Other Biochemistry, Biophysics, and Structural Biology Commons^™

Mitochondrial Reactive Oxygen Species In Lipotoxic Hearts Induces Post-Translational Modifications Of Akap121, Drp1 And Opa1 That Promote Mitochondrial Fission, Kensuke Tsushima, Heiko Bugger, Adam R. Wende, Jamie Soto, Gregory A. Jenson, Austin R. Tor, Rose Mcglauflin, Helena C. Kenny, Yuan Zhang, Rhonda Souvenir, Xiao X. Hu, Crystal L. Sloan, Renata O. Pereira, Vitor A. Lira, Kenneth W. Spitzer, Terry L. Sharp, Kooresh I. Shoghi, Genevieve C. Sparagna, Eva A. Rog-Zielinska, Peter Kohl, Oleh Khalimonchuk, Jean E. Schaffer, E. Dale Abel

Department of Biochemistry: Faculty Publications

Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood.

Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo.

Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation …

Ydj1 Governs Fungal Morphogenesis And Stress Response, And Facilitates Mitochondrial Protein Import Via Mas1 And Mas2, Jinglin L. Xie, Iryna Bohovych, Erin O.Y. Wong, Jean-Philippe Lambert, Anne-Claude Gingras, Oleh Khalimonchuk, Leah E. Cowen, Michelle D. Leach

Department of Biochemistry: Faculty Publications

Mitochondria underpin metabolism, bioenergetics, signalling, development and cell death in eukaryotes. Most of the ~1,000 yeast mitochondrial proteins are encoded in the nucleus and synthesised as precursors in the cytosol, with mitochondrial import facilitated by molecular chaperones. Here, we focus on the Hsp40 chaperone Ydj1 in the fungal pathogen Candida albicans, finding that it is localised to both the cytosol and outer mitochondrial membrane, and is required for cellular stress responses and for filamentation, a key virulence trait. Mapping the Ydj1 protein interaction network highlighted connections with co-chaperones and regulators of filamentation. Furthermore, the mitochondrial processing peptidases Mas1 and …

Identification Of Potential Tissue-Specific Cancer Biomarkers And Development Of Cancer Versus Normal Genomic Classifiers, Akram Mohammed, Greyson Biegert, Jiri Adamec, Tomáš Helikar

Department of Biochemistry: Faculty Publications

Machine learning techniques for cancer prediction and biomarker discovery can hasten cancer detection and significantly improve prognosis. Recent “OMICS” studies which include a variety of cancer and normal tissue samples along with machine learning approaches have the potential to further accelerate such discovery. To demonstrate this potential, 2,175 gene expression samples from nine tissue types were obtained to identify gene sets whose expression is characteristic of each cancer class. Using random forests classification and ten-fold cross-validation, we developed nine single-tissue classifiers, two multi-tissue cancer-versus-normal classifiers, and one multi-tissue normal classifier. Given a sample of a specified tissue type, the single-tissue …

Organ-Specific Regulation Of Atp7a Abundance Is Coordinated With Systemic Copper Homeostasis, Haarin Chun, Tracy Catterson, Heejeong Kim, Jaekwon Lee, Byung-Eun Kim

Department of Biochemistry: Faculty Publications

Copper (Cu) is an essential cofactor for various enzymatic activities including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Consequently, Cu dysregulation is associated with fatal neonatal disease, liver and cardiac dysfunction, and anemia. While the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation. Here we show, using Cu-deficient mouse models, that steadystate levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency …

Metalloproteases Of The Inner Mitochondrial Membrane, Roman M. Levytskyy, Iryna Bohovych, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

The inner mitochondrial membrane (IM) is among most protein-rich cellular compartments. The metastable IM sub-proteome where the concentration of proteins is approaching oversaturation creates a challenging protein folding environment with high probability for protein malfunction or aggregation. Failure to maintain protein homeostasis in such a setting can impair functional integrity of the mitochondria and drive clinical manifestations. The IM is equipped with a series of highly conserved, proteolytic complexes dedicated to the maintenance of normal protein homeostasis within this mitochondrial sub-compartment. Particularly important is a group of membrane-anchored metallopeptidases commonly known as m-AAA and i-AAA proteases, and the ATP-independent Oma1 …

The Role Of Interactions Of Long Non-Coding Rnas And Heterogeneous Nuclear Ribonucleoproteins In Regulating Cellular Functions, Xinghui Sun, Mohamed Sham Shihabudeen Haider Ali, Matthew Moran

Department of Biochemistry: Faculty Publications

Long non-coding RNAs (lncRNAs) are emerging as critical regulators of various biological processes and human diseases. The mechanisms of action involve their interactions with proteins, RNA and genomic DNA. Most lncRNAs display strong nuclear localization. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RNA-binding proteins that are important for multiple aspects of nucleic acid metabolism. hnRNPs are also predominantly expressed in the nucleus. This review discusses the interactions of lncRNAs and hnRNPs in regulating gene expression at transcriptional and post-transcriptional levels or by changing genomic structure, highlighting their involvements in glucose and lipid metabolism, immune response, DNA damage response, …

Identification And Metabolite Profiling Of Chemical Activators Of Lipid Accumulation In Green Algae, Nishikant Wase, Boqiang Tu, James Allen, Paul N. Black, Concetta Dirusso

Department of Biochemistry: Faculty Publications

Microalgae are proposed as feedstock organisms useful for producing biofuels and coproducts. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii, and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity, and 15 were selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG …

Physiological Evidence For Isopotential Tunneling In The Electron Transport Chain Of Methane-Producing Archaea, Nikolas Duszenko, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli. We found the quantity of MPh per …

Microbal Strans And Methods Of Making And Using, Nicole Roswitha Buan Murphy, Jennifer Catlett

Department of Biochemistry: Faculty Publications

Microbial strains are provided, as are methods of making and using Such microbial strains.

Pneb193-Derived Suicide Plasmids For Gene Deletion And Protein Expression In The Methane-Producing Archaeon, Methanosarcina Acetivorans, Mitchell T. Shea, Mary E. Walter, Nikolas Duszenko, Anne-Lise Ducluzeau, Jared Aldridge, Shannon K. King, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Gene deletion and protein expression are cornerstone procedures for studying metabolism in any organism, including methane-producing archaea (methanogens). Methanogens produce coenzymes and cofactors not found in most bacteria, therefore it is sometimes necessary to express and purify methanogen proteins from the natural host. Protein expression in the native organism is also useful when studying post-translational modifications and their effect on gene expression or enzyme activity. We have created several new suicide plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The new plasmids are derived from the commercially available E. coli plasmid, pNEB193, and cannot replicate …

Seasonal Switchgrass Ecotype Contributions To Soil Organic Carbon, Deep Soil Microbial Community Composition And Rhizodeposit Uptake During An Extreme Drought, Catherine E. Stewart, Damaris Roosendaal, Karolien Denef, Elizabeth Pruessner, Louise H. Comas, Gautam Sarath, Virginia L. Jin, Marty R. Schmer, Madhavan Soundararajan

Department of Biochemistry: Faculty Publications

The importance of rhizodeposit C and associated microbial communities in deep soil C stabilization is relatively unknown. Phenotypic variability in plant root biomass could impact C cycling through belowground plant allocation, rooting architecture, and microbial community abundance and composition. We used a pulse-chase ¹³C labeling experiment with compound-specific stable-isotope probing to investigate the importance of rhizodeposit C to deep soil microbial biomass under two switchgrass ecotypes (Panicum virgatum L., Kanlow and Summer) with contrasting root morphology. We quantified root phenology, soil microbial biomass (phospholipid fatty acids, PLFA), and microbial rhizodeposit uptake (¹³C-PLFAs) to 150 cm over …

Secs (Sinusoidal Endothelial Cells), Liver Microenvironment, And Fibrosis, Vaishaali Natarajan, Edward N. Harris, Srivatsan Kidambi

Department of Biochemistry: Faculty Publications

Liver fibrosis is awound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs …

Gene Duplication And Neo-Functionalization In The Evolutionary And Functional Divergence Of The Metazoan Copper Transporters Ctr1 And Ctr2, Brandon L. Logeman, L. Kent Wood, Jaekwoon Lee, Dennis J. Thiele

Department of Biochemistry: Faculty Publications

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu_ across both the plasma membrane and endomembranes. Although mammalian Ctr¹ functions as a Cu_ transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr², has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr¹ activity, but …

Carbon And Acyl Chain Flux During Stress-Induced Triglyceride Accumulation By Stable Isotopic Labeling Of The Polar Microalga Coccomyxa Subellipsoidea C169, James W. Allen, Concetta C. Dirusso, Paul N. Black

Department of Biochemistry: Faculty Publications

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO₂ balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-¹³C]glucose, ¹³CO₂, or D₂O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid …

Oxidative Stress, Metabolomics Profiling, And Mechanism Of Local Anesthetic Induced Cell Death In Yeast, Cory Honsinger Thomas Boone, Ryan A. Grove, Dana Adamcova, Javier Seravalli, Jiri Adamec

Department of Biochemistry: Faculty Publications

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6 h. Exposure of an LC₅₀ dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by …

Toxoplasma Dj-1 Regulates Organelle Secretion By A Direct Interaction With Calcium-Dependent Protein Kinase 1, Matthew A. Child, Megan Garland, Ian Foe, Peter Madzelan, Moritz Treeck, Wouter A. Van Der Linden, Kristina Oresic Bender, Evanthie Weerapana, Mark A. Wilson, John C. Boothroyd, Michael L. Reese

Department of Biochemistry: Faculty Publications

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-A resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, …

Significant Enhancement Of Fatty Acid Composition In Seeds Of The Allohexaploid, Camelina Sativa, Using Crispr/Cas9 Gene Editing, Wen Zhi Jhang, Isabelle M. Henry, Peter G. Lynagh, Lucia Comai, Edgar B. Cahoon, Donald P. Weeks

Department of Biochemistry: Faculty Publications

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. …