Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif.

Import of proteins into peroxisomes depends on PEX5, PEX13 and PEX14. By combining biochemical methods and structural biology, we show that the C-terminal SH3 domain of PEX13 mediates intramolecular interactions with a proximal FxxxF motif. The SH3 domain also binds WxxxF peptide motifs in the import receptor PEX5, demonstrating evolutionary conservation of such interactions from yeast to human. Strikingly, intramolecular interaction of the PEX13 FxxxF motif regulates binding of PEX5 WxxxF/Y motifs to the PEX13 SH3 domain. Crystal structures reveal how FxxxF and WxxxF/Y motifs are recognized by a non-canonical surface on the SH3 domain. The PEX13 FxxxF motif also mediates binding to PEX14. Surprisingly, the potential PxxP binding surface of the SH3 domain does not recognize PEX14 PxxP motifs, distinct from its yeast ortholog. Our data show that the dynamic network of PEX13 interactions with PEX5 and PEX14, mediated by diaromatic peptide motifs, modulates peroxisomal matrix import.

Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen.

The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.

PEX1 is essential for glycosome biogenesis and trypanosomatid parasite survival.

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.

Plastid Transient and Stable Interactions with Other Cell Compartments.

Plastids are organelles delineated by two envelopes playing important roles in different cellular processes such as energy production or lipid biosynthesis. To regulate their biogenesis and their function, plastids have to communicate with other cellular compartments. This communication can be mediated by metabolites, signaling molecules, and by the establishment of direct contacts between the plastid envelope and other organelles such as the endoplasmic reticulum, mitochondria, peroxisomes, plasma membrane, and the nucleus. These interactions are highly dynamic and respond to different biotic and abiotic stresses. However, the mechanisms involved in the formation of plastid-organelle contact sites and their functions are still far from being understood. In this chapter, we summarize our current knowledge about plastid contact sites and their role in the regulation of plastid biogenesis and function.

Integrative transcriptomic and proteomic profile revealed inhibition of oxidative phosphorylation and peroxisomes during renal interstitial fibrosis.

Effective therapies of chronic kidney disease (CKD) are lacking due to the unclear molecular pathogenesis. Previous single omics-studies have described potential molecular regulation mechanism of CKD only at the level of transcription or translation. Therefore, this study generated an integrated transcriptomic and proteomic profile to provide deep insights into the continuous transcription-translation process during CKD. The comprehensive datasets identified 14,948 transcripts and 6423 proteins, 233 up-regulated and 364 down-regulated common differentially expressed genes of transcriptome and proteome were selected to further combined bioinformatics analysis. The obtained results revealed reactive oxygen species (ROS) metabolism and antioxidant system due to imbalance of mitochondria and peroxisomes were significantly repressed in CKD. Overall, this study presents a valuable multi-omics analysis that sheds light on the molecular mechanisms underlying CKD. SIGNIFICANCE: Chronic kidney disease (CKD) is a progressive and irreversible condition that results in abnormal kidney function and structure, and is ranked 18th among the leading causes of death globally, leading to a significant societal burden. Hence, there is an urgent need for research to detect new, sensitive, and specific biomarkers. Omics-based studies offer great potential to identify underlying disease mechanisms, aid in clinical diagnosis, and develop novel treatment strategies for CKD. Previous studies have mainly focused on the regulation of gene expression or protein synthesis in CKD, thereby compelling us to conduct a meticulous analysis of transcriptomic and proteomic data from the UUO mouse model. Here, we have performed a unified analysis of CKD model by integrating transcriptomes and protein suites for the first time. Our study contributes to a deeper understanding of the pathogenesis of CKD and provides a basis for subsequent disease management and drug development.

Peroxisome deficiency underlies failures in hepatic immune cell development and antigen presentation in a severe Zellweger disease model.

Peroxisome biogenesis disorders (PBDs) represent a group of metabolic conditions that cause severe developmental defects. Peroxisomes are essential metabolic organelles, present in virtually every eukaryotic cell and mediating key processes in immunometabolism. To date, the full spectrum of PBDs remains to be identified, and the impact PBDs have on immune function is unexplored. This study presents a characterization of the hepatic immune compartment of a neonatal PBD mouse model at single-cell resolution to establish the importance and function of peroxisomes in developmental hematopoiesis. We report that hematopoietic defects are a feature in a severe PBD murine model. Finally, we identify a role for peroxisomes in the regulation of the major histocompatibility class II expression and antigen presentation to CD4+ T cells in dendritic cells. This study adds to our understanding of the mechanisms of PBDs and expands our knowledge of the role of peroxisomes in immunometabolism.

