BBA - Molecular Cell Research (v.1793, #11)
Editorial Board (i).
Cytokine signalling via gp130 in gastric cancer by M. Howlett; T.R. Menheniott; L.M. Judd; A.S. Giraud (1623-1633).
Cytokine signalling pathways that depend on gp130 are dysregulated in several epithelial cancers including gastric cancer. It has been established that blockade of SHP2 activation of MAPK signalling results in hyperactivation of STAT3 resulting in increased cell proliferation, angiogenesis, inflammation and inhibition of both immunocyte and epithelial cell apoptosis. Additionally, key genes regulated downstream of gp130 via MAPK activation such as the stomach-specific tumor suppressor gene tff1 are suppressed, contributing to the oncogenic outcome. The main cytokine driver of gp130 signalling in the stomach is IL-11, with IL-6 having little activity in the antral stomach in which most pathology initiates. IL-11 is up-regulated in both mouse and human gastric cancer and in pre-neoplastic mucosa. A characteristic gene signature specifically associated with IL-11 drive has been observed, although the prognostic value of the signature has not yet been assessed. Infection of human or mouse stomach with Helicobacter pylori, especially that expressing the CagA cytotoxin, produces constitutive MAPK activation, but also activated STAT3 and increases IL-11 expression. The possibility of designing and utilising small molecule inhibitors of either IL-11 or STAT3 activation may be worthwhile in developing new cancer therapeutics.
Keywords: gp130; Gastric cancer; IL-11; STAT3; Cytokine;
Insights into the molecular function of the inactivating mutations of B-Raf involving the DFG motif by Sonia Moretti; Valentina De Falco; Anna Tamburrino; Flavia Barbi; Maria Tavano; Nicola Avenia; Fausto Santeusanio; Massimo Santoro; Antonio Macchiarulo; Efisio Puxeddu (1634-1645).
BRAF gene mutations have been associated with human cancers. Among the naturally occurring mutations, two that involve amino acids of the conserved DFG motif in the activation loop (D594V and G596R), appear to be inactivating. Aim of this study was to analyze the molecular mechanisms involved in the loss of function of B-Raf inactivating mutation G596R. Furthermore, the ability of the B-Raf DFG motif mutants to generate heterodimers with C-Raf and the possible functional consequences of the B-Raf/C-Raf heterodimer formation was examined. Wet molecular experiments in HEK293T cells demonstrate that B-RafG596R is a kinase-impaired mutant. Molecular dynamics simulations show that the loss of function of B-RafG596R depends on a restraining effect of Arg596 on the catalytic residue Asp594, which results in the loss of the appropriate spatial localization and/or conformation of the latter necessary for anchoring ATP to the enzyme. Exploration of B-Raf/C-Raf heterodimer formation indicates the occurrence of functioning heterodimers in the case of all the DFG B-Raf mutants, independently from the expected differences in spatial conformation of the activation loop, although the transforming activity of the mutants appear negligible. In conclusion, this study delivers novel information on the functional properties of the B-Raf DFG motif inactivating mutants and on the mechanisms driving B-Raf/C-Raf heterodimerization and consequent C-Raf transactivation.
Keywords: BRAF mutation; B-RafV600E; B-RafD594V; B-RafG596R; Kinase activity; Molecular dynamics simulation;
Constitutive Gs-mediated, but not G12-mediated, activity of the 5-hydroxytryptamine 5-HT7(a) receptor is modulated by the palmitoylation of its C-terminal domain by Elena Kvachnina; Aline Dumuis; Jakub Wlodarczyk; Ute Renner; Maud Cochet; Diethelm W. Richter; Evgeni Ponimaskin (1646-1655).
