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An inter-subunit protein-protein interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin

ABSTRACT: Reptin forms multiple inter-subunit protein-protein interaction contacts. Rate-limiting motifs in ligand-dependent oligomer assembly are not defined. We set up a Reptin-self peptide scan assay to identify functional protein-protein interfaces that dominate in Reptin self assembly. The most dominant self-peptide binding mapped to the inter-subunit “rim” of the Reptin oligomer that is formed a tyrosine finger binding into an adjacent hydrophobic region in an adjacent subunit. Reptin binding to the tyrosine finger peptide motif is suppressed by Liddean or ADP suggesting that oligomer formation excludes the self-peptide from binding. HDX-mass spectrometry demonstrated that ATP suppressed deuteration at the dimer interface in both wt and Y340A Reptin. However, the Y340A mutation attenuated deuterium suppression of Reptin within the tyrosine finger in the presence of ligand. The tyrosine finger of Reptin interacts with a more shallow pocket in Pontin. Gel filtration demonstrated that the Y340A Reptin mutant does not form ADP-induced oligomers compared to the D299N mutant or wt- Reptin. The Y340A mutation shows increased AGR2 binding but decreased Pontin or p53 binding. These data indicate that the Y340 tyrosine “finger” plays an important role in stabilizing the Reptin oligomer in the presence of ligand. We propose that the Reptin interactome is regulated by ligand-dependent conversion between monomeric and oligomeric forms that is regulated by a divergent inter-subunit protein-protein interaction motif.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dominika Coufalová

LAB HEAD: Ted R. Hupp

PROVIDER: PXD008226 | Pride | 2019-04-23

REPOSITORIES: Pride

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Publications

An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin.

Coufalova Dominika D Remnant Lucy L Hernychova Lenka L Muller Petr P Healy Alan A Kannan Srinivasaraghavan S Westwood Nicholas N Verma Chandra S CS Vojtesek Borek B Hupp Ted R TR Houston Douglas R DR

Journal of proteomics 20190309

Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-peptide interface mapping to an inter-subunit "rim" of the hexamer bridged by Tyrosine-340. A Y340A mutation reduced ADP-dependent oligomer formation using a gel filtration assay, suggesting that Y340 for ...[more]

PMID: 30862565

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Project description:Collagen deposition is a key process during idiopathic pulmonary fibrosis (IPF); however, little is known about the dynamics of collagen formation during disease development. Tissue samples of early stages of human disease are not readily available and it is difficult to identify changes in collagen content, since standard collagen analysis does not distinguish between 'old' and 'new' collagen. Therefore, the current study aimed to (i) investigate the dynamics of new collagen formation in mice using bleomycin-induced lung fibrosis in which newly synthesized collagen was labelled with deuterated water and (ii) use this information to identify genes and processes correlated to new collagen formation from gene expression analysis. Lung fibrosis was induced in female C57BL/6 mice by bleomycin instillation and sacrificed. Animals were sacrificed at 1 to 5 weeks after fibrosis induction. Collagen synthesized during the week before sacrifice was labelled with deuterium by providing mice with deuterated drinking water. After sacrifice, lung tissue was collected for microarray analysis, determination of new collagen formation, and histology. Deuterated water labelling showed a strong increase in new collagen formation already during the first week after fibrosis induction and a complete return to baseline at five weeks. Correlation of new collagen formation data with gene expression data revealed fibrosis specific processes, of which proliferation was an unexpected one. This was confirmed by measuring cell proliferation and collagen synthesis simultaneously using deuterated water incorporation. Furthermore, new collagen formation strongly correlated with gene expression of e.g. elastin, tenascin C, MMP-14, lysyl oxidase, and type V collagen. These data demonstrate, using a novel combination of technologies, that proliferation and extracellular matrix production are correlated to the core process of fibrosis, i.e. the formation of new collagen. In addition, it identified genes directly correlated to fibrosis, thus providing more insight into the aetiology of IPF. Total RNA was obtained from mouse lungs at timepoint 0 as a control (n = 7) or timepoints 1 (n = 7), 2 (n = 6), 3 (n = 6), 4 (n = 6) or 5 (n = 6) weeks after bleomycin-instillation to induce lung fibrosis.

Dataset Information

An inter-subunit protein-protein interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin

Publications

An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin.

Similar Datasets

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