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Keratin-dependent regulation of progenitor keratinocyte identity and commitment to differentiation

角质形成细胞祖细胞身份和分化承诺的角蛋白依赖性调节

基本信息

批准号：
10666664
负责人：
Catherine Julia Redmond
金额：
$ 4.42万
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：
2022
资助国家：
美国
起止时间：
2022-07-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10666664
关键词：
Alleles Basal Cell Binding Biochemical Cancer Biology Cell Density Cell Differentiation process Cell Nucleus Cell Proliferation Cell Shape Cell-Matrix Junction Cells Cellular biology Chemicals Complex Confocal Microscopy Crowding Cues Cysteine Cytokeratin filaments Cytoplasm Data Dehydration Dermatologic Development Disease Epidermis Epithelium Equilibrium Etiology Failure Filament Functional disorder Future Genetic Growth Hair follicle structure Health Homeostasis Human Human papillomavirus 16 In Vitro Intermediate Filament Proteins Intermediate Filaments Investigation Keratin Knock-in Knockout Mice Knowledge Label Location Mechanics Medicine Modeling Modification Molecular Morphology Mus Mutation Nuclear Organ Pathogenesis Pathologic Pathology Phenotype Physiologic pulse Physiological Polymers Positioning Attribute Post-Translational Protein Processing Preventive measure Process Proteins Publishing Regulation Research Rod Role Scaffolding Protein Signal Transduction Site Skin Skin Cancer Skin Carcinogenesis Stains Stratified Squamous Epithelium Stratum Basale Stress Structure Stuttering Surface Testing Thymidine Tissues Tongue Training Transcription Coactivator Transgenes Transgenic Mice Transmission Electron Microscopy UV Radiation Exposure Ultraviolet Rays Viral Visualization Water Work analog cancer initiation dimer dimethylbenzanthracene disulfide bond experimental study follow-up in vivo insight keratin 5 keratinocyte keratinocyte differentiation molecular phenotype mouse model mutant novel null mutation pathogen postnatal development pre-doctoral prevent progenitor programs response skin squamous cell carcinoma stem stem cells tumor progression tumorigenesis tumorigenic

项目摘要

Abstract Keratins are intermediate filament (IF) forming proteins whose expression and post-translational modifications are tightly regulated in response to development, differentiation, stress, and disease. Keratin 14 (K14) is highly expressed in progenitor keratinocytes located in the basal layer of stratified squamous epithelia, including the skin epidermis. K14 and its obligate assembly partner keratin 5 (K5) form highly stable coiled-coil dimers that further polymerize into 10 nm keratin IF filaments in basal keratinocytes. The coiled-coiled structure of all IF proteins including K5/K14 heterodimers is made possible by long-range heptad repeats located within their defining, central rod domain. All IF proteins contain a 4-residue disruption in this heptad repeat at a specific location within the rod domain, known as the stutter region. K14 and select keratins expressed in surface epithelia contain an evolutionarily conserved cysteine residue in position 2 within the stuffer motif (mouse K14 C373). The functions of the stutter region and its conserved cysteine remain incompletely defined. Recent work in the Coulombe lab identified a role for K14 stutter cysteine in the regulation of epidermal homeostasis. Expanding upon prior a crystal structure determination and follow-up observations, we generated a Krt14 C373A mouse line, which shows altered K14-dependent disulfide bonding in the epidermis. The remarkable molecular phenotype of the Krt14 C373A mouse informed our current mechanism proposing that disulfide bonded K14 binds to the scaffolding protein 14-3-3σ, in a differentiation cue-dependent fashion, leading to sequestration and inactivation of the pro-growth transcriptional co-activator YAP1. As part of ongoing studies, we sought to accentuate the otherwise subtle Krt14 C373A gross phenotype, in contrast to its molecular phenotype, by generating a novel mouse line, Krt14 C373A/null. The Krt14 C373A/null mouse contains a single mutant Krt14 allele, resulting in reduced levels of K14 post-translationally modified at the stutter. The Krt14 C373A/null mouse phenotype presents with precocious post-natal development, hair follicle abnormalities, and crowded, irregular basal keratinocyte nuclei in the skin and tongue. From observations made so far using Krt14 C373A/null mice I hypothesize that the concentration and proportion of stutter cysteine-dependent disulfide bonded K14 within the keratin IF network of basal keratinocytes define basal keratinocyte identity and regulate their entry into terminal differentiation. Further, I hypothesize that alterations in the regulation of basal cell identity will promote skin cancer initiation and progression. This hypothesis proposes that basal keratinocyte identity is defined by the composition of the keratin filament network, including K14 and an additional type I keratin, K15, and that the proper balance of K15 and disulfide-bonded K14 prevents pathological growth of progenitor keratinocytes. Altogether the testing of these hypotheses, mechanism, and the Krt14 C373A/null mouse model will shed critical insight into the regulation of keratinocyte differentiation and permit more effective and targeted medicine towards the failure of homeostasis that underlies the complex pathology of skin cancer.

