Stimulation of Ribosomal Frameshifting by Cotranslational Membrane Protein Folding and Misfolding

摘要 蛋白质稳态网络依赖于众多的反馈机制来在不同的速率之间取得平衡。 蛋白质的合成和降解，这对于维持蛋白质稳态至关重要。适当调整 蛋白质合成的速率对于共翻译蛋白质折叠的保真度也至关重要，这需要 核糖体和各种分子伴侣之间的协调。这种翻译规则特别 由于翻译动力学的破坏，对于膜蛋白 (MP) 生物合成的保真度非常重要 似乎与共翻译错误折叠和过早降解相一致。尽管如此，目前 目前尚不清楚翻译机制如何检测并响应共翻译 MP 错误折叠。在最近的一次 通过对辛德毕斯病毒（SINV）结构多蛋白拓扑特性的研究，我们团队发现 易位子介导的新生多肽的膜整合刺激核糖体移码 翻译提前终止。这项工作揭示了共翻译（错误）折叠可以改变翻译 通过程序化核糖体移码 (PRF)，这通常被视为 RNA 介导的翻译 重新编码机制。在下文中，我们概述了表明易位子介导的 PRF 发生在 许多人类 MP 的翻译，包括几种容易错误折叠的 MP，例如囊性纤维化 跨膜电导调节器（CFTR）。我们提供了多种证据来证明 PRF CFTR 合成过程中的几个“检查点”可能发生，并表明已知会诱导的致病突变 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持续合成能力 响应新生链中构象转变的翻译。为了检验这个假设，我们将 评估改变共翻译 CFTR 折叠的突变和小分子如何影响 CFTR 的持续合成能力 每个 PRF 站点的翻译。为了获得对这种核糖体移码机制的结构见解，我们还将 扩展我们对 SINV 结构多蛋白的研究。绘制易位子介导的序列限制 PRF，我们通过深度突变测量了 2,003 个突变对核糖体移码效率的影响 扫描。我们的初步结果揭示了几个对 PRF 至关重要的结构特征，包括 新生跨膜结构域内的假定脂质结合面和核糖体内的螺旋片段 退出隧道。为了确定这些结构特征如何诱导 PRF，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来阐明这些结构特征。最后，我们将 利用这些见解来开发基于序列的能量预测，以提高积分内 PRF 的效率 议员们。我们还将描述几种与疾病相关的 MP 中假定的 PRF 位点，以验证这些 研究结果并探讨 PRF 在 MP 稳态中的潜在作用。这些调查将共同提供 对新型共翻译反馈机制和疾病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.