Citrate Regulates the Saccharomyces cerevisiae Mitochondrial GDP/GTP Carrier (Ggc1p) by Triggering Unidirectional Transport of GTP.

The yeast mitochondrial transport of GTP and GDP is mediated by Ggc1p, a member of the mitochondrial carrier family. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria in exchange for GDP generated in the matrix. ggc1Δ cells exhibit lower levels of GTP and increased levels of GDP in mitochondria, are unable to grow on nonfermentable substrates and lose mtDNA. Because in yeast, succinyl-CoA ligase produces ATP instead of GTP, and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space, Ggc1p is the only supplier of mitochondrial GTP required for the maturation of proteins containing Fe-S clusters, such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. In this work, it was demonstrated that citrate is a regulator of purified and reconstituted Ggc1p by trans-activating unidirectional transport of GTP across the proteoliposomal membrane. It was also shown that the binding site of Ggc1p for citrate is different from the binding site for the substrate GTP. It is proposed that the citrate-induced GTP uniport (CIGU) mediated by Ggc1p is involved in the homeostasis of the guanine nucleotide pool in the mitochondrial matrix.

Mannich base approach to 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone: A water-soluble prodrug for a multitarget inhibition of cholinesterases, beta-amyloid fibrillization and oligomer-induced cytotoxicity.

Targeting protein aggregation for the therapy of neurodegenerative diseases remains elusive for medicinal chemists, despite a number of small molecules known to interfere in amyloidogenesis, particularly of amyloid beta (Aß) protein. Starting from previous findings in the antiaggregating activity of a class of indolin-2-ones inhibiting Aß fibrillization, 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone 1 was identified as a multitarget inhibitor of Aß aggregation and cholinesterases with IC50s in the low µM range. With the aim of increasing aqueous solubility, a Mannich-base functionalization led to the synthesis of N-methylpiperazine derivative 2. At acidic pH, an outstanding solubility increase of 2 over the parent compound 1 was proved through a turbidimetric method. HPLC analysis revealed an improved stability of the Mannich base 2 at pH2 along with a rapid release of 1 in human serum as well as an outstanding hydrolytic stability of the parent hydrazone. Coincubation of Aß1-42 with 2 resulted in the accumulation of low MW oligomers, as detected with PICUP assay. Cell assays on SH-SY5Y cells revealed that 2 exerts strong cytoprotective effects in both cell viability and radical quenching assays, mainly related to its active metabolite 1. These findings show that 2 drives the formation of non-toxic, off-pathway Aß oligomers unable to trigger the amyloid cascade and toxicity.

Intra-mitochondrial Methylation Deficiency Due to Mutations in SLC25A26.

S-adenosylmethionine (SAM) is the predominant methyl group donor and has a large spectrum of target substrates. As such, it is essential for nearly all biological methylation reactions. SAM is synthesized by methionine adenosyltransferase from methionine and ATP in the cytoplasm and subsequently distributed throughout the different cellular compartments, including mitochondria, where methylation is mostly required for nucleic-acid modifications and respiratory-chain function. We report a syndrome in three families affected by reduced intra-mitochondrial methylation caused by recessive mutations in the gene encoding the only known mitochondrial SAM transporter, SLC25A26. Clinical findings ranged from neonatal mortality resulting from respiratory insufficiency and hydrops to childhood acute episodes of cardiopulmonary failure and slowly progressive muscle weakness. We show that SLC25A26 mutations cause various mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation, and the biosynthesis of CoQ10 and lipoic acid.

Functional and structural role of amino acid residues in the matrix alpha-helices, termini and cytosolic loops of the bovine mitochondrial oxoglutarate carrier.

The mitochondrial oxoglutarate carrier belongs to the mitochondrial carrier family and exchanges oxoglutarate for malate and other dicarboxylates across the mitochondrial inner membrane. Here, single-cysteine mutant carriers were engineered for every residue in the amino- and carboxy-terminus, cytoplasmic loops, and matrix alpha-helices and their transport activity was measured in the presence and absence of sulfhydryl reagents. The analysis of the cytoplasmic side of the oxoglutarate carrier showed that the conserved and symmetric residues of the mitochondrial carrier motif [DE]XX[RK] localized at the C-terminal end of the even-numbered transmembrane alpha-helices are important for the function of the carrier, but the non-conserved cytoplasmic loops and termini are not. On the mitochondrial matrix side of the carrier most residues of the three matrix alpha-helices that are in the interface with the transmembrane alpha-helical bundle are important for function. Among these are the residues of the symmetric [ED]G motif present at the C-terminus of the matrix alpha-helices; the tyrosines of the symmetric YK motif at the N-terminus of the matrix alpha-helices; and the hydrophobic residues M147, I171 and I247. The functional role of these residues was assessed in the structural context of the homology model of OGC. Furthermore, in this study no evidence was found for the presence of a specific homo-dimerisation interface on the surface of the carrier consisting of conserved, asymmetric and transport-critical residues.

Functional and structural role of amino acid residues in the odd-numbered transmembrane alpha-helices of the bovine mitochondrial oxoglutarate carrier.

The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle and gluconeogenesis. To establish amino acid residues that are important for function, each residue in the transmembrane alpha-helices H1, H3 and H5 was replaced systematically by a cysteine in a fully functional mutant carrier that was devoid of cysteine residues. The transport activity of the mutant carriers was measured in the presence and absence of sulfhydryl reagents. The observed effects were rationalized by using a comparative structural model of the OGC. Most of the residues that are critical for function are found at the bottom of the cavity and they belong to the signature motifs P-X-[DE]-X-X-[KR] that form a network of three inter-helical salt bridges that close the carrier at the matrix side. The OGC deviates from most other carriers, because it has a conserved leucine (L144) rather than a positively charged residue in the signature motif of the second repeat and thus the salt bridge network is lacking one salt bridge. Incomplete salt-bridge networks due to hydrophobic, aromatic or polar substitutions are observed in other dicarboxylate, phosphate and adenine nucleotide transporters. The interaction between the carrier and the substrate has to provide the activation energy to trigger the re-arrangement of the salt-bridge network and other structural changes required for substrate translocation. For substrates such as malate, which has only two carboxylic and one hydroxyl group, a reduction in the number of salt bridges in the network may be required to lower the energy barrier for translocation. Another group of key residues, consisting of T36, A134, and T233, is close to the putative substrate binding site and substitutions or modifications of these residues may interfere with substrate binding and ion coupling. Residues G32, A35, Q40, G130, G133, A134, G230, and S237 are potentially engaged in inter-helical interactions and they may be involved in the movements of the alpha-helices during translocation.

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study.

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.