Cytogenetically Balanced Reciprocal Translocation Could Hide Molecular Genomic Unbalances: Implications for Foetal Phenotype Correlation.

When an increased nuchal translucency (>3.00 mm) is observed during the echographic examination of a foetus in the first trimester of pregnancy, an increased risk of chromosomopathy is considered, and the pregnant woman is offered the possibility of an invasive investigation. Here, we focused our attention on prenatal diagnosis issues in cases of foetuses with cytogenetically balanced reciprocal translocations. We report the finding of a cytogenetically balanced, de facto genomically unbalanced translocation that poses a challenge in a case of prenatal diagnosis, changing the risk of Down syndrome in a Zellweger syndromic spectrum risk (PEX3 deletion). At term, a healthy baby was born. This case teaches that prenatal diagnosis in cases of foetuses at increased risk of chromosomal abnormality imperatively requires molecular investigation in addition to a morphological karyotype.

Germ cell-specific deletion of Pex3 reveals essential roles of PEX3-dependent peroxisomes in spermiogenesis.

Peroxisomes are organelles enclosed by a single membrane and are present in various species. The abruption of peroxisomes is correlated with peroxisome biogenesis disorders and single peroxisomal enzyme deficiencies that induce diverse diseases in different organs. However, little is known about the protein compositions and corresponding roles of heterogeneous peroxisomes in various organs. Through transcriptomic and proteomic analyses, we observed heterogenous peroxisomal components among different organs, as well as between testicular somatic cells and different developmental stages of germ cells. As Pex3 is expressed in both germ cells and Sertoli cells, we generated Pex3 germ cell- and Sertoli cell-specific knockout mice. While Pex3 deletion in Sertoli cells did not affect spermatogenesis, the deletion in germ cells resulted in male sterility, manifested as the destruction of intercellular bridges between spermatids and the formation of multinucleated giant cells. Proteomic analysis of the Pex3-deleted spermatids revealed defective expressions of peroxisomal proteins and spermiogenesis-related proteins. These findings provide new insights that PEX3-dependent peroxisomes are essential for germ cells undergoing spermiogenesis, but not for Sertoli cells.

Reciprocal Regulation of Peroxisome Biogenesis and Myogenic Factors Is Critical for Myogenesis.

Mitochondria (MITO) and peroxisomes (PEXO) are the major organelles involved in the oxidative metabolism of cells, but detailed examination of their dynamics and functional adaptations during skeletal muscle (SKM) development (myogenesis) is still lacking. In this study, we found that during myogenesis, MITO DNA, ROS level, and redox ratio increased in myotubes, but the membrane potential (Δψm) and ATP content reduced, implying that the MITO efficiency might reduce during myogenesis. The PEXO number and density both increased during myogenesis, which probably resulted from the accumulation and increased biogenesis of PEXO. The expression of PEXO biogenesis factors was induced during myogenesis in vitro and in utero, and their promoters were also activated by MyoD. Knockdown of the biogenesis factors Pex3 repressed not only the PEXO density and functions but also the levels of MITO genes and functions, suggesting a close coupling between PEXO biogenesis and MITO functions. Surprisingly, Pex3 knockdown by the CRISPRi system repressed myogenic differentiation, indicating critical involvement of PEXO biogenesis in myogenesis. Taken together, these observations suggest that the dynamics and functions of both MITO and PEXO are coupled with each other and with the metabolic changes that occur during myogenesis, and these metabolic couplings are critical to myogenesis.

Hypomorphic mutation of PEX3 with peroxisomal mosaicism reveals the oscillating nature of peroxisome biogenesis coupled with differential metabolic activities.

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options.

rs9390123 and rs9399451 influence the DNA repair capacity of lung cancer by regulating PEX3 and PHACTR2­AS1 expression instead of PHACTR2.

