Phosphorylation and acetylation of chromatin conjugate protein A24.

An analysis was made of the phosphorylation and acetylation of chromatin protein A24, a conjugate of histone 2A and ubiquitin. ³²P-orthophosphate was incorporated into phosphoserine of histone 2A and protein A24 in Novikoff hepatoma ascites cells in culture. The ratio of ³²P incorporation and the pattern of tryptic digestion of ³²P-labeled protein A24 indicated that the histone 2A component was phosphorylated and the ubiquitin component was not. Analysis of ε-N-acetyl lysine in protein A24, histone 2A and ubiquitin showed that while protein A24 and histone 2A were acetylated, ubiquitin was not. Apparently, even though it is conjugated with ubiquitin, the histone 2A portion of protein A24 has the same modifications as free histone 2A. The lack of modification of ubiquitin differs from that of high mobility group (HMG) non-histone chromatin proteins with which it is co-extracted from chromatin.     

Protein A24 lyase activity in nucleoli of thioacetamide-treated rat liver releases histone 2A and ubiquitin from conjugated protein A24

Isolated liver nucleoli from rats treated for 3 days with thioacetamide contained an enzyme activity which specifically degraded conjugate protein A24. Two-dimensional polyacrylamide gel electrophoresis indicated that the amount of protein A24 in chromatin decreased during incubation at 37 degrees C for 60 min with these nucleoli. Concomitantly, a marked increase was found in the content of free ubiquitin, the nonhistone component of protein A24. Incubation of 3H-labeled protein A24 with the thioacetamide-treated liver nucleoli resulted in the linear release of 3H-labeled histone 2A and 3H-free ubiquitin in the presence of phenylmethanesulfonyl fluoride (PMSF) for 2 h. Pretreatment of the nucleoli with trypsin or by heating at 80 degrees C for 10 min inhibited their ability to cleave protein A24. Protein A24 lyase catalyzes the reaction: protein A24 leads to histone 2A plus ubiquitin.     

Substrate recognition of isopeptidase: specific cleavage of the epsilon-(alpha-glycyl)lysine linkage in ubiquitin-protein conjugates

The structural chromatin protein A24 (uH2A) is a conjugate of histone H2A and a non-histone protein, ubiquitin. Eukaryotic cells contain an enzyme, generically termed isopeptidase, which can cleave A24 stoichiometrically into H2A and ubiquitin in vitro. Isopeptidase, free of proteinase activity, has been partially purified from calf thymus by ion-exchange chromatography, gel filtration and affinity chromatography, and analyzed for its substate specificity. There are three major types of isopeptide bonds besides the epsilon-(alpha-glycyl)lysine bond between H2A and ubiquitin; namely, the disulfide bridge, the aldol and aldimide bonds and the epsilon-(gamma-glutamyl)lysine crosslink. Under conditions where A24 was completely cleaved into H2A and ubiquitin, none of these naturally occurring isopeptide bonds was cleaved by isopeptidase. Furthermore, the bonds formed in vitro by transglutaminase reaction between casein and putrescine, through the gamma-NH2 of glutamine residue and the NH2 of putrescine, were not cleaved by the enzyme. The enzyme also failed to cleave the glycyl-lysyl and other orthodox peptide linkages within proteins. Among various proteins examined, the substrates for isopeptidase reaction were confined to conjugates between ubiquitin and other proteins, formed through epsilon-(alpha-glycyl)lysine bonds. Since ubiquitin released by isopeptidase is re-usable for an ATP-dependent conjugation with other proteins, its carboxyl terminal -Gly-Gly-COOH most likely is preserved intact, and is not blocked. These results suggest that isopeptidase specifically recognizes and cleaves the epsilon-(alpha-glycyl)lysine bond. A possible biological significance of this enzyme is discussed.     

Inhibition of protein A24 lyase by nitrosoureas

The protein A24 content of Ehrlich ascites tumor cells increased several-fold following treatment of cell cultures with nitrosoureas, but did not increase when other alkylating agents not containing carbamoyl moieties were tested. The same nitrosoureas and, in addition, 2-chloroethyl isocyanate inhibited an A24 lyase-containing cytoplasmic extract in cleaving protein A24 into histone H2A and ubiquitin. It appears that carbamoylation of A24 lyase by nitrosoureas inhibits the enzyme and is responsible for the measured increases in cellular protein A24 content due to reduced turnover of this protein.     