The 5-HT7 receptor is the most recently described member of the serotonin receptor family. This receptor is mainly expressed in the thalamus, hypothalamus as well as in the hippocampus and cortex. In the present study, we demonstrate that the mouse 5-hydroxytryptamine 5-HT7(a) receptor undergoes post-translational modification by the palmitate, which is covalently attached to the protein through a thioester-type bond. Analysis of protein-bound fatty acids revealed that the 5-HT7(a) receptor predominantly contains palmitic acid. Labelling experiments performed in the presence of agonists show that the 5-HT7(a) receptor is dynamically palmitoylated in an agonist-dependent manner and that previously synthesized receptors may be subjected to repeated cycles of palmitoylation/depalmitoylation. Mutation analysis revealed that cysteine residues 404 and 438/441 located in the C-terminal receptor domain are the main palmitoylation sites responsible for the attachment of 90% of the receptor-bound palmitate. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT7(a) receptors were indistinguishable from the wild-type for their ability to interact with Gs- and G12-proteins after agonist stimulation. However, mutation of the proximal palmitoylation site Cys404-Ser (either alone or in combination with Cys438/441-Ser) significantly increased the agonist-independent, Gs-mediated constitutive 5-HT7(a) receptor activity, while the activation of Gα12-protein was not affected. This demonstrates a functional importance of 5-HT7(a) dynamic palmitoylation for the fine tuning of receptor-mediated signaling.
Keywords: G-protein coupled receptor; 5-Hydroxytriptamine; Palmitoylation; Heterotrimeric G-protein;
Induction of glioma apoptosis by microglia-secreted molecules: The role of nitric oxide and cathepsin B by So-Young Hwang; Byong-Chul Yoo; Jae-won Jung; Eok-Soo Oh; Ji-Sun Hwang; Jin-A Shin; Song-Yi Kim; Seok-Ho Cha; Inn-Oc Han (1656-1668).
Microglia contributes significantly to brain tumor mass, particularly in astrocytic gliomas. Here, we examine the cytotoxic effects of soluble components secreted from microglia culture on glioma cells. Microglia conditioned culture medium (MCM) actively stimulated apoptotic death of glioma cells, and the effects of MCM prepared from LPS- or IFN-γ-activated microglia were more pronounced. The cytotoxic effects were glioma-specific in that primary cultured rat astrocytes were not affected by MCM. A donor of peroxynitrite induced glioma-specific cell death. In addition, NO synthase inhibitor suppressed glioma cell death induced by activated MCM, indicating that NO is one of the key molecules responsible for glioma cytotoxicity mediated by activated MCM. However, since unstimulated resting microglia produces low or very limited level of NO, MCM may contain other critical molecule(s) that induce glioma apoptosis. To identify the proteins secreted in MCM, proteomic analysis was performed on control or activated medium. Among over 200 protein spots detected by Coomassie blue staining, we identified 26 constitutive and 28 LPS- or IFN-γ-regulated MCM proteins. Several cathepsin proteases were markedly expressed, which were reduced upon activation. In particular, suppression of cathepsin B by the chemical inhibitors significantly reversed MCM-induced glioma cell death, implying a critical role of this protease in cytotoxicity. Our findings provide evidence on the functional implications of specific microglial-secreted proteins in glioma cytotoxicity, as well as a basis to develop a proteomic databank of both basal and activation-related proteins in microglia.
Keywords: Microglia; Glioma; Apoptosis;
The cytosolic domain of PEX3, a protein involved in the biogenesis of peroxisomes, binds membrane lipids by Manuel P. Pinto; Cláudia P. Grou; Marc Fransen; Clara Sá-Miranda; Jorge E. Azevedo (1669-1675).
According to current models, most newly synthesized peroxisomal intrinsic membrane proteins are recognized in the cytosol and targeted to the peroxisomal membrane by PEX19. At the organelle membrane the PEX19-cargo protein complex interacts with PEX3, a protein believed to possess only one transmembrane domain and exposing the majority of its polypeptide chain into the cytosol. In agreement with this topological model, a recombinant protein comprising the cytosolic domain of PEX3 can be purified in a soluble and monomeric form in the absence of detergents or other solubilizing agents. Here, we show that this recombinant protein actually precipitates when incubated with mild detergents, suggesting that this domain of PEX3 interacts with amphipathic molecules. Following this observation, we tested this recombinant protein in lipid-binding assays and found that it interacts strongly with liposomes inducing their flocculation or even partial solubilization. The implications of these findings are discussed.