Lung cancer is a common cancer type, and has the highest mortality rate in the world. A genome­wide association study suggests that the genetic marker rs9390123 is significantly associated with DNA repair capacity (DRC) in lung cancer. Analysis of the data derived from the 1000 Genomes Project indicated that there is another single nucleotide polymorphism (SNP), rs9399451, in strong linkage disequilibrium with rs9390123 in Caucasian individuals, thus suggesting that this SNP could be associated with DRC. However, the causal SNP and mechanism of DRC remain unclear. In the present study, dual luciferase assay results indicated that both SNPs are functional in lung cells. Through chromosome conformation capture, an enhancer containing the two functional SNPs was observed to bind the promoter of peroxisomal biogenesis factor 3 and phosphatase and actin regulator 2 antisense RNA 1 (PHACTR2­AS1). Knockdown of PHACTR2­AS1 could significantly influence lung cell proliferation, colony formation, migration and wound healing, which verified that PHACTR2­AS1 is a novel oncogene for lung cancer. Through chromatin immunoprecipitation, the transcription factor POU class 2 homeobox 1 (POU2F1) was identified to bind to the surrounding segments of these two SNPs, and their interaction was investigated. The present study identified the mechanism via which rs9390123 and rs9399451 could influence DRC.

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER.

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.

Novel Trypanocidal Inhibitors that Block Glycosome Biogenesis by Targeting PEX3-PEX19 Interaction.

Human pathogenic trypanosomatid parasites harbor a unique form of peroxisomes termed glycosomes that are essential for parasite viability. We and others previously identified and characterized the essential Trypanosoma brucei ortholog TbPEX3, which is the membrane-docking factor for the cytosolic receptor PEX19 bound to the glycosomal membrane proteins. Knockdown of TbPEX3 expression leads to mislocalization of glycosomal membrane and matrix proteins, and subsequent cell death. As an early step in glycosome biogenesis, the PEX3-PEX19 interaction is an attractive drug target. We established a high-throughput assay for TbPEX3-TbPEX19 interaction and screened a compound library for small-molecule inhibitors. Hits from the screen were further validated using an in vitro ELISA assay. We identified three compounds, which exhibit significant trypanocidal activity but show no apparent toxicity to human cells. Furthermore, we show that these compounds lead to mislocalization of glycosomal proteins, which is toxic to the trypanosomes. Moreover, NMR-based experiments indicate that the inhibitors bind to PEX3. The inhibitors interfering with glycosomal biogenesis by targeting the TbPEX3-TbPEX19 interaction serve as starting points for further optimization and anti-trypanosomal drug development.

Genome sequencing identifies a rare case of moderate Zellweger spectrum disorder caused by a PEX3 defect: Case report and literature review.

Defects in PEX3 are associated with a severe neonatal-lethal form of Zellweger spectrum disorder. We report two moderately affected siblings whose clinical and biochemical phenotypes expand the reported spectrum of PEX3-related disease. Genome sequencing of an adolescent male with progressive movement disorder, spasticity and neurodegeneration, and previous non-diagnostic plasma very-long chain fatty acid analysis, revealed a homozygous likely pathogenic missense variant in PEX3 [c.991G > A; p.(Gly331Arg)]. A younger sibling with significant motor decline since the age of three years was also subsequently found to be homozygous for the familial PEX3 variant. A comprehensive review of the scientific literature identified three additional families with non-lethal infantile- or childhood-onset PEX3-related disease, which together with this clinical report illustrate the potential for highly variable disease severity. Our findings demonstrate the diagnostic utility of genome-wide sequencing for identifying clinically and biochemically heterogeneous inherited metabolic disorders such as the peroxisome biogenesis disorders.

Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation.

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36-Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36-Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.

The AAA+ ATPase Msp1 is a processive protein translocase with robust unfoldase activity.

Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.

Pex3 is involved in the genetic regulation of Nr3c2 expression in the amygdala of mice.

