Biomedical subjects

Roger F Duncan

Publications and source records attributed to Roger F Duncan.

6 recordsLinked to original sources

PubMed2005

Signal transduction pathways leading to increased eIF4E phosphorylation caused by oxidative stress.

Phosphorylation of eIF4E is associated with increased activity of the translational machinery. Oxidative stress of resident vascular cells and macrophages potently enhances eIF4E phosphorylation. Oxidative stress activates numerous intracellular signaling pathways, including MAP-family kinase pathways and pathways leading to S6 kinase activation. The activation of MAP-family kinase pathways leads to the activation of Mnk and hence eIF4E phosphorylation, whereas the S6 kinase pathway is not involved, based on insensitivity to its inhibitors rapamycin and wortmannin. Ca-dependent pathways have been implicated in eIF4E phosphorylation, but the oxidative stress response pathway targeting eIF4E does not appear to require their participation. The results suggest that the potent activation of ERK and p38 protein kinases is sufficient to account for the enhanced eIF4E phosphorylation. Either is independently sufficient to effect the change, as neither PD098059 (Erk pathway inhibitor) nor SB202190 (p38 pathway inhibitor) alone can block the response, but when combined the response is almost completely abrogated. Mnk activation by oxidative stress leading to enhanced eIF4E phosphorylation may play a role in promoting stress-induced hyperproliferative diseases, such as smooth muscle cell proliferation and hypertrophy in cardiovascular disease, as the synthesis of several key regulators of cell growth has been shown to be held in check by moderation of eIF4E activity.

Animals↗

PubMed2005

Inhibition of Hsp90 function delays and impairs recovery from heat shock.

The induction of the heat shock response as well as its termination is autoregulated by heat shock protein activities. In this study we have investigated whether Hsp90 functional protein levels influence the characteristics and duration of the heat shock response. Treatment of cells with several benzoquinone ansamycin inhibitors of Hsp90 (geldanamycin, herbimycin A) activated a heat shock response in the absence of heat shock, as reported previously. Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock. Concurrently, the rate of Hsp synthesis and accumulation was substantially increased and prolonged. The cessation of heat shock protein synthesis did not occur until the levels of Hsp70 were substantially elevated relative to its standard threshold for autoregulation. The elevated levels of HSPS 22-28 (the small HSPS) and Hsp70 are not able to promote thermotolerance when Hsp90 activity is repressed by ansamycins; rather a suppression of thermotolerance is observed. These results suggest that a multicomponent protein chaperone complex involving both Hsp90 and Hsp70 signals the cessation of heat shock protein synthesis, the restoration of normal translation, and likely the establishment of thermotolerance. Impaired function of either component is sufficient to alter the heat shock response.

Animals↗

PubMed2004

Translational regulation of Hsp90 mRNA. AUG-proximal 5'-untranslated region elements essential for preferential heat shock translation.

Heat shock in Drosophila results in repression of most normal (non-heat shock) mRNA translation and the preferential translation of the heat shock mRNAs. The sequence elements that confer preferential translation have been localized to the 5'-untranslated region (5'-UTR) for Hsp22 and Hsp70 mRNAs (in Drosophila). Hsp90 mRNA is unique among the heat shock mRNAs in having extensive secondary structure in its 5'-UTR and being abundantly represented in the non-heat shocked cell. In this study, we show that Hsp90 mRNA translation is inefficient at normal growth temperature, and substantially activated by heat shock. Its preferential translation is not based on an IRES-mediated translation pathway, because overexpression of eIF4E-BP inhibits its translation (and the translation of Hsp70 mRNA). The ability of Hsp90 mRNA to be preferentially translated is conferred by its 5'-UTR, but, in contrast to Hsp22 and -70, is primarily influenced by nucleotides close to the AUG initiation codon. We present a model to account for Hsp90 mRNA translation, incorporating results indicating that heat shock inhibits eIF4F activity, and that Hsp90 mRNA translation is sensitive to eIF4F inactivation.

5' Untranslated Regions↗

PubMed2003

c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis.

The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis.

Animals↗

PubMed2003

Oxidative stress increases eukaryotic initiation factor 4E phosphorylation in vascular cells.

Dysregulated cell growth can be caused by increased activity of protein synthesis eukaryotic initiation factor (eIF) 4E. Dysregulated cell growth is also characteristic of atherosclerosis. It is postulated that exposure of vascular cells, such as endothelial cells, smooth muscle cells and monocytes/macrophages, to oxidants, such as oxidized low-density lipoprotein (oxLDL), leads to the elaboration of growth factors and cytokines, which in turn results in smooth muscle cell hyperproliferation. To investigate whether activation of eIF4E might play a role in this hyperproliferative response, vascular cells were treated with oxLDL, oxidized lipid components of oxLDL and several model oxidants, including H(2)O(2) and dimethyl naphthoquinone. Exposure to each of these compounds led to a dose- and time-dependent increase in eIF4E phosphorylation in all three types of vascular cells, correlated with a modest increase in overall translation rate. No changes in eIF4EBP, eIF2 or eIF4B modification state were observed. Increased eIF4E phosphorylation was paralleled by increased presence of eIF4E in high-molecular-mass protein complexes characteristic of its most active form. Anti-oxidants at concentrations typically employed to block oxidant-induced cell signalling likewise promoted eIF4E phosphorylation. The results of this study indicate that increased eIF4E activity may contribute to the pathophysiological events in early atherogenesis by increasing the expression of translationally inefficient mRNAs encoding growth-promoting proteins.

Animals↗

PubMed2002

Hepatitis C virus IRES-dependent translation is insensitive to an eIF2alpha-independent mechanism of inhibition by interferon in hepatocyte cell lines.

Interferon (IFN) in combination with ribavirin is the main treatment for hepatitis C virus (HCV) infection. The sensitivity or resistance of the virus to IFN has been linked to certain types of the interferon sensitivity determining region (ISDR) and PKR-eIF2alpha phosphorylation homology domain (PePHD) sequences in the NS5A and E2 regions of the viral genome, respectively. In search of the other potential mechanisms of HCV resistance to IFN, we tested the effect of IFN-alpha on translational activity of the HCV IRES in various cell types. Using bicistronic dual luciferase reporter RNAs in direct RNA transfection studies, we found that the cap-dependent translation was dramatically inhibited by IFN (5- to 16-fold), whereas HCV IRES translation was inhibited only marginally in two hepatoma cell lines, Huh7 and HepG2 cells. No difference in IFN sensitivity was observed among IRESs of genotypes 1a, 1b, and 2a. Translation under the control of encephalomyocarditis virus (EMCV) IRES was inhibited by IFN to the same extent as cap-dependent translation. In cells of nonhepatic origin (HeLa and Raji), however, HCV IRES-, EMCV IRES-, and cap-dependent translation were dramatically inhibited to similar levels. The PKR expression level was enhanced by IFN in all cells, but eIF2alpha phosphorylation level was not changed, probably due to the absence of double-stranded RNA species. There was also no evidence of RNase L activation. Therefore, inhibition of translation by IFN under these conditions was probably mediated by novel IFN-induced inhibitory pathways, independent of eIF2alpha phosphorylation, while HCV IRES was not subject to this inhibition in hepatoma cell lines. Thus, HCV IRES-driven translation was resistant to IFN-induced, eIF2alpha-independent inhibition in human hepatoma cells that are frequently used in studies on HCV replication. This may present a new potential mechanism of viral resistance to IFN treatment during the early steps of virus infection.

5' Untranslated Regions↗