Chromosomal protein A24 has a unique structure inasmuch as it contains histone 2A and a nonhistone polypeptide the sequence of which has been partially determined.

No abstract

NO participates in numerous biological events in a variety of cell types including activated glomerular mesangial cells. Many of these events appear to be independent of the known effects of NO on soluble guanylyl cyclase. NO derived from all major isoforms of NO synthase can Snitrosylate cysteine residues in target proteins, potentially altering their functional activities. Recent evidence suggests that S-nitrosylation is specific, is regulated, and may play an important regulatory role akin to phosphorylation. In the present study, the "biotin-switch" method of isolating S-nitrosylated proteins was coupled with twodimensional PAGE protein separation followed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting to identify target proteins for S-nitrosylation in murine mesangial cells treated with NO donors or appropriate controls. This approach resolved 790 protein spots. We analyzed the most abundant spots and identified 34 known proteins. Of these, 31 are unique S-nitrosylated proteins not previously identified, including signaling proteins, receptors and membrane proteins, cytoskeletal or cell matrix proteins, and cytoplasmic proteins. Prominent among these were peroxisome proliferator activated receptor ␥, uroguanylin, GTP-binding protein ␣, protein 14-3-3, NADPH-cytochrome P450 oxidoreductase, transcription factor IIA, melusin, mitosin, phospholipase A 2 -activating protein, and protein-tyrosine phosphatase. The in vivo induction of S-nitrosylation was assayed by treating mesangial cells with interleukin-1␤ followed by the biotinswitch and Western blot of selected targets. These results broaden our knowledge of potential signal transduction pathways and other cell functions mediated by NO Snitrosylation. Molecular & Cellular Proteomics 2: 156 -163, 2003.NO is synthesized from L-arginine by the nitric-oxide synthase (NOS) 1 isozymes. Neuronal NOS and endothelial NOS isozymes have restricted tissue distributions and are regulated in part by intracellular Ca 2ϩ transients. Inducible nitricoxide synthase (iNOS) is expressed in a number of cell types in mammals after induction by cytokines and/or lipopolysaccharide, and once expressed, it is active at resting levels of intracellular Ca 2ϩ (1). In the kidney, NO plays prominent roles in the homeostatic regulation of glomerular, vascular, and tubular function as well as a variety of fundamental cellular functions, including DNA replication, transcription, energy metabolism, and apoptosis (1-3). Recent studies provide clear evidence for participation of iNOS-generated NO in the induction, progression, or protection of several types of experimental and human glomerulonephritis. In human glomerulonephritis, iNOS gene expression has been described in glomerular mesangial cells as well as in local and infiltrating macrophages (4). Glomerular mesangial cells play a central role in maintaining the structure and function of the glomerular capillary ultrafiltration apparatus and, under physiological or pathological...

The data available at present indicates there are three distinct functions of ubiquitin, two of which are related to protein conjugation. The first of these has been extensively studied by our laboratory and others interested in nucleosomes and changes in chromatin states. The ubiquitin-histone (Ub-2A, Ub-2B) conjugation reaction now appears to be a very dynamic process. In the deconjugation (lyase) reaction, both the histone 2A and the ubiquitin are left intact and in a form which makes possible ready reconjugation. Accordingly, this may be a mechanism for "moment-to-moment" Control of the genome. The second function in which ubiquitin is conjugated involves proteolytic activity. This activity is correlated with protein turnover. In this process, the ubiquitin-protein conjugate apparently serves as a "signal" for the protease cleavage of the protein. The released ubiquitin is also intact and is probably available for reconjugation. In the third function, ubiquitin was suggested to serve as a "hormone". The studies thus far have been carried out primarily on induction of T- and B-lymphocytes, reduction or delay of Coombs' positivity and reduction of spleen weight. The precise physiological role of this reported function is still unclear, particularly because the ubiquitin used was probably not the physiologically active form.

Two-dimensional gel profiles of the 0.2 M H2SO4-soluble proteins of monomer nucleosomal fractions were found to contain protein A24. Protein A24 is of interest because it is composed of histone 2A and "ubiqnitin," apparently joined by an isopeptide linkage [Goldknopf, I. L. & Busch, H. (1977) Proc. NatL Acad. Sci. USA 74,[864][865][866][867][868]; Hunt, L. T. & Dayhoff, M. 0. (1977) Biochem. Biophys. Res. Commun. 74,[650][651][652][653][654][655]. Monomer nucleosomal fractions were obtained by sucrose densi: gradient centrifugation of micrococcal nuclease digests of rat liver nuclei. As shown by their DNA size, the monomer fractions were highly purified. Proteins A24 and Bu, another protein of unknown characteristics, were found along with histones 1, 2A, 2B, 3, and 4 in the monomer fractions in relative amounts similar to those found in extracts from whole nuclei and chromatin. Other-acid-soluble proteins found in the nuclear and chromatin extracts were essentially absent from the monomer fraction. Inasmuch-as protein A24 and Bu were found in lesser amounts than the histones, it is suggested that they are associated with specialized subsets of nucleosomes. Protein A24 (1), a conjugated chromatin protein (2), has a -branched structure in which one arm and the stem are composed of histone 2A (3) and the other arm contains "ubiquitin" (4-7). The carboxyl terminus of ubiquitin is attached to a Gly-Gly peptide which is in an isopeptide linkage to the E-NH2 group of lysine residue 119 of histone 2A (8). Lysine 119 is a part of the very basic carboxyl-terminal amino acid sequence of histone 2A (9, 10). The overall structure of protein A24 is (8): Ubiquitin-( X)-Gly-Gly 7Histone 2A:R-Lys-Lys-Thr-Glu-Ser-His-His-Lys-Ala-Lys-Gly-Lys. 119 129The presence of histone 2A in protein A24 as well as its histone-like chromatin binding properties (1) suggested that protein A24 may be a component of nucleosomes (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).In the present study, the 0.2 M H2SO4-soluble proteins of purified rat liver monomer nucleosomes were analyzed by two-dimensional polyacryla'mide gel electrophoresis. On the basis of their electrophoretic mobility (24)(25)(26), protein A24, protein Bu, and the histones were present in the nucleosome extracts. Proteins A24 and Bu were present in smaller amounts than the histones and may be associated with specialized subsets of nucleosomes. MATERIALS AND METHODSRat liver nuclei were isolated by centrifugation in 2.2 M sucrose/3 mM calcium acetate as described previously (27). The monomer nucleosome fraction was obtained by a modification of the procedure of Lacey and Axel (28) and Sahasrabuddhe and Van Holde (17). Nuclei were suspended in 25 ,uM CaCl2/5 mM sodium phosphate/i mM phenylmethylsulfonyl fluoride, pH 6.7, at an OD20 of 5.0. Digestion was at 370 for 10 min with micrococcal nuclease (Sigma grade VI) at 5 Mg/ml. One-fourth volume of 25 mM EDTA/1 mM phenylmethylsulfonyl fluoride, pH 7.0, was added to stop the reaction and the digest was concentrated by...