Repository logo

Browsing by Author "Santangelo, Thomas, committee member"

Now showing 1 - 4 of 4
  • Thumbnail Image
    ItemOpen Access
    Characterization of interactions of lipoquinone derivatives within model membrane systems
    (Colorado State University. Libraries, 2021) Bublitz, Gaia Rachel, author; Crans, Debbie, advisor; Cohen, Robert, advisor; Santangelo, Thomas, committee member; Roess, Deborah, committee member
    Menaquinones (MK) are electron carriers composed of a naphthoquinone moiety and an isoprene side chain of variable length and saturation. These molecules are the only quinone derivatives present in the electron transport systems of all Gram-positive bacteria and some Gram-negative anaerobes. Subsequently, MK plays a critical role in respiration for pathogens such as Staphylococcus aureus and Mycobacterium tuberculosis. Although the physiological function and relevance of MK as a redox cofactor have been established, its chemical interactions within the plasma membrane and the effects of these properties on MK-mediated electron transport are still obscure. These unknowns are reflected in existing literature, as MK is commonly depicted in an extended conformation, although in vitro and in vivo studies suggest that biomolecules with alkyl moieties assume folded conformations in native environments (Ko et al., 2011; Trembleau et al., 2003). In this study, we implemented 1D 1H and 2D 1H-1H NMR spectroscopic techniques to characterize the location and 3D conformation of MK-2 within a L-α-phosphatidylcholine liposome model. MK-2, a truncated menaquinone analog, was selected due to its limited rotational variability and previous characterization in a simple monolayer lipid system (Koehn et al., 2018). Our data suggests that MK-2 is largely incorporated into the phospholipid bilayer, with an aqueous subspecies residing at the polar membrane interface in a concentration-dependent manner. 2D NOESY spectroscopic analysis supports the interpretation that both the aqueous form and the membrane-associated form of MK-2 assume a folded conformation. These findings provide a reference for the study of the properties of MK derivatives with longer isoprene chains, which are analogous to functional MK variants in native environments.
  • Thumbnail Image
    ItemOpen Access
    Identification of the TPC2 interactome reveals TSPAN10 and OCA7 as key players in the biogenesis of melanosomes
    (Colorado State University. Libraries, 2023) Beyers, Wyatt, author; Di Pietro, Santiago, advisor; Amberg, Gregory, committee member; Santangelo, Thomas, committee member; Yao, Tingting, committee member
    Many specialized cell types gain their function through the generation of specialized organelles that make or store cell-specific biomolecules. A group of specialized organelles are called Lysosome Related Organelles (LROs) because they are derived from Golgi and endolysosomal compartments and their biogenesis depends on trafficking pathways and machinery shared with lysosomes, many have protein contents partially overlapping with lysosomes, and typically have low pH during stages of their maturation. One well-studied model LRO is the melanosome, the organelle in melanocytes and retinal pigment epithelial cells responsible for melanin pigment production in the eyes, hair, and skin, and defects in melanosome function lead to pigmentation diseases such as oculocutaneous albinism. Melanosome biogenesis is a complex process requiring ubiquitous membrane trafficking machinery to be repurposed for the differentiation of melanosomes from other endosomal compartments and specific delivery of melanosome synthesizing enzymes, Tyrosinase and Tyrosinase Related Proteins 1 and 2. Furthermore, correct melanosome maturation requires remodeling of the melanosome membrane, recycling of membrane trafficking machinery, generation of intraluminal amyloid fibrils with the correct structure for melanin packaging, tight pH control, as well as coordinated influx of copper, zinc, tyrosine, and cysteine for melanin synthesis. These processes require the temporospatial coordination of at least 100 known proteins, and probably dozens more remain