[Identification of Distinct Amino Acid Composition of Human Cruciform Binding Proteins].

Cruciform structures are preferential targets for many architectural and regulatory proteins, as well as a number of DNA binding proteins with weak sequence specificity. Some of these proteins are also capable of inducing the formation of cruciform structures upon DNA binding. In this paper we analyzed the amino acid composition of eighteen cruciform binding proteins of Homo sapiens. Comparison with general amino acid frequencies in all human proteins revealed unique differences, with notable enrichment for lysine and serine and/or depletion for alanine, glycine, glutamine, arginine, tyrosine and tryptophan residues. Based on bootstrap resampling and fuzzy cluster analysis, multiple molecular mechanisms of interaction with cruciform DNA structures could be suggested, including those involved in DNA repair, transcription and chromatin regulation. The proteins DEK, HMGB1 and TOP1 in particular formed a very distinctive group. Nonetheless, a strong interaction network connecting nearly all the cruciform binding proteins studied was demonstrated. Data reported here will be very useful for future prediction of new cruciform binding proteins or even construction of predictive tool/web-based application.

Spatio-temporal changes of SDF1 and its CXCR4 receptor in the dorsal root ganglia following unilateral sciatic nerve injury as a model of neuropathic pain.

There is a growing evidence that chemokines and their receptors play a role in inducing and maintaining neuropathic pain. In the present study, unilateral chronic constriction injury (CCI) of rat sciatic nerve under aseptic conditions was used to investigate changes for stromal derived factor-1 (SDF1) and its CXCR4 receptor in lumbal (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) from both sides of naïve, CCI-operated and sham-operated rats. All CCI-operated rats displayed mechanical allodynia and thermal hyperalgesia in hind paws ipsilateral to CCI, but forepaws exhibited only temporal changes of sensitivity not correlated with alterations in SDF1 and CXCR4 proteins. Naïve DRG displayed immunofluorescence for SDF1 (SDF1-IF) in the satellite glial cells (SGC) and CXCR4-IF in the neuronal bodies with highest intensity in small- and medium-sized neurons. Immunofluorescence staining and Western blot analysis confirmed that unilateral CCI induced bilateral alterations of SDF1 and CXCR4 proteins in both L4-L5 and C7-C8 DRG. Only lumbal DRG were invaded by ED-1+ macrophages exhibiting SDF1-IF while elevation of CXCR4-IF was found in DRG neurons and SGC but not in ED-1+ macrophages. No attenuation of mechanical allodynia, but reversed thermal hyperalgesia, in ipsi- and contralateral hind paws was found in CCI-operated rats after i.p. administration of CXCR4 antagonist (AMD3100). These results indicate that SDF1/CXCR4 changes are not limited to DRG associated with injured nerve but that they also spread to DRG non-associated with such nerve. Functional involvement of these alterations in DRG non-associated with injured nerve in neuropathic pain remains to be elucidated.

Binding of p53 and its core domain to supercoiled DNA.

We have compared the binding of human full-length p53 protein (p53; expressed in bacteria and insects) and its isolated core domain (p53CD, amino acids 94-312; expressed in bacteria) to negatively supercoiled (sc) DNA using gel electrophoresis and immunoblotting. Significant differences were observed; p53CD produced a relatively small and continuous retardation of scDNA, in contrast to the ladder of distinct bands formed by p53 in agarose gels. The ladder produced by full-length protein expressed in bacteria (p53b) was similar to that observed earlier with protein expressed in insect cells (p53i). Competition between scDNAs and their linearized (lin) forms showed a preference for scDNAs by both p53 and p53CD, but the ratios characterizing the distribution of the protein between sc and lin pBluescript DNAs were substantially higher for p53 (sc/lin > 60 in p53b) than for p53CD (sc/lin approximately 4). Strong binding of p53 to scDNA lacking the p53 consensus sequence may represent a new p53-binding mode, which we tentatively denote supercoil-selective (SCS) binding. This binding requires both the C-terminal domain and the core domain. Targets of this binding may include: (a) DNA segments defined both by the nucleotide sequence and local topology, and/or (b) strand crossings and/or bending. The binding preference of p53CD for scDNA may be due to the known nonspecific binding to internal single-stranded regions in scDNA (absent in relaxed DNA molecules) and/or to SCS binding albeit with reduced affinity due to the absence of contributions from other p53 domains.

Precise characterisation of monoclonal antibodies to the C-terminal region of p53 protein using the PEPSCAN ELISA technique and a new non-radioactive gel shift assay.

The development of human cancers is frequently associated with inactivation of the p53 tumour suppressor protein triggering cell cycle arrest or apoptosis in response to cellular stress. The p53 protein has been identified as a transcription factor with sequence-specific DNA binding properties. The DNA-binding activity is cryptic but can be modulated through the C-terminal region of the p53 protein by several different stimuli, including phosphorylation by casein kinase II (CKII), protein kinase C (PKC) or binding of the C-terminal monoclonal antibody PAb421. Monoclonal antibodies to the C-terminal region of p53 protein are able to activate the latent form of p53 and induce binding to DNA. To characterise such antibodies, we used a combination of the PEPSCAN ELISA procedure and a newly developed non-radioactive gel shift assay. Monoclonal antibodies from the Bp53 series displayed higher affinities for the human, rat and mouse p53 proteins than did the conventional antibody PAb421. In addition, these antibodies were able to activate the sequence-specific DNA binding functions in latent forms of p53 protein and, in contrast to PAb421, they were able to recognise both PKC phosphorylated and PKC non-phosphorylated forms of p53 protein. Our monoclonal antibodies recognising post-translationally modified target epitopes in the C-terminal region of p53 protein might assist the development of more effective molecules for p53-based cancer therapy.

