Q95399963
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Protease-activated receptors (PARs) belong to the G-protein-coupled receptor family, that are expressed in many body tissues especially in different epithelial cells, mast cells and also in neurons and astrocytes. PARs play different physiological roles according to the location of their expression. Increased evidence supports the importance of PARs activation during nociceptive signaling and in the development of chronic pain states. This short review focuses on the role of PAR2 receptors in nociceptive transmission with the emphasis on the modulation at the spinal cord level. PAR2 are cleaved and subsequently activated by endogenous proteases such as tryptase and trypsin. In vivo, peripheral and intrathecal administration of PAR2 agonists induces thermal and mechanical hypersensitivity that is thought to be mediated by PAR2-induced release of pronociceptive neuropeptides and modulation of different receptors. PAR2 activation leads also to sensitization of transient receptor potential channels (TRP) that are crucial for nociceptive signaling and modulation. PAR2 receptors may play an important modulatory role in the development and maintenance of different pathological pain states and could represent a potential target for new analgesic treatments.
- MeSH
- kationtové kanály TRP metabolismus MeSH
- lidé MeSH
- mícha metabolismus MeSH
- nádorová bolest metabolismus MeSH
- neuralgie metabolismus MeSH
- nocicepce * MeSH
- nociceptivní bolest metabolismus MeSH
- proteinkinasy metabolismus MeSH
- receptor PAR-2 metabolismus MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C-terminal DNA-binding site (CTDBS) is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodies mapping to epitopes at the protein C-terminus (to block binding within the CTDBS). In the absence of antibody, reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides, both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON, but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA, while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil-selective (SCS) binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence-specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress.
- MeSH
- lidé MeSH
- monoklonální protilátky metabolismus MeSH
- mutageneze cílená MeSH
- nádorový supresorový protein p53 metabolismus ultrastruktura MeSH
- oxidace-redukce MeSH
- sekvenční delece MeSH
- superhelikální DNA metabolismus ultrastruktura MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- vazebná místa protilátek MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
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.
