Now showing items 61-80 of 210

    • Investigating the Paxillin and Hic-5 Interactomes

      Turner, Christopher; Alpha, Kyle (2022-06)
      Focal adhesions are macromolecular structures that connect the cellular actin cytoskeleton to the surrounding extracellular matrix through transmembrane integrin receptors and the action of numerous proteins, including enzymes, signaling proteins, and scaffolding proteins. Paxillin and Hic-5 are two scaffolding adapter proteins that primarily localize to focal adhesions, as well as other intracellular regions including the nucleus and centrosome. Their signaling partners at focal adhesions and effects on the actin cytoskeleton have been well-characterized over the course of decades through rigorous biochemical studies, but broader analysis and comparison of the interactomes of these two closely related proteins has not yet been thoroughly pursued. In the introduction, recently described roles for paxillin and Hic-5 in regulating cell shape and invadopodia through actin dynamics will be described, in addition to recent discoveries regarding their interaction with the microtubule and intermediate filament cytoskeletons. This background will provide context to data presented in chapter two, in which paxillin and Hic-5 were used as baits for proximity labeling to compare their interactomes, to identify numerous potentially novel interactors for both proteins, and to confirm many previously known partners. Two interesting results of this analysis include a possible proximity interaction between both paxillin and Hic-5 with septin-7, and confirmation of a robust proximity reaction between paxillin and ponsin. In chapter three, another newly discovered interactor with paxillin will be characterized: the formin mDia1. These proteins are shown to bind in vitro and co-localize in vivo. Most interestingly, paxillin relieves mDia1 auto-inhibition to accelerate actin polymerization in in vitro TIRF microscopy assays. Finally, the significance of these results and avenues for future investigation will be discussed, including further confirmation of proximity interactors and experiments to better understand the mechanism by which paxillin interacts with mDia1.
    • Regulation of fibrosis through the ubiquitin pathway

      Bernstein, Audrey; Phillips, Andrew (2022-08)
      Scarring in the cornea obstructs the refraction of incoming light onto the retina causing visual disability. Both acute scarring and chronic fibrosis are characterized by an accumulation of disorganized extracellular matrix (ECM). Disorganized ECM is deposited into the wound by specialized cells termed myofibroblasts. Pathological myofibroblasts are characterized by the expression of the highly contractile alpha smooth muscle actin (a-SMA) and the av-family of integrins (avb1,b3, b5, b6). The persistence of myofibroblasts in a healing wound promotes an autocrine loop of TGFb activity, over contraction of tissue, deposition of fibrotic ECM proteins, and ultimately the generation of scar tissue. My work is focused on the relative contribute of the deubiquitinase, USP10 to scarring in the cornea. I found that after wounding an increase in the expression of USP10 leads to deubiquitination of integrins and a subsequent increase in integrin recycling and matrix deposition. Knockdown of USP10 in vivo after corneal wounding significantly reduced the presence of myofibroblasts and immune cells in the healing wound, and corneal scarring. Through a yeast 2-hybrid screen I also identified a novel USP10 interacting protein, the formin Daam1. I found that Daam1 sequesters USP10 on actin stress fibers inhibiting its activity. Under pathological conditions, the expression of both USP10 and Daam1 are increased. My data suggest that Daam1 acts as a cellular reservoir, adding a layer of homeostatic control over USP10 activity and integrin function. Although defects in protein degradation have been identified as a major contributor to many diseases, together, my studies indicate that protein degradation (ubiquitin) pathways need to be considered in the context of integrin biology and in the pathogenesis of fibrotic healing.
    • Characterization of the effects of steroid-resistant nephrotic syndrome associated MYO1E mutations on myosin 1e activity and podocyte functions

