Biochemistry electronic theses and dissertations (MU)
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The items in this collection are the theses and dissertations written by students of the Division of Biochemistry. Some items may be viewed only by members of the University of Missouri System and/or University of Missouri-Columbia. Click on one of the browse buttons above for a complete listing of the works.
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Item Novel roles of dynamin-related protein in iron-deficiency in Arabidopsis thaliana(University of Missouri--Columbia, 2024) Antoine-Mitchell, Alani; Heese, AntjePlants have developed sophisticated mechanisms to maintain optimal homeostasis for their survival in diverse environmental conditions. Among these mechanisms, the modulation of the plasma membrane (PM) proteome stands out as a crucial adaptation strategy. The PM serves as a dynamic interface essential for perceiving and adapting to extra- and intracellular stimuli, necessitating precise control over the composition of its proteome. One such control that is central to the fine-tuning of the PM protein composition is achieved through vesicular trafficking. Vesicular trafficking encompasses the sorting and trafficking of cargo proteins to and from the PM via the central intracellular sorting hub trans-Golgi network/early endosomes (TGN/EE). However, the roles of components involved in the post-Golgi trafficking of proteins required for critical cellular processes remain largely unknown. Here, we utilized the model plant Arabidopsis thaliana (Arabidopsis) as well as biochemical and transcriptomic approaches to uncover novel roles of the vesicular trafficking protein DYNAMIN-RELATED PROTEIN1A (DRP1A) in modulating the accumulation of proteins and cellular components essential for physiological processes including nutrient deficiency. In Arabidopsis, DRP1A is a plant-specific large GTPase that belongs to a family of sixteen dynamin-related proteins (DRPs). DRPs are divided into six subfamilies (DRP1-6) based on their domain structure and functions in fission of organelles and/or membrane vesicles. DRP1A is the best-studied of the five DRP1s (DRP1A-E) and studies have shown that loss-of-function drp1a mutants are defective in cytokinesis and constitutive bulk membrane endocytosis. Previous studies from our lab identified DRP1A in modulating the proper PM abundance of the leaf-specific immune receptor FLAGELLIN SENSING2 (FLS2) after elicitation with the bacterial pathogen-associated molecular pattern (PAMP) flg22. Underscoring its role as an endocytic accessory protein, drp1a mutants are impaired in the flg22-induced endocytosis of FLS2. DRP1A is also essential for the polar PM localization of root-specific hormone efflux carrier PINFORMED1 (PIN1) and the nutrient transporter BORON TRANSPORTER1 (BOR1) to specific subdomains of the PM. Given the significance of DRP1A in maintaining abundance and polar localization of PM proteins, we sought to identify other PMlocalized proteins that may be modulated by DRP1A. Vesicular trafficking regulates the PM abundance and polar localization of the root-specific iron (Fe) transporter IRON REGULATED TRANSPORTER1 (IRT1). While few vesicular trafficking components have been identified in this process, this study identified a novel role for DRP1A in modulating the abundance of IRT1 protein and mRNA under various growth conditions. We discovered that loss of DRP1A led to reduced PM accumulation of IRT1 which correlated with reduced Fe in root tissue of drp1a mutant seedlings as well as reduced chlorophyll content and protein accumulation of photosynthetic proteins that require Fe for their function and/or stability. Conversely, we uncovered that drp1a mutants showed increased protein accumulation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), potentially leading to increased intracellular reactive oxygen species (ROS). In turn, increased intracellular ROS may correlate with disrupting Fe-deficiency pathways, indicating a role for DRP1A in Fe homeostasis and stress responses. Under Fe-deficiency, IRT1 must be trafficked and reside in the soil-side of the PM, called outer domain, for uptake of Fe from the rhizosphere as needed; but IRT1 must be removed from the PM through endocytic internalization to prevent excess Fe uptake, which can be toxic. When exploring the physiological impact of loss of DRP1A during Fe-deficiency, we uncovered that under both continuous Fe-deficiency and induced Fedeficiency, drp1a mutants had reduced PM accumulation of IRT1 as well as reduced accumulation of PHOTOSYNTHETIC ELECTRON TRANSFER C, a photosynthetic protein that requires Fe as part of its Fe-S cluster to transport electrons from Photosystem II to Photosystem I. Additionally, we provide evidence that DRP1A played a role in modulating the PM abundance of RBOHD that appeared to be independent of induced Fe-deficiency. Taken these findings together, this study provides new insight on the role of DRP1A in modulating Fe-deficiency responses in plants, though much remains unknown about the underlying cellular and molecular mechanisms. Lastly, through qRT-PCR and RNA-seq analysis, we discovered that loss of DRP1A altered the transcriptional response of a subset of Fe-response genes, specifically the major Fe-deficiency transcription factor FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR (FIT) and its downstream targets IRT1 and FERRIC REDUCTASE OXIDASE2 (FRO2). Our findings revealed DRP1A's