Edgetic Perturbations Contribute to Phenotypic Variability in PEX26 Deficiency

Peroxisomes share metabolic pathways with other organelles and peroxisomes are embedded into key cellular processes. However, the specific function of many peroxisomal proteins remains unclear and restricted knowledge of the peroxisomal protein interaction network limits a precise mapping of this network into the cellular metabolism. Inborn peroxisomal disorders are autosomal or X-linked recessive diseases that affect peroxisomal biogenesis (PBD) and/or peroxisomal metabolism. Pathogenic variants in the PEX26 gene lead to peroxisomal disorders of the full Zellweger spectrum continuum. To investigate the phenotypic complexity of PEX26 deficiency, we performed a combined organelle protein interaction screen and network medicine approach and 1) analyzed whether PEX26 establishes interactions with other peroxisomal proteins, 2) deciphered the PEX26 interaction network, 3) determined how PEX26 is involved in further processes of peroxisomal biogenesis and metabolism, and 4) showed how variant-specific disruption of protein-protein interactions (edgetic perturbations) may contribute to phenotypic variability in PEX26 deficient patients. The discovery of 14 novel protein-protein interactions for PEX26 revealed a hub position of PEX26 inside the peroxisomal interactome. Analysis of edgetic perturbations of PEX26 variants revealed a strong correlation between the number of affected protein-protein interactions and the molecular phenotype of matrix protein import. The role of PEX26 in peroxisomal biogenesis was expanded encompassing matrix protein import, division and proliferation, and membrane assembly. Moreover, the PEX26 interaction network intersects with cellular lipid metabolism at different steps. The results of this study expand the knowledge about the function of PEX26 and refine genotype-phenotype correlations, which may contribute to our understanding of the underlying disease mechanism of PEX26 deficiency.

Hypothyroidism Intensifies Both Canonic and the De Novo Pathway of Peroxisomal Biogenesis in Rat Brown Adipocytes in a Time-Dependent Manner

Despite peroxisomes being important partners of mitochondria by carrying out fatty acid oxidation in brown adipocytes, no clear evidence concerning peroxisome origin and way(s) of biogenesis exists. Herein we used methimazole-induced hypothyroidism for 7, 15, and 21 days to study peroxisomal remodeling and origin in rat brown adipocytes. We found that peroxisomes originated via both canonic, and de novo pathways. Each pathway operates in euthyroid control and over the course of hypothyroidism, in a time-dependent manner. Hypothyroidism increased the peroxisomal number by 1.8-, 3.6- and 5.8-fold on days 7, 15, and 21. Peroxisomal presence, their distribution, and their degree of maturation were heterogeneous in brown adipocytes in a Harlequin-like manner, reflecting differences in their origin. The canonic pathway, through numerous dumbbell-like and “pearls on strings” structures, supported by high levels of Pex11β and Drp1, prevailed on day 7. The de novo pathway of peroxisomal biogenesis started on day 15 and became dominant by day 21. The transition of peroxisomal biogenesis from canonic to the de novo pathway was driven by increased levels of Pex19, PMP70, Pex5S, and Pex26 and characterized by numerous tubular structures. Furthermore, specific peroxisomal origin from mitochondria, regardless of thyroid status, indicates their mutual regulation in rat brown adipocytes.

Peroxisome Deficiency Dysregulates Fatty Acid Oxidization and Exacerbates Lipotoxicity in β Cells

