Mario García de Lacoba

The mitochondrial uncoupling proteins

SummaryThe uncoupling proteins (UCPs) are transporters, present in the mitochondrial inner membrane, that mediate a regulated discharge of the proton gradient that is generated by the respiratory chain. This energy-dissipatory mechanism can serve functions such as thermogenesis, maintenance of the redox balance, or reduction in the production of reactive oxygen species. Some UCP homologs may not act as true uncouplers, however, and their activity has yet to be defined. The UCPs are integral membrane proteins, each with a molecular mass of 31-34 kDa and a tripartite structure in which a region of around 100 residues is repeated three times; each repeat codes for two transmembrane segments and a long hydrophilic loop. The functional carrier unit is a homodimer. So far, 45 genes encoding members of the UCP family have been described, and they can be grouped into six families. Most of the described genes are from mammals, but UCP genes have also been found in fish, birds and plants, and there is also functional evidence to suggest their presence in fungi and protozoa. UCPs are encoded in their mature form by nuclear genes and, unlike many nuclear-encoded mitochondrial proteins, they lack a cleavable mitochondrial import signal. The information for mitochondrial targeting resides in the first loop that protrudes into the mitochondrial matrix; the second matrix loop is essential for insertion of the protein into the inner mitochondrial membrane. UCPs are regulated at both the transcriptional level and by activation and inhibition in the mitochondrion.

The yefM-yoeB Toxin-Antitoxin Systems of Escherichia coli and Streptococcus pneumoniae: Functional and Structural Correlation

Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM(Spn)) and toxin (YoeB(Spn)) products. We showed that overproduction of YoeB(Spn) is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB(Spn)-mediated toxicity could be reversed by the cognate antitoxin, YefM(Spn), but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

Characterization of a pathogenesis-related protein 4 (PR-4) induced in Capsicum chinense L3 plants with dual RNase and DNase activities

Resistance conferred by the L3 gene is active against most of the tobamoviruses, including the Spanish strain (PMMoV-S), a P1,2 pathotype, but not against certain strains of pepper mild mottle virus (PMMoV), termed as P1,2,3 pathotype, such as the Italian strain (PMMoV-I). PMMoV-S induces a hypersensitive reaction (HR) in C. chinense PI159236 plant leaves with the formation of necrotic local lesions and restriction of the virus at the primary infection sites. In this paper, a C. chinense PR-4 protein induced during both the compatible and the incompatible interactions has been identified. It was strongly associated with HR induction and to a lesser extent with the compatible interaction, but only in the later stages of infection. Moreover, it was found to accumulate during the necrogenic reaction induced by Potato virus X. The C. chinense PR-4 protein belongs to the PR-4 protein subgroup II, based on the absence of a hevein domain. Furthermore, it is shown that the purified protein does not have chitinase activity, as previously proposed for PR-4 proteins. Instead, it has both RNase and DNase activity, although its contribution to the bulk activity of nucleases in infected plants is very low.

The C-terminus of H-ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways

Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and Δ12-PGJ2 selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ2. Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

Identification of the mitochondrial carrier that provides Yarrowia lipolytica with a fatty acid-induced and nucleotide-sensitive uncoupling protein-like activity

Uncoupling proteins (UCPs) are mitochondrial carriers distributed throughout the eukaryotic kingdoms. While genes coding for UCPs have been identified in plants and animals, evidences for the presence of UCPs in fungi and protozoa are only functional. Here, it is reported that in the yeast Yarrowia lipolytica there is a fatty acid-promoted and GDP-sensitive uncoupling activity indicating the presence of a UCP. The uncoupling activity is higher in the stationary phase than in the mid-log growth phase. The in silico search on the Y. lipolytica genome led to the selection of two genes with the highest homology to the UCP family, XM_503525 and XM_500457. By phylogenetic analysis, XP_503525 was predicted to be an oxaloacetate carrier while XP_500457 would be a dicarboxylate carrier. Each of these two genes was cloned and heterologously expressed in Saccharomyces cerevisiae and the resulting phenotype was analyzed. The transport activity of the two gene products confirmed the phylogenetic predictions. In addition, only mitochondria isolated from yeasts expressing XP_503525 showed bioenergetic properties characteristic of a UCP: the proton conductance was increased by linoleic acid and inhibited by GDP. It is concluded that the XM_503525 gene from Y. lipolytica encodes for an oxaloacetate carrier although, remarkably, it also displays an uncoupling activity stimulated by fatty acids and inhibited by nucleotides.

