A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.

Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1-4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1-4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.

[Clinical characterization and genetic analysis of a newborn with chromosome 8q21.11 deletion syndrome].

OBJECTIVE: To explore the genetic etiology for a newborn with corneal opacity. METHODS: The neonate and her parents were subjected to routine G-banding chromosomal karyotyping analysis. Copy number variation (CNV) was analyzed with low-coverage whole-genome sequencing (WGS) and single nucleotide polymorphism microarray (SNP array). RESULTS: No karyotypic abnormality was found in the newborn and her parents. Low-coverage WGS has identified a de novo 5.5 Mb microdeletion at chromosome 8q21.11-q21.13 in the neonate, which encompassed the ZFHX4 and PEX2 genes. The result was confirmed by SNP array-based CNV analysis. CONCLUSION: The newborn was diagnosed with chromosome 8q21.11 deletion syndrome. ZFHX4 may be one of the key genes underlying this syndrome.

Deubiquitinating enzyme USP30 maintains basal peroxisome abundance by regulating pexophagy.

The regulation of organelle abundance is critical for cell function and survival; however, the mechanisms responsible are not fully understood. In this study, we characterize a role of the deubiquitinating enzyme USP30 in peroxisome maintenance. Peroxisomes are highly dynamic, changing in abundance in response to metabolic stress. In our recent study identifying the role of USP30 in mitophagy, we observed USP30 to be localized to punctate structures resembling peroxisomes. We report here that USP30, best known as a mitophagy regulator, is also necessary for regulating pexophagy, the selective autophagic degradation of peroxisomes. We find that overexpressing USP30 prevents pexophagy during amino acid starvation, and its depletion results in pexophagy induction under basal conditions. We demonstrate that USP30 prevents pexophagy by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2. Finally, we show that USP30 can rescue the peroxisome loss observed in some disease-causing peroxisome mutations, pointing to a potential therapeutic target.

Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse.

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.

PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation.

Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model.

A deleterious mutation in the PEX2 gene causes Zellweger syndrome in individuals of Ashkenazi Jewish descent.

Zellweger syndrome is known to be caused by numerous mutations that occur in at least 12 of the PEX genes. While phenotypes vary, many are severely debilitating, and death can result in affected newborns. Since the disease follows an autosomal recessive pattern of inheritance, carrier screening can be done for at-risk couples, but the number of potential mutations sites to screen can be daunting. Ethnicity-specific studies can help narrow this range by highlighting mutations that are present at higher percentages in certain populations. In this article, the carrier frequencies for two mutations causative of the severe Zellweger syndrome spectrum phenotype that occur in the PEX2 gene, c.355C>T and c.550del, were studied in individuals of Ashkenazi Jewish descent in order to advise on inclusion in existing carrier screening mutation panels for this population. The screening was performed for 2093 individuals through the use of TaqMan genotyping assays, real-time PCR, and allelic discrimination. Results indicated a carrier frequency of 0.813% (±0.385%) for the c.355C>T mutation and a carrier frequency of 0.00% (±0.00%) for the c.550del mutation. On the basis of these frequencies, we believe that the c.355C>T mutation should be considered for inclusion in carrier screening panels for the Ashkenazi population.

Cholesterol biosynthesis and ER stress in peroxisome deficiency.

