Protein Unfolding by Peptidylarginine Deiminase

Abstract

Peptidylarginine deiminases, which are commonly found in mammalian cells, catalyze the deimination of protein-bound arginine residues to citrullines. However, very little is known about their substrate requirements and the significance or consequences of this postsynthetic modification. We have explored this reaction in vitro with two known substrates filaggrin and trichohyalin. First, the degree and rate of modification of arginines to citrullines directly correlates with the structural order of the substrate. In filaggrin, which has little structural order, the reaction proceeded rapidly to >95% completion. However, in the highly ␣-helical protein trichohyalin, the reaction proceeded slowly 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 of the target arginines. Third, we show by gel electrophoresis, circular dichroism, and fluorescence spectroscopy that the reaction interferes with organized protein structure: the net formation of 10% citrulline results in protein denaturation. Cyanate modification of the lysines in model ␣-helix-rich proteins to homocitrullines also results in loss of organized structure. These data suggest that the ureido group on the citrulline formed by the peptidylarginine deiminase enzyme modification functions to unfold proteins due to decrease in net charge, loss of potential ionic bonds, and interference with H bonds.

Specific Citrullination Causes Assembly of a Globular S100A3 Homotetramer

“…Previous investigation has shown that this irreversible modification unfolds the protein structure of natural substrates, such as filaggrin in the skin epidermis and trichohyalin in the hair follicle (13), resulting in different outcomes. Citrullinated filaggrin disassociates from keratin filaments and is hydrolyzed into amino acids (13), whereas citrullinated trichohyalin becomes soluble and susceptible to cross-linking by another Ca 2ϩ -dependent enzyme, transglutaminase (16,17). In contrast, citrullinated S100A3 dimer assembled into a homotetramer.…”

“…Although this biochemical process is believed to be precisely regulated by the intracellular Ca 2ϩ concentration, there remains controversy regarding the mechanism by which PAD and transglutaminase could be activated in vivo, since both require a nearly millimolar level of Ca 2ϩ to exhibit their full activities in vitro (12,18). Several epithelial protein barrier components containing Ca 2ϩ -binding domains have been reported to be natural substrates of PAD (the mature filaggrin subunit, proteolytically processed from profilaggrin (13)) or of transgutaminase (S100A7, S100A10, and S100A11 (14,15)) in skin epidermis or of both these enzymes (trichohyalin (16,17)) in hair follicles. It has been proposed that the Ca 2ϩ ions bound to these proteins are presented to the Ca 2ϩ -dependent proteinmodifying enzymes (8,11); however, more direct evidence is necessary to support this notion.…”

Molecular Characterization of Peptidylarginine Deiminase in HL-60 Cells Induced by Retinoic Acid and 1α,25-Dihydroxyvitamin D3

“…Enzymatic deimination in vitro changes the functional properties of various proteins and alters their secondary and tertiary structures (1)(2)(3)(4). Deimination of keratins, filaggrin, and trichohyalin is involved in the process of keratinization of skin and hair (4 -9).…”

The Fate of Trichohyalin

“…Recently, we have shown that a bacterially expressed shorter form of human THH (THH domain 8, THH-8) is a substrate for the type 2 PAD enzyme (28), the substrate specificity of which has been shown to be very similar to that of the type 3 enzyme (20). Interestingly, this reaction converts the highly ␣-helical THH-8 to a disorganized structure (28).…”