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The role of an unconventional ribosomal protein in mitochondrial function
Abstracts
recoded ATP6 genes, and distinct 3′ untranslated regions (3′UTRs) to determine the combination that provides the best “rescue” or phenotypic improvement. We observe significant improvements in both longevity and locomotive impairment using a transgenic expression system, especially in flies that express a full-length algal ATP6. We have further tested Drosophila strains that contain different segments of the algal ATP6 (algal chimeras), and statistically significant improvements are again observed. We are planning to examine if the neuromuscular pathology observed in the mutant flies is alleviated in the transgenic flies with algal ATP6 expression. ATP6 synthase assays will also be performed to determine if and how much ATP6 production is resumed.
doi:10.1016/j.mito.2013.07.025
29 The role of an unconventional ribosomal protein in mitochondrial function Presenter: Tara Richman Tara R. Richman, Louis Scott, Stefan M.K. Davies, Oliver Rackham, Aleksandra Filipovska Western Australian Institute for Medical Research and Centre for Medical Research, The University of Western Australia, Perth, Western Australia 6000, Australia Body of Abstract: Mitochondria are ubiquitous organelles of eukaryotic cells that contain a circular double-stranded genome encoding 13 polypeptide subunits of the mitochondrial respiratory chain. The genes for these polypeptides are transcribed and translated in the mitochondrial matrix, using 22 tRNAs and 2 rRNAs encoded by the compact mitochondrial DNA. Consequently, mtDNA is dependent on nuclear encoded proteins for replication, repair, transcription and translation. Mammalian mitochondrial mRNAs generally begin at the start codon and lack conventional 5′ untranslated regions or ShineDalgarno sequences. In addition mitochondrial encoded proteins are hydrophobic and are likely to be co-translationally inserted into the membrane-embedded complexes providing unique constraints for their translation. Mitochondrial ribosomes must have evolved in an alternative way to regulate translation initiation and elongation to ensure accurate start codon recognition and facilitate protein complex assembly. Mammalian mitochondrial ribosomes are unique from bacterial and cytoplasmic ribosomes of eukaryotes because their ribosomal RNA has been reduced considerably and has been replaced by additional proteins. These proteins may have new functions in mitochondrial translation and recognition of mitochondrial mRNAs. We have identified an additional protein and found that it localizes to mitochondria, associates with mitochondrial ribosomes specifically, and has important roles in mitochondrial gene expression and consequently cellular energy metabolism.
doi:10.1016/j.mito.2013.07.026
30 Comparative aspects of bioenergetic profiling of tumor clinical material and cell cultures Igor Shevchuka, Andre Koita, Andrus Kaldmaaa, Natalja Timohhinaa, Vladimir Chekulayeva, Kersti Teppa, Jelena Bogovskajac, Vahur Valverec,d, Valdur Saksa,b, Tuuli Kaambrea a Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia b Laboratory of Fundamental and Applied Bioenergetics, Joseph Fourier University, Grenoble, France
907
c
Oncology and Hematology Clinic at the North Estonia Medical Centre, Tallinn, Estonia d Competence Centre for Cancer Research, Tallinn, Estonia Abstract: Today, there is only very limited information as to the bioenergetic function of mitochondria in human tumor cells in situ. In our study, we have analyzed quantitatively the cellular respiration in post-operational tissue samples taken from patients with human breast and colorectal cancers, comparative experiments were performed on murine neuroblastoma cells of the line Neuro-2a (N2a) both undifferentiated and differentiated by all-trans-retinoic acid. N2a cells display a Warburg phenotype characterized by accelerated aerobic glycolysis. We applied high resolution respirometry and the permeabilized cell techniques in combination with Metabolic Control Analysis (MCA) to detect possible OXPHOS defects in human breast and colorectal cancers; for this purpose, a corresponding flux control coefficient (FCC) was measured. MCA fulfills one promise of Systems Biology, i.e. to help understand how collectively molecular interaction properties lead to system level properties. We found that in the studied human tumors material OXPHOS prevails over glycolysis. Upon direct activation of mitochondrial respiration with exogenously added ADP, the activity of components of ATP synthasome and respiratory chain complexes was found to be significantly increased in human tumor cells in situ as compared to adjacent normal tissues as well as in N2a cells. For undifferentiated N2a cells the key sites of regulation of mitochondrial