Biochemistry, Biophysics, and Structural Biology Commons^™

The Mechanistic Basis Of Hemoglobin Adaptation In The High-Flying Barheaded Goose: Insights From Ancestral Protein Resurrection, Chandrasekhar Natarajan, Amit Kumar, Hideaki Moriyama, Roy E. Weber, Angela Fago, Jay F. Storz

School of Veterinary and Biomedical Sciences: Faculty Publications

The bar-headed goose (‘BHG’, Anser indicus) is renowned for its trans-Himalayan migratory flights, and the elevated hemoglobin (Hb)-O2 affinity of this species is thought to make a key contribution to its capacity for powered flight at elevations of ~9000 m. Here we revisit the molecular basis of this text-book example of biochemical adaptation. Previous hypotheses about the molecular basis of the evolved increase in Hb-O2 affinity were tested by engineering BHGspecific mutations into recombinant human Hb. This approach can provide important insights, but one problem with such ‘horizontal’ comparisons – swapping residues between proteins of contemporary species – is that …

Maporal Hantavirus Causes Mild Pathology In Deer Mice (Peromyscus Maniculatus), Amanda Mcguire, Kaitlyn Miedema, Joseph R. Fauver, Amber Rico, Tawfik Aboellail, Sandra L. Quackenbush, Ann Hawkinson, Tony Schountz

School of Veterinary and Biomedical Sciences: Faculty Publications

Rodent-borne hantaviruses can cause two human diseases with many pathological similarities: hantavirus cardiopulmonary syndrome (HCPS) in the western hemisphere and hemorrhagic fever with renal syndrome in the eastern hemisphere. Each virus is hosted by specific reservoir species without conspicuous disease. HCPS-causing hantaviruses require animal biosafety level-4 (ABSL-4) containment, which substantially limits experimental research of interactions between the viruses and their reservoir hosts. Maporal virus (MAPV) is a South American hantavirus not known to cause disease in humans, thus it can be manipulated under ABSL-3 conditions. The aim of this study was to develop an ABSL-3 hantavirus infection model using the …

Localization Of Cd8 T Cell Epitope Within Cardiac Myosin Heavy Chain-Α334-352 That Induces Autoimmune Myocarditis In A/J Mice, Chandirasegara Massilamany, Arunakumar Gangaplara, Rakesh H. Basavalingappa, Rajkumar A. Rajasekaran, Vahid Khalilzad-Sharghi, Zhongji Han, Shadi F. Othman, David Steffen, Jay Reddy

School of Veterinary and Biomedical Sciences: Faculty Publications

Background—Cardiac myosin heavy chain-α (Myhc), an intracellular protein expressed in the cardiomyocytes, has been identified as a major autoantigen in cardiac autoimmunity. In our studies with Myhc_334-352-induced experimental autoimmune myocarditis in A/J mice (H-2a), we discovered that Myhc_334-352, supposedly a CD4 T cell epitope, also induced antigen-specific CD8 T cells that transfer disease to naïve animals.

Methods and Results—In our efforts to identify the CD8 T cell determinants, we localized Myhc_338-348 within the full length-Myhc_334-352, leading to four key findings. (1) By acting as a dual epitope, Myhc_338-348 induces both …

Note On Ehrlichia Chaffeensis, Ehrlichia Ewingii, And “Borrelia Lonestari” Infection In Lone Star Ticks (Acari: Ixodidae), Nebraska, Usa, Amanda Maegli, J. Dustin Loy, Roberto Cortinas

School of Veterinary and Biomedical Sciences: Faculty Publications

The lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae), is established in southeastern Nebraska, yet the prevalence of tick-associated microorganisms is not known. An initial PCR-based analysis for Ehrlichia chaffeensis, Ehrlichia ewingii, and Borrelia infection in host-seeking adult ticks collected in southeast Nebraska was conducted. A total of 251 adult ticks collected in six sites in southeast Nebraska were tested. E. chaffeensis, E. ewingii, and Borrelia spp. were present, and the prevalence of each was approximately 1.6%. This study demonstrates that Ehrlichia spp. are present in Nebraska lone star tick populations.

