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 AIM 1.系统鉴定多种衰老模型中的细胞表面蛋白。 我们已经使用了一种改进的细胞表面蛋白标记的MS分析方法和两种模型的细胞衰老：复制性衰老（RS）和电离辐射诱导的衰老（IR-IS）。 表面蛋白通过巯基可裂解胺反应性生物素化标记，通过中性亲和素琼脂糖分离，并通过MS分析鉴定和定量。 我们鉴定了在衰老细胞RS（77）和IR-IS（144）的表面高度富集的几种蛋白质，而58种在两种比较之间共有。 58个共有蛋白的GO分析证实了58个共有蛋白中受体和跨膜蛋白的富集，包括高度富集的表面蛋白CD 44、CD 54和DPP 4。 这些和其他表面蛋白将被Cite-Seq、senolytics和senomorphics靶向。 AIM 2.使用表面蛋白，以便使用单细胞CITE-Seq（通过测序对转录组和表位进行细胞索引）对衰老细胞进行亚分类。 衰老细胞异质性代表了通过衰老治疗剂有效靶向它们的主要挑战。 我们和其他人已经表征了衰老转录组的异质性，这取决于细胞的起源和应激的类型（Hernana-Segura et al.，2017和Casella等人，2019年）。 然而，识别共享的表面蛋白提供了一个极好的机会，细分衰老细胞。 我们将使用AIM 1的表面蛋白来鉴定衰老细胞亚型。 我们的初步分析表明，与增殖细胞相比，CD 26和CD 106 mRNA在散装和单个衰老细胞中高度表达。 因此，我们探索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，CD 26和CD 106阳性细胞可能聚集在衰老细胞的特定亚群中。 未来计划： 1.验证和功能分析衰老相关的表面蛋白。 2.利用表面蛋白质鉴定衰老细胞的亚群。 3.以表面蛋白为靶点进行感官治疗。