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Role of keratin intermediate filaments in skin epithelial differentiation.

角蛋白中间丝在皮肤上皮分化中的作用。

基本信息

批准号：
10640121
负责人：
Pierre Coulombe
金额：
$ 42.68万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
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：
2021-08-09 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10640121
关键词：
Accounting Adaptor Signaling Protein Affect Alleles Architecture Automobile Driving Binding Binding Sites Biochemistry Bullous Congenital Ichthyosiform Erythroderma Cell physiology Cells Cessation of life Code Coupled Cysteine Cytokeratin filaments Cytoplasm Cytoskeletal Filaments Cytoskeletal Proteins Defect Disease Disulfides Ectoderm Endowment Epidermis Epidermolysis Bullosa Simplex Epithelial Cells Epithelium Equilibrium Exhibits Family Fostering Functional disorder Gene Expression Gene Family Genes Growth Hair follicle structure Homeostasis Human Impairment Individual Intermediate Filament Gene Intermediate Filament Proteins Intermediate Filaments Keratin Knock-in Knockout Mice Learning Link Maintenance Maps Mechanics Mediating Medicine Metabolism Mutation Phenotype Phosphotransferases Play Pliability Population Post-Translational Protein Processing Process Proliferating Property Protein Isoforms Proteins Regulation Research Role Signal Transduction Skin Societies Source Specific qualifier value Specificity Stress Structure Stuttering Surface Sweat Glands Testing Therapeutic Tissues Tooth structure Transcriptional Regulation Transgenic Mice appendage disease-causing mutation disulfide bond high throughput screening in vivo insight keratin 5 keratinocyte keratinocyte differentiation live cell imaging mechanical signal migration mouse model notch protein pachyonychia congenita progenitor protein function resilience response scaffold skin disorder transcriptomics

项目摘要

ABSTRACT Genetically-determined mutations that affect the sequence of keratin proteins account for a large number of skin epithelial disorders, many of which are rooted in defective cellular mechanics. Keratins are the most abundant proteins in surface epithelia like epidermis, where they primarily occur as intricate cytoplasmic networks of 10 nm wide intermediate filaments (IFs). Keratins are encoded by an evolutionarily conserved family of 54 genes subdivided into two major types (I and II). Pairwise regulation of type I and II keratin genes in epithelial cells reflects a strict heteropolymerization requirement during IF assembly. Specific pairs of keratin genes are regulated in an epithelial tissue-type and differentiation-specific fashion. One of the many roles so far elucidated for keratin IFs is to act as resilient yet pliable scaffolds that endow epithelial cells and tissues with the ability to sustain various stresses. A newly emerging, exciting role for keratin proteins is to regulate epithelial differentiation through the modulation of Hippo and Notch signaling. We recently discovered that keratin-dependent disulfide bonding plays a crucial role towards the intracellular organization and steady-state dynamics of keratin filaments in progenitor keratinocytes of epidermis, with an associated impact on the balance between proliferation and differentiation. This role entails a delicate interplay between disulfide bonding mediated by a specific residue known as the “stutter cysteine” in keratin 14 (K14), the adaptor protein 14-3-3sigma, and YAP1, a terminal effector of Hippo signaling. Since the stutter cysteine in K14 is conserved in several additional type I keratins expressed in skin epithelia we propose, as an overarching hypothesis, that keratins act as general regulators of Hippo signaling with an associated impact on differentiation and homeostasis in skin epithelia. In Aim 1, we will test the hypothesis that the stutter cysteine in K10 is responsible for proper regulation of YAP1 and Hippo signaling in the differentiating layers of epidermis, with direct relevance for the keratin disorder epidermolytic hyperkeratosis. In Aim 2 we will map the binding interface between 14-3-3sigma and each of K14 and K10, define the basis for the regulation the keratin/14-3-3 interactions, and identify the source of the signal that drives K14- and K10-dependent disulfide bonding in keratinocytes. We will also conduct a high-throughput screen comparing gene expression, at the single cell level, in control vs. Krt14 C373A transgenic mice, which exhibit an epidermal differentiation defect coupled to misregulation of YAP1. In Aim 3, finally, we will test the hypothesis that the stutter cysteine in K17 regulates YAP1 and Hippo signaling and the balance between proliferation and differentiation in hair follicles, sweat glands, tooth, and possibly in other appendages, with relevance for the disorder pachyonychia congenita. The proposed body of work lies on a robust premise and is poised to significantly advance our understanding of the significance of keratin protein function and regulation in vivo and the pathophysiology of keratin mutation-based skin diseases.

抽象的影响角蛋白序列的基因突变占很大一部分许多皮肤上皮疾病，其中许多根源于细胞力学缺陷。角蛋白是表面上皮细胞（如表皮）中最丰富的蛋白质，它们主要以复杂的细胞质形式存在 10 nm 宽的中间丝 (IF) 网络。角蛋白由进化上保守的编码 54 个基因家族分为两大类（I 和 II）。 I 型和 II 型角蛋白基因的成对调控上皮细胞中的异聚反映了 IF 组装过程中严格的异聚要求。特定的角蛋白对基因以上皮组织类型和分化特异性方式受到调节。众多角色之一目前已阐明的角蛋白 IF 是作为有弹性且柔韧的支架，赋予上皮细胞和组织具有承受各种压力的能力。角蛋白的一个新出现的、令人兴奋的作用是通过 Hippo 和 Notch 信号传导的调制。我们最近发现角蛋白依赖性二硫键对角蛋白丝的细胞内组织和稳态动力学起着至关重要的作用表皮角质形成细胞祖细胞，对增殖和增殖之间的平衡具有相关影响差异化。这种作用需要由特定残基介导的二硫键之间微妙的相互作用角蛋白 14 (K14) 中的“口吃半胱氨酸”、接头蛋白 14-3-3sigma 和 YAP1（末端蛋白） Hippo 信号传导的效应器。由于 K14 中的口吃半胱氨酸在其他几种 I 型角蛋白中是保守的在皮肤上皮细胞中表达，作为一个总体假设，我们提出角蛋白充当一般调节剂 Hippo 信号传导对皮肤上皮细胞分化和稳态的相关影响。在目标 1 中，我们将检验 K10 中的口吃半胱氨酸负责 YAP1 和 Hippo 的正确调节的假设表皮分化层中的信号传导，与角蛋白紊乱表皮松解症直接相关角化过度。在目标 2 中，我们将绘制 14-3-3sigma 与 K14 和 K10 之间的结合界面，定义角蛋白/14-3-3相互作用的调节基础，并确定信号源驱动角质形成细胞中 K14 和 K10 依赖性二硫键。我们还将进行高通量在单细胞水平上筛选比较对照小鼠和 Krt14 C373A 转基因小鼠的基因表达，其中表现出与 YAP1 失调相关的表皮分化缺陷。最后，在目标 3 中，我们将测试假设 K17 中的口吃半胱氨酸调节 YAP1 和 Hippo 信号传导以及两者之间的平衡毛囊、汗腺、牙齿以及可能的其他附属器的增殖和分化与先天性厚甲症的相关性。拟议的工作体系有一个坚实的前提，并且是有望显着增进我们对角蛋白功能和调节重要性的理解体内和基于角蛋白突变的皮肤病的病理生理学。