IU Indianapolis ScholarWorks :: Browsing by Author "Georgiadis, Millie"

Browsing by Author "Georgiadis, Millie"

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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.
Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex
(International Union of Crystallography, 2016-08-26) Chen, Qiujia; Georgiadis, Millie; Biochemistry and Molecular Biology, School of Medicine
The DNA-binding domain of SETMAR was successfully crystallized in a complex with its ancestral terminal inverted repeat and a variant of this sequence through a systematic approach, and initial Se SAD phasing was achieved through the judicious addition of Met residues., Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of the Hsmar1 transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestral Hsmar1 terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.
Developing Novel Methods to Identify RNA-Associated Mechanisms for Inheritance
(2020-11) Ettaki, Zacharia Nabil; Aoki, Scott T.; Georgiadis, Millie; Quilliam, Lawrence
Animals depend on inheriting non-genetic information early in life to grow and develop naturally. This inherited, non-genetic information was previously thought to be limited to DNA modifications and DNA binding proteins. But recent studies have expanded our understanding of inheritance to include RNA and RNA binding proteins. We currently lack methods to identify and enrich for RNA binding proteins that might be involved in providing non-genetic information from mother to daughter cells. Others have developed a method using modified enzyme tags to pulse-label proteins with small molecule fluorescent ligands and follow these proteins as they are inherited by cells. Here I characterized and tested the application of a fluorescent small molecule targeting antibody to enrich for these labeled proteins. I first tested the ability of this antibody to bind to fluorescent ligand-labeled enzymes. I determined that the antibody can efficiently bind to at least one of the labeled enzymes. Second, I determined crystallization conditions for the ligand binding antibody fragment. This thesis sets the stage for structure determination and to test whether this antibody can work in vivo to enrich for RNA binding proteins involved in the delivery of non-genetic information to cells.
Molecular cloning, heterologous expression, and steady-state kinetics of camplyobacter jejuni periplasmic nitrate reductase
(2020-08) Mintmier, Breeanna; Basu, Partha; Georgiadis, Millie; Deiss, Frédérique; Minto, Robert
Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they catalyze. The dimethyl sulfoxide reductase (DMSOR) family is the most diverse family of molybdoenzymes and, the members of this family catalyze a myriad of reactions that are important in microbial life processes. Periplasmic nitrate reductase (Nap) is an important member of the DMSO reductase family that catalyzes the reduction of nitrate (NO3-) to nitrite (NO2-), and yet the physiological role of Nap is not completely clear. Enzymes in this family can transform multiple substrates; however, quantitative information about the substrate preference is sparse and more importantly, the reasons for the substrate selectivity are not clear. Substrate specificity is proposed to be tuned by the ligands coordinating the molybdenum atom in the active site. As such, periplasmic nitrate reductase is utilized as a vehicle to understand the substrate preference and delineate the mechanistic underpinning of these differences. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum-coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic and biochemical properties of these variants will be discussed and compared with those of the native enzyme, providing quantitative information to understand the function.
Molecular dynamics simulations of spore photoproduct containing DNA systems
(2023-05) Hege, Mellisa; Pu, Jingzhi; Blacklock, Brenda; Georgiadis, Millie; Deng, Yongming
Bacterial endospores have been a topic of research interest over the last several decades given their high resistance to ultraviolet (UV) damage. Unlike vegetative bacterial cells, which form cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) as the major product upon UV irradiation, endospore bacteria form a spore photoproduct (5-(R-thyminyl)-5,6-dihydrothymine or SP) as the major product. Vegetative bacteria cells are subject to regular cell activities and processes such as division and deoxyribonucleic acid (DNA) replication, which are prone to damage from UV exposure. However, in endospores, which have a largely anhydrous inner environment, the DNA remains dormant when bound to spore-specific small acid-soluble proteins (SASP) and dipicolinic acid, making spores highly resistant to radiation, heat, desiccation, and chemical harm. During spore germination, SP lesions in DNA are repaired by a distinctive repair enzyme, spore photoproduct lyase (SPL). In this thesis, molecular dynamics (MD) simulations were carried out to (i) examine how the formation of the SP lesion in DNA affects the global and local structural properties of duplex DNA and (ii) study how this lesion is recognized and repaired in endospore. The first part of this work was focused on designing and developing a structurally and dynamically stable model for dinucleotide SP molecule (TpT), which was subsequently used as an SP patch incorporated into duplex DNA. Computationally, this requires modifications of the bond and nonbonded force field parameters. The stability of the patch and developed parameters was tested via solution-phase MD simulations for the SP lesion incorporated within the B-DNA dodecamer duplex (PDB 463B). The second part involved applying the new SP patch to simulate the crystallographic structure of the DNA oligomer containing SP lesions. Solution-phase MD simulations were performed for the SP-containing DNA oligomers (modeled based on PDB 4M94) and compared to the simulations of the native structure (PDB 4M95). Our analysis of the MD trajectories revealed a range of SP-induced structural and dynamical changes, including the weakened hydrogen bonds at the SP sites, increased DNA bending, and distinct conformational stability and distribution. In the third part of this thesis project, we carried out MD simulations of SP-containing DNA bound with SASPs to examine how the DNA interacts differently with SASP in the presence and absence of the SP lesion. The simulation results suggested decreased electrostatic and hydrogen bonding interactions between SASP and the damaged DNA at the SP site compared to the undamaged DNA-protein complex. In addition, decreased helicity percentage was observed in the SASPs that directly interact with the SP lesion. The last part of this this thesis work focused on the SP-dimer flipping mechanism, as the lesion is likely flipped out to its extrahelical state to be recognized and repaired by SPL. Using steered molecular dynamic (SMD) simulations and a pseudo-dihedral angle reaction coordinate, we obtained possible SP flipping pathways both in the presence and absence of SASP. Collectively, these simulation results lend new perspectives toward understanding the unique behavior of the SP lesion within the DNA duplex and the nucleoprotein complex. They also provide new insights into how the SP lesion is efficiently recognized and repaired during spore germination.
Small Molecule Inhibitors of GroEL That Disrupt Active Replication of Mycobacterium Tuberculosis and ESKAPE Bacteria
(2022-07) Tepper, Katelyn; Johnson, Steven M.; Georgiadis, Millie; Motea, Edward; Absalon, Sabrina
Globally, millions of people die every year due to complications involving infections from antibiotic-resistant bacteria. Of these infections, the most common organisms are Mycobacterium tuberculosis (Mtb) and a group of bacteria known as the ESKAPE pathogens (an acronym that stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter species). Unfortunately, as the need for antibiotics increases, industrial antibiotic development programs are drying up. However, unique antibiotic candidates targeting new pathways may be better for addressing antibacterial resistance. A target that is currently not the focus of any drug on the market is the bacterial GroEL chaperonin system. GroEL chaperonins are complex, oligomeric proteins that are upregulated in the cell under stressful conditions and prevent the misfolding and aggregation of other proteins. All bacteria have one homolog that performs protein folding functions – such is the case for E. coli and the ESKAPE bacteria – while others, like M. tuberculosis, contain additional GroEL isoforms that appear to perform non-canonical functions that are not well understood. The canonical isoforms are essential for survival under all conditions; thus, these chaperonins represent excellent targets for antibiotic development. This study aimed to identify inhibitors of GroEL in the ESKAPE bacteria and Mtb from a library of compounds with known antibiotic properties that was provided by the Medicines for Malaria Venture. Using two orthogonal assays that assess GroEL activity via its refolding of denatured enzymes Malate Dehydrogenase and Rhodanase, 37 inhibitors of E. coli GroEL were identified. Of these, 33 were examined in dose response testing in in vitro biochemical and cell viability assays. Compound 23 stood out in potency for inhibiting GroEL functions and actively-replicating Mtb bacteria, and thus a small panel of analogs were evaluated to develop structure-activity relationships (SAR) and study their mechanism. Two cysteine residues were identified as covalently modified by compound 23 and one of the lead analogs, giving insight into inhibitory sites on GroEL. Another lead analog bearing a nitrofuran moiety exhibited inhibition of actively-replicating E. coli, S. aureus, and Mtb bacteria. Importantly, this study identified new classes of GroEL inhibitors to explore for optimization as antibacterial candidates.
