A novel soluble protein factor with non-opioid dynorphin A-binding activity

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.     

SPCA1 pumps and Hailey-Hailey disease

Both the endoplasmic reticulum and the Golgi apparatus are agonist-sensitive intracellular Ca2+ stores. The Golgi apparatus has Ca2+-release channels and a Ca2+-uptake mechanism consisting of sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and secretory-pathway Ca2+-ATPases (SPCA). SPCA1 has been shown to transport both Ca2+ and Mn2+ in the Golgi lumen and therefore plays an important role in the cytosolic and intra-Golgi Ca2+ and Mn2+ homeostasis. Human genetic studies have provided new information on the physiological role of SPCA1. Loss of one functional copy of the SPCA1 (ATP2C1) gene causes Hailey-Hailey disease, a skin disorder arising in the adult age with recurrent vesicles and erosions in the flexural areas. Here, we review recent experimental evidence showing that the Golgi apparatus plays a much more important role in intracellular ion homeostasis than previously anticipated.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

NMR structures of salt-refolded forms of the 434-repressor DNA-binding domain in 6 M urea

The N-terminal 63-residue fragment of the phage 434-repressor, 434(1-63), has a well-defined globular fold in H(2)O solution, and is unfolded in 6 M urea at pH 7.5. In this study, 434(1-63) has been refolded by adding either 1.7 M NaCl or 0.47 M NaTFA to the solution in 6 M urea, and the NMR structures of both refolded forms have been determined. The two refolded forms have similar free energies of unfolding and are approximately 16 kJ/mol less stable than the protein in H(2)O solution. 434(1-63) refolded with NaCl exhibits NMR chemical shifts very similar to and a three-dimensional structure nearly identical to those of 434(1-63) in H(2)O solution. The protein refolded with NaTFA also has a similar global fold, but it shows local differences near Phe44, of which two different orientations of the aromatic ring are compatible with the experimental data. This local conformational polymorphism attracted our interest because hydrophobic contacts between two subdomains of residues 1-36 and 45-63 are mediated by the Phe44 side chain. Anion binding experiments suggest that this polymorphism is caused by binding of TFA(-) anions to a cluster of positively charged Arg and Lys residues located in the loop connecting the two subdomains, with apparent binding constants for TFA(-) (K(app)) on the order of 30 mM(-1).     

Hypothetical proteins with putative enzyme activity in human amnion, lymphocyte, bronchial epithelial and kidney cell lines

A myriad of predicted proteins have been described based upon nucleic acid sequences but the existence of these structures has not been confirmed at the protein level. The aim of the study was therefore to show expression of hypothetical proteins in several cell lines and to provide the analytical basis for their identification and characterisation. We used two-dimensional gel electrophoresis with in-gel digestion of high protein spots and subsequent MALDI-TOF analysis of cell lysates from human amnion, lymphocyte, bronchial epithelial and kidney cell lines. A pI range from 3 to 10 was selected and second dimension was run using 9-16% gradient gels. A series of structures that have not been described before at the protein level were identified in several cell lines and were assigned to major enzyme systems including proteolysis (proteases, peptidases, ubiquitin), intermediary metabolism and oxidoreductases. We conclude that the proteomic approach used serves as a suitable tool to verify the existence of predicted/hypothetical proteins. The herein identified enzymes may contribute to several pathways/cascades in the human organism. Furthermore, analytical data given are of major relevance as pIs, a prerequisite to find proteins in a map, cannot be predicted from nucleic acid sequences.     

Large-scale expansion of cytomegalovirus-specific cytotoxic T cells in suspension culture

Clinical trials in recent years involving the adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) have shown promise in restoring immunity against viral infection and reducing tumor burden in patients with solid and hematological malignancies. However, the large cell number required to achieve efficacy, 10(9) to 10(11), makes routine application of adoptive immunotherapy impractical. Investigation into new methods of CTL expansion may be useful in addressing this problem. Use of stirred suspension bioreactors are one such method that may allow large-scale T-cell expansion. Suspension cultures offer advantages over conventional static culture methods, including providing a homogeneous culture environment, and the potential for optimization and control of culture conditions. We generated cytomegalovirus (CMV)-specific CTL and investigated the potential of stirred bioreactor systems for expansion of large cell numbers. We found that CTL can be readily expanded ( > 200-fold) from cryopreserved stocks by nonspecific stimulation in the presence of allogeneic feeder cells and interleukin-2 (IL-2). Activated CTL inoculated into either suspension or static cultures could be subsequently expanded tenfold, and showed similar growth kinetics and metabolism independent of the culture vessel used. Furthermore, CTL remained specific for CMVpp65 peptide through the expansion phases, as demonstrated by pp65-tetramer staining ( > 95% tetramer(+)) and cytotoxicity assays. This study indicates that suspension reactor systems may be useful in large-scale expansion of antigen-specific CTL lines or clones, and may facilitate the advancement of routine adoptive immunotherapy.     

IRS-1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G-protein Gi

Platelet agonists initiate aggregation and secretion by activating receptors coupled to the G-protein G(q), thereby raising cytosolic Ca(2+), [Ca(2+)](i). The rise in [Ca(2+)](i) is facilitated via inhibition of cAMP formation by the inhibitory G-protein of adenylyl cyclase, G(i). Since insulin attenuates platelet activation, we investigated whether insulin interferes with cAMP regulation. Here we report that insulin (0.5-200 nmol/liter) interferes with agonist-induced increases in [Ca(2+)](i) (ADP, thrombin), cAMP suppression (thrombin), and aggregation (ADP). The effects of insulin are as follows: (i) independent of the P2Y(12) receptor, which mediates ADP-induced cAMP lowering; (ii) not observed during G(s)-mediated cAMP formation; (iii) unaffected by treatments that affect phosphodiesterases (3-isobutyl-1-methylxanthine); and (iv) not changed by interfering with NO-mediated regulation of cAMP degradation (N(G)-monomethyl-l-arginine). Hence, insulin might interfere with G(i). Indeed, insulin induces the following: (i) tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 (IRS-1) and G(i)alpha(2); (ii) co-precipitation of IRS-1 with G(i)alpha(2) but not with other G alpha subunits. Despite persistent receptor activation, the association of IRS-1 with G(i)alpha(2) is transient, being optimal at 5 min and 1 nmol/liter insulin, which is sufficient to suppress Ca(2+) signaling by ADP, and at 10 min and 100 nmol/liter insulin, which is required to suppress Ca(2+) signaling by thrombin. Epinephrine, a known platelet sensitizer and antagonist of insulin, abolishes the effect of insulin on [Ca(2+)](i), tyrosine phosphorylation of G(i)alpha(2), and aggregation by interfering with the phosphorylation of the insulin receptor beta subunit. We conclude that insulin attenuates platelet functions by interfering with cAMP suppression through IRS-1 and G(i).     

Localization of the serine protease-binding sites in the collagen-like domain of mannose-binding protein: indirect effects of naturally occurring mutations on protease binding and activation

Mutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs.     

Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry

As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R² ≥ 0.99 and standard deviation around 2% for the different positions. The limit of detection was ~5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.