Dataset Information

Complete and ubiquitinated proteome of the Legionella-containing vacuole within human macrophages.

ABSTRACT: Within protozoa or human macrophages Legionella pneumophila evades the endosomal pathway and replicates within an ER-derived vacuole termed the Legionella-containing vacuole (LCV). The LCV membrane-localized AnkB effector of L. pneumophila is an F-box protein that mediates decoration of the LCV with lysine(48)-linked polyubiquitinated proteins, which is essential for intravacuolar replication. Using high-throughput LC-MS analysis, we have identified the total and ubiquitinated host-derived proteome of LCVs purified from human U937 macrophages. The LCVs harboring the AA100/130b WT strain contain 1193 proteins including 24 ubiquitinated proteins, while the ankB mutant LCVs contain 1546 proteins with 29 ubiquitinated proteins. Pathway analyses reveal the enrichment of proteins involved in signaling, protein transport, phosphatidylinositol, and carbohydrate metabolism on both WT and ankB mutant LCVs. The ankB mutant LCVs are preferentially enriched for proteins involved in transcription/translation and immune responses. Ubiquitinated proteins on the WT strain LCVs are enriched for immune response, signaling, regulation, intracellular trafficking, and amino acid transport pathways, while ubiquitinated proteins on the ankB mutant LCVs are enriched for vesicle trafficking, signaling, and ubiquitination pathways. The complete and ubiquitinated LCV proteome within human macrophages illustrates complex and dynamic biogenesis of the LCV and provides a rich resource for future studies.

SUBMITTER: Bruckert WM

PROVIDER: S-EPMC4286187 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Complete and ubiquitinated proteome of the Legionella-containing vacuole within human macrophages.

Bruckert William M WM Abu Kwaik Yousef Y

Journal of proteome research 20141113 1

Within protozoa or human macrophages Legionella pneumophila evades the endosomal pathway and replicates within an ER-derived vacuole termed the Legionella-containing vacuole (LCV). The LCV membrane-localized AnkB effector of L. pneumophila is an F-box protein that mediates decoration of the LCV with lysine(48)-linked polyubiquitinated proteins, which is essential for intravacuolar replication. Using high-throughput LC-MS analysis, we have identified the total and ubiquitinated host-derived prote ...[more]

PMID: 25369898

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Project description:Protein ubiquitination plays an essential regulatory role within all eukaryotes. Large-scale analyses of ubiquitinated proteins are usually performed by combining affinity purification strategies with mass spectrometry. However, there is no reliable method to systematically differentiate ubiquitinated species from copurified unmodified components. Here we report a simple strategy for the large-scale validation of ubiquitination by reconstructing virtual Western blots for proteins analyzed by gel electrophoresis and mass spectrometry. Because protein ubiquitination, especially polyubiquitination, causes a dramatic shift of molecular weight, the difference between experimental and expected molecular weight was used to confirm the status of ubiquitination. Experimental molecular weight of putative yeast ubiquitin-conjugates was computed from the value and distribution of spectral counts in the gel using a Gaussian curve fitting approach. Unmodified proteins in yeast cell lysate were also analyzed as a control to assess the accuracy of the method. Multiple thresholds that incorporated the mass of ubiquitin and/or experimental variations were evaluated with respect to sensitivity and specificity. Ultimately, only approximately 30% of the candidate ubiquitin-conjugates were accepted based on the stringent filtering criteria, although they were purified under denaturing conditions. These accepted conjugates had an estimated false discovery rate of approximately 8% and primarily consisted of proteins larger than 100 kDa. Compared with another validation method (i.e., identification of ubiquitinated lysine sites), approximately 95% of the proteins with defined modification sites showed a convincing increase in molecular weight on the virtual Western blots. A second independent analysis indicated that the method can be simplified by excising fewer than ten gel bands. Therefore, this strategy establishes criteria necessary for the interpretation of ubiquitinated proteins.

Dataset Information

Complete and ubiquitinated proteome of the Legionella-containing vacuole within human macrophages.

Publications

Complete and ubiquitinated proteome of the Legionella-containing vacuole within human macrophages.

Similar Datasets

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