Recent Submissions

  • DevATLAS: A novel tool to monitor the sequence of neural circuit development and study neurodevelopmental disorders

    Brawner, Andrew (2025-04-04)
    Early postnatal brain development is the critical stage when the symptoms of many neurodevelopment disorders (NDDs) start manifesting. These functional deficits are often caused by abnormal neural circuit maturation without accompanying gross alteration to brain architecture, making it challenging to pinpoint disruptions in these NDD models. There is an urgent need for genetic tools to track the neural circuit maturation sequence on the whole brain level during the early postnatal period. One of the key driving factors of neural circuit maturation is neuronal activity. Our lab has developed DevATLAS, the Developmental Activation Timing-based Longitudinal Acquisition System, to overcome this challenge based on the immediate early gene Npas4 expression. Npas4 is selectively induced by neuronal activity, and its activation during development triggers activity-dependent synapse development, which is a critical step during the functional maturation of neural circuits. DevATLAS permanently labels neurons with tdTomato (tdT) as they are activated by neuronal activity to express Npas4. We demonstrate that DevATLAS captures the functional neural circuit maturation sequence across the whole brain during the early postnatal period. We also demonstrate that early environmental enrichment (EE) intervention can accelerate functional neural circuit development in the granule cells (GC) of the dentate gyrus (DG). Finally, we combine DevATLAS with the NDD model of Fragile-X Syndrome (FXS) and observe significant developmental perturbations in neural circuit maturation in multiple ASD-associated regions, including the dorsal striatum, primary motor cortex, medial prefrontal cortex, which can be associated with perturbed behaviors in juvenile FXS mice. Within the DG of FXS mice, we use DevATLAS to track perturbed neural circuit maturation with delayed emergence of contextual learning and memory with altered development of granule cell dendritic arbors and spines, as well as demonstrate how early EE can ameliorate. Our results indicate that DevATLAS can study neural circuit maturation in NDD models, enable researchers to gain an improved underlying NDD etiology, and ultimately help derive new therapeutic interventions to ameliorate NDD deficits.
  • Mitochondria-induced Bioenergetic Independent Stress Signaling in the Heart

    Arnav, Rana (2025-03-24)
    Mitochondria are well known for their function in providing energy supply to the cell. Aside from these "bioenergetic" functions, mitochondria perform many other essential processes. To accomplish these functions, mitochondria must import many proteins from the cytosol. This import process can sometimes become dysfunctional and induce a severe stress on the cell via the mistargeting of mitochondrial proteins to the cytosol. Our lab termed this specific type of cell stress as mitochondrial precursor overaccumulation stress (mPOS). Our work has focused on demonstrating that mPOS is able to occur in various models of disease, both in vivo and in vitro. In this thesis we demonstrate that mPOS can occur in the heart of mice and induce significant signaling and functional changes over the lifespan (chapter 2). Additionally, in a related work, we found that mitochondrial protein import clogging can induce mPOS in the central nervous system (CNS) and potentiate pathology in a mouse model of Parkinson's disease (appendix I). Most importantly, all the changes occurring in both animal models do not necessarily co-occur with bioenergetic deficiencies. The implication of this is that mPOS may be a bioenergetic independent mechanism liking mitochondrial dysfunction with tissue dysfunction. Speaking more generally, mPOS may occur in many clinically relevant conditions such as heart failure, normative ageing, muscle loss, and neurodegenerative diseases. This work and future work therefore aims to establish the basic mechanisms by which mPOS may occur within cells and how cells can in turn respond to this stress.
  • Engineering Generalized Protein-Based Biosensors for Molecular Detection and Clinical Applications

