Stimulation of Ribosomal Frameshifting by Cotranslational Membrane Protein Folding and Misfolding

共翻译膜蛋白折叠和错误折叠刺激核糖体移码

• 批准号: 10032886
• 负责人: Jonathan Patrick Schlebach
• 金额: $ 31.99万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10032886
• 关键词: Alphavirus; Base Sequence; Binding; Biochemical; Biochemistry; Cells; Chloride Channels; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Delta F508 mutation; Development; Disease; Elements; Equilibrium; FDA approved; Face; Feedback; Genetic; Human; Hydrophobicity; Integral Membrane Protein; Investigation; Link; Lipid Binding; Maintenance; Maps; Measures; Mechanics; Mediating; Membrane; Membrane Proteins; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; Pathogenicity; Pharmaceutical Preparations; Play; Polyproteins; Potential Energy; Property; Protein Analysis; Protein Biosynthesis; Proteins; Proteome; Quality Control; RNA; Reaction; Ribosomal Frameshifting; Ribosomes; Role; Series; Sindbis Virus; Site; Stimulus; Stress; Structural Models; Structure; Testing; Therapeutic; Translational Regulation; Translations; Transmembrane Domain; Work; base; conformational conversion; experimental study; improved; insight; knowledge base; mechanical force; molecular modeling; mutation screening; novel; polypeptide; premature; prevent; protein degradation; protein folding; protein misfolding; proteostasis; proteotoxicity; response; small molecule; transcriptome; virology

ABSTRACT The proteostasis network relies on numerous feedback mechanisms to strike a balance between the rates of protein synthesis and degradation, which is crucial for the maintenance of protein homeostasis. Proper tuning of the rate of protein synthesis is also critical for the fidelity of cotranslational protein folding, which requires coordination between the ribosome and various molecular chaperones. This translational regulation is especially important for the fidelity of membrane protein (MP) biosynthesis, as the disruption of translational dynamics appears to coincide with cotranslational misfolding and premature degradation. Nevertheless, it is currently unclear how the translational machinery detects and responds to the cotranslational MP misfolding. In a recent study of the topological properties of the Sindbis virus (SINV) structural polyprotein, our team found that the translocon-mediated membrane integration of the nascent polypeptide stimulates ribosomal frameshifting and the premature termination of translation. This work revealed that cotranslational (mis)folding can alter translation through programmed ribosomal frameshifting (PRF), which is typically viewed as an RNA-mediated translational recoding mechanism. In the following, we outline evidence suggesting translocon-mediated PRF occurs during the translation of many human MPs, including several misfolding-prone MPs such as the cystic fibrosis transmembrane conductance regulator (CFTR). We provide multiple lines of evidence that demonstrate that PRF can occur at several “checkpoints” during CFTR synthesis, and show that a pathogenic mutation known to induce cotranslational misfolding (ΔF508) stimulates ribosomal frameshifting and the premature termination of CFTR translation. Based on these findings, we hypothesize that PRF sites allow the ribosome to tune the processivity of translation in response to conformational transitions in the nascent chain. To test this hypothesis, we will assess how mutations and small molecules that alter cotranslational CFTR folding impacts the processivity of translation at each PRF site. To gain structural insights into this ribosomal frameshifting mechanism, we will also extend our studies on the SINV structural polyprotein. To map the sequence constraints of translocon-mediated PRF, we measured the effects of 2,003 mutations on the efficiency of ribosomal frameshifting by deep mutational scanning. Our preliminary results reveal several structural features that appear to be critical for PRF, including a putative lipid-binding face within a nascent transmembrane domain and a helical segment within the ribosomal exit tunnel. To determine how these structural features induce PRF, we propose a novel fusion of molecular modeling, cellular biochemistry, and virology experiments to elucidate these structural features. Finally, we will leverage these insights to develop sequence-based energetic predictions for the efficiency of PRF within integral MPs. We will also characterize putative PRF sites in several disease-linked MPs in order to validate these findings and explore the potential role of PRF in MP homeostasis. Together, these investigations will provide fundamental insights into a novel cotranslational feedback mechanism and the molecular basis of disease.

抽象的 Proteostasis网络依靠多种反馈机制来取得平衡 蛋白质合成和降解，这对于维持蛋白质稳态至关重要 蛋白质合成的速率对于需要旋晶的共转移的保真度至关重要，这需要 核糖体和各种分子伴侣之间的配位。 对于膜蛋白（MP）生物合成的保真度很重要，作为转化动力学的破坏 似乎与共转运的错误折叠和过早降解相吻合。 尚不清楚转化机械如何检测和响应惯用的MP Sindbis病毒（SINV）结构多蛋白的拓扑特性的研究，我们的团队发现了您 新生多肽的转运介导的膜整合刺激核糖体帧速率和 翻译的终止。 通过编程的核糖体框架（PRF），通常被视为RNA介导的翻译 记录机制。 许多人类国会议员的翻译，包括闭经率高的国会议员，例如囊性firosis 跨膜电导调节剂（CFTR）。 在CFTR合成过程中可能发生在几个“检查点”，并表明一种致病性诱导 共转运错误折叠（ΔF508）刺激核糖体框架和CFTR的预终止 翻译。根据发现，我们假设PRF站点允许Ribosis调整摄影。 响应新生链中的同意转变的翻译。 评估改变共透明CFTR折叠的突变和小分子如何影响 在每个PRF位点进行翻译。 扩展我们对SINV结构蛋白的研究。 PRF，我们测量了2,003个突变对深突变的核糖体移状效率的影响 扫描。 新生的跨膜结构域内的假定脂质结合面和核糖体内的螺旋段 出口隧道。确定结构特征如何诱导PRF 建模，细胞生物化学和病毒学实验，以阐明这些诱因。 利用Thesights为Prfin积分的有效性开发基于序列的能量预测 MPS。 调查结果并探索PRF在MP稳态中的潜在作用。 对新颖的共晶反馈机制和疾病的分子基础的基本见解。