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Complementary RNA and protein profiling identifies iron as a key regulator of mitochondrial biogenesis
Abstracts
hypothesis, we have first treated wild-type human fibroblasts at different concentrations of phenylbutyrate. Following drug treatment, cells were harvested for Western blot analysis using antibodies specific for the E1α subunit of the PDHC phosphorylated at each of the three phosphorylation sites of the E1 (Ser293, Ser232, and Ser300). Western blot showed a significant reduction in the levels of phosphorylated E1α subunit of PDHC in phenylbutyrate treated cells compared to untreated cells. Several reports show that phenylbutyrate cross the blood–brain-barrier and it has pharmacological effects in brain cells. Therefore, we next investigated the effect of phenylbutyrate in vivo on brain PDHC phosphorylation. To this end, we have given saline or phenylbutyrate (250 and 500 mg/kg/day, which are doses given to humans) orally by gavage to C57B6 wildtype mice and after three days of treatment the animals were sacrificed for analyses. Western blot on brain mitochondrial extracts showed that phenylbutyrate resulted in a significant reduction of the levels of phosphorylated E1α subunit of Pdhc compared to saline treated mice, while it did not have effect on total amount of E1α. Pdhc enzyme activity in brain mitochondrial extracts was increased, thus showing that phenylbutyrate enhances cerebral Pdhc enzyme activity in vivo in a disease relevant tissue. These results are extremely promising because they show that phenylbutyrate increases the activity of the Pdhc in the brain which is the most affected tissue in PDHC deficiency. Moreover, we found that phenylbutyrate increases PDHC residual enzyme activity in 3 out of 4 fibroblast cell lines from PDHC deficient patients. Given that phenylbutyrate is a drug already approved for use in humans and its safety profile is well characterized, our findings have the potential to be rapidly translated into treatment for patients with PDHC deficiency.
doi:10.1016/j.mito.2012.07.058
64 Complementary RNA and protein profiling identifies iron as a key regulator of mitochondrial biogenesis Presenter: Jarred W. Rensvold Jarred W. Rensvolda, Shao-En Ongb, Athavi Jeevananthana, Steven A. Carrc, Vamsi K. Moothac,d, David J. Pagliarinia a Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, United States b Department of Pharmacology, University of Washington, Seattle, WA 98195, United States c Broad Institute of MIT and Harvard, Cambridge, MA 02142, United States d Departments of Systems Biology and Medicine, Harvard Medical School, Boston, MA 02446, United States The production of mitochondria, known as mitochondrial biogenesis, involves the orchestrated transcription, translation, import and assembly of more than one thousand proteins encoded by two genomes. The number and composition of mitochondria varies across tissues and changes in response to environmental conditions and nutrient demands, indicating that mitochondrial biogenesis can be tailored to meet specific cellular requirements. Defects in this process are associated with a broad range of human diseases and age-related disorders. The transcriptional regulators that control mitochondrial biogenesis, including the peroxisome proliferator-activated receptor gamma, coactivator-1 (PGC-1) family of transcriptional coactivators and their associated transcription factors, are well described. However, other aspects of the cellular control of mitochondrial biogenesis remain less clear. These include post-transcriptional processes that control mitochondrial gene expression, peripheral pathways that are important
573
for supporting mitochondrial biogenesis and signals that communicate a need for mitochondrial restructuring. To better define the mitochondrial biogenesis program, we performed matched, quantitative SILAC (stable isotope labeling by amino acids in cell culture)-based proteomics and microarray analyses of mouse muscle cells following overexpression of PGC-1α. From these analyses we find that proteins involved in cellular iron homeostasis are highly regulated during mitochondrial biogenesis, and that depletion of cellular iron results in a coordinated downregulation of mitochondrial protein levels. Immunoblot and quantitative PCR analyses confirm that depletion of cellular iron through various mechanisms results in a rapid, dose-dependent decrease in nuclear- and mitochondrial-encoded gene expression and oxidative capacity in a variety of mammalian cell lines that is fully recoverable within 3–4 days following the reintroduction of iron. Furthermore, we find that the effects of iron deprivation are distinct from previously reported HIF-1α (hypoxia inducible factor)-driven reductions in mitochondrial biogenesis. Collectively, our work shows that cellular iron concentration is a key parameter in calibrating the mitochondrial biogenesis program and suggests that cellular iron deprivation initiates a reversible, adaptive transcriptional response to remodel the mitochondrial proteome and reduce mitochondrial respiratory function. Our data serve as a resource for investigating genes subject to post-transcriptional regulation, and for identifying additional auxiliary pathways that might be important for modulating the mitochondrial biogenesis program.
