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Molecular cellular proteomics [journal]
- Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 24.
Accurate annotation of protein-coding genes is one of the primary tasks upon completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high resolution mass spectrometry-based proteomic profiling of ten adult organs, whole adult fish body and two developmental stages of zebrafish (SAT line) in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses and ~69,000 high confidence transcripts were assembled from the RNA-Seq data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high quality transcriptomic and proteomic data were used to manually re-annotate the zebrafish genome. We report identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures which include novel exons, change in exon coordinates, change in frame of translation, translation in annotated UTRs and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.
- Broad range glycosidase activity profiling. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 23.
Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)-modelling, protein and lipid modification and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Consequently, glycosidases are poorly defined in plants and other organisms. Here, we apply activity-based glycosidase profiling using cell permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detect the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable on different organs, different plant species, in living cells and in subproteomes. We displayed the active state of previously uncharacterized glycosidases, one of which encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.
- Inhibition of Circulating Dipeptidyl Peptidase 4 Activity in Patients with Metastatic Prostate Cancer. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 23.
Cancer is responsible for many deaths and a major source of healthcare expenditures. Identification of new, non-invasive biomarkers may allow improving upon direct diagnostic or prognostic ability of already available tools. Here, we have taken an innovative approach by interrogating the activity of exopeptidases in serum of cancer patients with the aim to establish a distinction based on enzymatic function, instead of simple protein levels, as a means to biomarker discovery. We first analyzed two well-characterized mouse models of prostate cancer, each with a distinct genetic lesion, and established that broad exopeptidase and targeted aminopeptidase activity tests reveal proteolytic changes associated with tumor development. We also describe new peptide-based, freeze-frame reagents uniquely suited to probe the altered balance of selected aminopeptidases, as opposed to the full array of exopeptidases, and/or their modulators in patient serum or plasma. One particular proteolytic activity was impaired in animals with aggressive disease as compared to cancer-free littermates. The protease in question was identified as dipeptidyl peptidase 4 (DPP4) by analyzing selected knockout mice and evaluating the effect of specific inhibitors. DPP4 activity was also reduced in sera of patients with metastatic prostate cancer when compared to patients with localized disease or healthy controls. Yet no significant differences in DPP4 serum levels were observed, establishing the loss of activity was the result of impaired enzymatic function. Biochemical analysis indicated that reduced activity is not the result of post-translational modifications or allosteric changes but instead of a low-molecular weight inhibitor. After adjusting for age and total prostate-specific antigen, reduced DPP4 activity remained a significant predictor of cancer status. The results of this proof-of-principle study suggest that DPP4 activity may be a potential candidate as blood-based indicator for the presence of metastatic cancer of prostatic origin, either by itself or, more likely, to improve sensitivity and specificity of existing markers.
- A systematic proteomic analysis of Listeria monocytogenes house-keeping protein secretion systems. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 23.
Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. While great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analysed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein.
- Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 23.
One of the critical gaps in malaria transmission biology and surveillance is our lack of knowledge about Plasmodium falciparum gametocyte biology, especially sexual dimorphic development and how sex ratios that may influence transmission from the human to the mosquito. Dissecting this process has been hampered by the lack of sex-specific protein markers for the circulating, mature stage V gametocytes. The current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium, and therefore presumably for sex-specific genes as well. To better our understanding of gametocyte development and subsequent infectiousness to mosquitoes, we undertook a Systematic Subtractive Bioinformatic analysis (filtering) approach to identify sex-specific P. falciparum NF54 protein markers based on a comparison with the Dd2 strain, which is defective in producing males, and with syntenic male and female proteins from the reanalyzed and updated P. berghei (related rodent malaria parasite) gametocyte proteomes. This produced a short list of 174 male- and 258 female-enriched P. falciparum stage V proteins, some of which appear to be under strong diversifying selection, suggesting ongoing adaptation to mosquito vector species. We generated antibodies against three putative female-specific gametocyte stage V proteins in P. falciparum and confirmed either conserved sex-specificity or the lack of cross-species sex-partitioning. Finally, our study provides not only an additional resource for mass spectrometry-derived evidence for gametocyte proteins but also lays down the foundation for rational screening and development of novel sex-partitioned protein biomarkers and transmission-blocking vaccine candidates.
- A systems level analysis reveals transcriptomic and proteomic complexity in Ixodes ricinus midgut and salivary glands during early attachment and feeding. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 21.
Although pathogens are usually transmitted within the first 24-48 hours of attachment of the castor bean tick Ixodes ricinus, little is known about the tick biological responses at these earliest phases of attachment. Tick midgut and salivary glands are the main tissues involved in tick blood feeding and pathogen transmission but the limited genomic information for I. ricinus delays the application of high-throughput methods to study their physiology. We took advantage of the latest advances in the fields of Next Generation RNA-Sequencing and Label-free Quantitative Proteomics to deliver an unprecedented, quantitative description of the gene expression dynamics in the midgut and salivary glands of this disease vector upon attachment to the vertebrate host. 373 of 1510 total identified proteins had higher expression in the salivary glands, but only 110 had correspondingly high transcript levels in the same tissue. Furthermore, there was midgut-specific expression of 217 genes at both the transcriptome and proteome level. Tissue-dependent transcript, but not protein, accumulation was revealed for 552 of 885 genes. Moreover, we discovered the enrichment of tick salivary glands in proteins involved in gene transcription and translation which agrees with the secretory role of this tissue; this finding also agrees with the finding of lower tick t-RNA representation in the salivary glands when compared to the midgut. The midgut, in turn, is enriched in metabolic components and proteins that support its mechanical integrity in order to accommodate and metabolize the ingested blood. Beyond understanding the physiological events that support hematophagy by arthropod ectoparasites, we discovered more than 1500 proteins located at the interface between ticks, the vertebrate host and the tick-borne pathogens. Thus, our work significantly improves the knowledge of the genetics underlying the transmission lifecycle of this tick species which is an essential step for developing alternative methods to better control tick-borne diseases.
- Structural characterization by MSn of human milk glycans recognized by human rotaviruses. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 21.
We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains N155(G10P) and RV3(G3P) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans (HMG). The accompanying study by Yu et al, describes a HMG shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library (TGL) that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by MALDI MS. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the TGL for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that among the 28 glycan targets analyzed 27 unique structures were identified, and 23 of the HMGs recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of HMG provides significant insight into the repertoire of glycans comprising the human milk metaglycome.
- Human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 21.
Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated by multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate an HMG shotgun glycan microarray (SGM). To investigate the potential role of HMG as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG-SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains, N155(G10P) and RV3(G3P), and a bovine strain, B223(G10P). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using Metadata-Assisted Glycan Sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MSn analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8*-binding, many of which are novel HMGs, whose detailed structural assignments by MSn are described in the accompanying manuscript by Ashline et al. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures.
- High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture (SILAC), two-dimensional gel electrophoresis (2DE) and nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer. [Journal Article]
- Mol Cell Proteomics 2014 Jul; 13(7):1900.
- Progesterone receptor membrane component 1 is a functional part of the GLP-1 receptor complex in pancreatic beta cells. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Jul 20.
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Due to their direct stimulation of insulin secretion from pancreatic beta cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells (CHO) or MIN6 beta cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, Rab5c) were further validated by co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer (FRET) and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1 induced insulin secretion (GIIS) with the most robust effect. Knock-down of PGRMC1 in beta cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS while its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation and enhanced cell surface GLP-1R expression. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibiting adenylyl cyclase/EPAC signaling or the EGFR-PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGFR signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of beta cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.