Investigating the Broad Matrix-Gate Network in the Mitochondrial ADP/ATP Carrier through Molecular Dynamics Simulations.

The mitochondrial ADP/ATP carrier (AAC) exports ATP and imports ADP through alternating between cytosol-open (c-) and matrix-open (m-) states. The salt bridge networks near the matrix side (m-gate) and cytosol side (c-gate) are thought to be crucial for state transitions, yet our knowledge on these networks is still limited. In the current work, we focus on more conserved m-gate network in the c-state AAC. All-atom molecular dynamics (MD) simulations on a variety of mutants and the CATR-AAC complex have revealed that: (1) without involvement of other positive residues, the charged residues from the three Px[DE]xx[KR] motifs only are prone to form symmetrical inter-helical network; (2) R235 plays a determinant role for the asymmetry in m-gate network of AAC; (3) R235 significantly strengthens the interactions between H3 and H5; (4) R79 exhibits more significant impact on m-gate than R279; (5) CATR promotes symmetry in m-gate mainly through separating R234 from D231 and fixing R79; (6) vulnerability of the H2-H3 interface near matrix side could be functionally important. Our results provide new insights into the highly conserved yet variable m-gate network in the big mitochondrial carrier family.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity.

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

Acyl atractyligenin and carboxyatractyligenin glycosides from Antennaria rosea subsp. confinis.

Eight previously undescribed acyl atractyligenin and carboxyatractyligenin glycosides were isolated from whole Antennaria rosea subsp. confinis (Greene) R. J. Bayer (Compositae) [syn. Leontopodium leontopodioides (Willd.) Beauv. (Asteraceae)] plants and their structures were determined by spectroscopic and chemical methods. The compounds were trivially named leontopodiosides F-M. Seven of the compounds showed potent in vitro inhibitory activity toward pancreatic lipase with IC50 values ranging from 3.4 to 52.5 µM, suggesting that they participate in the previously observed effect this plant has in reducing triglyceride absorption in rats.

Systems pharmacology reveals the unique mechanism features of Shenzhu Capsule for treatment of ulcerative colitis in comparison with synthetic drugs.

In clinic, both synthetic drugs and Shenzhu Capsule (SZC), one kind of traditional Chinese medicines (TCMs), are used to treat ulcerative colitis (UC). In our study, a systems pharmacology approach was employed to elucidate the chemical and mechanism differences between SZC and synthetic drugs in treating UC. First, the compound databases were constructed for SZC and synthetic drugs. Then, the targets of SZC were predicted with on-line tools and validated using molecular docking method. Finally, chemical space, targets, and pathways of SZC and synthetic drugs were compared. Results showed that atractylenolide I, atractylone, kaempferol, etc., were bioactive compounds of SZC. Comparison of SZC and synthetic drugs showed that (1) in chemical space, the area of SZC encompasses the area of synthetic drugs; (2) SZC can act on more targets and pathways than synthetic drugs; (3) SZC can not only regulate immune and inflammatory reactions but also act on ulcerative colitis complications (bloody diarrhea) and prevent UC to develop into colorectal cancer whereas synthetic drugs mainly regulate immune and inflammatory reactions. Our study could help us to understand the compound and mechanism differences between TCM and synthetic drugs.

Identification of new highly selective inhibitors of the human ADP/ATP carriers by molecular docking and in vitro transport assays.

Mitochondrial carriers are proteins that shuttle a variety of metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. The mitochondrial ADP/ATP carriers (AACs) specifically translocate the ATP synthesized within mitochondria to the cytosol in exchange for the cytosolic ADP, playing a key role in energy production, in promoting cell viability and regulating mitochondrial permeability transition pore opening. In Homo sapiens four genes code for AACs with different tissue distribution and expression patterns. Since AACs are dysregulated in several cancer types, the employment of known and new AAC inhibitors might be crucial for inducing mitochondrial-mediated apoptosis in cancer cells. Albeit carboxyatractyloside (CATR) and bongkrekic acid (BKA) are known to be powerful and highly selective AAC inhibitors, able to induce mitochondrial dysfunction at molecular level and poisoning at physiological level, we estimated here for the first time their affinity for the human recombinant AAC2 by in vitro transport assays. We found that the inhibition constants of CATR and BKA are 4 nM and 2.0 µM, respectively. For finding new AAC inhibitors we also performed a docking-based virtual screening of an in-house developed chemical library and we identified about 100 ligands showing high affinity for the AAC2 binding region. By testing 13 commercially available molecules, out of the 100 predicted candidates, we found that 2 of them, namely suramin and chebulinic acid, are competitive AAC2 inhibitors with inhibition constants 0.3 µM and 2.1 µM, respectively. We also demonstrated that chebulinic acid and suramin are "highly selective" AAC2 inhibitors, since they poorly inhibit other human mitochondrial carriers (namely ORC1, APC1 and AGC1).