Keywords: PEX3; PEX19; Peroxisome; Membrane lipid; Biogenesis; Small unilamelar vesicle;
Identification of DH IC-2 as a HIF-1 independent protein involved in the adaptive response to hypoxia in tumor cells: A putative role in metastasis by Sebastien Pyr dit Ruys; Edouard Delaive; Catherine Demazy; Marc Dieu; Martine Raes; Carine Michiels (1676-1690).
The master regulator of the adaptive response to hypoxia is HIF-1. However, while some data show that HIF-1 can control more than 80% of the genes induced under hypoxia, other experiments clearly demonstrate that a part of the hypoxic response is independent of HIF-1. The goal of this study was to identify some of these HIF-1 independent factors and to investigate their functional role in the adaptation of tumor cells to hypoxia. We show that the cytoplasmic dynein intermediate chain 2 (DH IC-2), a component of an intracellular ATPase minus-end directed tubulin-based motile complex, was stabilized and post-translationally modified under hypoxia in a HIF-1 independent way. We identified this modification as a phosphorylation by protein kinase C, which is inhibited under hypoxia. In parallel, the migration of HepG2 cells was enhanced under hypoxia. Cell migration was also increased, to the same extent, by the invalidation of DH IC-2 using siRNA. Taken together, these results suggest that under hypoxia, a specific modification of DH IC-2 may modulate its activity, and in turn promote cell migration. These results are important to better understand cancer development since they highlight a HIF-1 independent mechanism, which may be involved in metastasis.
Keywords: Hypoxia; Cancer; Dynein; Metastasis; HIF-1 independent; Proteomics;
Rasosomes spread Ras signals from plasma membrane ‘hotspots’ by Merav Kofer-Geles; Irit Gottfried; Roni Haklai; Galit Elad-Zefadia; Yoel Kloog; Uri Ashery (1691-1702).
Ras proteins regulate cell growth, differentiation, and apoptosis from various cellular platforms. We have recently identified a novel potential signaling platform, the rasosome, which moves rapidly near the plasma membrane (PM) and in the cytosol, carrying multiple copies of palmitoylated Ras proteins. In the present study we demonstrate that rasosomes are unique entities distinct from PM nanoclusters or from endocytotic compartments. In addition, we examine whether rasosomes can act as regulated Ras signaling platforms. We show that a single rasosome simultaneously carries different types of Ras molecules in their active and inactive state, suggesting that rasosomes can upload and download Ras signals. Total internal reflection fluorescence (TIRF) microscopy combined with fast time-lapse and a new spatial analysis algorithm demonstrate that rasosome movement near the PM is restricted to distinctive areas, rasosomal ‘hotspots’, localized between actin filament cages. In addition, Ras-binding domain of Raf-1 (RBD) is recruited to Ras in rasosomal hotspots as revealed by bimolecular fluorescence complementation experiments. Interestingly, epidermal growth factor stimulates H/NRas activation on rasosomes and the subsequent recruitment of RBD to rasosomes. Moreover, we show that rasosomes are loaded with Ras downstream effectors and modulators. These findings establish that physiological stimulation originating from PM hotspots is transduced to rasosomes, which appear to serve as robust Ras signaling platforms that spread signals across the cell.
Keywords: Ras protein; Rasosomes; TIRF; Ras signaling; Actin mesh;
Yeast prion [PSI + ] lowers the levels of mitochondrial prohibitins by Jacek Sikora; Joanna Towpik; Damian Graczyk; Michał Kistowski; Tymon Rubel; Jaroslaw Poznanski; James Langridge; Chris Hughes; Michał Dadlez; Magdalena Boguta (1703-1709).
