Rapid identification and typing of listeria species by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic "fingerprint" mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.

Hypusination of eukaryotic initiation factor 5A (eIF5A): a novel therapeutic target in BCR-ABL-positive leukemias identified by a proteomics approach.

Inhibition of BCR-ABL tyrosine kinase with imatinib represents a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular BCR-ABL downstream targets, we analyzed differences in global protein expression in BCR-ABL-positive K562 cells treated with or without imatinib in vitro. Among the 19 proteins found to be differentially expressed, we detected the down-regulation of eukaryotic initiation factor 5A (eIF5A), a protein essential for cell proliferation. eIF5A represents the only known eukaryotic protein activated by posttranslational hypusination. Hypusination inhibitors (HIs) alone exerted an antiproliferative effect on BCR-ABL-positive and -negative leukemia cell lines in vitro. However, the synergistic dose-response relationship found for the combination of imatinib and HI was restricted to Bcr-Abl-positive cells. Furthermore, this synergistic effect was confirmed by cytotoxicity assays, cell-cycle analysis, and CFSE labeling of primary CD34+ CML cells. Specificity of this effect could be demonstrated by cotreatment of K562 cells with imatinib and siRNA against eIF5. In conclusion, through a comparative proteomics approach and further functional analysis, we identified the inhibition of eIF5A hypusination as a promising new approach for combination therapy in BCR-ABL-positive leukemias.

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus.

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.

Proteomic identification of the involvement of the mitochondrial rieske protein in epilepsy.

PURPOSE: Kindled seizures are widely used to model epileptogenesis, but the molecular mechanisms underlying the attainment of kindling status are largely unknown. Recently we showed that achievement of kindling status in the Sprague-Dawley rat is associated with a critical developmental interval of 25 +/- 1 days; the identification of this long, well-defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relation to normal and pathologic brain function. METHODS: By using proteomics on cerebral tissue from our new rat kindling model, we undertook a global analysis of protein expression in kindled animals. Some of the identified proteins were further investigated by using immunohistochemistry. RESULTS: We report the identification of a modified variant of the Rieske iron-sulfur protein, a component of the mitochondrial cytochrome bc1 complex, whose isoelectric point is shifted toward more alkaline values in the hippocampus of kindled rats. By immunohistochemistry, the Rieske protein is well expressed in the hippocampus, except in the CA1 subfield, an area of selective vulnerability to seizures in humans and animal models. We also noted an asymmetric, selective expression of the Rieske protein in the subgranular neurons of the dorsal dentate gyrus, a region implicated in neurogenesis. CONCLUSIONS: These results indicate that the Rieske protein may play a role in the response of neurons to seizure activity and could give important new insights into the molecular pathogenesis of epilepsy.

Determination of the peptide binding motif and high-affinity ligands for HLA-DQ4 using synthetic peptide libraries.

Juvenile idiopathic arthritis (JIA) is considered to be an autoimmune disease. Various human leukocyte antigen (HLA) associations for different subgroups of this heterogeneous disease have been found. For early-onset pauciarticular arthritis (now oligoarthritic JIA), a strong association with the HLA class II haplotype DQA1*0401/DQB1*0402 (DQ4) has been described. We determined the peptide-binding specificities of this HLA-DQ molecule by screening a synthetic acetylated nonapeptide amide library with one defined and eight random sequence positions. A characteristic binding motif could be deduced. By use of these data, we designed defined specific nonapeptides and identified high-affinity ligands binding to HLA-DQ4. The peptide binding motif of HLA-DQ4 is very similar to the motif of HLA-DQ7, also associated with oligoarthritic JIA. It is, however, different from binding motifs of neutral or protective HLA-DQ molecules. Our results further support the idea of differential peptide presentation in the pathogenesis of oligoarthritic JIA.

Cathepsin G, and not the asparagine-specific endoprotease, controls the processing of myelin basic protein in lysosomes from human B lymphocytes.

The asparagine-specific endoprotease (AEP) controls lysosomal processing of the potential autoantigen myelin basic protein (MBP) by human B lymphoblastoid cells, a feature implicated in the immunopathogenesis of multiple sclerosis. In this study, we demonstrate that freshly isolated human B lymphocytes lack significant AEP activity and that cleavage by AEP is dispensable for proteolytic processing of MBP in this type of cell. Instead, cathepsin (Cat) G, a serine protease that is not endogenously synthesized by B lymphocytes, is internalized from the plasma membrane and present in lysosomes from human B cells where it represents a major functional constituent of the proteolytic machinery. CatG initialized and dominated the destruction of intact MBP by B cell-derived lysosomal extracts, degrading the immunodominant MBP epitope and eliminating both its binding to MHC class II and a MBP-specific T cell response. Degradation of intact MBP by CatG was not restricted to a lysosomal environment, but was also performed by soluble CatG. Thus, the abundant protease CatG might participate in eliminating the immunodominant determinant of MBP. Internalization of exogenous CatG represents a novel mechanism of professional APC to acquire functionally dominant proteolytic activity that complements the panel of endogenous lysosomal enzymes.

Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner.

