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PubMed2006

Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis.

Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases, which is related to angiogenesis and inflammation processes. Here, we describe a proteomic screening for putative ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. Conditioned medium proteins of cultures of the two cell lines have been quantitatively compared by DIGE. Proteins showing differential levels have been identified by MS techniques leading to the finding of five potential new substrates of ADAMTS1: the basement membrane proteins nidogen-1 and -2, the desmosomal protein desmocollin-3, and the extracellular glycoproteins dystroglycan 1 and Mac-2-binding protein. Nidogen-1 and -2 have been further validated as substrates by immunochemical analysis. Our results demonstrate the utility of the DIGE proteomic technique for the discovery of specific substrates of matrix proteases.

ADAM Proteins↗

PubMed2025

Proximity Proteomics to Profile Ebola Virus Protein Interactome in Its Functional Context.

Proximity labeling-based proteomics (proximity proteomics) has emerged as a popular and versatile approach to illuminate the molecular interactions between viruses and their hosts. In this approach, a proximity labeling enzyme tag is fused to a bait protein and labels neighboring proteins with a chemical handle such as biotin, allowing for downstream affinity purification. Compared to another widely used technique, affinity purification coupled mass spectrometry, proximity proteomics enables the detection of low affinity or transient interactors that might have important functions in the viral life cycle. Further, proximity proteomics can identify interactors of a labile bait protein, of which affinity purification is technically challenging. Here, we describe a proximity proteomic protocol to identify cellular interactors of the Ebola virus polymerase. A similar strategy is readily applicable to elucidate the virus-host interactions for Marburg virus.

Ebolavirus↗

PubMed2026

Canine Tear Proteomics: A New Frontier in Veterinary Ophthalmology.

Canine tear proteomics is an emerging field with significant potential for advancing both veterinary ophthalmology and comparative biomedical research. Tears are a readily accessible, non-invasive biofluid that contain a mixture of proteins involved in ocular surface protection, immune defense, and intercellular signaling. In dogs, tear proteomics studies have revealed biomarkers associated with various ocular and non-ocular diseases including keratoconjunctivitis sicca (KCS), glaucoma, neoplasia, and diabetes mellitus. This review compiles all previous studies conducted on the comprehensive canine tear proteome and highlights some of the key tear proteomic studies in human biomedical research. Tear film composition, study design, technological advancements, and select tear proteins are discussed along with key protein alterations and their use as potential biomarkers of disease. Fundamental challenges, clinical implications, and future directions of this rapidly growing field are discussed in detail. As proteomic technology and analytical techniques continue to evolve, canine tear proteomics will become a valuable tool for the veterinary ophthalmologist, enabling the early identification and diagnosis of ocular disease as well as providing a means for monitoring treatment outcomes, disease progression, and overall prognosis for the canine patient.

Animals↗

PubMed2004

Proteomic and metabolomic analysis of vascular smooth muscle cells: role of PKCdelta.

Recent developments of proteomic and metabolomic techniques provide powerful tools for studying molecular mechanisms of cell function. Previously, we demonstrated that neointima formation was markedly increased in vein grafts of PKCdelta-deficient mice compared with wild-type controls. To clarify the underlying mechanism, we performed a proteomic and metabolomic analysis of cultured vascular smooth muscle cells (SMCs) derived from PKCdelta+/+ and PKCdelta-/- mice. Using 2-dimensional electrophoresis and mass spectrometry, we identified >30 protein species that were altered in PKCdelta-/- SMCs, including enzymes related to glucose and lipid metabolism, glutathione recycling, chaperones, and cytoskeletal proteins. Interestingly, nuclear magnetic resonance spectroscopy confirmed marked changes in glucose metabolism in PKCdelta-/- SMCs, which were associated with a significant increase in cellular glutathione levels resulting in resistance to cell death induced by oxidative stress. Furthermore, PKCdelta-/- SMCs overexpressed RhoGDIalpha, an endogenous inhibitor of Rho signaling pathways. Inhibition of Rho signaling was associated with a loss of stress fiber formation and decreased expression of SMC differentiation markers. Thus, we performed the first combined proteomic and metabolomic study in vascular SMCs and demonstrate that PKCdelta is crucial in regulating glucose and lipid metabolism, controlling the cellular redox state, and maintaining SMC differentiation.

Animals↗

PubMed2005

Comparative analysis of human intronless proteins.