Expression And Distribution Of Thiol-Regulating Enzyme Glutaredoxin 2 (Grx2) In Porcine Ocular Tissues, Bijaya Upadhyaya, Xiaoli Tian, Hongli Wu, Marjorie F. Lou

School of Veterinary and Biomedical Sciences: Faculty Publications

Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase’s distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were …

Autoimmunity In Picornavirus Infections, Chandirasegara Massilamany, Andreas Koenig, Jay Reddy, Sally Huber, Iwona Buskiewicz

School of Veterinary and Biomedical Sciences: Faculty Publications

Enteroviruses are small, non-enveloped, positive-sense singlestrand RNA viruses, and are ubiquitously found throughout the world. These viruses usually cause asymptomatic or mild febrile illnesses, but have a propensity to induce severe diseases including type 1 diabetes and pancreatitis, paralysis and neuroinflammatory disease, myocarditis, or hepatitis. This pathogenicity may result from induction of autoimmunity to organ-specific antigens. While enterovirus-triggered autoimmunity can arise from multiple mechanisms including antigenic mimicry and release of sequestered antigens, the recent demonstration of T cells expressing dual T cell receptors arising as a natural consequence of Theiler’s virus infection is the first demonstration of this autoimmune mechanism.

Genome-Wide Search For Host Association Factors During Ovine Progressive Pneumonia Virus Infection, Jesse Thompson, Fangrui Ma, Meghan Quinn, Shi-Hua Xiang

School of Veterinary and Biomedical Sciences: Faculty Publications

Ovine progressive pneumonia virus (OPPV) is an important virus that causes serious diseases in sheep and goats with a prevalence of 36% in the USA. Although OPPV was discovered more than half of a century ago, little is known about the infection and pathogenesis of this virus. In this report, we used RNA-seq technology to conduct a genome-wide probe for cellular factors that are associated with OPPV infection. A total of approximately 22,000 goat host genes were detected of which 657 were found to have been significantly up-regulated and 889 down-regulated at 12 hours post-infection. In addition to previously known …

From Crystal Structure Of Α-Conotoxin Gic In Complex With Ac-Achbp To Molecular Determinants Of Its High Selectivity For Α3Β2 Nachr, Bo Lin, Manyu Xu, Xiaopeng Zhu, Yong Wu, Xi Liu, Dongting Zhangsun, Yuanyan Hu, Shi-Hua Xiang, Igor E. Kasheverov, Victor I. Tsetlin, Xinquan Wang, Sulan Luo

School of Veterinary and Biomedical Sciences: Faculty Publications

Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking …

From Crystal Structure Of Α-Conotoxin Gic In Complex With Ac-Achbp To Molecular Determinants Of Its High Selectivity For Α3Β2 Nachr, Bo Lin, Manyu Xu, Xiaopeng Zhu, Yong Wu, Xi Liu, Dongting Zhangsun, Yuanyan Hu, Shi-Hua Xiang, Igor E. Kasheverov, Victor I. Tsetlin, Xianquan Wang, Sulan Luo

School of Veterinary and Biomedical Sciences: Faculty Publications

Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking …

Guidelines For The Use And Interpretation Of Assays For Monitoring Autophagy (3rd Edition), Daniel J. Klionsky, Rodrigo Franco, More Than 2,000 Co-Authors

School of Veterinary and Biomedical Sciences: Faculty Publications

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that …

Noninvasive Measurement Of Electrical Events Associated With A Single Chlorovirus Infection Of A Microalgal Cell, Seung-Woo Lee, Eun-Hee Lee, Gerhard Thiel, James L. Van Etten, Ravi Saraf

Department of Chemical and Biomolecular Engineering: Faculty Publications

Chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains a viral-encoded K⁺ channel imbedded in its internal membrane, which triggers host plasma membrane depolarization during virus infection. This early stage of infection was monitored at high resolution by recording the cell membrane depolarization of a single Chlorella cell during infection by a single PBCV-1 particle. The measurement was achieved by depositing the cells onto a network of one-dimensional necklaces of Au nanoparticles, which spanned two electrodes 70 μm apart. The nanoparticle necklace array has been shown to behave as a single-electron device at room temperature. The resulting electrochemical field-effect transistor …

