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PubMed2006

Serologic proteome analysis of Borrelia burgdorferi membrane-associated proteins.

Lyme disease, a global health concern, is caused by infection with Borrelia burgdorferi, B. afzelii, or B. garinii. The spirochete responsible for the disease in the United States is B. burgdorferi and is spread by the bite of an infected Ixodes tick. We utilized multiple two-dimensional gel techniques combined with proteomics to reveal the full humoral immune response of mice and Lyme patients to membrane-associated proteins isolated from Borrelia burgdorferi. Our studies indicated that a subset of immunogenic membrane-associated proteins (some new and some previously identified) was recognized by mice experimentally infected with Borrelia burgdorferi either by low-dose needle inoculation or by tick infestation. Moreover, the majority of these immunogenic membrane-associated proteins were recognized by sera from patients diagnosed with early-disseminated Lyme disease. These included RevA, ErpA, ErpP, DbpA, BmpA, FtsZ, ErpB, LA7, OppA I, OppA II, OppA IV, FlhF, BBA64, BBA66, and BB0323. Some immunogens (i.e., BBI36/38) were more reactive with sera from mice than Lyme patients, while additional membrane proteins (i.e., FlaB, P66, LA7, and Hsp90) were recognized more strongly with sera from patients diagnosed with early-localized, early-disseminated, or late (chronic)-stage Lyme disease. We were able to examine the humoral response in Lyme patients in a temporal fashion and to identify the majority of immunoreactive proteins as the disease progresses from early to late stages. This serologic proteome analysis enabled the identification of novel membrane-associated proteins that may serve as new diagnostic markers and, more importantly, as second-generation vaccine candidates for protection against Lyme disease.

Animals↗
PubMed2006

Characterization of the outer membrane proteome of Leptospira interrogans expressed during acute lethal infection.

Pathogenic Leptospira species adapt to a wide range of environmental conditions during disease transmission and infection. While the proteome of in vitro cultivated Leptospira has been characterized in several studies to date, relatively little is known of the proteome as expressed by Leptospira during disease processes. Isolates of Leptospira obtained from patients suffering the severe pulmonary form of leptospirosis cause acute lethal infection in guinea pigs and chronic asymptomatic infection in rats. Recent studies have demonstrated that protein and lipopolysaccharide constituents of Leptospira recovered from acutely infected guinea pig tissue differ from that of Leptospira in chronically infected rat tissue and in vitro cultivated Leptospira (J. E. Nally, E. Chow, M. C. Fishbein, D. R. Blanco, and M. A. Lovett, Infect. Immun. 73:3251-3260, 2005). In the current study, the proteome of Leptospira expressed during disease processes was characterized relative to that of in vitro cultivated Leptospira (IVCL) after enrichment for hydrophobic membrane proteins with Triton X-114. Protein samples were separated by two-dimensional gel electrophoresis, and antigens expressed during infection were identified by immunoblotting with monospecific antiserum and convalescent rat serum in addition to mass spectrometry. Results suggest a significant increase in the expression of the outer membrane protein Loa22 during acute infection of guinea pigs relative to other outer membrane proteins, whose expression is generally diminished relative to expression in IVCL. Significant amounts of LipL32 are also expressed by Leptospira during acute infection of guinea pigs.

Adaptation, Physiological↗
PubMed2006

Protein-protein interactions among Helicobacter pylori cag proteins.

Many Helicobacter pylori isolates contain a 40-kb region of chromosomal DNA known as the cag pathogenicity island (PAI). The risk for development of gastric cancer or peptic ulcer disease is higher among humans infected with cag PAI-positive H. pylori strains than among those infected with cag PAI-negative strains. The cag PAI encodes a type IV secretion system that translocates CagA into gastric epithelial cells. To identify Cag proteins that are expressed by H. pylori during growth in vitro, we compared the proteomes of a wild-type H. pylori strain and an isogenic cag PAI deletion mutant using two-dimensional difference gel electrophoresis (2D-DIGE) in multiple pH ranges. Seven Cag proteins were identified by this approach. We then used a yeast two-hybrid system to detect potential protein-protein interactions among 14 Cag proteins. One heterotypic interaction (CagY/7 with CagX/8) and two homotypic interactions (involving H. pylori VirB11/ATPase and Cag5) were similar to interactions previously reported to occur among homologous components of the Agrobacterium tumefaciens type IV secretion system. Other interactions involved Cag proteins that do not have known homologues in other bacterial species. Biochemical analysis confirmed selected interactions involving five of the proteins that were identified by 2D-DIGE. Protein-protein interactions among Cag proteins are likely to have an important role in the assembly of the H. pylori type IV secretion apparatus.

