IU Indianapolis ScholarWorks :: Browsing by Author "Mosley, Amber"

Browsing by Author "Mosley, Amber"

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12-lipoxygenase Promotes Macrophage Infiltration and Pancreatic Islet Dysfunction in the Vertebrate Models of Diabetes Pathogenesis
(2020-05) Kulkarni, Abhishek Anant; Harrington, Maureen; Mirmira, Raghavendra; Anderson, Ryan; Goebl, Mark; Mosley, Amber; Marrs, James
Diabetes is a morbid metabolic disorder that affects almost 500 million people worldwide. Although multiple factors contribute to diabetes pathogenesis, pancreatic islet inflammation and dysfunction are shared characteristics of its major forms. 12- lipoxygenase (12-LOX), an enzyme involved in lipid metabolism, has been implicated in islet inflammation. 12-LOX generates reactive oxygen species (ROS) that activate inflammation and serve as major contributors to islet dysfunction. Importantly, since ROS are transient moieties, they are challenging to study in vivo. Hence, establishing better animal models of ROS-mediated stress is critical to facilitate the discovery and preclinical testing of novel diabetes therapeutics. Here, I have adapted a zebrafish model of conditional β-cell injury, which is regulated by the administration of the prodrug metronidazole (MTZ), to study responses to ROS in vivo. I demonstrate that with MTZ treatment, ROS are generated within β-cells and subsequently exhibit recruitment of macrophages into the islet and induction of β-cell death. I utilized this model to uncover roles for macrophages and 12-LOX during islet injury. Excessive macrophage infiltration exacerbates islet inflammation and dysfunction. Interestingly, on the depletion of macrophages in zebrafish, I observed that β-cells recovered normal function upon cessation of prodrug treatment. This suggests that infiltrating macrophages promote maladaptive inflammation and premature removal of damaged β-cells. Thus, limiting the macrophage infiltration may be a therapeutic approach to restoring β-cell function. Based on the established roles of 12-LOX in other contexts, I hypothesized that its inhibition would prevent the localized infiltration of proinflammatory macrophages. To test this, I used both zebrafish and mouse models and observed a significant reduction in macrophage migration upon loss of 12- LOX activity. Furthermore, I found that expression of CXCR3, a crucial receptor regulating migration, was significantly reduced in 12-LOX loss-of-function macrophages. These data suggest a role for 12-LOX in macrophages, which is conserved across species. Collectively, my study reveals novel roles for 12-LOX in macrophage function and provides testable therapeutic targets for the resolution of inflammation-induced damage in the pancreatic islets.
Biochemical and pharmacological characterization of the Atg8 conjugation system in toxoplasma gondii
(2017-06-28) Varberg, Joseph M.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Mosley, Amber; Safa, Ahmad; Vasko, Michael R.
Toxoplasma gondii is an important human pathogen that infects millions of people worldwide and causing severe and potentially lethal disease in immunocompromised individuals. Recently, a homologue for the autophagy protein Atg8 (TgAtg8) was identified in Toxoplasma that is required for both canonical and noncanonical processes essential for parasite viability. Importantly, TgAtg8 functionality requires its conjugation to phosphatidylethanolamine through the activity of the Atg8 conjugation system. In this thesis, we characterized the proteins that interact with TgAtg8 and TgAtg3, a component of the Atg8 conjugation system, to further define their functions in Toxoplasma and identify opportunities for targeted inhibition of Atg8-related processes. We previously identified that TgAtg8 is acetylated at lysine 23 (K23) and assessed the role of this modification in this thesis. Using mutagenesis, we showed that K23 acetylation did not modulate the interaction with TgAtg3, but appeared to promote TgAtg8 protein stability. Additionally, endogenous mutation of K23 to the nonacetylatable amino acid arginine resulted in severe impairment of parasite replication and spontaneous differentiation into bradyzoites. To gain insight into the role of TgAtg8 in Toxoplasma biology, we next characterized TgAtg8 and TgAtg3 interacting proteins using affinity purification and mass spectrometry. We identified a novel group of interacting proteins that are unique to Toxoplasma, including the dynamin-related protein DrpC. Functional characterization of DrpC identified a potential role of TgAtg8 in trafficking of membrane from the Golgi to the nascent daughter parasites during replication. Lastly, we examined a group of small molecules recently identified as Atg3-Atg8 inhibitors in Plasmodium falciparum and assessed their activity against Toxoplasma. Although the compounds effectively inhibited Toxoplasma replication, they did so through novel mechanisms of action unrelated to the disruption of the TgAtg3-Atg8 interaction. Together, this work provides insight into the function of the Atg8 conjugation system in Toxoplasma that will help guide the future development of novel therapeutics targeting Atg8-related processes.
