UQCRC2-related mitochondrial complex III deficiency, about 7 patients.

Isolated complex III defect is a relatively rare cause of mitochondrial disorder. New genes involved were identified in the last two decades, with only a few cases described for each deficiency. UQCRC2, which encodes ubiquinol-cytochrome c reductase core protein 2, is one of the eleven structural subunits of complex III. We report seven French patients with UQCRC2 deficiency to complete the phenotype reported so far. We highlight the similarities with neoglucogenesis defect during decompensations - hypoglycaemias, liver failure and lactic acidosis - and point out the rapid improvement with glucose fluid infusion, which is a remarkable feature for a mitochondrial disorder. Finally, we discuss the relevance of coenzyme Q10 supplementation in this defect.

Interaction of picolinamide fungicide primary metabolites UK-2A and CAS-649 with the cytochrome bc1 complex Qi site: mutation effects and modelling in Saccharomyces cerevisiae.

BACKGROUND: Fenpicoxamid and florylpicoxamid are picolinamide fungicides targeting the Qi site of the cytochrome bc1 complex, via their primary metabolites UK-2A and CAS-649, respectively. We explore binding interactions and resistance mechanisms for picolinamides, antimycin A and ilicicolin H in yeast by testing effects of cytochrome b amino acid changes on fungicide sensitivity and interpreting results using molecular docking. RESULTS: Effects of amino acid changes on sensitivity to UK-2A and CAS-649 were similar, with highest resistance associated with exchanges involving G37 and substitutions N31K and L198F. These changes, as well as K228M, also affected antimycin A, while ilicicolin H was affected by changes at G37 and L198, as well as Q22E. N31 substitution patterns suggest that a lysine at position 31 introduces an electrostatic interaction with neighbouring D229, causing disruption of a key salt-bridge interaction with picolinamides. Changes involving G37 and L198 imply resistance primarily through steric interference. G37 changes also showed differences between CAS-649 and UK-2A or antimycin A with respect to branched versus unbranched amino acids. N31K and substitution of G37 by large amino acids reduced growth rate substantially while L198 substitutions showed little effect on growth. CONCLUSION: Binding of UK-2A and CAS-649 at the Qi site involves similar interactions such that general cross-resistance between fenpicoxamid and florylpicoxamid is anticipated in target pathogens. Some resistance mutations reduced growth rate and could carry a fitness penalty in pathogens. However, certain changes involving G37 and L198 carry little or no growth penalty and may pose the greatest risk for resistance development in the field. © 2022 Society of Chemical Industry.

Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly.

The protein complexes of the mitochondrial electron transport chain exist in isolation and in higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III2+IV). The association of complexes I, III and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) potently increased respirasome assembly and activity. By-passing DHODH inhibition via uridine supplementation decreases SC assembly by altering mitochondrial phospholipid composition, specifically elevated peroxisomal-derived ether phospholipids. Cell growth rates upon DHODH inhibition depend on ether lipid synthesis and SC assembly. These data reveal that nucleotide pools signal to peroxisomes to modulate synthesis and transport of ether phospholipids to mitochondria for SC assembly, which are necessary for optimal cell growth in conditions of nucleotide limitation.

Monomeric cocoa catechins enhance ß-cell function by increasing mitochondrial respiration.

A hallmark of type 2 diabetes (T2D) is ß-cell dysfunction and the eventual loss of functional ß-cell mass. Therefore, mechanisms that improve or preserve ß-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on ß-cell function. INS-1 832/13-derived ß-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract and with oligomeric or polymeric procyanidin-rich fraction demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in ß-cell fuel utilization. Mitochondrial complex III, IV and V components are up-regulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with nuclear factor, erythroid 2 like 2 (Nrf2) nuclear localization and expression of Nrf2 target genes including nuclear respiratory factor 1 (Nrf1) and GA binding protein transcription factor alpha subunit (GABPA), essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and up-regulation of genes critical for mitochondrial respiration, glucose-stimulated insulin secretion and ultimately improved ß-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients.

A gene-environment interaction analysis of plasma selenium with prevalent and incident diabetes: The Hortega study.

