Biochemistry, Biophysics, and Structural Biology Commons^™

Controlling Platelet Secretion To Modulate Hemostasis And Thrombosis, Smita Joshi

Theses and Dissertations--Molecular and Cellular Biochemistry

Upon vascular injury, activated blood platelets fuse their granules to the plasma membrane and release cargo to regulate the vascular microenvironment, a dynamic process central to platelet function in many critical processes including hemostasis, thrombosis, immunity, wound healing, angiogenesis etc. This granule- plasma membrane fusion is mediated by a family of membrane proteins- Soluble N-ethyl maleimide Attachment Receptor Proteins(SNAREs). SNAREs that reside on vesicle (v-SNAREs) /Vesicle-Associated Membrane Proteins(VAMPs) interact with target/t-SNAREs forming a trans-bilayer complex that facilitates granule fusion. Though many components of exocytic machinery are identified, it is still not clear how it could be manipulated to prevent …

Impact Of Phosphoinositides On Regulation Of K-Atp By Atp And Hydrogen Sulfide, Tyler Hendon

Theses and Dissertations

Hydrogen sulfide (H2S) reduces ischemia reperfusion (IR) injury by stimulating adenosine triphosphate (ATP) sensitive potassium channels (KATP) [1-5]. Demonstrating H2S stimulation is unique to KATP, as other inwardly rectifying potassium (Kir) channels demonstrate inhibition or are unaffected [6]. We recently showed that H2S inhibits Kir2 and Kir3 by decreasing channel sensitivity to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) [6]. Here, we test the hypothesis that H2S regulation of Kir6.2, a pore-forming subunit of the KATP channel, is also dependent on PIP2. Using whole-cell patch-clamp we show that H2S increases the activity of Kir6.2 channels expressed in HEK-293 cells. To study the …

Modulation Of Electron Transport By Metformin In Cardiac Protection: Role Of Complex I, Ahmed Abdul Hussein Mohsin

Theses and Dissertations

Modulation of mitochondrial complex I during reperfusion reduces cardiac injury. Complex I exists in two structural states: active (A) and deactive (D) with transition from A→D during ischemia. Reperfusion reactivates D→A with an increase in ROS production. Metformin preserves the D-Form. Our aim was to study the contribution of maintenance of deactivation of complex I during early reperfusion by metformin to protect against ischemia reperfusion injury. Our results showed that metformin decreased H9c2 cardiomyoblast apoptosis and total cell death following simulated ischemia for six hours followed by reoxygenation for twenty four hours compared to untreated cells. Reactive oxygen species (ROS) …

Development Of Septic Polysynovitis And Uveitis In Foals Experimentally Infected With Rhodococcus Equi, Laura Huber, Steeve Giguère, Londa J. Berghaus, Amanda Hanafi, Sarah Vitosh-Sillman, Sarah L. Czerwinski

School of Veterinary and Biomedical Sciences: Faculty Publications

Rhodococcus equi is one of the most important causes of disease in foals. Infection is typically characterized by pyogranulomatous pneumonia although extrapulmonary infections occur occasionally. Uveitis and polysynovitis have been reported in foals naturally infected with R. equi and are thought to be the result of an immune-mediated process. However, the pathogenesis of these conditions is poorly understood. The objectives of this study were to document the occurrence of uveitis and polysynovitis after experimental infection with R. equi and to determine if these disorders are the direct result of infection at these sites. Foals between 3 and 4 weeks of …

Alkaline Stabilization Of Manure Slurry Inactivates Porcine Epidemic Diarrhea Virus, Erin E. Stevens, Daniel N. Miller, Bethany A. Brittenham, Sarah J. Vitosh-Sillman, Bruce W. Brodersen, Virginia L. Jin, John D. Loy, Amy M. Schmidt

School of Veterinary and Biomedical Sciences: Faculty Publications

Hydrated lime manure treatment was evaluated to determine porcine epidemic diarrhea virus (PEDV) susceptibility to alkaline stabilization. At pH 10, PEDV decreased (quantitative polymerase chain reaction) and lost infectivity (swine bioassay). Although ammonium decreased above pH 9 (up to 25%), alkaline stabilization managed to control potential infection from manure sources.

