Expression, Structure And Function Of The Cornified Cell

层状鳞状上皮屏障功能的主要组成部分是角化细胞包膜（CE）。这是一种多组分10纳米厚的高度不溶性蛋白质层，在细胞终末分化过程中沉积在质膜的内表面。在表皮的情况下，一层5纳米厚的神经酰胺脂质（脂质包膜）附着在外表面。蛋白质包膜的不溶性在很大程度上是由于转谷氨酰胺酶的几个结构交联。研究蛋白质和脂质组分的生物学和组装是本实验室的主要工作。具体而言，我们正在研究：(i)从各种来源分离的ce中蛋白质的交联，以探索哪些蛋白质通过哪些谷氨酰胺和赖氨酸交联在一起，并提供结构和功能信息；（ii）几种关键结构蛋白，如loricrin、小脯氨酸富蛋白（SPR）家族、involucrin、envoplakin和periplakin；（iii）与CE共价结合的神经酰胺脂质；（iv）在培养的角质形成细胞中产生的CE组装的最早阶段；(v)尝试使用体外合成脂质囊泡（slv）模型系统重建ce样结构。CE蛋白包膜结构与组装。我们研究了从人包皮表皮角质层、未成熟终分化包皮表皮、培养的诱导终分化的人表皮角质形成细胞、小鼠前胃上皮和人口腔上皮中分离的ce的特征。我们使用控制蛋白水解来解剖分离ce，分离交联肽，并进行蛋白质测序。综上所述，我们的数据与CE组装沿着质膜在桥粒间位点开始的可能性是一致的，这种组装是通过天花素与自身的头对头和头对尾交联开始的，也可能是与包膜蛋白和外包膜蛋白的交联。不久之后，总苞叶蛋白沉积扩散到桥粒位点，因此沿细胞外围形成了连续的总苞叶蛋白层、包膜蛋白层和周膜蛋白层：这一层可能形成了CE组装后期的支架，包括大量沉积其他蛋白质，如loricrin和SPRs。我们在细菌中表达了人Loricrin，并利用它来表征其结构、生化特性和体外表皮谷氨酰胺转胺酶（TGase）的交联。通过生物物理测量，它在溶液中具有与其多个酪氨酸相关的某些结构。它是一个完整的TGase底物，因为它在体外反应中被所有三种表皮TGase寡聚，尽管具有不同的动力学效率，并且利用不同的谷氨酰胺和赖氨酸。通过比较体外使用的残基和体内使用的残基，我们可以得出结论，TGase 1和TGase 3都是体内氯丙林正确交联所必需的。我们发现，在体外交联后，氯丙胺变成紧凑的接近球形的形状，更容易溶解。由此我们可以推测，表皮ce含有大约两层氯丙林分子。此外，数据表明，转谷氨酰胺酶3的初步交联可能使蛋白质更具可溶性，从而允许易位到细胞外周，最终附着在生长的CE结构上。人类loricrin基因的近端启动子位于转录起始位点上方的第一个160 bp内。我们的数据表明，有多个正向和负向相互作用的元件赋予loricrin基因的紧致表皮特异性表达。小的富含脯氨酸的蛋白质

A major component of barrier function in stratified squamous epithelia is the cornified cell envelope (CE). This is a multi-component 10 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid envelope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the cross-linking of several structural by transglutaminases. Studies on the biology and assembly of the protein and lipid components are a major effort of this laboratory. Specifically, we are studying: (i) the cross-linking of proteins in CEs isolated from a variety of sources to explore which proteins are cross-linked together through which glutamines and lysines, and to provide information on structure and function; (ii) several key structural proteins such as loricrin, the small proline rich protein (SPR) families, involucrin, envoplakin and periplakin; (iii) the ceramide lipids which become covalently attached to the CE; (iv) the earliest stages of CE assembly produced in cultured keratinocytes; and (v) an attempt to recreate a CE-like structure using an in vitro synthetic lipid vesicle (slv) model system. CE protein envelope structure and assembly. We are studying the features of CEs isolated from human foreskin epidermal stratum corneum, immature terminally differentiating foreskin epidermis, cultured human epidermal keratinocytes induced to terminally differentiate, mouse forestomach epithelium, and human oral epithelia. We have used controlled proteolysis to dissect apart the CEs, separate cross-linked peptides, and perform protein sequencing. Together, our data are consistent with the possibility that CE assembly is initiated along the plasma membrane at interdesmosomal sites by head-to-head and head-to-tail cross-linking of involucrin to itself, and perhaps to envoplakin and periplakin. Shortly later, involucrin deposition spreads to desmosomal sites so that a continuous layer of involucrin, envoplakin and perhaps periplakin is formed along the cell periphery: this layer perhaps forms a scaffold for the later stages of CE assembly involving substantial deposition of other proteins such as loricrin and SPRs. Loricrin We have expressed human loricrin in bacteria and used it to characterize its structure, biochemical properties, and cross-linking by epidermal transglutaminases (TGase) in vitro. By biophysical measurements it has some structure in solution associated with its multiple tyrosines. It is a complete TGase substrate because it is oligomerized by all three epidermal TGases in in vitro reactions, although with different kinetic efficiencies, and utilization of different glutamines and lysines. From comparisons of the residues used in vitro with those used in vivo from sequencing of