Browsing by Subject "Metals"

Over the past two decades, a toolbox of mass spectrometry-based proteomic methods has been developed that enables the conformational properties of proteins and protein-ligand complexes to be probed in complex biological mixtures, from cell lysates to whole cells. The focus of this dissertation is the extension of these methodologies to the study of protein-gas and protein-metal interactions, an area of limited application. The goals of this work are two-fold. The first is to improve current mass spectrometry-based proteomic methods that measure protein folding stability, which is accomplished by the development of a chemo-selection strategy for proteolysis procedures and a “one-pot” approach that increases statistical significance while decreasing experiment costs. The second goal of this work is the application of these methodologies and others to the study of protein-gas and protein-metal interactions in complex biological mixtures (i.e., cell lysates), in which insights could be gained about gas and metal biological activities by surveying their interactions within a proteome. The first part of this dissertation describes in more detail the development and application of a semitryptic peptide enrichment strategy for proteolysis procedures (STEPP) that enables the isolation of information-rich semitryptic peptides. With the STEPP protocol, the number of semitryptic peptides increased by 5- to 10-fold and the amount of structural information was maximized in limited proteolysis experiments. The combination of the pulse proteolysis technique with a novel “one-pot” approach for data acquisition and analysis (one-pot STEPP-PP), resulted in false positive rates reaching close to zero (i.e., 0.09%) for a proof-of-principle drug target identification experiment for cyclosporine A and a yeast lysate. Described in the second part of this dissertation is the application of the improved proteolysis methodologies and others to multiple studies of protein-gas and protein-metal interactions on the proteomic scale. First, the development and application of protein stability measurements to the study of protein-gas interactions, specifically protein-xenon interactions, is described. A sample preparation protocol that was conducive to protein-gas binding studies is developed and validated against a known xenon-binding protein, metmyoglobin. Ultimately, this sample preparation protocol was employed in large-scale, proteome-wide SPROX and limited proteolysis experiments to identify xenon-interacting proteins in a yeast lysate. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known. Our survey of the proteome revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes and revealed correlations between the mechanisms by which ATP and Xe target proteins. Next, the application of one-pot STEPP-PP is described in the context of two research areas, both related to identifying the protein targets of metal-associated cell death processes. First described is the utilization of this technique in combination with protein expression level analysis to identify bacterial protein targets of copper delivered by small molecule ionophores. The protein folding stability and expression level profiles generated in this work enabled the effects of ionophore vs. copper to be distinguished and revealed copper-driven stability changes in proteins from processes spanning metabolism, translation, and cell redox homeostasis. The 159 differentially stabilized proteins identified in this analysis were significantly more numerous (by 3-fold) than the 53 proteins identified with differential expression levels. These results illustrate the unique information that protein stability measurements can provide to decipher metal-dependent processes in drug mode of action studies. The second application of the one-pot STEPP-PP methodology is to the study of Fe- and Zn-mediated sensitization to erastin-induced ferroptotic cell death. Our approach enabled differential protein expression and protein folding stability measurements to be made on RCC4 cells exposed to excess iron and zinc along with the ferroptosis-inducing molecule erastin. Of the protein targets identified, a few have known ties to pathways involved in ferroptotic cell death, while others have not been previously linked with ferroptosis. Future work aims at assaying the potential metal binding properties of these proteins, to connect them to their metal-enhancing ferroptosis effects. The final research area described in this dissertation is the development and application of a novel metal-induced protein precipitation (MiPP) approach which exploits the protein precipitation properties of metals to study proteins that are susceptible to metal overload. Total protein precipitation as a function of metal concentration was assayed across various proteomes (bacterial, fungal, and mammalian) and metals (copper, zinc, iron, etc). Copper-induced protein precipitation was measured within E. coli and C. albicans proteomes by coupling precipitation curves with a bottom-up proteomics readout. Proteome-wide precipitation studies revealed a wide distribution of copper precipitation midpoints for the identified proteins within these species. A fundamental understanding of the biophysical basis of susceptibility or tolerance to metal precipitation can potentially be garnered through more in-depth analysis of the proteins that fall significantly outside the average precipitation midpoint of each proteome.

Cellular damage due to oxidative stress is implicated in a wide variety of conditions including degenerative diseases like Alzheimer's and Parkinson's Diseases. One source of oxidative stress is the interaction of redox-active metals such as copper and iron with hydrogen peroxide to produce hydroxyl radicals. Preventing metal-induced oxidative stress by metal chelation is one potential approach to treat some of these diseases, but there remain significant challenges in designing chelators that target damaging metals while not disturbing healthy metal ion distribution.

To overcome this challenge, prochelators that are responsive to conditions of oxidative stress have been introduced. By designing ligands that only bind metal ions in the presence of oxidants, damaging metals can be bound and removed while not perturbing the metals necessary for cell function. Masking the phenol of a chelator with a boronic ester creates a prochelator that has little to no affinity for metal ions until exposure to H₂O₂ converts the prochelator to the chelator, which is then available to bind metal ions. Described here is the development of boronate-based prochelators that react with H₂O₂ to produce chelating agents of variable denticity, ranging from 2 to 6.