undiscovered. In this dissertation, I present the discovery of new proteins involved in the biogenesis of melanosomes. Proximity biotinylation by promiscuous biotin ligase enzymes followed by biotin pulldown and mass spectrometry has emerged as a powerful technique for the identification of protein-protein interactions, protein complex determination, and identification of organelle membrane proteomes. I utilized the melanosome localized cation channel, TPC2, genetically fused with the BioID2 biotin ligase, to identify proteins in proximity to TPC2 at the cytosolic surface of melanosome membranes of MNT1 melanoma cells. Through mass spectrometry analysis of biotinylated proteins enriched through Streptactin pulldown, a TPC2 proximity interactome was identified comprising over 200 proteins. Subsequent fluorescence confocal microscopy analysis confirmed several proteins, including PLD1, SV2A, TSPAN10, and OCA7/C10orf11/LRMDA all colocalize highly with TPC2-EGFP, confirming they are new melanosome proteins. In follow-up functional studies, TSPAN10 and OCA7 were confirmed to be involved in pigmentation, with severe melanin depletion in TSPAN10 or OCA7 knockout MNT1 cells. TSPAN10 and OCA7 both influence the processing of the PMEL protein, which is required for correct melanosome ultrastructure and for melanin packaging. Further investigation of TSPAN10 revealed it functions with the pigmentation associated metalloproteinase, ADAM10, and is required for ADAM10 expression and localization to endosomal compartments. On the other hand, OCA7 was found to work with the melanosome localized Rab proteins, Rab32 and Rab38, and regulates the pH of melanosomes. Thus, the newly defined TPC2 interactome in melanocytes was proven as a valuable dataset that robustly identifies new melanosome proteins. Chapter 1 of this dissertation provides a broad overview of membrane trafficking pathways, as well as a synopsis of the specific proteins and pathways involved in melanosome biogenesis and homeostasis. Chapter 2 investigates the TPC2 interactome in MNT1 cells, and it characterizes TSPAN10 as a new player in melanosome biogenesis. Finally, Chapter 3 provides a characterization of the OCA7 protein associated with oculocutaneous albinism type 7 and investigates OCA7 function using a newly generated OCA7 knockout cell model.
  • Thumbnail Image
    ItemEmbargo
    Spn1, Spt4, Spt5, and Spt6 preserve chromatin structure over promoters and open reading frames
    (Colorado State University. Libraries, 2024) Tonsager, Andrew Jordan, author; Stargell, Laurie A., advisor; Hansen, Jeffrey C., committee member; Santangelo, Thomas, committee member; Argueso, Juan Lucas, committee member
    The eukaryotic chromatin landscape presents formidable nucleosomal barriers for processes that require access to DNA, such as transcription. These barriers are overcome through the action of many factors, including histone chaperones Spn1, Spt5, Spt6, and FACT and transcription elongation factor Spt4. However, it is poorly understood how each contributes to this process. To ascertain the role that these factors play on preserving chromatin structure over the genome, this thesis has utilized micrococcal nuclease digestion followed by sequencing (MNase-seq) to analyze chromatin protections in the yeast genome in cells expressing numerous mutant alleles of these factors. Extensive characterization of MNase-protected fragments in a wide range of sizes established that the essential histone chaperone Spn1 preserves both nucleosomal and subnucleosomal structures over both promoters and open reading frames across the genome. Additional analyses from existing MNase-seq datasets demonstrated the extent to which Spn1 and other RNAPII-associated factors maintain nucleosome features over genes of varied characteristics. The study of factors described in this thesis is performed in living cells, which have been genetically modified to express mutant alleles of chromatin factors. This thesis also describes a course-based undergraduate research experience (CURE) developed to introduce upper-level biochemistry students to techniques in yeast genome engineering in an authentic research setting.