respiratory were found to be complex-I (FCC = 1.11) and complex-IV (FCC = 0.92), whereas for human breast cancer the main regulatory complexes are ANT (FCC =1.02), complex IV (FCC = 0.74), ATP synthase (FCC = 0.61), and inorganic phosphate carrier (FCC = 0.60). The reason of these high values of FCC(s) not diffusion restrictions. For the studied malignant cells, the sum of FCC(s) was found to be in the range from 3 to 5 and this exceeds substantially the theoretic value for linear systems −1. It is possible that the high FCC(s) are the common feature of neoplastic cells. That is possibly associated by the remodeling of mitochondrial respiratory chain due to the formation of large protein supercomplexes. Our work is in good accordance with series of works from Giorgio Lenaz laboratory. The authors showed by using MCA that respiratory complexes may kinetically behave as single supramolecular units with FCC approaching the unity for each component suggesting the existence of substrate channeling within supercomplexes. An analysis of the regulation of mitochondrial respiration showed that mitochondria in tumor cells have an increased affinity for exogenously added ADP in comparison with certain normal oxidative tissues that may be casually linked with a decreased expression of some cytoskeletal proteins. Bioenergetic profiling and the understanding of the cancer cell energy metabolism are of the greatest importance, since it can bring forward new effective targets for medical treatment and early diagnostics. doi:10.1016/j.mito.2013.07.027
31 Oxidative stress triggers mitochondrial mediated retrograde signaling by activating its translocation and function switching Presenter: Sumitra Miriyala Sumitra Miriyalaa, Manikandan Panchatcharama, Ines Batinic-Habereleb, Daret St. Clairc a Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences, Shreveport, LA, USA b Department of Radiation Oncology, Duke University Medical Center, Durham, NC, USA c Graduate Center for Toxicology, University of Kentucky, Lexington, KY, USA Mitochondria, apart from being the powerhouse of the cell by producing ATP, are major sites of ROS production and integrators of
recoded ATP6 genes, and distinct 3′ untranslated regions (3′UTRs) to determine the combination that provides the best “rescue” or phenotypic improvement. We observe significant improvements in both longevity and locomotive impairment using a transgenic expression system, especially in flies that express a full-length algal ATP6. We have further tested Drosophila strains that contain different segments of the algal ATP6 (algal chimeras), and statistically significant improvements are again observed. We are planning to examine if the neuromuscular pathology observed in the mutant flies is alleviated in the transgenic flies with algal ATP6 expression. ATP6 synthase assays will also be performed to determine if and how much ATP6 production is resumed.
doi:10.1016/j.mito.2013.07.025
29 The role of an unconventional ribosomal protein in mitochondrial function Presenter: Tara Richman Tara R. Richman, Louis Scott, Stefan M.K. Davies, Oliver Rackham, Aleksandra Filipovska Western Australian Institute for Medical Research and Centre for Medical Research, The University of Western Australia, Perth, Western Australia 6000, Australia Body of Abstract: Mitochondria are ubiquitous organelles of eukaryotic cells that contain a circular double-stranded genome encoding 13 polypeptide subunits of the mitochondrial respiratory chain. The genes for these polypeptides are transcribed and translated in the mitochondrial matrix, using 22 tRNAs and 2 rRNAs encoded by the compact mitochondrial DNA. Consequently, mtDNA is dependent on nuclear encoded proteins for replication, repair, transcription and translation. Mammalian mitochondrial mRNAs generally begin at the start codon and lack conventional 5′ untranslated regions or ShineDalgarno sequences. In addition mitochondrial encoded proteins are hydrophobic and are likely to be co-translationally inserted into the membrane-embedded complexes providing unique constraints for their translation. Mitochondrial ribosomes must have evolved in an alternative way to regulate translation initiation and elongation to ensure accurate start codon recognition and facilitate protein complex assembly. Mammalian mitochondrial ribosomes are unique from bacterial and cytoplasmic ribosomes of eukaryotes because their ribosomal RNA has been reduced considerably and has been replaced by additional proteins. These proteins may have new functions in mitochondrial translation and recognition of mitochondrial mRNAs. We have identified an additional protein and found that it localizes to mitochondria, associates with mitochondrial ribosomes specifically, and has important roles in mitochondrial gene expression and consequently cellular energy metabolism.
