Chitin synthesizing supercomplexes (ChiSC) in insects: analysis of constituents and functions

甲壳素纳米纤维是不同类型的细胞外基质（包括昆虫表皮和围食基质）的高度通用的结构组分。参与几丁质生物合成的蛋白质是开发环境友好型杀虫剂的有吸引力的目标。然而，这一研究领域的发展仍处于早期阶段，由于缺乏知识的精确组成的几丁质合成复合物和功能的几丁质形成所需的蛋白质，特别是在后催化步骤。我们假设几丁质生物发生依赖于几丁质合成超复合物（ChiSC），其联合收割机结合了协调几丁质纳米原纤维形成所需的不同非催化和催化功能。我们以前的工作表明，特定的几丁质酶，几丁质结合蛋白，几丁质脱乙酰酶（参与壳聚糖形成），并可能ABC运输物理相互作用的几丁质合成酶在几丁质生物合成。我们已经开始探索ChiSC成分的一些候选成分的结构、功能和调节。为了了解昆虫ChiSC的核心成分，我们将使用两种遗传学上遥远但已建立的几丁质生物合成模式害虫赤拟谷盗（Tribolium castaneum）和飞蝗（Locusta migratoria）进行比较研究。在中德合作伙伴的共同研究努力中，实现了以下四个目标：1）鉴定新的ChiSC组分并分析ChiSC的定位、组装和拆卸; 2）表征每个ChiSC组分的功能及其对鞘翅表皮中几丁质纳米纤维组织的影响; 3）CHS和ChiSC组分的功能性共表达，以及CHS活性和界面相互作用的生化分析; 4）单个ChiSC成分和诱变形式对几丁质纳米原纤维形成动力学的影响的高速AFM分析。预期的结果不仅将增加新的知识，几丁质生物合成的基本机制的组成和其在昆虫中的作用方式，但也可能揭示潜在的目标，开发新的杀虫剂干扰几丁质合成。

Chitin nanofibers are highly versatile structural components of different types of extracellular matrices including insect cuticles and peritrophic matrices. Proteins involved in the biosynthesis of chitin are attractive targets for the development of eco-friendly insecticides. However, the development of this research field is still in its early stage due to the lack of knowledge on the precise composition of chitin-synthesizing complexes and the function of proteins necessary for chitin formation particularly at post-catalytic steps. We hypothesize that chitin biogenesis depends on chitin synthesizing supercomplexes (ChiSC), which combine different non-catalytic and catalytic functions that are required to orchestrate chitin nanofibril formation. Our previous work suggests that specific chitinases, chitin-binding proteins, chitin deacetylases (involved in chitosan formation), and possibly ABC transporters physically interact with the chitin synthase during chitin biogenesis. We have already started to explore the structure, function and regulation of some of these candidates of ChiSC constituents. To learn about the core components of the insect ChiSC, we will perform a comparative study using two phylogenetically distant but well-established model insect pests for chitin biosynthesis, Tribolium castaneum and Locusta migratoria. In an joint research effort of the Chinese and German partners, the following four objectives are addressed: 1) Identification of novel ChiSC constituents and analyses of ChiSC localization, assembly and disassembly; 2) Characterization of the function of each ChiSC component and its influence on chitin nanofibril organization in elytral cuticles; 3) Functional co-expression of CHS and ChiSC constituents, and biochemical analysis of CHS activity and interfacial interactions; 4) High-speed AFM analyses of the effects of individual ChiSC constituents and mutagenized versions on the kinetics of chitin nanofibril formation. The expected outcomes will not only add new knowledge to the composition of the basal machinery of chitin biosynthesis and its mode of action in insects, but may also reveal potential targets for developing novel insecticides interfering with chitin synthesis.