Histones protease

Before our work [39], only one catalytic mechanism for zinc dependent HDACs has been proposed in the literature, which was originated from the crystallographic study of HDLP [47], a histone-deacetylase-like protein that is widely used as a model for class-I HDACs. In the enzyme active site, the catalytic metal zinc is penta-coordinated by two asp residues, one histidine residues as well as the inhibitor [47], Based on their crystal structures, Finnin et al. [47] postulated a catalytic mechanism for HDACs in which the first reaction step is analogous to the hydroxide mechanism for zinc proteases zinc-bound water is a nucleophile and Zn2+ is five-fold coordinated during the reaction process. However, recent experimental studies by Kapustin et al. suggested that the transition state of HDACs may not be analogous to zinc-proteases [48], which cast some doubts on this mechanism. 

As to the stoichiometry of the H3-H4-DNA particle, two complexes were identified an H3-H4 tetramer and an H3-H4 octamer, each associated with about 140 base pairs of DNA. The complexing of 140 base pairs of DNA with H3 and H4 resulted in the formation of nucleosome-like particles, as observed by the EM, and reported to have an s20base pairs (Bina-Stein and Simpson, 1977 Bina-Stein, 1978). These results differ from those of Simon et al. (1978) who report that at least two complexes of H3 H4-DNA can be obtained upon reconstitution of H3, H4, and 150 bp DNA. In this experiment both an octamer and a tetramer of H3-H4 were found bound to 150 base pairs of DNA, having sM,w equal to 10.4 and 7.5 for the octamer and tetramer, respectively. The stoichiometry of the complexes obtained is dependent on the histone-to-DNA ratio. At low ratios of histone to DNA the predominant species contains an H3-H4 tetramer per 150 base pairs of DNA. At a histone-to-DNA ratio of 1 1 the octamer prevails. The nuclease and protease digestion experiments (Camerini-Otero et al., 1976 Sollner-Webb et al., 1976) were performed at a histone-to-DNA ratio of 0.5, conditions which for 140-base-pair DNA would lead primarily to a tetrameric complex. Therefore, it seems that a tetramer of H3 H4 is sufficient for the generation of nuclease-resistant fragments similar to those of complete nucleosomes. Upon addition of H2A and H2B to the tetrameric complex, nucleosomes are formed. Addition of H3-H4 to the tetrameric complex resulted in an octameric complex which is similar in compaction to nucleosomes. H3-H4 tetramers and octamers were similarly found complexed with about 140 base pairs of DNA upon reconstitution of H3-H4 with SV40 DNA. Both complexes were reported to be able to fold 140 base pairs of DNA (Thomas and Oudet, 1979). 

ADP-ribosylation has also been implicated as a proteolytic antagonist during embryonic development [231]. Following fertilization in sea urchin, sperm-specific histones are degraded by the sperm-histone-selective (SpH) protease and subsequently replaced by cleavage stage histone variants. During this process, the maternal replacement histones are protected from proteolysis by ADP-ribosylation. 

Fig. 2. NAPI facilitates H2A, H2B release from nucleosomes that are on positively coiled DNA (A) but not negatively coiled DNA (B). The positively coiled DNA (6.0 kb) with a superhelical density of + 0.05 and negatively coiled DNA (6.0 kb) with a superhelical density of -0.05 were reconstituted with lysine, arginine-labeled histones H3, H4, H2A, H2B by NaCl dialysis from 2.0 M to 1.2 M to 0.6 M to 0.1 M NaCl over a 14 h period. The samples were incubated with NAPI at 35 °C for 5 min and applied to a 5-20% sucrose/100 mM NaCl/40 mM Tris, pH 7.8 gradient. After sedimentation at 200,000 X g for 5 h, fractions were collected and the distribution of DNA (bottom panel) was determined on agarose gel and the distribution of protein (top panel) on SDS-PAGE followed by fluorography. These data are unpublished observations (V. Levchenko and V. Jackson). The deg-H2A is degraded H2A in which a 15 amino acid peptide of the C terminal has been proteolytically removed. When H2A, H2B is no longer present in a nucleosome, the C terminal region is sensitive to proteolysis [126] from a protease which is a minor contaminate in the NAPI preparation.

MM Falk, PR Grigera, IE Bergmann, A Zibert, G Multhaup, E Beck. Foot and mouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3. J Virol 64 748-756, 1990. 

Proteins were then dissolved in 20 mL of 0.125 M Tris-HCl, pH 7.4 containing 1% (m/v) CHAPS and the protease inhibitor cocktail, and treated with CPLL. With several variations from the standard treatments such as other complementary extractions from seeds, the use of urea-containing extraction buffer, and an additional peptide ligand library, the total number of detected gene products was quite large. Two hundred and thirty one unique proteins were found in the pulp (vs. 56 described previously) and 61 in the seeds (vs. only four reported by the literature). In the latter case, the presence of seed storage proteins, oleosins, and histones were detected in the pulp, a thaumatin-like protein, an allergenic protein also named Ole el3, was confirmed. 

Some of the PolyP complexes with proteins are very important in cell regulatory processes. RNA polymerase isolated from the stationary-phase cells of E. coli was found to be closely bound with PolyP (Kusano and Ishihama, 1997). The ATP-dependent protease Lon formed a complex with PolyPs under degradation of ribosomal proteins at amino acids starvation (Kuroda et al, 2001). PolyP is able to compete with DNA for the DNA binding sites at histones (Schroder et al., 1999), while PolyPs can interact with non-histone proteins in the nucleus (Offenbacher and Kline, 1984). 

Ca -regulated serine proteases Histone-associated protease... 

Several E. coli heterologous gene expression systems based on the tac, T7, and Hlac promoters have been used successfrilly to obtain recombinant archaeal histones, by following the commercial protocols specified for each expression system. The purification protocol detailed above for native archaeal histones also works well for recombinant archaeal histones, although the protease inhil >itor phenylmethylsulfonyl fluoride (PMSF) must be added to a final concentration of 0.1 mAf during the DNase I digestion. Almost 100% purity can be achieved from E. coli crude extracts. 

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