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry Identification Of Moraxella Bovoculi And Moraxella Bovis Isolates From Cattle, Kara Robbins, Aaron M. Dickey, Michael L. Clawson, John Dustin Loy

School of Veterinary and Biomedical Sciences: Faculty Publications

Infectious bovine keratoconjunctivitis (IBK) is an economically significant disease caused by Moraxella bovis. Moraxella bovoculi, although not reported to cause IBK, has been isolated from the eyes of cattle diagnosed with IBK. Identification of M. bovis and M. bovoculi can be performed using biochemical or DNA-based approaches, both of which may be time consuming and inconsistent between laboratories. We conducted a comparative evaluation of M. bovoculi and M. bovis identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a database provided by Bruker Daltonics (termed the BDAL database), the BDAL database supplemented with spectra generated in …

Circadian Behavioral Responses To Light And Optic Chiasm-Evoked Glutamatergic Epscs In The Suprachiasmatic Nucleus Of Iprgc Conditional Vglut2 Knock-Out Mice, Michael G. Moldavan, Patricia J. Sollars, Michael R. Lasarev, Charles N. Allen, Gary E. Pickard

School of Veterinary and Biomedical Sciences: Faculty Publications

Intrinsically photosensitive retinal ganglion cells (ipRGCs) innervate the hypothalamic suprachiasmatic nucleus (SCN), a circadian oscillator that functions as a biological clock. ipRGCs use vesicular glutamate transporter 2 (vGlut2) to package glutamate into synaptic vesicles and light-evoked resetting of the SCN circadian clock is widely attributed to ipRGC glutamatergic neurotransmission. Pituitary adenylate cyclase-activating polypeptide (PACAP) is also packaged into vesicles in ipRGCs and PACAP may be coreleased with glutamate in the SCN. vGlut2 has been conditionally deleted in ipRGCs in mice [conditional knock-outs (cKOs)] and their aberrant photoentrainment and residual attenuated light responses have been ascribed to ipRGC PACAP release. However, …

Development Of A Multiplex Real-Time Pcr Assay Using Two Thermocycling Platforms For Detection Of Major Bacterial Pathogens Associated With Bovine Respiratory Disease Complex From Clinical Samples, John Dustin Loy, Laura Leger, Aspen M. Workman, Michael L. Clawson, Ece Bulut, Bing Wang

School of Veterinary and Biomedical Sciences: Faculty Publications

Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2–12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected …

Coordinated Regulation Of Transcription By Ccpa And The Staphylococcus Aureus Twocomponent System Hptrs, Joseph M. Reed, Sean Olson, Danielle F. Brees, Caitlin E. Griffin, Ryan A. Grove, Paul J. Davis, Stephen D. Kachman, Jiri Adamec, Greg A. Somerville

School of Veterinary and Biomedical Sciences: Faculty Publications

The success of Staphylococcus aureus as a pathogen is due in part to its ability to adapt to changing environmental conditions using signal transduction pathways, such as metaboliteresponsive regulators and two-component systems. S. aureus has a two-component system encoded by the gene pair sav0224 (hptS) and sav0223 (hptR) that regulate the hexose phosphate transport (uhpT) system in response to extracellular glucose-6-phosphate. Glycolytic intermediates such as glucose-6-phosphate are important carbon sources that also modulate the activity of the global metabolite-responsive transcriptional regulator CcpA. Because uhpT has a putative CcpA binding site in its promoter and …

Novel Docosahexaenoic Acid Ester Of Phloridzin Inhibits Proliferation And Triggers Apoptosis In An In Vitro Model Of Skin Cancer, Theodora Mantso, Dimitrios T. Trafalis, Sotiris Botaitis, Rodrigo Franco, Aglaia Pappa, H. P. Vasantha Rupasinghe, Mihalis I. Panayiotidis