BACKGROUND: Selenium and single-nucleotide-polymorphisms in selenoprotein genes have been associated to diabetes. However, the interaction of selenium with genetic variation in diabetes and oxidative stress-related genes has not been evaluated as a potential determinant of diabetes risk. METHODS: We evaluated the cross-sectional and prospective associations of plasma selenium concentrations with type 2 diabetes, and the interaction of selenium concentrations with genetic variation in candidate polymorphisms, in a representative sample of 1452 men and women aged 18-85 years from Spain. RESULTS: The geometric mean of plasma selenium levels in the study sample was 84.2µg/L. 120 participants had diabetes at baseline. Among diabetes-free participants who were not lost during the follow-up (N=1234), 75 developed diabetes over time. The multivariable adjusted odds ratios (95% confidence interval) for diabetes prevalence comparing the second and third to the first tertiles of plasma selenium levels were 1.80 (1.03, 3.14) and 1.97 (1.14, 3.41), respectively. The corresponding hazard ratios (95% CI) for diabetes incidence were 1.76 (0.96, 3.22) and 1.80 (0.98, 3.31), respectively. In addition, we observed significant interactions between selenium and polymorphisms in PPARGC1A, and in genes encoding mitochondrial proteins, such as BCS1L and SDHA, and suggestive interactions of selenium with other genes related to selenoproteins and redox metabolism. CONCLUSIONS: Plasma selenium was positively associated with prevalent and incident diabetes. While the statistical interactions of selenium with polymorphisms involved in regulation of redox and insulin signaling pathways provide biological plausibility to the positive associations of selenium with diabetes, further research is needed to elucidate the causal pathways underlying these associations.

Endogenic oxidative stress response contributes to glutathione over-accumulation in mutant Saccharomyces cerevisiae Y518.

Mechanisms of glutathione (GSH) over-accumulation in mutant Saccharomyces cerevisiae Y518 screened by ultraviolet and nitrosoguanidine-induced random mutagenesis were studied. Y518 accumulated higher levels of GSH and L-cysteine than its wild-type strain. RNA-Seq and pathway enrichment analysis indicated a difference in the expression of key genes involved in cysteine production, the GSH biosynthesis pathway, and antioxidation processes. GSH1, MET17, CYS4, GPX2, CTT1, TRX2, and SOD1 and the transcriptional activators SKN7 and YAP1 were up-regulated in the mutant. Moreover, Y518 showed a dysfunctional respiratory chain resulting from dramatically weakened activity of complex III and significant elevation of intracellular reactive oxygen species (ROS) levels. The supplementation of antimycin A in the culture of the parent strain showed equivalent changes of ROS and GSH level. This study indicates that defective complex III prompts abundant endogenic ROS generation, which triggers an oxidative stress response and upregulation of gene expression associated with GSH biosynthesis. This finding may be helpful for developing new strategies for GSH fermentation process optimization or metabolic engineering.

The perfect storm? Histiocytoid cardiomyopathy and compound CACNA2D1 and RANGRF mutation in a baby.

A female baby suffered from a rare association between histiocytoid cardiomyopathy, left ventricular non-compaction, and Wolff-Parkinson-White syndrome causing severe and recurrent arrhythmic storms. Antiarrhythmic drugs, radiofrequency ablation of Purkinje tissue, and sympathetic denervation were ineffective. The implant of a cardiac defibrillator allowed her to survive till heart transplant. Compound mutation of CACNA2D1 and RANGRF genes were found. To the best of our knowledge, this is the first comprehensive description of the concurrence of these two mutations and histiocytoid cardiomyopathy.

Tetramethylpyrazine ameliorates high glucose-induced endothelial dysfunction by increasing mitochondrial biogenesis.

