Expression, Structure And Function Of Trichohyalin

Trichohyalin(THH)是毛囊内根鞘细胞的主要分化产物，是毛发纤维的髓质，表达于表皮和其他各种硬化的复层鳞状上皮细胞和组织中。最初，我们提出，在这些组织中，它至少部分作为角蛋白中间丝相关蛋白发挥功能。我们特别感兴趣的是观察到THH是谷氨酰胺转氨酶(TGase)的底物，转谷氨酰胺酶(TGase)将其交联成聚合物，以及肽基精氨酸脱亚胺酶(PAD)将蛋白质结合的精氨酸转化为瓜氨酸。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，它在内根鞘等组织中的利用方式，以及这两个主要的合成后修饰的细节和结果。由于全长的人THH是非常难溶的(1微克/毫升)，我们在THH的细菌结构域8(约40%)(命名为THH-8)中表达了THH-8，用于这两个合成后修饰事件的研究。该产品含有大量的多肽重复序列，这是完整THH的典型特征，具有高度的α-螺旋结构(&gt；90%)，其溶解度约为50微克/毫升，适合于体外生化分析。我们发现，一种商用的PAD酶可以将THH-8中60%的精氨酸转化为瓜氨酸，并伴随着溶解度的显著增加。此外，由于圆二色谱，这导致THH-8的α-螺旋结构完全丧失。THH-8被所有三种已知存在于表皮中的TGase作为一个完整的底物使用，即THH-8既提供谷氨酰胺供体，又提供赖氨酸受体残基。然而，通过计算动力学参数，TGase 3酶利用它的效率最高。大约10%的谷氨酸用于交联，对大多数赖氨酸具有高度的特异性。此外，经过最大限度的PAD修饰后，TGase 3酶的动力学效率大大提高。在这种情况下，几乎所有的谷氨酸都可能部分地用于与所有赖氨酸的交联：也就是THH-8，并由此推断细胞中完整的THH在变性后成为更有效的底物。然而，最近又发现了TGase家族的另外三个成员。现在将以THH-8为底物对每种酶进行测试，以探索这些新酶在体内处理THH过程中的作用(如果有的话)。 然而，现有的数据表明，我们在小鼠毛囊中探索的THH合成后修饰的时间顺序模型。使用具有特定抗体的间接免疫荧光程序，我们发现THH的表达先于TGase 3酶的表达。其他数据表明，PAD酶在THH表达启动后表达，但在TGase 3表达之前表达。因此，我们建议在毛囊中，THH首先被PAD酶修饰，PAD酶使其变性，使其更易溶。然后，溶解的修饰的THH成为TGase 3酶的非常有效的底物，从而将其交联到高度不溶的复合体上。我们建议可以招募更多的赖氨酸进行交联，包括那些来自这些含有THH的上皮细胞的角蛋白中间丝的赖氨酸。这在毛囊内根鞘细胞中尤其重要，毛囊内根鞘细胞含有约三分之二的角蛋白细丝和三分之一的THH。通过这种方式，我们认为THH有效地作为一种丝间基质蛋白来加强和硬化组织。我们目前正在通过对从小鼠内根鞘和髓质组织中提取的多肽进行测序实验来验证这一假设。这些组织的小鼠THH和角蛋白角蛋白的序列现在是已知的。通过确定哪些蛋白质是相互交联的，我们可以第一次获得关于THH在这些细胞类型中的确切使用和功能的详细信息。这样的信息也可能有助于理解它在其他组织中的确切作用。它含有生物学上已知的任何蛋白质中带电残基的最高含量。特别是，有人提出，天然THH的特征a-螺旋结构是通过形成&gt；1盐桥/a-螺旋的3.5个残基/转角来稳定的：也就是说，THH可能是唯一已知的能够形成稳定的单链a-螺旋构象的蛋白质。我们已经合成了一个55个残基的合成肽，它与完整的THH具有保守的结构基序。对其进行了晶化处理，获得了良好的X射线衍射数据。解决结构问题迫在眉睫。人类和小鼠THH基因的近端启动子区域位于转录起始点上方的前160-175个碱基对。该区域包含一个重要的AP1位点，以及重叠的Ets样位点、Nf-KB样位点、Cre样位点和Sp1样位点，以及一些未知的抑制元件。这些位点的功能和协同作用已经在CAT构建的瞬时表达中被测试，这些构建物从出生3天的小鼠的毛囊中获得。这些区域在毛囊组织中的表达具有高度的特异性，因为它们在培养的其他上皮样细胞类型的表皮角质形成细胞中没有表达或只有非常低的表达水平。然而，老鼠和人类的序列和主题是非常不同的。然而，这些不同的元件协同作用，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确定那些负责毛囊特异性的元件(S)，因为这些基序将对研究其他毛囊表达的基因具有重要价值。

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.