Life Sciences Commons^™

Targeted Insertion Of Cysteine By Decoding Uga Codons With Mammalian Selenocysteine Machinery, Xue-Ming Xu, Anton A. Turanov, Bradley A. Carlson, Min-Hyuk Yoo, Robert A. Everley, Renu Nandakumar, Irina Sorokina, Steven P. Gygi, Vadim N. Gladyshev, Dolph L. Hatfield

Department of Biochemistry: Faculty Publications

Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA^[Ser]Sec and its insertion was dependent on the Sec insertion sequence element in the 3′ UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA^[Ser]Sec to generate Cys-tRNA^[Ser]Sec. Cys was inserted in vivo at UGA …

Lateral Blood Flow Velocity Estimation Based On Ultrasound Speckle Size Change With Scan Velocity, Tiantian Xu, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

Conventional (Doppler-based) blood flow velocity measurement methods using ultrasound are capable of resolving the axial component (i.e., that aligned with the ultrasound propagation direction) of the blood flow velocity vector. However, these methods are incapable of detecting blood flow in the direction normal to the ultrasound beam. In addition, these methods require repeated pulse-echo interrogation at the same spatial location. A new method has been introduced which estimates the lateral component of blood flow within a single image frame using the observation that the speckle pattern corresponding to blood reflectors (typically red blood cells) stretches (i.e., is smeared) if the …

The In Situ Function Of A Microbial Community Profiled By Ft-Ir: A Snapshot In Time, Ryan Roberts

Department of Biochemistry: Dissertations, Theses, and Student Research

Photographs of an ecosystem are an important tool in macro-community ecology. The photograph is a permanent record of species phenotype. In microbiology, biochemical activity provides the most descriptive information of an organism’s phenotype. A method for fingerprinting all biochemical activities occurring within a microbial community is analogous to a photograph. Infrared spectroscopy in the region between wavelengths 2500 to 20,000 nm (mid-IR) is a well established instrumental method for fingerprinting the total biochemical profile of axenic cultures. Spectra are complex and sensitive as demonstrated by the ability to discriminate between strains of bacteria, fungi, and algae. This thesis develops the …

Glycopeptidome Of A Heavily N-Glycosylated Cell Surface Glycoprotein Of Dictyostelium Implicated In Cell Adhesion, Christa L. Feasley, Jennifer M. Johnson, Christopher M. West, Catherine P. Chia

School of Biological Sciences: Faculty Publications

Genetic analysis has implicated the cell surface glycoprotein gp130 in cell interactions of the social amoeba Dictyostelium, and information about the utilization of the 18 N-glycosylation sequons present in gp130 is needed to identify critical molecular determinants of its activity. Various glycomics strategies, including mass spectrometry of native and derivatized glycans, monosaccharide analysis, exoglycosidase digestion, and antibody binding, were applied to characterize a nonanchored version secreted from Dictyostelium. s-gp130 is modified by a predominant Man8GlcNAc4 species containing bisecting and intersecting GlcNAc residues and additional high-mannose N-glycans substituted with sulfate, methyl-phosphate, and/or core R3-fucose. Site mapping confirmed the occupancy …

T10c12 Conjugated Linoleic Acid Causes Delipidation In 3t3-L1 Adipocytes And Mice, Shan Jiang

Department of Animal Science: Dissertations, Theses, and Student Research

T10c12 conjugated linoleic acid (t10c12 CLA) causes the reduction of triglyceride contents in adipocytes. T10c12 CLA’s delipidation effect is associated with decreased nutrient uptake, adipogenesis, lipogenesis, and increased energy expenditure, lipolysis, fatty acid oxidation in adipocytes. However, the molecular mechanisms of CLA’s delipidation effects are still unknown. AMP-activated protein kinase (AMPK), a central regulator of cellular energy levels, is activated by an increase in the cellular AMP:ATP ratio or various cellular stresses. We demonstrated that t10c12 CLA activated AMPK in 3T3-L1 adipocytes, leading to inhibition of anabolic biosynthesis and increase of energy expenditure. Strong activation of AMPK can induce an …

