Mechanisms of mRNA translation that modulate protein aggregation

项目摘要 我们的申请题为“调节蛋白质聚集的mRNA翻译机制”， 翻译机制调节新合成的蛋白质聚集的倾向。研究 酵母和哺乳动物细胞已经表明，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， 蛋白质聚集。我们研究四环素类抗生素如米诺环素如何预防 蛋白质聚集，揭示了用米诺环素预处理细胞也可以防止热休克诱导的蛋白质聚集。 聚合来然而，与完全阻断翻译的放线菌酮相反， 将所有翻译减少-25%。米诺环素作用机制的进一步研究 调节翻译揭示米诺环素结合核糖体的40 S亚基， “核糖体负荷”，定义为每个mRNA的核糖体数量。因此，米诺环素优先 减少由重质多核糖体翻译的高表达mRNA的翻译， 影响已经低表达的mRNA。基于这些发现，我们假设，翻译的高 密度多聚核糖体通过合成数百个拷贝的蛋白质， 同样的蛋白质。这增加了聚集的风险，因为数百个新生的 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 或者在生物信号如炎症信号显著改变基因表达的情况下 导致强烈的翻译活性。虽然年轻的生物体有足够的折叠能力， 吸收翻译的突然和强烈的增加，更古老的生物可能不会，因为折叠能力 随着年龄的增长逐渐下降。米诺环素治疗，通过减少多核糖体形成，减少聚集。 在这个应用程序中，我们将产生一个丰富的画像翻译翻译的翻译状态分析和核糖体 在热冲击诱导之前进行剖析。随后通过蛋白质组学对聚集体的分析将揭示 聚集蛋白的身份，使我们能够将翻译的各个方面，如核糖体负载， 聚合来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来干扰翻译 改变经历最高负荷的一组mRNA，将揭示翻译如何调节 新合成的蛋白质聚集。这些研究的成功完成将揭示新的 调节蛋白质聚集的机会，其可用于治疗开发，包括 设计保留其抗聚集作用但缺乏抗生素活性的真核四环素。

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.