We report proteomic analyses that establish the effect of cytoplasmic prion [PSI + ] on the protein complement of yeast mitochondria. A set of 44 yeast mitochondrial proteins whose levels were affected by [PSI + ] was identified by two methods of gel-free and label-free differential proteomics. From this set we focused on prohibitins, Phb1 and Phb2, and the mitochondrially synthesized Cox2 subunit of cytochrome oxidase. By immunoblotting we confirmed the decreased level of Cox2 and reduced mitochondrial localization of the prohibitins in [PSI + ] cells, which both became partially restored by [PSI + ] curing. The presence of the [PSI + ] prion also caused premature fragmentation of mitochondria, a phenomenon linked to prohibitin depletion in mammalian cells. By fractionation of cellular extracts we demonstrated a [PSI + ]-dependent increase of the proportion of prohibitins in the high molecular weight fraction of aggregated proteins. We propose that the presence of the yeast prion causes newly synthesized prohibitins to aggregate in the cytosol, and therefore reduces their levels in mitochondria, which in turn reduces the stability of Cox2 and possibly of other proteins, not investigated here in detail.
Keywords: Differential proteomics; Yeast mitochondria; Yeast prion; Prohibitin;
SPFH1 and SPFH2 mediate the ubiquitination and degradation of inositol 1,4,5-trisphosphate receptors in muscarinic receptor-expressing HeLa cells by Yuan Wang; Margaret M.P. Pearce; Danielle A. Sliter; James A. Olzmann; John C. Christianson; Ron R. Kopito; Stephanie Boeckmann; Christine Gagen; Gil S. Leichner; Joseph Roitelman; Richard J.H. Wojcikiewicz (1710-1718).
Inositol 1,4,5-trisphosphate (IP3) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. While it is clear that IP3 receptors are polyubiquitinated and are transferred to the proteasome by a p97-based complex, currently very little is known about the proteins that initially select activated IP3 receptors for ERAD. Here, we have transfected HeLa cells to stably express m3 muscarinic receptors to allow for the study of IP3 receptor ERAD in this cell type, and show that IP3 receptors are polyubiquitinated and then degraded by the proteasome in response to carbachol, a muscarinic agonist. In seeking to identify proteins that mediate IP3 receptor ERAD we found that both SPFH1 and SPFH2 (also known as erlin 1 and erlin 2), which exist as a hetero-oligomeric complex, rapidly associate with IP3 receptors in a manner that precedes polyubiquitination and the association of p97. Suppression of SPFH1 and SPFH2 expression by RNA interference markedly inhibited carbachol-induced IP3 receptor polyubiquitination and degradation, but did not affect carbachol-induced calcium mobilization or IκBα processing, indicating that the SPFH1/2 complex is a key player in IP3 receptor ERAD, acting at a step after IP3 receptor activation, but prior to IP3 receptor polyubiquitination. Suppression of SPFH1 and SPFH2 expression had only slight effects on the turnover of some exogenous model ERAD substrates, and had no effect on sterol-induced ERAD of endogenous 3-hydroxy-3-methylglutaryl-CoA reductase. Overall, these studies show that m3 receptor-expressing HeLa cells are a valuable system for studying IP3 receptor ERAD, and suggest that the SPFH1/2 complex is a factor that selectively mediates the ERAD of activated IP3 receptors.
Keywords: Inositol 1,4,5-trisphosphate receptor; SPFH1; SPFH2; Endoplasmic reticulum-associated degradation; Ubiquitin; Proteasome;
FGF1 nuclear translocation is required for both its neurotrophic activity and its p53-dependent apoptosis protection by Aida Rodriguez-Enfedaque; Sylvina Bouleau; Maryvonne Laurent; Yves Courtois; Bernard Mignotte; Jean-Luc Vayssière; Flore Renaud (1719-1727).
Fibroblast growth factor 1 (FGF1) is a differentiation and survival factor for neuronal cells both in vitro and in vivo. FGF1 activities can be mediated not only by paracrine and autocrine pathways involving FGF receptors but also by an intracrine pathway, which is an underestimated mode of action. Indeed, FGF1 lacks a secretion signal peptide and contains a nuclear localization sequence (NLS), which is consistent with its usual intracellular and nuclear localization. To progress in the comprehension of the FGF1 intracrine pathway in neuronal cells, we examined the role of the nuclear translocation of FGF1 for its neurotrophic activity as well as for its protective activity against p53-dependent apoptosis. Thus, we have transfected PC12 cells with different FGF1 expression vectors encoding wild type or mutant (Δ NLS) FGF1. This deletion inhibited both FGF1 nuclear translocation and FGF1 neurotrophic activity (including differentiation and serum-free cell survival). We also show that endogenous FGF1 protection of PC12 cells against p53-dependent cell death requires FGF1 nuclear translocation. Strikingly, wild type FGF1 is found interacting with p53, in contrast to the mutant FGF1 deleted of its NLS, suggesting the presence of direct and/or indirect interactions between FGF1 and p53 pathways. Thus, we present evidences that FGF1 may act by a nuclear pathway to induce neuronal differentiation and to protect the cells from apoptosis whether cell death is induced by serum depletion or p53 activation.
