Browsing by Subject "Mitochondria"
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Item Biochemical Pathways in Apoptosis(2005-05-03) Nijhawan, Deepak; Wang, XiaodongCaspases are a family of proteases that once activated execute apoptosis, a cellular suicide pathway. Activated caspases have a unique property to cleave and activate themselves. Once the first caspase is activated, it generates a chain reaction resulting in robust caspase activity and rapid death. The central question in apoptosis is to understand how the first caspase is activated. To address this question, we present an assay that recapitulates de novo caspase activation in vitro. We describe how this assay was used to purify three proteins that were sufficient to reconstitute caspase activation in vitro: Apaf-1, cytochrome c, and caspase-9. The mechanism of caspase activation in vivo, however, is complicated by these proteins subcelluar localization. In the living cell, Apaf-1 and caspase-9 are cytoplasmic whereas cytochrome c is mitochondria, however, during apoptosis, cytochrome is released to the cytoplasm. In vitro, cytochrome c induces the formation of a stable Apaf-1/caspase-9 complex and caspase-9 autoactivation suggesting that cytochrome c release from the mitochondria to the cytosol is the rate-limiting event that leads to caspase activation. This conclusion shifted the focus of our studies upstream from what initiates caspase activation to what triggers cytochrome c release. The second part of the dissertation uses a biochemical approach to identify how cytochrome c release is regulated after exposure to ultraviolet light. Ultraviolet light irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of such a response revealed that the initial step leading to cytochrome c release is the complete disappearance of the mRNA of Mcl-1, an anti-apoptotic member of the Bcl-2 family. This event leads to the elimination of Mcl-1 protein from cells due to the short half-life of its protein. The block or delay of Mcl-1 disappearance by either proteasome inhibitors or Mcl-1 over-expression prevents subsequent steps of this apoptotic pathway including the translocation of Bax and Bcl-xL from cytosol to mitochondria and dephosphorylation of BimEL on mitochondria. These sequential events lead to the oligomerization of Bax and Bak on the mitochondria, cytochrome c release and caspase activation.Item The Contextual Roles of Isocitrate Dehydrogenase-1 and Isocitrate Dehydrogenase-2 in Electron Transport Chain Complex III Deficiency(December 2021) Harris, Robert Charles; Chahrour, Maria; DeBerardinis, Ralph J.; Mishra, Prashant; Minassian, BergeInborn errors of metabolism provide excellent opportunities in translational research, because individual clinical cases can stimulate insights that lead to a deeper understanding of human metabolism and sometimes inform personalized patient care. Our clinical genetics team identified a female patient who presented at age 3 with recurrent episodes of metabolic decompensation involving hypoglycemia and hyperlactatemia, but for whom conventional testing could not identify the underlying molecular defect. Whole exome sequencing identified compound heterozygosity for novel, putatively pathogenic variants in the gene encoding Ubiquinol-Cytochrome C Reductase Core Protein II (UQCRC2), a nuclear-encoded subunit of electron transport chain complex III (ETCIII). Only a handful of UQCRC2-deficient patients have been described worldwide, and none have been subjected to a detailed metabolic analysis to understand the impact of defects in this protein. We used CRISPR-Cas9 gene editing to generate UQCRC2-deficient cells in order to model deficiency of this protein. Metabolomic profiling of the patient's plasma and UQCRC2 KO cell lines demonstrated strong overlap, including some abnormalities broadly characteristic of ETC defects and others that may be more specific for ETCIII defects. UQCRC2-deficient cells constitutively exhibit reductive glutamine metabolism catalyzed by reversible isoforms of isocitrate dehydrogenase (IDH1 and IDH2) and undergo rapid cell death when glucose becomes scarce. Reasoning that these cell lines could be used to address basic questions about the contextual roles of IDH1 and IDH2 in regulation of the reductive carboxylation pathway, they were used to create concomitant IDH1 and IDH2 knockout lines. Surprisingly, loss