Erythrocyte maturation through global remodeling of the proteome

通过蛋白质组的整体重塑实现红细胞成熟

• 批准号: 10211683
• 负责人: MARK D FLEMING
• 金额: $ 59.84万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211683
• 关键词: Actinin; Actins; Address; Anabolism; Automobile Driving; Binding; Biochemical; Biochemistry; Biological Process; Biology; Bypass; Cell Differentiation process; Cells; Cellular biology; Centrosome; Complex; Crystallins; Crystallography; Cytoskeletal Modeling; Cytoskeletal Proteins; Cytoskeleton; Data; Development; Dynein ATPase; Enzymes; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Eukaryotic Cell; Family; Generations; Genes; Genetic Structures; Genetic Translation; Globin; Goals; Hemoglobin; Hydrogen; In Vitro; Informatics; Lens Fiber; Leucine-Rich Repeat; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Microscopy; Microtubules; Mitochondrial Proteins; Motor Activity; Mus; Mutate; Myosin ATPase; Network-based; Phase; Physiological; Play; Process; Proteasome Binding; Proteins; Proteome; Proteomics; Radial; Regulatory Pathway; Reticulocytes; Ribosomes; Role; Signal Transduction; Specificity; Spectrin; Structure; System; Testing; Time; Translational Regulation; Translations; Tubulin; Ubiquitin; Ubiquitin Like Proteins; Ubiquitin-Conjugating Enzymes; Ubiquitination; Variant; Vesicle; Work; base; cell type; crosslink; dynactin; erythroid differentiation; fiber cell; genetic regulatory protein; interest; milligram; multicatalytic endopeptidase complex; mutant; novel; preservation; programs; protein crosslink; protein degradation; reconstitution; ribosome profiling; structural biology; transcriptome sequencing; transcriptomics; ubiquitin ligase

PROJECT SUMMARY / ABSTRACT As cells undergo extreme forms of terminal differentiation, they are able to accumulate specific proteins to exceptionally high levels–hundreds of milligrams per ml in the case of globins and crystallins. At the same time, almost all other cellular components are eliminated. How cells can carry out such vast programs of biosynthesis and degradation simultaneously has been almost a complete mystery. We proposed in 1995 that the ubiquitin-proteasome system (UPS) may play a central role in global proteome remodeling. Using murine reticulocytes, a uniquely powerful system to study global proteome remodeling, we found that indeed UBE2O, a ubiquitin-conjugating enzyme that is strongly induced in late erythroid differentiation, mediates the elimination of ribosomes and myriad other proteins via the proteasome. We proceeded to examine other UPS components that are strongly induced in erythroid cells, and found that TRIM10 eliminates dynactin, many myosins, actin crosslinking proteins, the erythroid regulator TMCC2, and COP1 vesicles; TRIM58 eliminates dynein and centrosomal proteins; the unique ubiquitin-like protein TBCEL specifically dismantles the tubulin cytoskeleton; and UBE2H, together with the GID complex, eliminates a broad set of mRNA-binding translational regulatory proteins while also promoting the elimination of many mitochondrial proteins. Thus, these UPS components have highly distinct specificities, each driving the elimination of different parts of the cell or proteome. This work uncovers a vast new regulatory pathway that appears to be central to the maturation of the erythrocyte. It also indicates a remarkable new capacity of the UPS: to effect global and developmentally controlled proteomic remodeling. In contrast to the above-described proteins, most UPS components disappear during erythroid maturation; thus, a highly specialized variant of the UPS mediates remodeling. Focusing on TBCEL, TRIM10, and TRIM58, we will use biochemical reconstitution, crystallography, and hydrogen exchange mass spectrometry to resolve specific mechanisms of degradation and degradation signals in target proteins. Cellular studies will focus on the cytoskeleton and on translational control as highlighted by our proteomic data. As the erythroblast matures into the red blood cell, its radial, microtubule-based cytoskeleton is replaced by an acentric actin-based network. We will characterize how the cytoskeleton functions during the unusual and to date uncharacterized transition period that takes place in the reticulocyte. We will then assess the impact of programmed elimination of tubulin and other cytoskeletal proteins on this cytoskeletal transformation. Although late erythroid cells are known to be characterized by extensive translational regulation, our findings indicate a new mechanism by which diverse translational regulators are themselves controlled. We will use our mutants to determine the impact of UBE2H-dependent ubiquitination on mRNA translation in reticulocytes, using RNA-Seq and Ribo-Seq in parallel. In summary, we propose that ubiquitin-dependent proteome remodeling is an important new aspect of the biology of eukaryotic cells, critical for the generation of highly differentiated cell types.

项目总结/摘要 当细胞经历极端形式的终末分化时，它们能够积累特定的蛋白质 到异常高的水平-在球蛋白和晶体蛋白的情况下每毫升数百毫克。在同一 几乎所有其他细胞成分都被消除了。细胞是如何执行如此庞大的 生物合成和降解同时进行几乎是一个完全的谜。我们在1995年提出， 泛素-蛋白酶体系统（UPS）可能在蛋白质组重构中起重要作用。使用鼠 网织红细胞，一个独特的强大的系统，研究全球蛋白质组重塑，我们发现，确实UBE 2 O，一个 在红系分化晚期强烈诱导的泛素结合酶，介导 核糖体和无数其他蛋白质。我们继续检查其他UPS组件 在红系细胞中被强烈诱导，并发现TRIM 10消除了dynactin，许多肌球蛋白，肌动蛋白， TRIM 58消除了动力蛋白， 中心体蛋白;独特的泛素样蛋白TBCEL特异性地破坏微管蛋白细胞骨架; 和UBE 2 H，连同GID复合物，消除了一系列广泛的mRNA结合的翻译调控， 蛋白质，同时也促进许多线粒体蛋白质的消除。因此，这些UPS组件具有 高度不同的特异性，每个驱动消除细胞或蛋白质组的不同部分。这项工作 揭示了一个巨大的新的调节途径，似乎是红细胞成熟的核心。它还 表明UPS的一个显着的新能力：影响全球和发育控制的蛋白质组学 重塑与上述蛋白质相反，大多数UPS成分在红细胞期消失 因此，UPS的高度专门化变体介导重塑。聚焦TBCEL、TRIM 10、 和TRIM 58，我们将使用生化重组，晶体学和氢交换质量 通过质谱分析来解析靶蛋白中的特定降解机制和降解信号。蜂窝 研究将集中在我们的蛋白质组学数据突出的细胞骨架和翻译控制。为 当成红细胞成熟为红细胞时，其放射状的、基于微管的细胞骨架被无着丝粒的 肌动蛋白网络我们将描述细胞骨架如何在不寻常的和迄今为止的功能 在网织红细胞中发生的未表征的过渡期。然后，我们将评估 微管蛋白和其他细胞骨架蛋白在这种细胞骨架转化中的程序性消除。虽然 已知晚期红系细胞具有广泛的翻译调节，我们的研究结果表明， 一种新的机制，通过这种机制，多种翻译调节因子自身受到控制。我们会利用变种人 使用RNA-Seq确定UBE 2 H依赖性泛素化对网织红细胞mRNA翻译的影响 和核糖核酸测序总之，我们认为，泛素依赖的蛋白质组重构是一个重要的 真核细胞生物学的新方面，对于高度分化的细胞类型的产生至关重要。