The availability of the complete genome sequences of Homo sapiens together with those of taxonomically diverse organisms provides an opportunity to carry out cross-species comparison. Comparisons of protein sequences from different organisms are significant source of information as these could help in answering questions regarding the fraction of proteins that are shared by humans and organisms representing the three domains of life, viz., archaea, bacteria, and eukaryota. In the present study, a comparative analysis of the proteins encoded by intronless genes in humans was undertaken. We identified 1125 human intronless proteins that are solely present in eukaryotic lineage. More than two-thirds of these eukaryotic specific proteins appear to be mammalia specific while a small fraction of proteins are conserved in bilateria and coelomata, indicating that diversification of these proteins occurred after the divergence of the major lineages of the eukaryotic crown group. A large fraction of mammalia specific proteins are enriched in proteins responsible for transport and binding, cell envelope, and housekeeping function particularly translation. Another 228 intronless proteins are observed that do not exhibit homology to any of the proteins in the database. The distribution of human intronless proteins suggests that lineage specific expansion is one of the most important sources of organizational diversity in crown-group eukaryotes. The presence of these eukaryotic as well as human specific intronless proteins provides the foundation for rapid analysis of some of the basic processes involved in human genome.

Animals↗

PubMed2006

Comparative immunoproteomics of identification and characterization of virulence factors from Helicobacter pylori related to gastric cancer.

Helicobacter pylori is an important risk factor of gastric cancer (GC). Although many H. pylori virulence factors have been reported, the pathogenic mechanism by which H. pylori infection causes GC remains unclear. The aims of this study were to identify GC-related antigens from H. pylori and characterize their roles in the development of GC. As GC and duodenal ulcer (DU) are considered clinically divergent, we compared two-dimensional immunoblots of an acid-glycine extract of H. pylori probed with serum samples from 15 patients with GC and 15 with DU to find GC-related antigens, which were subsequently identified by mass spectrometry. Many protein spots were recognized by more than one serum, and 24 of these were better recognized by GC sera. The proteins showing higher frequency of recognition in GC group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase alpha subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Of these proteins, GroES was identified as a dominant GC-related antigen with a much higher seropositivity of GC samples (64.2%, n = 95) compared with 30.9% for gastritis (n = 94) and 35.5% for DU (n = 124). GroES seropositivity was more commonly associated with antral GC than with non-antral GC (odds ratio = 2.7; 95% confidence interval, 1.1-6.7). In peripheral blood mononuclear cells, GroES stimulated production of interleukin (IL)-8, IL-6, granulocyte macrophage colony-stimulating factor, IL-1beta, tumor necrosis factor-alpha, cyclooxygenase-2, and prostaglandin E(2). Moreover when incubated with gastric epithelial cells, GroES induced expression of IL-8, cell proliferation, and up-regulation of c-jun, c-fos, and cyclin D1 but caused down-regulation of p27(Kip1). We conclude that GroES of H. pylori is a novel GC-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation.

Antigens, Bacterial↗

PubMed2005

Comparison of small- and large-scale expression of selected Pyrococcus furiosus genes as an aid to high-throughput protein production.

As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production.

Bacterial Proteins↗

PubMed2002

The K(A)/K(S) ratio test for assessing the protein-coding potential of genomic regions: an empirical and simulation study.

Comparative genomics is a simple, powerful way to increase the accuracy of gene prediction. In this study, we show the utility of a simple test for the identification of protein-coding exons using human/mouse sequence comparisons. The test takes advantage of the fact that in the vast majority of coding regions, synonymous substitutions (K(S)) occur much more frequently than nonsynonymous ones (K(A)) and uses the K(A)/K(S) ratio as the criterion. We show the following: (1) most of the human and mouse exons are sufficiently long and have a suitable degree of sequence divergence for the test to perform reliably; (2) the test is suited for the identification of long exons and single exon genes, which are difficult to predict by current methods; (3) the test has a false-negative rate, lower than most of current gene prediction methods and a false-positive rate lower than all current methods; (4) the test has been automated and can be used in combination with other existing gene-prediction methods.

Animals↗

PubMed2005

Active kinase proteome screening reveals novel signal complexity in cardiomyopathy.

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions.

Animals↗

PubMed2004

Data mining techniques for cancer detection using serum proteomic profiling.

