caspase cleavage motif, DEVD, within the N-termini of these cytokines. Here, we show that DEVD-modified IL-36α, IL-36β and IL-36γ cytokines were highly soluble and were readily processed and activated by caspase-3. Caspase-3-processed IL-36 family cytokines exhibited robust biological activity on a range of responsive cell types, including primary keratinocytes. We also generated specific polyclonal antibodies against all three IL-36 family members through immunization with purified recombinant IL-36 cytokines. The modified forms of IL-36 described herein will be useful for production of large quantities of biologically active IL-36 for structure and function studies on these important proinflammatory cytokines.
最近的证据有力地表明，白细胞介素-36（IL-36）细胞因子是皮肤屏障炎症的关键启动因子。IL-36细胞因子属于扩展的IL-1家族，与该家族的大多数成员相似，以无活性前体形式表达，需要蛋白水解加工才能激活。由于尚未报道负责激活IL-36亚家族成员的蛋白酶，我们开发了一种方法，通过在这些细胞因子的N端引入半胱天冬酶切割基序DEVD来生产具有生物活性的IL-36。在此，我们表明，DEVD修饰的IL-36α、IL-36β和IL-36γ细胞因子高度可溶，并且很容易被半胱天冬酶-3加工和激活。经半胱天冬酶-3加工的IL-36家族细胞因子在一系列反应性细胞类型（包括原代角质形成细胞）上表现出强大的生物活性。我们还通过用纯化的重组IL-36细胞因子免疫，产生了针对所有三种IL-36家族成员的特异性多克隆抗体。本文所述的IL-36修饰形式将有助于大量生产具有生物活性的IL-36，用于对这些重要促炎细胞因子的结构和功能研究。