School of Veterinary and Biomedical Sciences: Faculty Publications

Skin cancer is among the most common cancer types accompanied by rapidly increasing incidence rates, thus making the development of more efficient therapeutic approaches a necessity. Recent studies have revealed the potential role of decosahexaenoic acid ester of phloridzin (PZDHA) in suppressing proliferation of liver, breast, and blood cancer cell lines. In the present study, we investigated the cytotoxic potential of PZDHA in an in vitro model of skin cancer consisting of melanoma (A375), epidermoid carcinoma (A431), and non-tumorigenic (HaCaT) cell lines. Decosahexaenoic acid ester of phloridzin led to increased cytotoxicity in all cell lines as revealed by cell viability …

Whole Genome Sequencing Of Moraxella Bovoculi Reveals High Genetic Diversity And Evidence For Interspecies Recombination At Multiple Loci, Aaron M. Dickey, Gennie Schuller, John D. Loy, Michael L. Clawson

School of Veterinary and Biomedical Sciences: Faculty Publications

Moraxella bovoculi is frequently cultured from the ocular secretions and conjunctiva of cattle with Infectious Bovine Keratoconjunctivitis (IBK). Previous work has shown that single nucleotide polymorphism (SNP) diversity in this species is quite high with 81,284 SNPs identified in eight genomes representing two distinct genotypes isolated from IBK affected eyes (genotype 1) and the nasopharynx of cattle without clinical IBK signs (genotype 2), respectively. The goals of this study were to identify SNPs from a collection of geographically diverse and epidemiologically unlinked M. bovoculi strains from the eyes of IBK positive cattle (n = 183) and another from the eyes …

Differential Modulation Of Human Gabac‑Ρ1 Receptor By Sulfur‑Containing Compounds Structurally Related To Taurine, Lenin David Ochoa‑De La Paz, Martin González‑Andrade, Herminia Pasantes‑Morales, Rodrigo Franco, Rubén Zamora‑Alvarado, Edgar Zenteno, Hugo Quiroz‑Mercado, Roberto Gonzales‑Salinas, Rosario Gulias‑Cañizo

School of Veterinary and Biomedical Sciences: Faculty Publications

Background: The amino acid taurine (2-Aminoethanesulfonic acid) modulates inhibitory neurotransmitter receptors. This study aimed to determine if the dual action of taurine on GABAC- ρ1R relates to its structure. To address this, we tested the ability of the structurally related compounds homotaurine, hypotaurine, and isethionic acid to modulate GABAC- ρ1R.

Results: In Xenopus laevis oocytes, hypotaurine and homotaurine partially activate heterologously expressed GABAC- ρ1R, showing an increment in its deactivation time with no changes in channel permeability, whereas isethionic acid showed no effect. Competitive assays suggest that hypotaurine and homotaurine compete for the GABA-binding site. In addition, their effects were …

Using Art And Story To Explore How Primary School Students In Rural Tanzania Understand Planetary Health: A Qualitative Analysis, Elizabeth Vanwormer, Jesca Mlawa, Elizabeth Komba, Christopher Gustafson, Hilda Mrema, Jenny M. Dauer

School of Veterinary and Biomedical Sciences: Faculty Publications

Background The global planetary health community increasingly recognises the need to prepare students to investigate and address connections between environmental change and human health. As we strive to support education on planetary health themes for students of all ages, understanding students’ concepts of linkages between the health of people and animals, and their shared environments might advance educational approaches. Children living in villages bordering Ruaha National Park in Iringa Region, Tanzania, have direct experience of these connections as they share a water-stressed but biodiverse environment with domestic animals and wildlife. Livelihoods in these villages depend predominantly on crop and livestock …

Tellurite Resistance In Shiga Toxin-Producing Escherichia Coli , Gentry L. Lewis, Quentin R. Jorgensen, John Dustin Loy, Rodney A. Moxley

