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: 10273570
• 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
• 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/10273570
• 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; Cues; 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; 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.

摘要 影响角蛋白蛋白序列的基因决定的突变占很大一部分 许多皮肤上皮病，其中许多是根植于细胞机制缺陷。角蛋白是 最丰富的蛋白质在表面上皮细胞中，如表皮，它们主要以复杂的细胞质出现 由10纳米宽的中间细丝(IF)组成的网络。角蛋白是由进化上保守的 由54个基因组成的家族分为两个主要类型(I和II)。I型和II型角蛋白基因的成对调控 在上皮细胞中反映了在IF组装过程中严格的异聚要求。特定的角蛋白对 基因以一种上皮组织类型和分化特有的方式进行调控。作为众多角色中的一个 到目前为止，角蛋白IF被阐明是作为具有弹性但柔韧的支架，赋予上皮细胞和组织 具有承受各种压力的能力。 角蛋白蛋白的一个新出现的令人兴奋的作用是通过 河马和Notch信号的调制。我们最近发现，角蛋白依赖的二硫键 对角蛋白细丝的细胞内组织和稳态动力学起着至关重要的作用。 表皮祖细胞角质形成细胞，对增殖和细胞间平衡产生相关影响 差异化。这种作用需要在特定残基介导的二硫键之间产生微妙的相互作用 角蛋白14(K14)中的“卡顿半胱氨酸”(K14)、接头蛋白14-3-3-Sigma和末端的YAP1被称为 河马信号的效应器。由于K14中的口吃半胱氨酸在几种额外的I型角蛋白中是保守的 在皮肤上皮细胞中表达，我们提出，作为一个首要假设，角蛋白起着一般的调节作用。 河马信号对皮肤上皮细胞分化和动态平衡的相关影响。在目标1中，我们 将检验K10中的口吃半胱氨酸负责正确调节YAP1和河马的假设 表皮分化层中的信号与角蛋白紊乱的表皮松解症直接相关 角化过度。在目标2中，我们将映射14-3-3sigma与K14和K10中的每一个之间的结合接口， 确定调控角蛋白/14-3-3相互作用的基础，并确定 驱动角质形成细胞中依赖于K14和K10的二硫键。我们还将进行高吞吐量的 筛选比较对照和Krt14 C373A转基因小鼠在单细胞水平上的基因表达 表现出与YAP1调控失调有关的表皮分化缺陷。在目标3中，最后，我们将测试 假设K17中的口吃半胱氨酸调节YAP1和河马信号以及两者之间的平衡 毛囊、汗腺、牙齿的增殖和分化，可能还有其他附件 与先天性肥甲症的相关性。拟议的工作主体以可靠的前提为基础，并 准备显著地促进我们对角蛋白蛋白功能和调节的重要性的理解 活体和角蛋白突变皮肤病的病理生理学。