Keywords: FGF1; Differentiation; Serum-free survival; p53-dependent apoptosis; PC12 cell;
Microscopic dissection of the process of stress granule assembly by Ken Fujimura; Jun Katahira; Fumi Kano; Yoshihiro Yoneda; Masayuki Murata (1728-1737).
Stress granules (SGs) are mRNA triage sites that are formed in response to a variety of cellular stress. To study how SGs bring about the massive spatial compartmentalization, we monitored the localization of various RNA-binding proteins (RBPs) targeted to SGs upon exposure to stress. We discovered that concomitant with the onset of eIF2α phosphorylation, RBPs accumulate locally in the cytoplasm, which leads to increased inter-molecular interactions and the formation of robustly detergent-resistant foci. Subsequently, microtubules (MTs) mediate 1) the ordered spatial organization of SGs and 2) the recruitment of a set of nuclear-localized SG components to the cytoplasm. Meanwhile, MTs did not appear to be required for the maintenance of SG distribution after its assembly. Our data suggest that the process of SG formation is composed of MT-independent and -dependent pathways, which take place sequentially during stress response.
Keywords: Stress granule; Oxidative stress; RNA-binding protein; Microtubule;
Structural and functional diversity in the family of small heat shock proteins from the parasite Toxoplasma gondii by Natalia de Miguel; Nathalie Braun; Alexander Bepperling; Thomas Kriehuber; Andreas Kastenmüller; Johannes Buchner; Sergio O. Angel; Martin Haslbeck (1738-1748).
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones which prevent the nonspecific aggregation of non-native proteins. Five potential sHsps exist in the parasite Toxoplasma gondii. They are located in different intracellular compartments including mitochondria and are differentially expressed during the parasite's life cycle. Here, we analyzed the structural and functional properties of all five proteins. Interestingly, this first in vitro characterization of sHsps from protists showed that all T. gondii sHsps exhibit the characteristic properties of sHsps such as oligomeric structure and chaperone activity. However, differences in their quaternary structure and in their specific chaperone properties exist. On the structural level, the T. gondii sHsps can be divided in small (12–18 subunits) and large (24–32 subunits) oligomers. Furthermore, they differ in their interaction with non-native proteins. While some bind substrates tightly, others interact more transiently. The chaperone activity of the three more mono-disperse T. gondii sHsps is regulated by temperature with a decrease in temperature leading to the activation of chaperone activity, suggesting an adaption to specific steps of the parasite's life cycle.
Keywords: sHsp; α-crystallin; Toxoplasma gondii; Protein folding; Protein aggregation;
Histone deacetylase inhibitors repress macrophage migration inhibitory factor (MIF) expression by targeting MIF gene transcription through a local chromatin deacetylation by Jérôme Lugrin; Xavier C. Ding; Didier Le Roy; Anne-Laure Chanson; Fred C.G.J. Sweep; Thierry Calandra; Thierry Roger (1749-1758).