School of Veterinary and Biomedical Sciences: Faculty Publications

Potassium tellurite ( K₂TeO₃) is an effective selective agent for O157:H7 Shiga toxin-producing Escherichia coli (STEC), whereas tellurite resistance in non-O157 STEC is variable with information on O45 minimal. High-level K₂TeO₃ resistance in STEC is attributable to the ter gene cluster with terD an indicator of the cluster’s presence. Polymerase chain reactions for terD and K₂TeO₃ minimum inhibitory concentration (MIC) determinations in broth cultures were conducted on 70 STEC and 40 non-STEC control organisms. Sixty-six STEC strains (94.3%) were terD+ compared to 28 control organisms (70.0%; P < 0.001). The prevalence of terD in …

Epitope Mapping Of Serca2a Identifies An Antigenic Determinant That Induces Mainly Atrial Myocarditis In A/J Mice, Bharathi Krishnan, Chandirasegara Massilamany, Rakesh H. Basavalingappa, Arunakumar Gangaplara, Rajkumar Rajasekaran, Muhammad Z. Afzal, Vahid Khalilzad-Sharghi, You Zhou, Jean-Jack Riethoven, Shyam S. Nandi, Paras K. Mishra, Raymond A. Sobel, Jennifer L. Strande, David Steffen, Jay Reddy

School of Veterinary and Biomedical Sciences: Faculty Publications

Sarcoplasmic/endoplasmic reticulum Ca²⁺ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971–990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a …

Total Mercury Determination In Muscle And Liver Tissue Samples From Brazilian Amazon Fish Using Slurry Sampling, João Vitor De Queiroz, José Cavalcante Souza Vieira, Izabela Da Cunha Bataglioli, Alis Correia Bittarello, Camila Pereira Braga, Grasieli De Oliveira, Cilene Do Carmo Federici Padilha, Pedro De Magalhães Padilha

Department of Biochemistry: Faculty Publications

This paper presents a slurry sampling method for total mercury determination by graphite furnace atomic absorption spectrometry (GFAAS) in tissue of fish from the Amazon. The tissue samples were lyophilized and macerated, and then the slurry samples were prepared by putting 20 mg of tissue, added to a solution containing Triton X-100, Suprapur HNO₃, and zirconium nitrate directly in sampling vials of a spectrometer. Mercury standard solutions were prepared under the same conditions as the slurry samples. The slurry samples and the mercury standard solutions were sonicated for 20 s. Twenty microliters of slurry samples were injected into …

Cancerdiscover: An Integrative Pipeline For Cancer Biomarker And Cancer Class Prediction From High-Throughput Sequencing Data, Akram Mohammed, Greyson Biegert, Jiri Adamec, Tomáš Helikar

Department of Biochemistry: Faculty Publications

Accurate identification of cancer biomarkers and classification of cancer type and subtype from High Throughput Sequencing (HTS) data is a challenging problem because it requires manual processing of raw HTS data from various sequencing platforms, quality control, and normalization, which are both tedious and timeconsuming. Machine learning techniques for cancer class prediction and biomarker discovery can hasten cancer detection and significantly improve prognosis. To date, great research efforts have been taken for cancer biomarker identification and cancer class prediction. However, currently available tools and pipelines lack flexibility in data preprocessing, running multiple feature selection methods and learning algorithms, therefore, developing …

Towards The Synthetic Design Of Camelina Oil Enriched In Tailored Acetyl-Triacylglycerols With Medium-Chain Fatty Acids, Sunil Bansal, Hae Jin Kim, Gunnam Na, Megan E. Hamilton, Edgar B. Cahoon, Chaofu Lu, Timothy P. Durrett

Department of Biochemistry: Faculty Publications

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and …

Single-Molecule Unbinding Forces Between The Polysaccharide Hyaluronan And Its Binding Proteins, Fouzia Bano, Markku I. Tammi, David W. Kang, Edward N. Harris, Ralf P. Richter