The Genomics Education Partnership: Successful Integration Of Research Into Laboratory Classes At A Diverse Group Of Undergraduate Institutions, Christopher D. Shaffer, Consuelo Alvarez, Cheryl Bailey, Daron Barnard, Satish Bhalla, Chitra Chandrasekaran, Vidya Chandrasekaran, Hui-Min Chung, Douglas R. Dorer, Chunguang Du, Todd T. Eckdahl, Jeff L. Poet, Donald Frohlich, Anya L. Goodman, Yuying Gossner, Charles Hauser, Laura L.M. Hoopes, Diana Johnson, Christopher J. Jones, Marian Kaehler, Nighat Kokan, Olga R. Kopp, Gary A. Kuleck, Gerard Mcneil, Robert Moss, Jennifer L. Myka, Alexis Nagengast, Robert Morris, Paul J. Overvoorde, Elizabeth Shoop, Susan Parrish, Kelynne Reed, E. Gloria Regisford, Dennis Revie, Anne G. Rosenwald, Ken Saville, Stephanie Schroeder, Mary Shaw, Gary Skuse, Christopher Smith, Mary Smith, Eric P. Spana, Mary Spratt, Joyce Stamm, Jeff S. Thompson, Matthew Wawersik, Barbara A. Wilson, Jim Youngblom, Wilson Leung, Jeremy Buhler, Elaine R. Mardis, David Lopatto, Sarah C.R. Elgin

Department of Biochemistry: Faculty Publications

Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 …

Characterization Of Glycation Sites On Human Serum Albumin Using Mass Spectrometry, Omar S. Barnaby

Department of Chemistry: Dissertations, Theses, and Student Research

The modification of proteins by reducing sugars is a process that occurs naturally in the body. This process, which is known as glycation, has been linked to many of the chronic complications encountered during diabetes. Glycation has also been linked to changes in the binding of human serum albumin (HSA) to several drugs and small solutes in the body. While these effects are known, there is little information that explains why these changes in binding occur. The goal of this project was to obtain qualitative and quantitative information about glycation that occurs on HSA. The first section of this dissertation …

Role Of All Of The Prrsv Glycoproteins In Protective Immune Response, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions amongst each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by co-immunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that strong interaction exists between GP4 and GP5 proteins, although weak interactions …

Cell-Type Specific Distribution Of Chloride Transporters In The Rat Suprachiasmatic Nucleus, Michael A. Belenky, Patricia J. Sollars, David B. Mount, Seth L. Alper, Yosef Yarom, Gary E. Pickard

School of Veterinary and Biomedical Sciences: Faculty Publications

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use γ-aminobutyric acid (GABA) as a neurotransmitter, the principal inhibitory neurotransmitter in the adult central nervous system. Acting via the ionotropic GABAA receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl− influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function …

Charge Switch Nucleotides, John G. K. Williams, Gregory R. Bashford, Jiyan Chen, Dan Draney, Nara Narayanan, Bambi L. Reynolds, Pamela Sheaff

Biomedical Imaging and Biosignal Analysis Laboratory

The present invention provides compounds, methods and systems for sequencing nucleic acid using single molecule detection. Using labeled NPs that exhibit charge-switching behavior, single-molecule DNA sequencing in a microchannel sorting system is realized. In operation, sequencing products are detected enabling real-time sequencing as successive detectable moieties flow through a detection channel. By electrically sorting charged molecules, the cleaved product molecules are detected in isolation Without interference from unincorporated NPs and Without illuminating the polymerase-DNA complex.

Porcine Reproductive And Respiratory Syndrome Virus Nonstructural Protein 1Β Modulates Host Innate Immune Response By Antagonizing Irf3 Activation, Lalit Beura, Saumendra Sarkar, Byungjoon Kwon, Sakthivel Subramaniam, Clinton J. Jones, Asit K. Pattnaik, Fernando A. Osorio

School of Veterinary and Biomedical Sciences: Faculty Publications

Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-β) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, …

The Minor Envelope Glycoproteins Gp2a And Gp4 Of Porcine Reproductive And Respiratory Syndrome Virus Interact With The Receptor Cd163, Phani B. Das, Phat X. Dinh, Israrul H. Ansari, Marcelo De Lima, Fernando A. Osorio, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions with each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by coimmunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that a strong interaction exists between the GP4 and GP5 proteins, although …

Very-Long-Chain Fatty Acids Are Involved In Polar Auxin Transport And Developmental Patterning In Arabidopsis, Françoise Roudier, Lionel Gissot, Frédéric Beaudoin, Richard Haslam, Louise V. Michaelson, Jessica Marion, Diana Molino, Amparo Lima, Liên Bach, Halima Morin, Frédérique Tellier, Jean-Christophe Palauqui, Yannick Bellec, Charlotte Renne, Martine Miquel, Marco Dacosta, Julien Vignard, Christine Rochat, Jennifer E. Markham, Patrick Moreau, Jonathan Napier, Jean-Denis Faure

Department of Biochemistry: Faculty Publications

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of …

Cytotoxicity Of Bacterial-Derived Toxins To Immortal Lung Epithelial And Macrophage Cells, Dianne E. Peterson, Jayne M. Collier, Matthew E. Katterman, Rachel A. Turner, Mark R. Riley