Cholesterol biosynthesis is a multi-step process involving more than 20 enzymes in several subcellular compartments. The pre-squalene segment of the cholesterol/isoprenoid biosynthetic pathway is localized in peroxisomes. This review intends to highlight recent findings illustrating the important role peroxisomes play in cholesterol biosynthesis and maintenance of cholesterol homeostasis. Disruption of the Pex2 gene leads to peroxisome deficiency and widespread metabolic dysfunction. The Pex2(-/-) mouse model for Zellweger syndrome enabled us to evaluate the role of peroxisomes in cholesterol biosynthesis. These studies have shown that Pex2(-/-) mice exhibit low levels of cholesterol in plasma and liver. Pex2(-/-) mice were unable to maintain normal cholesterol homeostasis despite activation of SREBP-2, the master transcriptional regulator of cholesterol biosynthesis, and increased protein levels and activities of cholesterol biosynthetic enzymes. The SREBP-2 pathway remained activated even after normalization of hepatic cholesterol levels in response to bile acid feeding as well as in extrahepatic tissues and the liver of neonatal and longer surviving Pex2 mutants, where cholesterol levels were normal. Several studies have shown that endoplasmic reticulum (ER) stress can dysregulate lipid metabolism via SREBP activation independently of intracellular cholesterol concentration. We demonstrated that peroxisome deficiency activates endoplasmic reticulum stress pathways in Pex2(-/-) mice, especially the integrated stress response mediated by PERK and ATF4 signaling, and thereby leads to dysregulation of the SREBP-2 pathway. Our findings suggest that functional peroxisomes are necessary to prevent chronic ER stress and dysregulation of the endogenous sterol response pathway. The constitutive activation of ER stress pathways might contribute to organ pathology and metabolic dysfunction in peroxisomal disorder patients.

Peroxisome deficiency-induced ER stress and SREBP-2 pathway activation in the liver of newborn PEX2 knock-out mice.

Disruption of the Pex2 gene leads to peroxisome deficiency and widespread metabolic dysfunction. We previously demonstrated that peroxisomes are critical for maintaining cholesterol homeostasis, using peroxisome-deficient Pex2(-/-) mice on a hybrid Swiss Webster×129S6/SvEv (SW/129) genetic background. Peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, leading to dysregulation of the endogenous sterol response mechanism. Herein, we demonstrate a more profound dysregulation of cholesterol homeostasis in newborn Pex2(-/-) mice congenic on a 129S6/SvEv (129) genetic background, and substantial differences between newborn versus postnatal Pex2(-/-) mice in factors that activate ER stress. These differences extend to relationships between activation of genes regulated by SREBP-2 versus PPARα. The SREBP-2 pathway is induced in neonatal Pex2(-/-) livers from 129 and SW/129 strains, despite normal hepatic cholesterol levels. ER stress markers are increased in newborn 129 Pex2(-/-) livers, which occurs in the absence of hepatic steatosis or accumulation of peroxins in the ER. Moreover, the induction of SREBP-2 and ER stress pathways is independent of PPARα activation in livers of newborn 129 and SW/129 Pex2(-/-) mice. Two-week-old wild-type mice treated with the peroxisome proliferator WY-14,643 show strong induction of PPARα-regulated genes and decreased expression of SREBP-2 and its target genes, further demonstrating that SREBP-2 pathway induction is not dependent on PPARα activation. Lastly, there is no activation of either SREBP-2 or ER stress pathways in kidney and lung of newborn Pex2(-/-) mice, suggesting a parallel induction of these pathways in peroxisome-deficient mice. These findings establish novel associations between SREBP-2, ER stress and PPARα pathway inductions.

Elevation of hemopexin-like fragment of matrix metalloproteinase-2 tissue levels inhibits ischemic wound healing and angiogenesis.

OBJECTIVE: Matrix metalloproteinase-2 (MMP-2) degrades type IV collagen and enables endothelial cell (EC) migration during angiogenesis and wound healing. Peroxisomal biogenesis factor 2 (PEX2), a by-product of activated MMP-2 autocatalysis, competitively inhibits newly activated MMP-2 from EC surface binding and migration. We hypothesize that PEX2 is elevated during limb ischemia and contributes to poor wound healing, with decreased capillary density. METHODS: Western blot was used to identify PEX2 in the hind limbs of FVB/NJ mice with surgically induced ischemia. The PEX2 effect on healing was evaluated by calculating the area of exposed muscle after wounding the dorsum of mice and administering daily injections with human recombinant PEX2 (hrPEX2). Wounds were also injected with lentivirus-expressing PEX2 (PEX2-LV), harvested on postoperative day 7 and processed for staining. Epithelial gap was assessed with light microscopy. Capillary density was evaluated after wounding Tie2-green fluorescent protein (GFP)(+) transgenic FVB mice (ECs labeled green) and viral transduction with PEX2-LV. Wounds were harvested on postoperative day (POD) 7, frozen in liquid nitrogen, sectioned, and stained with Hoechst. Vessel density was assessed via fluorescence microscopy as the average number of capillaries/10 high-powered fields. Paired t test was used to assess differences between the groups. RESULTS: PEX2 was elevated 5.5 ± 2.0-fold (P = .005) on POD 2 and 2.9 ± 0.69-fold (P = .004) on POD 4 in gastrocnemius muscles of ischemic hind limbs. The wound surface area, or lack of granulation tissue and exposed muscle, decreased daily in all mice but was greater in the hrPEX2-treated mice by 12% to 16% (P < .004). Wounds in the control group were completely covered with granulation tissue by POD 3. Wounds injected with hrPEX2 were not completely covered by POD 7 but continued to have exposed muscle. Microscopic examination of wounds after PEX2-LV viral transduction demonstrated an average epithelial gap of 1.6 ± 0.3 vs 0.64 ± 0.3 µm in control wounds (P < .04). Wounds from Tie2-GFP mice had an average number of 3.8 ± 1.1 capillaries vs 6.9 ± 1.2 in control wounds (P < .007). CONCLUSIONS: Our study links elevated PEX2 to ischemia and poor wound healing. We demonstrate comparative PEX2 elevation in ischemic murine hind limbs. Less granulation tissue is produced and healing is retarded in wounds subjected to hrPEX2 or viral transduction with PEX2-LV. Microscopic examination shows the wounds exhibit fewer capillaries, supporting the hypothesis that PEX2 decreases angiogenesis.

Genome-wide association study identifies four loci associated with eruption of permanent teeth.

The sequence and timing of permanent tooth eruption is thought to be highly heritable and can have important implications for the risk of malocclusion, crowding, and periodontal disease. We conducted a genome-wide association study of number of permanent teeth erupted between age 6 and 14 years, analyzed as age-adjusted standard deviation score averaged over multiple time points, based on childhood records for 5,104 women from the Danish National Birth Cohort. Four loci showed association at P<5×10(-8) and were replicated in four independent study groups from the United States and Denmark with a total of 3,762 individuals; all combined P-values were below 10(-11). Two loci agreed with previous findings in primary tooth eruption and were also known to influence height and breast cancer, respectively. The two other loci pointed to genomic regions without any previous significant genome-wide association study results. The intronic SNP rs7924176 in ADK could be linked to gene expression in monocytes. The combined effect of the four genetic variants was most pronounced between age 10 and 12 years, where children with 6 to 8 delayed tooth eruption alleles had on average 3.5 (95% confidence interval: 2.9-4.1) fewer permanent teeth than children with 0 or 1 of these alleles.

Nonsense suppressor therapies rescue peroxisome lipid metabolism and assembly in cells from patients with specific PEX gene mutations.

Peroxisome biogenesis disorders (PBDs) are multisystemic autosomal recessive disorders resulting from mutations in PEX genes required for normal peroxisome assembly and metabolic activities. Here, we evaluated the potential effectiveness of aminoglycoside G418 (geneticin) and PTC124 (ataluren) nonsense suppression therapies for the treatment of PBD patients with disease-causing nonsense mutations. PBD patient skin fibroblasts producing stable PEX2 or PEX12 nonsense transcripts and Chinese hamster ovary (CHO) cells with a Pex2 nonsense allele all showed dramatic improvements in peroxisomal very long chain fatty acid catabolism and plasmalogen biosynthesis in response to G418 treatments. Cell imaging assays provided complementary confirmatory evidence of improved peroxisome assembly in G418-treated patient fibroblasts. In contrast, we observed no appreciable rescue of peroxisome lipid metabolism or assembly for any patient fibroblast or CHO cell culture treated with various doses of PTC124. Additionally, PTC124 did not show measurable nonsense suppression in immunoblot assays that directly evaluated the read-through of PEX7 nonsense alleles found in PBD patients with rhizomelic chondrodysplasia punctata type 1 (RCDP1). Overall, our results support the continued development of safe and effective nonsense suppressor therapies that could benefit a significant subset of individuals with PBDs. Furthermore, we suggest that the described cell culture assay systems could be useful for evaluating and screening for novel nonsense suppressor therapies.