摘要角蛋白是形成中间丝（IF）的蛋白质，其表达和翻译后修饰在发育、分化、压力和疾病中受到严格调控。角蛋白14（K14）是高度在位于复层鳞状上皮基底层的前体角质形成细胞中表达，包括皮肤表皮K14和它的专性装配伴侣角蛋白5（K5）形成高度稳定的卷曲螺旋二聚体，进一步在基底角质形成细胞中形成10 nm角蛋白IF丝。所有IF的螺旋-螺旋结构包括K5/K14异源二聚体的蛋白质通过位于其内部的长程七肽重复序列而成为可能。定义中心杆域。所有IF蛋白在该七肽重复序列中的特定位置处含有4个残基的破坏。在杆域内的位置，称为口吃区域。K14和选择角蛋白在表面上皮中表达在stuffer基序内的2位含有进化上保守的半胱氨酸残基（小鼠K14 C373）。的 Stutter区域及其保守的半胱氨酸的功能仍然不完全确定。联合国最近的工作 Coulombe实验室确定了K14 stutter半胱氨酸在调节表皮稳态中的作用。扩大在先前的晶体结构测定和后续观察的基础上，我们产生了Krt 14 C373 A小鼠，线，其显示表皮中K14依赖性二硫键的改变。令人瞩目的分子 Krt 14 C373 A小鼠的表型告知我们目前的机制，提出二硫键K14 以分化线索依赖的方式与支架蛋白14-3-3σ结合，导致螯合，促生长转录共激活因子YAP 1的失活。作为正在进行的研究的一部分，我们试图与其分子表型相反，通过以下方式强调其他细微的Krt 14 C373 A总体表型：产生新的小鼠品系Krt 14 C373 A/null。Krt 14 C373 A/null小鼠含有单一突变型Krt 14 等位基因，导致口吃时K14的后修饰水平降低。Krt 14 C373 A/null小鼠表型表现为出生后发育早熟，毛囊异常，拥挤，不规则皮肤和舌头中的基底角质细胞核。根据迄今为止使用Krt 14 C373 A/无效小鼠进行的观察，假设半胱氨酸依赖性二硫键K14的浓度和比例在基底角质形成细胞的角蛋白IF网络内，进入终端分化阶段。此外，我推测，在调节基底细胞的变化，身份将促进皮肤癌的发生和发展。这一假说提出，角质形成细胞的身份由角蛋白丝网络的组成来定义，包括K14和另外的 I型角蛋白K15，K15和二硫键结合的K14的适当平衡防止病理性生长角质形成细胞的祖细胞。总之，这些假设的测试，机制，和Krt 14 C373 A/null 小鼠模型将揭示角质形成细胞分化的调节，并允许更有效的以及针对皮肤癌复杂病理学基础的体内平衡失败的靶向药物。