Specific modulation of p53 binding to consensus sequence within supercoiled DNA by monoclonal antibodies.

Monoclonal antibodies (MAbs) were used to investigate the binding of insect cell-expressed, wild-type human p53 protein to the consensus sequence (p53CON) in a 474-bp DNA fragment and to supercoiled (sc) DNAs with and without p53CON. Supershifting of p53-DNA complexes by MAbs in agarose gels was applied to studies of activation of p53 for sequence-specific binding within scDNA. C-terminal specific antibody Bp53-10.1 activated the sequence-specific binding of p53 to p53CON within pPGM1 scDNA but did not influence binding of p53 to pBluescript scDNA (not containing p53CON). Incubation of p53 with DO-1 prior to addition of Bp53-10.1 prevented activation of p53 and induced dissociation of a portion of pPGM1 scDNA from the sequence-specific immune complex; no such dissociation was observed if pPGM1 scDNA was replaced by the 474-bp p53CON-containing DNA fragment.

Effects of Oxidation Agents and Metal Ions on Binding of p53 to Supercoiled DNA.

Summary Wild type human full length (f.1.) tumor suppressor p53 protein binds preferentially to super-coiled (sc) DNA in vitro both in the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on the agarose gel. Bands revealed by immunoblotting with antibody DO-1 corresponded to the ethidium stained retarded bands. The intensity and the number of bands of p53-scDNA complex were decreased by physiological concentrations of unchelated zinc ions. Nickel and cobalt ions inhibited binding of p53 to scDNA and to p53CON in linear DNA fragments less efficiently than zinc. Compared to the intrinsic zinc strongly bound to Cys 176, Cys 238, Cys 242 and His 179 in the p53 core domain, binding of additional Zn(2+) to p53 was much weaker as shown by an easy removal of the latter ions by low concentrations of EDTA. Oxidation of the protein with diamide resulted in a decrease of the number of the retarded bands. Under the same conditions, no binding of oxidized p53 to p53CON in a linear DNA fragment was observed. In agreement with the literature oxidation of f.1. p53 with diamide was irreversible and was not reverted by an excess of DTT. We showed that in the presence of 0.1 mM zinc ions, oxidation of p53 became reversible. Other divalent cations tested (cadmium, cobalt, nickel) exhibited no such effect. We suggested that the irreversibility of p53 oxidation was due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain (after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein) accompanied by a change in the p53 conformation. Binding of C-terminal anti-p53 antibody also protected bacterially expressed protein against irreversible loss of activity due to diamide oxidation. Binding the human p53 core domain (segment 94-312) to scDNA greatly differed from that observed with the full-length p53. The core domain did not posses the ability to bind strongly to many sites in scDNA regardless of the presence or absence of p53CON suggesting involvement of some other domain (probably C-terminal) in binding of the full-length p53 to scDNA. Supershift experiments using antibodies against p53 N- or C-terminus suggested that in oxidized p53, scDNA binding through the C-terminus gained importance.

Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments.

Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these hands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 microM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 microM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 microM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo.

Tumor suppressor protein p53 binds preferentially to supercoiled DNA.

Wild type human tumor suppressor protein p53 (expressed in insect cells) binds strongly to negatively supercoiled (sc) plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of scDNA in agarose gels and imaging by scanning force microscopy (SFM). The binding occurs both in the presence and absence of the p53 consensus sequence. At relatively low p53/DNA ratios, binding of p53 to scDNA results in the appearance of several retarded DNA bands on the gels, similar to a conventional topoisomer ladder generated enzymatically. However, after removal of p53 by deproteination, the original mobility of the scDNA is recovered, indicating that the reduction of torsional stress accompanying p53 binding does not reflect changes in linking number. In DNA samples partially relaxed by topoisomerase I p53 binds preferentially to the scDNA molecules with the largest negative superhelix density. SFM imaging of the p53/scDNA complex reveals a partial or total relaxation of the compact scDNA, the degree of which increases with the number of bound p53 molecules. Competition assays with linear DNA reveal a preference of p53 for scDNA. In addition, scDNA induces dissociation of p53 from a preformed complex with a DNA fragment (474 bp) containing the consensus sequence. We conclude that the affinity of p53 for negatively supercoiled DNA is greater than that for the consensus sequence in linear fragments. However, thermally denatured linearized plasmid DNA is efficient in competing for the binding of p53 to scDNA, although the first retarded band (presumed to contain one bound p53 molecule) is retained in the case of the plasmid containing the consensus sequence. Thus, it appears that interactions involving both the core domain and the C-terminal domain regulate the binding of p53 to scDNA. The above results are not restricted to human p53; the wild type rat p53 protein also results in the retardation of scDNA on agarose gels. The biological implications of the novel DNA binding activities of p53 are discussed.