The cytokine macrophage migration inhibitory factor plays a central role in inflammation, cell proliferation and tumorigenesis. Moreover, macrophage migration inhibitory factor levels correlate with tumor aggressiveness and metastatic potential. Histone deacetylase inhibitors are potent antitumor agents recently introduced in the clinic. Therefore, we hypothesized that macrophage migration inhibitory factor would represent a target of histone deacetylase inhibitors. Confirming our hypothesis, we report that histone deacetylase inhibitors of various chemical classes strongly inhibited macrophage migration inhibitory factor expression in a broad range of cell lines, in primary cells and in vivo. Nuclear run on, transient transfection with macrophage migration inhibitory factor promoter reporter constructs and transduction with macrophage migration inhibitory factor expressing adenovirus demonstrated that trichostatin A (a prototypical histone deacetylase inhibitor) inhibited endogenous, but not episomal, MIF gene transcription. Interestingly, trichostatin A induced a local and specific deacetylation of macrophage migration inhibitory factor promoter-associated H3 and H4 histones which did not affect chromatin accessibility but was associated with an impaired recruitment of RNA polymerase II and Sp1 and CREB transcription factors required for basal MIF gene transcription. Altogether, this study describes a new molecular mechanism by which histone deacetylase inhibitors inhibit MIF gene expression, and suggests that macrophage migration inhibitory factor inhibition by histone deacetylase inhibitors may contribute to the antitumorigenic effects of histone deacetylase inhibitors.
Keywords: Macrophage migration inhibitory factor; Histone deacetylase inhibitor; Trichostatin A; Chromatin; Transcription factor;
Visfatin through STAT3 activation enhances IL-6 expression that promotes endothelial angiogenesis by Jee-Young Kim; Yun-Hee Bae; Moon-Kyoung Bae; Su-Ryun Kim; Hyun-Joo Park; Hee-Jun Wee; Soo-Kyung Bae (1759-1767).
Signal transducer and activator of transcription 3 (STAT3) acts as a mediator and biomarker in endothelial activation. We have recently shown that a novel adipokine visfatin promotes endothelial angiogenesis. The present study was to determine whether visfatin affects STAT3 activity and to explore the potential target gene(s). Here, we found that visfatin induced the activation of STAT3, as characterized by increased tyrosine phosphorylation, nuclear translocation, and DNA-binding activity in human endothelial cells. In addition, visfatin significantly upregulated mRNA and protein levels of endothelial interleukin-6 (IL-6), which was blocked by a specific inhibitor of STAT3 signaling and by the transfection of siRNA specific for STAT3. Furthermore, visfatin-induced angiogenesis was reduced by the inhibition of STAT3 signaling or neutralization of IL-6 function, as measured by tube formation, rat aortic ring assay, and mouse Matrigel plug assay. Taken together, our results provide the first example of STAT3-dependent endothelial IL-6 induction by visfatin and of the role of IL-6 in mediating visfatin-induced angiogenesis.
Keywords: Visfatin; STAT3; IL-6; Angiogenesis; Human endothelial cell;
Regulation and function of the cytosolic viral RNA sensor RIG-I in pancreatic beta cells by Mónica García; Zeynep Dogusan; Fabrice Moore; Shintaro Sato; Gunther Hartmann; Decio L. Eizirik; Joanne Rasschaert (1768-1775).
Enteroviral infections are associated with type I diabetes. The mechanisms by which viruses or viral products such as double-stranded RNA (dsRNA) affect pancreatic beta cell function and survival remain unclear. We have shown that extracellular dsRNA induces beta cell death via Toll-like receptor-3 (TLR3) signaling whereas cytosolic dsRNA triggers the production of type I interferons and apoptosis via a TLR3-independent process. We presently examined expression of the intracellular viral RNA sensors, the RNA helicases RIG-I and MDA5, and documented the functionality of RIG-I in pancreatic beta cells.FACS-purified rat beta cells and islet cells from wild-type or TLR3−/− mice were cultured with or without the RIG-I-specific ligand 5′-triphosphate single-stranded RNA (5′triP-ssRNA), the synthetic dsRNA polyI:C (PIC) or 5′OH-ssRNA (negative control); the RNA compounds were added in the medium or transfected in the cells using lipofectamine. RIG-I and MDA5 expression were determined by real-time RT-PCR. NF-κB and IFN-β promoter activation were studied in the presence or absence of a dominant-negative form of RIG-I (DN-RIG-I). Both extracellular (PICex) and intracellular (PICin) PIC increased expression of RIG-I and MDA5 in pancreatic beta cells. TLR3 deletion abolished PICex-induced up-regulation of the helicases in beta cells but not in dendritic cells. PICin-induced NF-κB and IFN-β promoter activation were prevented by the DN-RIG-I. The RIG-I-specific ligand 5′triP-ssRNA induced IFN-β promoter activation and beta cell apoptosis. Our results suggest that the RIG-I pathway is present and active in beta cells and could contribute to the induction of insulitis by viral RNA intermediates.