Autosomal recessive cerebellar ataxia caused by mutations in the PEX2 gene.

OBJECTIVE: To expand the spectrum of genetic causes of autosomal recessive cerebellar ataxia (ARCA). CASE REPORT: Two brothers are described who developed progressive cerebellar ataxia at 3 1/2 and 18 years, respectively. After ruling out known common genetic causes of ARCA, analysis of blood peroxisomal markers strongly suggested a peroxisomal biogenesis disorder. Sequencing of candidate PEX genes revealed a homozygous c.865_866insA mutation in the PEX2 gene leading to a frameshift 17 codons upstream of the stop codon. PEX gene mutations usually result in a severe neurological phenotype (Zellweger spectrum disorders). CONCLUSIONS: Genetic screening of PEX2 and other PEX genes involved in peroxisomal biogenesis is warranted in children and adults with ARCA.

Peroxisome deficiency causes a complex phenotype because of hepatic SREBP/Insig dysregulation associated with endoplasmic reticulum stress.

Regulation of hepatic cholesterol biosynthesis, lipogenesis, and insulin signaling intersect at the transcriptional level by control of SREBP and Insig genes. We previously demonstrated that peroxisome-deficient PEX2-/- mice activate SREBP-2 pathways but are unable to maintain normal cholesterol homeostasis. In this study, we demonstrate that oral bile acid treatment normalized hepatic and plasma cholesterol levels and hepatic cholesterol synthesis in early postnatal PEX2 mutants, but SREBP-2 and its target gene expressions remained increased. SREBP-2 pathway induction was also observed in neonatal and longer surviving PEX2 mutants, where hepatic cholesterol levels were normal. Abnormal expression patterns for SREBP-1c and Insig-2a, and novel regulation of Insig-2b, further demonstrate that peroxisome deficiency widely affects the regulation of related metabolic pathways. We have provided the first demonstration that peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, especially the integrated stress response mediated by PERK and ATF4 signaling. Our studies suggest a mechanism whereby ER stress leads to dysregulation of the endogenous sterol response mechanism and concordantly activates oxidative stress pathways. Several metabolic derangements in peroxisome-deficient PEX2-/- liver are likely to trigger ER stress, including perturbed flux of mevalonate metabolites, altered bile acid homeostasis, changes in fatty acid levels and composition, and oxidative stress.

Identification of novel mutations in PEX2, PEX6, PEX10, PEX12, and PEX13 in Zellweger spectrum patients.

Mutations in each of the 13 identified human PEX genes are known to cause a peroxisomal biogenesis defect (PBD). Affected patients can be divided into two broad clinical spectra: the Zellweger spectrum, which accounts for about 80% of PBD patients, and the rhizomelia chondrodysplasia punctata (RCDP) spectrum. The clinical continuum of Zellweger spectrum patients extends from Zellweger syndrome (ZS) as the prototype and the most severe entity of this group to neonatal adrenoleukodystrophy (NALD) as an intermediate form and infantile Refsum (IRD) disease as the mildest variant. Characteristic features of ZS patients are dysmorphic features, severe neurological impairment, liver dysfunction, and eye and skeletal abnormalities. Similar but less severe clinical signs are seen in patients with NALD and IRD. In this study ten clinically and/or biochemically well-characterized patients with classical ZS were investigated for defects in all known human PEX genes. We identified two novel mutations in PEX2 (official symbol, PXMP3), two novel mutations in PEX6, two novel mutations in PEX10, one novel mutation in PEX12, and one novel mutation in PEX13.

Identification of trypanosomatid PEX19: functional characterization reveals impact on cell growth and glycosome size and number.

