Nuclear proteins nonhistone

The primary Junction of the nucleosomes is to condense DNA. Further condensation of nucleosome DNA requires nonhistone nuclear proteins. These proteins make up a scaffoldlike structure around an additional helix consisting of coiled nucleosomes. This produces a structure that resembles a solenoid, with six nucleosome subunits per turn. The solenoid structure can form large loops that give additional structure to the incipient chromosome. 

Histones and nonhistone nuclear proteins Ribosomal protein S6 elF (eukaryotic initiation factor) eEF (eukaryotic elongation factor)... 

All nuclear receptors have sequences known as domains of nuclear location (Picard et al. 1987). These sequences, rich in arginine and lysine, confer upon the many proteins that contain them the capacity to bind to nonhistone nuclear proteins. Receptors have up to four of these sequences, whose cooperation is necessary for nuclear location. When these sequences are exposed, the receptor... 

In isolated nuclei from rat liver and kidney, Ni2 + was bound to chromatin, polynucleosomes and to deproteinized DNA [339]. Ni2+ directly interacted with stable binding sites on the DNA molecule in chromatin and was associated with histone and nonhistone nuclear proteins [339, 340]. 

Nickel sulphate in peripheral blood T lymphocytes gave a marked increase of 32P label into nonhistone proteins [ 109], especially in the 30-40 kDa region. It was postulated that the increase in nuclear protein phosphorylation probably reflected an activation of the lymphocytes. 

Hunt, L.T., and Dayhoff, M.O. (1977). Amino-terminal sequence identity of ubiquitin and the nonhistone component of nuclear protein A24. Biochem. Biophys. Res. Commun. 74,650-655. 

Other nonhistone nuclear proteins. Polyacrylamide gel electrophoresis revealed more than 450 components in HeLa cell nuclei. Most are present in small amounts of <10,000 molecules per cell and are not detectable in cytoplasm.112 Among the more acidic proteins are many enzymes including RNA polymerases. There are also gene repressors, hormone-binding proteins, protein kinases, and topoi-somerases.113 Among the six most abundant nonhistone nuclear proteins in the rat are the cytoskeletal proteins myosin, actin, tubulin, and tropomyosin.114... 

Although the DNA-binding properties of several procaryotic topoisomerases have been well characterized, little information is currently available concerning eucaryotic enzymes. Some eucaryotic topoisomerases may be intimately associated with other nuclear proteins HeLa topoisomerases I and II have been found to be associated with chromatin (Javaherian and Liu, 1983 Liu et al., 1983b). HeLa topoisomerase I has been shown to bind to the nonhistone protein HMG17, which also stimulates DNA catenation by the enzyme (Javaherian and Liu, 1983 Tse et al., 1984). It has been suggested that type II topoisomerase is an important component of the chromosomal scaffold in interphase nuclei and mitotic chromosomes from chicken cell lines (Earnshaw et al., 1985). In addition, an ATP-dependent topoisomerase has been found associated with several other enzymes of DNA metabolism in a complex (termed the replitase complex) isolated from the nuclei of Chinese hamster embryo fibroblast cells (Noguchi et al., 1983). 

Both mono- and di-methylated arginine residues have been described in proteins such as myelin basic protein, myosin, heat shock proteins, nuclear proteins, ribosomal proteins, and tooth matrix proteins (reviewed in Paik and Kim, 1980). NG N O-dimethylarginine residues are found mainly in myelin basic proteins, while N, N -dimethylarginine residues are found largely in nonhistone nuclear proteins. 

It must be emphasized that this model does not explain the functions of heteromodification, whereby nuclear proteins other than the poly(ADP-ribose) polymerase also become covalently modified, including both histones and nonhistone proteins. 

A serum factor that was initially felt to represent a specific thymic hormone was termed SF (A. Astaldi et al., 1976 G. Astaldi et al., 1979, 1980 Facchini et al., 1982). Serum factor was shown to increase cAMP, to decrease TdT activity, to induce changes in nonhistone nuclear proteins in... 

Both histone and nonhistone proteins appear to be synthesized in the cytoplasm of sea urchin embryos, partly from maternal templates, and subsequently migrate to the cleavage nuclei (Kedes et al., 1969 Crane and Villee, 1971 Johnson and Hnilica, 1971 Moav and Nemer, 1971 Seale and Aronson, 1973a,b). Similar studies on amphibians have been scarce. The only direct analysis of nuclear proteins in amphibians is that reported by Asao (1969, 1972). Using a mixture of C-labeled amino acids, either injected into Triturus females during ovulation or... 

Several procedures have been developed for the isolation of ADP-rihosylated nuclear protein. In one described by Hayaishi and coworkers (162) ADP-ribosylated nuclear proteins were separated by affinity chromatography in 6 M guanidine- HCl on a dihydroxyhoryl polyacrylamide column (162). A very specific interaction of the borate residue with the cis diol portion of ribose rings of the ADP-ribose permits the selective isolation of ADP-ribosylated proteins. Applying this technique to rat liver nuclear proteins, they observed a distribution of ADP-ribosylation between histone and nonhistone proteins, with histone H2B (67%) and Hi (33%) being preferentially modified. 