Department of Biological Systems Engineering: Papers and Publications

Health risks associated with inhalation and deposition of biological materials have been a topic of great concern due to highly publicized cases of inhalation anthrax, of new regulations on the release of particulate matter, and to increased concerns on the hazards of indoor air pollution. Here, we present an evaluation of the sensitivity of two immortal cell lines (A549, human lung carcinoma epithelia) and NR8383 (rat alveolar macrophages) to a variety of bacterial-derived inhalation hazards and simulants including etoposide, gliotoxin, streptolysin O, and warfarin. The cell response is evaluated through quantification of changes in mitochondrial succinate dehydrogenase activity, release of …

Method For Identification Of Virulence Determinants, Raul G. Barletta, Beth Harris

School of Veterinary and Biomedical Sciences: Faculty Publications

Disclosed are methods for the determination of virulence determinants in bacteria and in particular bacteria of the genus Mycobacterium. Also disclosed are compositions and methods for stimulating an immune response in an animal using bacteria and virulence determinants identified by the methods of the present invention.

Udp-Glucose Dehydrogenase As A Novel Field-Specific Candidate Biomarker Of Prostate Cancer, Dali Huang, George P. Casale, Jun Tian, Subodh M. Lele, Vladimir M. Pisarev, Melanie A. Simpson, George P. Hemstreet Iii

Department of Biochemistry: Faculty Publications

Uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor for synthesis of glycosaminoglycans and proteoglycans that promote aggressive prostate cancer (PC) progression. The purpose of our study was to determine if the UGDH expression in normal appearing acini (NAA) from cancerous glands is a candidate biomarker for PC field disease/effect assayed by quantitative fluorescence imaging analysis (QFIA). A polyclonal antibody to UGDH was titrated to saturation binding and fluorescent microscopic images acquired from fixed, paraffin-embedded tissue slices were quantitatively analyzed. Specificity of the assay was confirmed by Western blot analysis and competitive inhibition …

Identification And Characterization Of Small Compound Inhibitors Of Human Fatp2, Angel Sandoval, Aalap Chokshi, Elliot D. Jesch, Paul N. Black, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

Fatty acid transport proteins (FATPs) are bifunctional proteins, which transport long chain fatty acids into cells and activate very long chain fatty acids by esterification with coenzyme A. In an effort to understand the linkage between cellular fatty acid transport and the pathology associated with excessive accumulation of exogenous fatty acids, we targeted FATP-mediated fatty acid transport in a high throughput screen of more than 100,000 small diverse chemical compounds in yeast expressing human FATP2 (hsFATP2). Compounds were selected for their ability to depress the transport of the fluorescent long chain fatty acid analogue, C₁-BODIPY-C₁₂. Among …

Mzm1 Influences A Labile Pool Of Mitochondrial Zinc Important For Respiratory Function, Aaron Atkinson, Oleh Khalimonchuk, Pamela Smith, Hana Sabic, David Eide, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Zinc is essential for function of mitochondria as a cofactor for

several matrix zinc metalloproteins. We demonstrate that a

labile cationic zinc component of low molecular mass exists in

the yeast mitochondrial matrix. This zinc pool is homeostatically

regulated in response to the cellular zinc status. This pool

of zinc is functionally important because matrix targeting of a

cytosolic zinc-binding protein reduces the level of labile zinc

and interferes with mitochondrial respiratory function. We

identified a series of proteins that modulate the matrix zinc

pool, one of which is a novel conserved mitochondrial protein

designated Mzm1. Mutant mzm1∆ cells …

Structural Basis For Feedback And Pharmacological Inhibition Of Saccharomyces Cerevisiae Glutamate Cysteine Ligase, Ekaterina I Biterova, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A ; R = 19.9%, R_free = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine- …

Cug Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms In Mouse Testes, Maxim Gerashchenko, Dan Su, Vadim Gladyshev

Department of Biochemistry: Faculty Publications

Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate …

Identification And Characterization Of Oxalate Oxidoreductase, A Novel Thiamine Pyrophosphate-Dependent 2-Oxoacid Oxidoreductase That Enables Anaerobic Growth On Oxalate, Elizabeth Pierce, Donald F. Becker, Stephen W. Ragsdale

Department of Biochemistry: Faculty Publications

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO₂ fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO₂ or bicarbonate. Like …

Methanogenesis By Methanosarcina Acetivorans Involves Two Structurally And Functionally Distinct Classes Of Heterodisulfide Reductase, Nicole R. Buan, William W. Metcalf

Department of Biochemistry: Faculty Publications

Biochemical studies have revealed two distinct classes of Coenzyme B-Coenzyme M heterodisulfide (CoB-S-S-CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methaneproducing archaea. A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane-bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic …

The Role Of Coa2 In Hemylation Of Yeast Cox1 Revealed By Its Genetic Interaction With Cox10, Megan Bestwick, Oleh Khalimonchuk, Fabien Pierrel, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Saccharomyces cerevisiae cells lacking the cytochrome c oxidase (CcO) assembly factor Coa2 are impaired in Cox1 maturation and exhibit a rapid degradation of newly synthesized Cox1. The respiratory deficiency of coa2 Δ cells is suppressed either by the presence of a mutant allele of the Cox10 farnesyl transferase involved in heme a biosynthesis or through impaired proteolysis by the disruption of the mitochondrial Oma1 protease. Cox10 with an N196K substitution functions as a robust gain-of-function suppressor of the respiratory deficiency of coa2 Δ cells but lacks suppressor activity for two other CcO assembly mutant strains, the coa1 Δ and shy1 …

Formation Of The Redox Cofactor Centers During Cox1 Maturation In Yeast Cytochrome Oxidase, Oleh Khalimonchuk, Megan Bestwick, Brigitte Meunier, Talina C. Watts, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The biogenesis of cytochrome c oxidase initiates with synthesis and maturation of the mitochondrionencoded Cox1 subunit prior to the addition of other subunits. Cox1 contains redox cofactors, including the low-spin heme a center and the heterobimetallic heme a₃:Cu_B center. We sought to identify the step in the maturation of Cox1 in which the redox cofactor centers are assembled. Newly synthesized Cox1 is incorporated within one early assembly intermediate containing Mss51 in Saccharomyces cerevisiae. Subsequent Cox1 maturation involves the progression to downstream assembly intermediates involving Coa1 and Shy1. We show that the two heme a cofactor sites …

Regulation Of Sealing Ring Formation By L-Plastin And Cortactin In Osteoclasts, Tao Ma, Kavitha Sadashivalah, Nandakumar Madayiputhiya, Meenakshi A. Chellaia

Department of Biochemistry: Faculty Publications

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be …

Quantitative Nuclear Proteomics Identifies Mtor Regulation Of Dna Damage Response, Sricharan Bandhakavi, Young-Mi Kim, Seung-Hyun Ro, Hongwei Xie, Getiria Onsongo, Chang-Bong Jun, Do-Hyung Kim, Timothy J. Griffin

Department of Biochemistry: Faculty Publications

Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed downregulation of nuclear abundance of many proteins involved in translation and RNA modification. …

N-Glycans On The Link Domain Of Human Hare/Stabilin-2 Are Needed For Hyaluronan Binding To Purified Ecto-Domain, But Not For Cellular Endocytosis Of Hyaluronan, Ed Harris, Simon Parry, Mark Sutton-Smith, Madhu S. Pandey, Maria Panico, Howard R. Morris, Stuart M. Haslam, Anne Dell, Paul H. Weigel

Department of Biochemistry: Faculty Publications

The hyaluronic acid receptor for endocytosis (HARE)/Stabilin- 2 is the primary systemic scavenger receptor for 13 ligands including hyaluronan (HA), heparin and chondroitin sulfates. Most ligand-binding sites are within the 190 kDa isoform, which contains ~25 kDa of N-glycans and is the C-terminal half of the full-length 315 kDa HARE. Glycoproteomic analyses of purified recombinant human 190-HARE ecto-domain identified a diverse population of glycans at 10 of 17 consensus sites. The most diversity (and the only sialylated structures) occurred at N²²⁸⁰, within the HA-binding Link domain. To determine if these N-glycans are required for HA binding, …

Functional Hybrid Rubisco Enzymes With Plant Small Subunits And Algal Large Subunits Engineered Rbcs Cdna For Expression In Chlamydomonas, Todor Genkov, Moritz Meyer, Howard Griffiths, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO₂ fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO₂/O₂ specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, …

Structure Of The Proline Utilization A Proline Dehydrogenase Domain Inactivated By N-Propargylglycine Provides Insight Into Conformational Changes Induced By Substrate Binding And Flavin Reduction, Dhiraj Srivastava, Weidong Zhu, William H. Johnson Jr., Christian P. Whitman, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it …

Induction Of Interferon And Interferon Signaling Pathways By Replication Of Defective Interfering Particle Rna In Cells Constitutively Expressing Vesicular Stomatitis Virus Replication Proteins, Debasis Panda, Phat X. Dinh, Lalit Beura, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

We show here that replication of defective interfering (DI) particle RNA in HEK293 cells stably expressing vesicular stomatitis virus (VSV) replication proteins potently activates interferon (IFN) and IFN signaling pathways through upregulation of IFN- promoter, IFN-stimulated response element (ISRE) promoter, and NF-κB promoter activities. Replication of DI particle RNA, not mere expression of the viral replication proteins, was found to be critical for induction of IFN and IFN signaling. The stable cells supporting replication of DI RNA described in this report will be useful in further examining the innate immune signaling pathways and the host cell functions in viral genome …