Preparation of chromosomal protein A24 (uH2A) by denaturing gel filtration and preparation of its free nonhistone component ubiquitin by ion-exchange chromatography

Chromosomal protein A24 (uH2A) is unique in that it comprises the nucleosomal core histone H2A in isopeptide linkage with the highly conserved, globular, and stable nonhistone protein ubiquitin. Some 10% of the chromatin complement of H2A is modified in this way and studies to elucidate a role for this modification have concentrated on observations requiring no purification of A24 due to the difficulty in isolating the protein in large and pure quantities. We describe a method for isolating A24 by chromatography on Pharmacia G-100 gel filtration medium under urea denaturing conditions. A24 prepared by this method is structurally intact and is available in the quantities required for studies of the behavior and influence of the protein on histone-histone, histone-DNA, and enzymatic interactions. In conjunction with this method we describe a procedure for the isolation of large quantities of free ubiquitin of far greater purity than previously reported.     

Assembly of semihistone A24.

Nucleosomal semihistone A24 (uH2A) is composed of histone H2A and ubiquitin peptides. The kinetics of incorporation of nascent H2A and ubiquitin into A24 of transformed chicken lymphocytes (MSB cells) have been examined by peptide mapping, COOH-terminus analysis, density labeling and isopycnic centrifugation of chromosomal proteins. We find that newly synthesized H2A is rapidly conjugated to ubiquitin. SDS-PAGE analysis of fractionated density gradients suggests, however, that newly synthesized ubiquitin becomes bound nonselectively to both new and preexisting H2A molecules.     

Structure of polyubiquitinated histone H2A

We have recently demonstrated that trout liver histones H2A, H2B, and H2A.Z can be polyubiquitinated [Davie, J.R., Delcuve, G.P., Nickel, B.E., Moyer, R., & Bailey, G. (1987) Cancer Res. 47, 5407-5410]. In the present study we determined the arrangement of the ubiquitin molecules in polyubiquitinated histone H2A. Trout liver chromatin fragments. which had histone H1 removed, were digested with Staphylococcus aureus (V8 strain) protease which cleaves specifically on the carboxyl side of glutamic acid residues under the conditions used. The V8 protease readily degraded histone H2A and ubiquitinated (u) H2A at equivalent rates. One site in H2A and uH2A, the peptide bond between Glu 121 and Lys 122, was cleaved, yielding protein species cH2A and cuH2A, respectively. None of the other nucleosomal histones (H2B, H2A.Z, H3, and H4) including uH2B and uH2A.Z were sensitive to digestion. Trout liver histones cleaved with either V8 protease, histone H2A specific protease, or cyanogen bromide were resolved by two-dimensional gel electrophoresis and ubiquitinated peptides detected with anti-ubiquitin IgG. The results suggest that the major arrangement of ubiquitin in polyubiquitinated H2A is a chain of ubiquitin molecules joined to each other by isopeptide bonds to a ubiquitin molecule that is attached to the epsilon-amino group of lysine 119 of histone H2A.     

Affinity of chromatin constituents for isopeptidase

Isopeptidase is a novel eukaryotic enzyme that cleaves a structural chromatin protein, A24, stoichiometrically into H2A and ubiquitin. To understand the rapid turnover of ubiquitin in mitosis as wells as the high specific activity of the enzyme associated with metaphase chromosomes, attempts were made to determine chromatin constituents that show high affinity for this enzyme. Endogenous protease-free isopeptidase was prepared from calf thymus and applied to a Sepharose 4B affinity column on which histones, DNA, NHCP and ubiquitin were respectively immobilized. The enzyme proved to bind only histones. To further determine which of the histone fractions is involved, affinity columns with each histone fraction were also used. The enzyme showed affinity for all histone fractions. However, the strength of affinity varied in the order H2A>H³ H2B≥H4?H1, being inversely correlated with the ratio of basic/acidic amino acids in these molecules. These results suggest that the turnover of A24 in mitosis is controlled, at least in part, by the affinity of enzyme for histones, and also that such affinity is caused by a mechanism which cannot be explained simply by the electrostatic interaction between negatively charged enzyme molecules and positively charged histones.     

The heterogeneity of rat high density lipoproteins.

Ubiquitin was first found in nuclei in protein A24 where its carboxyl terminal is covalently bound to histone 2A by an isopeptide linkage (Goldknopf, I. L. and Busch, H. (1977) Proc. Natl. Acad. Sci. USA $\text{74}$, 864–868). Two-dimensional polyacrylamide gel electrophoresis of the 0.4 N H₂SO₄ soluble proteins from fractionated rat liver chromatin showed that protein A24 and histones H1, H2A, H2B, H3 and H4 were present in fractions P1 and P2 and markedly diminished in relative amounts in fraction S2. Conversely, a spot designated Ub was found in fraction S2 along with an increased amount and number of non-histone proteins. The Ub spot was not found in chromatin fractions P1 and P2. Ub was identified as ubiquitin by migration on two-dimensional gels and after purification by preparative polyacrylamide gel electrophoresis by its methionine NH₂-terminal amino acid and its amino acid composition.     

