Biochemistry Commons^™

Structural Basis For Intrinsic Strand Displacement Activity Of Mitochondrial Dna Polymerase, Ashok Nayak, Viktoriia Sokolova, Sirelin Sillamaa, Karl Herbine, Juhan Sedman, Dmitry Temiakov

Department of Biochemistry and Molecular Biology Faculty Papers

Members of the Pol A family of DNA polymerases, found across all domains of life, utilize various strategies for DNA strand separation during replication. In higher eukaryotes, mitochondrial DNA polymerase γ relies on the replicative helicase TWINKLE, whereas the yeast ortholog, Mip1, can unwind DNA independently. Using Mip1 as a model, we present a series of high-resolution cryo-EM structures that capture the process of DNA strand displacement. Our data reveal previously unidentified structural elements that facilitate the unwinding of the downstream DNA duplex. Yeast cells harboring Mip1 variants defective in strand displacement exhibit impaired oxidative phosphorylation and loss of mtDNA, …

Artesunate Enhances The Efficacy Of Enzalutamide In Advanced Prostate Cancer, Xinyi Wang, Jinghui Liu, Fengyi Mao, Yifan Kong, Qiongsi Zhang, Chaohao Li, Daheng He, Chi Wang, Yanquan Zhang, Ruixin Wang, Sally R. Ellingson, Qiou Wei, Zhiguo Li, Xiaoqi Liu

Markey Cancer Center Faculty Publications

Prostate cancer (PCa) is one of the leading causes of death among men worldwide. Treatments targeting the androgen receptor pathway remain the standard therapy for PCa patients. Enzalutamide (ENZ), a second-generation androgen receptor inhibitor, was developed to treat castration-resistant prostate cancer. However, while patients initially respond to ENZ, drug resistance typically develops within a few months. Artesunate (ART), a semisynthetic derivative of the Artemisinin plant, is approved for antimalaria treatment. In this study, we conducted an FDA-approved drug screening and identified ART as a potential candidate for overcoming ENZ resistance in PCa. Mechanistically, ART induces the degradation of c-Myc, enhancing …

Overexpression And Biophysical And Functional Characterization Of A Recombinant Fgf21, Phuc Phan, Jason Hoang, Thallapuranam Krishnaswamy Suresh Kumar

Chemistry & Biochemistry Faculty Publications and Presentations

Fibroblast growth factor 21 (FGF21) is an endocrine FGF that plays a vital role in regulating essential metabolic pathways. FGF21 increases glucose uptake by cells, promotes fatty acid oxidation, reduces blood glucose levels, and alleviates metabolic diseases. However, detailed studies on its stability and biophysical characteristics have not been reported. Herein, we present the overexpression, biophysical characterization, and metabolic activity of a soluble recombinant FGF21 (rFGF21). The far-UV circular dichroism spectra of rFGF21 show a negative trough at 215 nm, indicating that the protein’s backbone predominantly adopts a β sheet conformation. rFGF21 shows intrinsic tyrosine fluorescence at 305 nm. Thermal …

The Role Of Secondary And Tertiary Structure In The Cap-Independent Translation Of Fgf-9 And Hif-1-Alpha, Amanda Michelle Whittaker

Dissertations, Theses, and Capstone Projects

Under normoxic conditions, eukaryotes initiate translation of RNA through eIF4E recognition of the 5’ cap. However, under cellular stress, eukaryotic translation must be initiated through a 4E-independent, or “cap-independent” mechanism, involving eukaryotic initiation factor 4G (eIF4G) binding directly to the 5’ untranslated regions (5’ UTR) of the RNA. eIF4G binding then recruits the ribosome to the transcript. While this mechanism is useful for translation of apoptotic transcripts and transcripts involved in cell survival, cap-independent translation is also utilized by oncogenic RNA for tumorigenesis. Previous work by our lab and others has categorized this recruitment and initiation mechanism as either internal-ribosome-entry-site …

Staying Sane In The Membrane: Neutral Sphingomyelinase 2 As A Master Regulator Of Plasma Membrane Ceramide, Zainuddin Quadri, Erhard Bieberich

Markey Cancer Center Faculty Publications

Ceramide, a key signaling sphingolipid in the plasma membrane, plays a pivotal role in fundamental cellular processes such as adhesion, polarity, and programmed cell death. The generation of plasma membrane ceramide is largely attributed to the activity of two types of sphingomyelinases: neutral sphingomyelinase 2 (nSMase2, Smpd3) and acid sphingomyelinase (aSMase, Smpd1). While many studies have explored ceramide generation following experimental activation of these enzymes, the mechanisms governing basal or steady- state ceramide levels have remained poorly understood. Using an innovative mass spectrometry approach developed in the Canals’ lab, the team has quantified the distinct contributions of nSMase2 and aSMase …

Anti-Cancer Compounds Based On Natural Imidazole Compounds, Autumn Forsythe, Victoria Hardy, Kerri L. Shelton

CSU Tower Day

We are developing a comprehensive review on imidazole-based compounds of natural origin, the most common groups of compounds, and their properties, such as the imidazole ring. The intent of this presentation is to learn about the compound’s occurrence, general/predicted properties, and advantages. The review will be a fluid development comparing imidazole derivatives of natural origin with antinociceptive/lethal properties from the last 5 years. This review would include both purely extracted products but also those modified by synthesis.

Characterizing And Targeting The Non-Catalytic Functions Of Phosphatase Of Regenerating Liver 3 (Prl-3) In Oncogenesis And Cancer Progression, Jeffery T. Jolly

Theses and Dissertations--Molecular and Cellular Biochemistry

Phosphatase of Regenerating Liver 3 (PRL-3) is frequently upregulated in various cancers and is associated with poor patient prognosis. Although traditionally studied for its phosphatase activity, PRL-3 also interacts with the CNNM family of magnesium transporters through its catalytic site, and these two functions are mutually exclusive at any given time. Most previous studies relied on a commonly used PRL-3 mutation that disrupts both phosphatase activity and CNNM binding, making it challenging to determine which function drives its oncogenic effects. To address this gap in the field, I utilized a panel of PRL-3 mutants that selectively disrupt either phosphatase activity …

Characterization Of The Erv41-Erv46 Complex As A Retrograde Receptor For Misfolded Secretory Proteins, John Andrew Fuesler

Dartmouth College Ph.D Dissertations

The Endoplasmic Reticulum (ER), the site of secretory protein biosynthesis

provides a favorable environment to promote polypeptide folding, protein

oligomerization and export. Although the ER contains many chaperones and other

factors that assist in protein folding, this process is error prone. Eukaryotes have

evolved protein quality control checkpoints to maintain homeostasis in the secretory

pathway, however these processes are imperfect and may result in proteotoxicity

when toxic levels or aggregates of proteins arise. Mutations in proteins involved in

trafficking in the early secretory pathway are closely associated with disease states.

Therefore, it is of great interest to further understand how …

Bridging The Gap Between Structural And Cellular Biology: Insight Into The Organization Of Glycolytic Enzymes In Cells, Heather Hansen

Graduate Theses, Dissertations, and Problem Reports (ETD)

Glycolytic enzymes are spatially organized in the cytoplasm, and are shown to localize with cellular structures such as the plasma membrane, F-actin, mitochondria, or associate independently within the cytosol. The assembly of these enzymes is hypothesized to regulate pathway flux to meet the energetic needs of the cell. However, it is unclear whether all of these assemblies have the same mechanism of formation or function in the cell. We investigated the role of glycolytic enzyme assemblies in cells, using the glycolytic “gatekeeper” enzyme phosphofructokinase-1 (PFK1). PFK1 is extensively regulated by multiple mechanisms, including over 20 allosteric ligands, post translational modifications, …

Exploring The Effects Of Road Salt On Earthworm Species, Alea Mry Koston

Williams Honors College, Honors Research Projects

This study examines the effects of two road salts, sodium chloride (NaCl) and calcium magnesium acetate (CMA), on the health of two earthworm species: Eisenia fetida and Lumbricus terrestris. Over a 4-week period, approximately 168 earthworms were exposed to varying concentrations of either NaCl, CMA, or mixed ratios of both, with a control group included for comparison. The experiment involved monitoring the changes in earthworm weight and measuring their contraction rates to determine physiological responses. Statistical analysis revealed no significant differences in weight or activity between NaCl and CMA treatments for either species. However, higher concentrations of NaCl resulted in …

Unleashing Serine Palmitoyltransferase In Test Tubes And Mice, Matthew W. Peart

Theses and Dissertations

Canonically known both for structural contributions to lipid bilayers and roles in cell signaling, the sphingolipids comprise a dynamic, multifaceted class of molecules which are studied to understand homeostatic cell biology as well as pathophysiology. Sphingolipids contribute to tissue-specific structures such as myelin, a multilayer membrane that allows for rapid conduction of action potentials along neuronal axons. All sphingolipids are downstream products of the rate-limiting and initiating enzyme in the de novo sphingolipid synthesis pathway, Serine palmitoyltransferase (SPT). Demonstrative of its status as the enzyme that catalyzes a crucial step in a biochemical pathway, SPT activity is strictly regulated. This …

Reversible Degradation Of Peptidoglycan By Lytic Transglycosylases, Aaron Devereaux

Theses and Dissertations (Comprehensive)

Antimicrobial resistance continues to be a burden on the global healthcare system with an estimated cost of billions of dollars and millions of deaths each year. Recently, there has been little to no development on new antibiotics to treat bacterial infection, and any that are developed become resisted to within a few years. Both Gram-positive and Gram-negative bacteria contain a mesh-like layer called peptidoglycan (PG) that surrounds their cells providing strength, cell shape, and protection from their environments. This layer is a polymer of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) connected via a b-1,4-glycosidic bond, with each strand being cross-linked …

P85Α Degradation Mediated By Ubiquitin E3 Ligase Fbxo21 Offers Therapeutic Potential In Leukemia, Kasidy K. Weber

Theses & Dissertations

Acute myeloid leukemia (AML) is a complex and heterogeneous disease characterized by the clonal expansion of myeloid blasts in the bone marrow. Despite significant therapeutic advances over the years, the prognosis for AML patients remains dismal, with high relapse rates and poor overall survival. The ubiquitin-proteasome system (UPS) plays a critical role in maintaining cellular homeostasis by regulating the degradation of proteins involved in essential processes such as cell cycle control, DNA repair, apoptosis, and various signaling pathways. Given this vital function, targeting ubiquitin E3 ligases within the UPS presents a promising strategy for developing more effective and targeted therapies …

Apoa2 Increases Cholesterol Efflux Capacity To Plasma Hdl By Displacing The C-Terminus Of Resident Apoa1, Snigdha Sarkar, Jamie Morris, Youngki You, Hannah Sexmith, Scott E. Street, Stephanie M. Thibert, Isaac K. Attah, Chelsea M. Hutchinson Bunch, Irina V. Novikova, James E. Evans, Amy S. Shah, Scott M. Gordon, Jere P. Segrest, Karin E. Bornfeldt, Tomas Vaisar, Jay W. Heinecke, W. Sean Davidson, John T. Melchior

Saha Cardiovascular Research Center Faculty Publications

Abstract The ability of high-density lipoprotein (HDL) to promote cellular cholesterol efflux is a more robust predictor of cardiovascular disease protection than HDL-cholesterol levels in plasma. Previously, we found that lipidated HDL containing both apolipoprotein A-I (APOA1) and A-II (APOA2) promotes cholesterol efflux via the ATP-binding cassette transporter (ABCA1). In the current study, we directly added purified, lipid-free APOA2 to human plasma and found a dose-dependent increase in whole plasma cholesterol efflux capacity. APOA2 likewise increased the cholesterol efflux capacity of isolated HDL with the maximum effect occurring when equal masses of APOA1 and APOA2 coexisted on the particles. Follow-up …

Untargeted Lipidomics Reveals Novel Hdl Metabotypes And Lipid-Clinical Correlates, Peer W. F. Karmaus, Scott M. Gordon, Marcus Y. Chen, Alison A. Motsinger-Reif, Rodney W. Snyder, Timothy R. Fennell, Suramya Waidyanatha, Reshan A. Fernando, Alan T. Remaley, Michael B. Fessler