Influence of Processing on the Content of Toxic Carboxyatractyloside and Atractyloside and the Microbiological Status of Xanthium sibiricum Fruits (Cang'erzi).

The dried ripe fruits of Xanthium sibiricum (Cang'erzi) are used in traditional Chinese medicine for the treatment of nasal congestion, nasal discharge, allergic rhinitis, sinusitis, and wind-cold headaches. Carboxyatractyloside and atractyloside are important constituents of the fruits because these diterpenoid glycosides are responsible for their toxicity. In order to evaluate procedures for reducing the amount of the more toxic carboxyatractyloside, the fruits were dried and heated with different methods. Carboxyatractyloside and atractyloside were analysed by a new reversed-phase high-performance liquid chromatographic method using liquid chromatography-diode array detector-tandem mass spectrometry analysis. The results revealed that temperature and drying methods have a strong influence on the content of carboxyatractyloside and atractyloside. Fruits which were treated at higher temperatures showed a lower content of carboxyatractyloside and an increased content of atractyloside, which is 50 times less toxic. This indicates that the roasting process can reduce toxicity effectively. The microbiological colonisation of Xanthium fruits is also reduced by roasting and by drying above 100 °C. For the safe use of Cang'erzi, the effect of processing should be monitored and analysis of carboxyatractyloside and atractyloside should be obligatory in quality control.

Analytical confirmation of Xanthium strumarium poisoning in cattle.

Xanthium strumarium, commonly referred to as "cocklebur," rarely causes poisoning in cattle. When mature, this robust, annual weed bears numerous oval, brownish, spiny burs. Only the seeds in the burs and young seedlings (cotyledonary leaves) contain the toxic principle, carboxyatractyloside. In the Frankfort district of the Free State Province of South Africa, a herd of 150 Bonsmara cows were allowed to graze on the banks of a small river, where mature cocklebur was growing. Four cows died while grazing in this relatively small area. Clinical signs ranged from recumbency, apparent blindness, and hypersensitivity to convulsive seizures. During necropsy, burs completely matted with ingesta were located in the rumen content. The most distinctive microscopic lesions were severe, bridging centrilobular to midzonal hepatocyte necrosis and hemorrhage. Ultrastructurally, periacinar hepatocytes were necrotic, and novel electron-dense cytoplasmic needle-like crystals were observed, often in close association with peroxisomes. Carboxyatractyloside concentrations were determined using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Carboxyatractyloside was present in rumen contents at 2.5 mg/kg; in burs removed from the rumen at 0.17 mg/kg; in liver at 66 ng/g, and was below the limit of quantitation in the kidney sample, estimated at approximately 0.8 ng/g. Based on the presence of the plants on the riverbank, the history of exposure, the clinical findings, the presence of burs in the rumen, and the microscopic and ultrastructural lesions, X. strumarium poisoning in the herd of cattle was confirmed and was supported by LC-HRMS.

2-O-ß-D-Glucopyranosyl-carboxyatractyligenin from Coffea L. inhibits adenine nucleotide translocase in isolated mitochondria but is quantitatively degraded during coffee roasting.

Atractyloside (1) and carboxyatractyloside (2) are well-known inhibitors of the adenine nucleotide translocase (ANT) in mitochondria, thus effectively blocking oxidative phosphorylation. Structurally related derivatives atractyligenin (3), 2-O-ß-D-glucopyranosyl-atractyligenin (4), 3'-O-ß-D-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-atractyligenin)-ß-D-glucopyranoside (5), and 2-O-ß-D-glucopyranosyl-carboxyatractyligenin (6) were isolated from raw beans of Coffea L. and the impact of 1-6 on ANT activity was evaluated in isolated mitochondria. Among the coffee components, 6 significantly inhibited ANT activity leading to reduced respiration. Quantitative analysis in commercial coffees, experimental roastings of coffee, and model experiments using purified compound 6 consistently revealed a complete degradation during thermal treatment. In comparison, raw coffee extracts were found to contain high levels of 6, which are therefore expected to be present in food products enriched with raw coffee extracts. This implies the necessity of analytically controlling the levels of 6 in raw coffee extracts when used as additives for food products.

The degradation mechanism of toxic atractyloside in herbal medicines by decoction.