Adenosine Triphosphatases↗
PubMed2006

Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes.

Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Delta lgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Delta lgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Delta lgt Delta prfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.

3T3 Cells↗
PubMed2006

Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation.

Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in developed countries, and yet little is known concerning the mechanisms by which this fastidious organism survives within its environment. We have demonstrated that C. jejuni 11168 can form biofilms on a variety of surfaces. Proteomic analyses of planktonic and biofilm-grown cells demonstrated differences in protein expression profiles between the two growth modes. Proteins involved in the motility complex, including the flagellins (FlaA, FlaB), the filament cap (FliD), the basal body (FlgG, FlgG2), and the chemotactic protein (CheA), all exhibited higher levels of expression in biofilms than found in stationary-phase planktonic cells. Additional proteins with enhanced expression included those involved in the general (GroEL, GroES) and oxidative (Tpx, Ahp) stress responses, two known adhesins (Peb1, FlaC), and proteins involved in biosynthesis, energy generation, and catabolic functions. An aflagellate flhA mutant not only lost the ability to attach to a solid matrix and form a biofilm but could no longer form a pellicle at the air-liquid interface of a liquid culture. Insertional inactivation of genes that affect the flagellar filament (fliA, flaA, flaB, flaG) or the expression of the cell adhesin (flaC) also resulted in a delay in pellicle formation. These findings demonstrate that the flagellar motility complex plays a crucial role in the initial attachment of C. jejuni 11168 to solid surfaces during biofilm formation as well as in the cell-to-cell interactions required for pellicle formation. Continued expression of the motility complex in mature biofilms is unusual and suggests a role for the flagellar apparatus in the biofilm phenotype.

Bacterial Adhesion↗
PubMed2006

Ctk complex-mediated regulation of histone methylation by COMPASS.

A comparative global proteomic screen identified factors required for COMPASS (complex of proteins associated with Set1)-mediated mono-, di-, and trimethylation of the fourth lysine of histone H3 (H3K4), which included components of a cyclin-dependent protein kinase (Ctk complex) that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II). Our results indicate that histone H3K4 methylation levels are regulated by the Ctk1, Ctk2, and Ctk3 components of the Ctk complex. We show that loss of Ctk1 kinase activity results in reduced histone H3K4 monomethylation levels, followed by a global increase in histone H3K4 trimethylation levels on chromatin. Ctk1 loss does not appear to have a substantial effect on histone H2B monoubiquitination levels or COMPASS and Paf1 complex phosphorylation. Our chromatin immunoprecipitation studies demonstrate that histone H3 eviction during active transcription is decelerated in a CTK1 deletion strain in response to reduced levels of Pol II recruitment. Our in vitro studies show that the onset of monomethylation on an unmethylated histone H3 by COMPASS is virtually immediate, while the onset of trimethylation occurs upon extended time of association between the histone tail and COMPASS. Our study suggests a role for the Ctk complex in the regulation of the pattern of H3K4 mono-, di-, and trimethylation via COMPASS.

Chromatin Immunoprecipitation↗
PubMed2005

Qualified predictions for microarray and proteomics pattern diagnostics with confidence machines.

We focus on the problem of prediction with confidence and describe a recently developed learning algorithm called transductive confidence machine for making qualified region predictions. Its main advantage, in comparison with other classifiers, is that it is well-calibrated, with number of prediction errors strictly controlled by a given predefined confidence level. We apply the transductive confidence machine to the problems of acute leukaemia and ovarian cancer prediction using microarray and proteomics pattern diagnostics, respectively. We demonstrate that the algorithm performs well, yielding well-calibrated and informative predictions whilst maintaining a high level of accuracy.

Algorithms↗
PubMed2004

An autoantibody-mediated immune response to calreticulin isoforms in pancreatic cancer.

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Aliquots of solubilized proteins from a pancreatic cancer cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which sera of individual patients were tested for primary antibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis, 33 patients with other cancers, and 15 healthy subjects were analyzed. Autoantibodies were detected against either one or two calreticulin isoforms identified by mass spectrometry in sera from 21 of 36 patients with pancreatic cancer. One of 18 chronic pancreatitis patients and 1 of 15 healthy controls demonstrated autoantibodies to calreticulin isoform 1; none demonstrated autoantibodies to isoform 2. None of the sera from patients with colon cancer exhibited reactivity against either of these two proteins. One of 14 sera from lung adenocarcinoma patients demonstrated autoantibodies to calreticulin isoform 1; 2 of 14 demonstrated autoantibodies to isoform 2. Immunohistochemical analysis of calreticulin in pancreatic/ampullary tumor tissue arrays using an isoform nonspecific antibody revealed diffuse and consistent cytoplasmic staining in the neoplastic epithelial cells of the pancreatic and ampullary adenocarcinomas. The detection of autoantibodies to calreticulin isoforms may have utility for the early diagnosis of pancreatic cancer.

Adenocarcinoma↗
PubMed2005

Possible detection of pancreatic cancer by plasma protein profiling.

The survival rate of pancreatic cancer patients is the lowest among those with common solid tumors, and early detection is one of the most feasible means of improving outcomes. We compared plasma proteomes between pancreatic cancer patients and sex- and age-matched healthy controls using surface-enhanced laser desorption/ionization coupled with hybrid quadrupole time-of-flight mass spectrometry. Proteomic spectra were generated from a total of 245 plasma samples obtained from two institutes. A discriminating proteomic pattern was extracted from a training cohort (71 pancreatic cancer patients and 71 healthy controls) using a support vector machine learning algorithm and was applied to two validation cohorts. We recognized a set of four mass peaks at 8,766, 17,272, 28,080, and 14,779 m/z, whose mean intensities differed significantly (Mann-Whitney U test, P < 0.01), as most accurately discriminating cancer patients from healthy controls in the training cohort [sensitivity of 97.2% (69 of 71), specificity of 94.4% (67 of 71), and area under the curve value of 0.978]. This set discriminated cancer patients in the first validation cohort with a sensitivity of 90.9% (30 of 33) and a specificity of 91.1% (41 of 45), and its discriminating capacity was further validated in an independent cohort at a second institution. When combined with CA19-9, 100% (29 of 29 patients) of pancreatic cancers, including early-stage (stages I and II) tumors, were detected. Although a multi-institutional large-scale study will be necessary to confirm clinical significance, the biomarker set identified in this study may be applicable to using plasma samples to diagnose pancreatic cancer.

Biomarkers, Tumor↗
PubMed2005

Characterization of 954 bovine full-CDS cDNA sequences.

BACKGROUND: Genome assemblies rely on the existence of transcript sequence to stitch together contigs, verify assembly of whole genome shotgun reads, and annotate genes. Functional genomics studies also rely on transcript sequence to create expression microarrays or interpret digital tag data produced by methods such as Serial Analysis of Gene Expression (SAGE). Transcript sequence can be predicted based on reconstruction from overlapping expressed sequence tags (EST) that are obtained by single-pass sequencing of random cDNA clones, but these reconstructions are prone to errors caused by alternative splice forms, transcripts from gene families with related sequences, and expressed pseudogenes. These errors confound genome assembly and annotation. The most useful transcript sequences are derived by complete insert sequencing of clones containing the entire length, or at least the full protein coding sequence (CDS) portion, of the source mRNA. While the bovine genome sequencing initiative is nearing completion, there is currently a paucity of bovine full-CDS mRNA and protein sequence data to support bovine genome assembly and functional genomics studies. Consequently, the production of high-quality bovine full-CDS cDNA sequences will enhance the bovine genome assembly and functional studies of bovine genes and gene products. The goal of this investigation was to identify and characterize the full-CDS sequences of bovine transcripts from clones identified in non-full-length enriched cDNA libraries. In contrast to several recent full-length cDNA investigations, these full-CDS cDNAs were selected, sequenced, and annotated without the benefit of the target organism's genomic sequence, by using comparison of bovine EST sequence to existing human mRNA to identify likely full-CDS clones for full-length insert cDNA (FLIC) sequencing. RESULTS: The predicted bovine protein lengths, 5' UTR lengths, and Kozak consensus sequences from 954 bovine FLIC sequences (bFLICs; average length 1713 nt, representing 762 distinct loci) are all consistent with previously sequenced mammalian full-length transcripts. CONCLUSION: In most cases, the bFLICs span the entire CDS of the genes, providing the basis for creating predicted bovine protein sequences to support proteomics and comparative evolutionary research as well as functional genomics and genome annotation. The results demonstrate the utility of the comparative approach in obtaining predicted protein sequences in other species.

