Expression, Structure And Function Of Trichohyalin

毛透明蛋白（Trichohyalin，THH）是毛囊内根鞘细胞（毛纤维的髓质）的主要分化产物，在表皮和多种其他硬化的复层鳞状上皮细胞和组织中表达。最初，我们提出，它的功能至少部分作为一个角蛋白中间丝相关蛋白在这些组织。我们特别感兴趣的是观察到THH是将其交联成聚合物的转氨酶（TGases）和将蛋白质结合的精氨酸转化为瓜氨酸的肽基精氨酸脱亚胺酶（PAD）的底物。我们研究的总体目的是了解THH基因的表达，THH蛋白可能的独特结构，它在组织中的利用方式，如内根鞘，以及它的这两个主要合成后修饰的细节和后果。（<1微克/ml），我们已经在细菌中表达了THH的结构域8（约40%）（命名为THH-8），用于研究这两种合成后修饰事件。该产物含有许多完整THH的典型肽重复序列，是高度α-螺旋的（>90%），并且其溶解度为约50微克/ml，使得其适合于体外生物化学测定。我们发现，市售PAD酶可将60%的THH-8精氨酸转化为瓜氨酸，同时溶解度显著增加。此外，通过圆二色性，这导致THH-8的α-螺旋结构的完全丧失。THH-8被已知存在于表皮中的所有三种TGase用作完全底物，即THH-8提供谷氨酰胺供体和赖氨酸受体残基。然而，通过计算动力学参数，TGase 3酶最有效地使用它。约10%的谷氨酰胺用于与大多数赖氨酸高特异性交联。此外，TGase 3酶的动力学效率在最大PAD修饰后大大增加。在这种情况下，几乎所有的谷氨酰胺都可以部分地用于与所有的赖氨酸交联：也就是说，THH-8，并且通过推断细胞中的完整THH，在变性后变成更有效的底物。然而，最近发现了TGase家族的另外三个成员。现在将使用THH-8作为底物来测试这些酶中的每一种，以探索这些新酶在体内THH加工中的作用。 然而，现存的数据表明，THH的合成后修饰的时间顺序的模型，我们已经探讨了在小鼠毛囊。使用特异性抗体的间接免疫荧光程序，我们发现THH表达先于TGase 3酶的表达。其他数据表明PAD酶在THH表达起始后但在TGase 3表达前表达。因此，在毛囊中，我们提出THH首先被PAD酶修饰，PAD酶使其变性并使其更可溶。然后，溶解的修饰THH成为TGase 3酶的非常有效的底物，从而将其交联成高度不溶性的复合物。我们建议，额外的赖氨酸可以招募交联，包括那些从角蛋白中间丝这些THH-含上皮细胞。这在毛囊的内根鞘细胞中尤其重要，其中含有约三分之二的角蛋白丝和三分之一的THH。通过这种方式，我们提出THH有效地作为丝状基质蛋白来增强和硬化组织。 目前，我们正在测试这一假设进行测序实验的肽回收小鼠内根鞘和髓质组织。现在已知小鼠THH和这些组织的角蛋白角蛋白的序列。通过识别哪些蛋白质交联在一起，我们可以首次获得有关THH在这些细胞类型中如何使用和发挥作用的详细信息。这些信息也可能有助于了解它在其他组织中的确切作用。 THH含有生物学中已知的任何蛋白质中最高含量的带电残基。特别地，提出天然THH的特征性α-螺旋结构通过形成>1个盐桥/3.5个残基/α-螺旋转角而稳定：也就是说，THH可能是唯一已知的可以形成稳定的单链α-螺旋构象的蛋白质的实例。我们已经做了一个55个残基的合成肽，共享完整的THH的保守结构基序。这已被结晶，并已获得良好的X射线衍射数据。一个解决的结构是迫在眉睫。 人类和小鼠THH基因的近端启动子区包括转录起始位点上方的前160-175 bp。该区域包含一个必需的AP 1位点以及重叠的ets样、NF-κ B、Cre样和Sp1样位点，以及一些未知的阻遏物元件。这些位点的功能性和协同相互作用已经在CAT构建体瞬时表达到从<3日龄新生小鼠收获的毛囊中进行了测试。这些区域在毛囊组织中赋予高度的表达特异性，因为它们在培养的其它类上皮细胞类型的表皮角质形成细胞中没有表达或仅具有非常低的表达水平。然而，小鼠和人类的序列和基序是完全不同的。然而，这些不同的元件协同作用以赋予组织特异性表达。因此，进一步的工作正在进行中，以探索这些协同作用的细节。我们特别感兴趣的是定义那些负责毛囊特异性的元件，因为这些基序在其他毛囊表达基因的研究中具有重要价值。

Trichohyalin (THH) is a major differentiation product of the inner root sheath cells of the hair follicle, the medulla of the hair fiber, and it is expressed in the epidermis and a variety of other hardened stratified squamous epithelial cells and tissues. Initially, we proposed that it functions at least in part as a keratin intermediate filament associated protein in these tissues. Of particular interest to us are the observations that THH is a substrate for transglutaminases (TGases), which cross-link it into polymers, and for the peptidylarginine deiminase (PAD) enzymes which convert protein-bound arginines to citrullines. The overall purpose of our studies are to understand the expression of the THH gene, the likely unique structure of THH protein, the way in which it is utilized in tissues such as the inner root sheath, and the details and consequences of these two major postsynthetic modifications of it. Because full-length human THH is very insoluble (<1 microgram/ml), we have expressed in bacteria domain 8 (about 40%) of THH (named THH-8) for use in the study of these two postsynthetic modification events. This product contains numerous peptide repeats which are typical of the intact THH, is highly a-helical (>90%), and its solubility is about 50 microgram/ml so that it is suitable for in vitro biochemical assays. We found that a commercially available PAD enzyme converts 60% of the arginines of THH-8 to citrullines, with a concomitant significant increase in solubility. Moreover, by circular dichroism, this results in the complete loss of the a-helical structure of THH-8. THH-8 is used by all three TGases known to be present in the epidermis as a complete substrate, that is, the THH-8 provides both the glutamine donor and lysine acceptor residues. However, by calculation of kinetic parameters, the TGase 3 enzyme uses it most efficiently. About 10% of the glutamines are used for crosslinking with high specificity to most of the lysines. Furthermore, the kinetic efficiency of the TGase 3 enzyme is greatly increased following maximal PAD modification. In