An adverse intrauterine environment impairs the development of pancreatic islets in the fetus and leads to insufficient β cell mass and β cell dysfunction. We previously reported that Pex14, a peroxin protein involved in the biogenesis and degradation of peroxisomes, is markedly reduced in the pancreas of an intrauterine growth restriction fetus and last into adulthood. Peroxisomes function in a wide range of metabolic processes including fatty acid oxidization, ROS detoxification, and anti-inflammatory responses. To elucidate the impact of downregulation of the Pex14 gene on β cell, Pex14 was knocked down by siRNA in INS-1 cells. Pex14 knockdown disturbed peroxisomal biogenesis and dysregulated fatty acid metabolism and lipid storage capability, thereby increased ROS level and blunted insulin secretion. Moreover, Pex14 knockdown upregulated inflammation factors and regulators of endoplasmic reticulum stress. The lipotoxicity of fatty acid (including palmitic acid and linoleic acid) in β cells was exacerbated by knockdown of Pex14, as indicated by H2O2 accumulation and increased programmed cell death. The present results demonstrate the vital role of Pex14 in maintaining normal peroxisome function and β cell viability and highlight the importance of a functional peroxisomal metabolism for the detoxification of excess FAs in β cells.

Knockdown of PEX16 Induces Autophagic Degradation of Peroxisomes

Peroxisome abundance is regulated by homeostasis between the peroxisomal biogenesis and degradation processes. Peroxin 16 (PEX16) is a peroxisomal protein involved in trafficking membrane proteins for de novo peroxisome biogenesis. The present study demonstrates that PEX16 also modulates peroxisome abundance through pexophagic degradation. PEX16 knockdown in human retinal pigment epithelial-1 cells decreased peroxisome abundance and function, represented by reductions in the expression of peroxisome membrane protein ABCD3 and the levels of cholesterol and plasmalogens, respectively. The activation of pexophagy under PEX16 knockdown was shown by (i) abrogated peroxisome loss under PEX16 knockdown in autophagy-deficient ATG5 knockout cell lines, and (ii) increased autophagy flux and co-localization of p62—an autophagy adaptor protein—with ABCD3 in the presence of the autophagy inhibitor chloroquine. However, the levels of cholesterol and plasmalogens did not recover despite the restoration of peroxisome abundance following chloroquine treatment. Thus, PEX16 is indispensable for maintaining peroxisome homeostasis by regulating not only the commonly known biogenesis pathway but also the autophagic degradation of peroxisomes.

Peroxisomes Implicated in the Biosynthesis of Siderophores and Biotin, Cell Wall Integrity, Autophagy, and Response to Hydrogen Peroxide in the Citrus Pathogenic Fungus Alternaria alternata

Little is known about the roles of peroxisomes in the necrotrophic fungal plant pathogens. In the present study, a Pex6 gene encoding an ATPase-associated protein was characterized by analysis of functional mutations in the tangerine pathotype of Alternaria alternata, which produces a host-selective toxin. Peroxisomes were observed in fungal cells by expressing a mCherry fluorescent protein tagging with conserved tripeptides serine-lysing-leucine and transmission electron microscopy. The results indicated that Pex6 plays no roles in peroxisomal biogenesis but impacts protein import into peroxisomes. The number of peroxisomes was affected by nutritional conditions and H2O2, and their degradation was mediated by an autophagy-related machinery termed pexophagy. Pex6 was shown to be required for the formation of Woronin bodies, the biosynthesis of biotin, siderophores, and toxin, the uptake and accumulation of H2O2, growth, and virulence, as well as the Slt2 MAP kinase-mediated maintenance of cell wall integrity. Adding biotin, oleate, and iron in combination fully restored the growth of the pex6-deficient mutant (Δpex6), but failed to restore Δpex6 virulence to citrus. Adding purified toxin could only partially restore Δpex6 virulence even in the presence of biotin, oleate, and iron. Sensitivity assays revealed that Pex6 plays no roles in resistance to H2O2 and superoxide, but plays a negative role in resistance to 2-chloro-5-hydroxypyridine (a hydroxyl radical-generating compound), eosin Y and rose Bengal (singlet oxygen-generating compounds), and 2,3,5-triiodobenzoic acid (an auxin transport inhibitor). The diverse functions of Pex6 underscore the importance of peroxisomes in physiology, pathogenesis, and development in A. alternata.

