Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.

The crustacean gill (Na(+),K(+))-ATPase: Allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na(+), K(+))-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na(+) and K(+) of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na(+),K(+))-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na(+) and K(+). However, in contrast to Na(+), a threshold K(+) concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na(+),K(+))-ATPase compared to the vertebrate enzyme. (C) 2008 Elsevier Inc. All rights reserved.

Evaluation of the Binding Properties of Maghemite Nanoparticle Surface-Coated with Meso-2-3-Dimercaptosuccinic Acid to Serum Albumin

In this study the interaction between magnetic nanoparticles (MNPs) surface-coated with meso-2,3-dimercaptosuccinic acid (DMSA) with both bovine serum albumin (BSA) and human serum albumin (HSA) was investigated. The binding of the MNP-DMSA was probed by the fluorescence quenching of the BSA and HSA tryptophan residue. Magnetic resonance and light microscopy analyses were carried out in in vivo tests using female Swiss mice. The binding constants (K(b)) and the complex stoichiometries (n) indicate that MNP-DMSA/BSA and MNP-DMSA/HSA complexes have low association profiles. After five minutes following intravenous injection of MNP-DMSA into mice`s blood stream we found the lung firstly target by the MNP-DMSA, followed by the liver in a latter stage. This finding suggests that the nanoparticle`s DMSA-coating process probably hides the thiol group, through which albumin usually binds. This indicates that biocompatible MNP-DMSA is a very promising material system to be used as a drug delivery system (DDS), primarily for lung cancer treatment.

The human sulfotransferase SULT1A1 gene is regulated in a synergistic manner by Sp1 and GA binding protein

Human sulfotransferase SULT1A1 is an important phase II xenobiotic metabolizing enzyme that is highly expressed in the liver and mediates the sulfonation of drugs, carcinogens, and steroids. Until this study, the transcriptional regulation of the SULT1A subfamily had been largely unexplored. Preliminary experiments in primary human hepatocytes showed that SULT1A mRNA levels were not changed in response to nuclear receptor activators, such as dexamethasone and 3-methylcolanthrene, unlike other metabolizing enzymes. Using HepG2 cells, the high activity of the TATA-less SULT1A1 promoter was shown to be dependent on the presence of Sp1 and Ets transcription factor binding sites (EBS), located within - 112 nucleotides from the transcriptional start site. The homologous promoter of the closely related SULT1A3 catecholamine sulfotransferase, which is expressed at negligible levels in the adult liver, displayed 70% less activity than SULT1A1. This was shown to be caused by a two-base pair difference in the EBS. The Ets transcription factor GA binding protein (GABP) was shown to bind the SULT1A1 EBS and could transactivate the SULT1A1 promoter in Drosophila melanogaster S2 cells. Cotransfection of Sp1 could synergistically enhance GABP-mediated activation by 10-fold. Although Sp1 and GABP alone could induce SULT1A3 promoter activity, the lack of the EBS on this promoter prevented a synergistic interaction between the two factors. This study reports the first insight into the transcriptional regulation of the SULT1A1 gene and identifies a crucial difference in regulation of the closely related SULT1A3 gene, which accounts for the two enzymes' differential expression patterns observed in the adult liver.

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.

Characterization of temperature dependent and substrate-binding cleft movements in Bacillus circulans family 11 xylanase: A molecular dynamics investigation

Background: Xylanases (EC 3.2.1.8) hydrolyze xylan, one of the most abundant plant polysaccharides found in nature, and have many potential applications in biotechnology. Methods: Molecular dynamics simulations were used to investigate the effects of temperature between 298 to 338 K and xylobiose binding on residues located in the substrate-binding cleft of the family 11 xylanase from Bacillus circulans (BcX). Results: In the absence of xylobiose the BcX exhibits temperature dependent movement of the thumb region which adopts an open conformation exposing the active site at the optimum catalytic temperature (328 K). In the presence of substrate, the thumb region restricts access to the active site at all temperatures, and this conformation is maintained by substrate/protein hydrogen bonds involving active site residues, including hydrogen bonds between Tyr69 and the 2` hydroxyl group of the substrate. Substrate access to the active site is regulated by temperature dependent motions that are restricted to the thumb region, and the BcX/substrate complex is stabilized by extensive intermolecular hydrogen bonding with residues in the active site. General significance: These results call for a revision of both the ""hinge-bending"" model for the activity of group 11 xylanases, and the role of Tyr69 in the catalytic mechanism. (C) 2009 Elsevier B.V. All rights reserved.

