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

几丁质纳米纤维是多种细胞外基质（包括昆虫角质层和周营养基质）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保型杀虫剂的有吸引力的靶点。然而，由于缺乏对几丁质合成复合物的精确组成和几丁质形成所需的蛋白质功能的了解，特别是在催化后步骤，这一研究领域的发展仍处于早期阶段。我们假设几丁质生物发生依赖于几丁质合成超配合物（ChiSC），它结合了协调几丁质纳米纤维形成所需的不同非催化和催化功能。我们之前的研究表明，在甲壳素生物生成过程中，特定的几丁质酶、几丁质结合蛋白、几丁质去乙酰化酶（参与壳聚糖的形成）以及可能的ABC转运蛋白与几丁质合成酶发生物理相互作用。我们已经开始探索一些候选的ChiSC组分的结构、功能和调控。为了了解昆虫chic的核心成分，我们将对两种系统发育距离较远但已建立的几丁质生物合成模式害虫——castaneum Tribolium和Locusta migratoria进行比较研究。在中德合作伙伴的联合研究中，解决了以下四个目标：1)鉴定新的ChiSC成分并分析ChiSC的定位，组装和拆卸；2)分析了chsic各组分的功能及其对表皮几丁质纳米纤维组织的影响；3) CHS和ChiSC组分的功能共表达、CHS活性和界面相互作用的生化分析；4)高速原子力显微镜（AFM）分析了单个chsic成分和诱变版本对几丁质纳米纤维形成动力学的影响。预期的结果不仅将为几丁质生物合成的基本机制及其在昆虫中的作用方式的组成提供新的知识，而且可能揭示开发干扰几丁质合成的新型杀虫剂的潜在靶点。

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.