In vitro import of peroxisome-targeting signal type 2 (PTS2) receptor Pex7p into peroxisomes

AbstractPex7p, the peroxisome-targeting signal type 2 (PTS2) receptor, transports PTS2 proteins to peroxisomes from the cytosol. We here established a cell-free Pex7p translocation system. In assays using post-nuclear supernatant fractions each from wild-type CHO-K1 and pex7 ZPG207 cells, 35S-labeled Pex7p was imported into peroxisomes. 35S-Pex7p import was also evident using rat liver peroxisomes. 35S-Pex7p was not imported into peroxisomal remnants from a pex5 ZPG231 defective in PTS2 import and pex2 Z65. When the import of 35S-Pex5pL was inhibited with an excess amount of recombinant Pex5pS, 35S-Pex7p import was concomitantly abrogated, suggesting that Pex5pL was a transporter for Pex7p, unlike a yeast cochaperone, Pex18p. 35S-Pex7p as well as 35S-Pex5p was imported in an ATP-independent manner, whilst the import of PTS1 and PTS2 cargo-proteins was ATP-dependent. Thereby, ATP-independent import of Pex7p implicated that Pex5p export requiring ATP hydrolysis is not a limiting step for its cargo recruitment to peroxisomes. PTS1 protein import was indeed insensitive to N-ethylmaleimide, whereas Pex5p export was N-ethylmaleimide-sensitive. Taken together, the cargo-protein translocation through peroxisomal membrane more likely involves another ATP-requiring step in addition to the Pex5p export. Moreover, upon concurrent import into peroxisomes, 35S-Pex5pL and 35S-Pex7p were detected at mutually distinct ratios in the immunoprecipitates each of the import machinery peroxins including Pex14p, Pex13p, and Pex2p, hence suggesting that Pex7p as well as Pex5p translocated from the initial docking complex to RING complex on peroxisomes

BAK regulates catalase release from peroxisomes

Loss of voltage-dependent anion channel 2 (VDAC2) leads to impaired peroxisome biogenesis in mammalian cells. Knockdown of BAK restores peroxisomal biogenesis in VDAC2-deficient cells, where BAK localization shifts from mitochondria to peroxisomes. Moreover, overexpression of BAK activators in wild-type cells permeabilizes peroxisomes in a BAK-dependent manner. Together, BAK most likely regulates peroxisomal membrane permeability

Pharmacologic rescue of an enzyme-trafficking defect in primary hyperoxaluria 1

Primary hyperoxaluria 1 (PH1; Online Mendelian Inheritance in Man no. 259900), a typically lethal biochemical disorder, may be caused by the AGT(P11LG170R) allele in which the alanine:glyoxylate aminotransferase (AGT) enzyme is mistargeted from peroxisomes to mitochondria. AGT contains a C-terminal peroxisomal targeting sequence, but mutations generate an N-terminal mitochondrial targeting sequence that directs AGT from peroxisomes to mitochondria. Although AGT(P11LG170R) is functional, the enzyme must be in the peroxisome to detoxify glyoxylate by conversion to alanine; in disease, amassed glyoxylate in the peroxisome is transported to the cytosol and converted to oxalate by lactate dehydrogenase, leading to kidney failure. From a chemical genetic screen, we have identified small molecules that inhibit mitochondrial protein import. We tested whether one promising candidate, Food and Drug Administration (FDA)-approved dequalinium chloride (DECA), could restore proper peroxisomal trafficking of AGT(P11LG170R). Indeed, treatment with DECA inhibited AGT(P11LG170R) translocation into mitochondria and subsequently restored trafficking to peroxisomes. Previous studies have suggested that a mitochondrial uncoupler might work in a similar manner. Although the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited AGT(P11LG170R) import into mitochondria, AGT(P11LG170R) aggregated in the cytosol, and cells subsequently died. In a cellular model system that recapitulated oxalate accumulation, exposure to DECA reduced oxalate accumulation, similar to pyridoxine treatment that works in a small subset of PH1 patients. Moreover, treatment with both DECA and pyridoxine was additive in reducing oxalate levels. Thus, repurposing the FDA-approved DECA may be a pharmacologic strategy to treat PH1 patients with mutations in AGT because an additional 75 missense mutations in AGT may also result in mistrafficking