Quinoline boronic acid pinanediol ester, or QBP, is a new bidentate prochelator introduced here that reacts with H₂O₂ with a rate of 0.22 M-1s-1 to produce 8-hydroxyquinoline, a known metal-binding agent. Results in Chapter 2 show that QBP can be activated in vitro under conditions that mimic early Alzheimer's Disease pathology where copper, amyloid beta peptide, and ascorbic acid exacerbate formation of reactive oxygen species. QBP does not bind metal ions, nor does it disaggregate metal-promoted amyloid beta peptide aggregates. However, the released 8-hydroxyquinoline sequesters copper from amyloid beta and both diminishes further formation of reactive oxygen species and inhibits further aggregation of amyloid-beta.

The syntheses and crystal structures of hexadentate prochelators are described in Chapter 3, along with their rates of oxidation in response to hydrogen peroxide exposure and their ability to protect against hydroxyl radicals formed in vitro by iron (or copper), ascorbic acid, and hydrogen peroxide. The hexadentate chelators are based on a tripodal architecture in which three phenol moieties are linked via nitrogens on three alkyl arms to a central nitrogen to provide an N₃O₃ donor set for metal complexation. Of three prochelator/chelator pairs prepared, the pair (trenBsalam/trensalam) with amine linkages was deemed most suitable for potential biological studies. The prochelator trenBsalam oxidizes at a rate of 0.72 M-1s-1 to produce the chelator trensalam in the presence of hydrogen peroxide. The transition metal coordination chemistry and metal ion affinities of trensalam were further studied in Chapter 4 by x-ray crystallography, UV/Vis spectroscopy and cyclic voltammetry.

The response of a series of bidentate prochelators to various oxidants, including hydrogen peroxide, superoxide, peroxynitrite and hypochlorite, was evaluated by UV/Vis spectroscopy in Chapter 5. Varying the diol that is appended to the boronic ester results in hydrogen peroxide oxidation rates ranging from 0.018 to 1.27 M-1s-1. Lastly, the stability of different boronic acid and diol combinations was probed by spectroscopic techniques and indicate that boronic esters formed with pinanediol form the most stable prochelators under physiological conditions.

Plastic pollution is ubiquitous in the ocean. Researchers are actively determining how plastic pollution harms marine animals and which measures should be used to reduce pollution. I use the four pathways to global sustainability (created by Folke et al., 2021) as a guiding framework for this dissertation because this framework outlines how society can develop sustainable practices to address environmental challenges, in this case, plastic pollution. I identify risks posed by plastic pollution to marine animals and characterize government and corporate responses to plastic pollution. My overarching goal is to use these results to inform stakeholders of gaps or mismatches in plastics governance and chart a path toward global plastics sustainability. In Chapter 1, I investigate plastic consumption in marine animals, using the sea anemone as a model animal. I find that anemones readily consume plastic (polyethylene and polyvinyl chloride) pellets and can extract metallic additives, specifically lead and tin, from plastic. More broadly, this research suggests that plastic pollution may be a novel pathway for heavy metals to enter the marine food web. In Chapter 2, I examine government responses to plastic pollution by qualitatively analyzing public policies at all levels of government, from local to international, adopted between the years 2000 and 2019. I show that governments are increasingly adopting policies over time. Policies frequently target plastic bags and macroplastics and infrequently target microplastics. In Chapter 3, I qualitatively analyze voluntary commitments to reduce plastic pollution made by the world’s largest companies. I report that most companies are aware of the unsustainability of plastic and responding, albeit insufficiently given the scope of the challenges. Company commitments often lack concrete deadlines and frequently focus on recycling, despite low global recycling rates, which obscures corporate accountability. In Chapter 4, I provide insight into the role that emerging technologies may play in plastics sustainability. I synthesize literature on the use of plastic waste in road construction and highlight key knowledge gaps such as plastic additive leaching, microplastic and nanoplastic generation, and road recyclability at end-of-life. The dissertation closes with Chapter 5, which summarizes articles from the special issue “Emerging Challenges and Solutions to Plastic Pollution,” published in Frontiers in Marine Science, and contextualizes my dissertation findings in the broader scientific literature. I close Chapter 5 by synthesizing key dissertation findings and suggesting areas of future research. I highlight that societal responses to plastic pollution have not prioritized addressing the plastics that pose the greatest risks to aquatic ecosystems, resulting in negative environmental consequences. This dissertation contributes to the field by demonstrating the harms posed by plastic pollution to sea anemones and qualitatively characterizes public and private sector measures aiming to reduce plastic pollution. More broadly, I highlight the role of interdisciplinary research in environmental problem-solving and charting a path toward global plastics sustainability.