LC-MS Based Platform Simplifies Access to Metabolomics for Peroxisomal Disorders

Peroxisomes are central hubs for cell metabolism and their dysfunction is linked to devastating human disorders, such as peroxisomal biogenesis disorders and single peroxisomal enzyme/protein deficiencies. For decades, biochemical diagnostics have been carried out using classical markers such as very long-chain fatty acids (VLCFA), which can be inconspicuous in milder and atypical cases. Holistic metabolomics studies revealed several potentially new biomarkers for peroxisomal disorders for advanced laboratory diagnostics including atypical cases. However, establishing these new markers is a major challenge in routine diagnostic laboratories. We therefore investigated whether the commercially available AbsoluteIDQ p180 kit (Biocrates Lifesciences), which utilizes flow injection and liquid chromatography mass spectrometry, may be used to reproduce some key results from previous global metabolomics studies. We applied it to serum samples from patients with mutations in peroxisomal target genes PEX1, ABCD1, and the HSD17B4 gene. Here we found various changes in sphingomyelins and lysophosphatidylcholines. In conclusion, this kit can be used to carry out extended diagnostics for peroxisomal disorders in routine laboratories, even without access to a metabolomics unit.

Peroxisomal Disorders and Their Mouse Models Point to Essential Roles of Peroxisomes for Retinal Integrity

Peroxisomes are multifunctional organelles, well known for their role in cellular lipid homeostasis. Their importance is highlighted by the life-threatening diseases caused by peroxisomal dysfunction. Importantly, most patients suffering from peroxisomal biogenesis disorders, even those with a milder disease course, present with a number of ocular symptoms, including retinopathy. Patients with a selective defect in either peroxisomal α- or β-oxidation or ether lipid synthesis also suffer from vision problems. In this review, we thoroughly discuss the ophthalmological pathology in peroxisomal disorder patients and, where possible, the corresponding animal models, with a special emphasis on the retina. In addition, we attempt to link the observed retinal phenotype to the underlying biochemical alterations. It appears that the retinal pathology is highly variable and the lack of histopathological descriptions in patients hampers the translation of the findings in the mouse models. Furthermore, it becomes clear that there are still large gaps in the current knowledge on the contribution of the different metabolic disturbances to the retinopathy, but branched chain fatty acid accumulation and impaired retinal PUFA homeostasis are likely important factors.

Proteome Analysis of USP7 Substrates Revealed Its Role in Melanoma Through PI3K/Akt/FOXO and AMPK Pathways

The ubiquitin-specific protease 7 (USP7), as a deubiquitinating enzyme, plays an important role in tumor progression by various mechanisms and serves as a potential therapeutic target. However, the functional role of USP7 in melanoma remains elusive. Here, we found that USP7 is overexpressed in human melanoma by tissue microarray. We performed TMT-based quantitative proteomic analysis to evaluate the A375 human melanoma cells treated with siRNA of USP7. Our data revealed specific proteins as well as multiple pathways and processes that are impacted by USP7. We found that the phosphatidylinositol-3-kinases/Akt (PI3K-Akt), forkhead box O (FOXO), and AMP-activated protein kinase (AMPK) signaling pathways may be closely related to USP7 expression in melanoma. Moreover, knockdown of USP7 in A375 cells, particularly USP7 knockout using CRISPR-Cas9, verified that USP7 regulates cell proliferation in vivo and in vitro. The results showed that inhibition of USP7 increases expression of the AMPK beta (PRKAB1), caspase 7(CASP7), and protein phosphatase 2 subunit B R3 isoform (PPP2R3A), while attenuating expression of C subunit of vacuolar ATPase (ATP6V0C), and peroxisomal biogenesis factor 11 beta (PEX11B). In summary, these findings reveal an important role of USP7 in regulating melanoma progression via PI3K/Akt/FOXO and AMPK signaling pathways and implicate USP7 as an attractive anticancer target for melanoma.