doi:10.1016/j.mito.2013.07.026
30 Comparative aspects of bioenergetic profiling of tumor clinical material and cell cultures Igor Shevchuka, Andre Koita, Andrus Kaldmaaa, Natalja Timohhinaa, Vladimir Chekulayeva, Kersti Teppa, Jelena Bogovskajac, Vahur Valverec,d, Valdur Saksa,b, Tuuli Kaambrea a Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia b Laboratory of Fundamental and Applied Bioenergetics, Joseph Fourier University, Grenoble, France
907
c
Oncology and Hematology Clinic at the North Estonia Medical Centre, Tallinn, Estonia d Competence Centre for Cancer Research, Tallinn, Estonia Abstract: Today, there is only very limited information as to the bioenergetic function of mitochondria in human tumor cells in situ. In our study, we have analyzed quantitatively the cellular respiration in post-operational tissue samples taken from patients with human breast and colorectal cancers, comparative experiments were performed on murine neuroblastoma cells of the line Neuro-2a (N2a) both undifferentiated and differentiated by all-trans-retinoic acid. N2a cells display a Warburg phenotype characterized by accelerated aerobic glycolysis. We applied high resolution respirometry and the permeabilized cell techniques in combination with Metabolic Control Analysis (MCA) to detect possible OXPHOS defects in human breast and colorectal cancers; for this purpose, a corresponding flux control coefficient (FCC) was measured. MCA fulfills one promise of Systems Biology, i.e. to help understand how collectively molecular interaction properties lead to system level properties. We found that in the studied human tumors material OXPHOS prevails over glycolysis. Upon direct activation of mitochondrial respiration with exogenously added ADP, the activity of components of ATP synthasome and respiratory chain complexes was found to be significantly increased in human tumor cells in situ as compared to adjacent normal tissues as well as in N2a cells. For undifferentiated N2a cells the key sites of regulation of mitochondrial respiratory were found to be complex-I (FCC = 1.11) and complex-IV (FCC = 0.92), whereas for human breast cancer the main regulatory complexes are ANT (FCC =1.02), complex IV (FCC = 0.74), ATP synthase (FCC = 0.61), and inorganic phosphate carrier (FCC = 0.60). The reason of these high values of FCC(s) not diffusion restrictions. For the studied malignant cells, the sum of FCC(s) was found to be in the range from 3 to 5 and this exceeds substantially the theoretic value for linear systems −1. It is possible that the high FCC(s) are the common feature of neoplastic cells. That is possibly associated by the remodeling of mitochondrial respiratory chain due to the formation of large protein supercomplexes. Our work is in good accordance with series of works from Giorgio Lenaz laboratory. The authors showed by using MCA that respiratory complexes may kinetically behave as single supramolecular units with FCC approaching the unity for each component suggesting the existence of substrate channeling within supercomplexes. An analysis of the regulation of mitochondrial respiration showed that mitochondria in tumor cells have an increased affinity for exogenously added ADP in comparison with certain normal oxidative tissues that may be casually linked with a decreased expression of some cytoskeletal proteins. Bioenergetic profiling and the understanding of the cancer cell energy metabolism are of the greatest importance, since it can bring forward new effective targets for medical treatment and early diagnostics. doi:10.1016/j.mito.2013.07.027
31 Oxidative stress triggers mitochondrial mediated retrograde signaling by activating its translocation and function switching Presenter: Sumitra Miriyala Sumitra Miriyalaa, Manikandan Panchatcharama, Ines Batinic-Habereleb, Daret St. Clairc a Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences, Shreveport, LA, USA b Department of Radiation Oncology, Duke University Medical Center, Durham, NC, USA c Graduate Center for Toxicology, University of Kentucky, Lexington, KY, USA Mitochondria, apart from being the powerhouse of the cell by producing ATP, are major sites of ROS production and integrators of