Rapid determination of tricarboxylic acid cycle enzyme activities in biological samples

<p>Abstract</p> <p>Background</p> <p>In the last ten years, deficiencies in tricarboxylic acid cycle (TCAC) enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. A prerequisite to the identification of disease-causing TCAC enzyme deficiencies is the availability of effective enzyme assays.</p> <p>Results</p> <p>We developed three assays that measure the full set of TCAC enzymes. One assay relies on the sequential addition of reagents to measure succinyl-CoA ligase activity, followed by succinate dehydrogenase, fumarase and, finally, malate dehydrogenase. Another assay measures the activity of α-ketoglutarate dehydrogenase followed by aconitase and isocitrate dehydrogenase. The remaining assay measures citrate synthase activity using a standard procedure. We used these assays successfully on extracts of small numbers of human cells displaying various severe or partial TCAC deficiencies and on frozen heart homogenates from heterozygous mice harboring an SDHB gene deletion.</p> <p>Conclusion</p> <p>This set of assays is rapid and simple to use and can immediately detect even partial defects, as the activity of each enzyme can be readily compared with one or more other activities measured in the same sample.</p

Deferiprone targets aconitase: Implication for Friedreich's ataxia treatment

<p>Abstract</p> <p>Background</p> <p>Friedreich ataxia is a neurological disease originating from an iron-sulfur cluster enzyme deficiency due to impaired iron handling in the mitochondrion, aconitase being particularly affected. As a mean to counteract disease progression, it has been suggested to chelate free mitochondrial iron. Recent years have witnessed a renewed interest in this strategy because of availability of deferiprone, a chelator preferentially targeting mitochondrial iron.</p> <p>Method</p> <p>Control and Friedreich's ataxia patient cultured skin fibroblasts, frataxin-depleted neuroblastoma-derived cells (SK-N-AS) were studied for their response to iron chelation, with a particular attention paid to iron-sensitive aconitase activity.</p> <p>Results</p> <p>We found that a direct consequence of chelating mitochondrial free iron in various cell systems is a concentration and time dependent loss of aconitase activity. Impairing aconitase activity was shown to precede decreased cell proliferation.</p> <p>Conclusion</p> <p>We conclude that, if chelating excessive mitochondrial iron may be beneficial at some stage of the disease, great attention should be paid to not fully deplete mitochondrial iron store in order to avoid undesirable consequences.</p

Impaired Nuclear Nrf2 Translocation Undermines the Oxidative Stress Response in Friedreich Ataxia

BACKGROUND: Friedreich ataxia originates from a decrease in mitochondrial frataxin, which causes the death of a subset of neurons. The biochemical hallmarks of the disease include low activity of the iron sulfur cluster-containing proteins (ISP) and impairment of antioxidant defense mechanisms that may play a major role in disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We thus investigated signaling pathways involved in antioxidant defense mechanisms. We showed that cultured fibroblasts from patients with Friedreich ataxia exhibited hypersensitivity to oxidative insults because of an impairment in the Nrf2 signaling pathway, which led to faulty induction of antioxidant enzymes. This impairment originated from previously reported actin remodeling by hydrogen peroxide. CONCLUSIONS/SIGNIFICANCE: Thus, the defective machinery for ISP synthesis by causing mitochondrial iron dysmetabolism increases hydrogen peroxide production that accounts for the increased susceptibility to oxidative stress

The Variability of the Harlequin Mouse Phenotype Resembles that of Human Mitochondrial-Complex I-Deficiency Syndromes

Background: Despite the considerable progress made in understanding the molecular bases of mitochondrial diseases, no effective treatments have been developed to date. Faithful animal models would be extremely helpful for designing such treatments. We showed previously that the Harlequin mouse phenotype was due to a specific mitochondrial complex I deficiency resulting from the loss of the Apoptosis Inducing Factor (Aif) protein. Methodology/Principal Findings: Here, we conducted a detailed evaluation of the Harlequin mouse phenotype, including the biochemical abnormalities in various tissues. We observed highly variable disease expression considering both severity and time course progression. In each tissue, abnormalities correlated with the residual amount of the respiratory chain complex I 20 kDa subunit, rather than with residual Aif protein. Antioxidant enzyme activities were normal except in skeletal muscle, where they were moderately elevated. Conclusions/Significance: Thus, the Harlequin mouse phenotype appears to result from mitochondrial respiratory chain complex I deficiency. Its features resemble those of human complex I deficiency syndromes. The Harlequin mouse hold

Integrity of the Saccharomyces cerevisiae Rpn11 protein is critical for formation of proteasome storage granules (PSG) and survival in stationary phase.

Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process

CCDC90A (MCUR1) Is a Cytochrome c Oxidase Assembly Factor and Not a Regulator of the Mitochondrial Calcium Uniporter

SummaryMitochondrial calcium is an important modulator of cellular metabolism. CCDC90A was reported to be a regulator of the mitochondrial calcium uniporter (MCU) complex, a selective channel that controls mitochondrial calcium uptake, and hence was renamed MCUR1. Here we show that suppression of CCDC90A in human fibroblasts produces a specific cytochrome c oxidase (COX) assembly defect, resulting in decreased mitochondrial membrane potential and reduced mitochondrial calcium uptake capacity. Fibroblasts from patients with COX assembly defects due to mutations in TACO1 or COX10 also showed reduced mitochondrial membrane potential and impaired calcium uptake capacity, both of which were rescued by expression of the respective wild-type cDNAs. Deletion of fmp32, a homolog of CCDC90A in Saccharomyces cerevisiae, an organism that lacks an MCU, also produces a COX deficiency, demonstrating that the function of CCDC90A is evolutionarily conserved. We conclude that CCDC90A plays a role in COX assembly and does not directly regulate MCU

Rpn5-GFP localization in proteasome assembly defect mutant cells in exponential and stationary growth phases.

<p>Wild type, <i>Δump1</i>, <i>Δrpn10</i> and <i>Δspg5</i> cells expressing Rpn5-GFP were grown in glucose containing rich medium (YPD) at 26°C and examined by fluorescence microscopy in the exponential growth phase (EP) and after 5 days in stationary phase (SP). Typical images of Rpn5-GFP localization are shown.</p

Proteasome subunits localization in exponential and stationary growth phases.

<p>Wild type, <i>rpn11-m1</i> and <i>rpn11-m5</i> cells expressing Rpn5-GFP (W303), Rpn1-GFP (W303) or Pre6-GFP (BY4741) were grown in glucose and adenine containing rich medium (YPDA) at 26°C and examined by fluorescence microscopy in the exponential growth phase (EP) and after 5 days in stationary phase (SP). Typical images of each subunit fused to GFP localization are shown. (CP/20S Core Particle).</p

Localization of Rpn5-GFP in proteasome mutants defective in 26S assembly/stability.

<p>(<b>A</b>) Wild type, <i>rpn11-m1</i> and <i>rpn11-m5</i> cells expressing Rpn5-GFP were grown in YPDA medium at 26°C during 8 days. For each time point (day), the OD_{600 nm} was monitored, the survival rate performed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070357#pone.0070357.s001" target="_blank">Figure S1</a>) and the localization of Rpn5-GFP fluorescence was scored as nuclear (blue bar), at the nuclear periphery (red bar) or as cytosolic dots (green bar; n>100 cells for each time point; two independent experiments; error bars report the differences between the two experiments). (*) indicate that the differences in the distribution of the Rpn5-GFP signal in the mutant cells are significant relative to the wild-type cells after statistical analyses (Pearson’s chi-squared test, P values <0.05). (<b>B</b>) Wild type and <i>rpn11-m5</i> cells producing Rpn5-GFP were grown in YPDA medium at 26°C during 7 days. Localization of Rpn5-GFP was scored as in (A) but every day from day 1 to day 5 and at day 7. (<b>C</b>) Comparison of Rpn5-GFP-cytosolic foci apparition between the wild type (grey) and the <i>rpn11-m5</i> mutant (black) for each day. Error bars represent the difference observed between the two experiments and (*) indicates that the difference between the two strains is significant (Fisher’s exact test, P values <0.05).</p