Tetramethylpyrazine (TMP) is an active compound isolated from a Chinese herbal prescription that is widely used in traditional Chinese medicine for the treatment of inflammatory and cardiovascular diseases. We have previously reported that TMP acts as a potent antioxidant protecting endothelial cells against high glucose-induced damages. However, the molecular mechanism responsible for the antioxidant effect of TMP remains to be elucidated. In this study, we show that TMP increases nitric oxide production in endothelial cells and promotes endothelium-dependent relaxation in rate aortic rings. The antioxidant effect of TMP appears attributable to its ability to activate the mitochondrial biogenesis, as reflected in an up-regulation of complex III and amelioration of mitochondrial membrane potential. Furthermore, TMP is able to reverse high glucose-induced suppression of SIRT1 and the biogenesis-related factors, including PGC-1α, NRF1 and TFAM, suggesting a new molecular mechanism underlying the protective effect of TMP on the endothelium.

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis.

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

Severe renal tubulopathy in a newborn due to BCS1L gene mutation: effects of different treatment modalities on the clinical course.

Very early onset Toni-Debré-Fanconi Syndrome, a disorder of proximal renal tubules of the kidney which results in the increased urinary excretion of glucose, amino acids, uric acid, phosphate and bicarbonate, could be the manifestation of various inborn errors. Defects of oxidative phosphorylation are a heterogeneous group of disorders with various clinical presentations. Recently, patients with early liver failure, renal tubulopathy and encephalopathy due to the mutations in the BCS1L gene coding for a structural protein in mitochondrial complex III have been described. Ten-day-old female newborn was referred to our clinic because of intractable acidosis. Physical examination revealed severe hypotonia, and hepatomegaly. The laboratory examinations revealed lactic acidosis, increased blood alanine, alanine aminotransferase and aspartate aminotransferase levels, generalized aminoaciduria and glucosuria. The tubular reabsorption of phosphate was reduced. Because of multisystem involvement, mitochondrial disease was suspected and the mutational analysis of the BCS1L gene revealed homozygous P99L mutation. As the patient was unresponsive to bicarbonate replacement, oral dichloroacetate and peritoneal dialysis, continuous high dose intravenous sodium bicarbonate therapy with a dose up to 1.25 mEq/kg/h was started. The patient got on well until the age of 9 months when she died of sepsis. It was stressed that high dose intravenous continuous sodium bicarbonate therapy could be an alternative treatment option in patients with severe acidosis and renal tubulopathy resistant to dichloroacetate and peritoneal dialysis. Patients with BCS1L mutations should be considered in the differential diagnosis of severe tubulopathy in the newborn period.

Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.

In this mini review, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the "forward" and "reverse" electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302C in R. capsulatus and Y278C in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra-molecular organization of bacterial and mitochondrial respiratory chains (i.e., respirasomes) and their potential disease-associated protective roles. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Aspirin extends the lifespan of Caenorhabditis elegans via AMPK and DAF-16/FOXO in dietary restriction pathway.

Aspirin has been revealed to have many beneficial effects for health since it was discovered as a nonsteroidal anti-inflammatory drug (NSAID) to treat pain and inflammation. Here, we investigated the molecular mechanism of aspirin on the lifespan extension of Caenorhabditis elegans. Our results showed that aspirin could extend the lifespan of C. elegans, and increase its health span and stress resistance. The extension of lifespan by aspirin requires DAF-16/FOXO, AMPK, and LKB1, but not SIR-2.1. Aspirin could not extend the lifespan of the mutants of eat-2, clk-1, and isp-1. Aspirin could marginally extend the lifespan of long-live insulin-like receptor mutant daf-2(e1370) III. Taken together, aspirin might act through a dietary restriction-like mechanism, via increasing the AMP:ATP ratio and activating LKB1, subsequently activating AMPK, which stimulates DAF-16 to induce downstream effects through a DAF-16 translocation independent manner.

ORFH79 impairs mitochondrial function via interaction with a subunit of electron transport chain complex III in Honglian cytoplasmic male sterile rice.