The mineralocorticoid receptor (Nr3c2) has received increased attention as an important stress-related gene. Here, we sought to uncover candidate genes regulating the expression of Nr3c2. Using a genetical genomics approach, we identified a significant trans-regulated expression quantitative trait locus (eQTL) at Chromosome 10 for Nr3c2 expression in the amygdala of BXD RI strains. We then examined genes upstream of the eQTL to identify likely regulatory candidates of Nr3c2 expression. Pex3 (peroxisomal) expression was highly correlated with that of Nr3c2, had a significant cis-regulated eQTL that mapped to the Nr3c2 eQTL region and thus emerged as the most likely regulatory candidate of Nr3c2 expression. In vitro studies showed that silencing of Pex3 by siRNA decreased Nr3c2 expression in HEK293T and SHSY5 cell lines while overexpression increased Nr3c2 expression. A relationship between the expression of these two genes was further supported by our observations that expression levels of Pex3 and Nr3c2 decreased in the amygdala of mice exposed to chronic unpredictable stress. Our findings provide insight into the genetic regulation of Nr3c2 expression and suggest a new role for Pex3 in stress responses. Future characterization of Pex3's role in the regulation of Nr3c2 expression and the pathways involved may lead to a better understanding of stress responses and risk for stress-related pathology.

The peroxisome biogenesis factors Pex3 and Pex19: multitasking proteins with disputed functions.

The peroxisomal membrane protein (PMP) Pex3 and its cytosolic interaction partner Pex19 have been implicated in peroxisomal membrane biogenesis. Although these peroxins have been extensively studied, no consensus has been reached yet on how they operate. Here, we discuss two major models of their function, namely, in direct insertion of proteins into the peroxisomal membrane or in formation of PMP-containing vesicles from the endoplasmic reticulum (ER). Pex3 can also recruit other proteins to the peroxisomal membrane (e.g., Inp1, Atg30, Atg36), thereby fulfilling roles in other processes such as autophagy and organelle retention. Recent studies indicate that Pex3 and Pex19 can also facilitate sorting of certain membrane proteins to other cellular organelles, including the ER, lipid droplets, and mitochondria.

Peroxisome development in yeast is associated with the formation of Pex3-dependent peroxisome-vacuole contact sites.

Using electron and fluorescence microscopy techniques, we identified various physical contacts between peroxisomes and other cell organelles in the yeast Hansenula polymorpha. In exponential glucose-grown cells, which typically contain a single small peroxisome, contacts were only observed with the endoplasmic reticulum and the plasma membrane. Here we focus on a novel peroxisome-vacuole contact site that is formed when glucose-grown cells are shifted to methanol containing media, conditions that induce strong peroxisome development. At these conditions, the small peroxisomes rapidly increase in size, a phenomenon that is paralleled by the formation of distinct intimate contacts with the vacuole. Localization studies showed that the peroxin Pex3 accumulated in patches at the peroxisome-vacuole contact sites. In wild-type cells growing exponentially on medium containing glucose, peroxisome-vacuole contact sites were never observed. However, upon overproduction of Pex3 peroxisomes also associated to vacuoles at these growth conditions. Our observations strongly suggest a role for Pex3 in the formation of a novel peroxisome-vacuole contact site. This contact likely plays a role in membrane growth as it is formed solely at conditions of strong peroxisome expansion.

Determinants of the assembly, integrity and maintenance of the endoplasmic reticulum-peroxisome tether.

Organelle tethering and intercommunication are crucial for proper cell function. We previously described a tether between peroxisomes and the endoplasmic reticulum (ER) that acts in peroxisome population control in the yeast, Saccharomyces cerevisiae. Components of this tether are Pex3p, an integral membrane protein of both peroxisomes and the ER and Inp1p, a connector that links peroxisomes to the ER. Here, we report the analysis of random Inp1p mutants that enabled identification of regions in Inp1p required for the assembly and maintenance of the ER-peroxisome tether. Interaction analysis between Inp1p mutants and known Inp1p-binding proteins demonstrated that Pex3p and Inp1p do not constitute the sole components of the ER-peroxisome tether. Deletion of these Inp1p interactors whose steady-state localization is outside of ER-peroxisome tethers affected peroxisome dynamics. Our findings are consistent with the presence of regulatory cues that act on ER-peroxisome tethers and point to the existence of membrane contact sites between peroxisomes and organelles other than the ER.

Intracellular communication between lipid droplets and peroxisomes: the Janus face of PEX19.

