Other Biochemistry, Biophysics, and Structural Biology Commons^™

Molecular Evolution Of Protein-Rna Mimicry As A Mechanism For Translational Control, Assaf Katz, Lindsey Solden, S. Betty Zou, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is a conserved ribosome-binding protein that structurally mimics tRNA to enable the synthesis of peptides containing motifs that otherwise would induce translational stalling, including polyproline. In many bacteria, EF-P function requires post-translational modification with (R)-β-lysine by the lysyl-tRNA synthetase paralog PoxA. To investigate how recognition of EF-P by PoxA evolved from tRNA recognition by aminoacyl-tRNA synthetases, we compared the roles of EF-P/PoxA polar contacts with analogous interactions in a closely related tRNA/synthetase complex. PoxA was found to recognize EF-P solely via identity elements in the acceptor loop, the domain of the protein that interacts with the …

Cytochrome B5 Reductase Encoded By Cbr1 Is Essential For A Functional Male Gametophyte In Arabidopsis, Laura L. Wayne, James G. Wallis, Rajesh Kumar, Jennifer E. Markham, John Browse

Department of Biochemistry: Faculty Publications

In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the gene encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 …

Direction Of Aminoacylated Transfer Rnas Into Antibiotic Synthesis And Peptidoglycan-Mediated Antibiotic Resistance, Jennifer Shepherd, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Prokaryotic aminoacylated‐transfer RNAs often need to be efficiently segregated between translation and other cellular biosynthetic pathways. Many clinically relevant bacteria, including Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa direct some aminoacylated‐tRNA species into peptidoglycan biosynthesis and/or membrane phospholipid modification. Subsequent indirect peptidoglycan cross‐linkage or change in membrane permeability is often a prerequisite for high‐level antibiotic resistance. In Streptomycetes, aminoacylated‐tRNA species are used for antibiotic synthesis as well as antibiotic resistance. The direction of coding aminoacylated‐tRNA molecules away from translation and into antibiotic resistance and synthesis pathways are discussed in this review.

Lipid Ii-Independent Trans Editing Of Mischarged Trnas By The Penicillin Resistance Factor Murm, Jennifer Shepherd, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches l-serine or l-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNAAla, as observed in other bacteria, and also transferred alanine, serine, and glycine to tRNA^Phe. S. pneumoniae tRNA^Phe has an unusual U4:C69 mismatch in its acceptor stem that …

Diversity Of Plant Methionine Sulfoxide Reductases B And Evolution Of A Form Specific For Free Methionine Sulfoxide, Dung Tien Le, Lionel Tarrago, Yasuko Watanabe, Alaattin Kaya, Byung Cheon Lee, Uyen Tran, Rie Nishiyama, Dmitri E. Fomenko, Vadim N. Gladyshev, Lam-Son Phan Tran

Department of Biochemistry: Faculty Publications

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed …

A Cell Simulator Platform: The Cell Collective, Tomáš Helikar, B. Kowal, J. A. Rogers

Department of Biochemistry: Faculty Publications

Diseases are often a result of multiple malfunctions in complex, nonlinear biological/biochemical networks. As such, these processes are far more complicated to understand because they tend to give rise to functions that are emergent in nature, i.e., higher-level (mal)functions that are more than the sum of their parts. Systems biology provides a new approach to understanding biological systems and diseases from a holistic perspective.

Divergent Protein Motifs Direct Ef-P Mediated Translational Regulation In Salmonella And Escherichia Coli, Steven J. Hersch, Mengchi Wang, S. Betty Zou, Kyung-Mee Moon, Leonard J. Foster, Michael Ibba, William Wiley Navarre

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the …

Rescuing Acetylcholinesterase From Nerve Agent Inhibition: Protein Dynamics Driven Drug Discovery, Aiyana M. Emigh, Brian Bennion

STAR Program Research Presentations

Severe morbidity and mortality consequences result from irreversible inhibition of human acetylcholinesterase by organophosphates (OPs). Oxime-based reactivators are currently the only available treatments but lack efficacy in the central nervous system (CNS) where the most damage occurs. Computational docking and molecular dynamics (MD) simulations reveal complex structural barriers that may reduce oxime efficacy. These results may guide future drug designs of more effective countermeasures.

Proline Mechanisms Of Stress Survival, Xinwen Liang, Lu Zhang, Sathish Kumar Natarajan, Donald F. Becker

Department of Biochemistry: Faculty Publications

Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria …

Microrna Function In Human Diseases, Sathish Kumar Natarajan, Mary Anne Smith, Cody J. Wehrkamp, Ashley M. Mohr, Justin L. Mott

Department of Biochemistry: Faculty Publications

MicroRNAs are emerging as a hot topic in research, and rightfully so. They show great promise as targets of treatment and as markers for common human diseases, such as cancer and metabolic diseases. In this review, we address some of the basic questions regarding micro- RNA function in human disease and the clinical significance of microRNAs. Specifically, micro- RNAs in epigenetics, cancer, and metabolic diseases are discussed, with examples taken from cholangiocarcinoma and nonalcoholic fatty liver disease.

The Hyaluronan Receptor For Endocytosis (Hare) Activates Nf-Κb-Mediated Gene Expression In Response To 40–400-Kda, But Not Smaller Or Larger, Hyaluronans, Madhu S. Pandey, Bruce A. Baggenstoss, Jennifer Washburn, Edward N. Harris, Paul H. Weigel

Department of Biochemistry: Faculty Publications

Background: HARE mediates systemic clearance of hyaluronan (HA), which turns over continuously in tissues.

Results: HARE uptake of 40–400-kDa, but not larger or smaller, HA stimulated NF-κB activation.

Conclusion: HA-HARE signal complexes activate NF-κB and gene transcription only with optimally sized HA.

Significance: HARE responsiveness to a narrow size range ofHAdegradation products may be a sensing system to detect tissue ECM stress.

Functional Convergence Of Structurally Distinct Thioesterases From Cyanobacteria And Plants Involved In Phylloquinone Biosynthesis, Fabienne Furt, William J. Allen, Joshua R. Widhalm, Peter Madzelan, Robert C. Rizzo, Gilles J. Basset, Mark A. Wilson

Department of Biochemistry: Faculty Publications

The synthesis of phylloquinone (vitamin K₁) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy- 2-naphthoyl-CoA (DHNA-CoA) to release 1,4- dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacterium Synechocystis (Slr0204) and the flowering plant Arabidopsis thaliana (AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNACoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases from Pseudomonas and Arthrobacter. Like the 4-HBA-CoA …

The Basis For Limited Specificity And Mhc Restriction In A T Cell Receptor Interface, Kurt H. Piepenbrink, Sydney J. Blevins, Daniel R. Scott, Brian M. Baker

Department of Biochemistry: Faculty Publications

αβ Tcell receptors (TCRs) recognize peptides presented by major histocompatibility complex (MHC) proteins using multiple complementarity-determining region (CDR) loops. TCRs display an array of poorly understood recognition properties, including specificity, crossreactivity and MHC restriction. Here we report a comprehensive thermodynamic deconstruction of the interaction between the A6 TCR and the Tax peptide presented by the class I MHC HLA-A*0201, uncovering the physical basis for the receptor’s recognition properties. Broadly, our findings are in conflict with widely held generalities regarding TCR recognition, such as the relative contributions of central and peripheral peptide residues and the roles of the hypervariable and germline …

Elucidating Causes Of Diporeia Decline In The Great Lakes Via Metabolomics: Physiological Responses After Exposure To Different Stressors, Suman Maity, Amber Jannasch, Jiri Adamec, James M. Watkins, Thomas Nalepa, Tomas O. Höök, Maria S. Sepúlveda

Department of Biochemistry: Faculty Publications

The benthic macroinvertebrate Diporeia spp. have been extirpated from many areas of the Laurentian Great Lakes, but the mechanisms underlying such declines are not fully understood. Diporeia declines coinciding with the invasion of exotic dreissenid mussels (zebra and quagga) have led to the hypothesis that Diporeia declines are a result of decreased food availability from increasing competition with dreissenids for diatoms. There is additional evidence that Diporeia are negatively affected when in close proximity to dreissenids, probably because of exposure to toxins present in the mussels’ pseudofeces. Diporeia are also known to be sensitive to anthropogenic contaminants (such as polychlorinated …

Selenodb 2.0: Annotation Of Selenoprotein Genes In Animals And Their Genetic Diversity In Humans, Frederic Romagne, Didac Santesmasses, Louise White, Gaurab K. Sarangi, Marco Mariotti, Ron Hübler, Antje Weihmann, Genis Parra, Vadim N. Gladyshev, Roderic Guigo, Sergi Castellano

Department of Biochemistry: Faculty Publications

SelenoDB (http://www.selenodb.org) aims to provide high-quality annotations of selenoprotein genes, proteins and SECIS elements. Selenoproteins are proteins that contain the amino acid selenocysteine (Sec) and the first release of the database included annotations for eight species. Since the release of SelenoDB 1.0 many new animal genomes have been sequenced. The annotations of selenoproteins in new genomes usually contain many errors in major databases. For this reason, we have now fully annotated selenoprotein genes in 58 animal genomes. We provide manually curated annotations for human selenoproteins, whereas we use an automatic annotation pipeline to annotate selenoprotein genes in other animal genomes. …

Comparative Metabolic Profiling Of Haberlea Rhodopensis, Thellungiella Halophyla, And Arabidopsis Thaliana Exposed To Low Temperature, Maria Benina, Toshihiro Obata, Nikolay Mehterov, Ivan Ivanov, Veselin Petrov, Valentina Toneva, Alisdair R. Fernie, Tsanko S. Gechev

