Modulation of Protein Biogenesis and Secretion by Natural Product Translocon Ligands

天然产物易位子配体对蛋白质生物发生和分泌的调节

• 批准号: 10735736
• 负责人: JANE E ISHMAEL
• 金额: $ 44.25万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735736
• 关键词: Binding; Biogenesis; Biological Assay; Cell secretion; Cells; Cellular Membrane; Chemicals; Chronic Disease; Client; Complex; Cryoelectron Microscopy; Cyanobacterium; Cytosol; Databases; Development; Diabetes Mellitus; Dimensions; Disease; Docking; Elements; Eukaryotic Cell; Family; Future; Goals; Homeostasis; Human; Inflammatory; Knowledge; Lateral; Length; Libraries; Ligand Binding; Ligands; Luciferases; Maintenance; Malignant Neoplasms; Mammalian Cell; Membrane; Membrane Proteins; Modeling; Molecular; Multi-Institutional Clinical Trial; NMR Spectroscopy; Natural Products; Nature; Nerve Degeneration; Organism; Pathway interactions; Peptide Synthesis; Pharmaceutical Preparations; Pharmacology; Phase; Phenotype; Physiological; Process; Production; Protein Biosynthesis; Protein Dynamics; Protein Import; Protein Inhibition; Protein Secretion; Proteins; Proteome; Reporter; Resolution; Ribosomes; Scheme; Series; Solid; Solid Neoplasm; Structure; Structure-Activity Relationship; Substrate Specificity; Testing; Therapeutic; Therapeutic Agents; Toxic effect; Translating; Translations; Variant; Work; Yeasts; cellular targeting; chemical synthesis; cheminformatics; fungus; human disease; inhibitor; interdisciplinary approach; lipophilicity; metabolomics; novel therapeutics; pathogenic bacteria; pharmacologic; polypeptide; proteostasis; scaffold; screening; secretory protein; small molecule; structural biology; therapeutic target; tool

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.

真核细胞中的大多数分泌蛋白和膜蛋白在核糖体停靠在分泌途径的起点后，要么跨内质网膜易位，要么整合到内质网膜中。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（NPs）选择性地和可逆地阻断。这些特殊的NP代谢物直接与Sec61易位通道结合，抑制内质网新生蛋白的共翻译易位，从而导致内质网和细胞质中细胞蛋白质稳态的丧失。确切地说，这组NP化合物是如何诱导选择性或广泛抑制蛋白质生物合成的不同程度的蛋白质底物选择性尚不清楚。在哺乳动物细胞中，维持蛋白质平衡是一个高度调控的过程，细胞分泌途径中稳态的丧失与癌症、神经变性和糖尿病等主要人类疾病有关。因此，我们发现新的强效NP Sec61抑制剂提供了一个机会，既可以了解蛋白质输入内质网分泌途径的机制基础，又可以提供一个新的Sec61配体库和基于这些自然界复杂化学实体的潜在药物先导。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实现以下两个目标：1)扩展和定义针对共翻译易位的非极性NP衍生大环的类别；2)利用结构活性关系分析（SAR）和低温电镜（cryogenic electron microscopy, cryoEM）阐明Sec61抑制的机制基础。在Aim 1中，现有的NP文库可能富含非极性中型大环，将使用表型和基于靶标的分析来筛选新的蛋白质稳态调制剂，以抑制sec61依赖性内质网易位。利用基于lcms2的代谢组学、核磁共振波谱和化学信息学技术，早期确定结构基序，将指导NP分子家族不同代表的固相肽合成，为结构生物学研究发现Sec61配体提供平台。在Aim 2中，我们提出通过分析具有不同Sec61底物特异性的密切相关的合成化合物组合的Sec61结合界面，使用冷冻电镜研究两个不同NP分子家族的特异性Sec61结合。这种多维度的方法将揭示针对治疗需要的细胞蛋白静止的可行性，同时避免由于分泌蛋白生物合成的非特异性抑制而产生的毒性。

项目成果

TSignal: a transformer model for signal peptide prediction.

• DOI: 10.1093/bioinformatics/btad228
• 发表时间: 2023-06-30
• 期刊: Bioinformatics (Oxford, England)

Design of Synthetic Surrogates for the Macrolactone Linker Motif in Coibamide A.

• DOI: 10.1021/acsmedchemlett.3c00232
• 发表时间: 2023-10-12
• 期刊: ACS MEDICINAL CHEMISTRY LETTERS
• 影响因子: 4.2
• 作者: Suzuki, Rikito;Mattos, Daphne R.;Kitamura, Takashi;Tsujioka, Rina;Kobayashi, Kazuya;Inuki, Shinsuke;Ohno, Hiroaki;Ishmael, Jane E.;McPhail, Kerry L.;Oishi, Shinya
• 通讯作者: Oishi, Shinya

Design of Coibamide A Mimetics with Improved Cellular Bioactivity.

• DOI: 10.1021/acsmedchemlett.1c00591
• 发表时间: 2022-01-13
• 期刊: ACS medicinal chemistry letters
• 影响因子: 4.2
• 作者: Kitamura T;Suzuki R;Inuki S;Ohno H;McPhail KL;Oishi S
• 通讯作者: Oishi S