Atractyloside (ATR) is found in many Asteraceae plants that are commonly used as medicinal herbs in China and other eastern Asian countries. ATR binds specifically to the adenine nucleotide translocator in the inner mitochondrial membrane and competitively inhibits ADP and ATP transport. The toxicity of ATR in medical herbs can be reduced by hydrothermal processing, but the mechanisms of ATR degradation are not well understood. In this study, GC-MS coupled with SPE and TMS derivatisation was used to detect ATR levels in traditional Chinese medicinal herbs. Our results suggest that ATR molecules were disrupted by decomposition, hydrolysis and saponification after heating with water (decoction) for a long period of time. Hydrothermal processing could decompose the endogenous toxic compounds and also facilitate the detoxification of raw materials used in the Chinese medicine industry.

Mortality of a captive axis deer (Axis axis) and a llama (Lama glama) due to ingestion of Wedelia glauca.

The current study describes a naturally occurring cluster of cases of Wedelia glauca intoxication. Seven of 14 axis deer (Axis axis) and 1 of 8 llamas (Lama glama) in a zoo of Buenos Aires province, Argentina, died suddenly after ingestion of a new batch of alfalfa (Medicago sativa) hay bales contaminated with the hepatotoxic plant W. glauca. Necropsies of 1 deer and 1 llama were performed. Pathological findings in both animals included severe diffuse acute centrilobular hepatocellular necrosis and hemorrhage, and clear yellowish translucent gelatinous edema on the wall of the gall bladder and the serosa of the choledochoduodenal junction. Fragments of W. glauca plants were identified in the hay based on the botanical characteristics of the leaves. Samples of gastric contents were examined by microhistological analysis, which identified epidermal fragments of W. glauca based on the presence of characteristic uniseriate glandular hairs (trichomes), confirming recent ingestion of W. glauca in both cases. The fragments were quantified and represented 5% of all examined vegetal fragments in the deer and 10% in the llama.

Structural characterization and antimicrobial evaluation of atractyloside, atractyligenin, and 15-didehydroatractyligenin methyl ester.

We report the first complete structure elucidation of the ent-kaurane diterpenoid glycoside atractyloside (1) by means of NMR and X-ray diffractometry techniques. Extensive one- and two-dimensional NMR experiments were employed to assign the proton and carbon signals of 1, and crystallography experiments established the configurations of all stereogenic centers. Furthermore, we present a novel semisynthetic route for the preparation of the highly cytotoxic aglycone derivative of 1, 15-didehydroatractyligenin methyl ester (3). All compounds were tested for their antibiotic activity against Enterococcus faecalis, Escherichia coli, and several strains of Staphylococcus aureus, including fluoroquinolone-resistant (SA1199B) and two epidemic MRSA (EMRSA-15 and -16) strains. Compound 3 exhibited moderate activity against all of the Staph. aureus strains with an MIC value of 128 mg/L.

[Atractylis gummifera: from poisoning to the analytic methods]. / Atractylis gummifera : de l'intoxication aux méthodes analytiques.

Atractylis L gummifera is a plant that causes every year serious and often deadly poisonings. In Morocco, 153 cases of poisoning have been recorded between January 1980 and June 1995 by the Moroccan Antipoison Centre. The ignorance by the clinicians, the fast evolution and the frequency of these poisonings are the origin of diagnosis problems. The solution of those problems is to detect atractyloside and carboxyatractyloside in the biologic liquids. Since several decades, some toxicological analytical methods were established in view of an assay. The aim of our paper is to describe the poisoning by this plant and to review the methods of toxicological analysis used from the colorimetric technique until the news recent chromatographic methods.

Unambiguous structure of atractyloside and carboxyatractyloside.

Atractyloside (ATR) was characterized in 1868 and until now structural studies on diterpenic moiety had been done through the characterization of ATR derivatives; while the glycosidic moiety seemed to be a ß-D-glucopyranose a recent crystal structure of the mitochondrial ATP/ADP carrier in complex with CATR showed an α-D-glucopyranose. We decided to re-examine the ATR and CATR structures by crystallographic study of ATR.

Conformational dynamics of the bovine mitochondrial ADP/ATP carrier isoform 1 revealed by hydrogen/deuterium exchange coupled to mass spectrometry.

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.

Native membrane proteins vs. yeast recombinant: an example: the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier (Ancp) has long been a paradigm for studies of the mitochondrial carrier family due to, among other properties, its natural abundance and the existence of specific inhibitors, namely, carboxyatractyloside (CATR) and bongkrekic acid (BA), which lock the carrier under distinct and stable conformations. Bovine Anc1p isolated in complex with CATR in the presence of an aminoxyde detergent (LAPAO) was crystallized and its 3D structure determined. It is the first mitochondrial carrier structure resolved at high resolution (2.2 A, as reported by Pebay-Peyroula et al. (Nature 426:39-44, 2003)). Analyses revealed a monomer while most of the biochemical studies led to hypothesize Ancp functions as a dimer. To address the structural organization issue, we engineered a mutant of the yeast Ancp that corresponds to a covalent homodimer in view of 3D structure determination. We compare in this chapter the purification yield and quality of the chimera tagged either with six histidines at its C-ter end or nine histidines at its N-ter. We show that, as expected, length and position of the tag are important criteria for qualitative purification. We also discuss the advantages and drawbacks of purifying Ancp either from a natural source or from engineered yeast cells.

Structural approaches of the mitochondrial carrier family.

The transport of solutes across the inner mitochondrial membrane is highly selective and necessitates membrane proteins mainly from the mitochondrial carrier family (MCF). These carriers are required for the transport of a variety of metabolites implicated in all the important processes occurring within the mitochondrial matrix. Due to its high abundance, the ADP/ATP carrier (AAC) is the member of the family that was studied most. It is the first mitochondrial carrier for which a high-resolution X-ray structure is known. The carrier was crystallized in the presence of a strong inhibitor, the carboxyatractyloside (CATR). The structure gives an insight not only into the overall fold of mitochondrial carriers in general but also into atomic details of the AAC in a conformation that is open toward the intermembrane space (IMS). Molecular dynamics simulations indicate the first events occurring to the carrier after the binding of ADP. A careful analysis of the primary sequences of all the carriers in light with the structure highlights properties of the protein that are related to the substrate.

Probing the interactions of carboxy-atractyloside and atractyloside with the yeast mitochondrial ADP/ATP carrier.

Mitochondrial ADP/ATP carriers are inhibited by two natural compounds, atractyloside (ATR) or carboxy-atractyloside (CATR), which differ by one carboxylate group. The interactions of the inhibitors with the carrier were investigated by single-molecule force spectroscopy. Transmembrane alpha helices of the ATR-inhibited carrier displayed heterogeneous mechanical and kinetic properties. Whereas alpha helix H2 showed the most brittle mechanical properties and lowest kinetic stability, alpha helix H5 was mechanically the most flexible and possessed a kinetic stability 9 orders of magnitude greater than that of alpha helix H2. In contrast, CATR-binding substantially increased the kinetic stability of alpha helix H2 and tuned the mechanical flexibility of alpha helices H5 and H6. NMR spectroscopy confirmed that the additional carboxylate group of CATR binds to the sixth alpha helix, indicating that the enhanced stability of H2 is mediated via interactions between CATR and H6.

Conformational dynamics of the mitochondrial ADP/ATP carrier: a simulation study.

The mitochondrial ADP/ATP carrier is a six helix bundle membrane transport protein, which couples the exit of ATP from the mitochondrial matrix to the entry of ADP. Extended (4x20 ns) molecular dynamics simulations of the carrier, in the presence and absence of bound inhibitor (carboxyatractyloside), have been used to explore the conformational dynamics of the protein in a lipid bilayer environment, in the presence and absence of the carboxyatractyloside inhibitor. The dynamic flexibility (measured as conformational drift and fluctuations) of the protein is reduced in the presence of bound inhibitor. Proline residues in transmembrane helices H1, H3 and H5 appear to form dynamic hinges. Fluctuations in inter-helix salt bridges are also observed over the time course of the simulations. Inhibitor-protein and lipid-protein interactions have been characterised in some detail. Overall, the simulations support a transport mechanism in which flexibility about the proline hinges enables a transition between a 'closed' and an 'open' pore-like state of the carrier protein.

Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies.

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier has been recently crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). In the crystal structure, the six-transmembrane helix bundle that defines the nucleotide translocation pathway is closed on the matrix side due to sharp kinks in the odd-numbered helices. The closed conformation is further sealed by the loops protruding into the matrix that interact through an intricate network of charge-pairs. To gain insight into its structural dynamics we performed molecular dynamics (MD) simulation studies of the ADP/ATP carrier with and without its cocrystallized inhibitor. The two trajectories sampled a conformational space around two different configurations characterized by distinct salt-bridge networks with a significant shift from inter- to intrarepeat bonding on the matrix side in the absence of CATR. Analysis of the geometrical parameters defining the transmembrane helices showed that even-numbered helices can undergo a face rotation, whereas odd-numbered helices can undergo a change in the wobble angle with a conserved proline acting as molecular hinge. Our results provide new information on the dynamical properties of the ADP/ATP carrier and for the first time yield a detailed picture of a stable carrier conformation in absence of the inhibitor.

Molecular, functional, and pathological aspects of the mitochondrial ADP/ATP carrier.

In providing the cell with ATP generated by oxidative phosphorylation, the mitochondrial ADP/ATP carrier plays a central role in aerobic eukaryotic cells. Combining biochemical, genetic, and structural approaches contributes to understanding the molecular mechanism of this essential transport system, the dysfunction of which is implicated in neuromuscular diseases.