specific role in potentially modulating the transcriptional response to Fe-deficiency in roots at the level of FIT during induced Fe-deficiency. These genes are root-specific Fe-response genes and leaf-specific Fe-response genes such as OLIGOPEPTIDE TRANSPORTER3 (OPT3) and POPEYE (PYE) were not significantly altered in the leaf, we potentially identified a role for DRP1A in propagating the yet unknown Fe-deficiency signal within the roots, leading to reduced FIT gene expression. These findings highlight an essential role of DRP1A in the modulating the abundance of membrane proteins and transcriptional regulation of a subset of genes many of which have roles in maintaining Fe-homeostasis. With increasing global food demands, understanding the role of vesicular trafficking in Fe uptake and utilization is critical for developing sustainable, nutrient-rich crops to combat malnutrition and food insecurity.Item Structural studies of domain of unknown function 507 (DUF507)(University of Missouri--Columbia, 2023) McKay, Cole Ethan; Tanner, John J.The crystal structure of the domain of unknown function family 507 protein from Aquifex aeolicus is reported (AaDUF507, UniProt O67633, 183 residues). The structure was determined in two space groups (C2221 and P3221) at 1.9 A resolution. The phase problem was solved by molecular replacement using an AlphaFold model as the search model. AaDUF507 is a Y-shaped a-helical protein consisting of an anti- parallel 4-helix bundle base and two helical arms that extend 30-A from base. The two crystal structures differ by a 25 degrees rigid body rotation of the C-terminal arm. The tertiary structure exhibits pseudo-twofold symmetry. The structural symmetry mirrors internal sequence similarity: residues 11-57 and 102-148 are 30 percent identical and 53 percent similar with an E-value of 0.002. In one of the structures, electron density for an unknown ligand, consistent with nicotinamide or similar molecule, may indicate a functional site. Docking calculations suggest potential ligand binding hot spots in the region between the helical arms. Structure- based query of the Protein Data Bank revealed no other protein with a similar tertiary structure, leading us to propose that AaDUF507 represents a new protein fold.Item Roles of post-Golgi vesicular trafficking components in plant growth and immunity(University of Missouri--Columbia, 2023) Mason, Kelly; Heese, Antje[EMBARGOED UNTIL 12/1/2024] Delineating the molecular mechanisms of plant immunity can help inspire novel ways to engineer more resistant plants which could contribute to reducing crop loss due to pathogenic infection. The plasma membrane (PM) is a crucial contact point for plant defense as various proteins localized to the PM function in the perception of pathogens within the apoplast, initiation and attenuation of immune signaling, and production of defense related components. Thus, during the preparation and execution of plant defense responses, the PM must be tightly controlled to ensure an effective immune response. One method of modulating the PM composition is through vesicular trafficking via clathrin-coated vesicles (CCVs). Vesicular trafficking is the coordinated movement of cargo from one organelle to another via membrane-bound vesicles. CCVs function in post-Golgi trafficking and form at both the PM and trans-Golgi network/ early endosome (TGN/EE). The TGN/EE serves as an important sorting station for CCV trafficking by ensuring that proteins are directed to the correct functional location. However, the roles of TGN/EE-localized vesicular trafficking components in coordinating cellular and biological processes remain largely unexplored. Here, we examine the role of four vesicular trafficking components using reverse genetics and biochemical approaches to examine whether individual or combinatorial loss of these components impacts plant growth or immunity in the model plant Arabidopsis. Firstly, I presented a protocol for the staining and automated quantification of callose deposition in Arabidopsis seedlings and mature leaves after elicitation. Callose deposition is a defense response that is widely utilized to investigate defects in the plant immune signaling pathway. For instance, I used this technique throughout Chapter 4 to investigate a preformed callose deposition defect in Arabidopsis mutant seedlings. Additionally, the quantification methodology is valuable to the broader research community as it could be applied to quantify other cellular features. Our lab previously identified the CCV component EPSIN1 (EPS1) as a positive regulator of plant immunity against the pathogenic bacterium Pseudomonas syringae pv tomato (Pto) DC3000. EPS1 is a monomeric clathrin adaptor localized to the TGN/EE implicated in trafficking to the PM and vacuole. However, we found that the TGN/EE- localized adaptor MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) does not have an apparent function in bacterial immunity against Pto DC3000. EPS1 and MTV1 are Epsin N-terminal Homology (ENTH)-domain containing adaptors that are predicted to form a conserved tertiary structure typical of the ENTH domain. Thus, we attribute diverse biological functions in immunity likely to differences in protein primary structure demonstrated by the low amino acid similarity. Previous results from our lab confirmed that EPS1 was in-complex with the clathrin coat component, CLATHRIN HEAVY CHAIN2 (CHC2). Additionally, CHC2 and EPS1 displayed a synergistic genetic interaction