Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.     

Thyroid receptor ligands. Part 2: Thyromimetics with improved selectivity for the thyroid hormone receptor beta

A set of thyromimetics having improved selectivity for TR-beta1 were prepared by replacing the 3'-isopropyl group of 2 and 3 with substituents having increased steric bulk. From this limited SAR study, the most potent and selective compounds identified were derived from 2 and contained a 3'-phenyl moiety bearing small hydrophobic groups meta to the biphenyl link. X-ray crystal data of 15c complexed with TR-beta1 LBD shows methionine 442 to be displaced by the bulky R3' phenyl ethyl amide side chain. Movement of this amino acid side chain provides an expanded pocket for the bulky side chain while the ligand-receptor complex retains full agonist activity.     

Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.     

Base-pairing systems related to TNA containing phosphoramidate linkages: synthesis of building blocks and pairing properties

As part of a project that aims at screening TNA-related oligonucleotide systems in which threose backbone units may have some or all of their oxygen functions replaced by nitrogen, two TNA analogs containing (2'NH)- and (3'NH)-phosphoramidate groups, respectively, in place of phosphodiester groups were synthesized. They show base-pairing properties that are very similar to those of TNA itself. We also synthesized 2',3'-diamino analogs of alpha-L-threofuranosyl mononucleosides, yet attempts to convert them to TNA analogs containing phosphodiamidate linker groups were not successful. Such 2',3'-diamino derivatives of threofuranosyl nucleosides may be of interest, however, as building blocks of TNA analogs that contain non-phosphorous linker groups.     

High resolution mass spectrometric alveolar proteomics: identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micropreparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products. The high resolution mass spectrometric proteome analysis should facilitate the unequivocal identification of subunits, aggregations, modifications and degradation products of surfactant proteins and hence contribute to the understanding of the mechanistic basis of lung disease pathogenesis.     

Major histocompatibility complex diversity influences parasite resistance and innate immunity in sticklebacks

Proteins of the major histocompatibility complex (MHC) play a central role in the presentation of antigens to the adaptive immune system. The MHC also influences the odour-based choice of mates in humans and several animal taxa. It has recently been shown that female three-spined sticklebacks (Gasterosteus aculeatus) aim at a moderately high MHC diversity in their offspring when choosing a mate. Do they optimize the immune systems of their offspring? Using three-spined sticklebacks that varied in their individual numbers of MHC class IIB molecules, we tested, experimentally, whether allelic diversity at the MHC influences parasite resistance and immune parameters. We found that sticklebacks with low MHC diversity suffered more from parasite infection after experimental exposure to Schistocephalus solidus tapeworms and Glugea anomala microsporidians. They also showed the highest proportion of granulocytes and the strongest respiratory burst reaction, which are correlates of innate immunity. This indicates a strong activity of the innate immune system after challenge by parasites when MHC diversity is suboptimal. Individuals with very high allelic diversity at the MHC seemed inferior to those with moderately high diversity. Such a pattern is consistent with theoretical expectations of an optimal balance between the number of recognizable antigens and self-tolerance.     

Structure of the large subunit of class Ib ribonucleotide reductase from Salmonella typhimurium and its complexes with allosteric effectors

The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors. The enzyme contains the characteristic ten-stranded alpha/beta-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands. Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes. The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation. This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site. R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes. We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound. The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2'-OH group to bind.     

Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling

Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85 alpha and p85 beta, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1-/- (p85 alpha-/-) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30% decrease in Akt activity, leading to decreased insulin-induced glucose uptake and anti-apoptosis. Pik3r2-/- (p85 beta-/-) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85 beta-/- cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85 alpha-/- cells and p85 beta-/- cells exhibit significantly increased insulin-induced glycogen synthase activation. p85 alpha-/- cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85 alpha, p85 beta, or a p85 mutant that does not bind to p110, indicating the existence of p85-dependent, but PI 3-kinase-independent, signaling pathway. Furthermore, a reduction of p85 beta specifically increases insulin receptor substrate-2 phosphorylation. Thus, p85 alpha and p85 beta modulate PI 3-kinase-dependent signaling by multiple mechanisms and transmit signals independent of PI 3-kinase activation.