Cytoplasmic male sterility (CMS) has attracted great interest because of its application in crop breeding. Despite increasing knowledge of CMS, not much is understood about its molecular mechanisms. Previously, orfH79 was cloned and identified as the CMS gene in Honglian rice, but how the ORFH79 protein causes pollen abortion is still unknown. Through bacterial two-hybrid library screening, P61, a subunit of the mitochondrial electron transport chain (ETC) complex III, was selected as a candidate that interacts with ORFH79. Bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation (coIP) assays verified their interaction inside mitochondria. Blue native polyacrylamide gel electrophoresis (BN-PAGE) and western blotting showed ORF79 and P61 colocalized in mitochondrial ETC complex III of CMS lines. Compared with the maintainer line, Yuetai B (YB), a significant decrease of enzyme activity was detected in mitochondrial complex III of the CMS line, Yuetai A (YA), which resulted in decreased ATP concentrations and an increase in the reactive oxygen species (ROS) content. We propose that the CMS protein, ORFH79, can bind to complex III and decrease its enzyme activity through interaction with P61. This defect results in energy production dysfunction and oxidative stress in mitochondria, which may work as retrograde signals that lead to abnormal pollen development.

Myocardial regulation of lipidomic flux by cardiolipin synthase: setting the beat for bioenergetic efficiency.

Lipidomic regulation of mitochondrial cardiolipin content and molecular species composition is a prominent regulator of bioenergetic efficiency. However, the mechanisms controlling cardiolipin metabolism during health or disease progression have remained elusive. Herein, we demonstrate that cardiac myocyte-specific transgenic expression of cardiolipin synthase results in accelerated cardiolipin lipidomic flux that impacts multiple aspects of mitochondrial bioenergetics and signaling. During the postnatal period, cardiolipin synthase transgene expression results in marked changes in the temporal maturation of cardiolipin molecular species during development. In adult myocardium, cardiolipin synthase transgene expression leads to a marked increase in symmetric tetra-18:2 molecular species without a change in total cardiolipin content. Mechanistic analysis demonstrated that these alterations result from increased cardiolipin remodeling by sequential phospholipase and transacylase/acyltransferase activities in conjunction with a decrease in phosphatidylglycerol content. Moreover, cardiolipin synthase transgene expression results in alterations in signaling metabolites, including a marked increase in the cardioprotective eicosanoid 14,15-epoxyeicosatrienoic acid. Examination of mitochondrial bioenergetic function by high resolution respirometry demonstrated that cardiolipin synthase transgene expression resulted in improved mitochondrial bioenergetic efficiency as evidenced by enhanced electron transport chain coupling using multiple substrates as well as by salutary changes in Complex III and IV activities. Furthermore, transgenic expression of cardiolipin synthase attenuated maladaptive cardiolipin remodeling and bioenergetic inefficiency in myocardium rendered diabetic by streptozotocin treatment. Collectively, these results demonstrate the unanticipated role of cardiolipin synthase in maintaining physiologic membrane structure and function even under metabolic stress, thereby identifying cardiolipin synthase as a novel therapeutic target to attenuate mitochondrial dysfunction in diabetic myocardium.

Enzymatic activities of isolated cytochrome bc1-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains.

Homodimeric structure of cytochrome bc1, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc1-like complexes containing fused cytochrome b subunits in which asymmetrically introduced mutations inactivated individual branches in various combinations. The structural asymmetry of those forms was confirmed spectroscopically. All the asymmetric forms corresponding to cytochrome bc1 with partial or full inactivation of one monomer retain high enzymatic activity but at the same time show a decrease in the maximum turnover rate by a factor close to 2. This strongly supports the model assuming independent operation of monomers. The cross-inactivated form corresponding to cytochrome bc1 with disabled complementary parts of each monomer retains the enzymatic activity at the level that, for the first time on isolated from membranes and purified to homogeneity preparations, demonstrates that intermonomer electron transfer through the bridge effectively sustains the enzymatic turnover. The results fully support the concept that electrons freely distribute between the four catalytic sites of a dimer and that any path connecting the catalytic sites on the opposite sides of the membrane is enzymatically competent. The possibility to examine enzymatic properties of isolated forms of asymmetric complexes constructed using the cytochrome b fusion system extends the array of tools available for investigating the engineering of dimeric cytochrome bc1 from the mechanistic and physiological perspectives.

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state.

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.

Novel ETF dehydrogenase mutations in a patient with mild glutaric aciduria type II and complex II-III deficiency in liver and muscle.

We describe a 22-year-old male who developed severe hypoglycemia and lethargy during an acute illness at 4 months of age and subsequently grew and developed normally. At age 4 years he developed recurrent vomiting with mild hyperammonemia and dehydration requiring frequent hospitalizations. Glutaric aciduria Type II was suspected based upon biochemical findings and managed with cornstarch, carnitine and riboflavin supplements. He did not experience metabolic crises between ages 4-12 years. He experienced recurrent vomiting, mild hyperammonemia, and generalized weakness associated with acute illnesses and growth spurts. At age 18 years, he developed exercise intolerance and proximal muscle weakness leading to the identification of multiple acyl-CoA dehydrogenase and complex II/III deficiencies in both skeletal muscle and liver. Subsequent molecular characterization of the ETFDH gene revealed novel heterozygous mutations, p.G274X:c.820 G > T (exon 7) and p.P534L: c.1601 C > T (exon 12), the latter within the iron sulfur-cluster and predicted to affect ubiquinone reductase activity of ETFDH and the docking of ETF to ETFDH. Our case supports the concept of a structural interaction between ETFDH and other enzyme partners, and suggests that the conformational change upon ETF binding to ETFDH may play a key role in linking ETFDH to II/III super-complex formation.

Molecular cloning, expression and characterization of bovine UQCC and its association with body measurement traits.

Ubiquinol-cytochrome c reductase complex chaperone (UQCC) involved in the development and maintenance of bone and cartilage is an important candidate gene for body measurement traits selection through marker-assisted selection (MAS). The expression of UQCC is upregulated in many human and animal models of height as well as other stature indexes. We have cloned the cDNA sequence coding UQCC gene in bovine. Genomic structural analysis indicated that bovine UQCC shares a high similarity with human UQCC. Furthermore, Real-Time PCR analysis show that the expression of bovine UQCC is remarkably different in diverse tissues, including high level expression in the spleen, heart and windpipe, and relatively low expression in other tissues. We also analyzed allele frequencies in different cattle breeds and an association study on the selected SNPs. SNP DraI A2691T in intron 1 and SNP Bsh1236I A3150G in intron 8 are significantly associated with Body Length (BL), Rump Length (RL), Chest Depth (CD) and Pin Bone Width (PBW). For the A2691T SNP marker, there are significant effects on the RL (p = 0.0001), CD (p = 0.0059) and PBW (p < 0.0001) in 679 individuals; with A3150G SNP marker, there are significant effects on the BL (p = 0.0047) and CD (p = 0.0454. Regarding association analysis of combination of the two SNPs, there are significant effects on the BL (p = 0.0215), CD (p = 0.0282) and PBW (p = 0.0329) in the total population. The results suggest that the UQCC gene is a candidate gene of body measurement traits in bovine reproduction and breeding, and provide data for establishing of an animal model using cattle to study big animal body type.

Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency.

Mutations in BCS1L, an assembly factor that facilitates the insertion of the catalytic Rieske Iron-Sulfur subunit into respiratory chain complex III, result in a wide variety of clinical phenotypes that range from the relatively mild Björnstad syndrome to the severe GRACILE syndrome. To better understand the pathophysiological consequences of such mutations, we studied fibroblasts from six complex III-deficient patients harboring mutations in the BCS1L gene. Cells from patients with the most severe clinical phenotypes exhibited slow growth rates in glucose medium, variable combined enzyme deficiencies, and assembly defects of respiratory chain complexes I, III, and IV, increased H(2)O(2) levels, unbalanced expression of the cellular antioxidant defenses, and apoptotic cell death. In addition, all patients showed cytosolic accumulation of the BCS1L protein, suggestive of an impaired mitochondrial import, assembly or stability defects of the BCS1L complex, fragmentation of the mitochondrial networks, and decreased MFN2 protein levels. The observed structural alterations were independent of the respiratory chain function and ROS production. Our results provide new insights into the role of pathogenic BCS1L mutations in mitochondrial function and dynamics.

In silico cloning, expression of Rieske-like apoprotein gene and protein subcellular localization in the Pacific oyster, Crassostrea gigas.

Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined, respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5'- and 3'-untranslated regions of 35 and 161 bp, respectively, with an open reading frame of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe-2S] cluster binding sites and highly matched-pair tertiary structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences, box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological function.