Silibinin inhibits expression of HIF-1α through suppression of protein translation in prostate cancer cells

Silibinin is a polyphenolic flavonoid isolated from the milk thistle (Silybum marianum) and is reported to exhibit anticancer properties. Recently, it has been reported that silibinin inhibits hypoxia-inducible factor-1α (HIF-1α) expression in cancer cells. However, the precise mechanism by which silibinin decreases HIF-1 expression is not fully understood. In this study, silibinin inhibited basal and hypoxia induced expression levels of HIF-1α protein in LNCaP and PC-3 prostate cancer cells, while the rate of HIF-1α protein degradation and mRNA levels were not affected. We found that the decrease in HIF-1 protein by silibinin correlated with suppression of de novo synthesis of HIF-1α protein. Silibinin inhibited global protein synthesis coincided with reduction of eIF4F complex formation and induction of phosphorylation of the translation initiation factor 2α (eIF-2α) which can cause inhibition of general protein synthesis. These results suggest that silibinin’s activity to inhibit HIF-1α protein expression is associated with the suppression of global protein translation.     

Two-step aminoacylation of tRNA without channeling in Archaea

Catalysis of sequential reactions is often envisaged to occur by channeling of substrate between enzyme active sites without release into bulk solvent. However, while there are compelling physiological rationales for direct substrate transfer, proper experimental support for the hypothesis is often lacking, particularly for metabolic pathways involving RNA. Here, we apply transient kinetics approaches developed to study channeling in bienzyme complexes to an archaeal protein synthesis pathway featuring the misaminoacylated tRNA intermediate Glu-tRNA^Gln. Experimental and computational elucidation of a kinetic and thermodynamic framework for two-step cognate Gln-tRNA^Gln synthesis demonstrates that the misacylating aminoacyl-tRNA synthetase (GluRS^ND) and the tRNA-dependent amidotransferase (GatDE) function sequentially without channeling. Instead, rapid processing of the misacylated tRNA intermediate by GatDE and preferential elongation factor binding to the cognate Gln-tRNA^Gln together permit accurate protein synthesis without formation of a binary protein-protein complex between GluRS^ND and GatDE. These findings establish an alternate paradigm for protein quality control via two-step pathways for cognate aminoacyl-tRNA formation.     

Liver growth, biosynthesis of cytidine nucleotides and level of cytochrome P-450 in rat liver after administration of alpha-hexachlorocyclohexane.

The biosynthesis of cytidine nucleotides and the level of microsomal cytochrome P-450 in intact and regenerating rat liver after repeated administration of alpha-hexachlorocyclohexane (alpha-HCH) were compared. In alpha-HCH treated animals the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides is suppressed. In 24-h regenerating liver the incorporation of labelled orotic acid into cytidine nucleotides is markedly activated; the degree of activation is lower in regenerating livers of alpha-HCH treated animals. The changes in the level of cytochrome P-450 vary inversely with the changes in the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides. The activity of cytidine triphosphate synthetase of liver cytosol increases shortly after the administration of alpha-HCH; uridine-cytidine kinase is enhanced in the later stages of the drug action. Within 15-45 min after the administration of alpha-HCH the uptake of [U-14 C] cytidine into the liver and its incorporation into RNA cytosine are increased. After the administration of the drug the uptake of [2-14 C] uridine and its incorporation into RNA uracil is also enhanced whereas its utilization for the synthesis of cytidine nucleotides of the acid-soluble extract as well as for the RNA cytosine are suppressed.     

A conserved domain of herpes simplex virus ICP34.5 regulates protein phosphatase complex in mammalian cells

ICP34.5, encoded by herpes simplex virus 1, is a protein phosphatase 1 (PP1) regulatory subunit that mediates dephosphorylation of the alpha subunit of translation initiation factor 2 (eIF2alpha). However, the mechanism of its action remains poorly understood. Here, we show that amino acid substitutions in the arginine-rich motif have differential effects on ICP34.5 activity. The phenotypes parallel with viral protein synthesis and cytopathic effects in virus infected cells. Besides the consensus PP1 binding motif, the Arg-motif appears to enhance the interaction between ICP34.5 and PP1. These results suggest that concerted action between the PP1 binding domain and the effector domain of ICP34.5 is crucial for eIF2alpha dephosphorylation and viral protein synthesis.     

Molecular architecture of leishmania EF-1alpha reveals a novel site that may modulate protein translation: a possible target for drug development

Elongation factor-1alpha plays an essential role in eukaryotic protein biosynthesis. Recently, we have shown by protein structure modeling the presence of a hairpin-loop of 12 amino acids in mammalian EF-1alpha that is absent in the leishmania homologue [D. Nandan, A. Cherkasov, R. Sabouti, T. Yi, N.E. Reiner, Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue, Biochem. Biophys. Res. Commun. 302 (2003) 646-652]. As a consequence of this deletion, an exposed region is available on the main body of leishmania EF-1alpha. Here we report the generation of an anti-EF-1alpha antibody (DN-3) which bound selectively to the exposed region of leishmania EF-1alpha, with no reactivity with human EF-1alpha. In a leishmania cell-free protein translation system, DN-3 substantially inhibited protein translation. A similar inhibitory effect was observed when a specific peptide based on the exposed region was used in the cell-free protein translation assay. The application of structure-based in silico methods to identify potential ligands to target the exposed region identified a small molecule that selectively attenuated in vitro translation using leishmania extracts. Moreover, this small molecule showed selective suppressive effect on multiplication of leishmania in culture. Taken together, these findings identify a novel, exposed region in leishmania EF-1alpha that may be involved in protein synthesis and a potential site for drug targeting.     

