Isaya, Grazia

Abstract Friedreich’s Ataxia (FRDA) is a neuromuscular degenerative disorder caused by trinucleotide expansions in the first intron of the frataxin (FXN) gene, resulting in insufficient levels of functional FNX protein. Deficits in FXN involve mitochondrial disruptions including iron-sulfur cluster synthesis and impaired energetics. These studies were to identify unique protein-protein interactions with FXN to better understand its function and design therapeutics. Two complementary approaches were employed, BioID and Co-IP, to identify protein interactions with FXN at the direct binding, indirect binding, and non-proximal levels. Forty-one novel protein interactions were identified by BioID and IP techniques. The FXN protein landscape was further analyzed incorporating both interaction type and functional pathways using a maximum path of 6 proteins with a potential direct interaction between FXN and NFS1. Probing the intersection between FXN-protein landscape and biological pathways associated with FRDA, we identified 41 proteins of interest. Peroxiredoxin 3 (Prdx3) was chosen for further analysis because of its role in mitochondrial oxidative injury. Our data has demonstrated the strengths of employing complementary methods to identify a unique interactome for FXN. Our data provides new insights into FXN function and regulation, a potential direct interaction between FXN and NFS1, and pathway interactions between FXN and Prdx3.

Frataxin is a conserved mitochondrial protein whose deficiency causes mitochondrial iron imbalance, energy deficit, and oxidative stress, leading to the progressive neurologic and cardiac disease Friedreich ataxia. We reported previously that the mature form of human frataxin is degraded to shorter products by an unknown protease, designated frataxin degrading peptidase (FDP). We hypothesized that FDP might represent a new mechanism to modulate mitochondrial iron balance via regulation of frataxin turnover. We have now purified FDP from different tissue sources. Interestingly, the enzyme corresponds to a known mitochondrial homodimeric oxidoreductase involved in energy metabolism and antioxidant defense. The presence of proteolytic activity has been confirmed with a recombinant form of the oxidoreductase produced in E. coli . The 3D structure of the enzyme suggests the presence of a Ser‐His‐Glu catalytic triad at the homodimer interface. Indeed, the proteolytic activity is stimulated by point mutations or other conditions that destabilize the homodimer, and is inactivated by certain serine protease inhibitors. Combining oxidoreductase and proteolytic functions in the same protein may be a mechanism to integrate changes in energy metabolism and redox status with mitochondrial iron balance. [Supported by the Muscular Dystrophy Association and a pre‐doctoral fellowship from the American Heart Association (0415379Z)].

Oxidative stress and damage is considered to be a significant causative factor in aging and neurodegenerative diseases. Concurrent with the clinical phenotypes associated with these diseases is mitochondrial dysfunction in the form of biochemical malfunctions that create or potentiate reactive oxygen species (ROS) production. Dihydrolipoamide dehydrogenase (DLD) is a mitochondrial multifunctional enzyme with a homodimeric structure, resolved as two monomers bridged by an interface domain. In its dimeric form, DLD plays a critical role in energy metabolism and has been suggested to have a destructive role as a generator of ROS in its monomeric form. The latter activity has been confirmed in prior studies in both mouse and yeast models. Decreased superoxide activity has been measured in DLD knockout (‐/‐) mice vs. WT mice (Starkov et al., 2004) and higher diaphorase activity with increased hydrogen peroxide levels (Vaubel et al., 2011) has been shown in DLD mutants expressed in yeast systems. Our lab using virtual screening and computational docking has designed a small molecule library containing 40 synthetic compounds that putatively bind to a pocket within the interface domain of DLD with the aim of stabilizing the dimer. Primary screening of the compound library using the diaphorase assay is in progress. Validation of hits will be performed using Differential Scanning Fluorimetry (DSF), X‐ray crystallography and other techniques including in‐vivo analyses. We hope that these studies will enhance our understanding of the physiological role of diaphorase activity and facilitate the development of therapeutics for pathologies in which diaphorase activity has a suggested damaging function.

Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.

BACKGROUND Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. METHODS Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 μL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. RESULTS The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%–13% and 9.8%–16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2–200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12–22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1–26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. CONCLUSIONS This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.

Abstract The mitochondrion, conventionally thought to be an organelle specific to energy metabolism, is in fact multifunctional and implicated in many diseases, including cancer. To evaluate whether mitochondria-related genes are associated with increased risk for prostate cancer, we genotyped 24 single-nucleotide polymorphisms (SNP) within the mitochondrial genome and 376 tagSNPs localized to 78 nuclear-encoded mitochondrial genes. The tagSNPs were selected to achieve ≥80% coverage based on linkage disequilibrium. We compared allele and haplotype frequencies in ∼1,000 prostate cancer cases with ∼500 population controls. An association with prostate cancer was not detected for any of the SNPs within the mitochondrial genome individually or for 10 mitochondrial common haplotypes when evaluated using a global score statistic. For the nuclear-encoded genes, none of the tagSNPs were significantly associated with prostate cancer after adjusting for multiple testing. Nonetheless, we evaluated unadjusted P values by comparing our results with those from the Cancer Genetic Markers of Susceptibility (CGEMS) phase I data set. Seven tagSNPs had unadjusted P ≤ 0.05 in both our data and in CGEMS (two SNPs were identical and five were in strong linkage disequilibrium with CGEMS SNPs). These seven SNPs (rs17184211, rs4147684, rs4233367, rs2070902, rs3829037, rs7830235, and rs1203213) are located in genes MTRR, NDUFA9, NDUFS2, NDUFB9, and COX7A2, respectively. Five of the seven SNPs were further included in the CGEMS phase II study; however, none of the findings for these were replicated. Overall, these results suggest that polymorphisms in the mitochondrial genome and those in the nuclear-encoded mitochondrial genes evaluated are not substantial risk factors for prostate cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3558–66)

Boddaert and colleagues show the positive effects of selective iron chelation in young patients with Friedreich ataxia, a devastating inborn error of mitochondrial iron metabolism.

The mitochondrial enzyme, dihydrolipoamide dehydrogenase (DLD), is essential for energy metabolism across eukaryotes. Here, conditions known to destabilize the DLD homodimer enabled the mouse, pig, or human enzyme to function as a protease. A catalytic dyad (S456–E431) buried at the homodimer interface was identified. Serine protease inhibitors and an S456A or an E431A point mutation abolished the proteolytic activity, whereas other point mutations at the homodimer interface domain enhanced the proteolytic activity, causing partial or complete loss of DLD activity. In humans, mutations in the DLD homodimer interface have been linked to an atypical form of DLD deficiency. These findings reveal a previously unrecognized mechanism by which certain DLD mutations can simultaneously induce the loss of a primary metabolic activity and the gain of a moonlighting proteolytic activity. The latter could contribute to the metabolic derangement associated with DLD deficiency and represent a target for therapies of this condition.