Dataset Information

Proteomic analysis of highly purified bovine F0F1 ATP Synthase

ABSTRACT: We performed a proteomic analysis of highly purified bovine F0F1 ATP synthase to assess the quality of the purification procedure adopted to isolate the protein complex and identify possible contaminant proteins.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Heart

SUBMITTER: Giorgio Arrigoni

LAB HEAD: Giorgio Arrigoni

PROVIDER: PXD015108 | Pride | 2019-09-25

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	18Jun2018_ATP_synthase_Replicate_1.raw	Raw
	18Jun2018_ATP_synthase_Replicate_2.raw	Raw
	19Jul2018_ATPSynthase_Replicate_3.raw	Raw
	19Jul2018_ATPSynthase_Replicate_4.raw	Raw
	19Jul2018_ATPSynthase_Replicate_5.raw	Raw

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Publications

Purified F-ATP synthase forms a Ca2+-dependent high-conductance channel matching the mitochondrial permeability transition pore.

Urbani Andrea A Giorgio Valentina V Carrer Andrea A Franchin Cinzia C Arrigoni Giorgio G Jiko Chimari C Abe Kazuhiro K Maeda Shintaro S Shinzawa-Itoh Kyoko K Bogers Janna F M JFM McMillan Duncan G G DGG Gerle Christoph C Szabò Ildikò I Bernardi Paolo P

Nature communications 20190925 1

The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversib ...[more]

PMID: 31554800

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Project description:Purpose: Core 3 derived glycans, a major type of O-glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy, because of loss of expression of functional β3-N-acetylglucosaminyltransferase-6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re-expressing core 3 synthase in pancreatic cancer cells that had lost expression. Experimental Design: We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. We therefore re-expressed in human pancreatic cancer cells (Capan-2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Results: Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared with vector control cells. Expression of core 3 O-glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the down regulation of expression of several genes including REG1α and FGFR3, and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. Conclusions: These findings indicate that expression of core 3 derived O-glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins. Two-condition experiment, Core 3 synthase stable expression (C3) vs. vector control (PLVX) cells. Biological replicates: 3 Core 3 synthase stable expression, 3 vector control, independently grown and harvested. One replicate per array.

Project description:The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o) sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.

Dataset Information

Proteomic analysis of highly purified bovine F0F1 ATP Synthase

Dataset's files

Publications

Purified F-ATP synthase forms a Ca<sup>2+</sup>-dependent high-conductance channel matching the mitochondrial permeability transition pore.

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