in preformed callose deposition that was independent of elevated salicylic acid. Here, I further investigated the preformed defense responses in chc2 eps1 double mutant seedlings by finding that, in contrast to the upregulation of the immune marker gene PATHOGENESIS-RELATED 1 (PR1), the preformed callose deposition was also independent of continuous light during growth. Notably, we uncovered that the preformed callose deposition was dependent on CALLOSE SYNTHASE12 (CALS12)/POWDERY MILDEW RESISTANT4 (PMR4), which may be linked to increased PMR4 protein at the PM of chc2 eps1 seedlings. We also found that preformed callose deposition, independent of elevated PR1 mRNA, did not contribute to bacterial immunity against Pto DC3000, further elucidating the role of preformed callose deposition. EPS1 also biochemically interacts with VESICLE TRANSPORT V-SNARE 11 (VTI11)/ZIG, so we isolated the eps1 zig double mutant to explore the genetic interaction in plant growth and immunity. We discovered that EPS1 and VTI11/ZIG behaved independently or redundantly depending on the different stages of growth or plant organ. Furthermore, EPS1 and VTI11/ZIG displayed a synergistic interaction in plant immunity against the pathogenic bacterium Pto DC3000. Notably, we uncovered a novel role for VTI11/ZIG in modulating the total protein abundance of the immune receptor FLAGELLIN SENSING 2 (FLS2), that will be explored further in future studies. Lastly, I generated tools that will be essential for investigating the physiological role of the ENTH domain of EPS1 in addition to examining biochemical interactions with EPS1. We successfully generated an antibody against the ENTH domain of EPS1 that will be useful for in planta Arabidopsis studies and potentially to confirm an orthologous maize EPS1 mutant. Additionally, I developed recombinant full-length and variant proteins of EPS1, VTI11, and VTI12 that can be utilized to investigate biochemical interactions after optimizing or redesigning the attempted pull-down assays. Altogether, this work advanced the limited understanding of TGN/EE-localized vesicular trafficking components in modulating various plant processes including plant growth, bacterial immunity, and alteration of the PM proteome.Item Structural and biochemical studies of virus : host interactions in HIV-1(University of Missouri--Columbia, 2023) Paz Herrera, Amanda MarĂa; Heng, XiaoAccording to 2021 UNAIDS estimates, as of 2020, 680,000 people died from AIDS-related illnesses and 38 million people globally were living with Human Immunodeficiency Virus (HIV) (1). Despite advances in antiretroviral therapy, HIV's ability to remain dormant within the host and its rapid drug resistance acquisition presents an urgent need for the development of new therapeutics (2). Reverse Transcriptase (RT), is an error-prone protein that catalyzes the conversion of genomic viral RNA into DNA that can be integrated into the host. Through this process, it incorporates mutations which lead to the emergence of drug resistant HIV (3). Understanding the molecular details of reverse transcription is paramount to identify new therapeutic targets. We have previously reported that host RNA helicase A (RHA/DHX9) is recruited into HIV-1 virions and promotes virion infectivity by enhancing the processivity of RT (4). Our preliminary studies have suggested that RHA is likely to form direct interactions with the reverse transcription complex. This dissertation focuses on characterizing interactions among key components in reverse transcription: HIV's genomic RNA, HIV RT, and host factor RHA. In addition, we examine the role of Rocaglamide (RocA) as a potential drug targeting RNA : viral protein interactions required for packaging and assembly. Our findings provide insights on binding interfaces that can be exploited for rational drug design.Item DotB : Legionella pneumophila's Dot/Icm Type IV Secretion System's AAA+ motor(University of Missouri--Columbia, 2023) Riley, Ciairra Jordan; Hannink, MarkThe Gram-negative bacteria Legionella pneumophila is the causative agent of a severe form of pneumonia known as Legionnaire's Disease. Legionella utilizes its Defect in organelle trafficking/Intracellular multiplication Type IV Secretion System (Dot/Icm T4SS) to secrete over 300 effector proteins into the cytoplasm of alveolar macrophages for infection. The Dot/Icm T4SS has a Type IV Coupling Complex (T4CC) which has been identified to help recruit effector proteins for translocation during infection. However, not all effector proteins are translocated by the T4CC. DotB is a cytosolic ATPase from the pathogenic Legionella pneumophila and is suggested to mediate translocation of specific effector proteins during infection. DotB is a member of the ATPases associated with diverse cellular activities (AAA+) protein family. Characterization of DotB thus far has revealed its ability to form a complex with other T4SS-components and to function as an ATPase that plays a role with translocation. However, much remains unknown about the function of DotB. Earlier studies suggest that DotB may not only function to translocate proteins but can also help mediate other processes such as plasmid transfer and cytotoxicity. Thus, there is a need to further study and understand what proteins DotB can translocate. This review gives insight into the functions DotB may have within Legionella and suggests experimental approaches to further characterize DotB's role in the translocation of effector proteins.