Biochemistry, Biophysics, and Structural Biology Commons^™

Genome-Wide Ribosome Profiling Reveals Complex Translational Regulation In Response To Oxidative Stress, Maxim V. Gerashchenko, Alexei V. Lobanov, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Information on unique and coordinated regulation of transcription and translation in response to stress is central to the understanding of cellular homeostasis. Here we used ribosome profiling coupled with next-generation sequencing to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae. Hydrogen peroxide treatment led to a massive and rapid increase in ribosome occupancy of short upstream ORFs, including those with non-AUG translational starts, and of the N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through …

Reduced Utilization Of Selenium By Naked Mole Rats Due To A Specific Defect In Gpx1 Expression, Marina V. Kasaikina, Alexei V. Lobanov, Mikalai I. Malinouski, Byung Cheon Lee, Javier Seravalli, Dmitri E. Fomenko, Anton A. Turanov, Lydia Finney, Stefan Vogt, Thomas J. Park, Richard A. Miller, Dolph L. Hatfield, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Naked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long life span (>28 years). It is also not known to develop cancer. In the current work, tissue imaging by x-ray fluorescence microscopy and direct analyses of trace elements revealed low levels of selenium in the MR liver and kidney, whereas MR and mouse brains had similar selenium levels. This effect was not explained by uniform selenium deficiency because methionine sulfoxide reductase activities were similar in mice and MR. However, glutathione peroxidase activity was an order of magnitude lower inMRliver and kidney …

A 4-Selenocysteine, 2-Selenocysteine Insertion Sequence (Secis) Element Methionine Sulfoxide Reductase From Metridium Senile Reveals A Non-Catalytic Function Of Selenocysteines, Byung Cheon Lee, Alexey V. Lobanov, Stefano M. Marino, Alaattin Kaya, Javier Seravalli, Dolph L. Hatfield, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-Rsulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently …

Escherichia Coli Thioredoxin-Like Protein Ybbn Contains An Atypical Tetratricopeptide Repeat Motif And Is A Negative Regulator Of Groel, Jiusheng Lin, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Many proteins contain a thioredoxin (Trx)-like domain fused with one or more partner domains that diversify protein function by the modular construction of new molecules. The Escherichia coli protein YbbN is a Trx-like protein that contains a C-terminal domain with low homology to tetratricopeptide repeat motifs. YbbN has been proposed to act as a chaperone or co-chaperone that aids in heat stress response andDNAsynthesis. We report the crystal structure of YbbN, which is an elongated molecule with a mobile Trx domain and four atypical tetratricopeptide repeat motifs. The Trx domain lacks a canonical CXXC active site architecture and is not …

Defining A Relationship Between Dietary Fatty Acids And The Cytochrome P450 System In A Mouse Model Of Fatty Liver Disease, Monika Gonzalez, Whitney Sealls, Elliot D. Jesch, M. Julia Brosnan, Istvan Ladunga, Xinxin Ding, Paul N. Black, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

Liver-specific ablation of cytochrome P450 reductase in mice (LCN) results in hepatic steatosis that can progress to steatohepatitis characterized by inflammation and fibrosis. The specific cause of the fatty liver phenotype is poorly understood but is hypothesized to result from elevated expression of genes encoding fatty acid synthetic genes. Since expression of these genes is known to be suppressed by polyunsaturated fatty acids, we performed physiological and genomics studies to evaluate the effects of dietary linoleic and linolenic fatty acids (PUFA) or arachidonic and decosahexaenoic acids (HUFA) on the hepatic phenotypes of control and LCN mice by comparison with a …

Functional Diversification Of Thylakoidal Processing Peptidases In Arabidopsis Thaliana, Shih-Chi Hsu, Joshua K. Endow, Nicholas J. Ruppel, Rebecca Roston, Amy J. Baldwin, Kentaro Inoue

Department of Biochemistry: Faculty Publications

Thylakoidal processing peptidase (TPP) is responsible for removing amino-terminal thylakoid-transfer signals from several proteins in the thylakoid lumen. Three TPP isoforms are encoded by the nuclear genome of Arabidopsis thaliana. Previous studies showed that one of them termed plastidic type I signal peptidase 1 (Plsp1) was necessary for processing three thylakoidal proteins and one protein in the chloroplast envelope in vivo. The lack of Plsp1 resulted in seedling lethality, apparently due to disruption of proper thylakoid development. The physiological roles of the other two TPP homologs remain unknown. Here we show that the three A. thaliana TPP isoforms …

Enzymatic Defects Underlying Hereditary Glutamate Cysteine Ligase Deficiency Are Mitigated By Association Of The Catalytic And Regulatory Subunits, Melanie Neely Willis, Yilin Liu, Ekaterina I. Biterova, Melanie A. Simpson, Heejeong Kim, Jaekwon Lee, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises

production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly

reduced levels of erythrocyte glutathione, leading to hemolytic anemia and in some cases,

impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a

catalytic (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate limiting step

in glutathione production. Four clinical missense mutations have been identified within GCLC:

Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of

these mutations on enzymatic function in vivo and in vitro …

The Lyr Protein Mzm1 Functions In The Insertion Of The Rieske Fe/S Protein In Yeast Mitochondria, Aaron Atkinson, Pamela Smith, Jennifer L. Fox, Tie-Shong Cui, Oleh Khalimonchuk, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The assembly of the cytochrome bc1 complex in Saccharomyces cerevisiae is shown to be conditionally dependent on a novel factor, Mzm1. Cells lacking Mzm1 exhibit a modest bc₁ defect at 30°C, but the defect is exacerbated at elevated temperatures. Formation of bc₁ is stalled in mzm1 Δ cells at a late assembly intermediate lacking the Rieske iron-sulfur protein Rip1. Rip1 levels are markedly attenuated in mzm1 Δ cells at elevated temperatures. Respiratory growth can be restored in the mutant cells by the overexpression of the Rip1 subunit. Elevated levels of Mzm1 enhance the stabilization of Rip1 through …

Rpir Homologues May Link Staphylococcus Aureus Rnaiii Synthesis And Pentose Phosphate Pathway Regulation, Yefei Zhu, Nandakumar Madayiputhiya, Marat R. Sadykov, Nandakumar Madayiputhiya, Thanh T. Luong, Rosmarie Gaupp, Chia Y. Lee, Greg Somerville

Department of Biochemistry: Faculty Publications

Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant …

In Vivo Liver Endocytosis Followed By Purification Of Liver Cells By Liver Perfusion, Sandhya Gopalakrishnan, Edward N. Harris

Department of Biochemistry: Faculty Publications

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure ¹ in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the …

Conformation And Ion Transport Of Neuronal Uncoupling Proteins: Ucp2, Ucp4, And Ucp5, Tuan Hoang

Theses and Dissertations (Comprehensive)

Located in the inner mitochondrial membrane of brown adipose tissue, uncoupling protein -1 (UCP1) dissipates the proton electrochemical gradient, causing reduction in the rate of ATP synthesis, and generates heat by non-shivering thermogenesis. Three other UCP homologs (UCP2, UCP4 and UCP5), expressed in neurons, are suggested to have potential roles in the function and protection of the central nervous system (CNS). Up to date, structural information for UCPs still remains limited. Extensive biochemical studies on UCP2 have provided adequate evidence for its participation in proton and anion transport. So far, no functional studies in proteoliposome systems have been performed on …

Synthesis, Linda H. Pardo, Linda H. Geiser, Mark E. Fenn, Charles T. Driscoll, Christine L. Goodale, Edith B. Allen, Jill S. Baron, Roland Bobbink, William D. Bowman, Christopher M. Clark, Bridget Emmett, Frank S. Gilliam, Tara Greaver, Sharon J. Hall, Erik A. Lilleskov, Lingli Liu, Jason A. Lynch, Knute Nadelhoffer, Steven S. Perakis, Molly J. Robin-Abbott, John L. Stoddard, Kathleen C. Weathers

Biological Sciences Faculty Research

Human activity in the last century has led to a substantial increase in nitrogen (N) emissions and deposition. This N deposition has reached a level that has caused or is likely to cause alterations to the structure and function of many ecosystems across the United States. One approach for quantifying the level of pollution that would be harmful to ecosystems is the critical loads approach. The critical load is dei ned as the level of a pollutant below which no detrimental ecological effect occurs over the long term according to present knowledge.

The objective of this project was to synthesize …

Phospholipid Analysis In Trypanosoma Cruzi, Melissa Rashonda Harris

Open Access Theses & Dissertations

Chagas disease, caused by the flagellate protozoan Trypanosoma cruzi is one of the most endemic and deadly infectious diseases. T.cruzi is distributed throughout most of South and Central America, where it infects 12 to 19 million people, with an annual incidence of 561,000. Another 35 million are exposed to infection, 2 to 3 million people have clinical symptoms of chronic Chagas disease, and about 45,000 of these may die every year due to cardiac failure. In addition to the growing number of incidences in other countries the disease is an emerging infectious disease in the U.S due to the migration …

Molecular Mechanisms Of Poly [Adp-Ribose] Polymerase-1 In Hiv-1 Infection, Daniel Reyes

Open Access Theses & Dissertations

Poly (ADP-ribose) polymerase-1 (PARP-1) is a cellular enzyme involved in genome stability and transcriptional regulation. The role of this protein in HIV-1 infection is largely controversial. Some reports indicated a fundamental role of PARP-1 in HIV-1 DNA integration and results from other laboratories do not support these conclusions. An important characteristic in all these experiments is that the HIV-1 target cells that were used express, in addition to PARP-1, the functional homologue PARP-2. We evaluated the role of PARP-1 in the chicken B lymphoblastoid cell line DT40. These cells naturally lack PARP-2 and support the early steps of HIV infection. …

Structural And Functional Investigation Of Φ-El-Chaperonin Mediated Protein Folding, Sudheer Kumar Molugu

Open Access Theses & Dissertations

Chaperonins are ubiquitous, sequence related protein complexes that aid in the folding of nascent and misfolded polypeptides in an ATP driven pathway. Recently a GroEL-like, 860 kilo Dalton chaperonin protein complex was identified and isolated from the bacteriophage EL, a virus that infects the Gram-negative bacterium Pseudomonas aeruginosa. The bacteriophage EL contains 201 predicted open reading frames and is the only known phage that encodes for its own chaperonin known as Φ-EL-chaperonin.