A novel peroxisome-related gene signature predicts clinical prognosis and is associated with immune microenvironment in low-grade glioma.

Low-grade glioma (LGG), a common primary tumor, mainly originates from astrocytes and oligodendrocytes. Increasing evidence has shown that peroxisomes function in the regulation of tumorigenesis and development of cancer. However, the prognostic value of peroxisome-related genes (PRGs) in LGG has not been reported. Therefore, it is necessary to construct a prognostic risk model for LGG patients based on the expression profiles of peroxisome-related genes. Our study mainly concentrated on developing a peroxisome-related gene signature for overall survival (OS) prediction in LGG patients. First, according to these peroxisome-related genes, all LGG patients from The Cancer Genome Atlas (TCGA) database could be divided into two subtypes. Univariate Cox regression analysis was used to find prognostic peroxisome-related genes in TCGA_LGG dataset, and least absolute shrinkage and selection operator Cox regression analysis was employed to establish a 14-gene signature. The risk score based on the signature was positively associated with unfavorable prognosis. Then, multivariate Cox regression incorporating additional clinical characteristics showed that the 14-gene signature was an independent predictor of LGG. Time-dependent ROC curves revealed good performance of this prognostic signature in LGG patients. The performance about predicting OS of LGG was validated using the GSE107850 dataset derived from the Gene Expression Omnibus (GEO) database. Furethermore, we constructed a nomogram model based on the gene signature and age, which showed a better prognostic power. Gene ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) analyses showed that neuroactive ligand-receptor interaction and phagosome were enriched and that the immune status was decreased in the high-risk group. Finally, cell counting kit-8 (CCK8) were used to detect cell proliferation of U251 and A172 cells. Inhibition of ATAD1 (ATPase family AAA domain-containing 1) and ACBD5 (Acyl-CoA binding-domain-containing-5) expression led to significant inhibition of U251 and A172 cell proliferation. Flow cytometry detection showed that ATAD1 and ACBD5 could induce apoptosis of U251 and A172 cells. Therefore, through bioinformatics methods and cell experiments, our study developed a new peroxisome-related gene signature that migh t help improve personalized OS prediction in LGG patients.

Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

Acetyl-CoA carboxylase 1 controls a lipid droplet-peroxisome axis and is a vulnerability of endocrine-resistant ER+ breast cancer.

Targeting aromatase deprives ER+ breast cancers of estrogens and is an effective therapeutic approach for these tumors. However, drug resistance is an unmet clinical need. Lipidomic analysis of long-term estrogen-deprived (LTED) ER+ breast cancer cells, a model of aromatase inhibitor resistance, revealed enhanced intracellular lipid storage. Functional metabolic analysis showed that lipid droplets together with peroxisomes, which we showed to be enriched and active in the LTED cells, controlled redox homeostasis and conferred metabolic adaptability to the resistant tumors. This reprogramming was controlled by acetyl-CoA-carboxylase-1 (ACC1), whose targeting selectively impaired LTED survival. However, the addition of branched- and very long-chain fatty acids reverted ACC1 inhibition, a process that was mediated by peroxisome function and redox homeostasis. The therapeutic relevance of these findings was validated in aromatase inhibitor-treated patient-derived samples. Last, targeting ACC1 reduced tumor growth of resistant patient-derived xenografts, thus identifying a targetable hub to combat the acquisition of estrogen independence in ER+ breast cancers.

High-content image screening to identify chemical modulators for peroxisome and ferroptosis.

BACKGROUND: The peroxisome is a dynamic organelle with variety in number, size, shape, and activity in different cell types and physiological states. Recent studies have implicated peroxisomal homeostasis in ferroptosis susceptibility. Here, we developed a U-2OS cell line with a fluorescent peroxisomal tag and screened a target-selective chemical library through high-content imaging analysis. METHODS: U-2OS cells stably expressing the mOrange2-Peroxisomes2 tag were generated to screen a target-selective inhibitor library. The nuclear DNA was counterstained with Hoechst 33342 for cell cycle analysis. Cellular images were recorded and quantitatively analyzed through a high-content imaging platform. The effect of selected compounds on ferroptosis induction was analyzed in combination with ferroptosis inducers (RSL3 and erastin). Flow cytometry analysis was conducted to assess the level of reactive oxygen species (ROS) and cell death events. RESULTS: Through the quantification of DNA content and peroxisomal signals in single cells, we demonstrated that peroxisomal abundance was closely linked with cell cycle progression and that peroxisomal biogenesis mainly occurred in the G1/S phase. We further identified compounds that positively and negatively regulated peroxisomal abundance without significantly affecting the cell cycle distribution. Some compounds promoted peroxisomal signals by inducing oxidative stress, while others regulated peroxisomal abundance independent of redox status. Importantly, compounds with peroxisome-enhancing activity potentiated ferroptosis induction. CONCLUSIONS: Our findings pinpoint novel cellular targets that might be involved in peroxisome homeostasis and indicate that compounds promoting peroxisomal abundance could be jointly applied with ferroptosis inducers to potentiate anticancer effect.

