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.

项目概要/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 球蛋白和晶状体蛋白的含量极高，达到每毫升数百毫克。同时 随着时间的推移，几乎所有其他细胞成分都被消除了。细胞如何执行如此庞大的程序 同时进行生物合成和降解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）可能在全球蛋白质组重塑中发挥核心作用。使用小鼠 网织红细胞是研究全局蛋白质组重塑的独特强大系统，我们发现确实是 UBE2O， 在红细胞分化晚期强烈诱导的泛素结合酶，介导消除 通过蛋白酶体处理核糖体和无数其他蛋白质。我们继续检查其他 UPS 组件 在红系细胞中强烈诱导，并发现 TRIM10 消除了动力蛋白、许多肌球蛋白、肌动蛋白 交联蛋白、红细胞调节因子 TMCC2 和 COP1 囊泡； TRIM58 消除动力蛋白并 中心体蛋白；独特的类泛素蛋白TBCEL特异性拆除微管蛋白细胞骨架； UBE2H 与 GID 复合物一起消除了一系列广泛的 mRNA 结合翻译调节 蛋白质，同时还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，每种都驱动消除细胞或蛋白质组的不同部分。这部作品 揭示了一条巨大的新调控途径，该途径似乎对红细胞的成熟至关重要。它还 表明 UPS 具有显着的新能力：影响全局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 成分在红细胞生成过程中消失。 成熟；因此，UPS 的高度专业化变体可以进行改造。专注于TBCEL、TRIM10、 和 TRIM58，我们将使用生化重建、晶体学和氢交换质量 光谱测定法来解决目标蛋白质中降解的特定机制和降解信号。蜂窝网络 正如我们的蛋白质组数据所强调的，研究将集中在细胞骨架和翻译控制上。作为 成红细胞成熟为红细胞，其放射状、基于微管的细胞骨架被无心的细胞骨架取代 基于肌动蛋白的网络。我们将描述细胞骨架在异常和迄今为止的情况下如何发挥作用 网织红细胞中发生的未表征的过渡期。然后我们将评估影响 在这种细胞骨架转化过程中，微管蛋白和其他细胞骨架蛋白被程序性消除。虽然 众所周知，晚期红细胞具有广泛的翻译调控特征，我们的研究结果表明 多种翻译调节因子自身受到控制的新机制。我们将使用我们的突变体 使用 RNA-Seq 确定 UBE2H 依赖性泛素化对网织红细胞 mRNA 翻译的影响 和 Ribo-Seq 并行。总之，我们认为泛素依赖性蛋白质组重塑是一个重要的 真核细胞生物学的新方面，对于高度分化细胞类型的生成至关重要。