Mitochondrial Import Of tRNA: Mechanistic Studies Of Post- Receptor Translocation

Mitochondrial Import Of tRNA: Mechanistic Studies Of Post Receptor Translocation Import of tRNA from the cytosol into the mitochondria of the kinetoplastid protozoon Leishmania is a multistep process consisting of the binding of tRNA to specific receptors, its transfer to an intermediate factor and translocation through the membrane. A functional RNA import complex (RIC) isolated from Leishmania mitochondria or reconstituted by cloned and expressed protein subunits are being employed in our laboratory to determine molecular basis of these steps. The RIC from mitochondria of the kinetoplastid protozoon Leishmania tropica induces translocation of tRNAs across artificial or natural membranes. tRNA import consists of a number of discrete steps beginning with the binding of the substrate to a receptor subunit RIC1 or RIC8A, followed by its transfer to a third subunit RIC9. Subsequently the tRNA passes into the vesicle interior presumably through a membrane embedded translocation channel, the composition and properties of which are largely unknown. Although the receptor binding and transfer steps have been characterized in terms of role of specific subunits, little is known about the final translocation step. Specifically the objective of my research was to reconstitute functional import pore complex on lipid bilayers and determine the permeability of such membrane vesicles by RIC channel under different biochemical and biophysical conditions with tRNA and other different small molecules, to carry out structural and functional studies of translocation by site directed mutagenesis of important subunit gene. These experiments provided detailed insights into the interaction of RIC with mitochondrial and other membranes. I have shown that subunits RIC6 and RiC9 polymerize on the membrane to form the hexamer (RIC6)3-(RIC9)3 in presence of RIC4A formed a R3 complex. The resultant complex R3 induced translocation of tRNA when the pH of the medium was lowered to ~6. This process was independent of ATP and sensitive to the protonophore m-chlorocarbonylcyanide phenylhydrazone (CCCP), and to the K+ ionophore valinomycin, but resistant to k+/h+ exchanger nigericin, indicating the requirement of a membrane potrential Δψm generated by transmembrane proton gradient. Indeed R3 mediated tRNA translocation could be induced at neutral pH by K+ diffusion potential of 60-90mV (negative inside). However, translocation was independent of tRNA sequence, and small molecules such as ATP, oligonucleotides, labeled amino acids could be taken up by R3 liposomes at pH6.0 in contrast to large molecules such as linearized plasmid DNA which fails the internalization process and shows the size specificity of R3 complex. My results indicated that the (RIC6)3-(RIC9)3 complex forms a voltage gated pore similar to mitochondrial protein import channels. To understand the critical residues involves in proton sensing points mutants of RIC6 subunits were generated in all the 6 cysteines and 6 histidine residues as some of those residues are involved in proton sensing in a homologous protein rieske fe-s protein of RIC6. I have found out the critical cysteine and histidines along the protein chain are responsible for proton sensing. Atomic force microscopy has been used to find out the nature of the reconstituted channel in vitro. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface groove of ~20 nm rim diameter and ~1 nm depth

Imipramine Exploits Histone Deacetylase 11 To Increase the IL-12/IL-10 Ratio in Macrophages Infected with Antimony-Resistant Leishmania donovani and Clears Organ Parasites in Experimental Infection

The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kalaazar patients infected with antimony-resistant Leishmania donovani (SbRLD). Previously we showed that MDR-1 upregulation in SbRLD infection is IL-10–dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from SbRLD-infected macrophages (SbRLD-Mfs) and favors accumulation of surrogates of antimonials. It inhibits IL-10–driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulateshistone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from SbRLDMfs. It abrogates SbRLD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in SbRLD-Mfs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in SbRLD-Mfs. Furthermore, exogenous rIL-12 inhibits intracellular SbRLD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in SbRLD-Mfs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ SbRLD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on SbRLD clearance from infected hosts

Translatome Regulation in Neuronal Injury and Axon Regrowth.

Transcriptional events leading to outgrowth of neuronal axons have been intensively studied, but the role of translational regulation in this process is not well understood. Here, we use translatome analyses by ribosome pull-down and protein synthesis characterization by metabolic isotopic labeling to study nerve injury and axon outgrowth proteomes in rodent dorsal root ganglia (DRGs) and sensory neurons. We identify over 1600 gene products that are primarily translationally regulated in DRG neurons after nerve injury, many of which contain a 5'UTR cytosine-enriched regulator of translation (CERT) motif, implicating the translation initiation factor Eif4e in the injury response. We further identified approximately 200 proteins that undergo robust de novo synthesis in the initial stages of axon growth. ApoE is one of the highly synthesized proteins in neurons, and its receptor binding inhibition or knockout affects axon outgrowth. These findings provide a resource for future analyses of the role of translational regulation in neuronal injury responses and axon extension

Translatome Regulation in Neuronal Injury and Axon Regrowth.

Transcriptional events leading to outgrowth of neuronal axons have been intensively studied, but the role of translational regulation in this process is not well understood. Here, we use translatome analyses by ribosome pull-down and protein synthesis characterization by metabolic isotopic labeling to study nerve injury and axon outgrowth proteomes in rodent dorsal root ganglia (DRGs) and sensory neurons. We identify over 1600 gene products that are primarily translationally regulated in DRG neurons after nerve injury, many of which contain a 5'UTR cytosine-enriched regulator of translation (CERT) motif, implicating the translation initiation factor Eif4e in the injury response. We further identified approximately 200 proteins that undergo robust de novo synthesis in the initial stages of axon growth. ApoE is one of the highly synthesized proteins in neurons, and its receptor binding inhibition or knockout affects axon outgrowth. These findings provide a resource for future analyses of the role of translational regulation in neuronal injury responses and axon extension

Locally translated mTOR controls axonal local translation in nerve injury

How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin β1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling

Nucleolin-Mediated RNA Localization Regulates Neuron Growth and Cycling Cell Size

How can cells sense their own size to coordinate biosynthesis and metabolism with their growth needs? We recently proposed a motor-dependent bidirectional transport mechanism for axon length and cell size sensing, but the nature of the motor-transported size signals remained elusive. Here, we show that motor-dependent mRNA localization regulates neuronal growth and cycling cell size. We found that the RNA-binding protein nucleolin is associated with importin β1 mRNA in axons. Perturbation of nucleolin association with kinesins reduces its levels in axons, with a concomitant reduction in axonal importin β1 mRNA and protein levels. Strikingly, subcellular sequestration of nucleolin or importin β1 enhances axonal growth and causes a subcellular shift in protein synthesis. Similar findings were obtained in fibroblasts. Thus, subcellular mRNA localization regulates size and growth in both neurons and cycling cells