81 research outputs found
Leishmania mitochondrial tRNA importers
No full textThe RNA Import Complex (RIC) is a multi-subunit protein complex from the mitochondria of the kinetoplastid protozoon Leishmania tropica that induces transport of tRNA across natural and artificial membranes. Leishmania, Trypanosoma and related genera of the order Kinetoplastidae are early diverging, a typical eukaryotes with unique RNA metabolic pathways, including the import of nucleus-encoded tRNAs into the mitochondrion to complement the deletion of all organelle-encoded tRNA genes. Biochemical and genetic studies of RIC are contributing to greater understanding of the mechanism of import. Additionally, RIC was shown to act as an efficient delivery vehicle for tRNA and other small RNAs into mitochondria within intact mammalian cells, indicating its applicability to the management of diseases caused by mitochondrial mutations
Effects of Transient Hypoxia versus Prolonged Hypoxia on Satellite Cell Proliferation and Differentiation In Vivo
No full textThe microenvironment of the injury site can have profound effects on wound healing. Muscle injury results in ischemia leading to short-term local hypoxia, but there are conflicting reports on the role of hypoxia on the myogenic program in vivo and in vitro. In our rat model of mitochondrial restoration (MR), temporary upregulation of mitochondrial activity by a cocktail of organelle-encoded RNAs results in satellite cell proliferation and initiation of myogenesis. We now report that MR leads to a transient hypoxic response in situ. Inhibition of hypoxia by lowering mitochondrial O2 consumption, either by respiratory electron transport inhibitors, or by NO-mediated inhibition of O2 binding to cytochrome c oxidase, resulted in exacerbation of inflammation. Lentivirus-mediated knockdown of hypoxia-inducible factor 1α (HIF1α) or of Notch signaling components had a similar effect, and pharmacologic inhibition of HIF or Notch reduced the number of proliferating Pax7+ cells. In contrast, a prolonged hypoxic response induced either by uncoupling of respiration from oxidative phosphorylation or through HIF stabilization by dimethyloxalylglycine (DMOG) had an immediate anti-inflammatory effect. Although significant satellite cell proliferation occurred in presence of DMOG, expression of differentiation markers was affected. These results emphasize the importance of transient hypoxia as opposed to prolonged hypoxia for myogenesis
Suppression of reactive oxygen species in cells with multiple mitochondrial DNA deletions by exogenous protein-coding RNAs
No full textOrganization and Chromosomal Localization of ß-Tubulin Genes in Leishmania Donovani
No full textThe genomic organization and chromosomal location of the ß-tubulin isogenes
in Leishmania donovani promastigotes has been studied by nucleic acid hybridization techniques using a cloned ß-tubulin gene. We have cloned a ß-tubulin gene fragment, 3·3 kbp long, from genomic DNA of Leishmania donovani using a heterologous ß-tubulin DNA as probe. Restriction maps of this clone have been prepared. It has been estimated that there are approximately 11-15 copies of the ß-tubulin genes per haploid genome. The majority of these isogenes are arranged in a tandem repeat with a length of 3·5 kbp on a single chromosome. In addition a few dispersed gene copies at different chromosomal loci were detected by pulse field gradient gel electrophoresis. Part of the internal coding region of the gene has been sequenced to confirm the identity of the ß-tubulin clone and is found to be nearly identical to that of Leishmania mexicana amazonensis
Import of RNA into Leishmania Mitochondria Occurs through Direct Interaction with Membrane-bound Receptors
No full textCytoplasmic tRNAs are imported into the kinetoplast
mitochondrion of Leishmania, but the mechanism of import
is unknown, particularly whether RNA is transferred
as a ribonucleoprotein complex through the protein
import pathway or by a distinct receptor-mediated
mechanism. Using isolated mitochondria, it was shown
that a small, importable RNA, which is structurally homologous to tRNA, binds rapidly, specifically, and with
high affinity to the mitochondrial surface in the absence
of soluble protein factors to form an import intermediate.
Two classes of binding site of apparent Kd 0.3 and 10
nM, respectively, were distinguished. tRNA from Leishmania,
but not yeast, competitively inhibited the binding.
Northwestern blot analysis revealed the presence of
a 15-kDa RNA binding protein on the mitochondrial surface.