Translational control of immunoglobulin synthesis. I. Repression of heavy chain synthesis

When myeloma cells are incubated at 25 °C the secretion of myeloma protein ceases within 20 minutes. The synthesis of heavy and light chains and the assembly into the completed 7 S immunoglobulin continue at over 40% of the synthetic rate at 37 °C, resulting in an increasing intracellular concentration of myeloma protein with time. When myeloma cells containing an increased myeloma protein pool were re-incubated at 37 °C, there was an initially decreased synthesis of H-chain² relative to L-chain or total protein. Whereas L-chain synthesis returned to the pre-25 °C synthetic rate within 15 minutes, the synthesis of H-chain required over 60 minutes to return to the pre-incubation rate.Myeloma cells maintained in exponential growth contain a larger intracellular pool of H₂L₂ than cells in late stationary phase. When both populations of cells were incubated at 25 °C and the synthesis of H and L-chain protein measured, a reduced synthesis of H-chain was again observed. Exponentially growing cells showed an 80% reduction of H-chain synthesis after 100 minutes at 25 °C. Stationary cells, with the reduced intracellular level of H₂L₂, required 210 minutes to effect an equivalent reduction of H-chain synthesis.The opposite effect on myeloma protein synthesis was observed following depletion of the H₂L₂ pool. The intracellular H₂L₂ pool was reduced by allowing secretion in the absence of protein synthesis. When protein synthesis was allowed to continue following the depletion, a stimulation of myeloma protein synthesis relative to total protein synthesis was observed.These experiments suggest a close relation between the intracellular level of H₂L₂ and the production of H-chain. From the rapidity of the repression and de-repression of H-chain synthesis, a regulation at the translational level is suggested.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

Synthesis of macromolecules and rubratoxin by Penicillium rubrum

The relationship between primary metabolism and biosynthesis of rubratoxin was studied with replacement cultures of Penicillium rubrum 3290. Synthesis of protein and RNA was measured by determining incorporation of [U¹⁴C]L-leucine and [2¹⁴C]-uridine into the respective components of the fungal biomass. Rubratoxin formation was measured by determining incorporation of [1¹⁴C]acetate into the toxin. Both protein and RNA were synthesized rapidly with synthesis increasing during 108 h of incubation and then decreasing rapidly. Rubratoxin formation increased up to 72 h, declined through 96 h, became maximal at 108 h, and then decreased rapidly. Cycloheximide, at 100 g/ml, moderately blocked accumulation of dry weight and protein synthesis by the mold; at 150 g/ml, cycloheximide completely blocked in vivo synthesis of protein. When cycloheximide was added to cultures after synthesis of toxin had begun, protein synthesis, but not toxin formation, was blocked. Inhibition of protein synthesis by cycloheximide was reversed by washing the drug out of mold cultures. Rubratoxin was formed throughout the incubation; a transitional phase, characteristic of secondary biosynthesis, was not observed.     

双链RNA激活的蛋白激酶(PKR)的克隆表达及其对丙型肝炎病毒蛋白合成的抑制

α干扰素为治疗丙型肝炎病毒(HCV)感染的主要药物,但部分患者呈干扰素耐受而不能获得持久的病毒阴转,其可能的原因之一是病毒通过其编码的蛋白(NS5A及E2)抑制干扰素诱导的抗病毒效应分子——双链RNA激活的蛋白激酶(PKR)的活性.而关于PKR是否在IFN-α抗HCV的机理中起抑制作用目前仍有争议.为研究PKR对HCV蛋白合成环节是否有抑制作用,通过构建野生型 PKR真核表达载体（pPKRwt）及主要起负性调节作用的缺失突变PKR真核表达载体（pPKRΔ6）,并将pPKRwt /pPKRΔ6 与HCV复制子RNA同时转染Huh7细胞进行共表达, 用Western印迹检测 HCV IRES 下游的NPTⅡ蛋白表达水平,与转染空载体的对照细胞及单用IFN-α处理的细胞相比较.结果显示：表达PKRwt的细胞中NPTⅡ蛋白水平低于转染空载体的对照细胞,但高于经IFN-α单独处理的细胞;表达PKRΔ6的细胞中NPTⅡ蛋白水平与对照细胞无明显差别,但PKRΔ能部分抵消IFN-α的抑制作用,说明在IFN-α抑制HCV IRES指导的蛋白合成中,PKR有一定的抑制作用,但可能还有其它的PKR非依赖机制参与.     