Saha Cardiovascular Research Center Faculty Publications

Plasma high-density lipoprotein (HDL), originally studied for its role in lipid transport, is now appreciated to have wide-ranging biological functions that become defective during disease. While >200 lipids have collectively been detected in HDL, published HDL lipidomic analyses in different diseases have commonly been targeted to prespecified subsets of lipids. Here, we report the results of untargeted lipidomic analysis of HDL isolated from 101 subjects referred for computed tomographic coronary imaging for whom multiple additional clinical and lipoprotein metadata were measured. Unsupervised clustering of the total HDL lipidome revealed that the subjects fell into one of two discrete groups, herein …

Dysfunction Of Polyamine Metabolism In Down Syndrome Related To Cellular Senescence And Β-Amyloid Production, Andres D. Sola

Electronic Theses and Dissertations

Polyamines (PA) are ubiquitous in cells and highly involved in several important cellular functions and thus the dysregulation of their biosynthetic pathway is potentially associated with the progression of age-related diseases like Alzheimer’s’ disease (AD). Though there is research describing how PA content changes in organisms as they age or in those experiencing rapid aging from increased rates of senescence, as in Down syndrome (DS), it is not clear how this connection occurs and whether modulating this pathway may reveal downstream effects on related disease progression. This study examines how the modulation of the PA biosynthetic pathway by inhibition of …

Hepatic Targets To Control Blood Glucose And Potentially Aid In The Treatment Of Diabetes, Rena A. Velissarios

PANDION: The Osprey Journal of Research and Ideas

Maintaining glucose homeostasis is extremely important to remain in good health and prevent disease. This homeostasis is achieved via the regulation of hepatic metabolic pathways like gluconeogenesis and glycogenesis by insulin, which is expressed during high blood glucose. Insulin is responsible for facilitating the uptake of glucose into other tissues from the blood, as well as signaling whether to stimulate or slow glucose metabolism in the liver. Hyperglycemia is one of the main effects of diabetes, a disease characterized by the body’s inability to produce or respond to insulin. Without insulin, hepatic glucose output continues and blood glucose accumulates, causing …

Characterization Of Stealth Liposome-Based Nanoparticles Encapsulating Acat1/Soat1 Inhibitor F26: Potential Therapeutics For Niemann-Pick Type C And Other Neurodegenerative Diseases., Junghoon Lee

Dartmouth College Ph.D Dissertations

Cholesterol dysregulation is implicated in neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and Niemann-Pick type C disease (NPCD). This study focused on evaluating the potent ACAT1 inhibitor, F26, as a therapeutic strategy for NPCD. F26 demonstrated superior ACAT1 inhibition and efficacy compared to the previously studied inhibitor, F12511, both in vitro and in vivo. Notably, F26 exhibited enhanced pharmacokinetic properties, including prolonged duration of action and better brain retention, making it a more promising candidate for treating neurodegenerative diseases.

F26 was encapsulated in a liposome-based nanoparticle system, DSPE-PEG2000 with phosphatidylcholine (PC), for enhancing its …

Effect Of Heat Stress On The Expression Of Circulating Cyto(Chemo)Kine And Inflammatory Markers In Broiler Chickens Selected For High- Or Low-Water Efficiency, Elizabeth S. Greene, Travis Tabler, Walter G. Bottje, Sara Orlowski, Sami Dridi

Poultry Science Faculty Publications and Presentations

Background:

Water scarcity is a current, significant global concern that will only increase under the pressure of climate change. Improving water efficiency of poultry is a new and promising area to help temper agriculture's future impact on fresh water availability. Here, we explored the effects of acute heat stress (HS) on circulating stress and inflammatory markers in 2 lines of broilers divergently selected for water efficiency. Methods:

Male chicks from low (LWE) and high water efficient (HWE) lines were raised in 12 environmental chambers (2 pens/chamber, 6 chambers/line, 20 birds/pen) under normal conditions until day 28. On day 29, birds …

Unraveling The Role Of Hid-1: Insights Into Sgii Regulation, Breanna L. Sellers

Electronic Theses and Dissertations

The regulated secretory pathway is important for the secretion of hormones and neuropeptides from large dense core vesicles (LDCVs). Dysregulation of the biogenesis of LDCVs can result in metabolic and neurological diseases. Recent studies have suggested that the peripheral membrane protein HID-1 plays a role in the sorting and storage of granins which contribute to LDCV biogenesis. To further understand how HID-1 influences the regulation of granins, we investigated potential HID-1 interactors. We determined that while HID-1 is likely functioning in the same pathway as USP25, a deubiquitinating enzyme (DUB), the cause of reduced cellular SgII levels is likely not …

Assessment Of Tripeptides Self-Assembly & Er Stress On Cell Viability And Exosome Secretion In Mda-Mb231, Azmat Parveen

Theses and Dissertations

This study delves into the effects of tripeptides KYpF and WYpK(NBD) on MDA-MB-231 cells, uncovering KYpF's ability to reduce cell proliferation and exosome secretion in peptide treated cells. Conversely, WYpK(NBD) demonstrated significant toxicity at 10 μM. These insights pave the way for optimizing peptide-based cellular treatments for exosome secretion purposes.

Elucidating The Roles Of Septin Proteins In Thermotolerance And Cell Wall Integrity In Cryptococcus Neoformans, Stephani S. Martinez Barrera

All Dissertations

Cryptococcus neoformans is a globally distributed fungal pathogen responsible for causing cryptococcal meningitis in immunocompromised individuals. This pathogenic yeast must adapt to changes in temperature upon entering the human host. Septin proteins are conserved filament-forming GTPases that assemble as higher-order complexes at the cell cortex to support cytokinesis and morphogenesis in fungal and animal cells. In C. neoformans, four septin homologs (Cdc3, Cdc10, Cdc11, and Cdc12) assemble at the mother-bud neck, contributing to cytokinesis through poorly understood mechanisms. C. neoformans strains lacking the septins Cdc3 or Cdc12 are viable at 25°C, but fail to proliferate at 37°C, and are …

Functional And Structural Analysis Of The Neimann-Pick Disease Type C Pathway To Include Caveolin-1, Anthony Michael Seat

Chemistry and Chemical Biology ETDs

Human disease is often thought of as an all or nothing prospect, either one has the disease or one does not. This does not bear out in clinical or personal experiences, instead demonstrating that disease occurs within a spectrum ranging from presumed unaffected to demonstrably and detrimentally affected.Neimann-Pick disease is one example of this spectrum look into diseased states, with multiple named versions of a phenotypically similar disease. We focus on Neimann-Picktype C (NPC), which is the result of a disruption in the efflux of cholesterol and sphingolipids from the endocytic pathway. NPC demonstrates this concept of a spectrum of …

Role Of Sds-22/Pp1 Phosphatase In Polarizing The Germplasm Of Caenorhabditis Elegans Embryo, Aparna Nurni Ravi

Dartmouth College Ph.D Dissertations

During asymmetric cell division, cell polarity and cell cycle are tightly coupled to ensure robust segregation of cell fate determinants and generation of cellular diversity. In the one-cell C. elegans embryo, multiple cell cycle kinases regulate cell polarization and posterior segregation of germline fate determinants (germplasm). For example, PLK-1 kinase inhibits the retention of its germplasm substrate POS-1 in the anterior, driving POS-1 segregation to the posterior (Han et al., 2018). Additionally, MBK-2 kinase disassembles P granules in the anterior through phosphorylation of the P granule scaffold MEG-3, helping to drive P granule segregation to the posterior (Wang et al., …

Use Of Cobalt(Iii) And Copper(Ii) Complexes With Thiosemicarbazone Ligands And Berberine Chloride As Anti-Cancer Agents Against Triple Negative Breast Cancer, Duaa Rashad Alajroush

Chemistry & Biochemistry Theses & Dissertations

It is a worrying fact that chemotherapeutic options for treating triple negative breast cancer are limited in nature, including the use of inorganic pharmaceuticals such as cisplatin and its analogues. It has been reported that cisplatin has undesirable side effects when used as a chemotherapeutic drug. As such, there is a need to use non-platinum-containing complexes as chemotherapeutic agents. Based on that issue, it has been reported by our research group and others that cobalt- and copper-containing complexes have shown great potential in anti-cancer properties when compared to cisplatin. In this research project, the cytotoxic effects of the following complexes: …