Characterization of Missense Variants in the RNA Exosome Associated with Pontocerebellar Hypoplasia
(2025-05) Runnebohm, Avery Morgan; Mosley, Amber; Cornett, Evan; Vilseck, Jonah; Angus, Steve
High-throughput sequencing has been essential in identifying genetic variants associated with disease. However, protein-level characterization of identified genetic variants is lacking, especially for rare genetic diseases. RNA sequencing and proteomics techniques were used to characterize missense variants associated with the rare neurodegenerative disorder pontocerebellar hypoplasia 1b (PCH1b). Several variants of EXOSC3, a subunit of the RNA exosome, have been identified in patients with PCH1b. We investigated three missense variants of EXOSC3, including one variant of uncertain significance (VUS). We compared the VUS with two pathogenic variants, one of which is the most common in the general population by allele frequency. The RNA exosome complex consists of 9 core subunits and 2 catalytic subunits and cooperates with several cofactors to guide its function. The RNA exosome is involved in diverse cellular functions including general RNA turnover, rRNA processing, and regulation of RNA splicing. We focused on variants in the EXOSC3 core subunit, identifying differences in protein abundance and stability changes within missense variant cell lines generated by CRISPR. While overall RNA-level changes were limited, we observed differences in rRNA processing and RNA abundance. Proteomics revealed significant decreases in RNA exosome subunit protein abundance, and interestingly, four spatially proximal subunits displayed a greater degree of reduced abundance suggesting altered interactions between / assembly of these subunits. We see potential compensation with increased protein abundance of the catalytic subunit DIS3 and several rRNA processing proteins. Comparison of the VUS with the known pathogenic variants highlights the similarities in the molecular phenotypes but determined that the effects are not as pronounced in the VUS. Ongoing experiments seek to rescue the phenotypes caused by the missense variants by proteasome inhibition and exogenous overexpression of reference or variant EXOSC3. Overall, these experiments illustrate that disease associated EXOSC3 variants have a continuum of functional changes that could explain the range of severity observed in PCH1b patients. By obtaining protein-level insights into exosome complex dynamics, we can design rescue experiments to alleviate dysfunction.
