Evaluation of antihypertensive drugs in combination with enzyme replacement therapy in mice with Pompe disease.

Pompe disease is caused by the deficiency of lysosomal acid α-glucosidase (GAA) leading to progressive myopathy. Enzyme replacement therapy (ERT) with recombinant human (rh) GAA has limitations, including inefficient uptake of rhGAA in skeletal muscle linked to low cation-independent mannose-6-phosphate receptor (CI-MPR) expression. PURPOSE: To test the hypothesis that antihypertensive agents causing muscle hypertrophy by increasing insulin-like growth factor 1 expression can increase CI-MPR-mediated uptake of recombinant enzyme with therapeutic effects in skeletal muscle. METHODS: Three such agents were evaluated in mice with Pompe disease (carvedilol, losartan, and propranolol), either with or without concurrent ERT. RESULTS: Carvedilol, a selective ß-blocker, increased muscle strength but reduced biochemical correction from ERT. Administration of drugs alone had minimal effect, with the exception of losartan that increased glycogen storage and mortality either by itself or in combination with ERT. CONCLUSION: The ß-blocker carvedilol had beneficial effects during ERT in mice with Pompe disease, in comparison with propranolol or losartan. Caution is warranted when prescribing antihypertensive drugs in Pompe disease.

Novel hyperoxidation resistance motifs in 2-Cys peroxiredoxins.

2-Cys peroxiredoxins (Prxs) modulate hydrogen peroxide (H2O2)-mediated cell signaling. At high H2O2 levels, eukaryotic Prxs can be inactivated by hyperoxidation and are classified as sensitive Prxs. In contrast, prokaryotic Prxs are categorized as being resistant to hyperoxidation and lack the GGLG and C-terminal YF motifs present in the sensitive Prxs. Additional molecular determinants that account for the subtle differences in the susceptibility to hyperoxidation remain to be identified. A comparison of a new, 2.15-Å-resolution crystal structure of Prx2 in the oxidized, disulfide-bonded state with the hyperoxidized structure of Prx2 and Prx1 in complex with sulfiredoxin revealed three structural regions that rearrange during catalysis. With these regions in hand, focused sequence analyses were performed comparing sensitive and resistant Prx groups. From this combinatorial approach, we discovered two novel hyperoxidation resistance motifs, motifs A and B, which were validated using mutagenesis of sensitive human Prxs and resistant Salmonella enterica serovar Typhimurium AhpC. Introduction and removal of these motifs, respectively, resulted in drastic changes in the sensitivity to hyperoxidation with Prx1 becoming 100-fold more resistant to hyperoxidation and AhpC becoming 800-fold more sensitive to hyperoxidation. The increased sensitivity of the latter AhpC variant was also confirmed in vivo These results support the function of motifs A and B as primary drivers for tuning the sensitivity of Prxs to different levels of H2O2, thus enabling the initiation of variable signaling or antioxidant responses in cells.

Kinetic analysis of structural influences on the susceptibility of peroxiredoxins 2 and 3 to hyperoxidation.

Mammalian 2-cysteine peroxiredoxins (Prxs) are susceptible to hyperoxidation by excess H2O2. The cytoplasmic family member Prx2 hyperoxidizes more readily than mitochondrial Prx3 due to slower dimerization of the sulfenic acid (SpOH) intermediate. Four variant amino acids near the C-terminus have been shown to contribute to this difference. We have performed kinetic analysis of the relationship between hyperoxidation and disulfide formation, using whole-protein MS and comparing wild-type (WT) Prx2 and Prx3 with tail-swap mutants in which the four amino acids were reversed. These changes make Prx3 more sensitive and Prx2 less sensitive to hyperoxidation and accounted for ∼70% of the difference between the two proteins. The tail swap mutant of Prx3 was also more susceptible when expressed in the mitochondria of HeLa cells. The hyperoxidized product at lower excesses of H2O2 was a semi-hyperoxidized dimer with one active site disulfide and the other a sulfinic acid. For Prx2, increasing the H2O2 concentration resulted in complete hyperoxidation. In contrast, only approximately half the Prx3 active sites underwent hyperoxidation and, even with high H2O2, the predominant product was the hyperoxidized dimer. Size exclusion chromatography (SEC) showed that the oligomeric forms of all redox states of Prx3 dissociated more readily into dimeric units than their Prx2 counterparts. Notably the species with one disulfide and one hyperoxidized active site was decameric for Prx2 and dimeric for Prx3. Reduction and re-oxidation of the hyperoxidized dimer of Prx3 produced hyperoxidized monomers, implying dissociation and rearrangement of the subunits of the functional homodimer.

Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma.

Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2--thioredoxin 2 (TRX2)--peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.

Molecular basis for the resistance of human mitochondrial 2-Cys peroxiredoxin 3 to hyperoxidation.

Peroxiredoxins (Prxs) detoxify peroxides and modulate H2O2-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1-4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H2O2 to form Cys sulfinic acid (Cys-SO2H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H2O2 and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.

Reduction of cysteine sulfinic acid in eukaryotic, typical 2-Cys peroxiredoxins by sulfiredoxin.

The eukaryotic, typical 2-Cys peroxiredoxins (Prxs) are inactivated by hyperoxidation of one of their active-site cysteine residues to cysteine sulfinic acid. This covalent modification is thought to enable hydrogen peroxide-mediated cell signaling and to act as a functional switch between a peroxidase and a high-molecular-weight chaperone. Moreover, hyperoxidation has been implicated in a variety of disease states associated with oxidative stress, including cancer and aging-associated pathologies. A repair enzyme, sulfiredoxin (Srx), reduces the sulfinic acid moiety by using an unusual ATP-dependent mechanism. In this process, the Prx molecule undergoes dramatic structural rearrangements to facilitate repair. Structural, kinetic, mutational, and mass spectrometry-based approaches have been used to dissect the molecular basis for Srx catalysis. The available data support the direct formation of Cys sulfinic acid phosphoryl ester and protein-based thiosulfinate intermediates. This review discusses the role of Srx in the reversal of Prx hyperoxidation, the questions raised concerning the reductant required for human Srx regeneration, and the deglutathionylating activity of Srx. The complex interplay between Prx hyperoxidation, other forms of Prx covalent modification, and the oligomeric state also are discussed.

Molecular basis of reduced pyridoxine 5'-phosphate oxidase catalytic activity in neonatal epileptic encephalopathy disorder.

Mutations in pyridoxine 5'-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5'-phosphate oxidase catalyzes the oxidation of pyridoxine 5'-phosphate to pyridoxal 5'-phosphate, the active cofactor form of vitamin B(6) required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is approximately 850-fold less efficient than the wild-type enzyme due to an approximately 192-fold decrease in pyridoxine 5'-phosphate affinity and an approximately 4.5-fold decrease in catalytic activity. There is also an approximately 50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 A crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a beta-strand-loop-beta-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5'-phosphate oxidase mutations.