The SLC25 Carrier Family: Important Transport Proteins in Mitochondrial Physiology and Pathology.

Members of the mitochondrial carrier family (SLC25) transport a variety of compounds across the inner membrane of mitochondria. These transport steps provide building blocks for the cell and link the pathways of the mitochondrial matrix and cytosol. An increasing number of diseases and pathologies has been associated with their dysfunction. In this review, the molecular basis of these diseases is explained based on our current understanding of their transport mechanism

Mitochondrial ADP/ATP Carrier in Dodecylphosphocholine Binds Cardiolipins with Non-native Affinity.

Biophysical investigation of membrane proteins generally requires their extraction from native sources using detergents, a step that can lead, possibly irreversibly, to protein denaturation. The propensity of dodecylphosphocholine (DPC), a detergent widely utilized in NMR studies of membrane proteins, to distort their structure has been the subject of much controversy. It has been recently proposed that the binding specificity of the yeast mitochondrial ADP/ATP carrier (yAAC3) toward cardiolipins is preserved in DPC, thereby suggesting that DPC is a suitable environment in which to study membrane proteins. In this communication, we used all-atom molecular dynamics simulations to investigate the specific binding of cardiolipins to yAAC3. Our data demonstrate that the interaction interface observed in a native-like environment differs markedly from that inferred from an NMR investigation in DPC, implying that in this detergent, the protein structure is distorted. We further investigated yAAC3 solubilized in DPC and in the milder dodecylmaltoside with thermal-shift assays. The loss of thermal transition observed in DPC confirms that the protein is no longer properly folded in this environment

Characterization of drug-induced human mitochondrial ADP/ATP carrier inhibition.

An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile

Exercise-based cardiac rehabilitation improves exercise capacity and health-related quality of life in people with atrial fibrillation: A systematic review and meta-analysis of randomised and non-randomised trials

This is the final version. Available on open access from BMJ Publishing Group via the DOI in this recordObjective The aim of this study was to undertake a contemporary review of the impact of exercise-based cardiac rehabilitation (CR) targeted at patients with atrial fibrillation (AF). Methods We conducted searches of PubMED, EMBASE and the Cochrane Library of Controlled Trials (up until 30 November 2017) using key terms related to exercise-based CR and AF. Randomised and non-randomised controlled trials were included if they compared the effects of an exercise-based CR intervention to a no exercise or usual care control group. Meta-analyses of outcomes were conducted where appropriate. Results The nine randomised trials included 959 (483 exercise-based CR vs 476 controls) patients with various types of AF. Compared with control, pooled analysis showed no difference in all-cause mortality (risk ratio (RR) 1.08, 95% CI 0.77 to 1.53, p=0.64) following exercise-based CR. However, there were improvements in health-related quality of life (mean SF-36 mental component score (MCS): 4.00, 95% CI 0.26 to 7.74; p=0.04 and mean SF-36 physical component score: 1.82, 95% CI 0.06 to 3.59; p=0.04) and exercise capacity (mean peak VO2: 1.59 ml/kg/min, 95% CI 0.11 to 3.08; p=0.04; mean 6 min walk test: 46.9 m, 95% CI 26.4 to 67.4; p<0.001) with exercise-based CR. Improvements were also seen in AF symptom burden and markers of cardiac function. Conclusions Exercise capacity, cardiac function, symptom burden and health-related quality of life were improved with exercise-based CR in the short term (up to 6 months) targeted at patients with AF. However, high-quality multicentre randomised trials are needed to clarify the impact of exercise-based CR on key patient and health system outcomes (including health-related quality of life, mortality, hospitalisation and costs) and how these effects may vary across AF subtypes.National Institute for Health Research (NIHR

Screening of candidate substrates and coupling ions of transporters by thermostability shift assays

Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms

Expanding the phenotype of de novo SLC25A4-linked mitochondrial disease to include mild myopathy.