Modeling the Transmembrane Arrangement of the Uncoupling Protein UCP1 and Topological Considerations of the Nucleotide-Binding Site

The uncoupling protein from brown adipose tissue (UCP1) is a mitochondrial proton transporter whose activity is inhibited by purine nucleotides. UCP1, like the other members of the mitochondrial transporter superfamily, is an homodimer and each subunit contains six transmembrane segments. In an attempt to understand the structural elements that are important for nucleotide binding, a model for the transmembrane arrangement of UCP1 has been built by computational methods. Biochemical and sequence analysis considerations are taken as constraints. The main features of the model include the following: (i) the six transmembrane α-helices (TMHs) associate to form an antiparallel helix bundle; (ii) TMHs have an amphiphilic nature and thus the hydrophobic and variable residues face the lipid bilayer; (iii) matrix loops do not penetrate in the core of the bundle; and (iv) the polar core constitutes the translocation pathway. Photoaffinity labeling and mutagenesis studies have identified several UCP1 regions that interact with the nucleotide. We present a model where the nucleotide binds deep inside the bundle core. The purine ring interacts with the matrix loops while the polyphosphate chain is stabilized through interactions with essential Arg residues in the TMH and whose side chains face the core of the helix bundle.

Structural constraints in expansion segments from a midge 26S rDNA

DNA sequences representing approximately 40% of the large-subunit rRNA gene from the lower dipteran Chironomus thummi were analyzed. Once aligned with their Drosophila counterparts, sequence and base content comparisons were carried out. Sequence identity was found to be high overall, except for six regions that displayed a local bias in nucleotide composition toward AT. These regions were identified as expansion segments D3, D4, D5, D6, D7a, and D12. Besides base sequence divergence, differences in length were observed between the respective variable domains of the two species, particularly for D7a. Prediction of secondary structure showed that the folding of the Chironomus expansion segments analyzed is in agreement with the general patterns proposed for eukaryotic LSU rRNA. The comparison with Drosophila revealed also that the Chironomus secondary structures of the variable domains are supported by multiple compensatory substitutions or even compensatory insertions. Chironomus D7a displayed an unusual structural feature with respect to the insect D7a models that have been inferred up to now. The structural constraint observed in the expansion segments of Diptera so distantly related as midges and Drosophila suggests that these regions contribute to some functional role. Concerning the D7a of insects so far analyzed, there can be, in addition to a conserved secondary structure, a nucleotide composition constraint that might be important for the process giving rise to the alpha and beta halves of the 26S rRNA.

Structural features of the plasmid pMV158‐encoded transcriptional repressor CopG, a protein sharing similarities with both helix‐turn‐helix and β‐sheet DNA binding proteins

The small transcriptional repressor CopG protein (45 amino acids) encoded by the streptococcal plasmid pMV158 was purified to near homogeneity. Gel filtration chromatography and analytical ultracentrifugation showed that the native protein is a spherical dimer of identical subunits. Circular dichroism measurements of CopG indicated a consensus average content of more than 50% α‐helix and 10–35% β‐strand and turns, which is compatible with the predicted secondary structure of the protein. CopG exhibited a prolonged intracellular half‐life, but deletions in regions other than the C‐terminal affected the global structure of the protein, severely reducing the half‐lives of the CopG variants. This indicates that CopG has a compact structure, perhaps constituted by a single domain. Molecular modeling of CopG showed a good fitting between the helix‐turn‐helix motifs of well‐known repressor proteins and a bihelical unit of CopG. However, modeling of CopG with ribbon‐helix‐helix class of DNA binding proteins also exhibited an excellent fit. Eleven out of the 12 replicons belonging to the pMV158 plasmid family could also encode Cop proteins, which share features with both helix‐turn‐helix and β‐sheet DNA binding proteins. Proteins 32:248–261, 1998.

Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

Graphical abstract

Putative porin of Bradyrhizobium sp. (Lupinus) bacteroids induced by glyphosate.

ABSTRACT Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed.