Biochemistry Commons^™

Rna Oxidation Adducts 8-Ohg And 8-Oha Change With Aβ42 Levels In Late-Stage Alzheimer's Disease, Adam M. Weidner, Melissa A. Bradley, Tina L. Beckett, Dana M. Niedowicz, Amy L.S. Dowling, Sergey V. Matveev, Harry Levine, Mark A. Lovell, M. Paul Murphy

Sanders-Brown Center on Aging Faculty Publications

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain …

Stomatin-Like Protein 2 Binds Cardiolipin And Regulates Mitochondrial Biogenesis And Function., Darah Christie, Caitlin D Lemke, Isaac M Elias, Luan A Chau, Mark G Kirchhof, Bo Li, Eric H Ball, Stanley D Dunn, Grant M Hatch, Joaquín Madrenas

Biochemistry Publications

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched …

Digeorge Critical Region 8 (Dgcr8) Is A Double-Cysteine-Ligated Heme Protein., Ian Barr, Aaron T. Smith, Rachel Senturia, Yanqiu Chen, Brooke D. Scheidemantle, Judith N. Burstyn, Feng Guo

Natural Sciences and Mathematics | Faculty Scholarship

All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 …

Decreased Stability And Increased Formation Of Soluble Aggregates By Immature Superoxide Dismutase Do Not Account For Disease Severity In Als., Kenrick A Vassall, Helen R Stubbs, Heather A Primmer, Ming Sze Tong, Sarah M Sullivan, Ryan Sobering, Saipraveen Srinivasan, Lee-Ann K Briere, Stanley D Dunn, Wilfredo Colón, Elizabeth M Meiering

Biochemistry Publications

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic …

Mechanisms Of Oxidant Generation By Catalase, Diane E. Heck, Michael Shakarjian, Hong-Duck Kim, Jeffrey Laskin, Anna M. Vetrano

NYMC Faculty Publications

The enzyme catalase converts solar radiation into reactive oxidant species (ROS). In this study, we report that several bacterial catalases (hydroperoxidases, HP), including Escherichia coli HP-I and HP-II also generate reactive oxidants in response to ultraviolet B light (UVB). HP-I and HP-II are identical except for the presence of NADPH. We found that only one of the catalases, HPI, produces oxidants in response to UVB light, indicating a potential role for the nucleotide in ROS production. This prompts us to speculate that NADPH may act as a cofactor regulating ROS generation by mammalian catalases. Structural analysis of the NADPH domains …

The Marine Sponge Metabolite Mycothiazole: A Novel Prototype Mitochondrial Complex I Inhibitor., J Brian Morgan, Fakhri Mahdi, Yang Liu, Veena Coothankandaswamy, Mika B. Jekabsons, Tyler A. Johnson, Koneni V. Sashidhara, Phillip Crews, Dale G. Nagle, Yu-Dong Zhou

Natural Sciences and Mathematics | Faculty Scholarship

A natural product chemistry-based approach was applied to discover small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1). A Petrosaspongia mycofijiensis marine sponge extract yielded mycothiazole (1), a solid tumor selective compound with no known mechanism for its cell line-dependent cytotoxic activity. Compound 1 inhibited hypoxic HIF-1 signaling in tumor cells (IC(50) 1nM) that correlated with the suppression of hypoxia-stimulated tumor angiogenesis in vitro. However, 1 exhibited pronounced neurotoxicity in vitro. Mechanistic studies revealed that 1 selectively suppresses mitochondrial respiration at complex I (NADH-ubiquinone oxidoreductase). Unlike rotenone, MPP(+), annonaceous acetogenins, piericidin A, and other complex I inhibitors, mycothiazole is a mixed polyketide/peptide-derived compound …

Mimosa: A System For Minimotif Annotation, Jay Vyas, Ronald J. Nowling, Thomas Meusburger, David P. Sargeant, Krishna Kadaveru, Michael R. Gryk, Vamsi Kundeti, Sanguthevar Rajasekaran, Martin Schiller

Life Sciences Faculty Research

BACKGROUND:

Minimotifs are short peptide sequences within one protein, which are recognized by other proteins or molecules. While there are now several minimotif databases, they are incomplete. There are reports of many minimotifs in the primary literature, which have yet to be annotated, while entirely novel minimotifs continue to be published on a weekly basis. Our recently proposed function and sequence syntax for minimotifs enables us to build a general tool that will facilitate structured annotation and management of minimotif data from the biomedical literature.

RESULTS:

We have built the MimoSA application for minimotif annotation. The application supports management of …

Human Cerebral Neuropathology Of Type 2 Diabetes Mellitus, Peter T. Nelson, Charles D. Smith, Erin L. Abner, Frederick A. Schmitt, Stephen W. Scheff, Gregory J. Davis, Jeffrey N. Keller, Gregory A. Jicha, Daron Davis, Wang-Xia Wang, Adria Hartman, Douglas G. Katz, William R. Markesbery

Pathology and Laboratory Medicine Faculty Publications

The cerebral neuropathology of Type 2 diabetes (CNDM2) has not been positively defined. This review includes a description of CNDM2 research from before the ‘Pubmed Era’. Recent neuroimaging studies have focused on cerebrovascular and white matter pathology. These and prior studies about cerebrovascular histopathology in diabetes are reviewed. Evidence is also described for and against the link between CNDM2 and Alzheimer's disease pathogenesis. To study this matter directly, we evaluated data from University of Kentucky Alzheimer's Disease Center (UK ADC) patients recruited while non-demented and followed longitudinally. Of patients who had come to autopsy (N = 234), 139 met …

Discovery Of Platelet-Type 12-Human Lipoxygenase Selective Inhibitors By High-Throughput Screening Of Structurally Diverse Libraries., Joshua D. Deschamps, Jeffrey T. Gautschi, Stephanie Whitman, Tyler A. Johnson, Nadine C. Gassner, Phillip Crews, Theodore R. Holman

Natural Sciences and Mathematics | Faculty Scholarship

Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors …

Heparin Modulates The 99-Loop Of Factor Ixa: Effects On Reactivity With Isolated Kunitz-Type Inhibitor Domains, Pierre F. Neuenschwander, Stephen R. Williamson, Armen Nalian, Kimberly J. Baker-Deadmond

Faculty Publications

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor …

Never Let Me Clone? Countering An Ethical Argument Against The Reproductive Cloning Of Humans, Yvette Pearson

Philosophy Faculty Publications

In the March 2006 issue of EMBO reports, Christof Tannert, a bioethicist at the Max Delbrück Research Centre in Berlin, Germany, presented a moral argument against human reproductive cloning on the basis of Immanuel Kant’s categorical imperative (Tannert, 2006). In this article, I address some problems with Tannert’s views and show that our concerns about this prospective procedure should prompt us to scrutinize carefully the conventional procreative practices and attitudes. Indeed, if we set aside objections that are grounded in genetic determinism, many of the offensive features of human cloning are identical to problems with procreation by more conventional means, …

The Kini Kinesin Kif2a Is Required For Bipolar Spindle Assembly Through A Functional Relationship With Mcak, Neil J. Ganem, Duane A. Compton

Dartmouth Scholarship

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore–microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle …

Minus-End Capture Of Preformed Kinetochore Fibers Contributes To Spindle Morphogenesis, Alexey Khodjakov, Lily Copenagle, Michael B. Gordon, Duane A. Compton, Tarun M. Kapoor

Dartmouth Scholarship

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient …

Cloning Of Human Acetyl-Coa Carboxylase-Beta And Its Unique Features., Joohun Ha, Jung-Kee Lee, Kyung-Sup Kim, Lee A. Witters, Ki-Han Kim

Dartmouth Scholarship

Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles. In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis. ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the …

A Novel Iron-Regulated Metal Transporter From Plants Identified By Functional Expression In Yeast., David Eide, Margaret Broderius, Jeanette Fett, Mary Lou Guerinot

Dartmouth Scholarship

Iron is an essential nutrient for virtually all organisms. The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Related sequences were also found in the genomes of rice, yeast, nematodes, and humans. In Arabidopsis, …