To understand the importance of Φ-EL-chaperonin in phage EL life cycle, the recombinant Φ-EL-chaperonin protein was expressed in Escherichia coli (E. coli) purified to homogeneity and the structure …

Inhibition Of Calcium Oxalate Calculi By Ndga - A Spectroscopic Study, Preethi Dacha

Open Access Theses & Dissertations

Formation of calculi in urinary tract leads to kidney stones. This process is known as Urolithiasis. Some of the causes of urolithiasis are metabolic disorders, genetic factors, and anatomic abnormalities in the urinary system. Nowadays, urolithiasis is very common disease in population of all over the world. The present study strives to understand calcium oxalate calculi formation as well as its inhibition by using Nordihydroguaiaretic acid (NDGA), which is a chemical extract of the Larrea Tridentata bush, a plant that is traditionally employed in the El paso region as a natural treatment for kidney stones and bladder diseases. Thus, in …

Free Radical Stress-Induced Parkinsonian Lewy-Like Aggregation Prevented Through Polyphenolic Phytochemical Analog Intervention: Implications For Subcellular Trafficking And Neurodegenerative Disorders, Rituraj Pal

Open Access Theses & Dissertations

Protein disulfide isomerase (PDI), the chief endoplasmic reticulum (ER)-resident oxidoreductase chaperone, is known to catalyze the maturation of disulfide-bond-containing proteins primarily through oxidation-reduction and isomerization functions. The rate-determining step in the oxidative regeneration path of disulfide-bond-containing proteins generally couples chemical thiol-disulfide-exchange reactions to a physical conformational folding reaction. I have determined the impact of PDI and its subdomains on the rate-determining step in ribonuclease A folding and on the physical structure-forming step of select ER-processed proteins including RNase A. This was facilitated through application of a novel chemical tool to exclusively populate native-disulfide-containing intermediates in unstructured forms. The described biochemical …

Synthesis, Self-Assembly And Biological Properties Of Self-Folding Cu(Ii) Coordination Polymers, Alejandro Arzola-Rubio

Open Access Theses & Dissertations

The synthesis of materials via molecular self-assembly is a powerful bottom-up approach for fabricating matter in the nano- and micro-meter scales. This methodology involves the spontaneous and reversible organization of small molecules that interact with one another to create larger structures driven by non-covalent interactions such as hydrogen bonding, hydrophobic forces and metal-ligand coordination interactions. In this thesis, the design, synthesis and characterization of a new set of molecules that spontaneously self-organize in water to form three-dimensional nanoscale toroidal structures was investigated. These supramolecular structures were designed with an intrinsic affinity to bind and condense DNA strands with the intent …

Identification And Characterization Of Fkbp52-Specific Inhibitors For The Treatment Of Prostate Cancer, Johanny Tonos De Leon

Open Access Theses & Dissertations

Steroid hormone receptors require the ordered assembly of various chaperone and cochaperone proteins in order to reach a functional state. The final stage in the receptor maturation process requires the formation of a mutimeric complex consisting of Hsp90 dimer, p23, and one of several large immunophilins. Studies conducted previously demonstrated that the large immunophilin FKBP52 acts to potentiate glucocorticoid, androgen, and progesterone receptor signaling pathways. The aim of these studies was to identify and characterize FKBP52-specific inhibitors that would not only serve as tools for the pharmacological analysis of FKBP52-receptor interactions, but may also lead to novel drugs with significant …

Changes In Apparent Molar Water Volume And Dkp Solubility Yield Insights On The Hofmeister Effect, Daryl K. Eggers, A. Y. Payumo, R. M. Huijon, D. D. Mansfield, L. M. Belk, A. K. Bui, A. E. Knight

Faculty Publications, Chemistry

This study examines the properties of a 4 × 2 matrix of aqueous cations and anions at concentrations up to 8.0 M. The apparent molar water volume, as calculated by subtracting the mass and volume of the ions from the corresponding solution density, was found to exceed the molar volume of ice in many concentrated electrolyte solutions, underscoring the nonideal behavior of these systems. The solvent properties of water were also analyzed by measuring the solubility of diketopiperazine (DKP) in 2.000 M salt solutions prepared from the same ion combinations. Solution rankings for DKP solubility were found to parallel the …