doi:10.1016/j.mito.2012.07.059
65 Structural studies of Dnm1/Drp1 provide mechanistic insight into mitochondrial fission Presenter: Jason A. Mears Frances Joan D. Alvarez, Louie Zhou, Jason A. Mears Department of Pharmacology, Center for Mitochondrial Disease, Cleveland Center of Membrane and Structural Biology, Case Western Reserve University, Cleveland, OH 44106-4965, United States Dynamin-related protein 1 (Drp1) belongs to a family of large GTPase proteins that regulate membrane dynamics and morphology. Drp1 localizes to mitochondrial constriction sites in vivo to facilitate outer membrane fission, and mutations that inhibit its activity lead to hyper-fused mitochondria in vivo. Direct inhibition of Drp1 protects against cell death by limiting increased mitochondrial fission associated with apoptosis. Previously, we have studied the yeast homolog of Drp1, Dnm1, using electron microscopy (EM) to determine its structural properties. We found that Dnm1 forms large (N100 nm in diameter) helical oligomers that constrict upon GTP hydrolysis to generate a contractile force on the underlying membrane (1). We are using similar methods to gain mechanistic insight into the mammalian mitochondrial fission complex, and several differences exist between the yeast and mammalian systems. We find that recombinant Drp1 forms stable tetramers, which represent the pre-assembled state of Drp1. The size of this complex (~330 kDa) provides a suitable target for 3D image reconstruction. Interactions with GTP analogs and/or synthetic liposomes promote additional Drp1 self-assembly into helical oligomers. The diameter of Drp1 helices in a GTP-bound state (~40 nm) places considerable strain on the underlying lipid bilayer. We are examining the effects of GTP hydrolysis on the Drp1 oligomer to determine how this structure promotes outer mitochondrial membrane fission. Future studies will examine interactions between Drp1 and partner proteins in the mitochondrial fission complex.
hypothesis, we have first treated wild-type human fibroblasts at different concentrations of phenylbutyrate. Following drug treatment, cells were harvested for Western blot analysis using antibodies specific for the E1α subunit of the PDHC phosphorylated at each of the three phosphorylation sites of the E1 (Ser293, Ser232, and Ser300). Western blot showed a significant reduction in the levels of phosphorylated E1α subunit of PDHC in phenylbutyrate treated cells compared to untreated cells. Several reports show that phenylbutyrate cross the blood–brain-barrier and it has pharmacological effects in brain cells. Therefore, we next investigated the effect of phenylbutyrate in vivo on brain PDHC phosphorylation. To this end, we have given saline or phenylbutyrate (250 and 500 mg/kg/day, which are doses given to humans) orally by gavage to C57B6 wildtype mice and after three days of treatment the animals were sacrificed for analyses. Western blot on brain mitochondrial extracts showed that phenylbutyrate resulted in a significant reduction of the levels of phosphorylated E1α subunit of Pdhc compared to saline treated mice, while it did not have effect on total amount of E1α. Pdhc enzyme activity in brain mitochondrial extracts was increased, thus showing that phenylbutyrate enhances cerebral Pdhc enzyme activity in vivo in a disease relevant tissue. These results are extremely promising because they show that phenylbutyrate increases the activity of the Pdhc in the brain which is the most affected tissue in PDHC deficiency. Moreover, we found that phenylbutyrate increases PDHC residual enzyme activity in 3 out of 4 fibroblast cell lines from PDHC deficient patients. Given that phenylbutyrate is a drug already approved for use in humans and its safety profile is well characterized, our findings have the potential to be rapidly translated into treatment for patients with PDHC deficiency.