Whereas receptor binding was resistant to heparin
and KCl, internalization was sensitive to both reagents.
These results are consistent with the presence of a direct
mechanism of receptor-mediated RNA import on
Leishmania mitochondria
The α-Subunit of Leishmania F1 ATP Synthase Hydrolyzes ATP in Presence of tRNA
No full textImport of tRNAs into the mitochondria of the kinetoplastid
protozoon Leishmania requires the tRNA-dependent hydrolysis
of ATP leading to the generation of membrane potential
through the pumping of protons. Subunit RIC1 of the inner
membrane RNA import complex is a bi-functional protein that
is identical to the �-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency
of the tRNA-dependent ATPase activity of the mitochondrial
inner membrane. Moreover, RIC1 knockdown extracts
failed to generate a membrane potential across reconstituted
proteoliposomes, as shown by a rhodamine 123 uptake assay,
but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase �-subunit that acts as the major energy transducer fortRNA import
Corrigendum to “Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K+ channel” [Biochim. Biophys. Acta 1828 (2013) 845–850]
No full textRole of terminal dipole charges in aggregation of α-helix pair in the voltage gated K+ channel
No full textAbstractThe voltage sensor domain (VSD) of the potassium ion channel KvAP is comprised of four (S1–S4) α-helix proteins, which are encompassed by several charged residues. Apart from these charges, each peptide α-helix having two inherent equal and opposite terminal dipolar charges behave like a macrodipole. The activity of voltage gated ion channel is electrostatic, where all the charges (charged residues and dipolar terminal charges) interact with each other and with the transmembrane potential. There are evidences that the role of the charged residues dominate the stabilization of the conformation and the gating process of the ion channel, but the role of the terminal dipolar charges are never considered in such analysis. Here, using electrostatic theory, we have studied the role of the dipolar terminal charges in aggregation of the S3b–S4 helix pair of KvAP in the absence of any external field (V=0). A system attains stability, when its potential energy reaches minimum values. We have shown that the presence of terminal dipole charges (1) change the total potential energy of the charges on S3b–S4, affecting the stabilization of the α-helix pair within the bilayer lipid membrane and (2) the C- and the N-termini of the α-helices favor a different dielectric medium for enhanced stability. Thus, the dipolar terminal charges play a significant role in the aggregation of the two neighboring α-helices
Effect of dielectric interface on charge aggregation in the voltage-gated K+ion channel
No full textThere is experimental evidence of many cases of stable macromolecular conformations with charged amino-acids facing lipid, an arrangement thought to be energetically unfavourable. Employing classical electrostatics, we show that, this is not necessarily the case and studied the physical basis of the specific role of proximity of charges to the dielectric interface between two different environments. We illustrate how self and induced energies due to the dielectric medium polarization, on either side of the interface, contribute differentially to the stability of a pair of charges and hence the mutual conformation of the S3b-S4 α-helix pair of the voltage-gated K(+) channel. We show that (1) a pair of opposite charges on either side of lipid-protein interface confers significant stability; (2) hydrophobic media has an important role in holding together two similar repelling charges; (3) dielectric interface has stabilizing effect on a pair of charges, when an ion is closer to its interface than its neighboring charge; (4) in spite of the presence of dielectric interface, there is a nonexistence of any dielectric effect, when an ion is equidistant from its image and neighboring charge. We also demonstrate that, variation in dielectric media of the surrounding environment confers new mutual conformations to S3b-S4 α-helices of voltage sensor domain at zero potential, especially lipid environment on the helix side, which improved stability to the configuration by lowering the potential energy. Our results provide an answer to the long standing question of why charges face hydrophobic lipid membranes in the stable conformation of a protein
The complexity of mitochondrial tRNA import
No full textImport of nucleus-encoded, cytoplasmic tRNAs into mitochondria to compensate evolutionary loss of the corresponding mitochondrial genes has been documented in a large number of species. Although the phenomenon has been known for more than 25 years, it was only recently that the mechanism of tRNA import started receiving the sustained attention of workers investigating yeast, protozoal and higher plant systems. The purpose of this review is to summarize recent developments that shed new light on the selectivity of the process, the identity of the import apparatus and the nature of the bioenergetic transactions leading to tRNA translocation, and to build a working model of the import complex suggested by these observations
- …