    Sekhon, Harsimranjit (2025-03-19)
    Protein-based conformational switches serve as powerful tools for the construction of biosensors and for the control of cellular processes. These proteins feature a binding domain that recognizes a specific analyte and is coupled to an output domain in such a way that the binding event causes the output domain to provide an observable signal. These signals can either be turn-on of fluorescence, luminescence, or enzymatic activity or consist of the sensor changing its color. A challenge in constructing these protein switches is finding binding domains capable of relaying a ligand binding event to the conformational change of an output domain. Generalized binding domains can address these challenges by providing a scaffold that can easily be modified to detect a different ligand. These generalized binding domains are small proteins with modifiable residues that can be selected to bind a ligand of choice, usually through phage display and similar selection techniques. Here, we present two approaches to make generalized protein switches. In the first approach, antibody mimetics nanobodies and monobodies are inserted in fluorescent proteins such that binding of their ligand causes an increase in fluorescence. This technique, named adaptable turn-on maturation (ATOM), was used to develop biosensors for WD-40 repeat protein 5 (WDR5), c-Abl src homology 2 (SH2) domain, hRas, postsynaptic density protein 95 (PSD95), gephyrin, HOMER1, and mCherry for use in mammalian cells. ATOM is, therefore, compatible with a variety of ligands due to its input domain being a generalized binding domain. Additionally, the ATOM mechanism can be used to convert many fluorescent proteins into biosensors. For demonstration, we made biosensors from Clover, mTurqoise, mTagRFP-t, mStayGold, mBaoJin, and GCaMP6s. In the second approach, we develop a luminescent protein switch from the enzyme nanoluciferase (nLucAFF) that switches color from green to blue upon DNA binding. We show that DNA-based devices can then be used to detect various ligands and relay that event to nLucAFF, which provides an output easily quantifiable by a cell phone. The nLucAFF protein was used to detect DNA sequences amplified from cytomegalovirus (CMV), dengue, and nCoV. Additionally, aptamers binding to serotonin and aptamers were used to detect these molecules by directing the nLucAFF color change. The initial version of nLucAFF was slow, dim, and had low sensitivity. These drawbacks were resolved in the next version, nLucAFF2, to achieve turn-on within 5 minutes and detect ligands down to 40 pM with a cell phone camera. The last chapter combines two ligand-binding domains to activate a small cytotoxic RNase, barnase, and paves the way for the development of multi-input protein switches that can potentially be generalized ligand-binding domains.
  • Nanobody development for therapeutically targeting Vacuolar H+-ATPases

    Knight, Kassidy (2025-03-18)
    The vacuolar H+-ATPase (V-ATPase, V1Vo) is a dedicated proton pump that is highly conserved amongst eukaryotes, and is necessary for pH homeostasis within subcellular compartments. The V-ATPase consists of two subcomplexes: the soluble V1 responsible for hydrolyzing ATP, and the membrane integral Vo responsible for proton translocation across membranes. V1 and Vo are each comprised of multiple subunits, A3B3CDE3FG3 and ac8c'c"def Voa1 respectively. Many basic cellular functions depend on the differential pH gradient across cellular membranes to operate properly, making regulation of V-ATPases through "reversible disassembly" immensely important. Global loss of V-ATPase activity is lethal to all mammalian cell types, while aberrant activity and incorrectly localized V-ATPase results in various disease states. Current therapeutics struggle to target specific V-ATPase populations, and as a potential solution to this problem we generated 94 nanobody clones against the yeast nanodisc reconstituted Vo (VoND). Nanobodies (Nbs) are the small 15 kDa VHH domain isolated from heavy-chain only antibodies that are known for their high specificity. In this dissertation we describe the characterization of three α-yeast VoND Nbs, N27, N125, and N2149. Using an ATPase assay, we found that N27, but not N125 or N21149, fully inhibited the activity of assembled V-ATPase. Contrastingly, N2149, but not N27 or N125, was found to inhibit the assembly of the two subcomplexes. BLI was used to identify the binding affinity of each Nb, with affinities being observed in the nM-pM range. High-resolution structures obtained from cryoEM revealed the subunit specificity of each Nb, with N27 and N125 found to bind the c-ring in different stoichiometries, and N2149 found to bind the d subunit. Furthermore, we determined that N125 has cross affinity for the human enzyme. Overall, this study provides evidence that novel nanobody mediated inhibition of assembly or activity of V-ATPases is an effective technique with broader implications of nanobody development into therapeutics.
  • Nanotherapeutics for Immune Modulation in Sepsis

    Messina, Jennifer (2025-03-04)
    Due to its complexity and heterogeneity, managing immune dysregulation in sepsis poses a significant clinical challenge. Thus, there is great demand to both improve our understanding of mediators of immune dysregulation in sepsis and develop nuanced therapeutic approaches to provide precise immune modulation for sepsis treatment. This thesis first investigates the novel phenomenon of cytokine charge disparity as a potential regulator of cytokine function. Then, two novel telodendrimer immune modulation approaches are presented as a personalized medicine strategy for sepsis. Through extensive database and literature review, we have established cytokine charge disparity as a potential mechanism for immune regulation. Using our versatile telodendrimers (TDs), we then optimized and validated our TD nanotrap approach for effective and selective targeting of plasma cytokines. Our lead selective TD nanotraps displayed charge selective cytokine targeting and our lead pan-affinitive TD nanotrap demonstrated superior cytokine removal efficacy compared to commercial resin control. Additionally, pan-affinitive TD nanotrap maintained efficacy across a wide range patient immune status, indicating promising therapeutic potential to reduce mortality risk associated with overwhelming cytokine profiles. To further expand our immune modulation tool set for sepsis treatment, we optimized our TD nanodrug for delivery of dimethyl itaconate (ITA) to control both hyperinflammation and pyroptosis. Encapsulating ITA into TD nanoparticles (ITA:TDNPs) resulted in a sustained-release profile and improved biocompatibility compared to free ITA. ITA:TDNPs more effectively inhibited both LPS- and LTA-induced inflammation and pyroptosis in macrophages compared to ITA or TDNP alone. Finally, ITA:TDNPs demonstrated superior therapeutic efficacy in both an IV LPS and polymicrobial cecal slurry sepsis model compared to individual therapies. Collectively, we have uncovered a novel phenomenon of cytokine charge disparity and validated it as a potential mechanism to regulate cytokine activity, as well as established it as targeting mechanism for effective immune modulation via charge selective TD nanotrap. We further developed an immune modulating TD nanodrug for ITA delivery to control both hyperinflammation and immune cell pyroptosis in sepsis. Through precise targeting of immune dysregulation in sepsis using a systematic multimodal TD therapeutic approach for personalized medicine, we may successfully improve patient outcomes in this devastating disease.
  • Mechanism of Mitochondria-Induced Muscle Atrophy During Aging

