Mechanisms of mRNA translation that modulate protein aggregation

项目摘要 我们的题为《调节蛋白质聚集的信使核糖核酸翻译机制》的申请研究了 翻译机制调节新合成的蛋白质聚集的倾向。研究项目： 酵母和哺乳动物细胞已经表明，用蛋白质抑制剂放线菌酮对细胞进行预处理 合成，防止热休克诱导的蛋白质聚集，揭示了新合成的蛋白质的脆弱性 蛋白质聚集在一起。我们研究像米诺环素这样的四环抗生素如何预防 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 聚合。然而，与环己亚胺完全阻断翻译相反，米诺环素只 总体转换减少-25%。米诺环素作用机制的进一步研究 调节翻译显示米诺环素与核糖体的40S亚单位结合并减少 “核糖体负荷量”，定义为每条核糖体的数量。因此，米诺环素优先 减少由重多聚体翻译的高表达的mRNAs的翻译，但只有很少的 对已经低表达的mRNAs的影响。基于这些发现，我们假设翻译由高 密度多聚体通过合成数百个拷贝的蛋白质折叠机器来筛选蛋白质折叠机器的能力 在短时间内获得相同的蛋白质。这增加了聚集的风险，因为数百个新生的 蛋白质在局部竞争相同的折叠因子。这种聚集的风险很可能在 应激或炎症信号等生物信号显著改变基因表达的情况下 在一个细胞中，导致强烈的翻译活动。虽然年轻的生物体有足够的折叠能力 吸收突然而强烈的翻译增加，较老的生物体可能不会像折叠能力那样 随着年龄的增长逐渐下降。米诺环素治疗，通过减少多聚体的形成，减少聚集。 在这个应用程序中，我们将通过翻译状态分析和核糖体生成丰富的翻译画像 在引发热休克之前的侧写。随后通过蛋白质组学对聚集体的分析将揭示 聚集蛋白的特性，并允许我们将翻译的各个方面联系起来，如核糖体负载 聚合。通过将细胞暴露于米诺环素以减少核糖体负荷或肿瘤坏死因子α而干扰翻译 为了改变经历最高负荷的一组mRNAs，将揭示翻译如何调节 新合成的蛋白质聚集在一起。这些研究的成功完成将揭示新的 调节蛋白质聚集的机会，可用于治疗开发，包括 真核四环素类药物的设计保留了它们的抗聚集作用但缺乏抗生素活性。

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.