Characterizing Senescent Cell Heterogeneity by Surface Proteins: Single-Cell CITE-Seq

通过表面蛋白表征衰老细胞异质性：单细胞 CITE-Seq

• 批准号: 10913075
• 负责人: Myriam Gorospe
• 金额: $ 15.57万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10913075
• 关键词: Age; Aging; Amines; Angiogenic Factor; Biotinylation; CD44 gene; Cell Aging; Cell Proliferation; Cell Surface Proteins; Cell membrane; Cell model; Cell physiology; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Classification; Dipeptidyl Peptidases; Dipeptidyl-Peptidase IV; Exhibits; Galactosidase; Growth; Heterogeneity; ICAM1 gene; Inflammaging; Inflammation; Inflammatory; Integral Membrane Protein; Intervention; Ionizing radiation; Label; Malignant Neoplasms; Maps; Membrane Proteins; Messenger RNA; Metalloproteases; Methods; Modeling; Organ; Pattern; Phenotype; Production; Proteins; RNA; Sepharose; Stress; Sulfhydryl Compounds; Surface; Tissues; Vascular Cell Adhesion Molecule-1; Work; cytokine; design; improved; neutravidin; overexpression; receptor; response; senescence; transcriptome

ONGOING WORK AIM 1. To systematically identify cell surface proteins in multiple models of senescence. We have used an improved cell surface protein-labeling method designed for MS analysis and two models of cellular senescence: replicative senescence (RS) and ionizing radiation-induced senescence (IR-IS). Surface proteins were labeled by thiol-cleavable amine-reactive biotinylation, isolated by neutravidin agarose, and identified and quantified by MS analysis. We identified several proteins highly enriched on the surface of senescent cells RS (77) and IR-IS (144), while 58 were shared between the two comparisons. GO analysis of the 58 shared protein confirmed the enrichment of receptor and transmembrane proteins in the 58 shared proteins, including the highly enriched surface proteins CD44, CD54, and DPP4. These and other surface proteins will be targeted for Cite-Seq, senolytics and senomorphics. AIM 2. To use surface proteins in order to subclassify senescent cells using single cell-CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing). Senescent cell heterogeneity represents a major challenge in targeting them efficiently by senotherapeutics. We and others have characterized the heterogeneity of the senescence transcriptome depending on the origin of the cell and the type of stress (Hernandez-Segura et al., 2017 and Casella et al., 2019). However, identifying shared surface proteins presents an excellent opportunity to subclassify senescent cells. We will use surface proteins from AIM 1 to identify senescent cell subtypes. Our preliminary analysis indicates that CD26 and CD106 mRNAs are highly expressed in bulk and single senescent cells compared to proliferating cells. Thus, we explored if these two surface proteins were highly expressed in distinct sub-populations of senescent cells. Interestingly, CD26- and CD106-positive cells may cluster in specific sub-populations of senescent cells. FUTURE PLANS: 1. To validate and functionally analyze senescence-associated surface proteins. 2. To use surface proteins to identify sub-populations of senescent cells. 3. To target surface proteins for senotherapeutics.

正在进行的工作 目的1。在多种衰老模型中系统地识别细胞表面蛋白。 我们使用了一种改进的细胞表面蛋白标记方法，设计用于MS分析和两个细胞衰老模型：复制衰老（RS）和电离辐射诱导的衰老（IR-IS）。 表面蛋白通过硫醇可辨别的胺反应生物素化标记，通过中性蛋白琼脂糖分离，并通过MS分析鉴定和量化。 我们确定了几种高度富集在衰老细胞Rs（77）和IR-IS（144）表面的蛋白质，而两个比较之间共享了58个蛋白质。 对58种共有蛋白的GO分析证实了58种共享蛋白质中受体和跨膜蛋白的富集，包括高度富集的表面蛋白CD44，CD54和DPP4。 这些和其他表面蛋白将针对引用，鼻溶剂和鼻孔形态。 AIM 2。使用表面蛋白，以使用单细胞 - cite-seq（通过测序对转录组和表位的细胞索引）进行亚分析细胞。 衰老细胞异质性代表了通过鼻疗法有效地靶向它们的主要挑战。 我们和其他人的特征是衰老转录组的异质性，具体取决于细胞的起源和压力的类型（Hernandez-Segura等，2017和Casella等，2019）。 但是，鉴定共享的表面蛋白提供了亚分析细胞的绝佳机会。 我们将使用AIM 1的表面蛋白来识别衰老细胞亚型。 我们的初步分析表明，与增殖细胞相比，CD26和CD106 mRNA在散装和单个衰老细胞中高度表达。 因此，我们探讨了这两种表面蛋白是否在衰老细胞的不同亚群中高度表达。 有趣的是，CD26和CD106阳性细胞可能会聚集在衰老细胞的特定亚群中。 未来计划： 1。验证和功能分析与衰老相关的表面蛋白。 2。使用表面蛋白来鉴定衰老细胞的亚群。 3。靶向表面蛋白用于鼻疗法。