this case, virtually all of the glutamines may be partially used for crosslinking to all of the lysines: that is, THH-8, and by inference intact THH in cells, becomes a more efficient substrate following denaturation. However, three additional members of the TGase family have been recently discovered. Each of these will now be tested using THH-8 as the substrate, in order to explore what role, if any, these new enzymes have in the processing of THH in vivo. Nevertheless, extant data suggest a model for the temporal order of the postsynthetic modifications of THH which we have explored in mouse hair follicles. Using indirect immunofluorescence procedures with specific antibodies, we found that THH expression precedes expression of the TGase 3 enzyme. Other data have shown that the PAD enzymes are expressed after the initiation of THH expression, but before TGase 3 expression. Therefore in the hair follicle, we propose that THH is first modified by PAD enzymes, which denature it and render it more soluble. Then the solubilized modified THH becomes a very efficient substrate for the TGase 3 enzyme which thereby cross-links it to a highly insoluble complex. We propose that additional lysines may be recruited for cross-linking, including those from the keratin intermediate filament of these THH-containing epithelia. This is especially important in the inner root sheath cells of the hair follicle, which contain about two-thirds keratin filaments and one-third THH. In this way, we propose that the THH effectively functions as an interfilamentous matrix protein to strengthen and harden the tissue. We are currently testing this hypothesis by performing sequencing experiments on peptides recovered from mouse inner root sheath and medulla tissues. The sequences of the mouse THH and keratin keratins of these tissues are now known. By identifying which proteins are cross-linked together, we can obtain for the first time detailed information on exactly how the THH is used and functions in these cell types. Such information may also aid in understanding the precise role of it in other tissues. THH contains the highest content of charged residues of any protein known in biology. In particular, it is proposed that the characteristic a-helical structure of native THH is stabilized by the formation of >1 salt bridges/3.5 residues/turn of the a-helix: that is, THH may be the only known example of a protein which can form a stable single-stranded a-helical conformation. We have made a 55-residue synthetic peptide that shares the conserved structural motifs of intact THH. This has been crystallized and good X-ray diffraction data have been obtained. A solved structure is imminent. The proximal promoter regions of the human and mouse THH genes encompass the first 160-175 bp above the transcription start site. This region contains an essential AP1 site as well as overlapping ets-like, NF-KB, Cre-like and Sp1-like sites, as well as some as yet unknown repressor elements. The functionality and synergistic interaction of these sites have been tested in transient expression of CAT constructs into hair follicles harvested from <3 day-old neonatal mice. These regions confer a high degree of specificity of expression in hair follicle tissue since they have no or only very low levels of expression in cultured epidermal keratinocytes of other epitheliod cell types. However, the mouse and human sequences and motifs are quite different. Nevertheless, these various elements operate synergistically to confer tissue specific expression. Thus further work is in progress to explore the details of these synergistic interactions. We are particularly interested in defining those element(s) responsible for hair-follicle specificity, as such motifs will be of great value in the study of other hair follicle-expressed genes.