Integrative Omics reveals changes in the cellular landscape of yeast without peroxisomes
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ABSTRACT: Peroxisomes are organelles that are crucial for cellular metabolism. However, these organelles play also important roles in non-metabolic processes, such as signalling. To uncover the consequences of peroxisome deficiency, we compared two extremes, namely Saccharomyces cerevisiae wild-type and pex3 cells, which lack functional peroxisomes, employing transcriptomics and quantitative proteomics technology. Cells were grown on acetate, a carbon source that involves peroxisomal enzymes of the glyoxylate cycle and does not repress peroxisomal proteins. Transcripts of peroxisomal β-oxidation genes and the corresponding proteins were enhanced in pex3 cells. Peroxisome-deficiency also caused reduced levels of membrane bound peroxins, while the soluble receptors Pex5 and Pex7 were enhanced at the protein level. In addition, we observed alterations in non-peroxisomal transcripts and proteins, especially mitochondrial proteins involved in respiration or import processes. Our results not only reveal the impact of the absence of peroxisomes in yeast, but also represent a rich resource of candidate genes/proteins that are relevant in peroxisome biology.
Project description:E. histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although the set of proteins (peroxins) responsible for peroxisome biogenesis was reduced to only seven members (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0,044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living M. balamuthi and P. schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.
Project description: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.
Project description: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.
Project description:Peroxisomes are essential but often underappreciated metabolic organelles. In this study, we demonstrate that exuberant interferon signaling reshapes the macrophage peroxisome compartment following severe COVID-19. To uncover the molecular characteristics underlying the protective role of macrophage peroxisomes after viral infection, we isolated RNA from wild-type (WT) and Pex5-deficient alveolar macrophages (AMs) with or without Poly(I:C) treatment in vitro, followed by bulk RNA sequencing. Our analysis revealed enhanced expression of inflammation-associated pathways and innate immune responses in Pex5-deficient AMs. However, these cells also exhibited attenuated activation of tissue development-related pathways and impaired mitochondrial functions, regardless of Poly(I:C) treatment.
Project description:Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information.
Project description:Peroxisomes are common eukaryotic cell organelles. So far, the inventory of the yeast peroxisomal proteome is still incomplete. To identify novel peroxisomal proteins, we compared yeast wild-type (WT) and PEX3 knockout cells, which lack functional peroxisomes by whole cell proteomics and transcriptomics. Cells were grown on acetate, a carbon source that involves peroxisomal enzymes of the glyoxylate cycle. Transcriptome analysis showed that, with the exception of transcripts of β-oxidation enzymes, the transcriptome of peroxisomal proteins was largely unchanged. In contrast, whole cell proteomics analysis revealed that peroxisome deficiency results in a reduction of the levels of peroxisomal membrane proteins. A set of non-peroxisomal membrane proteins showed a similar behavior. We therefore analyzed one of these proteins, the mitochondrial pyruvate carrier protein Mpc3, and show that Mpc3 has a dual localization on peroxisomes and mitochondria. In conclusion we present a novel approach to uncover new examples of dually localized membrane proteins.
Project description:Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver diseases induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, and this increase was more pronounced than in mitochondria or the cytosol, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.
Project description:This is a pioneer genome-wide study of localization of mRNAs to peroxisomes. The findings suggest that translation of a subset of peroxiomal proteins and other cellular metabolic enzymes may be spatially regulated by directing their encoding mRNAs to the surface of peroxisomes. The study provides foundation for more detailed dissection of mechanisms of RNA targeting to subcellular compartments. In this study we performed the genome-wide transcriptome analysis of peroxisome preparations from the mouse liver using microarrays. We demonstrate that RNA is absent inside peroxisomes, however it is associated at their exterior via the noncovalent contacts with the membrane proteins. We detect enrichment of specific sets of transcripts in two preparations of peroxisomes, purified with different degrees of stringency. Importantly, among these were mRNAs encoding bona fide peroxisomal proteins, such as peroxins and peroxisomal matrix enzymes involved in beta-oxidation of fatty acids and bile acid biosynthesis.
Project description:This project sought to identify the features in PEX5 required for interaction with the peroxisomal PEX2-PEX10-PEX12 ubiquitin ligase complex. Peroxisomes equilibrated with different PEX5 mutants (i.e., C11A, AH1, AH2) were purified from Xenopus egg extract, solubilized in digitonin, and PEX5-associated material was immunoprecipitated via a C-terminal FLAG tag. PEX5 without a FLAG tag was used as a negative control. PEX5-interacting peroxins were identified by LC-MS/MS at the Taplin Mass Spectrometry Facility at Harvard Medical School.
Project description:Peroxisomes are highly abundant in proximal tubules where peroxisomal enzymes have been proposed to play an important role in a variety of metabolic and antioxidant functions. This hypothesis was supported by human genetic studies that identified mutations leading to peroxisomal biogenesis deficiency, resulting in severe multi-organ damage (Zellweger’s spectrum disorders (ZSD)), including renal impairment. However, the role of proximal tubule peroxisomes in renal (patho)physiology remains uninvestigated. We addressed this question in mice with conditional ablation of peroxisomal biogenesis in the renal tubule. Our results demonstrate that renal tubular peroxisomes are dispensable for normal renal function and suggest that renal damage in ZSD patients is of extrarenal origin.