Differential regulation of expression of the multiple ADP/ATP translocase genes in human cells

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been investigated in cultured cell systems under different experimental conditions, using isoform- specific probes. In several human cell lines tested, i.e. HeLa, Hep3B, 143B, HL60, the T3 gene is expressed as a single 1300-nucleotide mRNA, whereas the T2 gene produces two species of mRNA, 1450 and 1600 nucleotides in size. These two species, which are present in HeLa cells in approximately equivalent amounts, were shown to derive from the use of two different polyadenylation signals. The gene for the muscle-specific isoform of ADP/ATP translocase, T1, was not found to be expressed in any of the cell lines investigated. The levels of T2 and T3 mRNAs in HeLa cells are differentially affected by the growth conditions. In fact, the T2 mRNA level remains relatively constant throughout the exponential and stationary phases, whereas the T3 mRNA level decreases progressively in the second half of the exponential phase and in the stationary phase down to less than 50%. This difference in quantitative behavior of the two mRNAs must reflect changes in their rates of synthesis, since their half-lives are very similar (t^1/2 = 5-6 h), with no significant growth-related differences. Treatment of HL60 cells with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid, two agents which induce cessation of cell proliferation and cell differentiation, resulted in a marked decrease in both T2 and T3 mRNA levels. Exposure of HeLa cells to chloramphenicol produced a pronounced decrease in the levels of both T2 and T3 mRNAs after 48 to 72 h of treatment. Half-life time measurements strongly suggested that this decrease reflected a reduction in the rate of synthesis of the two transcripts. Treatment of HeLa cells with dinitrophenol also produced a dramatic decrease in the steady state levels of both T2 and T3 mRNA, which, however, in contrast to the just mentioned situation, could be accounted for by a decrease in their metabolic stability. Control experiments indicated that the chloramphenicol- and dinitrophenol-induced changes were not a nonspecific consequence of mitochondrial dysfunction. The observations reported here clearly demonstrate that the expression of the multiple ADP/ATP translocase genes in human cells is sensitive to the cell physiological conditions, responding to the varying cellular demands by changes in the rate of synthesis or stability of their mRNAs

eEF1Bγ binds the Che-1 and TP53 gene promoters and their transcripts

Background: We have previously shown that the eukaryotic elongation factor subunit 1B gamma (eEF1Bγ) interacts with the RNA polymerase II (pol II) alpha-like subunit “C” (POLR2C), alone or complexed, in the pol II enzyme. Moreover, we demonstrated that eEF1Bγ binds the promoter region and the 3’ UTR mRNA of the vimentin gene. These events contribute to localize the vimentin transcript and consequentially its translation, promoting a proper mitochondrial network. Methods: With the intent of identifying additional transcripts that complex with the eEF1Bγ protein, we performed a series of ribonucleoprotein immunoprecipitation (RIP) assays using a mitochondria-enriched heavy membrane (HM) fraction. Results: Among the eEF1Bγ complexed transcripts, we found the mRNA encoding the Che-1/AATF multifunctional protein. As reported by other research groups, we found the tumor suppressor p53 transcript complexed with the eEF1Bγ protein. Here, we show for the first time that eEF1Bγ binds not only Che-1 and p53 transcripts but also their promoters. Remarkably, we demonstrate that both the Che-1 transcript and its translated product localize also to the mitochondria and that eEF1Bγ depletion strongly perturbs the mitochondrial network and the correct localization of Che-1. In a doxorubicin (Dox)-induced DNA damage assay we show that eEF1Bγ depletion significantly decreases p53 protein accumulation and slightly impacts on Che-1 accumulation. Importantly, Che-1 and p53 proteins are components of the DNA damage response machinery that maintains genome integrity and prevents tumorigenesis. Conclusions: Our data support the notion that eEF1Bγ, besides its canonical role in translation, is an RNA-binding protein and a key player in cellular stress responses. We suggest for eEF1Bγ a role as primordial transcription/translation factor that links fundamental steps from transcription control to local translatio

Mitochondrial RNA processing in health and disease

Mitochondria are often described as the powerhouses of the cell, providing the main cellular energy source in the form of adenosine triphosphate. Five enzyme complexes, collectively termed the oxidative phosphorylation system, use the reducing power from nutrients to synthesise adenosine triphosphate via cellular respiration. This energy conversion is dependent on factors encoded by the nuclear and mitochondrial genome, with the latter encoding 13 subunits within four of the five oxidative phosphorylation system complexes. Adenosine triphosphate synthesis is therefore under dual genetic control, and this thesis addresses mechanisms that control and regulate mitochondrial gene expression. The mitochondrial genome is transcribed as long, polycistronic premature transcripts, which need to undergo cleavage and maturation before they can be used for correct translation on mitochondrial ribosomes. However, the mechanisms of this RNA processing, as well as the mechanisms underlying mitochondrial RNA homeostasis, are not fully understood. Here I used the fruit fly, Drosophila melanogaster, to study factors involved in mitochondrial gene expression. In two studies I addressed the functions of factors involved in the mitochondrial degradosome, responsible for RNA turnover. Additionally, I addressed the role of polyadenylation in RNA degradation, and studied how the polyadenylation machinery affects mitochondrial translation. Finally, defects of mitochondrial gene expression can have severe clinical consequences and form an important part of human pathology. One study of this thesis validated the pathogenicity of mutations in a tRNA aminoacyl transferase gene, identified in two siblings suffering from mitochondrial disease

Action potentials as indicators of metabolic perturbations for temporal proteomic analysis

The single largest cause of compound attrition during drug development is due to inadequate tools capable of predicting and identifying protein interactions. Several tools have been developed to explore how a compound interferes with specific pathways. However, these tools lack the potential to chronically monitor the time dependent temporal changes in complex biochemical networks, thus limiting our ability to identify possible secondary signaling pathways that could lead to potential toxicity. To overcome this, we have developed an in silico neuronal-metabolic model by coupling the membrane electrical activity to intracellular biochemical pathways that would enable us to perform non-invasive temporal proteomics. This model is capable of predicting and correlating the changes in cellular signaling, metabolic networks and action potential responses to metabolic perturbation. The neuronal-metabolic model was experimentally validated by performing biochemical and electrophysiological measurements on NG108-15 cells followed by testing its prediction capabilities for pathway analysis. The model accurately predicted the changes in neuronal action potentials and the changes in intracellular biochemical pathways when exposed to metabolic perturbations. NG108-15 cells showed a large effect upon exposure to 2DG compared to cyanide and malonate as these cells have elevated glycolysis. A combinational treatment of 2DG, cyanide and malonate had a much higher and faster effect on the cells. A time-dependent change in neuronal action potentials occurred based on the inhibited pathway. We conclude that the experimentally validated in silico model accurately predicts the changes in neuronal action potential shapes and proteins activities to perturbations, and would be a powerful tool for performing proteomics facilitating drug discovery by using action potential peak shape analysis to determine pathway perturbation from an administered compound

Heat-shock pretreatment inhibits sorbitol-induced apoptosis in K562, U937 and HeLa cells.

The aim of this study was to determine whether heat-shock pretreatment exerted a protective effect against sorbitol-induced apoptotic cell death in K562, U937 and HeLa cell lines and whether such protection was associated with a decreased cytochrome c release from mithocondria and a decreased activation of caspase-9 and -3. Following heat-shock pretreatment (42 6 0.3C for 1 hr), these cell lines were exposed to sorbitol for 1 hr. Apoptosis was evaluated by DNA fragmentation, whereas caspase-9,-3 activation, cytochrome c release and heat-shock protein70 (HSP70) were assayed by Western Blot. Sorbitol exposure-induced apoptosis in these different cell lines with a marked activation of caspase-9 and caspase- 3, whereas heat-shock pretreatment before sorbitol exposure, induced expression of HSP70 and inhibited sorbitol-mediated cytochrome c release and subsequent activation of caspase-9 and caspase- 3. Similarly, overexpression of HSP70 in the three cell lines studied prevented caspase-9 cleavage and activation as well as cell death. Furthermore, we showed that the mRNA expression of iNOS decreased during both the heat-shock treatment and heat-shock pretreatment before sorbitol exposure. By contrast, the expression of Cu-Zn superoxide dismutase (SOD) and Mn-SOD proteins increased during heat-shock pretreatment before sorbitol exposure. We conclude that, heat-shock pretreatment protects different cell lines against sorbitol-induced apoptosis through a mechanism that is likely to involve SOD family members

Selective Manipulation of Tumour Cell Energy Metabolism

Rapidly growing, glycolytic tumours which are poorly differentiated were shown to contain functional mitochondria capable of oxidative phosphorylation. Mitochondria were isolated and assessed for use in screening potential antimitochondrial agents in vitro. As this model proved to be unsuitable, a second model, intact cells were employed for screening purposes. The effect of antimitochondrial agents on the metabolism of cultured tumour cells was compared to that in freshly isolated rat hepatocytes

Biochemical Aspects of Genetics

It would be impossible, in the time and space available, to cover all of the noteworthy advances that have been made in biochemical genetics during the past year. We have, therefore, selected for review a number of topics that are especially active at the moment and that promise to yield important new results in the near future. At the same time, we have tried to avoid duplicating the material of other chapters in this volume which are germane to biochemical genetics. We refer, in particular, to the chapters on Metabolism of Nucleic Acids (Macromolecular DNA and RNA); Nucleic Acids and Protein Biosynthesis; The Basic Proteins of Cell Nuclei; and Chemistry of Differentiation in Lower Organisms. The reader should consult these reviews, as well as the present one, for a fuller view of current activities in this field