Stimulation of Ribosomal Frameshifting by Cotranslational Membrane Protein Folding and Misfolding

摘要 蛋白质平衡网络依赖于众多的反馈机制，以在 蛋白质的合成和降解，这是维持蛋白质动态平衡的关键。适当地调整 蛋白质合成的速度对共翻译蛋白质折叠的保真度也是至关重要的，这需要 核糖体与各种分子伴侣之间的配位。这种翻译性的规定尤其 对膜蛋白(MP)生物合成的保真度很重要，因为翻译动力学的中断 似乎与共翻译错误折叠和过早降解相吻合。尽管如此，目前 不清楚翻译机制如何检测和响应协译MP的错误折叠。在最近一次 在研究辛德比斯病毒(SINV)结构多蛋白的拓扑性质时，我们的团队发现 转位子介导的新生多肽的膜整合刺激核糖体移码和 翻译的过早终止。这项工作揭示了共翻译(错位)折叠可以改变翻译 通过程序性核糖体移码(PRF)，这通常被视为RNA介导的翻译 重新编码机制。在下文中，我们概述了转位蛋白介导的PRF发生在 许多人类MPS的翻译，包括几个容易错误折叠的MPS，如囊性纤维化 跨膜电导调节器(CFTR)。我们提供了多条证据来证明PRF 可以发生在CFTR合成过程中的几个“检查点”，这表明一种已知的致病突变可以诱导 共翻译错误折叠(ΔF508)刺激核糖体移码和cftr提前终止 翻译。基于这些发现，我们假设PRF位点允许核糖体调节加工能力 翻译是对新生链中构象转变的反应。为了检验这一假设，我们将 评估改变共翻译cftr折叠的突变和小分子如何影响 在每个PRF站点进行翻译。为了从结构上深入了解这种核糖体移帧机制，我们还将 扩大我们对SINV结构多蛋白的研究。定位转座子介导的序列约束 PRF，我们测量了2,003个突变对深度突变核糖体移码效率的影响 扫描中。我们的初步结果揭示了几个似乎对PRF至关重要的结构特征，包括 核糖体中新生的跨膜区和螺旋片段中的一个假定的脂质结合面 出口隧道。为了确定这些结构特征是如何导致PRF的，我们提出了一种新的分子融合 建模、细胞生物化学和病毒学实验，以阐明这些结构特征。最后，我们会 利用这些洞察力来开发基于序列的能量预测，以提高PRF的整体效率 国会议员。我们还将在几个与疾病相关的MPS中鉴定假定的PRF位点，以验证这些 发现并探索PRF在MP动态平衡中的潜在作用。总而言之，这些调查将提供 对一种新的协译反馈机制和疾病的分子基础的基本见解。

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.