Genome-Wide Identification and Characterization of Tomato Fatty Acid ß-Oxidase Family Genes KAT and MFP.

Fatty acids and their derivatives play a variety of roles in living organisms. Fatty acids not only store energy but also comprise membrane lipids and act as signaling molecules. There are three main proteins involved in the fatty acid ß-oxidation pathway in plant peroxisomes, including acyl-CoA oxidase (ACX), multifunctional protein (MFP), and 3-ketolipoyl-CoA thiolase (KAT). However, genome-scale analysis of KAT and MFP has not been systemically investigated in tomatoes. Here, we conducted a bioinformatics analysis of KAT and MFP genes in tomatoes. Their physicochemical properties, protein secondary structure, subcellular localization, gene structure, phylogeny, and collinearity were also analyzed. In addition, a conserved motif analysis, an evolutionary pressure selection analysis, a cis-acting element analysis, tissue expression profiling, and a qRT-PCR analysis were conducted within tomato KAT and MFP family members. There are five KAT and four MFP family members in tomatoes, which are randomly distributed on four chromosomes. By analyzing the conserved motifs of tomato KAT and MFP family members, we found that both KAT and MFP members are highly conserved. In addition, the results of the evolutionary pressure selection analysis indicate that the KAT and MFP family members have evolved mainly from purifying selection, which makes them more structurally stable. The results of the cis-acting element analysis show that SlKAT and SlMFP with respect may respond to light, hormones, and adversity stresses. The tissue expression analysis showed that KAT and MFP family members have important roles in regulating the development of floral organs as well as fruit ripening. The qRT-PCR analysis revealed that the expressions of SlKAT and SlMFP genes can be regulated by ABA, MeJA, darkness, NaCl, PEG, UV, cold, heat, and H2O2 treatments. These results provide a basis for the involvement of the SlKAT and SlMFP genes in tomato floral organ development and abiotic stress response, which lay a foundation for future functional study of SlKAT and SlMFP in tomatoes.

Tissue-specific roles of peroxisomes revealed by expression meta-analysis.

Peroxisomes are primarily studied in the brain, kidney, and liver due to the conspicuous tissue-specific pathology of peroxisomal biogenesis disorders. In contrast, little is known about the role of peroxisomes in other tissues such as the heart. In this meta-analysis, we explore mitochondrial and peroxisomal gene expression on RNA and protein levels in the brain, heart, kidney, and liver, focusing on lipid metabolism. Further, we evaluate a potential developmental and heart region-dependent specificity of our gene set. We find marginal expression of the enzymes for peroxisomal fatty acid oxidation in cardiac tissue in comparison to the liver or cardiac mitochondrial ß-oxidation. However, the expression of peroxisome biogenesis proteins in the heart is similar to other tissues despite low levels of peroxisomal fatty acid oxidation. Strikingly, peroxisomal targeting signal type 2-containing factors and plasmalogen biosynthesis appear to play a fundamental role in explaining the essential protective and supporting functions of cardiac peroxisomes.

E3 Ligases Regulate Organelle Inheritance in Yeast.

Saccharomyces cerevisiae proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin-myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.

Protective effect of oleic acid against very long-chain fatty acid-induced apoptosis in peroxisome-deficient CHO cells.

