EXPRESSION, STRUCTURE AND FUNCTION OF TRICHOHYALIN

毛透明素（Trichohyalin， THH）是毛囊内根鞘细胞、毛纤维髓质的主要分化产物，在表皮及其他多种硬化层状鳞状上皮细胞和组织中表达。最初，我们提出它至少部分地在这些组织中作为角蛋白中间丝相关蛋白起作用。我们特别感兴趣的是观察到THH是转谷氨酰胺酶（TGases）的底物，它可以交联成聚合物，也是肽精氨酸脱亚胺酶（PAD）的底物，它可以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，以及它的两种主要合成后修饰的细节和后果。由于全长人THH非常不溶（<1 μ g/ml），我们在细菌结构域8中表达了约40%的THH（命名为THH-8），用于这两个合成后修饰事件的研究。该产品含有大量的肽重复，这是典型的完整THH，是高度a-螺旋（>90%），其溶解度约为50微克/毫升，因此适合体外生化分析。我们发现一种市售的PAD酶将THH-8中60%的精氨酸转化为瓜氨酸，同时显著提高了其溶解度。通过高效液相色谱分离多肽并进行氨基酸测序，我们发现除了谷氨酸残基两侧的精氨酸外，基本上所有的精氨酸都被定量地转化为瓜氨酸。此外，通过圆二色性，这导致THH-8的a-螺旋结构完全丧失。由于PAD酶（生物学上已知的四种）在许多细胞和组织中广泛表达，如果不是所有的细胞和组织，我们认为PAD酶通常具有变性蛋白质的功能。这可能在诸如细胞凋亡和程序性细胞死亡等事件中具有广泛的意义。该实验室未来的工作将针对两种PAD酶的细菌和/或杆状病毒表达，这些酶通常在上皮、表皮和毛囊中产生。这些将被转染到模型培养细胞中，以探索它们的功能和特性。已知存在于表皮中的所有三种tgase都使用THH-8作为完整的底物，也就是说，THH-8提供谷氨酰胺供体和赖氨酸受体残基。然而，通过动力学参数的计算，TGase 3酶最有效地利用了它。约10%的谷氨酰胺用于交联，对大多数赖氨酸具有高特异性。此外，在最大限度的PAD修饰后，TGase - 3酶的动力学效率大大提高。在这种情况下，几乎所有的谷氨酰胺都可以部分用于与所有赖氨酸交联：也就是说，THH-8，以及推断细胞中完整的THH，在变性后成为更有效的底物。这些数据为我们在小鼠毛囊中探索的THH合成后修饰的时间顺序提供了一个模型。使用特异性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表达起始之后表达，而在TGase 3表达之前表达。因此，在毛囊中，我们建议首先通过PAD酶修饰THH，使其变性并使其更具可溶性。然后，可溶解的改性THH成为TGase 3酶的非常有效的底物，从而交联到高度不溶的复合物上。我们提出可能会招募更多赖氨酸进行交联，包括来自这些含THH上皮的角蛋白中间丝的赖氨酸。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有效地作为一种纤维间基质蛋白来强化和硬化组织。为了支持这一假设，从前胃上皮收获的角化细胞包膜的蛋白质测序实验发现了THH和角蛋白丝之间的许多交联。因此，我们建议THH作为纤维间的交叉桥，旨在增加组织的抗拉强度和/或刚度。事实上，在组织中THH的表达水平与假定的组织物理需求之间存在明显的相关性。因此，正常人躯干表皮中THH含量很少或不含THH，包皮表皮中THH含量约为5%，鼠足垫和前胃中THH含量分别约为10%和20%，毛囊内根鞘细胞中THH含量高达三分之一左右。我们正在进行的实验是为了测试这个假设的各个方面。最近，我们从豚鼠毛囊内根鞘组织中回收了thh -角蛋白交联材料，以探索其组成蛋白之间的交联。我们的数据显示THH与自身、角蛋白IF以及已建立的细胞包膜结构蛋白（如SPRs、desmoplakin等）广泛交联。因此，我们认为THH在根内鞘细胞中至少有两种不同的作用，如果不是重叠的话：作为纤维间基质蛋白，以及作为该组织的细胞包膜屏障的组成部分。一个有待确定的问题是角蛋白IF在根鞘内表达的精确链组成。将进行额外的测序分析来解决这个问题。注意，在髓质的情况下，存在于许多类型的粗毛中，没有角蛋白丝。在这种情况下，THH似乎形成无定形沉积物，脱水后留下大的液泡空间。有人提出，这些空间在哺乳动物通过捕获空气来维持体温方面起着关键作用。四氢大麻酚是生物学中已知的蛋白质中电荷残基含量最高的。特别地，我们提出了天然THH特有的a-螺旋结构是通过形成>1盐桥/3.5个残基/转a-螺旋来稳定的：也就是说，THH可能是已知的唯一可以形成稳定单链a-螺旋构象的蛋白质。我们建议通过进行原子分辨率结构研究来检验这一假设。我们已经合成了一个55个残基的合成肽，它共享完整THH的保守结构基序。这种肽在0.5 mg/ml时可溶，基本上是完全a-螺旋的。最近，我们已经结晶了这种肽，并试图解决其结构正在进行中。THH基因的近端启动子区包含转录起始位点以上的第一个160 bp。该区域包含一个重要的AP1位点以及重叠的et -like、NF-KB和Sp1-like位点。这些位点的功能和协同相互作用已经通过CAT构建体在<3日龄新生小鼠毛囊中的瞬时表达进行了测试。这些区域在毛囊组织中具有高度的特异性表达，因为它们在培养的表皮角质形成细胞或其他上皮细胞类型中没有或只有非常低的表达水平。正在进行进一步的工作，以完成这些协同作用的细节。此外，在THH和侧聚蛋白基因的近端启动子区域有显著的核苷酸同源性。然而，类似的侧聚蛋白基因构建物转染到毛囊中显示很少或没有活性。这可能意味着在THH的近端启动子区域存在赋予毛囊组织特异性表达的因素，我们将继续表征这种可能性。

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 crosslink 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, and the details and consequences of these two major postsynthetic modifications of it. Because full-length humanTHH is very insoluble (<1 microg/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 microg/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. By fractionation of peptides by HPLC followed by amino acid sequencing, we found that essentially all arginines except those which are flanked by glutamic acid residues are quantitatively converted to citrullines. Moreover, by circular dichroism, this results in the complete loss of the a-helical structure of THH-8. Since PAD enzymes (four are now known in biology) are widely expressed in many if not all cells and tissues, we propose that PAD enzymes in general function to denature proteins. This may have broad significance in such events as apoptosis and programmed cell death. Future work in this laboratory now will be directed toward bacterial and/or baculovirus expression of two of the PAD enzymes typically made in epithelia, epidermis and hair follicle. These will be transfected into model cultured cells to explore their functions and properties. 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. These 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 crosslinks it to a highly insoluble complex. We propose that additional lysines may be recruited for crosslinking, 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. In support of this hypothesis, protein sequencing experiments of the cornified cell envelopes harvested from forestomach epithelium recovered numerous crosslinks between THH and keratin filaments. Therefore, we propose that THH functions as an interfilamentous crossbridger designed to add tensile strength and/or rigidity to a tissue. Indeed, there is an apparent correlation between the expression levels of THH in tissues and the presumed physical requirements of the tissue. Thus normal human trunk epidermis contains little or no THH, foreskin epidermis contains about 5% THH, rodent foot pad and forestomach contain about 10% and 20% respectively, and the inner root sheath cells of hair follicles contain up to about one-third THH. Our ongoing experiments are directed toward testing aspects of this hypothesis. Recently, we have recovered THH-keratin crosslinked material from guinea pig hair follicle inner root sheath tissue in order to explore the crosslinking between the constituent proteins. Our data reveal that THH is extensively crosslinked to itself, keratin IF, and to established cell envelope structural proteins such as SPRs, desmoplakin, etc. Thus we propose that THH serves at least two distinct if not overlapping roles in the inner root sheath cells: as an interfilamentous matrix protein, and as a component of the cell envelope barrier for this tissue. One remaining issue which remains to be determined is the precise chain composition of the keratin IF expressed in the inner root sheath. Additional sequencing analyses will be performed to address this question. Note that in the case of the medulla, present in many types of coarser hairs, there are no keratin filaments. In this case it appears that the THH forms amorphous deposits that dehydrate leaving large vacuolar spaces. It is proposed that these spaces are critically involved in maintenance of body temperature in mammals by entrapment of air. 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 propose to test this hypothesis by performing atomic resolution structural studies. We have made a 55-residue synthetic peptide that shares the conserved structural motifs of intact THH. This peptide is soluble at >5 mg/ml and is essentially completely a-helical. Very recently, we have crystallized this peptide and attempts to solve its structure are in progress. The proximal promoter region of THH gene encompasses the first 160 bp above the transcription start site. This region contains an essential AP1 site as well as overlapping ets-like, NF-KB and Sp1-like sites. 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. Further work is in progress to complete the details of these synergistic interactions. In addition, there is significant nucleotide homology in the proximal promoter regions of the THH and profilaggrin genes. However, similar profilaggrin gene constructs transfected into hair follicles show little or no activity. This may mean there are elements which confer hair follicle tissue specificity of expression in the proximal promoter region of THH, a possibility which we will continue to characterize.