CEs, we can conclude that both TGase 1 and TGase 3 are required for the correct cross-linking of loricrin in vivo. We have found that following cross-linking in vitro, loricrin becomes a compact near-spherical in shape and far more soluble. From this we can speculate that epidermal CEs contain about two layers of loricrin molecules. Further, the data suggest that preliminary cross-linking by the transglutaminase 3 enzyme may render the protein more soluble to allow transloaction to the cell periphery where it is eventually attached to the growing CE structure. The proximal promoter of the human loricrin gene resides within the first 160 bp above the transcription initiation start site. Our data indicate that there are multiple positively- and negatively-interacting elements which confer tight epidermal specific expression on the loricrin gene. Small proline rich proteins SPRs consist of four distinct families consisting of from one to 11 members. We have expressed one member of each of the human SPR1, SPR2 and SPR3 proteins for in vitro studies. By circular dichroism, they have little organized structure in solution. What structure is present can be attributed to the central proline-rich peptide repeats, and the signal strength is proportional to the number of repeats. The SPR proteins are also complete substrates in in vitro cross-linking reactions for the three TGases commonly expressed in the epidermis. In all cases of SPR proteins studied, the glutamines and lysines used for cross-linking are located only on the end domains, suggesting they may function as cross-bridging proteins. However, the details are different, which have provided a wealth of information on how the proteins may be used in tissues in vivo. Solution nmr structural studies on the SPR2 and 3 proteins have been performed. Unfortunately, these proteins have little organized structure in solution and only short range interactions were obtained. Nevertheless, the data suggest the central peptide repeat domains adopt novel omega-loop-like protein folds. We have explored the expression of the SPR1 and SPR2 families in mouse epithelia by use of immunocytochemistry, in situ hybridization and RT-PCR. Both families are differentially expressed in different epithelia. In the case of SPR1, the amount expressed in the epidermis varies widely with the site, from which we can conclude that the amounts of SPRs may influence the biomechanical properties of the epidermal body sites. Involucrin We have expressed and purified full-length human involucrin in bacteria and baculovirus systems and showed that it retains some but not all of its expected a-helical content. We are currently attempting crystallization trials in an effort to solve its three-dimensional structure. In addition, various modeling analyses have predicted that the central repeat motifs of members of the small proline rich family might associate with the repeat domain of involucrin. This model has several attractive features, in that it can explain the known in vivo and in vitro biochemical and cross-linking properties of both proteins. Experiments are in progress to test this possibility. Periplakin and Envoplakin These two relatively new members of the plakin family are intimately involved in the earliest stages of CE assembly in epithelia. We have direct evidence that sequences on their tails are involved in cross-linking to keratin filaments, and other CE proteins, as well as attachment of ceramide lipids. In order to explore this further, we have expressed portions of the rods, tails and rods + tails of each protein. Crystallization trials have been set up in an effort to obtain three-dimensional structural information. Also, these fragments will be used for studies with synthetic lipid vesicles to investigate their possible roles in the earliest stages of CE assembly.