Biochemistry Commons^™

Inhibition Of Protein Ubiquitination By Paraquat And 1-Methyl-4-Phenylpyridinium Impairs Ubiquitin-Dependent Protein Degradation Pathways, Juliana Navarro-Yepes, Anandhan Annadurai, Erin Bradley, Iryna Bohovych, Bo Yarabe, Annemieke De Jong, Huib Ovaa, You Zhou, Oleh Khalimonchuk, Betzabet Quintanilla-Vega, Rodrigo Franco

Department of Biochemistry: Faculty Publications

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson’s disease (PD). Ubiquitin (Ub), alpha [α]-synuclein, p62/sequestosome 1 and oxidized proteins are major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effect of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP⁺) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells …

Potassium And The K+/H+ Exchanger Kha1p Promote Binding Of Copper To Apofet3p Multi-Copper Ferroxidase, Xiaobin Wu, Heejeong Kim, Javier Seravalli, Joseph J. Barycki, P. John Hart, David W. Gohara, Enrico Di Cera, Won Hee Jung, Daniel J. Kosman, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K⁺) compartmentalization to the trans-Golgi network via Kha1p, a K⁺/H⁺ exchanger. K⁺ in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K⁺ is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K⁺ acts as a chemical factor in Fet3p maturation, a …

Analysis Of Oligomerization Properties Of Heme A Synthase Provides Insights Into Its Function In Eukaryotes, Samantha Swenson, Andrew Cannon, Nicholas J. Harris, Nicholas G. Taylor, Jennifer L. Fox, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Heme a is an essential cofactor for function of cytochrome c oxidase in the mitochondrial electron transport chain. Several evolutionarily conserved enzymes have been implicated in the biosynthesis of heme a, including the heme a synthase Cox15. However, the structure of Cox15 is unknown, its enzymatic mechanism and the role of active site residues remain debated, and recent discoveries suggest additional chaperone-like roles for this enzyme. Here, we investigated Cox15 in the model eukaryote Saccharomyces cerevisiae via several approaches to examine its oligomeric states and determine the effects of active site and human pathogenic mutations. Our results indicate that Cox15 …

Development Of A Chemical Genetic Screen To Determine Synergistic Compounds With Laromustine In Treating Glioblastoma Multiforme Cultured Cells, Ryan Weeks

Honors Theses

Laromustine is a chemotherapeutic sulfonylhydrazine prodrug used in clinical trials to treat acute myeloid leukemia (AML) and glioblastoma multiforme (GBM). While treatment of AML with laromustine has more demonstrative clinical success, there are enough promising data against GBM to pursue additional pre-clinical and clinical experiments. To determine the synergistic effects caused by treating cultured GBM cells with laromustine and a library of FDA-approved compounds, a chemical genetic screen was developed. To optimize the screen, optimal cultured GBM cell seed density, growth period and maximum well capacity were determined. The treatment period for a lethal dose of laromustine in cultured GBM …

Towards Structural Determination Of Human Α1-Glycine Receptor Allostery, Rathna Jyothi Veeramachaneni

Electronic Theses and Dissertations

Recent advances in technology have led to the determination of numerous notable structures of membrane proteins. While they provide valuable information about the structure of membrane proteins these studies often provide static images with potentially limited dynamics, and structural determination often requires truncation of flexible regions, and often utilizes bacterial homologs given the need for stable, heterologous overexpression. In order to better understand allostery at a molecular level, state-dependent crosslinking studies coupled with multidimensional mass spectrometry (MS) were conducted on glycine receptor (GlyR) stabilized in different allosteric states. Predominant allosteric states were stabilized using wild type or mutated receptor in …

Superior Inhibitory Control And Resistance To Mental Fatigue In Professional Road Cyclists, Kristy Martin, Walter Staiano, Paolo Mensaspa, Tom Hennessey, Samuele M. Marcora, Richard J. Keegan, Kevin G. Thompson, David T. Martin, Shona R. Halson, Ben Rattray

Research outputs 2014 to 2021

Purpose:

Given the important role of the brain in regulating endurance performance, this comparative study sought to determine whether professional road cyclists have superior inhibitory control and resistance to mental fatigue compared to recreational road cyclists.

Methods:

After preliminary testing and familiarization, eleven professional and nine recreational road cyclists visited the lab on two occasions to complete a modified incongruent colour-word Stroop task (a cognitive task requiring inhibitory control) for 30 min (mental exertion condition), or an easy cognitive task for 10 min (control condition) in a randomized, counterbalanced cross-over order. After each cognitive task, participants completed a 20-min time …

Metabolomics Enables Precision Medicine: “A White Paper, Community Perspective”, Richard D. Beger, Warwick B. Dunn, Michael A. Schmidt, Steven S. Cross, Jennifer A. Kirwan, Marta S. Cascante, Lorraine Brennan, David S. Wishart, Matej Orešič, Thomas Hankemeier, David Broadhurst, Andrew N. Lane, Karsten Suhre, Gabi Kastenmüller, Susan J. Sumner, Ines Thiele, Oliver E. Fiehn, Rima Kaddurah- Daouk

Research outputs 2014 to 2021

Introduction:Background to metabolomics:

Metabolomics is the comprehensive study of the metabolome, the repertoire of biochemicals (or small molecules) present in cells, tissues, and body fluids. The study of metabolism at the global or “-omics” level is a rapidly growing field that has the potential to have a profound impact upon medical practice. At the center of metabolomics, is the concept that a person’s metabolic state provides a close representation of that individual’s overall health status. This metabolic state reflects what has been encoded by the genome, and modified by diet, environmental factors, and the gut microbiome. The metabolic profile provides …

The Uyghur Population And Genetic Susceptibility To Type 2 Diabetes: Potential Role For Variants In Capn10, Apm1 And Fut6 Genes, Feifei Zhao, Dolikun Mamatyusupu, Youxin Wang, Honghong Fang, Hao Wang, Qing Gao, Hao Dong, Siqi Ge, Xinwei Yu, Jie Zhang, Lijuan Wu, Manshu Song, Wei Wang

Research outputs 2014 to 2021

Genome-wide association studies have successfully identified over 70 loci associated with the risk of type 2 diabetes mellitus (T2DM) in multiple populations of European ancestry. However, the risk attributable to an individual variant is modest and does not yet provide convincing evidence for clinical utility. Association between these established genetic variants and T2DM in general populations is hitherto understudied in the isolated populations, such as the Uyghurs, resident in Hetian, far southern Xinjiang Uyghur Autonomous Region, China. In this case–control study, we genotyped 13 single-nucleotide polymorphisms (SNPs) at 10 genes associated with diabetes in 130 cases with T2DM and 135 …

It Is All About (U)Biquitin: Role Of Altered Ubiquitin-Proteasome System And Uchl1 In Alzheimer Disease, Antonella Tramutola, Fabio Di Domenico, Eugenio Barone, Marzia Perluigi, D. Allan Butterfield

Chemistry Faculty Publications

Free radical-mediated damage to macromolecules and the resulting oxidative modification of different cellular components are a common feature of aging, and this process becomes much more pronounced in age-associated pathologies, including Alzheimer disease (AD). In particular, proteins are particularly sensitive to oxidative stress-induced damage and these irreversible modifications lead to the alteration of protein structure and function. In order to maintain cell homeostasis, these oxidized/damaged proteins have to be removed in order to prevent their toxic accumulation. It is generally accepted that the age-related accumulation of “aberrant” proteins results from both the increased occurrence of damage and the decreased efficiency …

Piperlongumine (Piplartine) And Analogues: Antiproliferative Microtubule-Destabilising Agents, Mary J. Meegan, Seema M. Nathwani, Brendan Twamley, Daniela M. Zisterer, Niamh O'Boyle

Articles

Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and Western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in MCF-7 breast cancer cells were depolymerised when treated with 1. We synthesised a number of analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic profile of this series, …

A Fret Investigation Into Molecular Mechanisms Of Cardiac Troponin Activation In Reconstituted Thin Filaments, Maria Eleni Moutsoglou

Electronic Theses and Dissertations

Cardiomyopathies (CM) are the leading cause of death in America, and can develop from mutations in sarcomeric proteins, leading to altered protein structure and function. Current therapies target upstream signaling pathways to treat the symptoms of heart failure, but are associated with increased mortality by affecting downstream signaling pathways and other muscle types. Rational drug design can develop therapies to treat CM at the protein level. However, a detailed knowledge of how sarcomeric proteins regulate muscle contraction is required. Muscle contraction occurs through a cyclic interaction between actin thin and myosin thick filaments, regulated by intracellular Ca2+ concentration. Troponin (Tn), …

Alcoholic Vs Non-Alcoholic Fatty Liver In Rats: Distinct Differences In Endocytosis And Vesicle Trafficking Despite Similar Pathology, Karuna Rasineni, Daniel D. Penrice, Sathish Kumar Natarajan, Mark A. Mcniven, Benita L. Mcvicker, Kusum K. Kharbanda, Carol A. Casey, Edward N. Harris

Department of Biochemistry: Faculty Publications

Background: Non-alcoholic and alcoholic fatty liver disease (NAFLD and AFLD, respectively) are major health problems, as patients with either condition can progress to hepatitis, fibrosis, and cirrhosis. Although histologically similar, key differences likely exist in these two models. For example, altered content of several vesicle trafficking proteins have been identified in AFLD, but their content in NAFLD is unknown. In this study, we compared select parameters in NAFLD and AFLD in a rat model.

Methods: We fed either Lieber- DeCarli liquid control or alcohol-containing (35 % as calories) diet (AFLD model) or lean or high-fat (12 or 60 % derived …

Atomic Resolution Experimental Phase Information Reveals Extensive Disorder And Bound 2-Methyl-2,4-Pentanediol In Ca2+-Calmodulin, Jiusheng Lin, Henry Van Den Bedem, Axel T. Brunger, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca²⁺-CaM, but no assignment of these features has been made. In addition, Ca²⁺-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 A resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca²⁺-CaM. …

An Evaluation Of New And Established Methods To Determine T-Dna Copy Number And Homozygosity In Transgenic Plants, Katarzyna Glowacka, Johannes Kromdijk, Lauriebeth Leonelli, Krishna K. Niyogi, Tom E. Clemente, Stephen P. Long

Department of Biochemistry: Faculty Publications

Stable transformation of plants is a powerful tool for hypothesis testing.Arapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations.Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher …

Study On Dioxygen Binding To Heme Using Scan Functional, Zegnet Yimer Muhammed

Open Access Theses & Dissertations

Density functional theory, a quantum mechanical based electronic structure method with GGA-PBE and MGG-SCAN functionals, are used to investigate the structure and energies of singlet, triplet, and quintet spin states of FeP (iron Porphyrin), FePIm (imidazole iron porphyrin), and FePImO2 (dioxygen imidazole iron porphyrin) systems. The binding and release of dioxygen to and from hemoglobin (Hb) are the most crucial reaction takes place in human body to sustain the existence of life. FePImO2 is used to model this phenomenon. When O2 binds to FePIm, the system undergoes a conformational change. i.e. from domed structure of FePIm in which the Fe …

Structure Function Studies Of Hiv-1 Protease, Bradley James Keusch

Wayne State University Theses

HIV-1 is the causative agent of the devastating human disease Acquired Immunodeficiency Syndome (AIDS). While much progress has been made over the past two decades, HIV-1 remains a major global health concern. HIV-1 protease is 99-amino acid homodimer aspartyl protease that is essential to the life cycle of HIV. This has rendered it an attractive and very successful drug target. However, due to the high error rate of the HIV -1 reverse transcriptase, drug resistance mutations in the protease can develop very rapidly in some patients, rendering current protease inhibitors (one of the main classes of drug in common antiretroviral …

Beta-Sheet Forming Peptides By Design : Control Of Folding And Applications, Gaius Takor

Legacy Theses & Dissertations (2009 - 2024)

The focus of the present research is the synthesis of polypeptides for the study of protein folding and misfolding and for the development of novel polypeptide-based optical antennas in nanotechnology. It is hypothesized that simple polypeptides can be used as models to mimic in vivo folding of globular proteins. Desired repetitive polypeptides were genetically encoded and expressed in E. coli using conventional methods and characterized using a variety of spectroscopic (including circular dichroism (CD), deep UV resonance Raman (DUVRR), UV-vis and fluorescence) and microscopic (atomic force microscopy (AFM) and transmission electron microscopy (TEM)) techniques. The polypeptides predominantly formed bilayer, fibrillar …

Investigation Of Enzymatically Synthesized Glycogen As A Novel Nanodendrimer For Therapeutic Delivery, Sarah Ann Engelberth

Legacy Theses & Dissertations (2009 - 2024)

The field of medicinal chemistry is ever expanding, designing and discovering new therapeutic strategies. Oftentimes, it is challenging for these therapeutics to undergo clinical translation due to ineffective administration or unwanted toxicity in vivo. As such, drug delivery vehicles are designed to overcome these hurdles, allowing for delivery to the site of action by improving biodistribution, protecting therapeutic cargo, and decreasing toxicity. The work presented here aims to investigate a naturally-derived carbohydrate nanodendrimer, enzymatically synthesized glycogen (ESG) for drug delivery. This nontoxic, highly-branched, glucose-based structure has interior void volumes to allow for cargo encapsulation as well as a large density …

The Chemistry Of New Garlic-Derived Organosulfur Compounds And The Molecular Basis Of Olfaction, Berenice Dethier

Legacy Theses & Dissertations (2009 - 2024)

Garlic is a very popular condiment that has been used around the world for centuries. It is also a source of a remarkably extensive range of organosulfur compounds, whose chemistry is the focus of this thesis. The central reaction in formation of these compounds is the enzymatic cleavage of alk(en)yl cysteine sulfoxides by alliinases, which leads to sulfenic acids. The latter can then undergo condensation and rearrangement into various organosulfur compounds. Three aspects of the chemistry of garlic have been investigated in this thesis.

Study Of Protein-Protein Interaction By Using In-Cell Nmr In Human Cells, Asma Salem M Aldousary

Legacy Theses & Dissertations (2009 - 2024)

We developed a new technology to study protein-protein interaction in mammalian cells. This technology is based high resolution of Nucleic Magnetic Resonance (NMR) spectroscopy. Using this technology we studied interaction between the receptor for advanced glycation endproducts (RAGE). RAGE- is a multiligand receptor of immunoglobulin receptor family that is activated by a multitude of ligands. Activation of RAGE results in signal transduction that leads to the inflammatory response implicated in the complications of Diabetes. RAGE is an emergent drug target that has been explored for the variety of pathologist including cancers, neurological disorders, inflammatory disease, and diabetes. and Intracellular effector …