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. 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 human THH is very insoluble (<1 mg/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 mg/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. 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 filaments 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 seems to be a direct correlation between the expression levels of THH in tissues and the apparent 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. Future experiments will be directed toward testing aspects of this hypothesis. In particular, we wish to harvest THH-keratin crosslinked material from inner root sheath tissue in order to explore the crosslinking between them. 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. Our initial experiments have 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 will be needed to complete the details of these synergistic interactions.