Biochemistry, Biophysics, and Structural Biology Commons^™

Analysis Of Leigh Syndrome Mutations In The Yeast Surf1 Homolog Reveals A New Member Of The Cytochrome Oxidase Assembly Factor Family, Megan Bestwick, Mi-Young Jeong, Oleh Khalimonchuk, Hyung Kim, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F²⁴⁹T and Y³⁴⁴D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G¹³⁷E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G¹³⁷E Shy1 mutant phenocopied shy1 Δ cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G137E …

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 …

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 …

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. …

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 …

Epsvr And Epmeta: Prediction Of Antigenic Epitopes Using Support Vector Regression And Multiple Server Results, Shide Liang, Dandan Zheng, Daron M. Standley, Bo Yao, Martin Zacharias, Chi Zhang

School of Biological Sciences: Faculty Publications

Background: Accurate prediction of antigenic epitopes is important for immunologic research and medical applications, but it is still an open problem in bioinformatics. The case for discontinuous epitopes is even worse - currently there are only a few discontinuous epitope prediction servers available, though discontinuous peptides constitute the majority of all B-cell antigenic epitopes. The small number of structures for antigen-antibody complexes limits the development of reliable discontinuous epitope prediction methods and an unbiased benchmark to evaluate developed methods.

Results: In this work, we present two novel server applications for discontinuous epitope prediction: EPSVR and EPMeta, where EPMeta …

Biochemical Characterization Of Human Mismatch Recognition Proteins Mutsα And Mutsβ, Lei Tian

University of Kentucky Doctoral Dissertations

The integrity of an organism's genome depends on the fidelity of DNA replication and the efficiency of DNA repair. The DNA mismatch repair (MMR) system, which is highly conserved from prokaryotes to eukaryotes, plays an important role in maintaining genome stability by correcting base-base mismatches and insertion/deletion (ID) mispairs generated during DNA replication and other DNA transactions. Mismatch recognition is a critical step in MMR. Two mismatch recognition proteins, MutSα (MSH2-MSH6 heterodimer) and MutSβ (MSH2-MSH3 heterodimer), have been identified in eukaryotic cells. MutSα and MutSβ have partially overlapping functions, with MutSα recognizing primarily base-base mismatches and 1-2 nt ID mispairs …

Explorations In Homeoviscous Adaptation And Mass Spectral Analysis Of Membrane Lipids, Michael Douglas Timmons

University of Kentucky Doctoral Dissertations

The focus of this dissertation is centered on the mass spectral analysis of lipids and changes occurring in keeping with the concept of homeoviscous adaptation [1]. Homeoviscous adaptation is the process of modification of membrane lipids in response to environmental stimuli [1]. Dissertation investigations applied this concept to prokaryotic and eukaryotic organisms, and expanded the perception of environmental factors from exogenous organic solvents to intracellular environment.

The field of lipidomics deals with the analysis of phospholipid and fatty acid components of membranes the changes that occur due to environmental stimuli and their biological significance [2-6]. The high sensitivity of mass …

Dissecting The Biosyntheses Of Gilvocarcins And Ravidomycins, Madan Kumar Kharel

University of Kentucky Doctoral Dissertations

Gilvocarcin V (GV) and ravidomycin (RMV) exhibit excellent antitumor activities in the presence of near-UV light at low concentration maintaining a low in vivo cytotoxicity. Although, the exact molecular mechanism for in vivo actions of these antibiotics has yet to be determined, a [2+2] cycloaddition reaction of the vinyl side chain with DNA thymidine residues in addition to the inhibition of topoisomerase II and DNAhistone H3 cross-linking are reported for the GV’s mechanism of action. Such activities have made these molecules interesting candidates for the biosynthetic investigation to generate analogues with improved activity/solubility. Previous biosynthetic studies have suggested that the …

Analysis Of The Crmp Gene In Drosophila: Determining The Regulatory Role Of Crmp In Signaling And Behavior, Deanna Hardt Morris

University of Kentucky Doctoral Dissertations

The mammalian genome encodes five collapsin response mediator protein (CRMP) isoforms. Cell culture studies have shown that the CRMPs mediate growth cone dynamics and neuron polarity through associations with a variety of signal transduction components and cytoskeletal elements. CRMP is also a member of a protein family including the presumably ancestral dihydropyrimidinase (DHP) protein that catalyzes the second step in pyrimidine degradation. In Drosophila, CRMP and DHP proteins are produced by alternatively spliced transcripts of the CRMP gene. The alternative protein forms have a 91% sequence identity, but unique expression patterns. CRMP is found exclusively in neuronal tissues and …

In Vivo Oxidative Stress In Alzheimer Disease Brain And A Mouse Model Thereof: Effects Of Lipid Asymmetry And The Single Methionine Residue Of Amyloid-Β Peptide, Miranda Lu Bader Lange

University of Kentucky Doctoral Dissertations

Studies presented in this dissertation were conducted to gain more insight into the role of phospholipid asymmetry and amyloid-β (Aβ)-induced oxidative stress in brain of subjects with amnestic mild cognitive impairment (aMCI) and Alzheimer disease (AD). AD is a largely sporadic, age-associated neurodegenerative disorder clinically characterized by the vast, progressive loss of memory and cognition commonly in populations over the age of ~65 years, with the exception of those with familial AD, which develop AD symptoms as early as ~30 years-old. Neuropathologically, both AD and FAD can be characterized by synapse and neuronal cell loss in conjunction with accumulation of …

Protein Folding, Aggregation And Unfolding In Monte Carlo Simulations, Sandipan Mohanty, Anders Irbäck, Simon Mitternacht, Giorgio Favrin, U. H.E. Hansmann

Michigan Tech Publications, Part 1

An implicit water all-atom model is used to study folding, aggregation and mechanical unfolding of small proteins. Physically reasonable results obtained for a variety of applications indicate healthy global properties of the interaction potential.

Stream Invertebrate Responses To A Catastrophic Decline In Consumer Diversity, Jose Checo Colón-Gaud, Matt R. Whiles, Karen R. Lips, Catherine M. Pringle, Susan Kilham, Roberto Brenes, Scot D. Peterson

Biology: Faculty Publications

Tadpoles are often abundant and diverse consumers in headwater streams in the Neotropics. However, their populations are declining catastrophically in many regions, in part because of a chytrid fungal pathogen. These declines are occurring along a moving disease front in Central America and offer the rare opportunity to quantify the consequences of a sudden, dramatic decline in consumer diversity in a natural system. As part of the Tropical Amphibian Declines in Streams (TADS) project, we examined stream macroinvertebrate assemblage structure and production for 2 y in 4 stream reaches at 2 sites in Panama. One site initially had healthy amphibians …

Plasticity Of Acquired Secondary Metabolites In Clathria Prolifera (Demospongia: Poecilosclerida): Putative Photoprotective Role Of Carotenoids In A Temperate Intertidal Sponge, Jonathan D. Dattelbaum, Drew Sieg, Malcolm Hill, Chris M. Manieri, Giles Thomson

Chemistry Faculty Publications

Several marine sponges sequester high concentrations of carotenoids in their tissues. The diversity of carotenoid compounds has been described in detail for a handful of species, but to date, little attention has been paid to natural variability in the concentration and constituency of carotenoid pools. Also lacking are experimental tests of some of the proposed adaptive benefits of carotenoids to the sponge. To address some of these deficits in our understanding of sponge ecology, we used a combination of analytic chemistry, field surveys, and manipulative experiments to determine what function these compounds might play. Attention was focused on the common, …

Low Cost Production Of Proinsulin In Tobacco And Lettuce Chloroplasts For Injectable Or Oral Delivery Of Functional Insulin And, Diane Burberry

Electronic Theses and Dissertations

Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, and C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to …

Acetazolamide-Induced Decrease Of Apical Fluid Flow In Choroid Plexus Is Independent Of The Concomitant Changes In Aquaporin-1 Expression, Pouya Alexander Ameli

Electronic Theses and Dissertations

Acetazolamide (AZA), the only drug approved for treatment of hydrocephalus, is effective in only 25-30% of patients while its effect on fluid flow in the choroid plexus (CP) is unknown. The drug reversibly inhibits Aquaporin 4 (AQP4), the most highly expressed „water pore‟ in the brain, and it is postulated that it reduces cerebrospinal fluid (CSF) production by modulating AQP1 (mostly found in the apical membrane of the CP). In this study, we sought to elucidate the effect of AZA on AQP1 and fluid flow in CP. Primary CP culture from p10 Sprague-Dawley rats and TRCSF-B cell line were grown …

Purification And Properties Of Amycolatopsis Mediterranei Dsm 43304 Lipase And Its Potential In Flavour Ester Synthesis, Dharmendra Dheeman, Gary Henehan, Jesus Maria Frias

Articles

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in 398 fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of and for p-nitrophenyl palmitate (p-NPP) under optimal temperature (60°C) and pH (8.0) conditions were 0.10 ± 0.01 mM and 2.53 ± 0.06 mmol/minmg, respectively. The purified …

The Distribution Of Gnrh And Erbeta In Pre- And Post-Pubertal Male Rats, Maysaa Nageeb

Master's Theses

The hypothalamic-pituitary- gonadal axis is intrinsic in regulating reproductive function. Each part orchestrates the synthesis and release of a key hormone. At the level of the hypothalamus, Gonadotropin- releasing hormone (GnRH) is released and it is the

most upstream regulator of sexual maturation. GnRH neurons express a type of estrogen receptor, ERbeta, which suggests that estradiol (E2) plays a direct role in modulating GnRH function (Herbison and Pape 2001). The objective of this study was to determine if gonadal steroid hormones regulate the expression of ERbeta and GnRH mRNA in the male brain during pubertal development. In-situ hybridization was used …

Developmental Characterization Of Cara Mitad: A Drosophila Nuclear Receptor Co-Regulator, Chhavi Chauhan

Dissertations

In insects and vertebrates, hormone titers drive cellular proliferation and differentiation events that guide proper development. Nuclear receptors (NR) respond to these hormone signals by activating cascades of gene expression, along with coregulator protein complexes. In Drosophila melanogaster, fluctuating titers of the steroid hormone ecdysone are responsible for coordinating the timing of organismal development. Despite major advances in our understanding of Drosophila NR activities, we lack essential knowledge of the coregulators that are required for their proper function.

We have recently identified the Drosophila cara mitad (cmi) (`dear half') gene. The deduced CMI protein is closely related to the N-terminal …

Lysine Specific Demethylase-1 And The Brahma Chromatin Remodeling Complex Regulate Conserved Signaling Pathways During Drosophila Wing Development, Brenda Jean Curtis

Dissertations

The conserved SWI/SNF chromatin remodeling complex uses the energy from ATP hydrolysis to alter local chromatin environments by disrupting DNA-histone contacts. The Drosophila SWI/SNF counterpart, the Brahma complex, has been shown to have an essential role in regulating the proper expression of many developmentally important genes, including those required for eye and wing tissue morphogenesis. A temperature sensitive mutation in one of the core subunits, SNR1 (SNF5/Ini1/SMARC B1), results in reproducible wing patterning phenotypes that can be enhanced and suppressed by extragenic mutations. SNR1 functions as a regulatory subunit to modulate chromatin remodeling activities of the Brahma complex on target …