Characterization Of The Interaction Between Basigin And The Pattern Recognition Receptor Tlr4, Josephine Michelle Brown

UNF Graduate Theses and Dissertations

Toll-like receptors (TLRs) are a major group of pattern recognition receptors expressed on the surface of immune cells that recognize molecular patterns associated with all classes of pathogenic microorganisms. TLR4 recognizes the lipopolysaccharide component of Gram-negative bacterial cell walls and is the only TLR known to induce signaling through both the MyD88 and TRIF pathways. Basigin, a ubiquitous cell adhesion molecule, is a member of the immunoglobulin superfamily that has the ability to influence cell signaling mediated by the MyD88 and TRIF pathways, the same signaling pathways induced by the TLR4 receptor protein. Analysis of the Basigin protein sequence indicates …

The Effect Of Posttranslational Modifications On Protein Aggregation, Morphology, And Toxicity, Mu Yang

Dissertations, Master's Theses and Master's Reports

Proteins are one of the most versatile macromolecules in the biological system. The function or activity of a protein highly depends on its 3D native structure. However, under stress, they are at risk of misfolding/aggregation, leading to formation of structures that can indicate loss of function or gain of toxicity. In severe cases, protein aggregation can result in many diseases, including neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis. Due to the heterogeneous nature of cellular environment and protein molecules, mechanism of in vivo folding and related toxicity still remains elusive. To have a better understanding of …

Effect Of Disulfide Bond Scrambling On Protein Stability, Aggregation, And Cytotoxicity, Colina Dutta

Dissertations, Master's Theses and Master's Reports

Proteins are nano-machines that carry out majority of the cellular functions. Thermodynamically they are functional and stable within a very narrow range (1 kcal/mol). External perturbations in the form of pH change, thermal, or oxidative/reducing stress can destabilize the protein resulting in misfolding and aggregation. Prolonged environmental stress can affect the cells adaptive response resulting in loss of ability to refold or recycle proteins. This can lead to accumulation of misfolded or aggregated proteins within the cell. Such accumulation of aggregated proteins have been associated with neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS), Parkinson’s, Huntington’s, and Alzheimer’s disease. There …

Sensing And Mapping Of Surface Hydrophobicity Of Proteins By Fluorescent Probes, Nethaniah Dorh

Dissertations, Master's Theses and Master's Reports

Surface hydrophobic interactions in proteins play a critical role in molecular recognition, influence biological functions, and play a central role in many protein misfolding diseases. As significance of surface hydrophobic interactions in age-related proteinopathies is becoming clear; it has led to an increased demand for better probes and tools to sense and characterize protein surface hydrophobicity. Current commercially available fluorescent probes such as 8-anilino-1-naphthalene sulfonic acid (ANS), 4,4′ -dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS), 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN), tetraphenylethene derivative, and Nile Red can sense proteins average hydrophobicity. However, probe limitations prevents their application for measuring the protein surface hydrophobicity. Some of the major deficiencies …

Enabling Method To Design Versatile Biomaterial Systems From Colloidal Building Blocks, Shalini Saxena, L. Andrew Lyon

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Development of materials with fine spatial control over topographical, mechanical, or chemical features has been investigated for a variety of applications. Here we present a method to fabricate an array of polyelectrolyte constructs including two-dimensionally and three-dimensionally patterned assemblies using both compressible and incompressible colloidal building blocks. This method eliminates prior constraints associated with specific chemistries, and can be used to develop modular, multi-component, patterned assemblies. In particular, development of constructs were investigated using microgels, which are colloidally stable hydrogel microparticles, polystyrene (PS) beads, and PS-microgel core-shell building blocks in conjunction with the polycation poly(ethyleneimine) (PEI). The topography, mechanical properties, …

Suppression Of Nlrp3 Inflammasome By Γ-Tocotrienol Ameliorates Type 2 Diabetes, Yongeun Kim, Wei Wang, Meshail Okla, Inhae Kang, Regis Moreau, Soonkyu Chung

