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。

• 批准号: 10462643
• 负责人: Pierre Coulombe
• 金额: $ 42.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/10462643
• 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; Crystallization; Cysteine; Cytokeratin filaments; Cytoplasm; Cytoskeletal Filaments; Cytoskeletal Proteins; Defect; Disease; Disulfides; Ectoderm; Epidermis; Epidermolysis Bullosa Simplex; Epithelial; Epithelial Cells; 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; Link; Maintenance; Maps; Mechanics; Mediating; Medicine; Metabolism; Modification; Mutation; Phenotype; Phosphotransferases; Plant Roots; Play; Pliability; Population; Process; 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; Translations; appendage; base; 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; response; scaffold; skin disorder; transcriptomics

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 影响角蛋白蛋白序列的遗传确定的突变解释了一个大的 皮肤上皮疾病的数量，其中许多根源于缺陷的细胞力学。角蛋白是 表面上皮中的大多数丰富蛋白（如表皮），它们主要以复杂的细胞质形式出现 10 nm宽的中间丝（IF）的网络。角蛋白由进化构成的编码 54个基因的家族分为两种主要类型（I和II）。 I型和II角蛋白基因的成对调节 在上皮细胞中，反映了IF组装过程中严格的杂聚合需求。角蛋白的特定对 基因以上皮组织类型和分化特异性的方式进行调节。众多角色之一 在角蛋白IF中阐明的是赋予上皮细胞和组织的弹性但柔韧的支架 具有维持各种压力的能力。 角蛋白蛋白的新出现，令人兴奋的作用是通过 河马和缺口信号的调节。我们最近发现角蛋白依赖于二硫键 在角质细丝的细胞内组织和稳态动力学中起着至关重要的作用 表皮的祖先角质形成细胞，对增殖和之间的平衡产生了相关的影响 分化。这个角色需要在特定居住介导的二硫键之间进行微妙的相互作用 在角蛋白14（K14）中称为“口吃半胱氨酸”，衔接蛋白14-3-3sigma和yap1，末端 河马信号的效应子。由于K14中的口吃半胱氨酸在几种额外的I角蛋白中配置 在皮肤上皮中表达，我们提出了一个总体假设，即角蛋白充当一般调节剂 河马信号传导，对皮肤上皮的分化和稳态的影响。在AIM 1中，我们 将检验以下假设：K10中的口吃半胱氨酸负责适当调节YAP1和河马 表皮层中层中的信号传导，与角蛋白疾病表皮溶解直接相关 高性病。在AIM 2中，我们将绘制14-3-3sigma和K14和K10的绑定界面， 定义调节的基础角蛋白/14-3-3相互作用，并确定信号的来源 在角质形成细胞中驱动K14-和K10依赖性二硫键。我们还将进行高通量 在对照与KRT14 C373A转基因小鼠中比较单细胞水平的基因表达的屏幕，该屏幕表达 表现出表皮分化缺陷，耦合YAP1的失误。在AIM 3中，最后，我们将测试 假设K17中的口吃半胱氨酸调节Yap1和河马信号以及之间的平衡 毛囊，汗水手套，牙齿以及其他附属物的增殖和差异 与Pachyonychia congenita的疾病相关。拟议的作品在于一个坚固的前提，是 被毒化以显着提高我们对角蛋白蛋白功能和调节意义的理解 体内和基于角蛋白突变的皮肤疾病的病理生理。