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Characterization of the mitochondrial proteome of human cells (MitoCoP)

ABSTRACT: The overall aim of the project is the definition and characterization of the mitochondrial proteome of human cells, referred to as MitoCoP. To this end, we combined the complementary strengths of subtractive proteomics, ImportOmics and subcellular protein profiling in a global multidimensional classification approach (see dataset PXD016924). In this part of the project, we analyzed the dynamics of the mitochondrial proteome in Huh7 cells following a pulsed SILAC approach.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Friedel Drepper

LAB HEAD: Prof. Dr. Bettina Warscheid

PROVIDER: PXD028169 | Pride | 2021-11-25

REPOSITORIES: Pride

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			Action	DRS
	ELITE-RSLC030174.raw	Raw
	ELITE-RSLC030175.raw	Raw
	ELITE-RSLC030176.raw	Raw
	ELITE-RSLC030177.raw	Raw
	ELITE-RSLC030178.raw	Raw

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Characterization of the mitochondrial proteome of human cells (MitoCoP)

Project description:The overall aim of the project is the definition and characterization of the mitochondrial proteome of human cells, referred to as MitoCoP. To this end, we combined the complementary strengths of subtractive proteomics, ImportOmics and subcellular protein profiling in a global multidimensional classification approach (see dataset PXD016924). In this part of the project, we (1) performed subtractive proteomics experiments of gradient-purified versus crude mitochondrial fractions prepared from multiple cell lines (multi-cell lines pM-vs-cM experiments), (2) studied the organization of mitochondrial proteins in large functional assemblies by Blue Native (BN) complexome profiling, and (3) analyzed the effect of LYRM9 knockout on mitochondrial protein complexes by BN-PAGE (LYRM9KO-vs-control experiment) to complement the characterization of MitoCoP.

2021-11-25 | PXD028149 | Pride

Identification of serine/threonine-specific phosphatases that dephosphorylate the co-chaperone BAG3 to mediate protein-protein interactions

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TOMM70 ImportOmics analysis of human mitochondria

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Definition and characterization of the mitochondrial proteome of human cells (MitoCoP)

Project description:Aim of this project was the definition and characterization of the mitochondrial proteome of human cells, referred to as MitoCoP. To this end, we combined the complementary strengths of subtractive proteomics, ImportOmics and subcellular protein profiling in a global multidimensional classification approach. This multifaceted human mitochondria-centered analysis was expanded to a functional characterization of the mitochondrial proteome. The association of proteins with mitochondrial membranes was assessed by quantitative proteomic and single-protein studies. Protein copy numbers per mitochondrial unit were determined followed by a thorough compilation and functional classification of a high-quality human mitochondrial proteome. We further examined the organization of mitochondrial proteins in large functional assemblies by Blue Native (BN) complexome profiling and characterized functional protein networks of so far not described mitochondrial constituents by quantitative affinity purification mass spectrometry (q-AP-MS) and biochemical analysis. And finally, we systematically studied mitochondrial proteome dynamics and established a comprehensive subunit-resolved turnover map of large membrane protein machineries and central biosynthesis pathways of human mitochondria.

2021-11-25 | PXD016924 | Pride

An Msp1-containing complex removes orphaned proteins in the mitochondrial outer membrane of trypanosomes (RNAi knockdown data)

Project description:The AAA-ATPase Msp1 extracts mislocalized outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pull down experiments, we show that trypanosomal Msp1 localizes to both the glycosome and the mitochondrial outer membrane, where it forms a stable complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of α-helically anchored mitochondrial outer membrane proteins, such as protein translocase subunits. Inhibition of complex assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double and triple RNAi cell lines combined with proteomic analyses, we demonstrate that both TbMsp1 and the trypanosomal homolog of a cytosolic AAA-ATPase, VCP, are implicated in this quality control pathway. Moreover, in the absence of TbVCP, three out of the four TbMsp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates, suggesting they act in concert with TbMsp1. pATOM36 is a functional analogue of the yeast MIM complex, and possibly of human MTCH2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.

2023-07-26 | PXD039634 | Pride

Transcription profiling of Streptococcus agalactiae strains 2603V/R and 515 and CsrR or CsrS isogenic mutants to better understand the CsrRS regulon

Project description:CsrRS is a two-component regulatory system that controls expression of multiple genes and affects virulence in group B Streptococcus (GBS), a major cause of neonatal sepsis and of invasive infection in chronically ill adults. To better understand the CsrRS regulon and its importance in GBS pathogenicity, we used GBS genomic microarrays to compare global gene expression in GBS strains 2603V/R and 515 with that in CsrR or CsrS isogenic mutants. These results and comparison with data reported previously for GBS strain NEM316 demonstrated a conserved CsrRS regulon of approximately 30 genes including those encoding beta-hemolysin, CAMP factor, and C5a-peptidase, as well as a variable repertoire of genes regulated in one or two of the three strains. Inactivation of CsrS had a similar but weaker effect at most regulated loci, but, in some cases, resulted in more pronounced or divergent regulation compared to CsrR. We conclude that CsrRS regulates a core group of genes including important virulence factors in diverse strains of GBS, but also displays marked variability in the repertoire of regulated genes and in the relative effects of CsrS signaling on CsrR-mediated gene regulation. Such variation is likely to play an important role in strain-specific adaptation of GBS to particular host environments and pathogenic potential in susceptible hosts.

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The Batten disease gene product CLN3 is required for the efflux of glycerophosphodiesters from lysosomes

Project description:The lysosome has many cellular roles, including degrading and recycling macromolecules and signaling to the mTORC1 growth regulator. Lysosomal dysfunction occurs in various human diseases, including common neurodegenerative diseases as well as monogenic lysosomal storage disorders (LSDs). For most LSDs the causal genes have been identified, but in many cases the function of the implicated gene is unknown. Here, we develop the LysoTag mouse line for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We apply it to the study of CLN3, a lysosomal transmembrane protein of unclear function whose loss causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and stable isotope tracing experiments show that CLN3 is required for their lysosomal egress. Loss of CLN3 also alters upstream glycerophospholipid catabolism in the lysosome. Our results suggest that CLN3 is a lysosomal effluxer of GPDs and reveal Batten disease as the first, to our knowledge, neurodegenerative LSD with a primary defect in glycerophospholipid metabolism.

2022-07-11 | PXD018624 | Pride

Protection of the prodomain α1-helix correlates with latency in the transforming growth factor-β family

Project description:The 33 members of the transforming growth factor beta (TGF-β) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of non-covalently associated prodomains and growth factors (GF). Pro-complexes from a subset of family members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, particularly because of large family diversity. Here, we have examined representative family members in negative stain electron microscopy (nsEM) and hydrogen deuterium exchange (HDX) to identify features that differentiate latent from non-latent members. nsEM showed three overall pro-complex conformations that differed in prodomain arm domain orientation relative to the bound growth factor. Two cross-armed members, TGF-β1 and TGF-β2, were each latent. However, among V-armed members, GDF8 was latent whereas ActA was not. All open-armed members, BMP7, BMP9, and BMP10, were non-latent. Family members exhibited remarkably varying HDX patterns, consistent with large prodomain sequence divergence. A strong correlation emerged between latency and protection of the prodomain α1-helix from exchange. Furthermore, latency and protection from exchange correlated structurally with increased α1-helix buried surface area, hydrogen bonds, and cation-pi bonds. Moreover, a specific pattern of conserved basic and hydrophobic residues in the α1-helix and aromatic residues in the interacting fastener were found only in latent members. Thus, this first comparative survey of TGF-β family members reveals not only diversity in conformation and dynamics but also unique features that distinguish latent members.

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A Novel Spectral Annotation Strategy Streamlines Reporting of mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-gamma

Project description:Mass spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4-hour exposure to systemic IFN-gamma. HCD collision energy experiments were first performed on the Obitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization.

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Project description:The goal of this study is to identify the targets of RVE8, a MYB-like transcription factor involved in the circadian clock in Arabidopsis. Analysis of 7 days old rve8-1 RVE8::RVE8:GR and rve8-1 seedlings treated with dexamethasone or mock identified genes responsive to RVE8 induction.The RVE8 up-regulated genes are enriched for evening-phased genes while the down-regulated genes are enriched for a morning phase. This study reveals that RVE8 is a master regulator of circadian gene expression in Arabidopsis. Transgenic line rve8-1 RVE8::RVE8:GR and rve8-1 treated with DEX or mock with three biological replicates each, 12 samples in total.

2013-04-15 | E-GEOD-38879 | biostudies-arrayexpress

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