Wheat sprout extract induces changes on 20S proteasomes functionality.

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols. The antioxidant properties of wheat sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells. The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin. The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. beta-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12-O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins.

Binding citrate/DNA in presence of divalent cations. Potential mimicry of acidic peptides/DNA interactions.

Citric acid whose structure is comparable to that of small acidic peptides, can bind to DNA in the presence of divalent cations (Cu2+, Fe2+, Zn2+, Mg2+). Citrate-DNA interaction occurs also in a cell homogenate and in this experimental model too requires the presence of natural divalent cations. In fact the addition of 2 mM EDTA to cell homogenate strongly decreases the DNA-citrate binding. The results demonstrate that divalent cations can act as bridges between two acidic molecules and that citric acid can mimic the structure of acidic peptides.

Competition between citrate and heptapeptide DDSDEEN binding to DNA in presence of divalent cations.

The binding of citrate and acidic peptide DDSDEEN with DNA in the presence of divalent cations is compared. Citric acid shows a higher number of binding sites on the DNA compared to the peptide; this is probably due to the bigger sitric hindrance of the peptide compared to the citric acid for the binding in the DNA grooves. Moreover. DNA preincubated with saturating amounts of citric acid is not available for the binding with successively added peptide. Therefore the peptide and citrate binding sites to some extent overlap.

Interleukin-1beta induces apoptosis in GL15 glioblastoma-derived human cell line.

Interleukin 1-beta (IL-1beta) induces apoptosis in a glioblastoma-derived human cell line, exhibiting a poorly differentiated astrocytic phenotype. The apoptotic effect was demonstrated by analyzing nuclear morphology, in situ DNA fragmentation, and by ELISA detection of cytoplasmatic nucleosomes. We correlated the degree of differentiation of GL15 cells with the apoptotic response: 1) 4',6-diamidino-2-phenylindole staining, combined with glial fibrillary acidic protein (GFAP) immunofluorescence, showed that the cells with apoptotic nuclei express low levels of GFAP; and 2) at 13 days of subculture, in a more differentiated state, GL15 cells did not respond with apoptosis to IL-1beta. In this cell line, nonrandom chromosome changes and the expression of SV40 early region have been previously shown. The involvement of p42/p44 mitogen-activated protein kinase (MAPK) pathway in the induction of apoptosis by IL-1beta was hypothesized. Previous studies have shown that SV40 small T antigen partially inhibits phosphatase 2A, leading to an enhancement of the steady-state activity of p42/p44 MAPK pathway. PD-098059, specific inhibitor of p42/p44 MAPK pathway, counteracts the apoptotic effect of IL-1beta, whereas SB-203580, specific inhibitor of p38 stress-activated protein kinase (SAPK) pathway, is ineffective. The imbalance between MAPK and SAPK pathways has been proposed as a key factor in determination of cell fate. Our results demonstrate that a further stimulation of p42/p44 MAPK pathway can constitute a death signal in tumor cells in which genomic damage and MAPK pathway control alterations occur.

Small acidic peptides and peptidomimetic molecules: cell growth and differentiation.

Small phosphorylated chromatin peptides exert a homeostatic regulation on gene expression which causes a strong inhibition of RNA synthesis and growth of neoplastic and fast-growing cells and a remarkable activation of metabolic pathways slowed down in ageing. By biochemical and mass spectrometry analysis, some molecular models of these peptides have been designed and synthesised. Recent studies show that it is possible to find peptidomimetic structures, such as citric acid, able to reproduce the antiproliferative effect. The mechanism of action has been investigated and partially clarified.

Differential scanning calorimetry of chromatin at different levels of condensation.

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 degrees C, while the other two occur at 63 degrees C and 74 degrees C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 degrees C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.

Molecular models of acidic peptides from pea bud chromatin and seminal plasma. Divalent cations-mediated interaction with DNA.

Small acidic peptides have been isolated from biological fluids (blood and seminal plasma) and from chromatin of several tissues. Their biological activity is related to the control of cell growth and gene expression. This work is an approach to the study of peptide structure-function relationship. Purified fractions from seminal plasma and pea bud chromatin were subjected to fast ion bombardment mass spectrometry. The results obtained were analyzed according to biochemical characteristics of the peptides studied and some possible molecular models have been designed. Two of the proposed sequences were synthesized and their biological activity assayed in cells and cell-free systems. The results demonstrate that the synthetic peptides are able to bind to DNA in the presence of divalent cations (Mg2+, Fe2+, Cu2+) with consequent inhibition of DNA transcription.

Binding of small phosphorylated chromatin peptides to DNA.

Low-molecular-weight peptides involved in gene expression and cell growth have been isolated from DNA preparation from eukaryotic cells. After phosphorylation with protein kinase CKII (pCKII) these peptides are able to bind to DNA in presence of divalent cations and salt/ethanol. This finding may explain the mechanism by which the peptides exert their activity.

Phosphorylation of the synthetic octapeptide pyroGlu-ASP-ASP-SER-ASP-GLU-GLU-ASN and binding to DNA in presence of divalent cations.

Small acidic peptides involved in gene expression have been isolated from prokaryotic and eukaryotic cells. Synthetic peptides, designed on the basis of native peptides characteristics, show a biological activity similar to that of native peptides in in vitro reconstituted systems. These synthetic peptides are able to bind to DNA in presence of divalent cations (Cu2+, Fe2+, Mg2+) and salt/ethanol.

Mass spectral and electrophoretic characterization of acidic peptides bound to chromatin of pea bud.