Very long-chain fatty acids (VLCFAs) are degraded exclusively in peroxisomes, as evidenced by the accumulation of VLCFAs in patients with certain peroxisomal disorders. Although accumulation of VLCFAs is considered to be associated with health issues, including neuronal degeneration, the mechanisms underlying VLCFAs-induced tissue degeneration remain unclear. Here, we report the toxic effect of VLCFA and protective effect of C18: 1 FA in peroxisome-deficient CHO cells. We examined the cytotoxicity of saturated and monounsaturated VLCFAs with chain-length at C20-C26, and found that longer and saturated VLCFA showed potent cytotoxicity at lower accumulation levels. Furthermore, the extent of VLCFA-induced toxicity was found to be associated with a decrease in cellular C18:1 FA levels. Notably, supplementation with C18:1 FA effectively rescued the cells from VLCFA-induced apoptosis without reducing the cellular VLCFAs levels, implying that peroxisome-deficient cells can survive in the presence of accumulated VLCFA, as long as the cells keep sufficient levels of cellular C18:1 FA. These results suggest a therapeutic potential of C18:1 FA in peroxisome disease and may provide new insights into the pharmacological effect of Lorenzo's oil, a 4:1 mixture of C18:1 and C22:1 FA.

Mitochondrial-derived vesicles in metabolism, disease, and aging.

Mitochondria are central hubs of cellular metabolism and are tightly connected to signaling pathways. The dynamic plasticity of mitochondria to fuse, divide, and contact other organelles to flux metabolites is central to their function. To ensure bona fide functionality and signaling interconnectivity, diverse molecular mechanisms evolved. An ancient and long-overlooked mechanism is the generation of mitochondrial-derived vesicles (MDVs) that shuttle selected mitochondrial cargoes to target organelles. Just recently, we gained significant insight into the mechanisms and functions of MDV transport, ranging from their role in mitochondrial quality control to immune signaling, thus demonstrating unexpected and diverse physiological aspects of MDV transport. This review highlights the origin of MDVs, their biogenesis, and their cargo selection, with a specific focus on the contribution of MDV transport to signaling across cell and organ barriers. Additionally, the implications of MDVs in peroxisome biogenesis, neurodegeneration, metabolism, aging, and cancer are discussed.

Studying the topology of peroxisomal acyl-CoA synthetases using self-assembling split sfGFP.

Peroxisomes are membrane-bounded organelles that contain enzymes involved in multiple lipid metabolic pathways. Several of these pathways require (re-)activation of fatty acids to coenzyme A (CoA) esters by acyl-CoA synthetases, which may take place inside the peroxisomal lumen or extraperoxisomal. The acyl-CoA synthetases SLC27A2, SLC27A4, ACSL1, and ACSL4 have different but overlapping substrate specificities and were previously reported to be localized in the peroxisomal membrane in addition to other subcellular locations. However, it has remained unclear if the catalytic acyl-CoA synthetase sites of these enzymes are facing the peroxisomal lumen or the cytosolic side of the peroxisomal membrane. To study this topology in cellulo we have developed a microscopy-based method that uses the previously developed self-assembling split superfolder (sf) green fluorescent protein (GFP) assay. We show that this self-assembling split sfGFP method can be used to study the localization as well as the topology of membrane proteins in the peroxisomal membrane, but that it is less suited to study the location of soluble peroxisomal proteins. With the method we could demonstrate that the acyl-CoA synthetase domains of the peroxisome-bound acyl-CoA synthetases SLC27A2 and SLC27A4 are oriented toward the peroxisomal lumen and the domain of ACSL1 toward the cytosol. In contrast to previous reports, ACSL4 was not found in peroxisomes.

The peroxisome: an update on mysteries 3.0.

Peroxisomes are highly dynamic, oxidative organelles with key metabolic functions in cellular lipid metabolism, such as the ß-oxidation of fatty acids and the synthesis of myelin sheath lipids, as well as the regulation of cellular redox balance. Loss of peroxisomal functions causes severe metabolic disorders in humans. Furthermore, peroxisomes also fulfil protective roles in pathogen and viral defence and immunity, highlighting their wider significance in human health and disease. This has sparked increasing interest in peroxisome biology and their physiological functions. This review presents an update and a continuation of three previous review articles addressing the unsolved mysteries of this remarkable organelle. We continue to highlight recent discoveries, advancements, and trends in peroxisome research, and address novel findings on the metabolic functions of peroxisomes, their biogenesis, protein import, membrane dynamics and division, as well as on peroxisome-organelle membrane contact sites and organelle cooperation. Furthermore, recent insights into peroxisome organisation through super-resolution microscopy are discussed. Finally, we address new roles for peroxisomes in immune and defence mechanisms and in human disorders, and for peroxisomal functions in different cell/tissue types, in particular their contribution to organ-specific pathologies.

Establishment and validation of a novel peroxisome-related gene prognostic risk model in kidney clear cell carcinoma.