      Krendel, Mira; Liu, Pei-Ju (2022-08)
      MYO1E mutations are associated with familial pediatric nephrotic syndrome, a disease in which pharmaceutical treatment is limited to immunosuppressive drugs such as steroids and cyclosporine but still often fails to prevent progression to end-stage renal disease. While Myo1e deficiency leads to glomerular filtration abnormality, including podocyte foot process effacement and glomerular basement membrane thickening, developing a precise treatment for this disease is hindered, as the underlying mechanisms linking MYO1E mutations and nephrotic syndrome are unclear. With the power of whole genome and exome sequencing, multiple novel Myo1e variants have been rapidly identified from SRNS cohorts. To determine whether a MYO1E mutation identified in patients is likely to be pathogenic or benign, we have characterized the functional effects of novel sequence variants in cultured podocytes. Differential protein degradation, localization, endocytic and motor activities of Myo1eT119I and Myo1eD388H have been discovered (Chapter 2). Specifically, even when expressed as a full-length protein, Myo1eT119I is deficient in localization to podocyte junctions and clathrin-coated vesicles (CCVs). Consistent with the hypothesis that Myo1eT119I is a loss-of-function mutant, cells expressing Myo1eT119I exhibit decreased CCV density and prolonged CCV lifetimes. The junctional and CCV localization of Myo1eD388H is not affected but it exhibits increased association with structures in the membrane-actin interface. Most importantly, Myo1eD388H is deficient in ATP hydrolysis and actin filament translocation. We have also characterized other MYO1E variants to provide cell-based evidence to assist in the curation of variants of uncertain significance (Chapter 3). Unexpectedly, while Myo1eD185G may be considered as a likely benign variant based on the population and computational predictions, it exhibits prolonged association with the podocyte junctions, while no junctional localization and dissociation abnormality was found in a likely deleterious variant, Myo1eR523W. We also discovered that localization of Myo1edel3094-7 to the podocyte junctions can be partially restored with a proteasomal inhibitor treatment, which may be considered as a potential treatment for patients with this variant. Finally, to follow up on the questions derived from Chapter 2, we examined our hypothesis that Myo1eD388H tail is constitutively active, we demonstrated the similar junctional exchange but differential endocytic activity of Myo1eD388H and Myo1eTAIL, revealing the critical regulation of Myo1e motor to tail domain when it comes to clathrin-mediated endocytosis (Appendix 1). Overall, the studies documented here have uncovered the differential molecular defects of steroid resistant nephrotic syndrome (SRNS)-associated Myo1e variants and further elucidated the underlying mechanisms of podocyte disease.
    • Examination of the effects of Myosin-1e expression on tumor behavior and computational analysis of MYO1E mutations associated with kidney disease

      Krendel, Mira; Plante, Eric (2022-08-19)
      Actin and actin cytoskeleton associated proteins are important for maintenance of cellular homeostasis and cellular processes such as cell polarization, cell movement, and endocytosis. The long-tailed class one myosin, Myosin-1e (Myo1e), is an actin dependent molecular motor that is found to be expressed in and contribute to the function of epithelial cells. Specific mutations of Myo1e are associated with dysfunction of specialized kidney epithelial cells known as podocytes and increased incidence of kidney disease. We found that disease associated mutation of Myo1e affects the conformation of key functional domains within the Myo1e protein structure using molecular dynamic simulations (Chapter 2). This finding leads to a more mechanistic understanding of dysfunctional Myo1e protein conformational pathways that may be associated with kidney disease. We also find that Myosin-1e expression influences the behavior of mammary tumors in mice (Chapter 3). Specifically, mammary epithelial cell oncogenic transformation induced using the MMTV-PyMT oncogene (mouse mammary tumor virus, polyoma middle T-antigen) was phenotypically altered in mice lacking Myo1e expression compared to Myo1e expressing mice. The tumor cells in mice lacking Myo1e were more differentiated and expressed transcriptomic profiles associated with tumor suppression compared to the more oncogenic profiles of Myo1e expressing cells. In-vitro analysis revealed a cell autonomous effect of Myo1e expression on cell-cell junctional strength and gene expression associated with differentiation (Chapter 3). Appendix chapters 1 and 2 discuss experiments examining Myo1e expression on in-vitro cell migration and epithelial to mesenchymal transition (EMT). Overall, experiments discussed in this thesis have uncovered insight into the effects of Myo1e expression on breast cancer progression, cell differentiation and motility. Moreover, experiments in this thesis also contribute to further understanding of the dynamics of protein conformation associated with disease mutations of a class one myosin.
    • The impact of mosquito salivary factors on Chikungunya virus transmission

      Thangamani, Saravanan; Esterly, Allen (2022-08-12)
      Vector-borne diseases affect an estimated 3.9 billion people in over 128 countries annually (WHO, 2017b). Mosquito-borne viruses, such as Chikungunya virus (CHIKV), are transmitted to human hosts through the saliva of female blood-feeding mosquitoes. During blood-feeding, salivary molecules enhance blood-feeding efficiency, dampen specific inflammatory signals, and enhance virus infection and dissemination. Skin is the interface where mosquito salivary molecules and viruses interact with the human immune system. Our long-term goal is to understand the impact of mosquito saliva on the skin and the role of salivary molecules in potentiating virus infection. This thesis aims to model the human skin bite site, identify mosquito salivary molecules present during virus infection, and investigate their impact on virus replication. We developed a human skin arbovirus infection model that was viable for four days ex vivo (Esterly et al., 2022a). Arbovirus infection in human skin ex vivo allowed flavivirus and alphavirus replication and dissemination. Using this model, we will investigate mosquito salivary factors and their impact on virus transmission. Next, we identified mosquito salivary factors highly expressed in Ae. aegypti salivary glands during CHIKV infection. We hypothesize that salivary proteins that contain secretion signals and are found to be highly expressed during CHIKV infection are delivered to the bite site during mosquito feeding. Through a combination of RNA-seq and miRNA-seq analysis, we identified several salivary factors, protein and small non-coding RNAs, which warrant further investigation. Lastly, we assessed the impact of identified and recombinantly expressed proteins or specific small RNA inhibitors on CHIKV replication in vitro relative to Ae. aegypti salivary gland extract (SGE). We found that both protein and small RNAs can influence CHIKV replication in a significant manner; however, the effect observed by mosquito saliva is a combination of multiple factors and will require more investigation to characterize fully. In summary, mosquito salivary factors work in concert to enhance virus replication and dissemination. Although individual factors may not recapitulate the total effect of SGE, the discovery and isolation of these factors further our understanding of arbovirus transmission. These studies highlight the complexity of mosquito salivary secretion and further our knowledge of mosquito-borne virus transmission. The following thesis will describe our most recent findings and contribution to understanding mosquito saliva's role during arbovirus transmission.
    • Biomimetic extracellular matrix hydrogels to model and investigate conventional outflow cell biology under normal and simulated glaucomatous conditions

      Herberg, Samuel; Li, Haiyan (2022-06)
      Dysfunction of the conventional outflow pathway (comprised of the trabecular meshwork (TM) and adjacent Schlemm's canal (SC)) is the principal cause of elevated intraocular pressure in primary open-angle glaucoma. Other in vitro TM model systems cannot accurately mimic the cell-extracellular matrix (ECM) interface, limiting their use for investigating glaucoma pathology. In this dissertation, we report a novel biomimetic hydrogel by mixing donor-derived human TM (HTM) cells with ECM proteins found in the native tissue. We demonstrated that this HTM hydrogel system allowed for investigation of actin arrangement, ECM remodeling, cell contractility, and HTM stiffness on a simulated tissue level (Chapter 2). Furthermore, we showed that TGFβ2-induced ERK signaling negatively regulates Rho-associated kinase-mediated phospho-myosin light chain expression and HTM cell contractility when cultured on soft ECM hydrogels but not on glass (Chapter 3). YAP and TAZ are important mechanotransducers implicated in glaucoma pathogenesis. We demonstrated that YAP/TAZ activity was upregulated by transforming growth factor beta 2 (TGFβ2) in both HTM and HSC cells cultured on/in ECM hydrogels (Chapters 4 and 5). It is widely accepted that the glaucomatous TM/SC interface is stiffer. To mimic the stiffness difference between diseased and healthy tissue, we utilized two different methods. In Chapter 4, riboflavin was used to facilitate secondary UV crosslinking of collagen fibrils and stiffen the matrix. We showed that ECM stiffening elevated YAP/TAZ activity in HTM cells through modulating focal adhesions and cytoskeletal rearrangement. In Chapter 6, we developed an ECM-alginate hybrid hydrogel system, which allowed for on-demand control over matrix stiffness during the culture of cells. We found that the stiffened matrix increased nuclear YAP and filamentous-actin fibers in HSC cells, which was completely reversed by matrix softening. We further demonstrated that YAP/TAZ inhibition could rescue HTM/HSC cell dysfunction induced by either TGFβ2 or stiff matrix (Chapters 4, 5, and 6). Finally, we showed that pharmacologic YAP/TAZ inhibition had promising potential to improve outflow facility in an ex vivo mouse eye perfusion model (Chapter 6). Collectively, we have developed bioengineered ECM hydrogels for modeling and investigation of conventional outflow cell-ECM interactions under normal and simulated glaucomatous conditions.
    • A dual to the death: using novel host-directed antivirals to promote death of HCMV-infected myeloid cells through apoptosis and necroptosis

