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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.

摘要影响角蛋白序列的基因决定的突变占大部分。许多皮肤上皮疾病，其中许多是植根于有缺陷的细胞力学。角蛋白是最丰富的蛋白质存在于表皮等表面上皮细胞中，主要以复杂的细胞质形式存在 10 nm宽的中间丝（IF）的网络。角蛋白由一种进化上保守的 54个基因的家族，分为两种主要类型（I和II）。I型和II型角蛋白基因的成对调节反映了IF组装过程中严格的异源聚合要求。特定角蛋白对基因以上皮组织类型和分化特异性方式调节。其中一个角色，目前已阐明的角蛋白IFs是作为弹性但柔韧的支架，赋予上皮细胞和组织能够承受各种压力角蛋白的一个新出现的令人兴奋的作用是通过调节上皮细胞的分化，调节Hippo和Notch信号传导。我们最近发现依赖角蛋白的二硫键对细胞内角蛋白丝的组织和稳态动力学起着至关重要的作用，表皮的前体角质形成细胞，对增殖和分化这一作用需要一个微妙的相互作用之间的二硫键介导的特定残基角蛋白14（K14）中称为“口吃半胱氨酸”的接头蛋白14-3-3sigma和YAP 1， Hippo Signaling信号由于K14中的stutter半胱氨酸在其他几种I型角蛋白中是保守的，作为一个总体假设，我们提出，角蛋白作为一般调节剂， Hippo信号传导与皮肤上皮细胞分化和稳态的相关影响。目标1：我将检验K10中的断续半胱氨酸负责YAP 1和Hippo的适当调节的假设。表皮分化层中的信号传导，与表皮炎性角蛋白疾病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，确定调节角蛋白/14-3-3相互作用的基础，并确定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的在单细胞水平上，在对照与Krt 14 C373 A转基因小鼠中比较基因表达，表现出与YAP 1失调相关的表皮分化缺陷。在目标3中，最后，我们将测试假设K17中的断续半胱氨酸调节YAP 1和Hippo信号传导以及毛囊、汗腺、牙齿和可能的其他附属物中的增殖和分化，与先天性厚甲症有关拟议的工作内容基于一个坚实的前提，准备大大推进我们对角蛋白功能和调节的重要性的理解在体内和病理生理学的角蛋白突变为基础的皮肤病。