Department of Biochemistry: Faculty Publications

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, …

Endosomal Escape Of Antisense Oligonucleotides Internalized By Stabilin Receptors Is Regulated By Rab5c And Eea1 During Endosomal Maturation, Colton M. Miller, W. Brad Wan, Punit P. Seth, Edward N. Harris

Department of Biochemistry: Faculty Publications

Second-generation (Gen 2) Antisense oligonucleotides (ASOs) show increased nuclease stability and affinity for their RNA targets, which has translated to improved potency and therapeutic index in the clinic. Gen 2 ASOs are typically modified using the phosphorothioate (PS) backbone modification, which enhances ASO interactions with plasma, cell surface, and intracellular proteins. This facilitates ASO distribution to peripheral tissues and also promotes cellular uptake after injection into animals. Previous work identified that Stabilin receptors specifically internalize PS-ASOs in the sinusoidal endothelial cells of the liver and the spleen. By modulating expression of specific proteins involved in the trafficking and maturation of …

Purification Of Hepatocytes And Sinusoidal Endothelial Cells From Mouse Liver Perfusion, Fatima Cabral, Colton M. Miller, Katrina M. Kudrna, Blake E. Hass, Jocelyn G. Daubendiek, Brianna M. Kellar, Edward N. Harris

Department of Biochemistry: Faculty Publications

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated …

A Tlr/Akt/Foxo3 Immune Tolerance–Like Pathway Disrupts The Repair Capacity Of Oligodendrocyte Progenitors, Taasin Srivastava, Parham Diba, Justin M. Dean, Fatima Banine, Daniel Shaver, Matthew Hagen, Xi Gong, Weiping Su, Ben Emery, Daniel L. Marks, Edward N. Harris, Bruce Baggentoss, Paul H. Weigel, Larry S. Sherman, Stephen A. Back

Department of Biochemistry: Faculty Publications

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter …

3-O Sulfation Of Heparin Leads To Hepatotropism And Longer Circulatory Half-Life, Colton M. Miller, Yongmei Xu, Katrina M. Kudrna, Blake E. Hass, Brianna M. Kellar, Andrew W. Egger, Jian Liu, Edward N. Harris

Department of Biochemistry: Faculty Publications

Introduction: Heparins are common blood anticoagulants that are critical for many surgical and biomedical procedures used in modern medicine. In contrast to natural heparin derived from porcine gut mucosa, synthetic heparins are homogenous by mass, polymer length, and chemistry.

Materials & methods: Stable cell lines expressing the human and mouse Stabilin receptors were used to evaluate endocytosis of natural and synthetic heparin. We chemoenzymatically produced synthetic heparin consisting of 12 sugars (dodecamers) containing 14 sulfate groups resulting in a non-3-O sulfated structure (n12mer). Half of the n12mer was modified with a 3-O sulfate on a single GlcNS sugar producing the …

Characterization Of A Recurrent Missense Mutation In The Forkhead Dna-Binding Domain Of Foxp1, Tyler B. Johnson, Keegan Mechels, Ruth Ellen Anderson, Jacob T. Cain, David A. Sturdevant, Stephen Braddock, Hailey Pinz, Mark A. Wilson, Megan Landsverk, Kyle J. Roux, Jill M. Weimer

Department of Biochemistry: Faculty Publications

Haploinsufficiency of Forkhead box protein P1 (FOXP1), a highly conserved transcription factor, leads to developmental delay, intellectual disability, autism spectrum disorder, speech delay, and dysmorphic features. Most of the reported FOXP1 mutations occur on the C-terminus of the protein and cluster around to the forkhead domain. All reported FOXP1 pathogenic variants result in abnormal cellular localization and loss of transcriptional repression activity of the protein product. Here we present three patients with the same FOXP1 mutation, c.1574G>A (p.R525Q), that results in the characteristic loss of transcription repression activity. This mutation, however, represents the first reported FOXP1 mutation that does …

Editorial: Structure And Function Of Chloroplasts, Rebecca L. Roston, Juliette Jouhet, Fei Yu, Hongbo Gao

