Distinct proteasome subpopulations in the alveolar space of patients with the acute respiratory distress syndrome.

There is increasing evidence that proteasomes have a biological role in the extracellular alveolar space, but inflammation could change their composition. We tested whether immunoproteasome protein-containing subpopulations are present in the alveolar space of patients with lung inflammation evoking the acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage (BAL) supernatants and cell pellet lysate from ARDS patients (n = 28) and healthy subjects (n = 10) were analyzed for the presence of immunoproteasome proteins (LMP2 and LMP7) and proteasome subtypes by western blot, chromatographic purification, and 2D-dimensional gelelectrophoresis. In all ARDS patients but not in healthy subjects LMP7 and LMP2 were observed in BAL supernatants. Proteasomes purified from pooled ARDS BAL supernatant showed an altered enzyme activity ratio. Chromatography revealed a distinct pattern with 7 proteasome subtype peaks in BAL supernatant of ARDS patients that differed from healthy subjects. Total proteasome concentration in BAL supernatant was increased in ARDS (971 ng/mL ± 1116 versus 59 ± 25; P < 0.001), and all fluorogenic substrates were hydrolyzed, albeit to a lesser extent, with inhibition by epoxomicin (P = 0.0001). Thus, we identified for the first time immunoproteasome proteins and a distinct proteasomal subtype pattern in the alveolar space of ARDS patients, presumably in response to inflammation.

Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn's disease.

BACKGROUND AND AIMS: The aetiopathogenesis of Crohn's disease, an inflammatory bowel disease (IBD), is not yet fully understood. Autoimmune mechanisms are thought to play a role in the development of Crohn's disease, but the target antigens and the underlying pathways have not been sufficiently identified. METHODS: Based on data from immunoblotting and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry, the major antigenic target of pancreatic autoantibodies (PABs), which are specific for Crohn's disease, was identified. Specificity of autoantibody reactivity was confirmed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) using purified rat and human recombinant GP2 synthesised in transiently transfected mammalian HEK 293 cells. Real-time polymerase chain reaction (rt-PCR) and IIF were used to detect mRNA and antigen localisation in human colon biopsies. RESULTS: The major zymogen granule membrane glycoprotein 2 (GP2) was identified as the autoantigen of PABs in Crohn's disease. PAB-positive sera from patients with Crohn's disease (n = 42) displayed significantly higher IgG reactivity to rat GP2 in ELISA than either PAB-negative sera (n = 31), or sera from patients with ulcerative colitis (n = 49), or sera from blood donors (n = 69) (p<0.0001, respectively). Twenty-eight (66%) and 18 (43%) of 42 PAB-positive sera demonstrated IgG and IgA reactivity to human recombinant GP2 in IIF, respectively. Patients with PAB-negative Crohn's disease (n = 31) were not reactive. GP2 mRNA transcription was significantly higher in colon biopsies from patients with Crohn's disease (n = 4) compared to patients with ulcerative colitis (n = 4) (p = 0.0286). Immunochemical staining confirmed GP2 expression in human colon biopsies from patients with Crohn's disease. CONCLUSION: Anti-GP2 autoantibodies constitute novel Crohn's disease-specific markers, the quantification of which could significantly improve the serological diagnosis of IBD. The expression of GP2 in human enterocytes suggests an important role for anti-GP2 response in the pathogenesis of Crohn's disease.

Association of citrullinated proteins with synovial exosomes.

OBJECTIVE: In addition to releasing proteins and mediators, cells also release membrane vesicles (exosomes and apoptotic blebs) into the extracellular environment. Apoptotic blebs contain multiple autoantigens, but few data are available concerning the protein content of exosomes. Exosomes are formed during an immune response and can directly stimulate T cells or bind to dendritic cells. The aim of this study was to identify the nature of synovial exosomes from patients with different rheumatic diseases and to examine their potential autoantigenic content, which may be involved in the induction of an autoimmune response. METHODS: Synovial exosomes from patients with rheumatoid arthritis (RA), patients with reactive arthritis, and patients with osteoarthritis were purified, analyzed by electron microscopy, and labeled with immunogold to detect IgG and IgM molecules. Autoantigen content was identified by 2-dimensional electrophoresis-immunoblotting and subsequent mass spectrometry. In order to investigate the presence of citrullinated proteins, immunoblotting with anticitrulline antibodies was performed. RESULTS: Citrullinated proteins were observed in all exosome preparations, in contrast to other autoantigenic proteins (e.g., BiP and heterogeneous nuclear RNP A2) that were previously observed in RA and other autoimmune diseases. These citrullinated proteins included the fibrin alpha-chain fragment, fibrin beta-chain, fibrinogen beta-chain precursor, fibrinogen D fragment, and the Sp alpha (CD5 antigen-like protein) receptor. Purification of synovial exosomes led to the detection of citrullinated fibrinogen and citrullinated Sp alpha associated with IgM and IgG. CONCLUSION: Synovial exosomes contain citrullinated proteins, which are known to be autoantigens in RA. Although immune mechanisms in which exosomes carry citrullinated peptides could play an important role in the induction and distribution of citrullinated proteins, there must be a specific recognition of these proteins that is unique to the RA immune system.