At present the events triggering and the mechanism controlling muscle differentiation are not well understood they may include changes in chromatin structure, gene amplification, and specific DNA and/or protein modification. One such modification, namely, poly(ADP-ribosyl)ation of nuclear proteins has recently attracted much attention and several studies have indicated that the enzyme poly(ADP-ribose) synthetase plays a role in cellular differentiation [6-9]. This enz)mie is a nuclear chromatin-associated protein which catalyzes covalent modification of both histone and nonhistone protein acceptors (for reviews see [10-13]). The synthetase is activated by DNA strand breaks and it has been suggested that DNA fragmentation and the consequent increase in poly(ADP-ribose) activity are obligatory events for chick muscle differentiation [6]. 

Fig. 2. Quantification of the increase in t PJNAD labeling of nuclear and chromosomal proteins resulting from incubation with DMS. Autoradiograms, such as those in Fig. 1, were scanned with a Cary spectrophotometer equipped with a gel scanning stage. The relative weights of the tracings were measured for incorporation by total proteins ( ), nonhistones (O), and the 116,000 dalton protein (x). Panel A, nuclear proteins from cells treated in growth medium panel B, cells treated in isolation buffer, panel C, metaphase chromosome proteins from cells incubated with mutagen in chromosome isolation buffer.

Apparently, all nuclear proteins participate in the uptake of the labeled amino acids as indicated by specific activities studies of nonhistone and histone-type protein. However, although the activity of nonhistone proteins was high, the amino acid uptake by histones was relatively low. The specific activity of the histones is only one-third the specific activity of the total protein. These basic experiments of the in vitro amino acid incorporation suggested that proteins are actively synthesized in the nuclear preparation. The cytoplasm is believed to provide the amino acids for the nucleus. 

In mammalian cells, direct evidence for cAMP s role in modulating transcription is unavailable, and the proposition that cyclic AMP affects transcription by phosphorylating histone or nonhistone nuclear proteins is premature since such an effect has not been convincingly established, and it is not known whether such phosphorylation is involved in transcription. Similarly, an effect of cAMP on translation in eukaryotes is questionable. Both inhibitory and stimulatory effects have been claimed. All effects of cAMP in cells are more likely to be traced to direct action on enzymes namely, the conversion of an inactive to an active form, or vice versa. 

Histones and nonhistone (acid) nuclear proteins have continued to attract attention, mainly from the standpoint of their possible role in the regulation of genetic activity of the nucleus and chromosome, i.e., in the phenomena of selective repression and de-repression. 

Most of the DNA of animal cells is found in the nucleus, where DNA is the major constituent of the chromosomes. On the other hand, most of the RNA is located in the cytoplasm. Nuclear DNA exists as a thin, double helix only 2 nm wide. The double helix is folded and complexed with protein to form chromosomal strands approxim-ately 100 to 200 nm in diameter. Each chromosome contains a single DNA duplex. The human chromosomes vary in size the smallest contains approximately 4.6 X 10 base pairs of DNA, and the largest 2.4 X 10 base pairs. In contrast, the Escherichia coli chromosome has 4.5 x 106 base pairs. The DNA of die chromosomes is tightly packed and associated with both histone and nonhistone proteins. 

Nuclear DNA in eukaryotes is found in chromatin associated with histones and nonhistone proteins. The basic packaging unit of chromatin is the nucleosome (Figure 1-1-13) ... 

The family of HDAC enzymes has been named after their first substrate identified, i.e., the nuclear histone proteins. Histone proteins (H2A, H2B, H3 and H4) form an octamer complex, around which the DNA helix is wrapped in order to establish a condensed chromatin structure. The acetylation status of histones is in a dynamic equilibrium governed by histone acetyl transferases (HATs), which acetylate and HDACs which are responsible for the deacetylation of histone tails (Fig. 1). Inhibition of the HDAC enzyme promotes the acetylation of nucleosome histone tails, favoring a more transcriptionally competent chromatin structure, which in turn leads to altered expression of genes involved in cellular processes such as cell prohferation, apoptosis and differentiation. Inhibition of HDAC activity results in the activation of only a limited set of pre-programmed genes microarray experiments have shown that 2% of all genes are activated by structmally different HDAC inhibitors [1-5]. In recent years, a growing number of additional nonhistone HDAC substrates have been identified, which will be discussed in more detail below. 

Many proliferation-associated antigens have been reported as clinically useful indicators of proliferative activity (1). Of these, the so-called proliferating cell nuclear antigen (PCNA) and Ki-67 have been identified as the most useful in both immunohistochemistry (see Chapter 27) and flow cytometry (FCM). PCNA is an auxiliary protein to DNA polymerase 8 (2,3) and is intimately associated with DNA replication, but also DNA repair (4,5). Ki-67 is a large protein associated with nuclear nonhistone proteins (6,7), and is expressed in all actively proliferating cells (8,9). Expression of these two proteins, in a cell population should equate to the growth fraction, i.e., the proportion of cells involved in an active cell cycle. However, there are apparent inconsistencies when these two proteins have been compared with one another (10) and with other methods of assessing cell proliferation (11). 

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