Department of Biochemistry: Faculty Publications

The primary energy resource of life on earth is the sun, whose energy is captured in the form of usable carbons by a process called photosynthesis. Photosynthesis occurs within a cellular organelle adapted to that purpose, called the chloroplast. Chloroplasts are unique metabolic and sensory organelles restricted to plants, algae, and a few protists. In this special topic, we aimed to gather new research, hypotheses, and reviews that would help us to better understand the important role of chloroplasts in all photosynthetic organisms. We were fortunate enough to have submissions from many talented chloroplast researchers. This topic contains a total …

Identification Of Genes Encoding Enzymes Catalyzing The Early Steps Of Carrot Polyacetylene Biosynthesis, Lucas Busta, Won Cheol Yim, Evan William Labrant, Peng Wang, Lindsey Grimes, Kiah Malyszka, John C. Cushman, Patricia Santos, Dylan K. Kosma, Edgar B. Cahoon

Department of Biochemistry: Faculty Publications

Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We …

Role Of Selenof As A Gatekeeper Of Secreted Disulfide-Rich Glycoproteins, Sun Hee Yim, Robert A. Everley, Frank A. Schildberg, Sang-Goo Lee, Andrea Orsi, Zachary R. Barbati, Kutay Karatepe, Dmitry E. Fomenko, Petra A. Tsuji, Hongbo R. Luo, Steven P. Gygi, Roberto Sitia, Arlene H. Sharpe, Dolph L. Hatfield, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDPglucose: glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi …

Visualizing The Invisible: A Guide To Designing, Printing, And Incorporating Dynamic 3d Molecular Models To Teach Structure–Function Relationships, Michelle E. Howell, Karin V. Van Dijk, Christine S. Booth, Tomáš Helikar, Brian A. Couch, Rebecca Roston

Department of Biochemistry: Faculty Publications

Understanding the intricate relationship between macromolecular structure and function represents a central goal of undergraduate biology education (1–3). In teaching complex three-dimensional (3D) concepts, instructors typically depend on static two-dimensional (2D) textbook images or computer-based visualization software, which can lead to unintended misconceptions (4–6). While chemical and molecular kits exist, these models cannot handle the size and detail of macromolecules. Consequently, students may graduate in the life sciences without understanding how structure underlies function or acquiring skills to translate between 2D and 3D molecular models (5, 7).

Structural Basis For The Substrate Inhibition Of Proline Utilization A By Proline, David A. Korasick, Travis A. Pemberton, Benjamin W. Arentson, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of L-proline to L-glutamate using spatially separated proline dehydrogenase (PRODH) and L-glutamate-y-semialdehyde dehydrogenase (GSALDH) active sites. Substrate inhibition of the coupled PRODH-GSALDH reaction by proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.15 Å resolution crystal structure of Bradyrhizobium japonicum PutA complexed with proline. Proline was discovered in five locations remote from the PRODH active site. Most notably, strong electron density indicated that proline bound tightly to the …

Lncrna Meg3 Protects Endothelial Function By Regulating The Dna Damage Response, Mohamed Sham Shihabudeen Haider Ali, Xiao Cheng, Matthew Moran, Stefan Haemmig, Michael J. Naldrett, Sophie Alvarez, Mark W. Feinberg, Xinghui Sun

Department of Biochemistry: Faculty Publications

The role of long non-coding RNAs (lncRNAs) in regulating endothelial function through the DNA damage response (DDR) remains poorly understood. In this study, we demonstrate that lncRNA maternally expressed gene 3 (Meg3) interacts with the RNA binding protein polypyrimidine tract binding protein 3 (PTBP3) to regulate gene expression and endothelial function through p53 signaling a major coordinator of apoptosis and cell proliferation triggered by the DDR. Meg3 expression is induced in endothelial cells (ECs) upon p53 activation. Meg3 silencing induces DNA damage, activates p53 signaling, increases the expression of p53 target genes, promotes EC apoptosis, and inhibits EC proliferation. Mechanistically, …