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Label-free shotgun proteomics of tmRNA substrates enriched by immunoprecipitation

ABSTRACT: We used a combination of genetic and proteomic approaches to characterize tmRNA (ssrA) activity in the genome-reduced bacterium Mycoplasma pneumoniae. For this, we generated tmRNA mutants encoding a tag resistant to proteolysis. Endogenous protein tagging by the mutant tmRNA gene (ssrAmk) was then examined by immunoprecipitation (IP) enrichment followed by LC-MS/MS analysis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mycoplasma Pneumoniae (strain Atcc 29342 / M129)

SUBMITTER: Marc Weber

LAB HEAD: Luis Serrano

PROVIDER: PXD023973 | Pride | 2021-08-23

REPOSITORIES: Pride

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			Action	DRS
	160725_S_RBLS_01_01_FASP_45pto.msf	Msf
	160725_S_RBLS_01_01_FASP_45pto.raw	Raw
	160725_S_RBLS_02_01_FASP_45pto.msf	Msf
	160725_S_RBLS_02_01_FASP_45pto.raw	Raw
	160926_RBLS_ProteomeDiscoverer.msf	Msf

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Publications

Widespread ribosome stalling in a genome-reduced bacterium and the need for translational quality control.

Burgos Raul R Weber Marc M Gallo Carolina C Lluch-Senar Maria M Serrano Luis L

iScience 20210816 9

Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, <i>Mycoplasma pneumoniae</i> (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show <i>in vivo</i> that in MPN the sole quality control cytoplasmic protease (Lon) d ...[more]

PMID: 34485867

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Label-free shotgun proteomics of Triton X-100 insoluble fractions of Lon and FtsH conditional mutants of Mycoplasma pneumoniae under depleting and inducing conditions

Project description:Using Mycoplasma pneumoniae as a model organism, we conditionally depleted the two essential ATP-dependent proteases (Lon and FtsH) of this bacterium, by engineering strains carrying a Lon and/or FtsH inducible expression locus. An integrative comparative study combining label-free shotgun proteomics and RNA-seq allowed us to decipher the global cellular response to Lon and FtsH depletion and to define protease substrates in this genome-reduced organism. This particular dataset corresponds to the analysis of the Triton X-100 insoluble fractions of Lon and FtsH mutants grown under inducing and depleting conditions. The dataset for the whole cell lysate is available under the identifier PXD016343.

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Label-free shotgun proteomics of Mycoplasma pneumoniae grown in serum-free medium

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Proteome wide measurements of protein half-lives in Mycoplasma pneumoniae

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Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification

Project description:Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types for proteogenomic-based discovery of neoantigens. By using an optimized computational approach, we discover a large number of tumor-specific and tumor-associated antigens. To create a pipeline for the identification of neoantigens in our cohort, we combine DNA and RNA sequencing with MS-based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity and an in-depth validation process. We detect a broad variety of non-canonical HLA-binding peptides in the majority of patients demonstrating partially immunogenicity. Our validation process allows for the selection of 32 potential neoantigen candidates. The majority of neoantigen candidates originates from variants identified in the RNA data set, illustrating the relevance of RNA as a still understudied source of cancer antigens. This study underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.

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Identification of potential mitochondrial CLPXP protease interactors and substrates

Project description:Maintenance of mitochondria is achieved by several mechanisms, including the regulation of mitochondrial proteostasis. The matrix protease CLPXP, involved in protein quality control, has been implicated in ageing and disease. However, particularly due to the lack of knowledge of CLPXP’s substrate spectrum, only little is known about the pathways and mechanisms controlled by this protease. Here we report the first comprehensive identification of potential mitochondrial CLPXP in vivo interaction partners and substrates using a combination of tandem affinity purification and differential proteomics. This analysis reveals that CLPXP in the fungal ageing model Podospora anserina is mainly associated with metabolic pathways in mitochondria, e.g. components of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle as well as subunits of electron transport chain complex I. These data suggest a possible function of mitochondrial CLPXP in the control and/or maintenance of energy metabolism. Since bioenergetic alterations are a common feature of neurodegenerative diseases, cancer, and ageing, our data comprise an important resource for specific studies addressing the role of CLPXP in these adverse processes.

2016-07-27 | PXD003264 | Pride

Identification of human GRHL1 interactors and post-translational modifications by LC-MS/MS

Project description:Aim of the project was to identify interactors and post-translational modifications of recombinant 3xFLAG-6xHis-tagged human GRHL1, tandem-affinity purified from isolated HEK293 nuclei treated with DMSO as control or with the phytochemical papaverine.

2021-11-02 | PXD021768 | Pride

RNA-seq of Mycoplasma pneumoniae grown in serum-free medium

Project description:We developed a serum and animal component-free medium capable of supporting robust growth of the genome-reduced bacterium Mycoplasma pneumoniae. Here, we performed RNA-seq expression analysis of the wild-type strain grown in serum-free vB13 medium.

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A non-canonical BRD9-containing BAF chromatin remodeling complex regulates naïve pluripotency in mouse embryonic stem cells

Project description:The mammalian BRG1-associated factors (BAF) complex is a multi-subunit chromatin remodeling complex that is an important component of the embryonic stem cell (ESC) transcriptional regulatory network. However, the role of individual subunits in BAF complex targeting and function needs to be elucidated. Here, we find that the Bromodomain containing protein 9 (BRD9) defines a smaller, non-canonical BAF complex in mouse ESCs that is distinct from the canonical embryonic stem cell BAF (esBAF) and the polybromo-associated BAF (PBAF) complexes. This BRD9-containing BAF complex, or BBAF complex, uniquely incorporates the BRD4-interacting chromatin remodeling associated protein-like (BICRAL) or its paralog BICRA and lacks several esBAF subunits including BAF47, ARID1A and BAF57. We demonstrate that BBAF and esBAF complexes are targeted to different features of the genome and are co-bound with different sets of pluripotency transcription factors. Specifically, BBAF complexes co-localize with key regulators of naïve pluripotency, KLF4 and Sp5, on the genome. Consistent with this, we provide evidence that BBAF’s specific function is to regulate the transcription of genes involved in the maintenance of naïve pluripotency, including Nanog and Prdm14. Additionally, we show that BRD9 is displaced from chromatin by the selective BRD9 bromodomain inhibitor, I-BRD9, and that this leads to changes in target gene expression. Together, our results provide evidence for the identification of a new BAF complex, and demonstrate functionally specific roles for BAF complex assemblies in maintaining the transcriptional network of pluripotency.

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