Dataset Information

Affinity proteomics on PEX1 and PEX1-G843D in HCT116 Cells

ABSTRACT: The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 and HsPEX6 disrupt peroxisomal matrix protein import and are the leading cause of Peroxisome Biogenesis Disorders (PBDs). The most common disease-causing mutation in PEX1 is the HsPEX1G843D allele, which results in a reduction of peroxisomal protein import. Here we demonstrate that in vitro the homologous yeast mutant, ScPex1G700D, reduces the stability of Pex1’s active D2 ATPase domain and impairs assembly with Pex6, but can still form an active AAA-ATPase motor. In vivo, ScPex1G700D exhibits only a slight defect in peroxisome import. We generated model human HsPEX1G843D cell lines and show that PEX1G843D is rapidly degraded by the proteasome, but that induced overexpression of PEX1G843D can restore peroxisome import. Additionally, we found that the G843D mutation reduces PEX1’s affinity for PEX6, and that impaired assembly is sufficient to induce degradation of PEX1WT. Lastly, we found that fusing a deubiquitinase to PEX1G843D significantly hinders its degradation in mammalian cells.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colonocyte, Cell Culture

SUBMITTER: Connor Sheedy

LAB HEAD: Brooke Meghan Gardner

PROVIDER: PXD061295 | Pride | 2025-04-28

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Gardener_fragpipe_tims11292021_protxml	Other
	HCT_analysis.tdf_bin	Other
	NP1-A3_analysis.tdf_bin	Other
	NP1-WT_analysis.tdf_bin	Other
	P1C-WT_analysis.tdf_bin	Other

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Publications

PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome.

Sheedy Connor J CJ Chowdhury Soham P SP Ali Bashir A BA Miyamoto Julia J Pang Eric Z EZ Bacal Julien J Tavasoli Katherine U KU Richardson Chris D CD Gardner Brooke M BM

The Journal of biological chemistry 20250328 5

The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 and HsPEX6 disrupt peroxisomal matrix protein import and are the leading cause of peroxisome biogenesis disorders. The most common disease-causing mutation in PEX1 is the HsPEX1G843D allele, which results in a reduction of peroxisomal protein import. Here, we demonstrate that the homologous yeast mutant, ScPex1G700D, reduces the stability of Pex1's active D2 ATPase do ...[more]

PMID: 40158855

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Project description:Background: Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. Two similar matrix-degrading metalloproteases, ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each have mild cardiac anomalies, but their combined genetic inactivation is precluded by tight linkage. We coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and mechanisms in mouse cardiac development. Methods: ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows covering myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant mouse mutants. Mass spectrometry-based N-terminomics was used to identify relevant substrates. Results: Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which we also observed in mice with ADAMTS cleavage-resistant versican. Subsequently, combined knockout impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect independently of versican proteolysis. N-terminomics of combined ADAMTS knockout and wild-type hearts identified a cleaved glypican-6 peptide only in the wild-type and showed that ADAMTS1 and ADAMTS5 each cleaved glypican-6. Paradoxically, ADAMTS1 and ADAMTS5 inactivated hearts lacked glypican-6 despite unaltered Gpc6 transcription. Gpc6-/- mice demonstrated similar rotational defects as the combined ADAMTS knockout and both had reduced Hedgehog signaling. Conclusions: ADAMTS1 and ADAMTS5 ensure proper cardiac development via cleavage of distinct proteoglycans, each with independent roles in cardiac development. Whereas versican clearance in cardiac jelly is required for proper ventricular cardiomyogenesis, glypican-6 cleavage may activate/stabilize this cell-surface proteoglycan which is required for Hedgehog signaling during outflow tract development.

Project description:Aims Leveraging the tolerogenic nature of their natural clearance pathway, we aim to exploit red blood cells (RBCs) as an antigen delivery system to achieve persistent exposure to ADAMTS13-derived peptides, promoting antigen-specific attenuation of autoreactive CD4+ T cells and induction of regulatory T cells. Background Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare and potentially fatal autoimmune disease caused by a severe ADAMTS13 functional deficiency mediated by autoantibodies targeting ADAMTS13. Despite high survival rates achieved with current treatments, approximately 40% of patients experience relapses. Ensuring longer-lasting recovery by restoring immune tolerance towards ADAMTS13 remains a significant unmet need. Conclusion(s) Our strategy offers a modular and effective method for targeting antigenic peptides to RBCs for transfusion. Antigen-decorated RBCs are efficiently phagocytosed by macrophages and facilitate the presentation of FINVAPHAR on MHC II molecules, giving macrophages the ability to target ADAMTS13-specific T cells. Based on our results we propose that RBCs loaded with ADAMTS13-derived peptides containing immunodominant T cell epitopes represent a promising strategy for promoting tolerance in patients with iTTP. Methods A fusion peptide comprising the TAT cell-penetrating peptide and an immunodominant ADAMTS13-derived T cell epitope (FINVAPHAR core sequence) was designed. Binding of the fusion peptide to RBCs was monitored by flow cytometry and Imagestream. To examine whether this approach can facilitate antigen-presentation on MHC II molecules, peptide-loaded RBCs were fed to macrophages, followed by isolation of HLA-DR-peptide complexes to study the presented peptide repertoire via mass spectrometry. Results We found that the fusion peptide binds to RBCs in a concentration-dependent manner. Peptide-bound RBCs were efficiently phagocytosed by macrophages. Mass spectrometric analysis revealed that phagocytosis of peptide-loaded RBCs by macrophages results in the presentation of FINVAPHAR-containing sequences of varying lengths on MHCII molecules from HLA-DRB1*11 donors, confirming functional antigen presentation

Dataset Information

Affinity proteomics on PEX1 and PEX1-G843D in HCT116 Cells

Dataset's files

Publications

PEX1<sup>G843D</sup> remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Affinity proteomics on PEX1 and PEX1-G843D in HCT116 Cells

Dataset's files

Publications

PEX1&lt;sup&gt;G843D&lt;/sup&gt; remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

PEX1<sup>G843D</sup> remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome.