S100B protein is elevated in the brains of patients with early stages of Alzheimer's disease and Down's syndrome. S100A4 is correlated with the development of metastasis. Both proteins bind to p53 tumor suppressor. We found that both S100B and S100A4 bind to the tetramerization domain of p53 (residues 325-355) only when exposed in lower oligomerization states and so they disrupt the tetramerization of p53. In addition, S100B binds to the negative regulatory and nuclear localization domains, which results in a very tight binding to p53 protein sequences that exposed the tetramerization domain in their C terminus. Because the trafficking of p53 depends on its oligomerization state, we suggest that S100B and S100A4 could regulate the subcellular localization of p53. But, the differences in the way these proteins bind to p53 could result in S100B and S1004 having different effects on p53 function in cell-cycle control.affinity ͉ binding ͉ fluorescence anisotropy ͉ S100A4 ͉ S100B T he tumor suppressor p53 protein is a homotetrameric transcription factor that regulates several cellular processes. In response to a subset of stresses, p53 prevents tumorigenic transformation through the induction of cell cycle arrest or apoptosis. Its crucial role is ref lected in that Ͼ50% of human cancers contain mutations in this gene (1-4). The protein is divided into four main functional domains: the N-terminal transactivation domain (residues 15-29), the core domain, which contains the specific DNA binding activity (residues 102-292), the tetramerization domain (residues 325-355), and the negative regulatory domain (residues 367-393) ( Fig. 1a) (5).In an unstressed cell, p53 tumor suppressor is maintained at low levels. When the cell is challenged by stress, however, p53 is activated through posttranslational modifications that increase its stability. The regulation of protein stability is one of the most effective mechanisms of controlling p53 function. Key to this process is MDM2, an E3 ligase that targets p53 for ubiquitination (2, 6). As p53 works essentially as a transcription factor, nuclear transport and retention are also crucial regulators of p53 activity. p53 is a nucleocytoplasmic shuttling protein that contains both nuclear localization sequences (NLSs) and nuclear export sequences (NESs); the latter are recognized by the nuclear transport factor CRM-1 (6-10). Tetramerization has also been found to be relevant in p53 activation (11). p53 binds to its DNA response elements most efficiently as a tetramer, and tetrameric p53 is most effective for transactivation (12)(13)(14)(15)(16)(17). These three levels of control of p53 activity (stability, cellular localization, and tetramerization) are tightly cross-regulated. For instance, tetrameric p53 is less efficient at entering the nucleus than monomeric p53 (10), p53 tetramerization occludes a NES signal, thereby ensuring nuclear retention of the DNA-binding form of p53 (9), and nuclear import and export are essential for MDM2-mediated p53 degradation (18).The S100 protein fa...

HDM2 is a negative regulator of p53 that inhibits its transcriptional activity and subjects it to degradation by an E3 ligase activity. The primary binding site for HDM2 on p53 is located in its N-terminal domain. A second site on the p53 core domain (p53C) binds to an unidentified site in HDM2. We found that this site is in its acidic domain and part of the zinc finger domain by examining the interaction of full-length and domain constructs of p53 with the N-terminal region of HDM2 and peptide arrays derived from the full-length protein. NMR spectroscopy showed that peptides derived from this region of HDM2 bound to residues in the specific DNA-binding site of p53C. The peptides were displaced from the site by gadd45 sequencespecific DNA. Phosphorylation of single amino acids in the central domain of HDM2 did not abolish the interaction between the HDM2-derived peptides and p53C. We speculate that this second binding site helps in stabilizing the interaction between HDM2 and p53 during p53 degradation.isothermal titration calorimetry ͉ Mdm2 ͉ NMR T he tumor suppressor protein p53 is important in maintaining genome stability and in preventing cancer development (1, 2). In response to various stress signals, p53 mediates cell-cycle arrest and apoptosis (3). p53 is a homotetramer consisting of an N-terminal transactivation domain, proline-rich regulatory domain, DNA-binding core domain (p53C), tetramerization domain, and C-terminal negative regulatory domain. Its major regulator, HDM2, is induced by p53 and acts as a feedback inhibitor (4). HDM2 regulates the activity of p53 in at least three ways. First, the N-terminal domain of HDM2 binds directly to p53's transactivation domain and inhibits its transcriptional function (5). Second, HDM2 acts as a ubiquitin ligase, targeting p53 and promoting its degradation (6, 7). Third, upon binding, HDM2 exports p53 from the nucleus to the cytoplasm (8).The interaction between peptides derived from the p53 N terminus and the HDM2-N-terminal domain has been extensively studied (9-12), and several compounds have been proposed to abolish this interaction (13-16). Recently, a new HDM2-binding site has been reported in p53C that plays a regulatory role in modulating p53 ubiquitination (17), although the exact binding site on HDM2 involved in this interaction has not yet been identified. There are conflicting speculations on its location at either the HDM2 N terminus (18) or in the acidic domain (19).Here, we examined the interaction of full-length and domain constructs of p53 with the N-terminal region of HDM2 and peptide arrays derived from the full-length protein. We found that the HDM2 N-terminal domain interacts only with p53's N-terminal domain, whereas from the peptide-array screen, we identified a second binding site for p53C located in the central domain (residues 221-302) of HDM2. We measured the binding affinity of the peptides to p53C by analytical ultracentrifugation. We identified the binding site on p53C by NMR and fluorescence anisotropy to be its DNA-binding site....

