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A genome-wide screen for peroxisome regulators connects peroxisomes with Wnt signaling through RNF146 and tankyrase

ABSTRACT: Peroxisomes are membrane-bound organelles that help cells specialize their metabolism. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. Thus, we performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that transcriptional inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), dramatically reduced the import of proteins into peroxisomes. The observed RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerase tankyrase, which binds the peroxisomal membrane protein PEX14. We propose a model in which RNF146 and tankyrase regulate peroxisome import efficiency by tuning PARsylation of proteins at the peroxisome membrane. Interestingly, we found that perturbations to peroxisomes altered tankyrase’s selection of substrates, including the beta-catenin destruction complex component AXIN1. The loss of peroxisomes caused tankyrase and RNF146-dependent degradation of AXIN1 and a concomitant increase of beta-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation, but also a novel role in bridging peroxisome function with Wnt/beta-catenin signaling during development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE266892 | GEO | 2024/06/01

REPOSITORIES: GEO

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Project description:Several proteomics studies have been performed in the past in order to define the mammalian peroxisomal proteome. During the last decade, it has as well become evident, that a significant number of proteins is shared between several subcellular compartments and that specialized proteins subsets are constituents of membrane contact zones, which bridge two adjacent organelle to allow metabolite exchange. In this respect, a number of proteins, which have been considered as mere contaminants in previous studies, might be indeed proteins, which are to a minor extent indeed truly associated with peroxisomes. Ongoing technical improvements in mass spectrometry (MS) allow to identify and to quantify the enrichment of such less abundant proteins in individual fractions. Accordingly, this study reassessed the proteome of mouse liver peroxisomes by parallel isolation of peroxisomes from a mitochondria- and a microsome-enriched pre-fraction combining density gradient centrifugation with a SWATH-MS quantitative proteomics approach to unveil novel peroxisomal or peroxisome-associated proteins. In total, 1071 proteins from the MS-samples were identified in amounts, which allowed their quantification in order to assess their enrichment in either high density peroxisomal or low-density gradient fractions, containing the bulk of organelle material. Combining the data from isolations from the mitochondria- and microsome-enriched prefractions allowed to identify specific protein clusters characteristic for mitochondria, the ER and peroxisomes. Among the proteins, which were significantly enriched in the peroxisomal cluster, were several proteins, which were not previously associated with the organelles implying that they are novel peroxisomal candidates. In order to validate the organelle proteomics strategy, five of the candidates were selected for further colocalization studies. The peroxismal import of two candidates, HTATIP2 and PAFAH2, which contain potential peroxisome targeting sequences 1, could be confirmed by overexpression in HepG2 cells. Two other candidates, SAR1B and PDCD6, which are known from ER exit sites, did not directly colocalize with peroxisomes but were found to reside at ER sites which often surrounded peroxisomes. Hence, both proteins might concentrate at peroxisome-ER membrane contact sites, which might be co-purified with peroxisomes. Intriguingly, the last candidate, OCIA domain-containing protein 1, was previously described to decrease mitochondrial branching and network formation. In this work, we not only validate its secondary peroxisomal localization but also observed a reduction in peroxisome numbers in response OCIAD1 expression. Hence, OCIAD1 appears to be a novel protein, which has an impact on both mitochondrial and peroxisomal maintenance.

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A genome-wide screen for peroxisome regulators connects peroxisomes with Wnt signaling through RNF146 and tankyrase

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