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

基本信息

批准号：
10598561
负责人：
MARK D FLEMING
金额：
$ 55.7万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
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/10598561
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 Generations Genes Genetic 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 Phase Physiological Play Process Proteasome Binding Proteins Proteome Proteomics Radial Regulatory Pathway Reticulocytes Ribosomes Role Signal Transduction Specificity Spectrin System Testing Time Translational Regulation Translations Tubulin Ubiquitin Ubiquitin Like Proteins Ubiquitin-Conjugating Enzymes Ubiquitination Variant Vesicle Work cell type crosslink dynactin erythroid differentiation fiber cell genetic regulatory protein interest mRNA Translation milligram multicatalytic endopeptidase complex mutant novel preservation programs protein crosslink protein degradation reconstitution ribosome profiling structural biology transcriptome sequencing transcriptomics ubiquitin ligase

项目摘要

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 /摘要随着细胞经历极端形式的末端分化，它们能够积累特定的蛋白质对于球蛋白和结晶蛋白而言，每毫升的数百毫克含量很高。同样时间，消除了几乎所有其他细胞成分。细胞如何执行如此庞大的程序生物合成和退化几乎是一个完全的谜。我们在1995年提议泛素 - 蛋白酶体系统（UPS）可能在全球蛋白质组重塑中起核心作用。使用鼠网状细胞是一种研究全球蛋白质组重塑的独特功能的系统，我们发现确实是ube2o，一个泛素结合酶在红细胞病晚期分化中强烈诱导，介导消除核糖体和其他蛋白质通过蛋白酶体。我们开始检查其他UPS组件在红细胞细胞中强烈诱导的，发现TRIM10消除了dynactin，许多肌动蛋白，肌动蛋白交联蛋白，红细胞调节剂TMCC2和COP1蔬菜； TRIM58消除了动力蛋白和中心蛋白；独特的泛素样蛋白TBCEL专门拆除了微管蛋白的细胞骨架。和UBE2H，与GID复合物一起消除了一组广泛的mRNA结合翻译调节蛋白质同时还促进了消除许多线粒体蛋白。那就是这些UPS组件高度不同的规格，每个规格都可以消除细胞或蛋白质组的不同部位。这项工作揭示了一条巨大的新调节途径，这似乎是红细胞成熟的核心。也是如此表示UPS的非凡新能力：实现全球和开发的蛋白质组学重塑。与上述蛋白质相反，大多数UPS组件在红斑期间消失成熟；因此，UPS的高度专业化变体介导了重塑。专注于tbcel，trim10，和TRIM58，我们将使用生化重构，晶体学和氢交换质量靶蛋白中降解和降解信号的特定机制的光谱法。细胞研究将重点放在细胞骨架和转化控制上，如我们的蛋白质组学数据所强调。作为红细胞成熟到红细胞中，其径向，基于微管的细胞骨架被分散替代基于肌动蛋白的网络。我们将表征细胞骨架在异常和迄今为止的功能在网状细胞中发生的未表征过渡期。然后，我们将评估在这种细胞骨架转化上，编程消除微管蛋白和其他细胞骨架蛋白。虽然已知晚期红细胞细胞的特征是广泛的翻译调节，我们的发现表明潜水员翻译调节器本身可以控制的新机制。我们将使用我们的突变体使用RNA-Seq确定Ube2H依赖性泛素化对网状细胞中mRNA翻译的影响和Ribo-Seq并行。总而言之，我们建议泛素依赖性蛋白质组重塑是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。