Characterizing the Unfolded Protein Response by Changes in Protein Thermal Stability
(2023-09) McCracken, Neil Andrew; Mosley, Amber; Wek, Ron; Evans-Molina, Carmella; Georgiadis, Millie; Quinney, Sara
The Unfolded Protein Response (UPR) protects eukaryotic cells from the threat of excessive protein flux into the Endoplasmic Reticulum (ER). UPR sentries PERK, Ire1 and ATF6 detect unfolded protein in the ER and alert the cell of the condition. Downstream pathways increase translation of select responders while simultaneously decreasing the global protein load in order that toxic protein aggregates do not form in the cell. While this warning system has been characterized over several decades through extensive reporting of UPR impact on transcript and protein abundance, little is known about the biophysical changes that occur to proteins as part of the UPR in the context of the cellular environment. An understanding of how the UPR affects the folding, stability and protein oligomerization is vital for describing subtle but important changes that occur and contribute to maladaptive physiology in diseases including diabetes, cancer, and neurodegeneration. I propose that deficiencies in characterizing the UPR can be overcome by using thermal shifts assays (TSA) that quantify changes in protein stability post stimuli. Findings described herein show the utility of the biophysical thermal shift assay in characterizing the UPR. Thermal shift assays (TSA) measure susceptibility of proteins to denature upon heat treatment and consequently detect changes in protein structure, modification, and interactions in the cellular environment. Previously unobserved protein relationships related to the UPR were detected using TSA. These workflows were improved through more strategic upstream sampling and downstream data analysis through creation of the publicly available InflectSSP program. Observed UPR phenomena during N-linked glycosylation inhibition and UPR induction include protein degradation, changes in stability of N-linked glycosylation enzymes, and transcriptional targets canonical to the UPR. Stability changes in proteins downstream of PERK were also observed in experiments where PERK genetic ablation was combined with UPR induction. Finally, the thermal shift assay was used to develop a “signature” for the UPR that holistically describes the ER stress response. Results described in this dissertation provide an improved perspective of the UPR along with an approach that can be used to identify novel targets for therapeutic intervention of the UPR.
classCleaner: A Quantitative Method for Validating Peptide Identification in LC-MS/MS Workflows
(2020-05) Key, Melissa Chester; Boukai, Benzion; Ragg, Susanne; Katz, Barry; Mosley, Amber
Because label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics infers the peptide sequence of each measurement, there is inherent uncertainty in the identity of each peptide and its originating protein. Removing misidentified peptides can improve the accuracy and power of downstream analyses when differences between proteins are of primary interest. In this dissertation I present classCleaner, a novel algorithm designed to identify misidentified peptides from each protein using the available quantitative data. The algorithm is based on the idea that distances between peptides belonging to the same protein are stochastically smaller than those between peptides in different proteins. The method first determines a threshold based on the estimated distribution of these two groups of distances. This is used to create a decision rule for each peptide based on counting the number of within-protein distances smaller than the threshold. Using simulated data, I show that classCleaner always reduces the proportion of misidentified peptides, with better results for larger proteins (by number of constituent peptides), smaller inherent misidentification rates, and larger sample sizes. ClassCleaner is also applied to a LC-MS/MS proteomics data set and the Congressional Voting Records data set from the UCI machine learning repository. The later is used to demonstrate that the algorithm is not specific to proteomics.
The Contribution of Pdx1-Bound Chromatin Remodelers in Controlling β-Cell Differentiation and Function
(2022-12) Davidson, Rebecca Kelly; Spaeth, Jason; Evans-Molina, Carmella; Mosley, Amber; Mastracci, Teresa; Balakrishnan, Lata
Understanding β-cell development and function is essential for generating more effective treatment options for individuals with diabetes. A key player in pancreatogenesis, islet development, and mature β-cell function is the Pdx1 transcription factor (TF). Pdx1 activity is modulated through interactions with various coregulators, including the Swi/Snf chromatin remodeling and Nucleosome Remodeling and Deacetylase (NuRD) complexes. Loss of one Swi/Snf ATPase subunit, Brg1, in early pancreatogenesis reduces final pancreas mass, and β-cell-specific deletion of both subunits, Brg1 and Brm, leads to glucose intolerance and loss of insulin production in the β-cell. Here, we hypothesized Swi/Snf governs endocrine progenitor cell development and postnatal islet function. To test this, we generated conditional murine knockouts of Brg1 (Brg1Δendo;Brm+/-), Brm (Brg1Δendo/+;Brm-/-), or both subunits (DKOΔendo) during endocrine cell development. No DKOΔendo mice were recovered at weaning, and loss of Brg1 but not Brm led to severe glucose intolerance, ad-lib fed hyperglycemia, and reduced insulin levels by four weeks of age. Brg1Δendo;Brm+/- mice had fewer islets and compromised insulin secretion. Together, these data suggest that loss of Brg1 during endocrine cell development has negative impacts on postnatal islet function, with loss of both Brg1 and Brm being early postnatal lethal. Pdx1 has been shown to also interact with the Chd4 helicase subunit of the NuRD complex. Here, we demonstrate Pdx1:Chd4 interactions are increased under stimulatory conditions and hypothesize that Chd4 modulates expression of Pdx1-bound genes critical for β-cell function. To test this, we generated a tamoxifen inducible, β-cell-specific Chd4 knockout mouse model (Chd4Δβ). Four weeks following Chd4 removal, Chd4Δβ mutants were glucose intolerant with severe insulin secretion defects. Additionally, Chd4Δβ islets contained fewer mature insulin granules and secreted more proinsulin. RNA-sequencing from Chd4Δβ β-cells identified numerous upregulated (eg Hk2, Mycl) and downregulated genes (eg MafA, Chga, Chgb, Slc2a2). Through ATAC-sequencing, we discovered several differentially accessible genomic regions, including Chd4-bound and Pdx1-controlled MafA Region 3, which had reduced accessibility in Chd4Δβ β-cells. Lastly, we demonstrate that CHD4 impacts human β-cell function and PDX1:CHD4 interactions were reduced in human donor β-cells with type 2 diabetes, demonstrating loss of these interactions is a significant feature of diabetes pathogenesis.
Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells
(Elsevier, 2013) Khanna, May; Imasaki, Tsuyoshi; Chikwana, Vimbai M.; Perez-Miller, Samantha; Hunter, Gerald O.; Mosley, Amber; Takagi, Yuichiro; Hurley, Thomas D.; Biochemistry and Molecular Biology, School of Medicine
We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.
Genetic and Functional Dissection of Neurodegeneration: Multiomic Analysis of Genetic Risk Variants in Pontocerebellar Hypoplasia 1B and Alzheimer's Disease
(2025-05) Wijeratne, H. R. Sagara; Mosley, Amber; Kim, Jungsu; Baucum, A. J.; Vilseck, Jonah
Each neurodegenerative diseases have a combination of features such as neuronal death, protein aggregation, inflammation, cytoskeletal abnormalities, altered proteostasis, synaptic defects, RNA/DNA defects, and altered energetics. While genetic variants are crucial to disease pathogenesis, the molecular mechanisms linking the genotype to these hallmarks remain unclear. We investigate two risk genes linked to different neurodegenerative diseases, pontocerebellar Hypoplasia 1B (PCH1b) and Alzheimer’s disease (AD), using a multiomic approach to uncover their genetic and functional underpinnings. PCH1b is caused by variants in an RNA exosome complex subunit EXOSC3. These variants disrupt RNA processing, leading to neuronal dysfunction and progressive neurodegeneration. By analyzing cell lines carrying a variant in between two RNA-binding domains of EXOSC3, we found significant changes in RNA abundance across multiple RNA classes and showing enrichment of transcripts containing AU-rich elements. Molecular dynamics simulations of EXOSC3 indicate that the variants produce an unstructured EXOSC3 isoform, possibly prone to degradation. Proteomics reveals altered protein abundance and thermal stability in specific RNA exosome subunits, particularly affecting the MPP6 cofactor. Apolipoprotein E (APOE) is the strongest genetic risk factor for sporadic AD though its exact molecular effects are not fully understood. Thermal proteome profiling of astrocytes from APOE knockout mice showed that APOE deficiency alters the thermal stability of mitochondrial proteins, particularly in Complex I of the electron transport chain. The functional consequences of this stability change is an increased ATP-linked respiration in APOE-deficient astrocytes, suggesting a shift in mitochondrial activity. These findings provide new insights into how APOE impacts mitochondrial function and protein stability, emphasizing thermal proteomic profiling as a powerful tool for studying neurodegenerative diseases. Multiomics analyses effectively link genotype to phenotype in neurodegenerative diseases by uncovering molecular alterations that define disease-specific hallmarks, such as RNA homeostasis changes in PCH1b and mitochondrial alteration in AD, offering valuable insights into their pathophysiology and therapeutic targets.
The impact of the termination override mutation on the activity of SSU72
(2016-12-19) McCracken, Neil Andrew; Mosley, Amber; Wek, Ronald; Goebl, Mark
Ssu72, an RNA Pol II CTD phosphatase that is conserved across eukaryotes, has been reported to have a wide array of genetic and physical associations with transcription factors and complexes in RNA transcription. Catalytic mutants of Ssu72 are lethal across many eukaryotes, and mutations to non-catalytic sites in SSU72 phosphatase have been shown to lower function. One spontaneous mutation of the SSU72 gene in Saccharomyces cerevisiae (A to C nucleotide mutation resulting in an L84F mutation in the coded protein) was shown to have transcription termination deficiency (termination override or TOV). This SSU72 mutation was suggested by Loya et al. to cause a lowering of the phosphatase activity of the protein and consequently affect proper termination. In research reported herein, an investigation was completed through in-vitro and ex-vivo approaches with the goal of understanding the impact of the SSU72 TOV mutation on the observed phenotype in S. cerevisiae. It can be concluded from work presented in this report that the SSU72 TOV mutation does not cause a decrease in in-vitro phosphatase activity as compared to wild type. Evidence presented even suggests an increase in phosphatase activity as compared to wild type Ssu72. One model for the observed responses in transcription termination is that the phenylalanine substitution in Ssu72 leads to cooperative interactions with proline residues in the CTD. It is proposed that the corresponding increase in Ssu72 phosphatase activity limits RNA Pol II CTD association with termination factors, such as Nrd1, thus causing deficient transcription termination.
Investigations into the function of Elp3 in Toxoplasma gondii
(2017-05-04) Padgett, Leah Rausch; Arrizabalaga, Gustavo; Jerde, Travis; Mosley, Amber; Nass, Richard M.; Sullivan, William J., Jr.
The parasite Toxoplasma gondii causes life-threatening infection in immunocompromised individuals. Our lab has determined that Toxoplasma Elongator protein-3 (TgElp3) is required for parasite viability. While catalytic domains are conserved, TgElp3 is the only component of the six-subunit Elongator complex present in Toxoplasma; moreover, TgElp3 localizes to the outer mitochondria membrane (OMM). These unusual features suggest that TgElp3 may have unique roles in parasite biology that could be useful in drug targeting. The goals of this thesis were to determine the function of TgElp3 and how the protein traffics to the OMM. In other species, Elp3 mediates lysine acetylation of histones and alphatubulin, and its radical S-adenosyl methionine (rSAM) domain is important for the formation of tRNA modifications, which enhance translation efficiency and fidelity. Given its location, histones would not be an expected substrate, and we further determined that tubulin acetylation in Toxoplasma is mediated by a different enzyme, TgATAT. We found that overexpression of TgElp3 at the parasite’s mitochondrion results in a significant replication defect, but overexpression of TgElp3 lacking the transmembrane domain (TMD) or with a mutant rSAM domain is tolerated. We identified one such modification, 5-methoxycarbonylmethyl-2thiouridine (mcm5S2U) that is likely mediated by TgElp3. These findings signify the importance of TgElp3’s rSAM domain for protein function, and confirms TgElp3 activity at the OMM is essential for Toxoplasma viability as previously reported. To determine how TgElp3 traffics to the OMM, we performed a bioinformatics survey that discovered over 50 additional “tail-anchored” proteins present in Toxoplasma. Mutational analyses found that targeting of these TA proteins to specific parasite organelles was strongly influenced by the TMD sequence, including charge of the flanking C-terminal sequence.

Browsing by Author "Mosley, Amber"

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