PREDBALB/c: a system for the prediction of peptide binding to H2(d) molecules, a haplotype of the BALB/c mouse

PREDBALB/c is a computational system that predicts peptides binding to the major histocompatibility complex-2 (H2(d)) of the BALB/c mouse, an important laboratory model organism. The predictions include the complete set of H2(d) class I ( H2-K-d, H2-L-d and H2-D-d) and class II (I-E-d and I-A(d)) molecules. The prediction system utilizes quantitative matrices, which were rigorously validated using experimentally determined binders and non-binders and also by in vivo studies using viral proteins. The prediction performance of PREDBALB/c is of very high accuracy. To our knowledge, this is the first online server for the prediction of peptides binding to a complete set of major histocompatibility complex molecules in a model organism (H2(d) haplotype). PREDBALB/c is available at http://antigen.i2r.a-star.edu.sg/predBalbc/.

Mapping of suramin binding sites on the group IIA human secreted phospholipase A(2)

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein. namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phosphohpase A(2) inhibitor. (C) 2009 Elsevier Inc. All rights reserved.

The 2.0 angstrom crystal structure of a pocilloporin at pH 3.5: The structural basis for the linkage between color transition and halide binding

The pocilloporin Rtms5 and an engineered variant Rtms5(H146S) undergo distinct color transitions (from blue to red to yellow to colorless) in a pH-dependent manner. pK(a) values of 4.1 and 3.2 were determined for the blue (absorption lambda(max), 590 nm) to yellow (absorption lambda(max), similar to 453 nm) transitions of Rtms5 and Rtms5H(146). The pK(a) for the blue-yellow transition of Rtms5H(146S) increased by 1.4 U in the presence of 0.1 M KI, whereas the pK(a) for the same transition of Rtms5 was relatively insensitive to added halides. To understand the structural basis for these observations, we have determined to 2.0 A resolution the crystal structure of a yellow form of Rtms5(H146S) at pH 3.5 in the presence of iodide. Iodide was found occupying a pocket in the structure with a pH of 3.5, forming van der Waals contacts with the tyrosyl moiety of the chromophore. Elsewhere, it was determined that this pocket is occupied by a water molecule in the Rtms5(H141S) structure (pH 8.0) and by the side chain of histidine 146 in the wild-type Rtms5 structure. Collectively, our data provide an explanation for the observed linkage between color transitions for Rtms5(H146S) and binding to halides.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

Binding and internalization of the melanocyte stimulating hormone receptor ligand [Nle(4), D-Phe(7)]alpha-MSH in B16 melanoma cells

The current study aims to ascertain the fate of the melanocyte stimulating hormone (MSH) receptor and its ligand [Nle(4), D-Phe(7)]alpha-MsH (NDP-MSH) following binding to murine B16 melanoma cells. Cells were incubated with [I-125]-NDP-MSH for up to 180 min and binding, internalization and degradation determined. Intracellular trafficking of the radiolabel was assessed !using Percoll density gradient centrifugation of homogenized cells. Receptor down-regulation and receptor mRNA levels were also measured over 96 hr after exposure to 1 mu M ligand. NDP-MSH accumulation increased with time in a temperature-dependent manner and was inhibited by excess peptide. The ligand was rapidly internalized and translocated to the lysosomal compartment where it was degraded. Internalization was accompanied by a loss or down-regulation of cell surface receptors, suggesting internalization of the NDP-MSH-receptor complex. No recycling of the receptors between the plasma membrane and intracellular compartments could be detected in this cell-hue. Approximately 15% of the surface receptors were resistant to down-regulation, possibly indicating receptor heterogeneity. Down-regulation persisted ibr up to 96 hr and was accompanied by a decrease in MSH receptor mRNA levels 48 hr after treatment. However, before this time, transcript levels were the same in treated and control cells. In contrast to what was seen with NDP-MSH, cell surface receptors removed with trypsin wc:re rapidly replaced. These results show that NDP-MSH not only induced MSH receptor :internalization but also inhibited receptor turnover, resulting in a prolonged down-regulation. It is concluded that, in B16 cells, the MSH receptor undergoes ligand-dependent internalization, resulting in a prolonged down-regulation. Copyright (C) 1996 Elsevier Science Ltd