Department of Nutrition and Health Sciences: Faculty Publications

The Nod-like receptor 3 (NLRP3) inflammasome is an intracellular sensor that sets off the innate immune system in response to microbial-derived and endogenous metabolic danger signals. We previously reported that γ-tocotrienol (γT3) attenuated adipose tissue inflammation and insulin resistance in diet-induced obesity, but the underlying mechanism remained elusive. Here, we investigated the effects of γT3 on NLRP3 inflammasome activation and attendant consequences on type 2 diabetes. γT3 repressed inflammasome activation, caspase-1 cleavage, and interleukin (IL) 1β secretion in murine macrophages, implicating the inhibition of NLRP3 inflammasome in the anti-inflammatory and antipyroptotic properties of γT3. Furthermore, supplementation of leptin-receptor KO mice …

The Role Of Microrna In Cardioprotection: Ischemic Preconditioning And Mesenchymal Stem Cell Paracrine Effects, Kristin Luther

Dissertations

Changes in gene expression and protein levels are an important aspect of cardioprotection in which short non-coding RNA known as miRNA may play a key regulatory role. We investigated the functions of several miRNAs in the context of two cardioprotective stimuli, ischemic preconditioning (IPC) and mesenchymal stem cell (MSC) paracrine effects. We hypothesized that downregulation of a set of miRNAs (miR-148a/b, miR-30b, and let-7a*) augments expression of protective heat shock proteins during IPC, and that MSC exosomes transfer miR-21 to cardiomyocytes, resulting in downregulation of pro-apoptotic genes and reduction of infarct size.

IPC increased the level of Hsp70, Hsp90, and …

Regulation Of The Subunits Of Hypoxia Inducible Factors By Sprouty2 And Its Impact On Different Biological Processes, Kristin Caroline Hicks

Dissertations

The hypoxia inducible factors (HIF1α, HIF2α, HIF1β) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (Spry2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation via different mechanisms. Because of the seemingly opposite actions of the HIF and Spry2 on cellular processes, we hypothesized that Spry2 decreases the protein levels of HIF1α, HIF2α and HIF1β by enhancing the proximity of the HIF subunit to an ubiquitin ligase capable of degrading the subunit. Focusing on HIF1α as a prototypical alpha subunit, in a variety of tumor derived cell lines, Spry2 decreases the protein …

Heart And Sole: The Functional Role Of Fast-Skeletal Myosin Binding Protein-C In Cardiac And Skeletal Muscle, Brian Leei Lin

Dissertations

The goal of my dissertation was to compare and contrast the function of all three major isoforms of Myosin Binding Protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C, and cMyBP-C, respectively), with a focus on the least characterized isoform, fsMyBP-C. Using a variety of ex vivo, in vitro, and in silico methods, my research demonstrated that the N-terminal region of all MyBP-C isoforms bind to actin and shift tropomyosin, thus activating the thin filament during contraction. Furthermore, each isoform differentially activated the thin filament over isoform-specific ranges of Ca2+: slow-skeletal activates at low Ca2+, fast-skeletal activates at higher Ca2+, and …

The Role Of Oxidative Stress In The Mechanisms Of Ammonia-Induced Brain Swelling And Tolerance In The Goldfish (Carassius Auratus), David F. Jones Lisser Mr.

Theses and Dissertations (Comprehensive)

Toxic build-ups of ammonia can cause potentially fatal brain swelling in mammals, but such swelling is reversible in the anoxia- and ammonia-tolerant goldfish (Carassius auratus). The mechanisms of ammonia-induced brain swelling and tolerance remain elusive, but several studies have suggested a role for reactive oxygen species (ROS), which may damage proteins and lipids in the plasma membrane of astrocytes in the brain. As a result, osmotic gradients across cell membranes may be altered leading to water uptake by astrocytes and swelling. While a role for ROS has been proposed in mammals, no studies have addressed this question in …

The Kinetic Characterization Of Two Putative Galactosidases, Bt2857 And Sco6594, And The Preliminary Analyses Of Sco6595-97, Maria Ngo

Theses and Dissertations (Comprehensive)

