Ekaterina A. Smirnova

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C12R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H+ ions was generated in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C12R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C12R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C12R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.

Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

Natural causes of programmed death of yeast Saccharomyces cerevisiae

The existence of cell death program in unicellular organisms has been reported for a number of species. Nevertheless, the question why the ability to commit suicide has been maintained throughout evolution is far from being solved. While it is believed that altruistic death of individual yeast cells could be beneficial for the population, it is generally not known (i) what is wrong with the individuals destined for elimination, (ii) what is the critical value of the parameter that makes a cell unfit and (iii) how the cell monitors this parameter. Studies performed on yeast Saccharomyces cerevisiae allow us to hypothesize on ways of possible solutions of these problems. Here we argue that (a) the main parameter for life-or-death decision measured by the cell is the degree of damage to the genetic material, (b) its critical value is dictated by quorum sensing machinery, and (c) it is measured by monitoring delays in cell division.

Evolutionary and genetic analyses of mitochondrial translation initiation factors identify the missing mitochondrial IF3 in S. cerevisiae

Mitochondrial translation is essentially bacteria-like, reflecting the bacterial endosymbiotic ancestry of the eukaryotic organelle. However, unlike the translation system of its bacterial ancestors, mitochondrial translation is limited to just a few mRNAs, mainly coding for components of the respiratory complex. The classical bacterial initiation factors (IFs) IF1, IF2 and IF3 are universal in bacteria, but only IF2 is universal in mitochondria (mIF2). We analyse the distribution of mitochondrial translation initiation factors and their sequence features, given two well-propagated claims: first, a sequence insertion in mitochondrial IF2 (mIF2) compensates for the universal lack of IF1 in mitochondria, and secondly, no homologue of mitochondrial IF3 (mIF3) is identifiable in Saccharomyces cerevisiae. Our comparative sequence analysis shows that, in fact, the mIF2 insertion is highly variable and restricted in length and primary sequence conservation to vertebrates, while phylogenetic and in vivo complementation analyses reveal that an uncharacterized S. cerevisiae mitochondrial protein currently named Aim23p is a bona fide evolutionary and functional orthologue of mIF3. Our results highlight the lineage-specific nature of mitochondrial translation and emphasise that comparative analyses among diverse taxa are essential for understanding whether generalizations from model organisms can be made across eukaryotes.

The Rise and Fall of Jasmonate Biological Activities

Jasmonates (JAs) constitute a major class of plant regulators that coordinate responses to biotic and abiotic threats and important aspects of plant development. The core biosynthetic pathway converts linolenic acid released from plastid membrane lipids to the cyclopentenone cis-oxo-phytodienoic acid (OPDA) that is further reduced and shortened to jasmonic acid (JA) in peroxisomes. Abundant pools of OPDA esterified to plastid lipids also occur upon stress, mainly in the Arabidopsis genus. Long thought to be the bioactive hormone, JA only gains its pleiotropic hormonal properties upon conjugation into jasmonoyl-isoleucine (JA-Ile). The signaling pathway triggered when JA-Ile promotes the assembly of COI1-JAZ (Coronatine Insensitive 1-JAsmonate Zim domain) co-receptor complexes has been the focus of most recent research in the jasmonate field. In parallel, OPDA and several other JA derivatives are recognized for their separate activities and contribute to the diversity of jasmonate action in plant physiology. We summarize in this chapter the properties of different bioactive JAs and review elements known for their perception and signal transduction. Much progress has also been gained on the enzymatic processes governing JA-Ile removal. Two JA-Ile catabolic pathways, operating through ω-oxidation (cytochromes P450) or conjugate cleavage (amido hydrolases) shape signal dynamics to allow optimal control on defense. JA-Ile turnover not only participates in signal attenuation, but also impact the homeostasis of the entire JA metabolic pathway.

tRNA mitochondrial import in yeast: Mapping of the import determinants in the carrier protein, the precursor of mitochondrial lysyl-tRNA synthetase.

Mitochondria of many species import of nuclear DNA-encoded tRNAs. This widely spread but poorly studied phenomenon proved to be a promising tool for mitochondrial transfection. In yeast Saccharomyces cerevisiae, one cytosolic tRNAs(Lys) is partially targeted into mitochondria. Previous studies have shown that binding of this tRNA to its putative protein carrier, the precursor of mitochondrial lysyl-tRNA synthetase (preMsk1p), IIb class aminoacyl-tRNA synthetase, was a pre-requisite of import. In this work, we identify the hinge region with two adjacent helices H5 and H7 to be responsible for mitochondrial targeting of the tRNA and characterize preMsk1p versions with altered tRK1 import capacities.

Alkylrhodamines enhance the toxicity of clotrimazole and benzalkonium chloride by interfering with yeast pleiotropic ABC-transporters.