OBJECTIVE: To determine the disease relevance of a novel de novo dominant variant in the SLC25A4 gene, encoding the muscle mitochondrial adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier, identified in a child presenting with a previously unreported phenotype of mild childhood-onset myopathy. METHODS: Immunohistochemical and western blot analysis of the patient's muscle tissue were used to assay for the evidence of mitochondrial myopathy and for complex I-V protein levels. To determine the effect of a putative pathogenic p.Lys33Gln variant on ADP/ATP transport, the mutant protein was expressed in Lactococcus lactis and its transport activity was assessed with fused membrane vesicles. RESULTS: Our data demonstrate that the heterozygous c.97A>T (p.Lys33Gln) SLC25A4 variant is associated with classic muscle biopsy findings of mitochondrial myopathy (cytochrome c oxidase [COX]-deficient and ragged blue fibers), significantly impaired ADP/ATP transport in Lactococcus lactis and decreased complex I, III, and IV protein levels in patient's skeletal muscle. Nonetheless, the expression levels of the total ADP/ATP carrier (AAC) content in the muscle biopsy was largely unaffected. CONCLUSIONS: This report further expands the clinical phenotype of de novo dominant SLC25A4 mutations to a childhood-onset, mild skeletal myopathy, without evidence of previously reported clinical features associated with SLC25A4-associated disease, such as cardiomyopathy, encephalopathy or ophthalmoplegia. The most likely reason for the milder disease phenotype is that the overall AAC expression levels were not affected, meaning that expression of the wild-type allele and other isoforms may in part have compensated for the impaired mutant variant

Methodological challenges in measuring vaccine effectiveness using population cohorts in low resource settings.

Post-licensure real world evaluation of vaccine implementation is important for establishing evidence of vaccine effectiveness (VE) and programme impact, including indirect effects. Large cohort studies offer an important epidemiological approach for evaluating VE, but have inherent methodological challenges. Since March 2012, we have conducted an open prospective cohort study in two sites in rural Malawi to evaluate the post-introduction effectiveness of 13-valent pneumococcal conjugate vaccine (PCV13) against all-cause post-neonatal infant mortality and monovalent rotavirus vaccine (RV1) against diarrhoea-related post-neonatal infant mortality. Our study sites cover a population of 500,000, with a baseline post-neonatal infant mortality of 25 per 1000 live births. We conducted a methodological review of cohort studies for vaccine effectiveness in a developing country setting, applied to our study context. Based on published literature, we outline key considerations when defining the denominator (study population), exposure (vaccination status) and outcome ascertainment (mortality and cause of death) of such studies. We assess various definitions in these three domains, in terms of their impact on power, effect size and potential biases and their direction, using our cohort study for illustration. Based on this iterative process, we discuss the pros and cons of our final per-protocol analysis plan. Since no single set of definitions or analytical approach accounts for all possible biases, we propose sensitivity analyses to interrogate our assumptions and methodological decisions. In the poorest regions of the world where routine vital birth and death surveillance are frequently unavailable and the burden of disease and death is greatest We conclude that provided the balance between definitions and their overall assumed impact on estimated VE are acknowledged, such large scale real-world cohort studies can provide crucial information to policymakers by providing robust and compelling evidence of total benefits of newly introduced vaccines on reducing child mortality

Modelling the free energy profile of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier catalyses the equimolar exchange of adenosine di- and tri-phosphates. It operates by an alternating access mechanism in which a single substrate-binding site is made available either to the mitochondrial matrix or the intermembrane space through conformational changes. These changes are prevented in the absence of substrate by a large energy barrier due to the need for sequential disruption and formation of a matrix and cytoplasmic salt bridge network that are located on either side of the central cavity. In analogy to enzyme catalysis, substrate lowers the energy barrier by binding tighter in the intermediate state. Here we provide an in-silico kinetic model that captures the free energy profile of these conformational changes and treats the carrier as a nanomachine moving stochastically from the matrix to cytoplasmic conformation under the influence of thermal energy. The model reproduces the dependency of experimentally determined kcat and KM values on the cytoplasmic network strength with good quantitative accuracy, implying that it captures the transport mechanism and can provide a framework to understand the structure-function relationships of this class of transporter. The results show that maximum transport occurs when the interaction energies of the cytoplasmic network, matrix network and substrate binding are approximately equal such that the energy barrier is minimized. Consequently, the model predicts that there will be other interactions in addition to those of the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier