Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome

<p>Abstract</p> <p>Background</p> <p>Brucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand <it>Brucella abortus </it>virulence, we consolidated the proteomic data collected and compared it to publically available genomic data.</p> <p>Results</p> <p>The proteomic data was compiled from several independent comparative studies of <it>Brucella abortus </it>that used either outer membrane blebs, cytosols, or whole bacteria grown in media, as well as intracellular bacteria recovered at different times following macrophage infection. We identified a total of 621 bacterial proteins that were differentially expressed in a condition-specific manner. For 305 of these proteins we provide the first experimental evidence of their expression. Using a custom-built protein sequence database, we uncovered 7 annotation errors. We provide experimental evidence of expression of 5 genes that were originally annotated as non-expressed pseudogenes, as well as start site annotation errors for 2 other genes.</p> <p>Conclusions</p> <p>An essential element for ensuring correct functional studies is the correspondence between reported genome sequences and subsequent proteomics studies. In this study, we have used proteomics evidence to confirm expression of multiple proteins previously considered to be putative, as well as correct annotation errors in the genome of <it>Brucella abortus </it>strain 2308.</p

Immunopeptidomic Data Integration to Artificial Neural Networks Enhances Protein-Drug Immunogenicity Prediction

Recombinant DNA technology has, in the last decades, contributed to a vast expansion of the use of protein drugs as pharmaceutical agents. However, such biological drugs can lead to the formation of anti-drug antibodies (ADAs) that may result in adverse effects, including allergic reactions and compromised therapeutic efficacy. Production of ADAs is most often associated with activation of CD4 T cell responses resulting from proteolysis of the biotherapeutic and loading of drug-specific peptides into major histocompatibility complex (MHC) class II on professional antigen-presenting cells. Recently, readouts from MHC-associated peptide proteomics (MAPPs) assays have been shown to correlate with the presence of CD4 T cell epitopes. However, the limited sensitivity of MAPPs challenges its use as an immunogenicity biomarker. In this work, MAPPs data was used to construct an artificial neural network (ANN) model for MHC class II antigen presentation. Using Infliximab and Rituximab as showcase stories, the model demonstrated an unprecedented performance for predicting MAPPs and CD4 T cell epitopes in the context of protein-drug immunogenicity, complementing results from MAPPs assays and outperforming conventional prediction models trained on binding affinity data.Fil: Barra, Carolina. Technical University of Denmark; DinamarcaFil: Ackaert, Chloe. No especifíca;Fil: Reynisson, Birkir. Technical University of Denmark; DinamarcaFil: Schockaert, Jana. No especifíca;Fil: Jessen, Leon Eyrich. Technical University of Denmark; DinamarcaFil: Watson, Mark. No especifíca;Fil: Jang, Anne. No especifíca;Fil: Comtois Marotte, Simon. No especifíca;Fil: Goulet, Jean Philippe. No especifíca;Fil: Pattijn, Sofie. No especifíca;Fil: Paramithiotis, Eustache. No especifíca;Fil: Nielsen, Morten. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

AbstractBiomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC=0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Can Mediate Degradation of the Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1)

<div><p>Elevated LDL-cholesterol (LDLc) levels are a major risk factor for cardiovascular disease and atherosclerosis. LDLc is cleared from circulation by the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin 9 (PCSK9) enhances the degradation of the LDLR in endosomes/lysosomes, resulting in increased circulating LDLc. PCSK9 can also mediate the degradation of LDLR lacking its cytosolic tail, suggesting the presence of as yet undefined lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a role for the transmembrane-domain of the LDLR in mediating its PCSK9-induced internalization and degradation. Recent findings from our laboratory also suggest a role for PCSK9 in enhancing tumor metastasis. We show herein that while the LDLR is insensitive to PCSK9 in murine B16F1 melanoma cells, PCSK9 is able to induce degradation of the low density lipoprotein receptor-related protein 1 (LRP-1), suggesting distinct targeting mechanisms for these receptors. Furthermore, PCSK9 is still capable of acting upon the LDLR in CHO 13-5-1 cells lacking LRP-1. Conversely, PCSK9 also acts on LRP-1 in the absence of the LDLR in CHO-A7 cells, where re-introduction of the LDLR leads to reduced PCSK9-mediated degradation of LRP-1. Thus, while PCSK9 is capable of inducing degradation of LRP-1, the latter is not an essential factor for LDLR regulation, but the LDLR effectively competes with LRP-1 for PCSK9 activity. Identification of PCSK9 targets should allow a better understanding of the consequences of PCSK9 inhibition for lowering LDLc and tumor metastasis.</p></div

PCSK9 acts on the LDLR independent of the receptor's CT and TMD.

<p><b>A</b>) Generation of chimeric truncated LDLR-V5 constructs. Schematic representation of the LDLR, LDLR lacking its CT (ΔCT), and ΔCT in which the LDLR TMD was swapped with that of ACE2 (ΔCT_TMDace2) or VLDLR (ΔCT_TMDvldlr). All constructs contained a C-terminal V5-tag. <b>B</b>) Expression in HEK293 cells. WT and chimeric LDLR constructs were transfected in HEK293 cells. Construct expression was assessed by immunoblotting with mAb-V5. Both mature and immature forms of the LDLR were detected. β-actin was used as a loading control. <b>C)</b> PCSK9 induces LDLR degradation independent of the LDLR's CT and TMD. LDLR, ΔCT, and the ΔCT_TMDace2 and ΔCT_TMDvldlr chimeric constructs were expressed in HEK293 cells. Twenty-four hours post-transfection, the cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media, which contains both full length PCSK9 and its furin cleaved product at Arg₂₁₈, PCSK9-ΔN₂₁₈<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064145#pone.0064145-Benjannet4" target="_blank">[33]</a>. Cells were lysed in 1x RIPA and subjected to Western blot analysis. LDLR and PCSK9 were detected with mAb-V5. β-actin was employed as a loading control. The ability of PCSK9 to induce degradation of the LDLR constructs was quantified using NIH ImageJ software and calculated relative to treatment with pIRES conditioned media. Data are representative of at least three independent experiments. <b>D</b>) PCSK9 reduces cell surface LDLR levels independent of the receptor's CT and TMD. To assess the ability of PCSK9 added exogenously to HEK293 cells expressing the LDLR or its chimeric constructs, transfected cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media. Subsequently, surface LDLR was quantified by FACS analysis. The values obtained after treatment with PCSK9 are represented graphically relative to treatment with control pIRES. Data are representative of at least three independent experiments. Error bars represent SEM. *, <i>p</i><0.05 (Student's t test).</p