ADP-ribosylation of nuclear protein A24

Nuclear protein A24, which is composed of histone H2A and ubiquitin, a nonhistone protein, joined by an isopeptide linkage [Goldknopf and Busch (1977) $\text{Proc}$. $\text{Natl}$. $\text{Acad}$. $\text{Sci}$. $\text{USA}$$\text{74}$, 864–868], is found to be ADP-ribosylated in isolated rat liver nuclei.     

H2B ubiquitylation: the end is in sight

Historically, the first eukaryotic protein found to be modified by ubiquitin was H2A, originally isolated from HeLa cells in 1975 by Harrison Busch and coworkers as a histone-like, nonhistone chromosomal protein called A24. Ubiquitylated histones have subsequently been found in many eukaryotic species, and to date, the core histones H2A, H2B, H3, the linker histone H1, and the histone variant H2A.Z are known to carry this modification. Although first on the scene, it was only recently that studies on histone ubiquitylation have enjoyed a renaissance. Part of the reason for the relatively slow pace of research on this fascinating histone modification was the absence of a good genetic system with which to study its cellular roles. This changed in 2000, when histone H2B was found to be ubiquitylated in the budding yeast S. cerevisiae, an organism with a low histone gene copy number and highly tractable genetics. Another factor was the almost exclusive focus of research on the role of polyubiquitylation in protein turnover. Because histones are generally monoubiquitylated, a form of the modification that is not associated with protein degradation, the significance of this minimalist ubiquitin conjugation was not heavily pursued. But perhaps the key reason for the renewed interest in histone ubiquitylation was the unexpected discovery of the past year that ubiquitylated H2B plays an important role in the trans-histone methylation of histone H3, a modification with close ties to the regulation of gene expression. This review will highlight some of the recent findings on the regulation and cellular roles of H2B ubiquitylation in yeast.     

Biosynthesis of a ubiouitin-related peptide in rat brain and in human and mouse pituitary tumors

A biosynthetic labeled peptide structurally related to the thymic peptide ubiquitin was first identified fortuitously in bovine pars intermedia cells in regard to its partial NH₂ terminal amino acid sequence (Met 1, Leu 8, 15 and Lys 6, 11, 27, 29, 33) after a protein segment data bank search. A peptide with the same behavior on carboxymethylcellulose chromatography and polyacrylamide gel electrophoresis has been purified after labeling experiments in two areas of rat brain, hypothalamus and striatum, and in a mouse and a human ACTH-secreting pituitary tumors. It represents about 1 to 10% of the total labeled proteins in the various experiments. Its identity with the above mentioned bovine pituitary peptide was confirmed by microsequence analysis with respect to Met 1, Lys 6, 11 in hypothalmus, Met 1 in striatum, and Lys 6, 11, 27, 29, 33 in the two pituitary tumors. The availability of standard purified ubiquitin allowed us to show that labeled and cold peptides have the same electrophoretic mobility and elution volume on Sephadex G-50 chromatography this further confirms their identity. Possible interests of such a biosynthetic characterization of a ubiquitin-related peptide are discussed, particularly in view of the structural relationship of ubiquitin to the non histone component of nuclear protein A-24, and as a test of tissue viability and biosynthetic efficiency in our in vitro biosynthetic systems.     

N-terminal amino acid sequence of leukemia derived growth factor (LGF) from human erythroleukemia cell culture

