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      Quantitative Membrane Proteomics Reveals New Cellular Targets of Viral Immune Modulators

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      PLoS Pathogens

      Public Library of Science

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          Abstract

          Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma–associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif–deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.

          Synopsis

          Viral immune modulators often target multiple cellular proteins for destruction. Presumably, this strategy enables viral pathogens to optimize evasion of multiple immune responses. To systematically identify such host cell targets in an unbiased fashion, Bartee et al. applied recently developed quantitative proteomics methods to identify novel targets for K5. K5 belongs to a family of viral ubiquitin ligases found in gamma-herpesviruses and poxviruses that target multiple cellular transmembrane proteins for destruction. Using stable isotope labeling combined with tandem mass spectrometry, the authors compared the abundance of proteins in membrane preparations from cells that expressed K5 to that in cells without K5. In their experiments, three novel membrane proteins (BST-2, Syntaxin-4, and ALCAM) were consistently found in lower abundance in K5-expressing cells. Importantly, the authors were able to confirm the K5-dependent downregulation of all of these proteins in independent experiments and by independent methods. ALCAM was chosen for a more in-depth analysis to firmly demonstrate that this protein is downregulated by K5 in a manner similar to other known targets. This proof-of-principle study demonstrates that novel targets of viral immune modulators can be identified with quantitative proteomics.

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          Most cited references55

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          Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

          Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
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            Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.

            We present a statistical model to estimate the accuracy of peptide assignments to tandem mass (MS/MS) spectra made by database search applications such as SEQUEST. Employing the expectation maximization algorithm, the analysis learns to distinguish correct from incorrect database search results, computing probabilities that peptide assignments to spectra are correct based upon database search scores and the number of tryptic termini of peptides. Using SEQUEST search results for spectra generated from a sample of known protein components, we demonstrate that the computed probabilities are accurate and have high power to discriminate between correctly and incorrectly assigned peptides. This analysis makes it possible to filter large volumes of MS/MS database search results with predictable false identification error rates and can serve as a common standard by which the results of different research groups are compared.
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              Direct analysis of protein complexes using mass spectrometry.

              We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                ppat
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                October 2006
                27 October 2006
                : 2
                : 10
                Affiliations
                [1]Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, United States of America
                University of California San Francisco, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: fruehk@ 123456ohsu.edu
                Article
                06-PLPA-RA-0223R2 plpa-02-10-13
                10.1371/journal.ppat.0020107
                1626102
                17238276
                fa65885d-dc94-452a-81b3-c540b5998593
                Copyright: © 2006 Bartee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 14
                Categories
                Research Article
                Biochemistry
                Cell Biology
                Immunology
                Molecular Biology - Structural Biology
                Virology
                In Vitro
                Viruses
                None
                Custom metadata
                Bartee E, McCormack A, Früh K (2006) Quantitative membrane proteomics reveals new cellular targets of viral immune modulators. PLoS Pathog 2(10): e107. DOI: 10.1371/journal.ppat.0020107

                Infectious disease & Microbiology

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