Modulation of Protein Biogenesis and Secretion by Natural Product Translocon Ligands

真核细胞中的大多数分泌蛋白和膜蛋白在核糖体对接在分泌途径的起点后，要么跨过内质网膜，要么整合到内质网膜上。这一过程可以被真菌、蓝藻和人类致病菌产生的一小群大环天然产物选择性和可逆地阻断。这些专门的NP代谢物直接与Sec61转运子通道结合，以抑制内质网新生蛋白的共翻译易位，导致内质网和胞浆中细胞蛋白平衡的丧失。这组NP化合物如何通过不同程度的蛋白质底物选择性地选择性或广泛地抑制蛋白质生物合成尚不清楚。蛋白质平衡的维持在哺乳动物细胞中是一个高度调控的过程，细胞分泌途径中动态平衡的丧失与人类主要疾病如癌症、神经退行性变和糖尿病有关。因此，我们发现了新的有效的NP Sec61抑制剂，这为了解蛋白质输入内质网分泌途径的机制提供了机会，并基于这些来自大自然的复杂化学实体提供了新的Sec61配体和潜在的药物先导。我们计划利用包括NP发现、大环固相肽合成、药理学、化学和结构生物学在内的多学科方法来追求以下两个目标：1)扩大和定义针对共翻译易位的非极性NP衍生大环的类别；2)通过结构活性关系(SAR)和低温电子显微镜(CryoEM)的分析来阐明Sec61抑制的机制基础。在目标1中，现有的NP文库可能富含非极性的中等大环，将使用表型和基于目标的分析方法筛选新的蛋白平衡调节剂，以抑制Sec61依赖的ER转位。利用基于LCMS2的代谢组学、核磁共振波谱和化学信息学早期确定结构基序将指导NP分子家族中不同代表的固相肽的合成，为结构生物学研究提供发现Sec61配体的平台。在目标2中，我们建议用冷冻EM法通过分析具有不同Sec61底物特异性的密切相关合成化合物的组间Sec61结合界面来研究两个不同NP分子家族的Sec61结合。这一多维方法将揭示靶向细胞蛋白平衡以满足治疗需求的可行性，同时避免由于非特异性抑制分泌蛋白生物合成而产生的毒性。

The majority of secreted and membrane proteins in eukaryotic cells are either translocated across or integrated into the ER membrane after the ribosome has docked at the start of the secretory pathway. This process can be selectively and reversibly blocked by a small group of macrocyclic natural products (NPs) produced by fungi, cyanobacteria and human pathogenic bacteria. These specialized NP metabolites bind directly to the Sec61 translocon channel to inhibit co-translational translocation of nascent proteins at the ER resulting in a loss of cellular proteostasis both in the ER and cytosol. Exactly how this group of NP compounds induces either selective or broad inhibition of protein biosynthesis with varying degrees of protein substrate selectivity is not clear. Maintenance of proteostasis is a highly regulated process in mammalian cells and the loss of homeostasis in the cellular secretory pathway is implicated in major human diseases such as cancer, neurodegeneration and diabetes. Thus, our discovery of new potent NP Sec61 inhibitors presents an opportunity to both understand the mechanistic basis of protein import into the ER secretory pathway and provide a reservoir of new Sec61 ligands and potential drug leads based on these complex chemical entities from Nature. We plan to utilize a multidisciplinary approach that includes NP discovery, solid-phase peptide synthesis of macrocycles, pharmacology, chemical and structural biology to pursue the following two aims: 1) Expand and define the class of nonpolar NP-derived macrocycles that target cotranslational translocation; 2) Elucidate the mechanistic basis of Sec61 inhibition using analysis of structure activity relationships (SAR) and cryogenic electron microscopy (cryoEM). In Aim 1, existing NP libraries likely to be rich in non-polar medium-sized macrocycles will be screened for new proteostasis modulators using phenotypic and target-based assays for Sec61-dependent inhibition of ER translocation. Early determination of structural motifs using LCMS2-based metabolomics, NMR spectroscopy and cheminformatics will guide the solid phase peptide synthesis of divergent representatives of NP molecular families to provide a platform for discovery of Sec61 ligands for structural biology studies. In Aim 2 we propose to use cryo-EM to study the specific Sec61 binding of two different NP molecular families by analyzing the Sec61 binding interface of suites of closely related synthetic compounds that have different Sec61 substrate specificity. This multidimensional approach will reveal the feasibility of targeting cellular proteostasis for therapeutic needs, while avoiding toxicities due to non-specific inhibition of secretory protein biosynthesis.