OBJECTIVE: Pathological changes in an organ or tissue may be reflected in proteomic patterns in serum. It is possible that unique serum proteomic patterns could be used to discriminate cancer samples from non-cancer ones. Due to the complexity of proteomic profiling, a higher order analysis such as data mining is needed to uncover the differences in complex proteomic patterns. The objectives of this paper are (1) to briefly review the application of data mining techniques in proteomics for cancer detection/diagnosis; (2) to explore a novel analytic method with different feature selection methods; (3) to compare the results obtained on different datasets and that reported by Petricoin et al. in terms of detection performance and selected proteomic patterns. METHODS AND MATERIAL: Three serum SELDI MS data sets were used in this research to identify serum proteomic patterns that distinguish the serum of ovarian cancer cases from non-cancer controls. A support vector machine-based method is applied in this study, in which statistical testing and genetic algorithm-based methods are used for feature selection respectively. Leave-one-out cross validation with receiver operating characteristic (ROC) curve is used for evaluation and comparison of cancer detection performance. RESULTS AND CONCLUSIONS: The results showed that (1) data mining techniques can be successfully applied to ovarian cancer detection with a reasonably high performance; (2) the classification using features selected by the genetic algorithm consistently outperformed those selected by statistical testing in terms of accuracy and robustness; (3) the discriminatory features (proteomic patterns) can be very different from one selection method to another. In other words, the pattern selection and its classification efficiency are highly classifier dependent. Therefore, when using data mining techniques, the discrimination of cancer from normal does not depend solely upon the identity and origination of cancer-related proteins.

Biomarkers, Tumor↗

PubMed2005

Proteome of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis.

We report on the modification of the Aedes aegypti larval proteome following infection by the microsporidian parasite Vavraia culicis. Mosquito larvae were sampled at 5 and 15 days of age to compare the effects of infection when the parasite was in two different developmental stages. Modifications of the host proteome due to the stress of infection were distinguished from those of a more general nature by treatments involving hypoxia. We found that the major reaction to stress was the suppression of particular protein spots. Older (15 days) larvae reacted more strongly to infection by V. culicis (46% of the total number of spots affected; 17% for 5 days larvae), while the strongest reaction of younger (5 days) larvae was to hypoxia for pH range 5-8 and to combined effects of infection and hypoxia for pH range 3-6. MALDI-TOF results indicate that proteins induced or suppressed by infection are involved directly or indirectly in defense against microorganisms. Finally, our MALDI-TOF results suggest that A. aegypti larvae try to control or clear V. culicis infection and also that V. culicis probably impairs the immune defense of this host via arginases-NOS competition.

Aedes↗

PubMed2025

Sex differences in cerebrospinal fluid proteomics of patients with restless legs syndrome.

STUDY OBJECTIVES: The pathobiology of restless legs syndrome (RLS) remains poorly understood, complicating effective treatment. This observational cross-sectional study aimed to identify a cerebrospinal fluid proteomic signature of RLS and to explore sex-specific differences in cerebrospinal fluid proteomics. METHODS: Cerebrospinal fluid samples were collected from 22 untreated RLS patients and 18 controls, matched for age, body mass index, and sex. Proteomic analysis was conducted using the SOMAscan platform, assessing over 7000 peptides. RESULTS: Eight proteins were differentially abundant between patients and controls, with CRP and JAML increased, and TAPBPL and IL1RL1 decreased. Pathway analysis highlighted significant involvement in immune response, coagulation, and cytoskeletal regulation. Analyses were then carried out using sex stratification, comparing men and women separately. Sex-specific analyses revealed more pronounced proteomic alterations in males (68 differentially abundant proteins vs. control males) than in females (17 proteins). Gene enrichment analysis revealed that men with RLS had more involvement in gene regulation and epigenetic factors than control males and women with restless legs syndrome had greater involvement in systemic inflammatory and vascular processes than control females. CONCLUSIONS: This study identified a cerebrospinal fluid proteomic signature in RLS, implicating immune and inflammatory pathways in the disease's pathophysiology. Significant sex differences in protein level suggest potential sex-specific mechanisms in RLS, warranting further investigation. These findings contribute to the current understanding of RLS and could inform future therapeutic strategies.

Humans↗

PubMed2000

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Animals↗

PubMed2003

The potential for proteomic definition of stem cell populations.

Embryonic and adult stem cell populations have great potential value in medicine, and hematopoietic stem cells are already being used in transplantation. Definition of these populations to increase our understanding of the programs that control differentiation, self-renewal, and possibly plasticity would be of great interest. The relative quantitation of transcriptional activity in stem cells and other populations has defined a profile of gene expression activity in stem cells. Confirmation that these differences have an impact on protein levels within stem cells via their complete protein complement and protein interactions will enable further understanding of regulatory processes in these cells. The recent developments in proteomics and their potential application to the definition of the stem cell proteome are discussed, and examples are given. Advances in mass spectrometry, subcellular prefractionation protocols, and electrophoresis that make stem cell proteomics a tractable problem are discussed. Beyond the proteome per se, advances in post-translational modification profiling mean that comparative analysis of phosphorylation patterns between stem cells and other populations can be approached.

