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
基本信息
- 批准号:10334403
- 负责人:
- 金额:$ 30.51万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AlphavirusBase SequenceBindingBiochemicalBiochemistryCellsChloride ChannelsComplexCystic FibrosisCystic Fibrosis Transmembrane Conductance RegulatorDefectDelta F508 mutationDevelopmentDiseaseElementsEquilibriumFDA approvedFaceFeedbackGeneticHumanHydrophobicityIntegral Membrane ProteinInvestigationLinkLipid BindingMaintenanceMapsMeasuresMechanicsMediatingMembraneMembrane ProteinsMolecularMolecular ChaperonesMolecular ConformationMutationPathogenicityPharmaceutical PreparationsPlayPolyproteinsPotential EnergyPropertyProtein AnalysisProtein BiosynthesisProteinsProteomeQuality ControlRNAReactionRibosomal FrameshiftingRibosomesRoleSeriesSindbis VirusSiteStimulusStressStructural ModelsStructureTestingTherapeuticTranslational RegulationTranslationsTransmembrane DomainWorkbaseconformational conversionexperimental studyimprovedinsightknowledge basemechanical forcemolecular modelingmutation screeningnovelpolypeptideprematurepreventprotein degradationprotein foldingprotein misfoldingproteostasisproteotoxicityresponsesmall moleculetranscriptomevirology
项目摘要
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.
摘要
蛋白质稳态网络依赖于许多反馈机制来在蛋白质稳态的速率之间取得平衡。
蛋白质的合成和降解,这对维持蛋白质的稳态至关重要。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是至关重要的,这需要
核糖体和各种分子伴侣之间的协调。这种翻译调节尤其是
重要的是膜蛋白(MP)生物合成的保真度,因为翻译动力学的破坏
似乎与共翻译错误折叠和过早降解相一致。然而,目前
目前还不清楚翻译机制如何检测和响应共翻译MP错误折叠。在最近的一
通过对辛德毕斯病毒(SINV)结构多蛋白拓扑特性的研究,我们的研究小组发现,
新生多肽的translocon介导的膜整合刺激核糖体移码,
翻译的提前终止。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,我们概述了表明translocon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,如囊性纤维化
跨膜传导调节因子(CFTR)。我们提供了多条证据,证明PRF
可以发生在CFTR合成过程中的几个“检查点”,并表明已知诱导
共翻译错误折叠(ΔF508)刺激核糖体移码和CFTR的提前终止
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
对新生链中构象转变的反应。为了验证这个假设,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
在每个PRF站点进行翻译。为了深入了解这种核糖体移码机制,我们还将
扩展了我们对SINV结构多蛋白的研究。为了定位translocon介导的
PRF,我们测量了2,003个突变对核糖体移码效率的影响,通过深度突变,
扫描。我们的初步结果揭示了几个结构特征,似乎是关键的PRF,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
出口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,开发基于序列的能量预测,以提高脉冲重复频率在积分型系统中的效率
宪兵。我们还将描述几种疾病相关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。这些调查将提供
对一种新的共翻译反馈机制和疾病的分子基础的基本见解。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Jonathan Patrick Schlebach其他文献
Jonathan Patrick Schlebach的其他文献
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{{ truncateString('Jonathan Patrick Schlebach', 18)}}的其他基金
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:
10536635 - 财政年份:2021
- 资助金额:
$ 30.51万 - 项目类别:
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 - 财政年份:2021
- 资助金额:
$ 30.51万 - 项目类别:
Topological Energetics and the Cellular Quality Control of Integral Membrane Proteins
完整膜蛋白的拓扑能量学和细胞质量控制
- 批准号:
10220073 - 财政年份:2018
- 资助金额:
$ 30.51万 - 项目类别:
Topological Energetics and the Cellular Quality Control of Integral Membrane Proteins
完整膜蛋白的拓扑能量学和细胞质量控制
- 批准号:
10437748 - 财政年份:2018
- 资助金额:
$ 30.51万 - 项目类别:
Structural Basis for the Partitioning of C99 into Liquid-Ordered Membrane Domains
C99 划分为液序膜域的结构基础
- 批准号:
8856220 - 财政年份:2014
- 资助金额:
$ 30.51万 - 项目类别:
Structural Basis for the Partitioning of C99 into Liquid-Ordered Membrane Domains
C99 划分为液序膜域的结构基础
- 批准号:
8717279 - 财政年份:2014
- 资助金额:
$ 30.51万 - 项目类别:
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