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.

项目摘要/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 达到异常高的水平--珠蛋白和晶体蛋白的含量为每毫克数百毫克。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 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)可能在全球蛋白质组重构中发挥核心作用。用小鼠 网织红细胞，一个研究全球蛋白质组重构的独特而强大的系统，我们发现确实是UBE2O，一个 泛素结合酶在红系分化晚期被强烈诱导，介导消除 核糖体和无数其他蛋白质通过蛋白酶体。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TRIM10消除动力蛋白，许多肌球蛋白，肌动蛋白 交联蛋白、红系调节剂TMCC2和COP1囊泡；TRIM58消除动力蛋白和 中心体蛋白；唯一的泛素样蛋白TBCEL特异性地分解微管蛋白细胞骨架； UBE2H与GID复合体一起，消除了一系列广泛的与mRNA结合的翻译调控 同时也促进了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，每一种都驱动细胞或蛋白质组不同部分的消除。这部作品 揭示了一条广阔的新的调控途径，这似乎是红细胞成熟的中心。它还 表明UPS的一项非凡的新能力：影响全球和发育控制的蛋白质组 改建。与上述蛋白质相反，大多数UPS成分在红系期间消失 成熟；因此，UPS的高度专门化变体调节重塑。重点关注TBCEL、TRIM10、 和TRIM58，我们将使用生化重组、结晶学和氢交换质量 光谱来解析特定的降解机制和目标蛋白质中的降解信号。蜂窝 正如我们的蛋白质组学数据所强调的，研究将集中在细胞骨架和翻译控制上。作为 红细胞成熟为红细胞，其放射状的、以微管为基础的细胞骨架被无着丝粒取代 基于肌动蛋白的网络。我们将描述细胞骨架在不寻常时期和到目前为止的功能。 发生在网织红细胞中的未描述的过渡期。然后，我们将评估 程序性地消除微管蛋白和其他细胞骨架蛋白对细胞骨架的转化。虽然 已知晚期红系细胞的特征是广泛的翻译调节，我们的发现表明 不同的翻译调节者自身受到控制的新机制。我们会用我们的变种人 用RNA-Seq技术确定UBE2H依赖的泛素化对网织红细胞中mRNA翻译的影响 和Ribo-Seq并行。综上所述，我们认为泛素依赖的蛋白质组重构是一种重要的 真核细胞生物学的新方面，对产生高度分化的细胞类型至关重要。