Eustache Paramithiotis

A prion protein epitope selective for the pathologically misfolded conformation

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of β-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.

Modulation of the phagosome proteome by interferon-gamma.

Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-γ (IFN-γ), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-γ-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-γ includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-γ-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-γ-activated macrophages.

Intracellular Adaptation of Brucella abortus

Macrophages were infected with virulent Brucella abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration; adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strains adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections.

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

BackgroundBrucellosis 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 Brucella abortus virulence, we consolidated the proteomic data collected and compared it to publically available genomic data.ResultsThe proteomic data was compiled from several independent comparative studies of Brucella abortus 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.ConclusionsAn 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 Brucella abortus strain 2308.

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

Biomarkers 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.

Protein biomarker quantification by mass spectrometry

The protein biomarker field is becoming increasingly interested in multiple reaction monitoring mass spectrometry (MRM-MS) assays for biomarker tests. Originally developed years ago and used extensively to quantify small molecules, this technique is now being adapted to peptides. A summary is presented of MRM-MS techniques for quantification of protein biomarkers, including biomarker panels, and clinical applications of this approach. A survey of the current literature relating to the use of MRM-MS to quantify protein biomarker panels was conducted. Future directions for MRM-MS include qualification and verification of candidate protein biomarkers. Furthermore, the analytical characteristics of the MRM-MS approach make it ideally suited for the clinical laboratory as an assay for biomarker tests.

Cdc42 and Rac1 activity is reduced in human pheochromocytoma and correlates with FARP1 and ARHGEF1 expression

Among small GTPases from the Rho family, Cdc42, RAC, and Rho are well known to mediate a large variety of cellular processes linked with cancer biology through their ability to cycle between an inactive (GDP-bound) and an active (GTP-bound) state. Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP for GTP to generate the activated form, whereas the GTPase-activating proteins (GAPs) catalyze GTP hydrolysis, leading to the inactivated form. Modulation of Rho GTPase activity following altered expression of RHO-GEFs and/or RHO-GAPs has already been reported in various human tumors. However, nothing is known about the Rho GTPase activity or the expression of their regulators in human pheochromocytomas, a neuroendocrine tumor (NET) arising from chromaffin cells of the adrenal medulla. In this study, we demonstrate, through an ELISA-based activity assay, that Rac1 and Cdc42 activities decrease in human pheochromocytomas (PCCs) compared with the matched adjacent non-tumor tissue. Furthermore, through quantitative mass spectrometry (MS) approaches, we show that the expression of two RHO-GEF proteins, namely ARHGEF1 and FARP1, is significantly reduced in tumors compared with matched non-tumor tissue, whereas ARHGAP36 expression is increased. Moreover, siRNA-based knockdown of ARHGEF1 and FARP1 in PC12 cells leads to a significant inhibition of Rac1 and Cdc42 activities, respectively. Finally, a principal component analysis (PCA) of our dataset was able to discriminate PCC from non-tumor tissue and indicates a close correlation between Cdc42/Rac1 activity and FARP1/ARHGEF1 expression. Altogether, our findings reveal for the first time the importance of modulation of Rho GTPase activities and expression of their regulators in human PCCs.

An evaluation of multidimensional fingerprinting in the context of clinical proteomics

Multidimensional fingerprinting (MDF) utilizes measurable peptide characteristics to identify proteins. In this study, 3‐D fingerprinting, namely, parent protein molecular weight, peptide mass, and peptide retention time on RPLC, is used to identify 331 differentially expressed proteins between normal and human colon cancer plasma membrane samples. A false discovery rate (FDR) procedure is introduced to evaluate the performance of MDF on the colon cancer dataset. This evaluation establishes a false protein identification rate below 15% for this dataset. Western blot analysis is performed to validate the differential expression of the MDF‐identified protein VDAC1 on the original tissue samples. The limits of MDF are further assessed by a simulation study where key parameters such as database size, query size, and mass accuracy are varied. The results of this simulation study demonstrate that fingerprinting with three dimensions yields low FDR values even for large queries on the complete human proteome without the need for prior peptide sequencing by tandem mass spectrometry. Specifically, when mass accuracy is 10 ppm or lower, full human proteome searches can achieve FDR values of 10% or less.

Identification of Host Proteins Predictive of Early Stage Mycobacterium tuberculosis Infection

The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age ≥ 12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p < 0.05) differentially expressed proteins were identified across all comparisons. Proteins associated with cellular immunity and lipid metabolism were induced early after Mtb infection. One hundred and fifty-nine proteins were selected for a targeted mass spectrometry assay. A set of longitudinal samples from 52 TST-negative subjects who converted to TST-positive or remained TST-negative were analyzed, and multivariate logistic regression was used to identify unique protein panels able to predict TST conversion with cross-validated AUC > 0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy.

Reply to “Properties of a disease-specific prion probe”

To the editor: In a recently published article1, Paramithiotis et al. describe antibodies specific for the prion Tyr-Tyr-Arg (YYR) repeat motif. These antibodies interact with the pathological isoform of the prion protein (PrPSc), but not with the normal cellular isoform (PrPC). Because of this restricted specificity, they suggest that YYR-specific antibodies could be useful for the diagnosis and treatment of prion diseases (Fig. 1). The monoclonal antibodies, all of the IgM isotype, were produced by immunizing mice with a synthetic peptide (CYYRRYYRYY). When coupled to magnetic beads, these YYR-specific antibodies immunoprecipitate PrPSc much more efficiently than PrPC. Notably, the Paramithiotis study did not rely on antibodies to YYR for specific detection of PrP. Their immunoblots were not ultimately probed with PrPSc-specific antibodies, but rather with ‘regular’ antibodies. The latter can detect PrP (but do not distinguish between PrPSc and PrPC) in a precipitate that could include any protein containing solvent-accessible tyrosine and arginine residues. This report is notably similar to that of Korth et al.2, who described a PrpSc-specific IgM (designated 15B3) after immunizing with full-length recombinant bovine PrP. The 15B3 epitope consists of three separate, linear segments of PrP (15B3-1, 15B3-2 and 15B3-3). The YYR epitope (bold) identified by Paramithiotis et al. is included in or located near two of the 15B3 segments (underlined): GSDYEDRYYR (15B3-1) and YYRPVDQYS (15B3-2). Thus, these two independent studies relying on the same method of immunoprecipitation have identified similar IgM antibodies interacting with the same region on PrP, and possibly with the same YYR motifs. The new reagents described could put an end to the quest for a PrPSc-specific antibody. Because the authors envision therapeutic use of the described antibodies, however, it seems appropriate to emphasize that YYR-specific antibodies could interact with any protein with tyrosine and arginine residues on its surface. In regard to PrPSc detection, it should be noted that no diagnostic application of 15B3 has been reported since the report of Korth et al. was published in in 1997. The availability of new diagnostic tests sensitive enough to ensure the protection of public health is an important issue3. For the design of such tests, there remains the choice between high-affinity antibodies that recognize both PrPSc and PrPC but require prior elimination of PrPC, and lower-affinity but PrPSc-specific antibodies.