Chemistry & Biochemistry Doctoral Work

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Apoptosis-Inducing Factor Mediates Metabolism and Oxidative Stress Signaling through Functionally Uncoupled Mechanisms to the Benefit of Tumorigenesis
(North Dakota State University, 2019) Scott, Andrew
Cancer is one of the leading causes of death in the world, arising when cells accumulate genomic mutations that render them incapable of controlling their own proliferation and survival. While substantial improvements in patient outcome have been achieved over the years, completely eliminating tumorigenic cells remains a significant challenge, and some cancer types (such as pancreatic cancer) have not shown meaningfully improved patient survival rates despite our advancements in understanding cancer cell biology and treatment. Genetic targeting approaches to the treatment of cancer have yielded some success, but have shown rather disappointing results overall. In contrast to the diversity of genetic aberrations within tumor cells (making treatment difficult), cancers share common phenotypes that are more universally targetable. As cancers evolve within their biological environment to become aggressive, the net effects of their genetic alterations lead to 1) decreased sensitivity to cell death cues, 2) altered oxidative state and redox-dependent signaling, and 3) dramatically increased demand for energy production and biosynthesis (altered metabolism). The work of this study shows that in pancreatic cancer cells reversible metabolic adaptation alters oxidative stress sensitivity and triggers persistent drug resistance, demonstrating the interplay of the above three cellular activities. Further experimentation identifies the mitochondrial protein apoptosis-inducing factor (AIF) as an essential signaling node functioning at the convergence of all three of these processes. In response to cellular metabolic cues, AIF controls the ability of the mitochondria to produce energy required for tumorigenic growth, invasion, and survival while functioning as mitochondrial translation factor. Distinct from this pivotal metabolic role, AIF serves as a transmitter of extra-mitochondrial redox signaling cues regulating pro-tumor gene expression programs that are lethal without AIF. Finally, AIF exhibits involvement in atypical death pathways associated with the cell death regulators PGAM5 and XIAP. Remarkably, these AIF activities are functionally dissociable and demonstrate the multifunctional nature of AIF in control of tumorigenesis. Overall this study defines a variety of diverse AIF activities at the intersection of multiple tumorigenic phenotypes, furthering our understanding of how cancer cells converge upon different aggressive characteristics and revealing potential vulnerabilities for therapeutic intervention.
Biochemical and Epidemiological Analysis of Mycobacterium Avium Subspecies Paratuberculosis and Investigation of its Relationship to Crohn's Disease in Humans
(North Dakota State University, 2011) Uzoigwe, Jacinta Chinwe
Background: Crohn's disease is a chronic inflammatory disease of the intestine in humans, with no known cause. Johne's disease is a chronic intestinal disease of ruminants caused by Mycobacterium avium suhspecies paratuberculosis (MAP), and has some features similar to Crohn's disease. Although MAP has been purported to play an etiologic role in Crohn's disease, this causal link is still under debate. Objective: The overall aim of this project is to analyze MAP strains from different hosts (cattle, sheep and humans) and regions in North Dakota by biochemical and epidemiological methods, in order to better understand the pathogenesis and epidemiology of MAP strains and the relationship between MAP and Crohn's disease. The specific aims of this research are the following: Aim 1. Investigate the epidemiological evidence for MAP as a cause of Crohn's disease. Aim 2. Conduct a comparative causality study to investigate whether MAP or other enteric pathogens cause Crohn's disease. Aim 3. Evaluate the occurrence of MAP infections in cattle in North Dakota, 1995-2005. Aim 4. Analyze MAP strains from symptomatic and asymptomatic cattle. Aim 5. Investigate the biochemical variations of rapid and slow growing MAP strains. Aim 6. Evaluate MAP strains from low shedders and high shedders. Methods: MAP isolates were analyzed by biochemical methods of gas chromatography, high performance liquid chromatography and mass spectrometry. In addition, extensive literature review was performed to (1) determine the epidemiologic causal link between MAP and Crohn' s disease and (2) determine whether MAP or other enteric pathogens cause Crohn's disease. Results: Results from our study indicated the availablity of epidemiologic evidence supporting the causal role of MAP in Crohn's disease. It was also demonstrated that MAP is the most implicated organism in the etiology of Crohn's disease when compared to other infectious agents. Investigation of the occurrence of MAP infection in North Dakota showed an increase in number of MAP cases reported, with seasonal trends. Biochemical typing of MAP strains from symptomatic and asymptomatic cattle indicated that the symptoms status of isolates was significantly associated with mass spectra patterns and shedder status (p < 0.05). However, the association between symptoms status and HPLC and GC patterns was not significant (p > 0.05). Investigation of biochemical variations of rapid and slow growing MAP strains showed associations between the biochemical variability of MAP strains and their growth rate and presence of symptoms in the source cattle. Evaluation of MAP strains of different shedding characteristics by univariate logistic regression revealed that the shedder status of isolates was significantly associated with growth rate of isolates, symptom status, and source regions, but not with mass spectra patterns of isolates. Conclusion: Overall, this study strengthens the theories of strain sharing, intraspecies and interspecies transmission, and supports an association between MAP and Crohn's disease. In addition, the understanding of the biochemical variation among MAP isolates will help in the future design of diagnostics, therapeutics and vaccines for Johne's and Crohn's diseases.
Biochemical and Functional Characterization of Rpa2 N-Terminal Phosphorylation during DNA Repair and Checkpoint Adaptation in Saccharomyces Cerevisiae
(North Dakota State University, 2018) Wilson, Timothy Michael
In response to DNA damage, a signaling cascade is activated within cells that promotes cell cycle arrest in order to provide adequate time for DNA repair to occur. Replication Factor A (RFA) is an essential, heterotrimeric complex that acts as a primary sensor of DNA damage, localizes to sites of and binds to broken DNA, and serves as a platform for several DNA damage repair proteins. RFA-coated ssDNA leads to activation of the sensor kinase Mec1. Activation of Mec1 leads directly to phosphorylation of the checkpoint regulator Rad53, which in turn activates several downstream effectors that halt cell growth and promote DNA repair. In response to a permanent, irreparable DNA lesion yeast cells undergo Rad53-dependent checkpoint arrest for approximately 12 hours. After this prolonged arrest, cell cycle restart occurs sometime between 12-15 hours, promoting mitosis despite the presence of an unrepaired lesion. This aberrant checkpoint exit is known as checkpoint adaptation. Checkpoint adaptation deficiency, or the inability to override the established Rad53-dependent checkpoint, can be conferred through deletion of non-essential but important genes involved in DNA damage repair (e.g., ku70, rdh54). The molecular mechanism(s) by which checkpoint adaptation occurs remains poorly understood; however, a single point mutation (K45E) in the Rfa1 subunit of the RFA complex is capable of rescuing adaptation deficiencies. During the DNA damage response, the RFA complex is known to be post-translationally modified through phosphorylation, ubiquitination, and sumoylation. Despite robust characterization of these modifications to RFA, the physiological role of phosphorylation within the N-terminal (NT) domain of the second subunit of the RFA complex, Rfa2, remains elusive. My studies using phospho-mimetic Rfa2 mutants demonstrates that phosphorylation of the NT is: 1) dispensable for activation of the Rad53-dependent checkpoint, 2) is readily detected during the temporal window within which checkpoint adaptation occurs and 3) promotes checkpoint adaptation in all adaptation-deficient mutations tested. Furthermore, analysis of Rfa2 phospho-mutants in sensor kinase deletions mec1 and tel1 reveal phosphorylation of the Rfa2 NT may directly impede Mec1 activity, thereby inhibiting maintenance of the Rad53-dependent checkpoint and providing a mechanism by which cells can escape an established DNA damage-dependent checkpoint.
Characterization of RPA2 N-terminal Function in the DNA Damage Response in Saccharomyces Cerevisiae
(North Dakota State University, 2015) Ghospurkar, Padmaja Laxman
In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. A key factor involved in the DNA damage response is the heterotrimeric protein complex Replication Protein A (RPA), which is not only essential for the repair of damaged DNA, but also is post-translationally modified on at least two of the three subunits in response to DNA damage by checkpoint kinases. Of particular interest is the 32-kDa subunit, called Rpa2, which is hyper-phosphorylated on its serine/threonine-rich N-terminus following DNA damage in human cells. This unstructured N-terminus is often referred to as the phosphorylation domain (PD) and is conserved amongst eukaryotic Rpa2 subunits, including Rfa2 in Saccharomyces cerevisiae. In this work we aim to characterize the function of Rfa2 N-terminus (Rfa2 NT) in DNA damage response and develop yeast as a tool to study human RPA. With the help of mutagenesis we developed Rfa2 NT extreme mutants, which showed that the phosphorylation of Rfa2 NT is dispensable in DNA damage response. However, the presence of Rfa2 NT is essential for cells to survive in stressed condition indicating an uncharacterized function. We further discovered seven S/T sites are responsible for the damage sensitive phenotype of Rfa2 NT extreme mutants. And the phosphorylation affects protein interaction of RFA complex. Although, the phosphorylation event of Rfa2 NT is dispensable in S. cerevisiae the cells have conserved the ability to phosphorylate Rfa2 N terminus. With the help Rfa2 NT fusion mutants we showed that S. cerevisiae could phosphorylate N terminus from seven different eukaryotic species. Hence, we successfully developed yeast as a tool to study Rpa2 phosphorylation amongst various eukaryotic species.
Delineating Signaling Mechanisms Involved in Lymphocyte Chemotaxis, Immune Homeostasis and Allergic Asthma
(North Dakota State University, 2015) Wanjara, Steve Biko
The vasoactive intestinal peptide (VIP) signaling axis constitutes VIP and its two G protein coupled receptors (GPCR) termed vasoactive intestinal/pituitary adenylate cyclase activating polypeptide (VPAC) 1 and 2. This signaling axis regulates numerous biological actions within the endocrine system, the nervous system and the immune system. Working as a gut hormone, VIP can increase cAMP signaling within beta-islet cells of the pancreas to impact insulin production. As a neurotransmitter, it acts as a master circadian regulator controlling light and dark cycling. Lastly, VIP regulates immune processes such as activation, chemotaxis, development and cytokine secretion. The focus of my doctoral research was to delineate VIP signaling mechanisms controlling immunity. We aimed at understanding: 1.) the molecular mechanism of VIP-induced T cell trafficking 2.) ability for VPAC2 signaling to regulate immune homeostasis and 3.) a phenotype of a B cell subset during asthma, an immune pathology devoid of VIP protein due to excessive protease activity. Methods employed utilized isolated primary mouse immune cells to measure a VIP-induced signaling pathway centered on the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, by qPCR and chemotaxis assays. Flow cytometry to enumerate immune cell numbers in VPAC2 deficient mice was done to accomplish aim 2. Lastly, using a published in vivo allergic asthma mouse model, we used qPCR, immunoblotting and flow cytometry analyses to measure expression of Hyaluronic acid binding proteins on B cells. Results from these studies revealed that VIP signaling in T cells is regulated by EGFR as inhibitors against its enzymatic activity abolished T cell movement towards VIP. Immune cell numbers were lowered as a consequence of VPAC2 deficiency, suggesting its involvement in homeostasis. Lastly, a unique B cell population homing to asthmatic lung secretes an anti-inflammatory mediator, TGF-beta, through the HA binding protein called RHAMM. Collectively, these data emphasize the importance of VIP signaling in the immune system controlling cell migration and homeostasis.
Investigating Selected Mechanisms of Modulation of BECN1-mediated Autophagy
(North Dakota State University, 2019) Li, Yue
Autophagy is a lysosomal degradation pathway wherein cytoplasmic components not needed by or harmful to the cell are degraded and recycled. BECN homologs are key autophagy proteins consisting of an intrinsically disordered region (IDR), flexible helical domain (FHD), coiled-coil domain (CCD) and β-α repeated, autophagy-specific domain (BARAD). Diverse proteins modulate autophagy by binding BECN1. Understanding the mechanisms by which these proteins regulate BECN1-mediated autophagy is important for developing therapeutics targeting these proteins. Toward this goal, we have developed purification protocols for multi-domain BECN1 fragments to explore the conformational flexibility and interactions. We show that a BECN1 helix transitions between mutually exclusive packing states, wherein it either forms part of the CCD homodimer or packs against the BARAD, but predominantly packs against the BARAD. The same set of residues on this helix contribute to the CCD homodimer or packing with the BARAD, and mutation of these residues abrogates starvation-induced up-regulation of autophagy. Next, we show the equatorial groove of GAPR-1 may be responsible for binding BECN1. The five conserved residues lining the GAPR-1 equatorial groove are essential for the interaction, as mutation of these residues disrupts GAPR-1:BECN1 interaction. We also solved the structure of this pentad mutant, which indicates the changes in the equatorial groove and the improved dimerization of pentad mutant likely abrogates BECN1-binding. We then show that BH3D is not required for BECN1 to up-regulate autophagy, though it is required for binding BCL2 homologs. Therefore, we investigated the interactions between BH3D-containing BECN1 fragments and the BCL2 homolog, M11. BECN1 regions outside the BH3D increase binding to M11 by 5-10 fold. In addition, M11-binding increases flexibility of the nuclear export sequence (NES). Further, homodimerization and thermostability of BECN1 BH3D-FHD-CCD increases upon M11-binding. Lastly, the M11:BH3D-FHD-CCD complex appears to fluctuate between two major types of conformations, which may be mediated by the increased flexibility of BECN1 NES upon binding M11. Lastly, we investigated the interactions between BH3D-containing BECN1 fragments and Bcl-XL. Our results indicate that BECN1 regions outside the BH3D do not affect BECN1 interaction with Bcl-XL. Together, these studies are important for better understanding how proteins down-regulate BECN1-mediate autophagy.
Investigating the Role of BECN1 Conformationally Flexible Regions and Invariant Cys-x-x-Cys Motifs in Autophagy
(North Dakota State University, 2020) Mukhopadhyay, Shreya
Autophagy is an essential catabolic cellular homeostasis process conserved in all eukaryotes. BECN1, a key autophagy protein involved in autophagosome nucleation, comprises of a large, poorly conserved, N-terminal intrinsically disordered region (IDR); a flexible helical domain; a coiled-coil domain (CCD) that forms anti-parallel homodimers in the absence of other interacting partners; and a β-α repeated autophagy specific domain (BARAD). The IDR of higher eukaryotes includes a BCL2 homology 3 domain (BH3D), which undergoes dramatic disorder-to-helix transitions upon binding to anti-apoptotic and anti-autophagic BCL2s. We show that the BH3D is not required for starvation-induced autophagy upregulation suggesting that BCL2-binding to the BH3D does not directly impede a pro-autophagy function of the BH3D, rather it may impact structure, oligomerization, interactions and function of other BECN1 domains. CCD C-terminal residues, named the overlap helix (OH), pack in two mutually-exclusive states stabilized by the same interface residues: against either the partner helix in a CCD dimer or the BARAD. We show that mutation of these interface residues abrogates starvation-induced autophagy upregulation. Together with our complementary structural studies, this suggests that autophagy-inactive BECN1 adopts conformations preventing the BARAD from membrane-association, with BECN1 heterodimerization with ATG14 or UVRAG disrupting this inhibitory conformation. In the BECN1 homodimer, the OH packs against a nuclear export signal sequence (NES) at the N-terminus of the partner CCD. We show that when released from this interaction, the NES can interact with the complex of the nuclear exporter, Chromosomal Region Maintenance 1 protein and a GTP-bound small G-protein, Ran. This interaction is essential for BECN1 export to the cytoplasm, and for autophagy. Two invariant CxxC motifs bookend the IDR. We find that both CxxC motifs are required, but the intervening IDR is less important, for starvation-triggered upregulation of autophagy. We demonstrate that BECN1 binds Zn2+ in a 1:1 molar ratio. Further, mutation of the invariant cysteines or treatment with reducing agent abrogates Zn2+ co-ordination, demonstrating that the invariant CxxC motifs are responsible for binding Zn2+. We use diverse biophysical methods to show that Zn2+-binding impacts the conformation and structural transitions of the BECN1 IDR, thereby playing an important role in regulating autophagy.
Ligand Binding and Catalysis in Selected Sirtuin Isozymes
(North Dakota State University, 2016) Yu, Junru
Due to their intimate roles in survival, longevity as well as pathogenesis via “epigenetic” and “metabolic” regulatory mechanisms, sirtuins have gained considerable interest toward undertaking detailed biochemical/biophysical studies. The present study was designed to ascertain the mechanistic details of ligand binding and catalysis in selected sirtuin isozymes (viz., SIRT1 and SIRT5) from the point of view of designing isozyme selective inhibitors as potential therapeutics. By screening of the in-house synthesized compounds, two barbiturate derivatives were identified as the SIRT5 selective inhibitors. These, along with some of known inhibitors of SIRT1 and SIRT5, namely, MH5-75, nicotinamide, suramin were investigated by a combination of spectroscopic, kinetic, and thermodynamic techniques. The influence of the sirtuin inhibitors in modulating the structural features of the enzymes were ascertained by CD spectroscopic, lifetime fluorescence, and thermal denaturation studies using wild-type and selected site-specific mutant enzymes. The experimental data revealed that the substrate selectivity and inhibitory features in SIRT5 were manifested via the mutual cooperation between Y102 and R105 residues of the enzyme, and the overall catalytic feature of the enzyme was modulated by changes in the protein structure. Whereas the stoichiometry of SIRT1 to suramin remained invariant as 1:1, that of SIRT5 to suramin increased from 1:1 to 2:1 upon increase in the molar ratio of the enzyme to the ligand. A comparative account of the experimental data presented herein sheds light on the structural-functional differences between SIRT1 and SIRT5, leading to the design of isozyme selective inhibitors as therapeutic tools for the treatment of sirtuin associated diseases.
Structural Basis for the Regulation of a Conserved TonB-dependent Iron Transport System via Cell-surface Signaling in Gram-negative Bacteria
(North Dakota State University, 2017) Jensen, Jaime Lea
Cell-surface signaling (CSS) pathways are highly conserved systems in Gram-negative bacteria that allow the cell to efficiently respond to environmental stimuli through transcriptional regulation. Three distinct proteins are involved in this process: an outer membrane (OM) protein that senses the extracellular signal, an inner membrane (IM) sigma regulator protein that transmits the signal from the OM protein to the cytoplasm, and an extracytoplasmic function (ECF) sigma factor that initiates transcription of stimulus response genes. One such CSS pathway regulates bacterial iron acquisition- an essential process for bacterial survival and pathogenesis. Under iron-limited conditions, expression of the OM transporter is upregulated by signal transduction through the IM protein to the sigma factor. The goal of this work is to provide a structural rationale for distinctive signal transduction through the CSS pathway that regulates ferric siderophore uptake in Gram-negative bacteria, by structurally characterizing these proteins from Pseudomonas capeferrum, with a focus on the IM protein, PupR. The solution structures of an OM transporter, PupA, an OM transducer, PupB, a PupAPupB-NTSD chimera, and the OM proteins with the PupR C-terminal cell-surface signaling domain (CCSSD) were probed by SEC-SAXS to examine global architectural differences amongst the OM proteins. The X-ray crystal structure of the PupB N-terminal signaling domain (NTSD):PupR-CCSSD complex was determined. The PupB-NTSD exhibits a conserved βαβ-repeat motif. Unexpectedly, the CCSSD subdomain contains the same fold, which is the first time this fold had been identified at a protein’s C-terminus. The other subdomain of the CCSSD, designated the C-terminal juxtamembrane (CJM) subdomain, has a novel, β-solenoid-like motif. Analysis of the CCSSD by CD spectroscopy and SEC-SAXS indicated that the domain is highly flexible, and is significantly stabilized by the PupB-NTSD. Concurrently, the PupB-NTSD structure was determined by NMR, and contrasted with published NTSDs to evaluate structural variation that may account for disparities in functionality. The PupR N-terminal anti-sigma domain (ASD) was solved by X-ray crystallography and presents as a dimer in solution- the first description of a transmembrane ASD to assume an oligomeric form. Structural characterization of these proteins suggests novel implications for CSS through the TonB-dependent ferric siderophore uptake pathway.

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Browsing Chemistry & Biochemistry Doctoral Work by browse.metadata.program "Biochemistry"