of either enzyme did not suppress cell growth or survival under nutrient-replete conditions. However, we identified differential effects on reductive metabolism and on cell survival under nutrient deprivation. Specifically, we find that IDH1 is primarily responsible for reductive glutamine metabolism in these models of ETCIII deficiency. When glucose and glutamine become scarce, IDH2 supports cell survival while IDH1 becomes detrimental. We speculate that chronic IDH1-dependent utilization of reducing equivalents limits cell survival under nutritional stress. These findings present the first report of a context where wild-type IDH1 activity is detrimental to cell survival.Item Defining a Novel Role for Hypoxia Inducible Factor-2 Alpha (HIF-2a)/EPAS1 : Maintenance of Mitochondrial and Redox Homeostasis(2005-12-20) Oktay, Yavuz; Garcia, Joseph A.The Epas1 gene encodes HIF-2alpha , a member of the Hypoxia Inducible Factor family of transcriptional regulators. The biological role for HIF-2alpha has been elusive due to embryonic lethality of the initial Epas1-/- mouse strains. Our lab reported the generation of the first viable Epas1-/- mice using a genetic breeding strategy. Adult Epas1-/- mice exhibit gross, histological, biochemical, and molecular evidence consistent with mitochondrial dysfunction. Similarities between Epas1 and Sod2 deficient strains suggest a biochemical etiology, increased oxidative stress, as well as a molecular etiology, decreased Sod2 gene expression, for the mitochondrial dysfunction in Epas1-/- mice. Consistent with this hypothesis, Sod2 gene expression is reduced in Epas1-/- mice whereas HIF-2a induces Sod2 gene promoter in transient transfection studies. Further studies revealed impaired mitochondrial respiration, sensitized mitochondrial permeability transition pore opening, increased electron transport chain activity and reduced mitochondrial aconitase activity. Given that it is the most sensitive enzymatic marker for oxidative stress, aconitase inhibition may explain impaired respiration. Also, redox balance in Epas1-/- liver is disturbed: the reduced cytoplasmic environment, and a relative oxidized environment for mitochondria from Epas1-/- liver implies a role for HIF-2a in maintenance of cellular redox balance. All these data suggest that HIF-2a is essential for maintenance of mitochondrial function, reactive oxygen species detoxification, and redox balance.Item Insights into the Metabolic Regulation by GATOR1 in Response to Amino Acid Signaling(2017-07-27) Chen, Jun; Liu, Yi; Tu, Benjamin; Phillips, Margaret A.; Goodman, Joel M.The GATOR1/SEACIT complex consisting of Iml1-Npr2-Npr3 inhibits Target of Rapamycin Complex 1 (TORC1) in response to amino acid insufficiency. In glucose medium, yeast mutants lacking the function of this complex grow poorly in the absence of amino acid supplementation, despite hallmarks of increased TORC1 signaling. Such mutants perceive they are amino acid-replete and thus repress metabolic activities that are important for achieving this state. I find that npr2∆ mutants have defective mitochondrial TCA cycle activity and retrograde response. Supplementation of glutamine, and especially aspartate, which are nitrogen-containing forms of TCA cycle intermediates, rescue growth of npr2∆ mutants. These amino acids are then consumed in biosynthetic pathways that require nitrogen to support proliferative metabolism. Our findings reveal that negative regulators of TORC1 such as GATOR1/SEACIT regulate the cataplerotic synthesis of these amino acids from the TCA cycle in tune with the amino acid and nitrogen status of cells.Item Mitochondrial Fission with Function Impairment in Burn Serum Treated C2C12 Cells(2016-01-19) Sehat, Alvand; Song, Juquan; Kumar, Puneet; Cai, Anthony; Huebinger, Ryan M.; Carlson, Deborah, L.; Zang, Qun S.; Wolf, Steven E.BACKGROUND: Burn patients suffer muscle mass loss associated with a hypercatabolic status. Mitochondria dynamics cycle is affected by metabolic status, and mitochondrial fission mediated high glucose induced cell death. Mitochondria function impairment associated with muscle mass loss has been observed in severe burn patients. We hypothesize that severe burn impaired muscle mass loss is associated with increased mitochondria fission with function impairment. The study was to investigate mitochondrial dynamics in response to burn serum stimulation. METHODS: Murine myoblast C2C12 cells were treated with DMEM media containing 10% rat serum isolated either from 40% TBSA scald burn rats, or control rats. Mitochondria was labeled with 3nM of MitoTracker Green FM dye, and live cell images were taken sequentially under a Nikon Ti Eclipse Confocal microscope. Cell lysates were collected for molecular biological analysis. Mitochondrial function was evaluated with Enzo Mito-ID membrane potential cytotoxicity kit. Target protein signals from cell lysate were detected by SDS-PAGE and western blot analysis. RESULTS: Mitochondrial morphology maintained the elongated linear shape in C2C12 cells when treated with 10% control rat serum. In contrast, when cells were treated with 10% burn serum, mitochondria reduced the elongated linear shape at 24 to 48 hours, and the florescent dye diffused at 72 hours. The cell florescent images showed an increase in circularity and fragmentation of mitochondria in C2C12 cells with burn serum stimulation. Meanwhile mitochondrial membrane potential decreased with 6hr post-burn serum stimulation. Western blot data showed that mitofusion-1 (Mfn1) significantly decreased in C2C12 cells with burn serum stimulation, confirming the observation of mitochondrial fission in response to burn serum. Cell death marker caspase 3 increased its expression in C2C12 cells with burn serum stimulation, suggesting a superfluous cell death in skeletal muscle after burn. CONCLUSION: Our results show an increase in the mitochondria fission/fusion ratio in C2C12 cells stimulated with burn serum isolated 6 hours after burn. The mechanism of mitochondrial fission with function impairment leading to muscle death is under investigation.Item [News](1977-01-18) Land, ChrisItem [News](1988-06-10) Fenley, BobItem [News](1973-08-03) Fenley, BobItem Reductive Carboxylation Is a Novel Pathway of Glutamine Metabolism That Supports the Growth of Tumor Cells with Metabolic Defects(2013-10-23) Mullen, Andrew Robbins; Burgess, Shawn C.; Abrams, John M.; Pearson, Gray W.; DeBerardinis, Ralph J.In growing cancer cells, oxidative metabolism of glucose and glutamine in the mitochondria provide precursors needed for de novo synthesis of proteins, nucleic acids and lipids. Yet, a subset tumors harbor genetic mutations in the electron transport chain or tricarboxylic acid cycle that disable normal oxidative mitochondrial function. Importantly, it has been unknown how these cells generate the biosynthetic precursors required for growth. To address this, I used models of mitochondrial dysfunction in isogenic cancer cell lines and studied their metabolism using a combination of Gas Chromatography- Mass Spectrometry and Nuclear Magnetic Resonance spectroscopy. In all cases, mitochondrial dysfunction stimulated a novel pathway of glutamine metabolism, characterized by reversal of the canonical tricarboxylic acid cycle, termed reductive carboxylation; providing a plausible mechanism for how cancer cells with mitochondrial defects generate biosynthetic precursors required for growth. To gain mechanistic insight into how this unusual pathway was regulated I carried out a targeted metabolomics analysis in our isogenic tumor cell models. This led to the striking discovery that cells engaged in the reductive carboxylation pathway also operate an additional metabolic pathway that, at first glance, would appear to be superfluous and inefficient. Functional characterization of this second pathway revealed, however, that its activity was necessary for the optimal function of the reductive carboxylation pathway. In summary, this work has given us insights into how cancer cells are able to grow in the context of defective mitochondria. Additionally, this has exposed a potential Achilles ’ heel that might be used to selectively kill tumors which rely on this pathway for growth.Item Regulation of Cytochrome C Release in UV-Induced Apoptosis(2006-05-16) Traer, Elie; Mumby, Marc C.Apoptosis, or programmed cell death, is vitally important for maintaining cellular homeostasis. When damaged cells do not appropriately enact the cell death program they have the potential to accumulate genetic mutations and become cancerous. Therefore, a mechanistic understanding of how cells decide to die would provide the foundation for drug discovery to specifically target cancer cells. Current genotoxic chemotherapeutics, and other sources such as ultraviolet (UV) radiation, damage DNA, leading to induction of apoptosis through the mitochondrial pathway. Cytochrome c is released from the mitochondria, binds dATP and Apaf-1, and together they form a structure called the apoptosome. The apoptosome then binds and activates caspase-9, which cleaves caspase-3 and other caspases resulting the morphological changes characteristic to apoptosis. To understand how UV causes apoptosis, an assay was developed to reproduce cytochrome c release in vitro. This assay revealed that UV radiation causes a rapid decrease of the anti-apoptotic protein Mcl-1 in HeLa cells. Reduction of Mcl-1 on the mitochondria causes in vitro release of cytochrome c from mitochondria three hours before in vivo release of cytochrome c is observed, i.e. the mitochondria are primed to release cytochrome c. Removal of Mcl-1 is required for UV-induced apoptosis, but it is not sufficient to induce apoptosis. This means that, in addition to mitochondrial priming, there is another required event, a second hit. It was reasonable to think that this required second hit might be bound specifically by Mcl-1. To that end, Mcl-1 was found to bind BimEL with far greater affinity than any other pro-apoptotic Bcl-2 family member. In addition, BimEL is dephosphorylated after UV in an ERK1/2-dependent manner. Despite perfectly fitting the profile of the second hit, BimEL and ERK1/2 phosphorylation do not have any affect on induction of caspases after UV. These results have helped gain insight into the regulation of cytochrome c release, which remains the most perplexing and important question in apoptosis.Item Regulation of Metabolic Processes by Micrornas and Class I Histone Deacetylases(2013-01-17) Carrer, Michele; Olson, Eric N.Obesity is a medical condition resulting from accumulation of excess body fat that affects more than 30% of the adult population in the U.S. Obesity-related pathological conditions include heart disease, stroke, type 2 diabetes and certain types of cancer. Despite the high incidence and the elevated social costs, the molecular basis of obesity and associated metabolic syndrome are still poorly understood. Yet, the need for novel therapeutic approaches for the treatment and prevention of obesity remains. In humans and animal model of disease, hallmarks of obesity include dysregulation of genes involved in mitochondrial function, lipid uptake and lipid storage. The dynamic and modifiable regulation of transcriptional pathways that control mitochondrial function and adipogenesis, as well as additional aspects of mammalian metabolism, will provide new approaches for pharmacological intervention in obesity. Thus, the modulation of epigenetic histone modifications and microRNA functions represents a potentially powerful approach for the treatment of metabolic disorders. We show that the Ppargc1b gene, which encodes the PGC-1β protein, also co-transcribes two microRNAs, miR-378 and miR-378*. Mice lacking miR-378/378* are resistant to high fat diet-induced obesity and display enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues. Taken together, our findings reveal that miR-378 and miR-378* function as integral components of the regulatory circuit formed by PGC-1beta and nuclear hormone receptors to control the overall oxidative capacity and energy homeostasis of insulin-target tissues. MiR-378/378* mutant mice do not display overt phenotypes under normal laboratory conditions, whereas their phenotypes become apparent under conditions of stress, in this case in response to excessive caloric intake. Thus, pharmacological modulation of miR-378/378* function might represent an effective approach in the treatment of obesity. In obese humans and mice, the unused caloric energy resulting from excessive net caloric intake is converted to triglycerides and stored in adipocytes for further usage. Lipid accumulation within adipocytes is under the control of a cascade of transcription factors that interact with histone acetyltransferases and deacetylases. We show that histone deacetylase inhibitors efficiently block adipocyte differentiation in vitro. Furthermore, through a loss-of-function approach, we provide evidence that histone deacetylases 1 and 2 play redundant and requisite roles in adipogenesis. In conclusion, we unveiled previously unrecognized roles for miR-378/378* in the control of mitochondrial metabolism and energy homeostasis, and for histone deacetylases in the control of adipocyte differentiation.Item [Southwestern News](1997-02-21) Steeves, Susan A.Item Structural Basis for the Activation of RIG-I/MAVS Antiviral Immune Signaling(2015-04-09) Xu, Hui; Rice, Luke M.; Chen, Zhijian J.; Jiang, Qiu-Xing; Rosen, Michael K.; Liu, QinghuaRetinoic acid inducible gene-I (RIG-I) is a key cytosolic pathogen RNA sensor that activates mitochondrial antiviral signaling protein (MAVS) to trigger rapid innate immune responses. Using RNAs of different lengths as model ligands, we showed that RIG-I oligomerized on dsRNA in an ATP hydrolysis-dependent and dsRNA length-dependent manner, which correlated with the strength of type-I interferon (IFN-I) activation. The obtained negative stain EM structure of full-length RIG-I in complex with a 5'ppp stem-loop RNA and the crystal structure of RIG-I/Ub complex elucidated a two-step oligomerization and conformational change of RIG-I for activation. RIG-I oligomers nucleate MAVS through homotypic interaction of the N-terminal caspase activation and recruitment domains (CARDs) and induce the formation of prion-like aggregates. The obtained cryoEM structure of left-handed helical filaments of MAVS CARD revealed specific interfaces between individual CARD subunits that are dictated by a combination of electrostatic and hydrophobic interactions and hydrogen bonding. Point mutations at multiple locations of these interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidated the structural mechanism of RIG-I activation by RNA and K63-linked ubiquitin chains as well as the activation of MAVS through polymerization, revealing a highly efficient signaling cascade for viral RNA sensing.Item Understanding Host Antiviral Signaling Pathways(2006-09-25) Bhargava, Rashu; Chen, Zhijian J.The innate immune response is the first line of defense against viral infections. Recent studies have revealed two immune receptor systems that detect virally-derived nucleic acids and trigger signaling pathways which lead to the activation of transcription factors like nuclear factor-kappa B (NF-kappa B), interferon regulatory factor 3 (IRF3) and interferon regulatory factor 7 (IRF7). These transcription factors regulate the synthesis of protective cytokines including type I interferons. The first detection system includes members of the toll-like receptor family (TLR3, TLR7, TLR8 and TLR9) that reside in the endosome and signal through the associated adaptor proteins. The second pathway uses retinoic acid inducible gene - I (RIG-I) to detect cytosolic viral double-stranded RNA. RIG-I signals to NF-kappa B and IRFs through its N-terminal CARD domains. An important area of research is to identify the downstream host factors that participate in these pathways and to determine the mechanism by which they function. Here, I describe the development of an in vitro biochemical assay to detect the activation of a key kinase involved in the activation of IRF3. This assay will be a valuable tool for identifying not only the specific IRF3 kinase, but also other molecular components involved in the antiviral signaling pathway. A bioinformatics approach led to the identification of a CARD domain containing protein, termed MAVS (Mitochondrial AntiViral Signaling protein) that functions downstream of RIG-I in the antiviral signaling pathway. Besides the N-terminal CARD domain, MAVS also contains a hydrophobic C-terminal transmembrane domain that targets the protein to the outer membrane of the mitochondria. The mitochondrial localization of MAVS is essential for signaling by MAVS. The importance of mitochondrial localization of MAVS for its function is underscored by the finding that hepatitis C virus (HCV) shuts down the host innate immune system by cleaving MAVS off the mitochondria. HCV achieves this through the action of NS3/4A, a virally encoded serine protease, which cleaves MAVS before the transmembrane domain at Cys-508. Prior to the identification of MAVS, the focus of antiviral research was on understanding the regulation of key transcription factors by established cytosolic signaling cascades. Our findings have established an important role for mitochondria in regulating antiviral immunity and represent a new paradigm in understanding the host-pathogen relationship.