The membrane proteome of Halobacterium salinarum

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha-helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2-DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2-DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.     

A novel beta-peptidyl aminopeptidase (BapA) from strain 3-2W4 cleaves peptide bonds of synthetic beta-tri- and beta-dipeptides

A novel bacterial strain that was capable of growing on the beta-tripeptide H-betahVal-betahAla-betahLeu-OH as the sole carbon and nitrogen source was isolated from an enrichment culture. On the basis of physiological characterization, partial 16S rRNA sequencing, and fatty acid analysis, strain 3-2W4 was identified as a member of the family Sphingomonadaceae. Growth on the beta-tripeptide and the beta-dipeptide H-betahAla-betahLeu-OH was observed, and emerging metabolites were characterized. Small amounts of a persisting metabolite, the N-acetylated beta-dipeptide, were identified in both media. According to dissolved organic carbon measurements, 74 to 80% of the available carbon was dissimilated. The beta-peptide-degrading enzyme was purified from the crude cell extract of cells from strain 3-2W4 grown on complex medium. The enzyme was composed of two subunits, and the N-terminal sequences of both were determined. With this information, it was possible to identify the complete nucleotide sequence and to deduce the primary structure of the gene bapA. The gene encoded a beta-peptidyl aminopeptidase (BapA) of 402 amino acids that was synthesized as preprotein with a signal sequence of 29 amino acids. The enzyme was cleaved into two subunits (residues 30 to 278 and 279 to 402). It belonged to the N-terminal nucleophile (Ntn) hydrolase superfamily.     

Effect of tumor-associated mutant E-cadherin variants with defects in exons 8 or 9 on matrix metalloproteinase 3

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. The cell adhesion molecule E-cadherin is frequently down-regulated or mutated in tumors. In addition to down-regulation of cell adhesion, degradation of the extracellular matrix by matrix metalloproteinases is necessary for tumor cell spread. To investigate a possible link between E-cadherin and matrix metalloproteinase 3 (MMP-3), we examined expression of MMP-3 in human MDA-MB-435S cells transfected with wild-type (wt) or three different tumor-associated mutant E-cadherin variants with alterations in exons 8 or 9, originally identified in gastric carcinoma patients. In the presence of wt E-cadherin, the MMP-3 protein level was decreased in cellular lysates and in the supernatant where a secreted form of the protein is detectable. Down-regulation of MMP-3 was not found in MDA-MB-435S transfectants expressing mutant E-cadherin variants which indicates that E-cadherin mutations interfere with the MMP-3 suppressing function of E-cadherin. The mechanism of regulation of MMP-3 by E-cadherin is presently not clear. We have previously found that cell motility is enhanced by expression of the mutant E-cadherin variants used in this study. Here, we found that application of the synthetic inhibitor of MMP-3 NNGH and small interfering RNA (siRNA) directed against MMP-3 reduce mutant E-cadherin-enhanced cell motility. Taken together, our results point to a functional link between MMP-3 and E-cadherin. MMP-3 is differentially regulated by expression of wt or mutant E-cadherin. On the other hand, MMP-3 plays a role in the enhancement of cell motility by mutant E-cadherin. Both observations may be highly relevant for tumor progression since they concern degradation of the extracellular matrix and tumor cell spread.     

Expression of PHA polymerase genes of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its effect on PHA formation

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.     

The matrilins--adaptor proteins in the extracellular matrix

The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.     

Ligation of CD28 by its natural ligand CD86 in the absence of TCR stimulation induces lipid raft polarization in human CD4 T cells

Stimulation of resting CD4 T cells with anti-CD3/CD28-coated beads leads to rapid polarization of lipid rafts (LRs). It has been postulated that a major role of costimulation is to facilitate LR aggregation. CD86 is up-regulated or expressed aberrantly on immune cells in a wide array of autoimmune and infectious diseases. Using an Ig fusion with the extracellular domain of CD86 (CD86Ig) bound to a magnetic bead or K562 cells expressing CD86, we demonstrated that ligation of CD28 by its natural ligand, but not by Ab, induced polarization of LRs at the cell-bead interface of fresh human CD4 T cells in the absence of TCR ligation. This correlated with activation of Vav-1, increase of the intracellular calcium concentration, and nuclear translocation of NF-kappaB p65, but did not result in T cell proliferation or cytokine production. These studies show, for the first time, that LR polarization can occur in the absence of TCR triggering, driven solely by the CD28/CD86 interaction. This result has implications for mechanisms of T cell activation. Abnormalities in this process may alter T and B cell tolerance and susceptibility to infection.     

Purification and characterization of a chemotolerant alcohol dehydrogenase applicable to coupled redox reactions

The purification and characterization of an organic solvent tolerant, NADH-dependent medium-chain secondary alcohol dehydrogenase (denoted sec-ADH "A") from Rhodococcus ruber DSM 44541 is reported. The enzyme can withstand elevated concentrations of organic solvents, such as acetone (up to 50% v/v) and 2-propanol (up to 80% v/v). Thus, it is ideally suited for the preparative-scale enantioselective oxidation of sec-alcohol and the asymmetric reduction of ketones, using acetone and 2-propanol, respectively, as cosubstrates for cofactor-regeneration via a coupled-substrate approach. The homodimeric protein was found to bear tightly bound zinc and displayed a molecular mass of 38 kDa per subunit as determined by SDS gel electrophoresis. The optimal temperature ranged from 30-50 degrees C and the half-life at 50 degrees C was 35 h. In addition, excellent storage stability was found. The pH optimum for reduction is pH 6.5; pH 9.0 is preferred for oxidation. The enzyme followed a sequential reaction mechanism. The substrates are medium chain sec-alcohols or (omega-1)-ketones; primary alcohols or aldehydes are not accepted.     

Association of the DTNBP1 locus with schizophrenia in a U.S. population

Linkage and association studies have recently implicated dystrobrevin-binding protein 1 (DTNBP1) in the etiology of schizophrenia. We analyzed seven previously tested DTNBP1 single-nucleotide polymorphisms (SNPs) in a cohort of 524 individuals with schizophrenia or schizoaffective disorder and 573 control subjects. The minor alleles of three SNPs (P1578, P1763, and P1765) were positively associated with the diagnosis of schizophrenia or schizoaffective disorder in the white subset of the study cohort (258 cases, 467 controls), with P1578 showing the most significant association (odds ratio 1.76, P =.0026). The same three SNPs were also associated in a smaller Hispanic subset (51 cases, 32 controls). No association was observed in the African American subset (215 cases, 74 controls). A stratified analysis of the white and Hispanic subsets showed association with the minor alleles of four SNPs (P1578, P1763, P1320, and P1765). Again, the most significant association was observed for P1578 (P =.0006). Haplotype analysis supported these findings, with a single risk haplotype significantly overrepresented in the white sample (P =.005). Our study provides further evidence for a role of the DTNBP1 gene in the genetic etiology of schizophrenia.