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

Abstract

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.

Fig. 1.

DECA (10 μM) attenuates protein translocation into mitochondria. (A) Structure of MitoBloCK-12, the chemical name of which is dequalinium chloride (DECA). (B) Isolated WT yeast mitochondria were preincubated with vehicle (1% DMSO) or DECA (2 μM, 4 μM, 10 μM) for 15 min, followed by the import of radiolabeled Su9-Ura3. Aliquots were removed after 5 min, 10 min, and 15 min, and trypsin was added to remove nonimported precursor. Samples were subsequently treated with trypsin inhibitor and were separated by SDS/PAGE and developed by autoradiography. As a negative control for import, the membrane potential was inhibited by CCCP treatment. A 10% standard is included. p, precursor; m, mature. (C) MitoDsRed (DsRed targeted to the mitochondrial matrix) was transiently expressed in CHO-K1 cells that were treated with 1% DMSO or DECA as indicated. At 24 h posttransfection, cells were fixed and stained with anti-TOMM20 antibody, and images were taken. (Scale bar: 20 μm.)

Fig. 2.

DECA (10 μM) does not decrease the membrane potential. CHO-K1 cells were treated with the indicated concentrations of DECA for 4 h. Cells were stained with the membrane potential-dependent dye MitoTracker CMXRos, fixed, and incubated with anti-TOMM20 antibody. (Scale bar: 20 μm.)

Fig. 3.

DECA treatment stops mitochondrial mislocalization of mutant AGT^P11LG170R and restores peroxisomal targeting. (A) CHO-K1 cells that were transiently transfected with AGT WT or AGT^P11LG170R constructs and then were treated with DECA or DMSO as indicated. At 48 h posttransfection, cells were fixed and stained with antibodies against AGT and mtHsp70. (B) CHO-K1 cells were transiently cotransfected with AGT WT or AGT^P11LG170R mutant and EGFP-SKL to mark peroxisomes. At 48 h posttransfection, cells were fixed and stained with anti-AGT antibody. (Scale bar: 20 μM.)

Fig. 4.

CCCP treatment blocks AGT^P11LG170R translocation into mitochondria but does not restore peroxisomal targeting. As in Fig. 3A, but cells were treated with the indicated concentration of CCCP. (Scale bar: 20 μM.)

Fig. 5.

DECA ameliorates oxalate accumulation in a PH1 model in CHO-K1 cells. CHO-K1 cells stably expressing glycolate oxidase with AGT WT (WT) or AGT^P11LG170R were treated with 2 μM DECA and 10 μM pyridoxal HCl for 4 d. After drug treatment, cells were incubated with 200 μM glycolate for 4 h, followed by oxalate measurement. The number 1 was set as the oxalate level of WT cells treated with DMSO. Average oxalate level ± SD of n = 3 trials. N.S., not significant. A paired t test was used to assess significance.