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Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation
Abstracts
complex I and complex II substrates simultaneously feed electrons into the respiratory chain. We asked if the REF-mediated ROS generation can occur when both complex I and complex II substrates are used together. Inhibition of complex I with rotenone decreases the REF-induced ROS generation. Overexpression of STAT 3 targeted to mitochondria (MLSTAT3E mouse) decreases complex I activity but maintains the inner mitochondrial membrane potential (Δψ). We used mitochondria isolated from STAT3E mouse heart to test if only partial inhibition of complex I affects the REF-induced ROS generation. The REF-induced ROS generation still occurred when glutamate and succinate were used together as substrates (Table), supporting the notion that the REF-induced ROS generation was independent of the NADH/NAD+ ratio. The REF-induced ROS generation was not increased in MLSTAT3E mouse compared to wild type [mean± SEM, 1592 ± 147 (wild type, pmol/mg/30 min) vs. 1846 ± 448 (MLSTAT3E), p = NS, n = 4]. These results indicated that partial inhibition of complex I alone did not prevent REF-mediated ROS generation. Complete depolarization of the Δψ by dinitrophenol (DNP) prevented REF-induced ROS generation (Table). Only a 16% decrease in Δψ by DIDS (an ion channel inhibitor) also prevented REF-induced ROS generation (Table). DIDS did not affect the glutamate oxidation in the isolated mitochondria (data not shown), indicating that the decreased ROS generation by DIDS was not due to decreased complex I activity. Taken together, the Δψ contributed a key role in REF-mediated ROS generation. The REF-induced ROS generation was less likely to occur in vivo in that Δψ is depolarized to support constant ADP phosphorylation. The increased ROS generation from mitochondria contributes to myocardial injury during ischemia–reperfusion. Cardiac ischemia damages the electron transport chain that led to decreased complex I activity and partially depolarized Δψ. Therefore, the REF-induced ROS generation should not occur during ischemia–reperfusion. Thus, the REF-mediated ROS generation is an unlikely potential source of cardiac injury during reperfusion.
Rat heart
Succinate
Succinate (5 mM)
Succinate (5 mM) + DNP
Succinate (5 mM) + DIDS
Mitochondria
(5 mM)
(0.3 mM)
(50 μM)
H2O2 (pmol/mg/30 min)
958 ± 112
+ glutamate (5 mM) 990 ± 107
37 ± 10*
35 ± 11*
Mean ± SEM: *p b 0.05 vs. succinate or succinate + glutamate. N = 5 in each group.
doi:10.1016/j.mito.2012.07.083
93 Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation Presenter: Steven G. Hershman Steven G. Hershmana,b,c, Elena J. Tuckerd,f, Caroline Köhrerg, Casey A. Belcher-Timmea,b,c, Jinal Patelc, Olga A. Goldbergera,b,c, John Christodoulouh,i,j, Jonathon M. Silbersteink, Matthew McKenziel, Michael T. Ryanm,n, Alison G. Comptond, Caterina Garoneo,p,q, Beatriz Garcia-Diazo, Salvatore DiMauroo, Jacob D. Jaffec, Steven A. Carrc, Sarah E. Calvoa,b,c, Uttam L. RajBhandaryg, David R. Thorburnd,e,f, Vamsi K. Moothaa,b,c a Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA b Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA c Broad Institute, Cambridge, MA 02142, USA d Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, VIC 3052, Australia
583
e Genetic Health Services Victoria, Royal Children's Hospital, Melbourne, VIC 3052, Australia f Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia g Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA h Genetic Metabolic Disorders Research Unit, Children's Hospital at Westmead, Sydney, NSW 2006, Australia i Discipline of Paediatrics and Child Health, University of Sydney, Sydney, NSW 2006, Australia j Discipline of Genetic Medicine, University of Sydney, Sydney, NSW 2006, Australia k Department of Neurology, Princess Margaret Hospital for Children, Perth, WA 6008, Australia l Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Melbourne, VIC 3168, Australia m Department of Biochemistry, La Trobe University, Melbourne, VIC 3086, Australia n ARC Centre of Excellence for Coherent X-Ray Science, La Trobe University, Melbourne, VIC 3086, Australia o Department of Neurology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA p Human Genetics Joint PhD Programme, University of Turin, 10125 Turin, Italy q Human Genetics Joint PhD Programme, University of Bologna, 40125 Bologna, Italy
Of the ~90 protein components of the oxidative phosphorylation (OXPHOS) machinery, 13 are encoded by the mitochondrial DNA (mtDNA) and translated within the organelle. Defects in mitochondrial protein synthesis lead to combined OXPHOS deficiency. Although the mtDNA encodes two of the ribosomal and 20 of the transfer RNAs, all remaining components of the mitochondrial translational machinery are encoded by nuclear genes and imported into the organelle. To date, mutations in more than ten different nuclear genes have been shown to cause defective mitochondrial translation in humans. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified mutations in the mitochondrial methionyl-tRNA formyltransferase (MTFMT) in unrelated children presenting with combined OXPHOS deficiency and Leigh syndrome. The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by MTFMT to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)); however, the requirement of formylation for initiation in human mitochondria is still under debate. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of MettRNA(Met) formylation. This work was funded by NIGMS (GM077465 and GM097136) and was published in the September 2011 issue of Cell Metabolism. doi:10.1016/j.mito.2012.07.084
94 A systems biology approach to mitochondrial disease in C. elegans Presenter: Phil G. Morgan Margaret M. Sedenskya, Marni J. Falkb, Eugene Kolkera, Roger Higdona, Chris Newgardc, Phil G. Morgana