Summary A human erythroleukemia cell line, K-562 T1, was adapted to a protein-free chemically defined medium (1); that is, the medium does not contain any proteins such as exogenous hormones, growth factors, serum and serum albumin. The K-562 T1 cells which can proliferate in a protein-free medium are one of the model systems suitably supporting the autocrine hypothesis (2), which claims that cancer cells produce and respond to their own growth factors (3). The K-562 T1 cells were cultured in a protein-free medium at large scale and the growth factors were purified from the conditioned medium. It was found that K-562 T1 cells produce at least two growth factors; one is LGF-I (leukemia-derived growth factor-I) which can stimulate the proliferation of a wide range of human leukemia cell lines and the other is LGF-II (leukemia-derived growth factor-II), which can contribute to the growth of fibroblasts. LGF-I was purified using QAE-Sephadex, Bio Gel P-60 and Mono S FPLC. The purified protein was found to be homogenous by SDS-polyacrylamide gel electrophoresis and NH₂-terminal sequence analysis. The molecular weight of LGF-I was 20,000 by SDS-polyacrylamide gel electrophoresis. The 30 NH₂-terminal residues of LGF-I are the same as that of ubiquitin. Ubiquitin is a protein found in eukaryotic cells with molecular weight of 8,600. In the nucleus ubiquitin is conjugated to histone 2A to form the nuclear protein A24 which may play a role in regulation of chromatin structure (3), and in the cytoplasm is part of an ATP-dependent non-lysosomal proteolytic pathway (4). However, its physiological significance has not yet been fully resolved. Ubiquitin purified from bovine thymus did not show cell proliferating activity for any cells tested. The results suggest that LGF-I is a new autocrine growth factor with a molecular weight of 20,000 daltons, containing ubiquitin at the NH₂-terminal end. This work was supported by funds obtained under the Research and Development Project of Basic Technologies for Future Industries from the Ministry of International Trade and Industry of Japan. Editor’s Statement Identification of sequences identical to ubiquitin associated with the leukemia-derived growth factor described in this report is particularly intriguing considering recent reports of association of ubiquitin with surface membrane receptors of lymphocytes and fibroblasts. References: SCIENCE vol. 231, pgs, 823–829; NATURE vol. 323, pgs 226–232, 1986. David W. Barnes     

Stimulation of ATP-dependent proteolysis requires ubiquitin with the COOH-terminal sequence Arg-Gly-Gly

It was previously shown that ubiquitin is very similar to the polypeptide cofactor of the ATP-dependent protein degradation system from rabbit reticulocytes (Wilkinson, K. D., Urban, M. K., and Haas, A. L. (1980) J. Biol. Chem. 255, 7529-7532). We have extended this work to show that the peptic peptide maps are identical for bovine ubiquitin and the polypeptide cofactor isolated from human erythrocytes. It was noted however that ubiquitin preparations were less active in stimulating proteolysis than preparations of the polypeptide cofactor. This decreased activity has been shown to be due to the presence of an inactive form of ubiquitin in some preparations. The two forms of ubiquitin are separable by high performance liquid chromatography. The active form of ubiquitin has the COOH-terminal sequence -Arg-Gly-Gly at residues number 74 to 76. The inactive form terminates in -Arg74 as previously reported in the sequence studies of ubiquitin. Limited tryptic digestion of active ubiquitin yields the inactive, later eluting form and the dipeptide glycylglycine. This preteolytic cleavage apparently occurs during purification from most tissues. We thus propose reserving the term ubiquitin for the intact 76-amino acid sequence and designating the 74-amino acid sequence as ubiquitin-t to indicate its derivation by a tryptic-like protease cleavage. This 76-residue sequence is consistent with the covalent structure of protein A-24, a conjugate where carboxyl group of the COOH-terminal glycylglycine of ubiquitin is linked by an amide bond to the epsilon-amino group of Lys-119 of histone H2A. Thus, the structural requirements of the protein and ubiquitin molecules are identical for formation of protein A-24 and for forming the covalent conjugates thought to be intermediates in ATP-dependent protein degradation.     

Ubiquitin-mediated degradation of histone H3 does not require the substrate-binding ubiquitin protein ligase, E3, or attachment of polyubiquitin chains

Radioiodinated histone H3 was incubated with ubiquitin, the ubiquitin-activating enzyme E1, and one of three ubiquitin carrier proteins, reticulocyte E2(20K) or E2(32K) or the yeast RAD6 product. Although the resulting ubiquitin-histone conjugates were synthesized in the absence of the substrate-binding protein E3, they were nevertheless degraded by purified rabbit reticulocyte 26 S protease. In contrast, unmodified histone H3 remained intact upon challenge with the 26 S ubiquitin/ATP-dependent enzyme. Conjugates produced by the RAD6 protein were better proteolytic substrates than those formed by reticulocyte E2 unless ubiquitin molecules with altered lysines were used for conjugate synthesis. Substitution of methylated ubiquitin or ubiquitin molecules in which lysine 48 was converted to arginine by site-directed mutation produced histone conjugates that were degraded at slow but measurable rates. Since methylated ubiquitin molecules are incapable of forming branched polyubiquitin chains, these results demonstrate that neither ubiquitin "trees" nor the substrate binding factor E3 is absolutely required for ubiquitin-dependent degradation of histone H3 in vitro.