13-107 Sigma-AldrichCore Histones

In the budding yeast Saccharomyces cerevisiae, the lysine acetyltransferase NuA4 has been linked to a host of cellular processes through the acetylation of histone and non-histone targets. To discover proteins regulated by NuA4-dependent acetylation, we performed genome-wide synthetic dosage lethal screens to identify genes whose overexpression is toxic to non-essential NuA4 deletion mutants. The resulting genetic network identified a novel link between NuA4 and septin proteins, a group of highly conserved GTP-binding proteins that function in cytokinesis. We show that acetyltransferase-deficient NuA4 mutants have defects in septin collar formation resulting in the development of elongated buds through the Swe1-dependent morphogenesis checkpoint. We have discovered multiple sites of acetylation on four of the five yeast mitotic septins, Cdc3, Cdc10, Cdc12 and Shs1, and determined that NuA4 can acetylate three of the four in vitro. In vivo we find that acetylation levels of both Shs1 and Cdc10 are reduced in a catalytically inactive esa1 mutant. Finally, we determine that cells expressing a Shs1 protein with decreased acetylation in vivo have defects in septin localization that are similar to those observed in NuA4 mutants. These findings provide the first evidence that yeast septin proteins are acetylated and that NuA4 impacts septin dynamics.

Acetylation is an essential post-translational modification in which an acetyl group is covalently conjugated to a protein substrate. Histone acetylation was first proposed nearly half a century ago by Dr. Vincent Allfrey. Subsequent studies have shown that acetylated core histones are often associated with transcriptionally active chromatin. Acetylation at lysine residues of histone tails neutralizes the positive charge, which decreases the binding ability to DNA and increases the accessibility of transcription factors and co-activators to the chromatin template. In addition to histones, a number of non-histone substrates are acetylated. Acetylation of non-histone proteins governs biological processes, including cellular proliferation and survival, transcriptional activity, and intracellular trafficking. We demonstrated that acetylation of transcription factors can regulate cellular growth. Further, we have shown that nuclear receptors are acetylated at a phylogenetically conserved motif. Since our initial observations with the estrogen and androgen receptors, more than a dozen nuclear receptors have been shown to function as substrates for acetyltransferases with a variety of new methods (Fig. 11.1). This chapter focuses on the protocol used in the studies of NR acetylation and de-acetylation. We will discuss the potential pitfalls of each method.

Chromatin structure is important for controlling gene expression, but mechanisms underlying chromatin remodeling are not fully understood. Here we report that an FKBP (FK506 binding protein) type immunophilin, AtFKBP53, possesses histone chaperone activity and is required for repressing ribosomal gene expression in Arabidopsis. The AtFKBP53 protein is a multidomain FKBP with a typical peptidylprolyl isomerase (PPIase) domain and several highly charged domains. Using nucleosome assembly assays, we showed that AtFKBP53 has histone chaperone activity and the charged acidic domains are sufficient for the activity. We show that AtFKBP53 interacts with histone H3 through the acidic domains, whereas the PPIase domain is dispensable for histone chaperone activity or histone binding. Ribosomal RNA gene (18S rDNA) is overexpressed when AtFKBP53 activity is reduced or eliminated in Arabidopsis plants. Chromatin immunoprecipitation assay showed that AtFKBP53 is associated with the 18S rDNA gene chromatin, implicating that AtFKBP53 represses rRNA genes at the chromatin level. This study identifies a new histone chaperone in plants that functions in chromatin remodeling and regulation of transcription.

The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund's adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund's adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.

Barrier to autointegration factor (BAF) is an essential conserved double-stranded DNA-binding protein in metazoans. BAF binds directly to LEM domain nuclear proteins (e.g. LAP2, Emerin, and MAN1), lamin A, homeodomain transcription factors, and human immunodeficiency virus type 1-encoded proteins. BAF influences higher order chromatin structure and is required to assemble nuclei. BAF also facilitates retroviral preintegration complex insertion into target DNA in vitro, through unknown mechanisms. We report that BAF binds directly and selectively to linker histone H1.1 (among three subtypes tested) and core histone H3 with affinities of approximately 700 nm and approximately 100-200 nm, respectively, in vitro and in vivo. Mutations at the bottom and top surfaces of the BAF dimer disrupted or enhanced, respectively, this binding and affected H1 and H3 similarly. Biochemical studies showed that C-terminal residues 108-215 of histone H1.1 and the N-terminal tail plus helix alphaN in the core of histone H3.1 were each necessary and sufficient to bind BAF. Based on its interactions with histones and DNA, we propose BAF might bind nucleosomes in vivo.

Metabotropic glutamate (mGlu) receptors have been implicated in a number of physiological and pathological responses to glutamate, but the exact role of group I mGlu receptors in causing postischaemic injury is not yet clear. In this study, we examined whether the recently-characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) could reduce neuronal death in vitro, following oxygen-glucose deprivation (OGD) in murine cortical cell and rat organotypic hippocampal cultures, and in vivo, after global ischaemia in gerbils. When present in the incubation medium during the OGD insult and the subsequent 24 h recovery period, AIDA and CBPG significantly reduced neuronal death in vitro. The extent of protection was similar to that observed with the nonselective mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] and with typical ionotropic glutamate (iGlu) receptor antagonists. Neuroprotection was also observed when AIDA or CBPG were added only after the OGD insult was terminated. Neuronal injury was not attenuated by the inactive isomer (-)MCPG, but was significantly enhanced by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid [(1S,3R)-ACPD] and the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (3,5-DHPG). The antagonists (+)MCPG, AIDA and CBPG were also neuroprotective in vivo, because i. c.v. administration reduced CA1 pyramidal cell degeneration examined 7 days following transient carotid occlusion in gerbils. Our results point to a role of mGlu1 receptors in the pathological mechanisms responsible for postischaemic neuronal death and propose a new target for neuroprotection.

Recombinant human erythropoietin (rhuEpo)-specific mouse monoclonal antibodies (MoAbs) have been produced and characterized. All antibodies were specifically reactive with rhuEpo in enzyme-linked immunosorbent assay (ELISA). Epitope exclusion studies showed three distinct epitope regions, A, B, and C, recognized by neutralizing MoAbs. An additional epitope region D was recognized by non-neutralizing MoAbs. Antibodies defining an epitope region competed with each other for binding sites, but did not compete with antibodies defining a different epitope region. Group B antibodies were able to compete for the receptor binding site on rhuEpo with a soluble human Epo-receptor-lg fusion protein. No single peptide sequences were found to specifically interact either with group B MoAbs or with the rhuEpo-receptor. Therefore, it is suggested that epitope region B and the receptor binding site share binding determinants that are primarily composed of conformational epitopes. Because group A and group C antibodies did not compete with the receptor for binding to the receptor binding site of the rhuEpo molecule, it is suggested that neutralization via epitope regions A and C is mediated through binding inhibition caused by conformational changes, transmuting the binding site(s) for the receptor. Conversely, binding to the receptor seems to induce conformational changes in the hormone molecule, eliminating epitopes for group A and C antibodies.