Wee1 And Cell Size Control In Fission Yeast By The Protein Kinase Cdr2, Rachel Berg-Murante

Dartmouth College Ph.D Dissertations

The mechanisms that govern cell size have long been topics of study in the field of cell biology. In eukaryotic cells this size control is tied to checkpoints, a set threshold of minimum necessary growth linked to cyclin dependent kinase activity regulation. In the fission yeast Schizosaccharomyces pombe, the Cdk1 regulatory network is conserved, and G2/M represents the major size checkpoint. Prior to mitosis, Cdk1 is inhibited by phosphorylation applied by Wee1 during G2 phase. Once S. pombe cells have satisfied the size checkpoint, Cdk1 is activated through dephosphorylation by Cdc25. Wee1 is a dose-dependent regulator of mitotic entry …

Adipose-Derived Mesenchymal Stem Cells And Their Derived Epidermal Progenitor Cells Conditioned Media Ameliorate Skin Aging In Rats, Mohamed M. Kamal, Omar Ibrahim Badr, Tasnim Mahmoud

Pharmacy

BACKGROUND: Skin alterations are among the most prominent signs of aging, and they arise from both intrinsic and extrinsic factors that interact and mutually influence one another. The use of D-galactose as an aging model in animals has been widely employed in anti-aging research. Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) are particularly promising for skin anti-aging therapy due to their capacity for effective re-epithelization and secretion of various growth factors essential for skin regeneration. Accordingly, we aimed to examine the potential utility of Ad-MSCs as a therapy for skin anti-aging. METHODS: In this study, we isolated and characterized adipose-derived mesenchymal …

Characterization Of Oxidized Tau Protein Aggregation And Inhibition Of Tau Aggregation Via Map2, Brad Krzesinski

Electronic Theses and Dissertations

Neurofibrillary tangles (NFT) composed of the microtubule stabilizing protein Tau are a pathological hallmark of Alzheimer's disease (AD) and many other dementia related diseases specified as Tauopathies. Due to alternative mRNA splicing, intrinsically disordered Tau exists as six separate isoforms in the adult human brain where they are categorized by the lack or inclusion of the second repeat in the microtubule binding domain (3R or 4R respectively). Isoforms are subcategorized by the inclusion or lack thereof, one or two N-terminal inserts. Many studies show that Tau aggregates into amyloid fibrils and these fibrils can break into small oligomeric units known …

Quantitative Proteomic Strategies To Determine Substrate Specificities Of Phosphoprotein Phosphatases, Hieu Trung Nguyen

Dartmouth College Ph.D Dissertations

Reversible phosphorylation is a crucial regulatory mechanism of cellular signaling pathways. Being the most prevalent post-translational modification (PTM) in the cells, with over 75% of all proteins detected to be phosphorylated, phosphorylation regulates a significant number of important cellular processes that have implications in various diseases. Phosphorylation is carried out by protein kinases, which have been extensively studied. However, the opposite reaction, carried out by protein phosphatases, has lagged significantly, exposing a gap of knowledge that is required to be investigated to delineate the kinase-substrate-phosphatase relationship. Phosphoprotein phosphatase family (PPPs), containing seven members of phospho-Serine (pS) and phospho-Threonine (pT) phosphatases, …

Towards A New Role Of Mitochondrial Hydrogen Peroxide In Synaptic Function, Cliyahnelle Z. Alexander

Student Theses and Dissertations

Aerobic metabolism is known to generate damaging ROS, particularly hydrogen peroxide. Reactive oxygen species (ROS) are highly reactive molecules containing oxygen that have the potential to cause damage to cells and tissues in the body. ROS are highly reactive atoms or molecules that rapidly interact with other molecules within a cell. Intracellular accumulation can result in oxidative damage, dysfunction, and cell death. Due to the limitations of H₂O₂ (hydrogen peroxide) detectors, other impacts of ROS exposure may have been missed. HyPer7, a genetically encoded sensor, measures hydrogen peroxide emissions precisely and sensitively, even at sublethal levels, during …