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

摘要 角蛋白是一种中间丝形成蛋白，其表达和翻译后修饰 受到严格调控以应对发育、分化、压力和疾病。角蛋白14(K14)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及其专性组装伙伴角蛋白5(K5)形成高度稳定的卷曲二聚体， 在基底层角质形成细胞中进一步聚合成10 nm角蛋白IF丝。所有IF的盘绕结构 包括K5/K14异源二聚体在内的蛋白质是由位于其 定义，中心杆域。所有IF蛋白在特定的七肽重复序列中包含4个残基的破坏 杆域内的位置，称为卡顿区域。K14和选择性角蛋白在表面上皮细胞中的表达 在填充物基序(小鼠K14 C373)内的第2位含有进化保守的半胱氨酸残基。这个 口吃区及其保守的半胱氨酸的功能仍未完全确定。最近的工作在 库仑实验室确认了K14口吃半胱氨酸在调节表皮动态平衡中的作用。正在扩张 根据先前的晶体结构测定和后续观察，我们产生了一只Krt14 C373A小鼠 显示了表皮中依赖于K14的二硫键的变化。非凡的分子 Krt14 C373A小鼠的表型告诉我们目前的机制，认为二硫键结合了K14 以依赖于分化线索的方式与支架蛋白14-3-3σ结合，导致隔离和 促生长转录共激活子YAP1的失活。作为正在进行的研究的一部分，我们试图 通过以下方式突出Krt14 C373A大体表型，而不是其分子表型 产生一个新的小鼠品系Krt14 C373A/空。Krt14 C373A/空小鼠含有单一突变体Krt14 等位基因，导致口吃时翻译后修改的K14水平降低。Krt14 C373A/空鼠标 表型表现为出生后发育早，毛囊异常，拥挤，不规则 皮肤和舌头中的基底层角质形成细胞核。从目前为止使用Krt14 C373A/空小鼠所做的观察来看， 假设口吃依赖半胱氨酸的二硫键的浓度和比例与K14结合 在角蛋白中，如果基础角质形成细胞网络定义了基础角质形成细胞的身份，并调节其 进入末端分化。此外，我假设基底细胞调节的变化 认同感将促进皮肤癌的发生和发展。这一假设提出了基本的 角质形成细胞的特性由角蛋白细丝网络的组成来定义，包括K14和另外一个 I型角蛋白，K15，K15和二硫键K14的适当平衡可防止病理性生长 祖细胞角质形成细胞。所有这些假设、机制和Krt14 C373A/NULL的测试 小鼠模型将对角质形成细胞分化的调控提供关键的见解，并允许更有效 以及针对作为皮肤癌复杂病理基础的动态平衡失败的靶向药物。

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.