The necessity of functional proteomics: protein species and molecular function elucidation exemplified by in vivo alpha A crystallin N-terminal truncation.

Ten years after the establishment of the term proteome, the science surrounding it has yet to fulfill its potential. While a host of technologies have generated lists of protein names, there are only a few reported studies that have examined the individual proteins at the covalent chemical level defined as protein species in 1997 and their function. In the current study, we demonstrate that this is possible with two-dimensional gel electrophoresis (2-DE) and mass spectrometry by presenting clear evidence of in vivo N-terminal alpha A crystallin truncation and relating this newly detected protein species to alpha crystallin activity regulation by protease cleavage in the healthy young murine lens. We assess the present state of technology and suggest a shift in resources and paradigm for the routine attainment of the protein species level in proteomics.

Eye lens proteomics.

The eye lens is a fascinating organ as it is in essence living transparent matter. Lenticular transparency is achieved through the peculiarities of lens morphology, a semi-apoptotic process where cells elongate and loose their organelles and the precise molecular arrangement of the bulk of soluble lenticular proteins, the crystallins. The 16 crystallins ubiquitous in mammals and their modifications have been extensively characterized by 2-DE, liquid chromatography, mass spectrometry and other protein analysis techniques. The various solubility dependant fractions as well as subproteomes of lenticular morphological sections have also been explored in detail. Extensive post translational modification of the crystallins is encountered throughout the lens as a result of ageing and disease resulting in a vast number of protein species. Proteomics methodology is therefore ideal to further comprehensive understanding of this organ and the factors involved in cataractogenesis.

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions.

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.

Separation and identification of human heart proteins.

Heart failure is not a uniform disease entity, but a syndrome with various causes, including hypertension, ischemia and congenital heart disease, cardiomyopathy, myocarditis and intoxication. During the recent years a number of molecular and cellular alterations have been identified in the diseased heart, but a direct causative link between these changes and functional impairment, medical responsiveness, progression of the disease and the patients' outcome remains to be established. After an accumulation of large amounts of DNA sequence data in genomic projects, scientists have now turned their attention to the central executors of all programs of life, the proteins. In complementation of the genomic initiatives, proteomics based approaches have lined up not only for large-scale identification of proteins and their post-translational modifications, but also to study the function of protein complexes, protein-protein interactions and regulatory and signalling cascades in the cellular network. In concert with genomic data functional proteomics will hold the key for a better understanding and therapeutical management of cardiovascular diseases in the future.

Immunization with gp96 from Listeria monocytogenes-infected mice is due to N-formylated listerial peptides.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.

Proteome analysis of bacterial pathogens.

Combining two-dimensional electrophoresis with mass spectrometry resulted in a powerful technology ideally suited to recognize and identify proteins of pathogenic microorganisms. This classical proteome analysis is now complemented by capillary chromatography/mass spectrometry combinations, miniaturization by chip technology and protein interaction investigations. Comparative proteomics is used to reveal vaccine candidates and pathogenicity factors. Immunoproteomics identifies specific and nonspecific antigens. For the management of the huge data amounts, bioinformatics is a valuable instrument for the construction of complex protein databases.

Proteome analysis of the common human pathogen Helicobacter pylori.

The common human pathogen Helicobacter pylori is the major cause of gastric and duodenal ulcers. Based on the complete genome sequences of two independent isolates more than 1800 protein species have been resolved by two-dimensional gel electrophoresis and more than 200 of them have been identified (http://www.mpiib-berlin.mpg.de/2D-PAGE). Using these data, a large range of research areas including strain fingerprinting, protein composition and subcellular localization, gene regulation, and pathogen-host interactions have been investigated. The results that have been obtained led to a more detailed understanding of the Helicobacter biology and pathology and open further interesting fields for future work.

Identification of acidic, low molecular mass proteins of Mycobacterium tuberculosis strain H37Rv by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.

Matrix-assisted laser desorption/ionization-mass spectrometry peptide mass mapping and nano-electrospray ionization tandem mass spectrometry were used to identify acidic, low molecular mass proteins of Mycobacterium tuberculosis strain H37Rv. Proteins were extracted from whole cell lysates of mycobacteria, separated by high resolution two-dimensional electrophoresis (2-DE) and analysed by mass spectrometry (MS). Silver-stained 2-DE patterns resolved about 1800 distinct protein species, 190 of which had an observed isoelectric point and molecular mass in the range of pH 4 to 6 and 6 to 15 kDa, respectively. Seventy-six spots from this range were excised from Coomassie Brilliant Blue G250-stained gels and analysed by MS, from which 72 were identified. These spots were shown to represent products of as many as 50 different protein-coding genes. Ten genes gave rise to more than one protein species. Eleven spots contained more than one protein. The present study led to the identification of 15 mycobacterial proteins with assigned putative functions, 28 conserved hypothetical proteins and one unknown protein. Most proteins of the latter two groups had previously been predicted at the DNA level only. Six additional spots were shown to comprise proteins encoded by open reading frames that have not been predicted for M. tuberculosis H37Rv by genomic investigations.

Tyrosine phosphorylation patterns and size modification of the Helicobacter pylori CagA protein after translocation into gastric epithelial cells.

Helicobacter pylori is one of the most common bacterial pathogens that causes a variety of gastric diseases. During infection, the immuno-dominant H. pylori CagA protein is translocated and tyrosine-phosphorylated in gastric epithelial cells. We compared tyrosine phosphorylation patterns of five CagA variants by two-dimensional electrophoresis (2-DE) and immunoblotting studies. Tyrosine-phosphorylated CagA was detected as two distinct protein species in strains P12, P227, G27 and 26695 suggesting that two tyrosine residues of CagA can be phosphorylated both separately and simultaneously. Prediction programs revealed the presence of three putative tyrosine phosphorylation motifs in the sequences of CagA. Mutations in these motifs were identified suggesting that only two putative phosphorylation-relevant tyrosines are present in each CagA variant. CagA of strain J99 was found to be unique because essential codons were mutated in each of the three motifs and, consequently, revealed no tyrosine phosphorylation signals at all. These findings support the view that CagA from different H. pylori strains can be tyrosine-phosphorylated at one or two out of three predicted positions. Additionally, truncated CagA protein species of about 100-105 kDa (p100CagA) have been detected after infection with some of the H. pylori strains. The isoelectric point determined by both 2-DE and sequence analysis suggested that p100CagA represents the amino (N)-terminal part of the protein. Translocation, tyrosine phosphorylation and size modification of CagA might be involved in host signal transduction and development of gastric disease.

Identification of a tyrosine-phosphorylated 35 kDa carboxy-terminal fragment (p35CagA) of the Helicobacter pylori CagA protein in phagocytic cells: processing or breakage?

Helicobacter pylori is a very common bacterial pathogen that causes gastric disease by inducing the infiltration of immune cells as an initial event. Virulent H. pylori strains express a type IV secretion system composed of several virulence (Vir) proteins encoded by the cag pathogenicity island (cag PAI). During infection of phagocytic cells (U937, Josk-M and J774A.1) we have detected a de novo tyrosine-phosphorylated protein (p35p-Tyr) with sizes of 30 kDa, 38 kDa or 40 kDa, depending on the H. pylori strain. p35p-Tyr occurrence required functional virB4, virB7, virB10, virB11, virD4 and cagA (cytotoxin-associated gene A) genes encoded by the cag PAI suggesting that p35p-Tyr is a bacterial protein of variable size. We have biochemically purified p35p-Tyr from infected U937 cells. Tryptic peptides of p35p-Tyr determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) identified the carboxy (C)-terminal part of the H. pylori CagA protein. Subsequent analysis by two-dimensional electrophoresis (2-DE) and immunoblotting using anti-CagA antibodies revealed the presence of three stable CagA protein species in phagocytes: (i) 130-140 kDa full-length CagA (p135CagA), (ii) a 100-105 kDa fragment (p100CagA) and (iii) a 30-40 kDa fragment (p35CagA). Unlike p135CagA, p35CagA and p100CagA were also detected in much lower amounts in H. pylori without host cell contact. Therefore, breakage or processing leads to the production of p35CagA and p100CagA, a process that is enhanced after translocation into host cells. MALDI-MS data and the isoelectric point determined by both 2-DE and sequence analysis suggested that p35CagA represents the C-terminal part of CagA and p100CagA corresponds to the remaining amino (N)-terminal fragment. The possible function of CagA in host signal transduction and development of gastric disease is discussed.

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Proteome analysis of rat hepatomas: carcinogen-dependent tumor-associated protein variants.

Proteome analysis led to the identification and characterization of tumor-associated protein variants by two-dimensional electrophoresis and mass spectrometry. We focused on comparing the influence of genotoxic nitroso compounds N-methyl-N-nitrosourea, diethylnitrosamine and N-nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor-inducing agents on the protein pattern of rat hepatomas. We found several tumor-associated variants that represent members of the aldo-keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor-associated protein, rat aldose reductase-like protein-1 (rARLP-1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP-1 were detected in nitroso compound-induced rat hepatomas, while rat aldo-keto reductase protein-c (Rak-c), a novel tumor-associated variant (65% sequence identity with 3alpha-hydroxysteroid dehydrogenase) was discovered in N-methyl-N-nitrosourea-induced hepatomas only. 3Alpha-hydroxysteroid dehydrogenase and delta4-3-ketosteroid-5beta-reductase, both liver-specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) and delta4-3-ketosteroid-5beta-reductase (5beta-Red) might be replaced in hepatomas by tumor-associated proteins that are often present in the embryonal state, like the rARLPs or the Rak-c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo-keto reductase superfamily have not been found in Nafenopin-induced hepatomas. The pattern of tumor-associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.

Proteome study of colorectal carcinogenesis.

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.

Proteomics reveals open reading frames in Mycobacterium tuberculosis H37Rv not predicted by genomics.

Genomics revealed the sequence of 3924 genes of the H37Rv strain of Mycobacterium tuberculosis. Proteomics complements genomics in showing which genes are really expressed, and here we show the expression of six genes not predicted by genomics, as proved by two-dimensional electrophoresis and matrix-assisted laser desorption ionization and nano-electrospray mass spectrometry.

Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

Echocardiographic Doppler evaluation of left ventricular diastolic filling in older, highly trained male endurance athletes.

Previously published data have suggested that endurance training does not retard the normative aging impairment of early left ventricular diastolic filling (LVDF). Those studies, suggesting no effect of exercise training, have not examined highly trained endurance athletes or their LVDF responses after exercise. We therefore compared LVDF characteristics in a group of older highly trained endurance athletes (n = 12, mean age 69 years, range 65-75) and a group of sedentary control subjects (n = 12, mean age 69 years, range 65-73) with no cardiovascular disease. For all subjects, M-mode and Doppler echocardiographic data were obtained at rest. After baseline studies, subjects underwent graded, maximal cardiopulmonary treadmill exercise testing using a modified Balke protocol. Breath-by-breath respiratory gas analysis and peak exercise oxygen consumption (VO(2)max) measurements were obtained. Immediately after exercise and at 3-6 minutes into recovery, repeat Doppler echocardiographic data were obtained for determination of LVDF parameters. VO(2)max (44 +/-6.3 vs 27+/-4.2 ml/kg/min, P<0.001), oxygen consumption at anaerobic threshold (35+/-5.4 vs 24+/-3.8 ml/kg/min, P<0.001), exercise duration (24+/-3 vs 12+/-6 minutes, P<0.001), and left ventricular mass index (61+/-13 vs 51+/-7.8 kg/m(2), P<0.05) were greater in endurance athletes than in sedentary control subjects, whereas body mass index was lower (22+/-1.7 vs 26+/-3.4 kg/m(2), P<0.001). No differences in any of the LVDF characteristics were observed between the groups with the exception of a trend toward a lower atrial filling fraction at rest in the endurance athlete group versus the control subjects (P = 0.07). High-intensity endurance exercise training promotes exceptional peak exercise oxygen consumption and cardiovascular stamina but does not appear to alter normative aging effects on left ventricular diastolic function.