Other Chemistry Commons^™

Response To Replication Stress And Maintenance Of Genome Stability By Wrn, The Werner Syndrome Protein, David K. Orren, Amrita Machwe

Markey Cancer Center Faculty Publications

Werner syndrome (WS) is an autosomal recessive disease caused by loss of function of WRN. WS is a segmental progeroid disease and shows early onset or increased frequency of many characteristics of normal aging. WRN possesses helicase, annealing, strand exchange, and exonuclease activities and acts on a variety of DNA substrates, even complex replication and re- combination intermediates. Here, we review the genetics, biochemistry, and probably physiological functions of the WRN protein. Although its precise role is unclear, evidence suggests WRN plays a role in pathways that respond to replication stress and maintain genome stability particularly in telomeric regions.

Myeloid-Derived Suppressor Cell Mitochondrial Fitness Governs Chemotherapeutic Efficacy In Hematologic Malignancies, Saeed Daneshmandi, Jee Eun Choi, Qi Yan, Cameron R. Macdonald, Manu Pandey, Mounika Goruganthu, Nathan Roberts, Prashant K. Singh, Richard M. Higashi, Andrew N. Lane, Teresa W-M Fan, Jianmin Wang, Philip L. Mccarthy, Elizabeth A. Repasky, Hemn Mohammadpour

Markey Cancer Center Faculty Publications

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via β2-adrenergic receptor (β2-AR) activation leads to improved MDSC function through metabolic reprogram- ing, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of …

A Protein-Based Therapeutic Combination For The Treatment Of Hard-To-Heal Wounds, Graham L. Strauss

USF Tampa Graduate Theses and Dissertations

Chronic wounds present many clinical challenges in relation to the successful treatment and closure of the damaged tissue. Most current treatment methods focused on one or two aspects to drive wound closure, while most chronic wounds are multifactorial environments with many of those dependencies relying on the termination of one another to effectively gain tissue construction, closure, and full skin thickness and composition. Natural wound healing processes allude to potential biologics that can impede the chronic breakdown of tissue, while restoring deposition of new tissue, and effectively leading to a healed wound. Proteases secreted by the body’s immune system lay …

Purification And Activity Assay Optimization Of Bacterial Catalase From Psychrobacter Koprana, Sydney Willis

Honors Program Theses

To date, catalase protein from bacterial species is not the most widely used commercial catalase. Catalase, the enzyme that breaks down hydrogen peroxide in most living organisms, is an invaluable enzyme in the food, agriculture, and pharmaceutical industries. In 2019, a novel species of a psychrotolerant bacterium was discovered in Cold Harbor, NY. This species, Psychrobacter koprana, exhibits highly active catalase. Preliminary data from activity assays demonstrates that P. koprana catalase has the same activity as commercially available catalase. After purification and activity assay optimization, the activity of this catalase has the potential to be more active than other easily …

A Nosy Neighbor: Purification And Functional Characterization Of Lpg2149, Ashley M. Holahan

The Journal of Purdue Undergraduate Research

Ubiquitination is a process that marks proteins for various cell-signaling pathways, namely protein degradation and other processes. Th ese pathways are essential in a wide array of cellular processes, including defense mechanisms against invading pathogens. Th e ubiquitination process is universally found in all eukaryotic organisms, including plants and animals, and thus plays a vital role in cellular homeostasis. Recently, more discoveries have been made on prokaryotic effector proteins that hijack the ubiquitination system even when they do not possess a ubiquitin system of their own. MavC, also known as lpg2147 (Gan, Nakayasu, Hollenbeck, & Luo, 2019; Puvar et al., …

Investigating Chitosan Modified With Triethylammonium Butanamide And Triethylphosphonium Butanamide As Non-Viral Gene Delivery Vectors By Examining Cytotoxicity And Transfection Efficiency, Deborah C. Ehie

Graduate Theses/Dissertations

Gene therapy is a very challenging field, especially with new emerging genetic disorders. Chitosan (CS), due to chitosan’s flexibility, biocompatibility, and biodegradability, has been of interest in the world of gene therapy especially as researchers are gravitating towards non-viral vectors due to the problems caused by viral vectors. Nevertheless, there are still issues regarding solubility, cellular uptake of cargos being transported in vitro or in vivo, increased cytotoxicity levels, as well as many other things that prevent chitosan from being an efficient gene delivery agent. Here I present five derivatives of chitosan, which were all modified with either triethylphosphonium …

Antifungal Defense Molecules From Bacterial Symbionts Of North American Trachymyrmex Ants, Georgia Scherer

CMC Senior Theses

Defensive symbioses, in which microbes provide molecular defenses for an animal host, hold great potential as untapped sources of therapeutically useful antibiotics. Fungus-growing ants use antifungal defenses from bacterial symbionts to suppress pathogenic fungi in their nests. Preliminary chemical investigations of symbiotic bacteria from this large family of ants have uncovered novel antifungal molecules with therapeutic potential, such as dentigerumycin and selvamicin.

In this study, the bacterial symbionts of North American Trachymyrmex fungus-growing ants are investigated for antifungal molecules. Plate-based bioassays using ecologically-relevant fungal pathogens confirmed that these bacteria have antifungal activity. In order to purify and identify the antifungal …

Characterization Of The Dimerization Domains On The Mannose-6-Phosphate/Insulin-Like Growth Factor Ii Receptor, Tyler Degener

Theses/Capstones/Creative Projects

The mannose-6-phosphate/insulin-like growth factor II (M6P/IGF2) receptor is a transmembrane protein known to sequester growth factors from the extracellular matrix. This behavior suggests a mechanism of tumor suppression. Structurally, the receptor’s extracellular region is segmented into 15 homologous repeats, which are divided further into 5 triplet domains, labelled 1-3, 4-6, 7-9, 10-12, and 13-15. What is notable about the triplets is their propensity to form dimers with triplets on a second M6P/IGF2 receptor. In fact, previous studies indicate that this protein functions optimally when dimerized. Thus, the purpose of this experiment is to characterize these domain interactions. Using a urea …

Kcnq1/Kcne1 Interaction In The Cardiac Iks Channel And Its Physiological Consequences, Jiajing Xu

McKelvey School of Engineering Graduate Student Theses & Dissertations

Dynamic conformational changes of ion channel proteins during activation gating determine their function as carriers of current. The relationship between these molecular movements and channel function over the physiological timescale of the action potential (AP) has not been fully established due to limitations of existing techniques. We constructed a library of possible cardiac IKs protein conformations and applied a combination of protein segmentation and energy linearization to study this relationship computationally. Simulations reproduced the effects of the beta-subunit (KCNE1) on the alpha-subunit (KCNQ1) dynamics and function, observed in experiments. Mechanistically, KCNE1 increased the probability of “visiting” conducting pore conformations on …

Advancing Professional Development Strategies For Chemistry And Biochemistry Undergraduates, Christian A. Brown

Senior Theses

In the Department of Chemistry and Biochemistry at the University of South Carolina (the Department), most curriculum-driven professional development is reserved for third- and fourth-year students. Classroom learning occurs at all levels, but the context comes late. Early exposure to career options and hands-on training is imperative for undergraduates to obtain maximum experiential learning. The purpose of this project is to advance the current professional development strategies of the Department, specifically targeting second- and third-year students. The study surveyed students' current involvement in professional development activities. The data on extracurricular experience and post-graduation interest was used to design a workshop …

Specific Binding Affinity Of The Non-Catalytic Domain Of Eukaryotic Like Type Ib Topoisomerase Of Vaccinia Virus, Benjamin R. Reed

Dissertations, Theses, and Capstone Projects

Topoisomerases are ubiquitous proteins that alter supercoiling in double stranded DNA (dsDNA) during transcription and replication and. vaccinia and the closely related poxvirus variola virus, at 314 amino acids in length, encode the smallest of the type I topoisomerases(TopIB). TopIB is a two domain protein that recognizes the sequence 5’-T/CCCTT, cleaves at the 3’-end and relaxes supercoiling through rotation. The C-terminal domain (CTD) alone contains the catalytic activity and specificity. Deletion of the N-terminal domain results in a greatly reduced rate of relaxation and rapid dissociation. Biochemical data suggests that the N-terminal domain (NTD) is important for pre-cleavage binding and …

Investigations Into The Molecular Mechanisms Of Bacterial Pathogen–Host Interactions: Construction Of A Dual Plasmid System For Incorporation Of Unnatural Amino Acids Into Pseudomonas Syringae Pv. Tomato Dc3000, Scotty D. Raber

Department of Chemistry: Dissertations, Theses, and Student Research

A dual plasmid system for the incorporation of unnatural amino acids into plant pathogen, Pseudomonas syringae pv. tomato DC3000, has been designed. This invention is expected to allow (a) mutations of proteins synthesized by the bacterium, P. syringae pv. tomato DC3000, that can capture molecular targets, especially for such modified proteins secreted by the phytopathogen into the host plant cells of A. thaliana and S. lycopersicum, (b) expression of biological probes in the bacterial species to monitor changes in redox, nutritional, and other small molecule states over pre-, post- and in situ disease stages, and secretion of such biological …

Dna Damage Responses In Progeroid Syndromes Arise From Defective Maturation Of Prelamin A, Michael Sinensky, Y. Liu, A. Rusinol, Y. Wang, Y. Zou

Faculty Publications, Biological Sciences

The genetic diseases Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) arise from accumulation of farnesylated prelamin A because of defects in the lamin A maturation pathway. Both of these diseases exhibit symptoms that can be viewed as accelerated aging. The mechanism by which accumulation of farnesylated prelamin A leads to these accelerated aging phenotypes is not understood. Here we present evidence that in HGPS and RD fibroblasts, DNA damage checkpoints are persistently activated because of the compromise in genomic integrity. Inactivation of checkpoint kinases Ataxia-telangiectasia-mutated (ATM) and ATR (ATM- and Rad3-related) in these patient cells can partially overcome their …

Farnesylated Lamins, Progeroid Syndromes And Farnesyl Transferase Inhibitors, Michael Sinensky, A. E. Rusinol

Faculty Publications, Biological Sciences

Three mammalian nuclear lamin proteins, lamin B1, lamin B2 and the lamin A precursor, prelamin A, undergo canonical farnesylation and processing at CAAX motifs. In the case of prelamin A, there is an additional farnesylation-dependent endoproteolysis, which is defective in two congenital diseases: Hutchinson-Gilford progeria (HGPS) and restrictive dermopathy (RD). These two diseases arise respectively from defects in the prelamin A substrate and the enzyme (ZmpSte24) that processes it. Recent work has shed light on the roles of the lamin proteins and the enzymes involved in their farnesylation-dependent maturation. Other experimental work, including mouse model studies, have examined the possibility …

Reduced Macrophage Apoptosis Is Associated With Accelerated Atherosclerosis In Low-Denstiy Lipoprotein Receptor-Null Mice, Michael Sinensky, J. Liu, D. P. Thweke, Y. R. Su, M. F. Linton, S. Fazio

Faculty Publications, Biological Sciences

Objective— The majority of apoptotic cells in atherosclerotic lesions are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains unclear. Elevated expression of Bax, one of the pivotal proapoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol-treated mouse macrophages. In this study, we examined the effect of Bax deficiency in bone marrow-derived leukocytes on the development of atherosclerosis in low-density lipoprotein receptor-null (LDLR−/−) mice. Methods and Results— Fourteen 8-week-old male LDLR−/− mice were lethally irradiated and reconstituted with either wild-type (WT) …

Expression Of Prelamin A Confers Sensitivity Of Dna Biosynthesis To Lovastatin On F9 Teratocarcinoma Cells, Michael Sinensky, T. Mclain, K. Fantle

Faculty Publications, Biological Sciences

No abstract provided.

The Processing Pathway Of Prelamin A, Michael Sinensky, K. Fantle, M. Trujillo, T. Mclain, A. Kupfer, M. Dalton

Faculty Publications, Biological Sciences

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subsequent to prenylation observed in other CAAX box proteins-the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine. To …

Isoprenylation Is Required For The Processing Of The Lamin A Precursor, Michael Sinensky, L. A. Beck, T. J. Hosick

Faculty Publications, Biological Sciences

The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. …

Incorporation Of A Product Of Mevalonic Acid Metabolism Into Proteins Of Chinese Hamster Ovary Nuclei, Michael Sinensky, L. A. Beck, T. Hosick

Faculty Publications, Biological Sciences

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. …

Adaptative Alteration In Phospholipid Composition Of Plasma Membranes From A Somatic Cell Mutant Defective In The Regulation Of Cholesterol Biosynthesis, Michael Sinensky

Faculty Publications, Biological Sciences

A somatic cell mutant (CR1) of a Chinese hamster ovary cell (CHO-K1) which has previously been shown to be defective in the regulation of cholesterol biosynthesis accumulates more cholesterol than the parental cell line in plasma membranes. Although such an increase in membrane cholesterol should lead to an increase in the order parameter of these membranes, as measured with an electron spin resonance spin probe, the order parameters of mutant and wild-type plasma membranes are identical--apparently because of an adaptive alteration in membrane phospholipid composition. The phospholipid compositions of mutant and wild-type cell plasma membranes are compared and the mutant …