PROTEIN UNFOLDING BY PEPTIDYLARGININE DEIMINASE - SUBSTRATE-SPECIFICITY AND STRUCTURAL RELATIONSHIPS OF THE NATURAL SUBSTRATES TRICHOHYALINAND FILAGGRIN

Peptidylarginine deiminases, which are commonly found in mammalian cel ls, catalyze the deimination of protein-bound arginine residues to cit rullines. However, very little is known about their substrate requirem ents and the significance or consequences of this postsynthetic modifi cation. We have explored this reaction in vitro with two known substra tes filaggrin and trichohyalin. First, the degree and rate of modifica tion of arginines to citrullines directly correlates with the structur al order of the substrate. In filaggrin, which has little structural o rder, the reaction proceeded rapidly to >95% completion. However, in t he highly a-helical protein trichohyalin, the reaction proceeded slowl y to about 25% and could be forced to a maximum of about 65%. Second, the rate and degree of modification depends on the sequence location o f the target arginines. Third, we show by gel electrophoresis, circula r dichroism, and fluorescence spectroscopy that the reaction interfere s with organized protein structure: the net formation of greater than or equal to 10% citrulline results in protein denaturation. Cyanate mo dification of the lysines in model alpha-helix-rich proteins to homoci trullines also results in loss of organized structure. These data sugg est that the ureido group on the citrulline formed by the peptidylargi nine deiminase enzyme modification functions to unfold proteins due to decrease in net charge, loss of potential ionic bonds, and interferen ce with H bonds.