      Chan, Gary; Cheung, Jennifer (2022-06)
      Human cytomegalovirus (HCMV) is a ubiquitous member of the betaherpesvirus family, with seroprevalence rates ranging from 40-100% worldwide. Although primary infection is asymptomatic in most immunocompetent patients, HCMV is a significant cause of morbidity and mortality in the immunosuppressed and immunonaїve, including transplant recipients, patients with AIDS, and developing fetuses in utero. The diverse clinical presentations of HCMV are attributable to the pervasive systemic dissemination and extensive cellular tropism of the virus. Peripheral blood monocytes are believed to be the key cells responsible for HCMV dissemination from the initial site of infection to distant organ systems. Monocytes are normally short-lived, surviving for only 48 h in circulation before undergoing apoptosis. Previous work from our lab has shown that HCMV circumvents the short lifespan of monocytes by inducing a noncanonical activation of Akt to upregulate the expression of antiapoptotic proteins, thereby prolonging survival of infected monocytes. HCMV promotes survival in the absence of viral replication and lytic gene expression, rendering current direct-acting antivirals ineffective against quiescently infected monocytes. There are currently no antivirals that target quiescent or latent HCMV infection. We hypothesize that targeting host proteins that are essential for HCMV's induction of monocyte survival mechanisms will reduce viability of infected monocytes, ultimately reducing systemic viral dissemination. The studies in this thesis investigate novel host-directed antivirals targeted at two different cellular factors and their efficacy against quiescent HCMV infection in monocytes. The first host protein under scrutiny as an antiviral target is Sirtuin 2 (Sirt2), an NAD+-dependent deacetylase. Treatment with novel Sirt2 inhibitors promoted death of HCMV-infected monocytes as a cellular antiviral defense response through two concurrent regulated death pathways: apoptosis and necroptosis. HCMV has developed mechanisms to impede both death pathways, but inhibition of Sirt2 relieves the viral obstructions on both pathways by disrupting HCMV's unique phosphorylation on Akt. The second host protein targeted as a potential antiviral strategy is Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins. HCMV-infected monocytes are dependent on Akt-dependent upregulation of Mcl-1 for survival during early infection. Treatment with Mcl-1 inhibitors blocked interaction between Mcl-1 and proapoptotic protein Bak, reducing viability of infected monocytes. Subsequent testing of Mcl-1 inhibitors in an ex vivo skin organ culture system resulted in a decrease in HCMV-infected cells that crawled out of the skin tissue, suggesting that Mcl-1 inhibition may reduce viral dissemination. Our studies lay down the groundwork for the investigation of novel host-directed antivirals, an approach that successfully targets quiescent HCMV infection in peripheral blood monocytes for the first time. Expanding the study of host-directed antivirals may bring the field one step closer to the possibility of a comprehensive antiviral regimen that is effective against all stages of viral infection.
    • Rational design of a genetically encoded fluorescent protein color switch using a modular, entropy-driven mechanism