Inhibitory effect of thyroxine on carbonic anhydrase B isozyme biosynthesis in rabbit reticulocyte lysates.

Biosynthesis of rabbit red cell carbonic anhydrase isozyme B and C was demonstrated in reticulocyte cell-free lysates by the specific immunoprecipitin reaction. Using this homologous protein synthesis system, it was found that 10^?5 to 10^?7 M thyroxine preferentially inhibited the synthesis of carbonic anhydrase B isozyme without affecting that of C isozyme. These results suggested that this inhibitory action of the protein synthesis by thyroxine may be responsible for the decreased level of the B type isozymes in human hyperthyroidism or experimental hyperthyroidism of rabbits.     

Suppression of hypoxia inducible factor-1alpha (HIF-1alpha) by YC-1 is dependent on murine double minute 2 (Mdm2)

Inhibition of HIF-1alpha activity provides an important strategy for the treatment of cancer. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) has been identified as an anti-HIF-1alpha drug in cancer therapy with unclear molecular mechanism. In the present study, we aimed to investigate the effect and mechanism of YC-1 on HIF-1alpha in a hepatocellular carcinoma cell line under hypoxic condition, which was generated by incubating cells with 0.1% O(2). The phenotypic and molecular changes of cells were determined by cell proliferation assay, apoptosis assay, luciferase promoter assay, and Western blot analysis. YC-1 arrested tumor cell growth in a dose-dependent manner, whereas it did not induce cell apoptosis. Hypoxia-induced upregulation of HIF-1alpha was suppressed by YC-1 administration. YC-1 inhibited HIF-1alpha protein synthesis under normoxia and affected protein stability under hypoxia. YC-1 suppressed the expression of total and phosphorylated forms of murine double minute 2 (Mdm2), whereas this inhibitory effect was blocked by overexpression of Mdm2. In conclusion, YC-1 suppressed both protein synthesis and stability of HIF-1alpha in HCC cells, and its inhibitory effects on HIF-1alpha were dependent on Mdm2.     

C/EBP-α, involvement of a novel transcription factor in leptin-induced VCAM-1 production in mouse chondrocytes

Leptin and vascular cell adhesion molecules-1 (VCAM-1) are two important mediators in obesity-related osteoarthritis, while the molecular mechanism linking leptin to VCAM-1 production is still obscure. Here we show that leptin upregulates VCAM-1 mRNA and protein levels in a time- and dose-dependent manner. Mechanistically, leptin induces VCAM-1 promoter activity by increasing the expression of C/EBP-α and facilitating its binding to a newly identified element in the VCAM-1 gene. Gain or loss of function studies reveal a regulatory role of C/EBP-α on VCAM-1 expression. Finally, elevated plasma leptin level correlates to increased C/EBP-α and VCAM-1 production in chondrocytes from obese mice.     

Effects of Temperature on Promastigotes of Several Species of Leishmania

ABSTRACT. Six Leishmania species were studied comparatively, in order to determine the influence of temperature "in vitro" on differentiation, infectivily and protein synthesis. Differentiation ocurred in a heterogeneous manner, even in species that produce similar clinical manifestations. Thus, no association could be found between thermosensitivity and disease. The association between expression of proteins and increasing temperatures was analyzed at 34° C by polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE), using different incubation times, and employing a technique involving metabolic incorporation of [³⁵S]-methionine. Protein synthesis was very similar in all the New World species apart from L. amazonensis , which expressed a protein of approximately 80 kDa when incubated at 34° C for 2 hours. All the tested species had in common the expression of a 70 kDa protein. Differences, however, were observed in relation to the time interval for protein expression. in L. chagasi , synthesis was detected after 30 minutes of incubation at 34° C, while L. braziliensis required 1 hour at the same temperature. The "in vivo" and "in vitro" infectivity of the differentiated forms was also analyzed, but no significant differences were observed.     

Heat-shock inhibits protein synthesis and eIF-2 activity in cultured cortical neurons

Stress, such as heat-shock, hypoxia and hypoglycemia, inhibits the initiation of protein synthesis. The effects of heat-shock on protein synthesis, eucaryotic initiation factor 2 (eIF-2) activity, protein kinase C (PKC), and casein kinase II (CKII) activities were studied in primary cortical neuronal cultures. In neurons exposed to heat-shock at 44°C for 20 min, protein synthesis is inhibited by more than 80%, and is accompanied by a 60% decrease in eIF-2 activity. Steady state PKC and CK II activities were not affected by heat-shock. Vanadate (200 M), a protein phosphotyrosine phosphatase inhibitor, partially prevented the depression of eIF-2 activity during heat-shock, and increased CKII activity by 90%. In contrast, staurosporine (62nM), a protein kinase C inhibitor, did not affect eIF-2 activity. We conclude that heat-shock causes a change in the phosphorylation/ dephosphorylation of regulatory proteins leading to a depressed eIF-2 activity and protein synthesis in neurons.