Department of Biochemistry: Faculty Publications

Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desiccation but also with ability to withstand low temperatures and freezing stress. In order to identify biochemical strategies which contribute to Haberlea's remarkable stress tolerance, the metabolic reconfiguration of H. rhodopensis during low temperature (4^◦C) and subsequent return to optimal temperatures (21^◦C) was investigated and compared with that of the stress tolerant Thellungiella halophyla and the stress sensitive Arabidopsis thaliana. Metabolic analysis by GC-MS revealed intrinsic differences in the metabolite levels of the three species even at 21^◦C. H. rhodopensis …

Rapid Detection And Quantification Of Triacylglycerol By Hplc–Elsd In Chlamydomonas Reinhardtii And Chlorella Strains, Naoko Kobayashi, Eric A. Noel, Austin Barnes, Julian Rosenberg, Concetta C. Dirusso, Paul N. Black, George A. Oyler

Department of Biochemistry: Faculty Publications

Triacylglycerol (TAG) analysis and quantification are commonly performed by first obtaining a purified TAG fraction from a total neutral lipid extract using thinlayer chromatography (TLC), and then analyzing the fatty

acid composition of the purified TAG fraction by gas chromatography (GC). This process is time-consuming, labor

intensive and is not suitable for analysis of small sample sizes or large numbers. A rapid and efficient method for monitoring oil accumulation in algae using high performance liquid chromatography for separation of all lipid classes combined with detection by evaporative light scattering (HPLC–ELSD) was developed and compared to the conventional TLC/GC method. TAG …

Probing Arabidopsis Chloroplast Diacylglycerol Pools By Selectively Targeting Bacterial Diacylglycerol Kinase To Suborganellar Membranes, Bagyalakshmi Muthan, Rebecca Roston, John E. Froehlich, Christoph Benning

Department of Biochemistry: Faculty Publications

Diacylglycerol (DAG) is an intermediate in metabolism of both triacylglycerols and membrane lipids. Probing the steady-state pools of DAG and understanding how they contribute to the synthesis of different lipids is important when designing plants with altered lipid metabolism. However, traditional methods of assaying DAG pools are difficult, because its abundance is low and because fractionation of subcellular membranes affects DAG pools. To manipulate and probe DAG pools in an in vivo context, we generated multiple stable transgenic lines of Arabidopsis (Arabidopsis thaliana) that target an Escherichia coli DAGkinase (DAGK) to each leaflet of each chloroplast envelope membrane. …

Reporters For The Analysis Of N-Glycosylation In Candida Albicans, Shahida Shahana, Hector M. Mora-Montes, Luis Castillo, Iryna Bohovych, Chirag C. Sheth, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

A large proportion of Candida albicans cell surface proteins are decorated post-translationally by glycosylation. Indeed N-glycosylation is critical for cell wall biogenesis in this major fungal pathogen and for its interactions with host cells. A detailed understanding of N-glycosylation will yield deeper insights into host-pathogen interactions. However, the analysis of N-glycosylation is extremely challenging because of the complexity and heterogeneity of these structures. Therefore, in an attempt to reduce this complexity and facilitate the analysis of N-glycosylation, we have developed new synthetic C. albicans reporters that carry a single N-linked glycosylation site derived from Saccharomyces cerevisiae Suc₂. These …

A Comprehensive, Multi-Scale Dynamical Model Of Erbb Receptor Signal Transduction In Human Mammary Epithelial Cells, Tomáš Helikar, Naomi Kochi, Bryan Kowal, Manjari Dimri, Mayumi Naramura, Srikumar M. Raja, Vimla Band, Hamid Band, Jim A. Rogers

Department of Biochemistry: Faculty Publications

The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1) have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins …

Sbml Qualitative Models: A Model Representation Format And Infrastructure To Foster Interactions Between Qualitative Modelling Formalisms And Tools, Claudine Chaouiya, Duncan Bérenguier, Sarah M. Keating, Aurélien Naldi, Martijn P. Van Iersel, Nicolas Rodriguez, Andreas Dräger, Finja Büchel, Thomas Cokelaer, Bryan Kowal, Benjamin Wicks, Emanuel Gonçalves, Julien Dorier, Michel Page, Pedro T. Monteiro, Axel Von Kamp, Ioannis Xenarios, Hidde De Jong, Michael Hucka, Steffen Klamt, Denis Thieffry, Nicolas Le Novère, Julio Saez-Rodriguez, Tomáš Helikar

Department of Biochemistry: Faculty Publications

Background: Qualitative frameworks, especially those based on the logical discrete formalism, are increasingly used to model regulatory and signalling networks. A major advantage of these frameworks is that they do not require precise quantitative data, and that they are well-suited for studies of large networks. While numerous groups have developed specific computational tools that provide original methods to analyse qualitative models, a standard format to exchange qualitative models has been missing.

Results: We present the Systems Biology Markup Language (SBML) Qualitative Models Package (“qual”), an extension of the SBML Level 3 standard designed for computer representation of qualitative …