  • Thumbnail Image
    ItemOpen Access
    Studies on selenium hyperaccumulator Stanleya pinnata and nonaccumulator Stanleya elata (Brassicaceae): functional characterization of selenate transporter SULTR1;2 in yeast and development of a micropropagation protocol
    (Colorado State University. Libraries, 2017) Guignardi, Zackary S., author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Santangelo, Thomas, committee member
    Stanleya pinnata is an herbaceous perennial species in the family Brassicaceae native to the western United States. This species is classified as a selenium (Se) hyperaccumulator, and can be found thriving on Se-rich soils. Selenium hyperaccumulators are plant species that have the capacity to accumulate Se over 1,000 mg kg-1 dry weight in their tissues, concentrations toxic to non-accumulator plant species as well as to herbivores and pathogens, which may explain why plants hyperaccumulate Se. Due to the chemical similarity of Se to sulfur (S), Se is believed to be transported and metabolized by the same proteins and enzymes, including sulfate transporters and the sulfate assimilation pathway. Selenate (SeO42-), the predominant available form of Se in soil, is transported into the roots mainly via the high-affinity membrane transporter SULTR1;2. While most plants do not appear to discriminate between selenate and sulfate, and the two compounds compete for uptake, selenate uptake in Se hyperaccumulators is less inhibited by high sulfate concentrations. Since SULTR1;2 is the main portal of entry for selenate into the plant, it may be hypothesized that SULTR1;2 from the Se hyperaccumulator S. pinnata has intrinsic properties that allow this species to discriminate between sulfate and selenate and preferentially take up selenate. One of the objectives of this thesis project was to test this hypothesis, by means of functional characterization of SULTR1;2 from S. pinnata and from control species Stanleya elata, and Arabidopsis thaliana in the YSD1 yeast mutant which lacks its native sulfate transporters. A secondary objective in this thesis project was to develop a micropropagation protocol for Stanleya. In order to effectively study Se hyperaccumulation in a laboratory setting, sufficient numbers of S. pinnata and S. elata plants need to be available. However, due to low rates of seed germination, vernalization requirements, self-incompatibility, and ineffectiveness of propagation by cuttings, conventional propagation methods via seed or vegetative cuttings severely limit the number of plants that can be cultivated at a time. In order to overcome these limits, a tissue culture micropropagation protocol for leaf explants of S. pinnata and S. elata was developed. This protocol will allow for the rapid reproduction of both Stanleya species, not only to be used in laboratory experiments, but also in industrial applications such as Se phytoremediation projects, as well as for horticultural and native landscaping purposes. The first chapter of this thesis reviews plant Se uptake and metabolism, offering an overview of the current understanding of the Se assimilation pathway in plants, including mechanisms of accumulation and tolerance unique to Se hyperaccumulators. This chapter also outlines key proteins and enzymes in the Se assimilation pathway that are candidates for future experiments to determine the mechanisms of Se hyperaccumulation. The second chapter describes the results from yeast studies, characterizing the selenate and sulfate transport capabilities of SULTR1;2 from hyperaccumulator S. pinnata and non-accumulators S. elata, and A. thaliana, and their selenate specificity, as judged from the effects of sulfate competition on selenate uptake. Interestingly, yeast transformed with SULTR1;2 from S. pinnata (SpSultr1;2) showed less inhibition of selenate uptake by high sulfate concentration, indicating that this species' selenate selectivity may be facilitated by the SULTR1;2 protein. While apparently more Se-specific, yeast transformed with SpSultr1;2 overall took up less Se when compared to yeast expression SULTR1;2 from non-accumulators. It is feasible that a mutation that changes the substrate specificity of SpSULTR1;2 also reduced its overall activity. In S. pinnata, SpSultr1;2 transcript was found in earlier studies to be ~10-fold up-regulated when compared to S. elata, which may compensate for decreased activity. Identification of a selenate-specific transporter has applications for Se phytoremediation and biofortification. Constitutive overexpression of a hyperaccumulator selenate transporter in other plant species may increase their uptake of Se, even in the presence of high environmental S levels. The third chapter of this thesis outlines the development of a fast and efficient tissue culture micropropagation protocol for S. pinnata and S. elata. Through the testing of multiple concentrations of hormones on in vitro callus formation, shoot induction and elongation, and root formation, followed by ex vitro acclimatization, both species of Stanleya were shown to be very amenable to micropropagation. Both exhibited rapid callus, shoot, and root induction under a wide range of 1-napthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), and indole-3-butyric acid (IBA) concentrations. Future experiments could explore the genetic transformation of S. elata plants with genes from S. pinnata to test their importance for Se accumulation and tolerance in this related non-accumulating species. This micropropagation protocol also opens up the possibility to cultivate the Stanleya species at a large scale for multiple applications including biofortification, phytoremediation, and native landscaping.