ABC-transporters with broad substrate specificity are responsible for pathogenic yeast resistance to antifungal compounds. Here we asked whether highly hydrophobic chemicals with delocalized positive charge can be used to overcome the resistance. Such molecules efficiently penetrate the plasma membrane and accumulate inside the cells. We reasoned that these properties can convert an active efflux of the compounds into a futile cycle thus interfering with the extrusion of the antibiotics. To test this, we studied the effects of several alkylated rhodamines on the drug resistance of yeast Saccharomyces cerevisiae We found that octylrhodamine synergetically increases toxicity of Pdr5p substrate-clotrimazole, while the others were less effective. Next, we compared the contributions of three major pleiotropic ABC-transporters (Pdr5p, Yor1p, Snq2p) on the accumulation of the alkylated rhodamines. While all of the tested compounds were extruded by Pdr5p, Yor1p and Snq2p showed narrower substrate specificity. Interestingly, among the tested alkylated rhodamines, inactivation of Pdr5p had the strongest effect on the accumulation of octylrhodamine inside the cells, which is consistent with the fact that clotrimazole is a substrate of Pdr5p. As alkylated rhodamines were shown to be non-toxic on mice, our study makes them potential components of pharmacological antifungal compositions.

Dynamics of Jasmonate Metabolism upon Flowering and across Leaf Stress Responses in Arabidopsis thaliana

The jasmonic acid (JA) signaling pathway plays important roles in adaptation of plants to environmental cues and in specific steps of their development, particularly in reproduction. Recent advances in metabolic studies have highlighted intricate mechanisms that govern enzymatic conversions within the jasmonate family. Here we analyzed jasmonate profile changes upon Arabidopsis thaliana flower development and investigated the contribution of catabolic pathways that were known to turnover the active hormonal compound jasmonoyl-isoleucine (JA-Ile) upon leaf stress. We report a rapid decline of JA-Ile upon flower opening, concomitant with the massive accumulation of its most oxidized catabolite, 12COOH-JA-Ile. Detailed genetic analysis identified CYP94C1 as the major player in this process. CYP94C1 is one out of three characterized cytochrome P450 enzymes that define an oxidative JA-Ile turnover pathway, besides a second, hydrolytic pathway represented by the amido-hydrolases IAR3 and ILL6. Expression studies combined with reporter gene analysis revealed the dominant expression of CYP94C1 in mature anthers, consistent with the established role of JA signaling in male fertility. Significant CYP94B1 expression was also evidenced in stamen filaments, but surprisingly, CYP94B1 deficiency was not associated with significant changes in JA profiles. Finally, we compared global flower JA profiles with those previously reported in leaves reacting to mechanical wounding or submitted to infection by the necrotrophic fungus Botrytis cinerea. These comparisons revealed distinct dynamics of JA accumulation and conversions in these three biological systems. Leaf injury boosts a strong and transient JA and JA-Ile accumulation that evolves rapidly into a profile dominated by ω-oxidized and/or Ile-conjugated derivatives. In contrast, B. cinerea-infected leaves contain mostly unconjugated jasmonates, about half of this content being ω-oxidized. Finally, developing flowers present an intermediate situation where young flower buds show detectable jasmonate oxidation (probably originating from stamen metabolism) which becomes exacerbated upon flower opening. Our data illustrate that in spite conserved enzymatic routes, the jasmonate metabolic grid shows considerable flexibility and dynamically equilibrates into specific blends in different physiological situations.

Accumulation of dodecyltriphenylphosphonium in mitochondria induces their swelling and ROS-dependent growth inhibition in yeast

Hydrophobic cations with delocalized charge are used to deliver drugs to mitochondria. However, micromolar concentrations of such compounds could be toxic due to their excessive accumulation in mitochondria. We studied possible pathophysiological effects of one such cation, i.e. dodecyltriphenylphosphonium (C12-TPP), in the yeast Saccharomyces cerevisiae. First, we found that C12-TPP induces high-amplitude mitochondrial swelling. The swelling can be prevented by addition of protonophorous uncoupler FCCP or antioxidant alpha-tocopherol, but not other tested antioxidants (N-acetylcysteine and Trolox). Second, FCCP prevents ROS-sensitive fluorescent dye (dichlorofluorescein diacetate) staining of yeast treated with C12-TPP. We also showed that all tested antioxidants partially restore the growth inhibited by C12-TPP. The latter points that ROS rather than the mitochondria swelling limit the growth rate.

Dodecyltriphenylphosphonium inhibits multiple drug resistance in the yeast Saccharomyces cerevisiae.

Multiple drug resistance pumps are potential drug targets. Here we asked whether the lipophilic cation dodecyltriphenylphosphonium (C12TPP) can interfere with their functioning. First, we found that suppression of ABC transporter gene PDR5 increases the toxicity of C12TPP in yeast. Second, C12TPP appeared to prevent the efflux of rhodamine 6G - a fluorescent substrate of Pdr5p. Moreover, C12TPP increased the cytostatic effects of some other known Pdr5p substrates. The chemical nature of C12TPP suggests that after Pdr5p-driven extrusion the molecules return to the plasma membrane and then into the cytosol, thus effectively competing with other substrates of the pump.