5' Untranslated Regions↗
PubMed2026

IL17 signaling promotes oocyte developmental competence acquisition during maturation.

BACKGROUND: Defects in the acquisition of oocyte developmental competence during the maturation process causes subfertility or infertility in animals and humans. Understanding the regulatory mechanisms of oocyte maturation is essential for reproductive biology and medicine. Follicular fluid (FF) is an important microenvironment governing oocyte maturation. METHODS: A tandem mass tags (TMT)-based comparative FF proteomic analysis was employed to identify FF proteins that are potentially crucial for oocyte maturation. A very large number of pig and mouse oocytes (approximately 20,000) and embryos (over 13,000, including somatic cell nuclear transfer, parthenogenetic activation, and in vitro fertilization embryos) were used to investigate the effects of identified FF proteins on in vitro oocyte maturation and subsequent in vitro and in vivo embryo development. RNA sequencing, quantitative PCR, enzyme-linked immunosorbent assays, and immunofluorescence were used to study the expression patterns and action mechanisms of identified FF proteins in oocytes. In addition, intra-oocyte levels of glutathione and reactive oxygen species were measured to assess redox homeostasis. RESULTS: Interleukin 17D (IL17D) was identified as an important FF protein and it is significantly upregulated in porcine FF during oocyte maturation. IL17D promotes oocyte maturation by enhancing bidirectional communication between oocytes and cumulus cells, via upregulating CX43 expression and transzonal projections, which helps to maintain oocyte redox homeostasis and nuclear-cytoplasmic synchrony. IL17D treatment of oocytes enhances subsequent in vitro and in vivo full-term embryo development by modulating lipid metabolism and histone modification reprogramming. IL17D exerts its function via activating IL17 signaling through binding to CD93. Two other IL17 family members, IL17A and IL17F, also enhance oocyte maturation quality. IL17D displays a conserved expression pattern and function in pig and mouse oocytes. CONCLUSIONS: This study reveals the critical roles of IL17D in regulating oocyte developmental competence acquisition during maturation by activating IL17 signaling. The findings provide valuable insights into the molecular mechanisms underlining oocyte developmental potential acquisition and may help to develop methods for efficient production of oocytes for assisted reproduction.

Animals↗
PubMed2004

The application of proteomics to the human alcoholic brain.

Alcoholism results in changes in the human brain that reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of proteins in key cells and brain areas. Described here is a proteomics-based approach aimed at determining the identity of proteins in the superior frontal cortex (SFC) of the human brain that show different levels of expression in autopsy samples taken from healthy and long-term alcohol abuse subjects. Soluble protein fractions constituting pooled samples combined from SFC biopsies of four well-characterized chronic alcoholics (mean consumption > 80 g ethanol/day throughout adulthood) and four matched controls (<20 g/day) were generated. Two-dimensional electrophoresis was performed in triplicate on alcoholic and control samples and the resultant protein profiles analyzed for differential expression. Overall, 182 proteins differed by the criterion of twofold or more between case and control samples. Of these, 139 showed significantly lower expression in alcoholics, 35 showed significantly higher expression, and 8 were new or had disappeared. To date, 63 proteins have been identified using MALDI-MS and MS-MS. The finding that the expression level of differentially expressed proteins is preponderantly lower in the alcoholic brain is supported by recent results from parallel studies using microarray mRNA transcript.

Aged↗
PubMed2005

Differential proteomics for studying Drosophila immunity.

Here, we report the identification of proteins associated with the immune response of Drosophila by analyzing the hemolymph profiles after infection. Two-dimensional difference gel electrophoresis was used to study the secretome in the hemolymph of Drosophila larvae. Shortly after induction with lipopolysaccharides, we identified 10 proteins, which we designated "Drosophila instantly released immune proteins". Infection with Micrococcus luteus or Saccharomyces cerevisiae induced 20 and 19 differential protein spots, respectively. Next to known immune proteins, new candidates that require further investigation were identified.

Animals↗
PubMed2004

Comparison of gene expression in intra-abdominal and subcutaneous fat: a study of men with morbid obesity and nonobese men using microarray and proteomics.

Extent of intra-abdominal fat had significant linear relations with six metabolic coronary risk factors: systolic and diastolic blood pressure, fasting blood concentrations of glucose, high density lipoprotein (HDL) cholesterol, triglyceride, and plasminogen activator inhibitor-1. Tumor necrosis factor-alpha and adiponectin can be biological mediators from the intra-abdominal fat to the metabolic coronary risk factors. Complementarily, we describe a new study that will analyze the gene expression in intra-abdominal and subcutaneous fat on mRNA and protein level using high throughput methods. The study will elucidate further whether intra-abdominal obesity is the common denominator for the different components of the metabolic syndrome.

Adipose Tissue↗
PubMed2005

Histomathematical analysis of clinical specimens: challenges and progress.

Proteomic analysis of clinical tissue specimens is a difficult undertaking. Described here is a multiplex study of protein expression levels in histological sections of human prostate that addresses many of the associated challenges. Whole-mount sections from 10 prostatectomy specimens were studied using 15 antibodies, immunohistochemical staining, digital imaging, and mathematical analysis of the data sets. The approach was successful in stratifying cell lineages present in the samples based on proteomic patterns, including differentiating normal epithelium from cancer. This strategy likely will be a useful method for extending the number of proteins that can be analyzed in clinical cancer specimens using currently available laboratory techniques.

Epithelial Cells↗
PubMed2003

The proteome: anno Domini 2002.

We present some current definitions related to functional and structural proteomics and the human proteome, and we review the following aspects of proteome analysis: Classical 2-D map analysis (isoelectric focusing (IEF) followed by SDS-PAGE); Quantitative proteomics (isotope-coded affinity tag (ICAT), fluorescent stains) and their use in e.g., tumor analysis and identification of new target proteins for drug development; Electrophoretic pre-fractionation (how to see the hidden proteome!); Multidimensional separations, such as: (a) coupled size-exclusion and reverse-phase (RP)-HPLC; (b) coupled ion-exchange and RP-HPLC; (c) coupled RP-HPLC and RP-HPLC at 25/60 degrees C; (d) coupled RP-HPLC and capillary electrophoresis (CE); (e) metal affinity chromatography coupled with CE; Protein chips. Some general conclusions are drawn on proteome analysis and we end this review by trying to decode the glass ball of the aruspex and answer the question: "Quo vadis, proteome"?

Animals↗
PubMed2006

Two-dimensional gels for toxicological drug discovery applications.

Two-dimensional gel electrophoresis (2DGE) continues to be a useful approach to study protein expression. Although liquid chromatographic and mass spectrometric approaches that overcome some of the limitations and labour intensity of 2DGE are increasingly popular, this electrophoretic approach still has exceptional relevance in toxicology. Despite the technical challenges, pharmacologists/toxicologists continue to use gel-based proteomics to assess the biological and health effects of chemical treatment and exposure. This brief review addresses the use of 2DGE-based proteomics in drug development and toxicology, emphasising its unique strengths and weaknesses, and considers recent developments in this strategy that have evolved to directly confront the issues of dynamic range and reproducibility that have previously limited the overall use of 2D electrophoresis.

Animals↗
PubMed2006

Embryonic stem cell proteomics.

Human embryonic stem cells potentially represent an unlimited source of cells and tissues for regenerative medicine. Understanding signaling events that drive proliferation and specialization of these cells into various differentiated derivatives is of utmost importance for controlling their behavior in vitro. Major progress has been made in unraveling these signaling events with large-scale studies at the transcriptional level, but analysis of protein expression, interaction and modification has been more limited, since it requires different strategies. Recent advances in mass spectrometry-based proteomics indicate that proteome characterization can contribute significantly to our understanding of embryonic stem cell biology. In this article, we review mass spectrometry-based studies of human and mouse embryonic stem cells and their differentiated progeny, as well as studies of conditioned media that have been reported to support self-renewal of the undifferentiated cells in the absence of the more commonly used feeder cells. In addition, we make concise comparisons with related transcriptome profiling reports.

Animals↗