Keywords: Diabetes; RNA helicases; Toll-like receptor 3; Apoptosis;
Synthesis of cytochrome c oxidase subunit 1 is translationally downregulated in the absence of functional F1F0-ATP synthase by Ileana C. Soto; Flavia Fontanesi; Melvys Valledor; Darryl Horn; Rajiv Singh; Antoni Barrientos (1776-1786).
The mitochondrial F1F0-ATP synthase or ATPase is a key enzyme for aerobic energy production in eukaryotic cells. Mutations in ATPase structural and assembly genes are the primary cause of severe human encephalomyopathies, frequently associated with a pleiotropic decrease in cytochrome c oxidase (COX) activity. We have studied the structural and functional constraints underlying the COX defect using Saccharomyces cerevisiae genetic and pharmacological models of ATPase deficiency. In both yeast Δatp10 and oligomycin-treated wild type cells, COX assembly is selectively impaired in the absence of functional ATPase. The COX biogenesis defect does not involve a primary alteration in the expression of the COX subunits as previously suggested but in their maturation and/or assembly. Expression of COX subunit 1, however, is translationally regulated as in most bona fide COX assembly mutants. Additionally, the COX defect in oligomycin-inhibited ATPase-deficient yeast cells, but not in atp10 cells could be partially prevented by partially dissipating the mitochondrial membrane potential using the uncoupler CCCP. Similar results were obtained with oligomycin-treated and ATP12-deficient human fibroblasts respectively. Our findings imply that fully assembled ATPase and its proton pumping function are both required for COX biogenesis in yeast and mammalian cells through a mechanism independent of Cox1p synthesis.
Keywords: Mitochondria; Cytochrome c oxidase assembly; F1F0-ATPase; Cox1p translational regulation; Mitochondrial membrane potential;
Evidence for the association of luteinizing hormone receptor mRNA-binding protein with the translating ribosomes during receptor downregulation by Bindu Menon; Helle Peegel; K.M.J. Menon (1787-1794).
Luteinizing hormone receptor (LHR) mRNA is post-transcriptionally regulated during ligand-induced downregulation. This process involves interaction of LHR mRNA with a specific mRNA-binding protein (LRBP), identified as mevalonate kinase (MVK), resulting in inhibition of translation followed by targeting the ribonucleoprotein complex to accelerated degradation. The present study investigated the endogenous association of LRBP with the translational machinery and its interaction with LHR mRNA during LH/hCG-induced downregulation. Ovaries were collected from rats that were injected with the ligand, hCG, to induce downregulation of LHR mRNA expression. Western blot analysis showed significantly higher levels of LRBP in polysomes from downregulated ovaries compared to controls. Western blot analysis of ribosome-rich fractions from FPLC-assisted gel filtration of post-mitochondrial supernatants confirmed the presence of LRBP in translating ribosomes isolated from the downregulated state but not from controls. The association of LRBP with LHR mRNA in the downregulated polysomes was demonstrated by immunoprecipitation with LRBP antibody followed by qPCR analysis of the associated RNA. Increased association of LHR mRNA with LRBP during downregulation was also demonstrated by subjecting the polysome-associated RNAs to oligo(dT) cellulose chromatography followed by immunoprecipitation and qPCR analysis. Additionally, analysis of in vitro translation of LHR mRNA showed increased inhibition of translation by polysomes from downregulated ovaries compared to control. This study provides strong in vivo and in vitro evidence to show that during ligand-induced downregulation, LRBP translocates to ribosomes and associates with LHR mRNA to form an untranslatable ribonucleoprotein complex and inhibits LHR mRNA translation, paving the way to its degradation.
Keywords: Luteinizing hormone receptor; mRNA decay; mRNA-binding protein; Ribosomes; Translational inhibition;