BACKGROUND: Kidney clear cell carcinoma (KIRC) is the most common subtype of renal cell carcinoma. Peroxisomes play a role in the regulation of tumorigenesis and cancer progression, yet the prognostic significance of peroxisome-related genes (PRGs) remains rarely studied. The study aimed to establish a novel prognostic risk model and identify potential biomarkers in KIRC. METHODS: The significant prognostic PRGs were screened through differential and Cox regression analyses, and LASSO Cox regression analysis was performed to establish a prognostic risk model in the training cohort, which was validated internally in the testing and entire cohorts, and further assessed in the GSE22541 cohort. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to explore the function and pathway differences between the high-risk and low-risk groups. The relationship between risk score and immune cell infiltration levels was evaluated in the CIBERSORT, ESTIMATE and TIMER databases. Finally, potential biomarkers were identified and validated from model genes, using immunohistochemistry. RESULTS: Fourteen significant prognostic PRGs were identified using multiple analyses, and 9 genes (ABCD1, ACAD11, ACAT1, AGXT, DAO, EPHX2, FNDC5, HAO1, and HNGCLL1) were obtained to establish a prognostic model via LASSO Cox regression analysis. Combining the risk score with clinical factors to construct a nomogram, which provided support for personalized treatment protocols for KIRC patients. GO and KEGG analyses highlighted associations with substance metabolism, transport, and the PPAR signaling pathways. Tumor immune infiltration indicated immune suppression in the high-risk group, accompanied by higher tumor purity and the expression of 9 model genes was positively correlated with the level of immune cell infiltration. ACAT1 has superior prognostic capabilities in predicting the outcomes of KIRC patients. CONCLUSIONS: The peroxisome-related prognostic risk model could better predict prognosis in KIRC patients.

Peroxisomal hydrogen peroxide signaling: A new chapter in intracellular communication research.

Hydrogen peroxide (H2O2), a natural metabolite commonly found in aerobic organisms, plays a crucial role in numerous cellular signaling processes. One of the key organelles involved in the cell's metabolism of H2O2 is the peroxisome. In this review, we first provide a concise overview of the current understanding of H2O2 as a molecular messenger in thiol redox signaling, along with the role of peroxisomes as guardians and modulators of cellular H2O2 balance. Next, we direct our focus toward the recently identified primary protein targets of H2O2 originating from peroxisomes, emphasizing their importance in unraveling the complex interplay between peroxisomal H2O2 and cell signaling. We specifically focus on three areas: signaling through peroxiredoxin redox relay complexes, calcium signaling, and phospho-signaling. Finally, we highlight key research directions that warrant further investigation to enhance our comprehension of the molecular and biochemical mechanisms linking alterations in peroxisomal H2O2 metabolism with disease.

Analysis of the Mouse Hepatic Peroxisome Proteome-Identification of Novel Protein Constituents Using a Semi-Quantitative SWATH-MS Approach.

Ongoing technical and bioinformatics improvements in mass spectrometry (MS) allow for the identifying and quantifying of the enrichment of increasingly less-abundant proteins in individual fractions. Accordingly, this study reassessed the proteome of mouse liver peroxisomes by the parallel isolation of peroxisomes from a mitochondria- and a microsome-enriched prefraction, combining density-gradient centrifugation with a semi-quantitative SWATH-MS proteomics approach to unveil novel peroxisomal or peroxisome-associated proteins. In total, 1071 proteins were identified using MS and assessed in terms of their distribution in either high-density peroxisomal or low-density gradient fractions, containing the bulk of organelle material. Combining the data from both fractionation approaches allowed for the identification of specific protein profiles characteristic of mitochondria, the ER and peroxisomes. Among the proteins significantly enriched in the peroxisomal cluster were several novel peroxisomal candidates. Five of those were validated by colocalization in peroxisomes, using confocal microscopy. The peroxisomal import of HTATIP2 and PAFAH2, which contain a peroxisome-targeting sequence 1 (PTS1), could be confirmed by overexpression in HepG2 cells. The candidates SAR1B and PDCD6, which are known ER-exit-site proteins, did not directly colocalize with peroxisomes, but resided at ER sites, which frequently surrounded peroxisomes. Hence, both proteins might concentrate at presumably co-purified peroxisome-ER membrane contacts. Intriguingly, the fifth candidate, OCIA domain-containing protein 1, was previously described as decreasing mitochondrial network formation. In this work, we confirmed its peroxisomal localization and further observed a reduction in peroxisome numbers in response to OCIAD1 overexpression. Hence, OCIAD1 appears to be a novel protein, which has an impact on both mitochondrial and peroxisomal maintenance.