Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. Its gene is a susceptibility locus for rheumatoid arthritis. Here we present the crystal structure of Ca(2+)-free wild-type PAD4, which shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminal domain forms an alpha/beta propeller structure. Five Ca(2+)-binding sites, none of which adopt an EF-hand motif, were identified in the structure of a Ca(2+)-bound inactive mutant with and without bound substrate. These structural data indicate that Ca(2+) binding induces conformational changes that generate the active site cleft. Our findings identify a novel mechanism for enzyme activation by Ca(2+) ions, and are important for understanding the mechanism of protein citrullination and for developing PAD-inhibiting drugs for the treatment of rheumatoid arthritis.

Peptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca 2؉ dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent proteintagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45-74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophore A23187, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed.A family of peptidylarginine deiminases (PAD) 1 (EC 3.5.3.15) catalyzes the conversion of arginine residues in proteins into citrulline residues in the presence of calcium ion. Four types of rodent PADs, I, II, III, and IV, and of human PADs, I, II, III, and V, are known (1-8). Rodent PAD IV is most closely related to human PAD V. The structures of these PADs are relatively conserved in the C-terminal two-thirds of the sequence and diverge more distantly in the N-terminal one-third of the sequence. Rodent enzymes have different substrate specificities toward synthetic substrates and different tissue distributions (9, 10). Biochemical and immunocytochemical studies have suggested the involvement of PAD I in the terminal differentiation of epidermis (11)(12)(13), that of PAD II in myelination and demyelination of central nerve axons (14,15), and that of PAD III in the keratinization of hair follicles (8,16,17). Information on the biological functions of mouse PAD IV and human PAD V is rather limited. In addition, increased amounts of deiminated myelin basic proteins are in the afflicted area of brains of patients with multiple sclerosis, and prevalent autoantibodies recognizing citrulline residues as an epitope of autoantigen in patients with rheumatoid arthritis have suggested the pathological involvement of PAD in diseases (15, 18 -21).PAD V was first found in HL-60 cells when cells were induced to differentiate into granulocytes (HL-60 granulocytes) by all-trans-retinoic acid (RA) and differentiate into monocytes by 1␣,25-dihydroxyvitamin D 3 (7). PAD V was also found in peripheral blood granulocytes (22). PAD V in HL-60 cells can be activated to deiminate nuclear proteins of nucleop...

Small RNA-mediated regulation of iPS cell generationThe generation of induced pluripotent stem cells is limited by the low reprogramming efficiency of somatic cells. Here, three clusters of miRNAs are shown to enhance reprogramming efficiency by targeting the TGF-β and p53 pathways, which inhibit the process.

Macrophages infiltrate adipose tissue in obesity and are involved in the induction of inflammation, thereby contributing to the development of obesity-associated metabolic disorders. Here, we show that the macrophage-derived soluble protein AIM is endocytosed into adipocytes via CD36. Within adipocytes, AIM associates with cytosolic fatty acid synthase (FAS), thereby decreasing FAS activity. This decreases lipid droplet size, stimulating the efflux of free fatty acids and glycerol from adipocytes. As an additional consequence of FAS inhibition, AIM prevents preadipocyte maturation. In vivo, the increase in adipocyte size and fat weight induced by high-fat diet (HFD) was accelerated in AIM-deficient (AIM(-)(/-)) mice compared to AIM(+/+) mice. Moreover, injection of recombinant AIM in AIM(-)(/-) mice suppresses the increase in fat mass induced by HFD. Interestingly, metabolic rates are comparable in AIM(-)(/-) and AIM(+/+) mice, suggesting that AIM specifically influences adipocyte status. Thus, this AIM function in adipocytes may be physiologically relevant to obesity progression.

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...