Glycosomes are peroxisome-like organelles present in trypanosomatid pathogens. These organelles compartmentalize glycolysis, among other reactions, and are essential in both bloodstream and procyclic form Trypanosoma brucei. Peroxins (PEXs) are proteins necessary for biogenesis of peroxisomes and glycosomes. In each assembled trypanosomatid genome, we identified a predicted protein with approximately 20% sequence identity to human PEX19, a protein required for insertion of peroxisomal membrane proteins (PMPs) into the membrane. Functional analysis demonstrated that these proteins are indeed PEX19 orthologues. Like other PEX19s, T. brucei and Leishmania major PEX19 GFP fusion proteins are predominantly cytosolic. We further showed that LmPEX19 interacts with the glycosomal membrane protein PEX2 in the yeast two-hybrid system. Partial knockdown of TbPEX19 slowed parasite growth, particularly when glucose was present. Immunofluorescence and electron microscopic studies revealed biogenesis defect as evidenced by a sharp reduction in the number of glycosomes. Surprisingly, a four-fold increase in the size of the remaining glycosomes was observed. We propose that this phenotype of fewer but larger glycosomes results from the reduction in import of glycosomal membrane proteins.

Analysis of human Pex19p's domain structure by pentapeptide scanning mutagenesis.

Pex19p, a primarily cytosolic protein, is essential for the biogenesis of numerous peroxisomal membrane proteins (PMPs); however, its precise function is unclear. Pex19p might function as a PMP-specific chaperone, a cycling PMP-receptor protein, a PMP membrane insertion factor, or an association/dissociation factor of membrane-associated protein complexes. Alternatively, Pex19p might act as a multifunctional peroxin and participate in a number of these activities. Here, we have employed transposon mutagenesis to generate a library of human pex19 alleles coding for Pex19p variants containing random in-frame pentapeptide insertions. A total of 87 different variants were characterized to identify functionally important regions. These studies revealed that Pex19p has a tripartite domain structure consisting of: (i) an amino-terminal domain that binds to Pex3p and is essential for docking at the peroxisome membrane; (ii) a central domain that competes with Pex5p and Pex13p for binding to Pex14p and may play a role in the assembly of PTS-receptor docking complexes; and (iii) a carboxy-terminal domain that interacts with multiple PMPs including Pex3p, Pex11pbeta, Pex12p, Pex13p, Pex16p, and Pex26p. Whether the latter interactions constitute the chaperone or transport functions (or both), remains to be determined. Finally, our observation that Pex19p contains two distinct binding sites for Pex3p suggests that the peroxin may bind PMPs in multiple places and for multiple purposes.

Novel mutations in the PEX2 gene of four unrelated patients with a peroxisome biogenesis disorder.

The peroxisome biogenesis disorders (PBDs) form a genetically and clinically heterogeneous group of disorders due to defects in at least 11 distinct genes. The prototype of this group of disorders is Zellweger syndrome (ZS) with neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) as milder variants. Common to PBDs are liver disease, variable neurodevelopmental delay, retinopathy and perceptive deafness. PBD patients belonging to complementation group 10 (CG10) have mutations in the PEX2 gene (PXMP3), which codes for a protein (PEX2) that contains two transmembrane domains and a zinc-binding domain considered to be important for its interaction with other proteins of the peroxisomal protein import machinery. We report on the identification of four PBD patients belonging to CG10. Sequence analysis of their PEX2 genes revealed 4 different mutations, 3 of which have not been reported before. Two of the patients had homozygous mutations leading to truncated proteins lacking both transmembrane domains and the zinc-binding domain. These mutations correlated well with their severe phenotypes. The third patient had a homozygous mutation leading to the absence of the zinc-binding domain (W223X) and the fourth patient had a homozygous mutation leading to the change of the second cysteine residue of the zinc-binding domain (C247R). Surprisingly, the patient lacking the domain had a mild phenotype, whereas the C247R patient had a severe phenotype. This might be due to an increased instability of PEX2 due to the R for C substitution or to a dominant negative effect on interacting proteins.