doi:10.1016/j.mito.2012.07.058
64 Complementary RNA and protein profiling identifies iron as a key regulator of mitochondrial biogenesis Presenter: Jarred W. Rensvold Jarred W. Rensvolda, Shao-En Ongb, Athavi Jeevananthana, Steven A. Carrc, Vamsi K. Moothac,d, David J. Pagliarinia a Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, United States b Department of Pharmacology, University of Washington, Seattle, WA 98195, United States c Broad Institute of MIT and Harvard, Cambridge, MA 02142, United States d Departments of Systems Biology and Medicine, Harvard Medical School, Boston, MA 02446, United States The production of mitochondria, known as mitochondrial biogenesis, involves the orchestrated transcription, translation, import and assembly of more than one thousand proteins encoded by two genomes. The number and composition of mitochondria varies across tissues and changes in response to environmental conditions and nutrient demands, indicating that mitochondrial biogenesis can be tailored to meet specific cellular requirements. Defects in this process are associated with a broad range of human diseases and age-related disorders. The transcriptional regulators that control mitochondrial biogenesis, including the peroxisome proliferator-activated receptor gamma, coactivator-1 (PGC-1) family of transcriptional coactivators and their associated transcription factors, are well described. However, other aspects of the cellular control of mitochondrial biogenesis remain less clear. These include post-transcriptional processes that control mitochondrial gene expression, peripheral pathways that are important
573
for supporting mitochondrial biogenesis and signals that communicate a need for mitochondrial restructuring. To better define the mitochondrial biogenesis program, we performed matched, quantitative SILAC (stable isotope labeling by amino acids in cell culture)-based proteomics and microarray analyses of mouse muscle cells following overexpression of PGC-1α. From these analyses we find that proteins involved in cellular iron homeostasis are highly regulated during mitochondrial biogenesis, and that depletion of cellular iron results in a coordinated downregulation of mitochondrial protein levels. Immunoblot and quantitative PCR analyses confirm that depletion of cellular iron through various mechanisms results in a rapid, dose-dependent decrease in nuclear- and mitochondrial-encoded gene expression and oxidative capacity in a variety of mammalian cell lines that is fully recoverable within 3–4 days following the reintroduction of iron. Furthermore, we find that the effects of iron deprivation are distinct from previously reported HIF-1α (hypoxia inducible factor)-driven reductions in mitochondrial biogenesis. Collectively, our work shows that cellular iron concentration is a key parameter in calibrating the mitochondrial biogenesis program and suggests that cellular iron deprivation initiates a reversible, adaptive transcriptional response to remodel the mitochondrial proteome and reduce mitochondrial respiratory function. Our data serve as a resource for investigating genes subject to post-transcriptional regulation, and for identifying additional auxiliary pathways that might be important for modulating the mitochondrial biogenesis program.
doi:10.1016/j.mito.2012.07.059
65 Structural studies of Dnm1/Drp1 provide mechanistic insight into mitochondrial fission Presenter: Jason A. Mears Frances Joan D. Alvarez, Louie Zhou, Jason A. Mears Department of Pharmacology, Center for Mitochondrial Disease, Cleveland Center of Membrane and Structural Biology, Case Western Reserve University, Cleveland, OH 44106-4965, United States Dynamin-related protein 1 (Drp1) belongs to a family of large GTPase proteins that regulate membrane dynamics and morphology. Drp1 localizes to mitochondrial constriction sites in vivo to facilitate outer membrane fission, and mutations that inhibit its activity lead to hyper-fused mitochondria in vivo. Direct inhibition of Drp1 protects against cell death by limiting increased mitochondrial fission associated with apoptosis. Previously, we have studied the yeast homolog of Drp1, Dnm1, using electron microscopy (EM) to determine its structural properties. We found that Dnm1 forms large (N100 nm in diameter) helical oligomers that constrict upon GTP hydrolysis to generate a contractile force on the underlying membrane (1). We are using similar methods to gain mechanistic insight into the mammalian mitochondrial fission complex, and several differences exist between the yeast and mammalian systems. We find that recombinant Drp1 forms stable tetramers, which represent the pre-assembled state of Drp1. The size of this complex (~330 kDa) provides a suitable target for 3D image reconstruction. Interactions with GTP analogs and/or synthetic liposomes promote additional Drp1 self-assembly into helical oligomers. The diameter of Drp1 helices in a GTP-bound state (~40 nm) places considerable strain on the underlying lipid bilayer. We are examining the effects of GTP hydrolysis on the Drp1 oligomer to determine how this structure promotes outer mitochondrial membrane fission. Future studies will examine interactions between Drp1 and partner proteins in the mitochondrial fission complex.