Ssu72 and Rtr1 Serine 5 Phosphates and Their Role in NNS and CPF Transcription Termination
(2020-05) Victorino, Jose Fabian; Mosley, Amber; Roach, Peter; Georgiadis, Millie; Liu, Yunlong; Arrizabalaga, Gustavo
Polyadenylation dependent transcription termination is dependent on the Cleavage and Polyadenylation Factor complex (CPF) which is essential for the termination and processing of mature RNA. Polyadenylation (PolyA) independent transcription termination is carried out by the NNS (Nrd1-Nab3-Sen1) termination pathway, which helps regulate termination and processing of non-coding RNA (ncRNA). The disruption of these pathways can impact expression of nearby genes, both protein coding and noncoding. Recruitment of termination pathway components is achieved through a domain unique to the largest subunit of RNA Polymerase II (RNAPII) referred to as the Cterminal domain (CTD), which contains a repeating heptad sequence, Y1S2P3T4S5P6S7, and acts as a docking site for transcription regulatory proteins. Ssu72 is a serine 5 phosphatase and an essential member of the CPF complex. Rtr1 is also a serine 5 phosphatase, but its mechanism of action is less well characterized. Both Rtr1 and Ssu72 regulate transcription machinery recruitment through control of the phosphorylation status of the CTD. My studies have focused on Rtr1 and Ssu72 mutants in yeast which show evidence of transcription termination related phenotypes. Chromatin immunoprecipitation of RNAPII followed by exonuclease treatment (ChIP-exo) studies provide evidence of RNAPII transcription continuing through termination sites at ncRNA genes as a result of a hyperactive Ssu72-L84F mutant, while an RTR1 knockout results in increased premature RNAPII transcription termination. Northern blots and RNA sequencing confirm premature transcription termination and decreased total RNA expression in the RTR1 knockout and increased length of ncRNA transcripts as well as total RNA expression in the Ssu72-L84F mutant. Mass spectrometry analysis has identified changes in the protein-protein interactions (PPI) within the CPF complex in the Ssu72-L84F mutant and decreased PPIs between different transcription machinery in RTR1 knockout cells. My results show that the CTD phosphatases Rtr1 and Ssu72 play unique roles in the regulation of RNAPII termination in eukaryotes.
Using Chemical Probes to Define the Role of Aldehyde Dehydrogenase 1A in a Breast Cancer Model
(2022-09) Takahashi, Cyrus; Hurley, Thomas; Georgiadis, Millie; Harrington, Maureen; Hawkins, Shannon; Wek, Ronald
The aldehyde dehydrogenase (ALDH) superfamily comprises a group of NAD(P)+-dependent enzymes that catalyze the conversion of aldehydes to their corresponding carboxylic acids. Of the nineteen human ALDH enzymes, members of the ALDH1A subfamily consisting of ALDH1A1, ALDH1A2, and ALDH1A3 have attracted interest as markers of cancer stem cells (CSCs) in several cancer types including lung, breast, and ovarian. CSCs represent a distinct subpopulation of highly tumorigenic cells that promote metastasis, recurrence, and resistance to conventional cancer therapies. The increased expression and activity of ALDH1A in CSCs is well-documented, as is the correlation between ALDH1A and a more aggressive cancer phenotype with poorer treatment outcomes. However, the actual functional role of ALDH1A in the context of CSCs has yet to be clearly defined. Elucidating this role will lead to a greater understanding of CSC biology and evaluate ALDH1A as a potential anti-CSC therapeutic target. In this study, previously developed and characterized selective small-molecule inhibitors of ALDH1A were used in conjunction with global transcriptomic, proteomic, and metabolomic analyses to identify pathways that could potentially establish a link between ALDH1A activity and early events in CSC formation in a triple-negative breast cancer (TNBC) model. These approaches revealed that ALDH1A inhibition is associated with mitochondrial and metabolic dysfunction and perturbation of the electron transport chain. ALDH1A inhibition also resulted in an increase in markers of endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), specifically mediated through the Protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway. These effects appear to occur independently of both the canonical function of ALDH1A in detoxifying reactive aldehydes as well as its potential metabolic contribution through the generation of NADH. Together, these results suggest a separate role for ALDH1A in TNBC CSCs in protecting against ER stress that warrants further study.

Browsing by Author "Georgiadis, Millie"

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