Mechanisms of mRNA translation that modulate protein aggregation

调节蛋白质聚集的 mRNA 翻译机制

• 批准号: 9585954
• 负责人: Michael Petrascheck
• 金额: $ 29.03万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9585954
• 关键词: Age; Alzheimer' s Disease; Amyloid beta-Protein; Antibiotics; Attenuated; Binding; Biological; Cells; Cycloheximide; Data; Disease; Elements; Environment; Etiology; Eukaryota; Gene Expression; Genetic; Genetic Translation; Heat shock proteins; Heat-Shock Proteins 70; Heat-Shock Response; Image; Inflammatory; Investigation; Link; Mammalian Cell; Messenger RNA; Minocycline; Minor; Modeling; Molecular Chaperones; Organism; Parkinson Disease; Phenotype; Polyribosomes; Portraits; Protein Biosynthesis; Protein-Folding Disease; Proteins; Proteomics; Ribosomes; Risk; Set protein; Signal Transduction; Stress; TNF gene; Techniques; Tetracyclines; Therapeutic; Time; Translating; Translation Process; Translations; Work; Yeasts; alpha synuclein; base; density; design; experience; inhibitor/antagonist; neuron loss; prevent; programs; protein aggregate; protein aggregation; protein folding; proteostasis; ribosome profiling; therapeutic development

Project Summary Our application entitled “Mechanisms of mRNA translation that modulate protein aggregation” investigates how mechanisms of translation modulate the propensity of newly synthesized proteins to aggregate. Studies in yeast and mammalian cells have shown that pretreatment of cells with cycloheximide, an inhibitor of protein synthesis, prevents heat shock-induced protein aggregation, revealing a vulnerability of newly synthesized proteins to aggregate. Our mechanistic work investigating how tetracyclic antibiotics like minocycline prevent protein aggregation, revealed that pre-treating cells with minocycline also prevents heat shock-induced protein aggregation. However, in contrast to cycloheximide, which blocks translation completely, minocycline only reduces over-all translation by -25%. Further investigations into the mechanism by which minocycline modulates translation revealed minocycline to bind to the 40S subunit of the ribosome and to reduce `ribosomal load,' defined as the number of ribosomes per mRNA. Consequently, minocycline preferentially reduces translation of highly expressed mRNAs that are translated by heavy polysomes but has very little effect on already lowly expressed mRNAs. Based on these findings, we hypothesize that translation by high density polysomes strains the capacity of the protein folding machinery by synthesizing hundreds of copies of the same protein in a short period of time. This increases the risk of aggregation as hundreds of nascent proteins locally compete for the same folding factors. This risk of aggregation is likely to be intensified during stress or in cases in which biological signals such as inflammatory signals dramatically alter gene expression in a cell, leading to intense translational activity. While young organisms have sufficient folding capacity to absorb a sudden and intense increase in translation, older organisms might not, as the folding capacity has progressively declined with age. Minocycline treatment, by reducing polysome formation, reduces aggregation. In this application we will generate a rich portrait of translation by translational state analysis and ribosome profiling before the induction of a heat shock. Subsequent analysis of the aggregates by proteomics will reveal the identity of the aggregating proteins and allow us to link aspects of translation such as ribosomal load to aggregation. Perturbing translation by exposing the cells to minocycline to reduce ribosomal load, or to TNFα to alter the set of mRNAs experiencing the highest load, will reveal how translation modulates the propensity of newly synthesized proteins to aggregate. The successful completion of these studies will reveal new opportunities to modulate protein aggregation that can be exploited for therapeutic development including the design of eukaryotic tetracyclines retaining their anti-aggregation effects but lacking antibiotic activity.

项目概要 我们的申请题为“调节蛋白质聚集的 mRNA 翻译机制”，研究了如何 翻译机制调节新合成蛋白质聚集的倾向。 酵母和哺乳动物细胞表明，用放线菌酮（一种蛋白质抑制剂）预处理细胞 合成，防止热休克诱导的蛋白质聚集，揭示了新合成的脆弱性 我们的机制研究米诺环素等四环抗生素如何预防。 蛋白质聚集，表明用米诺环素预处理细胞也可以防止热休克诱导的蛋白质 然而，与完全阻断翻译的放线菌酮相比，米诺环素仅能聚集。 进一步研究米诺环素的作用机制，使总体翻译减少-25%。 调节翻译显示米诺环素与核糖体的 40S 亚基结合并减少 “核糖体负荷”定义为每个测试的 mRNA 的核糖体数量，优先使用米诺环素。 减少由重多核糖体翻译的高表达 mRNA 的翻译，但几乎没有 对已经低表达的 mRNA 的影响 基于这些发现，我们追求高表达的翻译。 密度多核糖体通过合成数百个拷贝来增强蛋白质折叠机制的能力 相同的蛋白质在短时间内聚集，这增加了数百个新生蛋白质聚集的风险。 蛋白质局部竞争相同的折叠因子，这种聚集的风险可能会在过程中加剧。 压力或炎症信号等生物信号显着改变基因表达的情况 在细胞中，导致强烈的翻译活动，而年轻的生物体具有足够的折叠能力 吸收突然而强烈的翻译增加，较老的生物体可能不会，因为折叠能力已经 随着年龄的增长，米诺环素治疗通过减少多核糖体的形成而逐渐减少。 在此应用程序中，我们将通过翻译状态分析和核糖体生成丰富的翻译图像 随后通过蛋白质组学对聚集体进行分析将揭示。 聚集蛋白的身份，并允许我们将翻译的各个方面（例如核糖体负载）与 通过将细胞暴露于米诺环素以减少核糖体负荷或暴露于 TNFα 来扰乱翻译。 改变经历最高负载的 mRNA 集，将揭示翻译如何调节 新合成的蛋白质聚集体的成功完成将揭示新的内容。 调节蛋白质聚集的机会可用于治疗开发，包括 真核四环素的设计保留了其抗聚集作用但缺乏抗生素活性。