Endocytic proteolysis represents a major functional component of the major histocompatibility complex class II antigen-presentation machinery. Although transport and assembly of class II molecules in the endocytic compartment are well characterized, we lack information about the pattern of endocytic protease activity along this pathway. Here, we used chemical tools that visualize endocytic proteases in an activity-dependent manner in combination with subcellular fractionation to dissect the subcellular distribution of the major cathepsins (Cat) CatS, CatB, CatH, CatD, CatC, and CatZ as well as the asparagine-specific endoprotease (AEP) in human B-lymphoblastoid cells (BLC). Endocytic proteases were distributed in two distinct patterns: CatB and CatZ were most prominent in early and late endosomes but absent from lysosomes, and CatH, CatS, CatD, CatC, and AEP distributed between late endosomes and lysosomes, suggesting that CatB and CatZ might be involved in the initial proteolytic attack on a given antigen. The entire spectrum of protease activity colocalized with human leukocyte antigen-DM and the C-terminal and N-terminal processing of invariant chain (Ii) in late endosomes. CatS was active in all endocytic compartments. Surprisingly and in contrast with results from dendritic cells, inhibition of CatS activity by leucine-homophenylalanine-vinylsulfone-phenol prevented N-terminal processing of Ii but did not alter the subcellular trafficking or surface delivery of class II complexes, as deferred from pulse-chase analysis in combination with subcellular fractionation and biotinylation of cell-surface protein. Thus, BLC contain distinct activity patterns of proteases in endocytic compartments and regulate the intracellular transport and surface-delivery of class II in a CatS-independent manner.

Modulation of the endosomal and lysosomal distribution of cathepsins B, L and S in human monocytes/macrophages.

Endosomal and lysosomal fractions of human monocytes/macrophages prepared from buffy coats were analyzed for activities of cathepsins B, L and S, and expression of cathepsin proteins along with major histocompatibility complex class I and class II molecules under control and immunomodulatory conditions. While the total activity of cathepsins B, L, and S together remained unchanged in lysates of control cells during culture for 72 h, the subcellular distribution of cathepsin activities underwent a shift from a predominantly endosomal localization in freshly isolated cells to a lysosomal pattern after 72 h of culture. Interferon-gamma treatment for 72 h resulted in an upregulation of both major histocompatibility complex proteins and cathepsins with differential changes in cathepsin B, L and S activities in endosomes versus lysosomes. These changes suggest a remodeling of the endocytic machinery and imply different functions of cathepsins B, L and S during monocyte differentiation.

Cathepsin S activity is detectable in human keratinocytes and is selectively upregulated upon stimulation with interferon-gamma.

Keratinocytes are an integral component of the skin immune system and function as nonprofessional antigen-presenting cells in pathophysiologic conditions when they express major histocompatibility complex class II molecules, e.g., in psoriasis. In order to analyze further this function we investigated the activity of cathepsin S in comparison with cathepsins B and L. These enzymes were suggested to be involved in antigen presentation. Specific catalytic activities of these cathepsins were determined fluorometrically by hydrolysis of a synthetic substrate (Z-Phe-Arg-7-amido-4-methylcoumarin) in subcellular fractions of human keratinocytes. It was found that the human keratinocyte cell line HaCaT exhibits activities of all three cathepsins investigated. Endosomal/lysosomal compartments show highest cathepsin activities. Normal human keratinocytes in primary culture show a comparable pattern of cathepsin activities. In contrast to this, in syngeneic Epstein-Barr virus-transformed B cells the level of cathepsin B activity was found to be 10% of that in the corresponding keratinocytes, whereas the activities for cathepsins L and S were in a similar range. Interferon-gamma stimulation of primary keratinocytes and HaCaT cells resulted in a selective upregulation of the cathepsin S activity, the extent of which was very similar. The mechanism of this upregulation was demonstrated as induction at the mRNA and protein levels. This report documents that cathepsin S in human keratinocytes is selectively upregulated, in parallel to major histocompatibility complex class II molecules, in response to a pro-inflammatory cytokine. Our observations support the concept of keratinocytes functioning as nonprofessional antigen-presenting cells in states of inflammation.

Human alpha-defensins HNPs-1, -2, and -3 in renal cell carcinoma: influences on tumor cell proliferation.

The alpha-defensins human neutrophil peptides (HNPs)-1, -2, and -3 have been described as cytotoxic peptides with restricted expression in neutrophils and in some lymphocytes. In this study we report that HNPs-1, -2, and -3 are also expressed in renal cell carcinomas (RCCs). Several RCC lines were found to express mRNA as well as the specific peptides of HNP-1, -2, and -3 demonstrated by reverse transcriptase-polymerase chain reaction, mass spectrometric, and flow cytometric analyses. At physiological concentrations HNPs-1, -2, and -3 stimulated cell proliferation of selected RCC lines in vitro but at high concentrations were cytotoxic for all RCC lines tested. As in RCC lines, alpha-defensins were also detected in vivo in malignant epithelial cells of 31 RCC tissues in addition to their expected presence in neutrophils. In most RCC cases randomly, patchy immunostaining of alpha-defensins on epithelial cells surrounding neutrophils was seen, but in six tumors of higher grade malignancy all tumor cells were diffusely stained. Cellular necrosis observed in RCC tissues in association with extensive patches of HNP-1, -2, and -3, seemed to be related to high concentrations of alpha-defensins. The in vitro and in vivo findings suggest that alpha-defensins are frequent peptide constituents of malignant epithelial cells in RCC with a possible direct influence on tumor proliferation.

Characterization of legumain.

The mammalian legumain, also called asparaginyl endopeptidase (AEP), is critically involved in the processing of bacterial antigens for MHC class II presentation. In order to investigate the substrate specificity of AEP in the P1' position, we created a peptide library and digested it with purified pig kidney AEP. Digestion was less efficient only when proline was in the P1' position. Maximum AEP activity was found in lysosomal fractions of different types of antigen presenting cells (APC). When the multiple sclerosis-associated autoantigen myelin basic protein (MBP) was digested with AEP, the immunodominant epitope 83-99 was destroyed. Myoglobin as an alternative substrate was AEP resistant. These results suggest an important, but not necessarily critical role for AEP in lysosomal antigen degradation.