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

Abstract

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyteinducing agent 1␣,25-dihydroxyvitamin D 3 . We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50 -55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophageinduced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.Peptidylarginine deiminases (PADs) 1 (protein-arginine deiminase, protein L-arginine iminohydrolase, EC 3.5.3.15) are a family of post-translational modification enzymes which convert arginine residues to citrulline residues in the presence of calcium ion. Enzymatic deimination in vitro changes the functional properties of various proteins and alters their secondary and tertiary structures (1-4). Deimination of keratins, filaggrin, and trichohyalin is involved in the process of keratinization of skin and hair (4 -9). Deiminated keratins and filaggrin are found in the cornified layer of the epidermis and deiminated trichohyalin is localized in the medulla of hair and the inner root sheath of hair follicles and these modifications are tightly linked to cell-specific stages of epidermis differentiation and hair follicle development (5-9). Extensively deiminated forms of myelin basic protein are also found in normal infant brain and in demyelinated areas of brain with multiple sclerosis, and this deimination is thought to be associated with immature myelination (10, 11). We reported a correlation between deimination of vimentin in mouse peritoneal macrophages and ionomycin-induced apoptosis (12). Deimination of a 70-kDa nuclear protein in cultured keratinocytes associated with apoptosis was also reported recently (13). All these findings suggest involvements of PAD in biological as well as pathological processes. There are at least three types of PAD in various rodent tissues which seem to be cell type specific (3, 14 -16). Their substrate specificities for BAEE and Bz-L-Arg and their antigenic properties are different. PAD type II purified from rat muscle has been well characterized. It is also pres...

Inhibitors and Inactivators of Protein Arginine Deiminase 4: Functional and Structural Characterization,

“…As noted above, the calcium dependence of inactivation likely arises because PAD4 is a calcium-dependent enzyme whose in vivo activity can be regulated by the addition of a calcium ionophore (19). In vitro, calcium binding is known to cause a conformational change that moves Cys645 and His471 into positions that are competent for catalysis and presumably reaction with these inactivators.…”

Histone Deimination As a Response to Inflammatory Stimuli in Neutrophils

“…One intriguing histone posttranslational modification is the deimination of specific arginines in histones H2A, H3, and H4 (2)(3)(4). The conversion of arginine to citrulline, an atypical amino acid that lacks arginine's positive charge, is accomplished by a member of the peptidylarginine deiminase (PAD) 3 family of enzymes, PAD4 (5). Although PAD4-mediated modifications of histones are involved in regulating chromatin structure and gene expression in a variety of cell types (6 -8), PAD4 may assume an additional role in eosinophil and neutrophil responses to infections.…”

Anticancer Peptidylarginine Deiminase (PAD) Inhibitors Regulate the Autophagy Flux and the Mammalian Target of Rapamycin Complex 1 Activity

“…PAD4 is the only PAD family member containing a nuclear localization signal and citrullinates many substrates including histones (e.g. H3, H2A, and H4), p300/CREBbinding protein (CBP), nucleophosmin, ING4, and nuclear lamin C to exert various functions (15)(16)(17)(18)(19). Genome-wide association and pathology studies have implicated PAD4 in the etiology of rheumatoid arthritis and cancers in human patients (20 -23).…”