      Loh, Stewart; John, Anna (2022-06)
      Engineered protein conformational switches have applications in cellular and in vitro biosensing, molecular diagnostics and artificial signaling systems in synthetic biology. They broadly consist of an input module and an output module that communicate via a conformational change. The overarching goal of this thesis is to tackle two major challenges in protein switch design - signal transduction, by coupling a target recognition domain to an output domain to produce a robust change in signal in addition to modularity, which allows the facile creation of sensors binding novel targets. Here, we attempted to test a rational design strategy that exploits two key protein engineering principles (1) loop entropy, by which long insertions into a loop of a host protein destabilizes the host due to an entropic cost associated with loop closure unless the inserted sequence adopts a folded structure; and (2) alternate frame folding (AFF), which allows a protein - green fluorescent protein variants(GFP), in this case - to switch between two mutually exclusive folds. Toward this goal, we first studied the effect of loop entropy at two different insertion sites in a GFP variant (chapter 2) using a well-characterized ribose binding protein as the input domain. We provide stability measurements using circular dichroism and fluorescence data to support our hypothesis of the application of the loop entropy principle in a GFP beta barrel scaffold. To provide a proof-of-concept of the combination of loop entropy and the AFF mechanism in a genetically encodable GFP scaffold, we chose an unstable, circularly permuted FK506-binding protein (cpFKBP) as the input recognition domain and inserted it in one of the two mutually exclusive folds of the GFP-AFF fusion protein (chapter 3). Upon addition of ligand, binding induced folding of the cpFKBP domain effects a conformational change in which the tenth beta strand of GFP exchanges, replacing Thr203 (green state) with Tyr203 (yellow state). We confirmed this mechanism in vitro by a ratiometric change in fluorescence output and observed that the process is slow and irreversible. We elucidate the biophysical principles underlying this mechanism by using denaturant and temperature to modulate the relative populations of the two folds in vitro. We also observed a faster and higher intensiometric response in mammalian cells which may be attributed to an alternate mechanism. We then harnessed this intensiometric response in a single fold of the fluorescent protein combined with a previously engineered monobody scaffold capable of binding a variety of targets (chapter 4). Altogether this work may have the potential to create a novel class of fluorescent protein biosensors comparable to existing single fluorescent protein-based biosensors currently available.
    • Targeting SHIP paralogs to promote microglial effector function in the CNS

      Thomas, Stephen J.; Pedicone, Chiara (2022-06)
      The two SH2-containing inositol 5'-phosphatases , SHIP1 (INPP5D) and SHIP2 (INPPL1), play an essential role in modulation of cellular signaling by transforming the PI3K product PI(3,4,5)P3 into PI(3,4)P2. PI3K signaling triggers activation of downstream signaling cascades that drive survival, effector functions, differentiation, and proliferation. SHIP1 can also mask the cytoplasmic tails of key receptors or their adaptor proteins such as DAP12, thus preventing PI3K recruitment to Trem2, a critical receptor for microglial function. Several GWAS studies correlated single nucleotide polymorphisms (SNPs) in INPP5D with Alzheimer's Disease (AD). However, it remains unclear whether these SNPs are deleterious or protective in AD and how they alter SHIP1 protein expression. SHIP2 overexpression has also been correlated with AD, suggesting that both SHIP1 and SHIP2 might be therapeutic targets. To study how SHIP1 and SHIP2 modulate microglial functions we used small molecule inhibitors and agonists of these enzymes. In our initial study we found that both SHIP paralogs are expressed in murine microglia and that Pan- SHIP1/2 inhibition increases lysosomal size and enhances microglial phagocytosis of Ab1-42 fibrils and dead neurons using both flow cytometry and confocal microscopy. Our lead Pan-SHIP1/2 inhibitor, K161, showed Blood Brain Barrier penetration as detected in the cerebral cortex of treated mice with mass spectrometry. K161 treatment of WT mice showed no difference in microglial frequency or lysosomal content in vivo; however, we observed a significant 2 increase in Ab1-42 and dead neurons phagocytosis ex vivo in microglia in K161- treated mice versus controls. Subsequently, we discovered a novel and highly potent SHIP1 selective agonist (K306) via artificial intelligence guided computational screening. We found that K306 can reduce the release of inflammatory cytokines in macrophages and microglial cells stimulated with LPS or Ab1-42. Interestingly, K306 didn't alter microglial phagocytic uptake of cargo, but did promote degradation of phagocytosed lipid-laden cargo - defining a novel role of SHIP1 in degradation of lipid cargo in microglia. These results highlight the importance of SHIP1 and SHIP2 in microglial biology and their modulation as therapeutics in different stages of neurodegenerative disease where microglia play a major role, such as AD.
    • Targeting wild-type and mutant p53 for cancer treatment

      Stewart N Loh; Blayney, Alan John (2021)