    Brennan, Nicholas (2025-02-27)
    Mitochondrial dysfunction is strongly associated with aging-related degenerative diseases including muscle atrophy. However, whether bioenergetic defects are the sole drivers of mitochondria-induced muscle atrophy remains unknown. The Chen lab discovered that various forms of mitochondrial damage can disrupt protein import, leading to the toxic accumulation of unimported mitochondrial precursors in the cytosol. This causes a stress termed mitochondrial Precursor Over-accumulation Stress (mPOS). A mouse model of mPOS was developed in which the mitochondrial inner membrane protein, ANT1, was overexpressed to saturate the protein import machinery. Ant1Tg/+ mice were found to have a severe muscle wasting phenotype. The overarching goal of this dissertation is to investigate the mechanism by which mPOS drives muscle wasting and its implications for normative muscle aging. The findings presented in this thesis led to three conclusions. First, we identified a novel mitochondria-to-lysosomal proteostatic axis through which mPOS induces lysosomal damage. Lysosomal damage subsequently causes the release proteolytic enzymes, which leads to excessive protein degradation and subsequent progressive muscle atrophy. Importantly, we found that this pathway operates independently of mitochondrial respiratory complex activity and reactive oxygen species (ROS) production. Second, we demonstrated the presence of mPOS in physiologically aged muscle. Sarcopenic muscle exhibited phenotypes similar to those found in Ant1Tg/+ mice, evidenced by overlapping transcriptional and proteomic profiles, and lysosomal damage. These findings indicate that mitochondria-induced changes to autophagic activity may play a central role in the pathogenesis of sarcopenia. However, considering the overall protein content of muscle is elevated during aging, we propose that reduced protein quality, rather than absolute protein content, drives sarcopenia. We therefore termed this phenomenon Muscle Atrophy Independent of Protein Content (MAIPC). Finally, we explored additional cellular factors that affect proteostasis and muscle mass maintenance. We found that the GCN2 kinase, a well-established activator of the Integrated Stress Response (ISR), plays a role in protecting myofibers from mPOS-induced stress and muscle wasting in Ant1Tg/+ muscle. Interestingly, we found that this effect is ISR-independent. The data presented in this dissertation provide valuable insights into the mechanistic role of mitochondrial dysfunction in both normative aging and chronic muscle wasting conditions. Our findings conclude that mitochondria-induced muscle atrophy is induced by mechanisms that extend beyond bioenergetic defects. By characterizing these alternative pathways, this work opens new avenues for therapeutic strategies targeting mitochondrial stress in chronic muscle wasting conditions.
  • Non-neutralizing Antibodies in the Complex Dance of Dengue Clearance and Immune Avoidance

    Waldran, Mitchell (2025-02-13)
    Dengue virus (DENV) is endemic in over 100 countries causing widespread morbidity and mortality. Approximately 400 million people are infected annually with one of the four immunologically and genetically distinct serotypes of DENV, resulting in 100 million symptomatic cases and at least 40,000 deaths. While the mechanisms behind the pathophysiology of severe DENV infection are complex and incompletely understood, it has been previously suggested that antibodies directed against the DENV envelope (E) protein can facilitate antibody dependent enhancement (ADE) of the virus during secondary DENV infections, increasing the number of infected cells and the clinical severity of infection in an exposed individual. However, there are other functional roles for antibodies outside neutralization of the virion. In this thesis, we describe the roles of non-neutralizing antibodies during DENV-infection. We show that IgG and IgA non-structural protein 1 (NS1)- and E-reactive antibodies are capable of mediating monocytic phagocytosis of DENV-infected cells. We show that secreted NS1 (sNS1) acts as immunological chaff and abrogates NS1-reactive antibody-mediated phagocytosis. We also begin to investigate the potential of phagocytosis of DENV-infected cells to lead to lead to infection of phagocytic monocytes. The findings described in these studies have implications in therapeutics and vaccinations targeting both NS1 and E protein.
  • Unveiling cell-type-specific transcriptome and genetic regulation in postmortem brains of schizophrenia patients