Bacteroides thetaiotaomicron is a prolific bacterium found in the distal intestinal tract of humans that possesses the ability to breakdown complex polysaccharides through the release of carbohydrate-active enzymes (CAZymes). Overall, the saccharolytic prowess of B. thetaiotaomicron presents an intriguing model for understanding microbial-host nutrient exchange within the gut and to identify novel mechanisms for accessing carbohydrates. The successful expression, purification, and preliminary kinetic characterization of BT2857 are reported. BT2857 was found to be enzymatically active against the artificial substrate, 4-nitrophenyl β-D-galactopyranoside (pNP-Gal); suggesting its role as a putative β-galactosidase and allowed for the quantification of BT2857 activity by the determination …

Comparative Proteomic Analysis Of Extracellular Vesicles From Prostate Cancer-Derived Cell Lines, Gloria Polanco

Open Access Theses & Dissertations

Prostate cancer (PCa) is the leading non-cutaneous malignancy and the second deadliest among American men. PCa mortality rates among African American men are much higher than any other ethnic group, and the same is true for men of African ancestry world-wide. There is also a lack of reliable diagnostic markers and effective treatment options. Extracellular vesicles (EVs) have been observed to play an important role in cancer processes such as promotion of tumor growth and metastasis. They are also a promising source of diagnostic markers. This study addresses these problems by studying the proteome of EVs derived from PCa cells …

Identifying Non-Classical Active Sites As A Tool For Enzyme Inhibition, Marisol Serrano

Open Access Theses & Dissertations

Chagas disease, caused by the parasite Trypanosoma cruzi, is an endemic life-threatening disease that affects mainly the heart. It remains the leading cause of heart failure in Latin American countries. Since current treatments against this parasite are highly toxic and somewhat ineffective, novel and more efficacious types of interventions are desired. Cruzain, identified as the major cathepsin for T. cruzi, plays a major role in the parasite's life cycle; making this enzyme very attractive for potential trypanocidal drugs discovery. The recombinant cruzain is synthesized as a zymogenic pro-protein (PCZN) which possesses a pro domain and a catalytic domain. In this …

A Requirement For Y841 In Jak3 Enzymatic Activity And Hematopoietic Cancers, George Steven Martinez

Open Access Theses & Dissertations

A medical need exists for successfully treating people afflicted with leukemia, especially those who develop drug resistant forms. Relapse leukemia cases are particularly high within Hispanic populations where this disease is among the most frequently occurring cancer. Fourteen somatic mutations have been reported in Janus tyrosine kinase 3 (Jak3), including M511I and A573V, from patients with various forms of leukemia. To monitor drug sensitivity, a model system was developed. Indeed, many of these mutations have been shown to possess transforming ability in cell lines such as the IL-3 dependent pro-B cell line Ba/F3. As such, Ba/F3 cells were transformed to …

Mitochondrial Function Assessed By 31p Mrs And Bold Mri In Non-Obese Type 2 Diabetic Rats, Yuchi Liu, Xunbai Mei, Jielei Li, Nicola Lai, Xin Yu

Electrical & Computer Engineering Faculty Publications

The study aims to characterize age-associated changes in skeletal muscle bioenergetics by evaluating the response to ischemia-reperfusion in the skeletal muscle of the Goto-Kakizaki (GK) rats, a rat model of non-obese type 2 diabetes (T2D). ³¹P magnetic resonance spectroscopy (MRS) and blood oxygen level-dependent (BOLD) MRI was performed on the hindlimb of young (12 weeks) and adult (20 weeks) GK and Wistar (control) rats. ³¹P-MRS and BOLD-MRI data were acquired continuously during an ischemia and reperfusion protocol to quantify changes in phosphate metabolites and muscle oxygenation. The time constant of phosphocreatine recovery, an index of mitochondrial oxidative capacity, …

Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis, Lorenzo Alamo, Dan Qi, Willy Wriggers, Antonio Pinto, Jingui Zhu, Aivett Bilbao, Richard E. Gillilan, Songnian Hu, Raúl Padrón

Mechanical & Aerospace Engineering Faculty Publications

Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a …