In order to adapt to environmental changes, such as nutrient availability, cells have to orchestrate multiple metabolic pathways, which are catalyzed in distinct specialized organelles. Lipid droplets (LDs) and peroxisomes are both endoplasmic reticulum (ER)-derived organelles that fulfill complementary functions in lipid metabolism: Upon nutrient supply, LDs store metabolic energy in the form of neutral lipids and, when energy is needed, supply fatty acids for oxidation in peroxisomes and mitochondria. How these organelles communicate with each other for a concerted metabolic output remains a central question. Here, we summarize recent insights into the biogenesis and function of LDs and peroxisomes with emphasis on the role of PEX19 in these processes.

Pex3 and Atg37 compete to regulate the interaction between the pexophagy receptor, Atg30, and the Hrr25 kinase.

Macroautophagy/autophagy is a highly conserved process in which subcellular components destined for degradation are sequestered within autophagosomes. The selectivity of autophagy is determined by autophagy receptors, such as Pichia pastoris Atg30 (autophagy-related 30), which controls the selective degradation of peroxisomes (pexophagy) through the assembly of a receptor-protein complex (RPC). Previously, we proved that the peroxisomal acyl-CoA-binding protein, Atg37, and the highly conserved peroxin, Pex3, are required for RPC formation and efficient pexophagy. Here, we describe how Atg37 and Pex3 regulate the assembly and activation of the pexophagic RPC. We demonstrate that Atg30 requires both Atg37 and Pex3 to recruit Atg8 and Atg11 to the pexophagic RPC, because Atg37 depends on Pex3 for its localization at the peroxisomal membrane. We establish that due to close proximity of Atg37- and Pex3-binding sites in the middle domain of Atg30, the binding of these proteins to Atg30 is mutually exclusive within this region. We also show that direct binding of Pex3 and Atg37 to Atg30 regulates its phosphorylation by the Hrr25 kinase, negatively and positively, respectively. Based on these results we present a model that clarifies the assembly and activation of the pexophagic RPC through the phosphoregulation of Atg30.

Biochemical and genetic characterization of an unusual mild PEX3-related Zellweger spectrum disorder.

Patients with PEX3 mutations usually present with a severe form of Zellweger spectrum disorder with death in the first year of life. Whole exome sequencing in adult siblings with intellectual disability revealed a homozygous variant in PEX3 that abolishes the normal splice site. A cryptic acceptor splice site is activated and an in-frame transcript with a deletion is produced. This transcript translates into a protein with residual activity explaining the relatively mild peroxisomal abnormalities and clinical phenotype.

Saccharomyces cerevisiae cells lacking Pex3 contain membrane vesicles that harbor a subset of peroxisomal membrane proteins.

Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.

Assessing Pexophagy in Mammalian Cells.

In mammalian cells several hundred peroxisomes are maintained by a balance between the biogenesis and turnover by peroxisome homeostasis. Pexophagy, a form of autophagy specific for peroxisomes, is the main pathway for peroxisome degradation, but molecular mechanisms of mammalian pexophagy are largely unknown. This is due to the lack of well-established pexophagy-inducing conditions in mammalian cells. Recently, several conditions that induce pexophagy were described for mammalian cells, involving ubiquitin and adaptor proteins of autophagy. In this chapter, we describe the protocol for Pex3-induced pexophagy, the more readable and highly inducible pexophagy condition in mammalian cells.

Novel PEX3 Gene Mutations Resulting in a Moderate Zellweger Spectrum Disorder.

BACKGROUND: Peroxisome biogenesis disorders (PBDs) may have a variable clinical expression, ranging from severe, lethal to mild phenotypes with progressive evolution. PBDs are autosomal recessive disorders caused by mutations in PEX genes, which encode proteins called peroxins, involved in the assembly of the peroxisome. Patient Description: We herein report a patient who is currently 9 years old and who is compound heterozygous for two novel mutations in the PEX3 gene. RESULTS: Mild biochemical abnormalities of the peroxisomal parameters suggested a Zellweger spectrum defect in the patient. Sequence analysis of the PEX3 gene identified two novel heterozygous, pathogenic mutations. CONCLUSION: Mutations in PEX3 usually result in a severe, early lethal phenotype. We report a patient compound heterozygous for two novel mutations in the PEX3 gene, who is less affected than previously reported patients with a defect in the PEX3 gene. Our findings indicate that PEX3 defects may cause a disease spectrum similar as previously observed for other PEX gene defects.