complex I and complex II substrates simultaneously feed electrons into the respiratory chain. We asked if the REF-mediated ROS generation can occur when both complex I and complex II substrates are used together. Inhibition of complex I with rotenone decreases the REF-induced ROS generation. Overexpression of STAT 3 targeted to mitochondria (MLSTAT3E mouse) decreases complex I activity but maintains the inner mitochondrial membrane potential (Δψ). We used mitochondria isolated from STAT3E mouse heart to test if only partial inhibition of complex I affects the REF-induced ROS generation. The REF-induced ROS generation still occurred when glutamate and succinate were used together as substrates (Table), supporting the notion that the REF-induced ROS generation was independent of the NADH/NAD+ ratio. The REF-induced ROS generation was not increased in MLSTAT3E mouse compared to wild type [mean± SEM, 1592 ± 147 (wild type, pmol/mg/30 min) vs. 1846 ± 448 (MLSTAT3E), p = NS, n = 4]. These results indicated that partial inhibition of complex I alone did not prevent REF-mediated ROS generation. Complete depolarization of the Δψ by dinitrophenol (DNP) prevented REF-induced ROS generation (Table). Only a 16% decrease in Δψ by DIDS (an ion channel inhibitor) also prevented REF-induced ROS generation (Table). DIDS did not affect the glutamate oxidation in the isolated mitochondria (data not shown), indicating that the decreased ROS generation by DIDS was not due to decreased complex I activity. Taken together, the Δψ contributed a key role in REF-mediated ROS generation. The REF-induced ROS generation was less likely to occur in vivo in that Δψ is depolarized to support constant ADP phosphorylation. The increased ROS generation from mitochondria contributes to myocardial injury during ischemia–reperfusion. Cardiac ischemia damages the electron transport chain that led to decreased complex I activity and partially depolarized Δψ. Therefore, the REF-induced ROS generation should not occur during ischemia–reperfusion. Thus, the REF-mediated ROS generation is an unlikely potential source of cardiac injury during reperfusion.
Rat heart
Succinate
Succinate (5 mM)
Succinate (5 mM) + DNP
Succinate (5 mM) + DIDS
Mitochondria
(5 mM)
(0.3 mM)
(50 μM)
H2O2 (pmol/mg/30 min)
958 ± 112
+ glutamate (5 mM) 990 ± 107
37 ± 10*
35 ± 11*
Mean ± SEM: *p b 0.05 vs. succinate or succinate + glutamate. N = 5 in each group.
doi:10.1016/j.mito.2012.07.083
93 Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation Presenter: Steven G. Hershman Steven G. Hershmana,b,c, Elena J. Tuckerd,f, Caroline Köhrerg, Casey A. Belcher-Timmea,b,c, Jinal Patelc, Olga A. Goldbergera,b,c, John Christodoulouh,i,j, Jonathon M. Silbersteink, Matthew McKenziel, Michael T. Ryanm,n, Alison G. Comptond, Caterina Garoneo,p,q, Beatriz Garcia-Diazo, Salvatore DiMauroo, Jacob D. Jaffec, Steven A. Carrc, Sarah E. Calvoa,b,c, Uttam L. RajBhandaryg, David R. Thorburnd,e,f, Vamsi K. Moothaa,b,c a Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA b Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA c Broad Institute, Cambridge, MA 02142, USA d Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, VIC 3052, Australia
583
e Genetic Health Services Victoria, Royal Children's Hospital, Melbourne, VIC 3052, Australia f Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia g Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA h Genetic Metabolic Disorders Research Unit, Children's Hospital at Westmead, Sydney, NSW 2006, Australia i Discipline of Paediatrics and Child Health, University of Sydney, Sydney, NSW 2006, Australia j Discipline of Genetic Medicine, University of Sydney, Sydney, NSW 2006, Australia k Department of Neurology, Princess Margaret Hospital for Children, Perth, WA 6008, Australia l Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Melbourne, VIC 3168, Australia m Department of Biochemistry, La Trobe University, Melbourne, VIC 3086, Australia n ARC Centre of Excellence for Coherent X-Ray Science, La Trobe University, Melbourne, VIC 3086, Australia o Department of Neurology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA p Human Genetics Joint PhD Programme, University of Turin, 10125 Turin, Italy q Human Genetics Joint PhD Programme, University of Bologna, 40125 Bologna, Italy
Of the ~90 protein components of the oxidative phosphorylation (OXPHOS) machinery, 13 are encoded by the mitochondrial DNA (mtDNA) and translated within the organelle. Defects in mitochondrial protein synthesis lead to combined OXPHOS deficiency. Although the mtDNA encodes two of the ribosomal and 20 of the transfer RNAs, all remaining components of the mitochondrial translational machinery are encoded by nuclear genes and imported into the organelle. To date, mutations in more than ten different nuclear genes have been shown to cause defective mitochondrial translation in humans. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified mutations in the mitochondrial methionyl-tRNA formyltransferase (MTFMT) in unrelated children presenting with combined OXPHOS deficiency and Leigh syndrome. The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by MTFMT to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)); however, the requirement of formylation for initiation in human mitochondria is still under debate. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of MettRNA(Met) formylation. This work was funded by NIGMS (GM077465 and GM097136) and was published in the September 2011 issue of Cell Metabolism. doi:10.1016/j.mito.2012.07.084
94 A systems biology approach to mitochondrial disease in C. elegans Presenter: Phil G. Morgan Margaret M. Sedenskya, Marni J. Falkb, Eugene Kolkera, Roger Higdona, Chris Newgardc, Phil G. Morgana