Pharmacologic rescue of an enzyme-trafficking defect in primary hyperoxaluria 1

Primary hyperoxaluria 1 (PH1; Online Mendelian Inheritance in Man no. 259900), a typically lethal biochemical disorder, may be caused by the AGT(P11LG170R) allele in which the alanine:glyoxylate aminotransferase (AGT) enzyme is mistargeted from peroxisomes to mitochondria. AGT contains a C-terminal peroxisomal targeting sequence, but mutations generate an N-terminal mitochondrial targeting sequence that directs AGT from peroxisomes to mitochondria. Although AGT(P11LG170R) is functional, the enzyme must be in the peroxisome to detoxify glyoxylate by conversion to alanine; in disease, amassed glyoxylate in the peroxisome is transported to the cytosol and converted to oxalate by lactate dehydrogenase, leading to kidney failure. From a chemical genetic screen, we have identified small molecules that inhibit mitochondrial protein import. We tested whether one promising candidate, Food and Drug Administration (FDA)-approved dequalinium chloride (DECA), could restore proper peroxisomal trafficking of AGT(P11LG170R). Indeed, treatment with DECA inhibited AGT(P11LG170R) translocation into mitochondria and subsequently restored trafficking to peroxisomes. Previous studies have suggested that a mitochondrial uncoupler might work in a similar manner. Although the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited AGT(P11LG170R) import into mitochondria, AGT(P11LG170R) aggregated in the cytosol, and cells subsequently died. In a cellular model system that recapitulated oxalate accumulation, exposure to DECA reduced oxalate accumulation, similar to pyridoxine treatment that works in a small subset of PH1 patients. Moreover, treatment with both DECA and pyridoxine was additive in reducing oxalate levels. Thus, repurposing the FDA-approved DECA may be a pharmacologic strategy to treat PH1 patients with mutations in AGT because an additional 75 missense mutations in AGT may also result in mistrafficking

Mutations in Novel Peroxin Gene PEX26 That Cause Peroxisome-Biogenesis Disorders of Complementation Group 8 Provide a Genotype-Phenotype Correlation

The human disorders of peroxisome biogenesis (PBDs) are subdivided into 12 complementation groups (CGs). CG8 is one of the more common of these and is associated with varying phenotypes, ranging from the most severe, Zellweger syndrome (ZS), to the milder neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD). PEX26, encoding the 305-amino-acid membrane peroxin, has been shown to be deficient in CG8. We studied the PEX26 genotype in fibroblasts of eight CG8 patients—four with the ZS phenotype, two with NALD, and two with IRD. Catalase was mostly cytosolic in all these cell lines, but import of the proteins that contained PTS1, the SKL peroxisome targeting sequence, was normal. Expression of PEX26 reestablished peroxisomes in all eight cell lines, confirming that PEX26 defects are pathogenic in CG8 patients. When cells were cultured at 30°C, catalase import was restored in the cell lines from patients with the NALD and IRD phenotypes, but to a much lesser extent in those with the ZS phenotype, indicating that temperature sensitivity varied inversely with the severity of the clinical phenotype. Several types of mutations were identified, including homozygous G89R mutations in two patients with ZS. Expression of these PEX26 mutations in pex26 Chinese hamster ovary cells resulted in cell phenotypes similar to those in the human cell lines. These findings confirm that the degree of temperature sensitivity in pex26 cell lines is predictive of the clinical phenotype in patients with PEX26 deficiency

Adaptation of a Genetic Screen Reveals an Inhibitor for Mitochondrial Protein Import Component Tim44.

Diverse protein import pathways into mitochondria use translocons on the outer membrane (TOM) and inner membrane (TIM). We adapted a genetic screen, based on Ura3 mistargeting from mitochondria to the cytosol, to identify small molecules that attenuated protein import. Small molecule mitochondrial import blockers of the Carla Koehler laboratory (MB)-10 inhibited import of substrates that require the TIM23 translocon. Mutational analysis coupled with molecular docking and molecular dynamics modeling revealed that MB-10 binds to a specific pocket in the C-terminal domain of Tim44 of the protein-associated motor (PAM) complex. This region was proposed to anchor Tim44 to the membrane, but biochemical studies with MB-10 show that this region is required for binding to the translocating precursor and binding to mtHsp70 in low ATP conditions. This study also supports a direct role for the PAM complex in the import of substrates that are laterally sorted to the inner membrane, as well as the mitochondrial matrix. Thus, MB-10 is the first small molecule modulator to attenuate PAM complex activity, likely through binding to the C-terminal region of Tim44