Structural features of a class of chromatin peptides are studied in the aim of understanding their mechanism of action. They have been reported as a family of small acidic peptides that can affect cell proliferation and RNA transcription. Mass spectrometry analysis has suggested some molecular models of possible sequences that might be present in this group of peptides. These sequences have been synthesised and their chromatographic and electrophoretic behaviour is compared with that obtained from peptides extracted from pea bud chromatin. In this way electric charge and hydrophilic properties of the native peptides are evaluated. On the basis of these data and those obtained from further mass spectrum analysis new models for native peptides are proposed.

Phosphopeptides derived from in vitro phosphorylated E. coli RNA polymerase bind to DNA and affect DNA transcription.

E. Coli RNA polymerase was phosphorylated with protein kinase CKII and allowed to bind to pBR322. After digestion of the RNA polymerase-pBR322 complex with proteinase K, the phosphopeptides that remained bound to DNA were extracted and analyzed. These phosphopeptides are able to bind again to DNA and to inhibit transcription.

Dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn inhibits the proliferation rate of HL-60 cells.

Small acidic phosphorylated chromatin peptides show regulatory activity on gene expression. The peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn, synthesized on the basis of structural and biochemical studies, shows functional properties in vitro (phosphorylation by casein kinase II, control of DNA transcription by RNA polymerase II, inhibition of proliferation and promotion of differentiation in some cell lines) very similar to those of native chromatin peptides. In this report we show that the dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn remarkably inhibits cell growth of the HL-60 cell line. The biological effect of the peptide seems to be considerably higher than that shown by the nondansylated peptide, and it cannot be attributed to a toxic effect of the Dns group. The measurement of uptake of 3H-labelled Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn demonstrates that it is unable to pass through the HL-60 cell membrane. It is our considered opinion that the addition of hydrophobic groups to the peptide N-terminus should increase the biological activity by improving its transport through the cellular membrane.

Synthetic octapeptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn promotes differentiation in promyelocytic HL-60 cell line.

The activity of the octapeptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn in the control of cell growth and differentiation of human myeloblastic leukemia cells HL-60 is reported. Treatment with peptide slightly slows down the rate of cellular proliferation and this effect becomes more evident in cells grown for several weeks in the presence of the effector. An enhanced effect (40-50% inhibition respect to the control) is found in reversibly permeabilized cells and after 1% DMSO is added to the medium. Moreover the presence of peptide markedly increases the percentage of cells differentiated by DMSO and RA. The effect in DMSO-induced cells is more evident than that observed in RA-induced cells. This in agreement with our hypothesis that DMSO facilitates the peptide entry and its effect is due to an intracellular action.

Human topoisomerase I is phosphorylated in vitro on its amino terminal domain by protein kinase NII.

Topoisomerase I purified from HeLa cells was phosphorylated in vitro with protein kinase NII (pkNII) purified from calf thymus: this phosphorylation was inhibited by heparin. A peptide containing a sequence corresponding to a putative pkNII phosphorylation site in topoisomerase I was synthesized and phosphorylated with pkNII. HPLC and two-dimensional analysis show identity between the synthetic phosphorylated peptide and one topoisomerase I phosphopeptide indicating Ser10 as one of the in vitro pkNII phosphorylation sites in topoisomerase I.

Molecular models of small phosphorylated chromatin peptides. Structure-function relationship and regulatory activity on in vitro transcription and on cell growth and differentiation.

We previously reported the isolation of low molecular weight phosphorylated peptides from the chromatin of several tissues. The chromatin peptides show a regulatory activity on DNA in vitro transcription and on cell growth and differentiation. In this paper, we report a molecular model of the native peptides designed according to the structural information obtained by means of biochemical and mass spectrometry analysis: pyroGlu-Ala-Gly-Glu-Asp-Ser(P)-Asp-Glu-Glu-Asn. This or very similar sequences are present in many transcription factors; on the basis of the structural model we presented and of related protein sequences, we have synthesized the peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn. This peptide affects transcription rate in reconstituted systems in vitro and in isolated nuclei; moreover, it inhibits the growth of HL60 cells with a parallel stimulus of differentiation.

Possible specific activation of RNA synthesis in PC-12 cell isolated nuclei by small acidic peptides.

Three synthetic peptides, pyro-Glu-Ala-Gly-Glu-Ser-Glu-Asp (Pep A), pyro-Glu-Ala-Gly-Glu-Glu-Glu-Ser-Asn (Pep B), and pyro-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn (Pep C), bear sequences possibly belonging to components of a naturally occurring family of strongly related small acidic chromatin peptides involved in regulation of gene expression. In a crude nuclear fraction and in purified nuclei from PC-12 cells, Pep A and Pep B activate RNA synthesis, specifically acting on the RNA polymerase II transcription system. On the other hand, Pep C shows an inhibitory effect on RNA synthesis in purified nuclei but an activation in the crude nuclear fraction. Control experiments show that the serum thymic factor does not affect RNA synthesis in the crude nuclear fraction or in purified nuclei. A possible regulation by peptide phosphorylation via casein kinase II (more active in purified nuclei than in the crude nuclear fraction) is discussed.

Synthetic octapeptide pyroGLU-ASP-ASP-SER-ASP-GLU-GLU-ASN controls DNA transcription in vitro by RNA polymerase II.

The effect of the synthetic octapeptide pyroGLU-ASP-ASP-SER-ASP-GLU-GLU-ASN (phosphorylated by casein kinase II, CKII) on DNA transcription by RNA polymerase II has been studied. The peptide contains the acidic carboxy-terminus heptapeptide of the largest subunit of RNA polymerase II, which has been demonstrated to be a phosphorylation site for CKII. The aim of this work is to obtain some insights about the possible role of this domain in RNA polymerase II activity and DNA binding. Results demonstrated that the phosphorylated octapeptide causes strong inhibition of transcription of calf thymus DNA or pSVL SV40 plasmid DNA by RNA polymerase II, when used at concentrations between 0.4-4 micrograms/ml.