Animals↗

PubMed2005

Upregulation and dephosphorylation of cofilin: modulation by CD44 variant isoform in human colon cancer cells.

Colon cancer is among the leading causes of cancer death in North America. Dysregulation of the crypt homeostasis is evident in the early stage of colon cancer. Moreover, cytoskeletal rearrangement of actin also plays a crucial role in morphological changes during apoptosis. CD44, an adhesion and anti-apoptotic molecule is overexpressed in colon cancer and is known to interact with certain cytoskeletal proteins. In this study, we used the human colon cancer cell line SW620, which does not express CD44 but stably transfected with standard, 3-10v and 8-10v variant isoforms of CD44. By two-dimensional isoelectric focusing (2DIEF), we found an increasing concentration of a 21-kDa protein in SW620 colon cancer cells transfected with CD44 3-10v, as compared to cells transfected with an empty vector. Mass spectrometry (MS) and proteomic analysis of this protein identified the peptide fragment (YALYDATYETK) of 11 amino acids in length spanning residue 82 to 92 of cofilin, a widely distributed 21-kDa actin-modulating protein. Western blot analysis of lysates from cells expressing CD44 variant isoforms 3-10v had increased level of expression of cofilin compared to vector control consistent with our finding by 2DIEF. Also, immunocytochemistry showed that cofilin expression in colonic epithelial cells was greater in cells transfected with CD44 3-10v, as compared to vector controls. We observed that the phosphorylated form of cofilin is downregulated in cells expressing the 3-10v isoform of CD44 both by Western blot and immunocytochemistry. Cofilin expression is thus mechanistically associated with CD44 expression and its 3-10v isoform. Dephosphorylation of cofilin could bring about directional motility of cells that could have important implications to the proliferation and motility of colonic epithelial cells in cancer.

Actin Depolymerizing Factors↗

PubMed2005

A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics.

The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies.

Animals↗

PubMed2005

Proteomic analysis of oxidatively modified proteins induced by the mitochondrial toxin 3-nitropropionic acid in human astrocytes expressing the HIV protein tat.

The human immunodeficiency virus (HIV)-Tat protein has been implicated in the neuropathogenesis of HIV infection. However, its role in modulating astroglial function is poorly understood. Astrocyte infection with HIV has been associated with rapid progression of dementia. Intracellularly expressed Tat is not toxic to astrocytes. In fact, intracellularly expressed Tat offers protection against oxidative stress-related toxins such as the mitochondrial toxin 3-nitroproprionic acid (3-NP). In the current study, human astrocytes expressing Tat (SVGA-Tat) and vector controls (SVGA-pcDNA) were each treated with the irreversible mitochondrial complex II inhibitor 3-NP. Proteomics analysis was utilized to identify changes in protein expression levels. By coupling 2D fingerprinting and identification of proteins by mass spectrometry, actin, heat shock protein 90, and mitochondrial single-stranded DNA binding protein were identified as proteins with increased expression, while lactate dehydrogenase had decreased protein expression levels in SVGA-Tat cells treated with 3-NP compared to SVGA-pcDNA cells treated with 3-NP. Oxidative damage can lead to several events including loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, ultimately leading to neuronal death. Identification of specific proteins protected from oxidation is a crucial step in understanding the interaction of Tat with astrocytes. In the current study, proteomics also was used to identify proteins that were specifically oxidized in SVGA-pcDNA cells treated with 3-NP compared to SVGA-Tat cells treated with 3-NP. We found beta-actin, calreticulin precursor protein, and synovial sarcoma X breakpoint 5 isoform A to have increased oxidation in control SVGA-pcDNA cells treated with 3-NP compared to SVGA-Tat cells treated with 3-NP. These results are discussed with reference to potential involvement of these proteins in HIV dementia and protection of astrocytes against oxidative stress by the HIV virus, a prerequisite for survival of a viral host cell.

Astrocytes↗

PubMed2006

Serum proteomic signature for cystic fibrosis using an antibody microarray platform.

Antibody microarrays are a new proteomic technology, which we have developed as a platform for identifying a cystic fibrosis (CF)-specific serum proteomic signature. Serum samples from CF patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to CF. We find that the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the NFkappaB signaling pathway, and in proteins that may be selectively expressed in CF-affected tissues such as lung and intestine. In several instances, we validate the data from the antibody microarrays by quantitative analysis with Reverse Capture Protein Microarrays. We conclude that antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from CF and control patients.

Adolescent↗