We investigated the ways S100B, S100A1, S100A2, S100A4, and S100A6 bind to the different oligomeric forms of the tumor suppressor p53 in vitro, using analytical ultracentrifugation and multiangle light scattering. It is established that members of the S100 protein family bind to the tetramerization domain (residues 325-355) of p53 when it is uncovered in the monomer, and so binding can disrupt the tetramer. We found a stoichiometry of one dimer of S100 bound to a monomer of p53. We discovered that some S100 proteins could also bind to the tetramer. S100B bound the tetramer and also disrupted the dimer by binding monomeric p53. S100A2 bound monomeric p53 as well as tetrameric, whereas S100A1 only bound monomeric p53. S100A6 bound more tightly to tetrameric than to monomeric p53. We also identified an additional binding site for S100 proteins in the transactivation domain (1-57) of p53. Based on our results and published observations in vivo, we propose a model for the binding of S100 proteins to p53 that can explain both activation and inhibition of p53-mediated transcription. Depending on the concentration of p53 and the member of the S100 family, binding can alter the balance between monomer and tetramer in either direction.The S100 family is a highly conserved group of more than 20 members of small, acidic calcium-binding proteins in vertebrates (1). They are called S100 because they remain soluble in 100% ammonium sulfate at neutral pH (2). S100 proteins are dimers or form higher oligomers (3, 4). They have intracellular functions such as the regulation of protein phosphorylation, the regulation of calcium homeostasis, cell survival, proliferation, and differentiation, as well as extracellular functions, for example, as attractors for leukocytes and macrophages, neurite outgrowth, or the induction of apoptosis (5-8). Further, the expression of several S100 proteins has been linked to metastasis (9) and different kinds of melanomas and carcinomas (8). Nevertheless, the molecular mechanism of action of the S100 proteins is not fully understood.The tumor suppressor p53 is a crucial factor in the development of cancer. It acts as the central inducer of apoptosis and cell cycle arrest (10, 11). Posttranslational modifications and interaction with proteins regulate its activity (12)(13)(14). The interaction with the tumor suppressor protein p53 is a common feature of the S100 proteins (15-19). We previously demonstrated that S100 proteins generally bind to the tetramerization domain (residues 325-355) of p53, whereas only a subset can bind its negative regulatory domain (residues 367-393) (16,20). S100B, S100A2, S100A4, and S100A6 have been reported to influence p53-mediated transcription, but the effect remains controversial because some studies show a stimulating effect, whereas others claim that S100 proteins inhibit the transcriptional activity of p53 (17-19, 21, 22). We previously showed that oligomerization of p53 weakens the binding to S100B and S100A4, and it was deduced that S100 proteins inhibit the oligo...

Edited by Wilhelm JustProteostasis, the controlled balance of protein synthesis, folding, assembly, trafficking and degradation, is a paramount necessity for cell homeostasis. Impaired proteostasis is a hallmark of ageing and of many human diseases. Molecular chaperones are essential for proteostasis in eukaryotic cells, and their function has traditionally been linked to protein folding, assembly and disaggregation. More recent findings suggest that chaperones also contribute to key steps in protein degradation. In particular, Hsp70 has an essential role in substrate degradation through the ubiquitin-proteasome system, as well as through different autophagy pathways. Accumulated knowledge suggests that the fate of an Hsp70 substrate is dictated by the combination of partners (cochaperones and other chaperones) that interact with Hsp70 in a given cell context.