    Dai, Rujia (2024-12-12)
    Schizophrenia is a complex psychiatric disorder with a poorly understood etiology. This dissertation addresses three critical questions in schizophrenia research: identifying involved cell types, characterizing their transcriptomic changes, and elucidating how these changes mediate genetic risk. After rigorous evaluation, we conducted a comprehensive analysis of cell-type-specific gene expression in postmortem brains of schizophrenia patients and controls with single-cell RNA sequencing and cell deconvolution methods. Our findings provide compelling evidence for the involvement of upper-layer neurons and multiple non-neuronal cell types in schizophrenia. We observed significant alterations in synaptic function, neurodevelopment, immune response, and vascular transport within their respective cell types. Notably, we demonstrate that genetic risk for schizophrenia is predominantly enriched in neurons, particularly upper-layer neurons, with partial enrichment in oligodendrocyte precursor cells and vascular cells. This cell-type-specific approach offers novel insights into the molecular underpinnings of schizophrenia, potentially bridging the gap between genetic risk factors and clinical manifestations. By highlighting key genes and pathways, our study establishes a robust foundation for future research and opens avenues for innovative preventive and therapeutic approaches.
  • Mucosal Innate Immunity of Human Surfactant Protein A Genetic Variants against SARS-CoV-2 Infection

    Jacob, Ikechukwu (2024-11-18)
    More than 7 million people have died of the coronavirus disease-2019 (COVID-19) since first reported in December of 2019. Infection in some patients manifests as life-threatening ALI/ARDS, multi-organ dysfunctions, and/or death characterized by active viral replication and profound inflammatory cell influx into tissues/organs. SARS coronavirus-2 (SARS-CoV-2) infects human angiotensin-converting enzyme 2 (hACE2)-expressing cells through its spike protein (S protein). The S protein is highly glycosylated and could be a target for lectins. Surfactant protein A (SP-A) is a collectin, expressed by lung alveolar type II cells and other mucosal epithelial cells; it plays a crucial role in innate immunity and inflammatory regulation. SP-A modulates pathogenic infection and disease severity by binding to microbial and host glycoproteins to alter infectivity and regulate host inflammation. The human SP-A gene is located on chromosome 10q22-23, which contains two functional genes SP-A1 and SP-A2 (gene names: SFTPA1 and SFTPA2), and a pseudogene. SP-A1 and SP-A2 are highly polymorphic and consist of several genetic variants, such as SP-A1 (variants 6A2, 6A4) and SP-A2 (variants 1A0, 1A3). It has been demonstrated that these variants have differential antiviral and immunoregulatory capacities in response to various viral infections. The goal of this study was to investigate the mechanistic role of human SP-A variants in response to SARS-CoV-2 infection and COVID-19 susceptibility and severity. The results from this study showed that native human SP-A can bind SARS-CoV-2 S protein, receptor-binding domain (RBD), and hACE2 in a dose-dependent manner. A decrease in S protein and RBD binding was observed in the presence of EDTA and sugars, indicating that the SP-A carbohydrate-recognition domain (CRD) mediates S protein binding in a calcium-dependent manner. We further showed that human SP-A can attenuate viral infectivity in susceptible host cells, evidenced by the dose-dependent reduction in viral load in infected cells. These results suggest that human SP-A can bind SARS-CoV-2 S protein, RBD, and hACE2 to attenuate SARS-CoV-2 infectivity in susceptible host cells. Next, we examined the variations in antiviral and immunoregulatory roles of human SP-A variants in response to SARS-CoV-2 infection. The binding studies showed that in vitro-expressed SP-A variants differentially interact with S protein. Moreover, cells inoculated with SARS-CoV-2 pretreated with the 1A0 variant had a more reduced virus titer than those pretreated with the 6A2 variant, indicative of their differential antiviral capacities. These findings from in vitro studies demonstrated that human SP-A and their genetic variants directly interact with viral S protein to differentially modulate SARS-CoV-2 infectivity. To perform in vivo study, six genetically modified double-hTG mouse lines, expressing both hACE2 and the respective SP-A variants: (hACE2/6A2 (6A2), hACE2/6A4 (6A4), hACE2/1A0 (1A0), and hACE2/1A3 (1A3), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were generated and challenged intranasally with 103 PFU SARS-CoV-2 (Delta) or saline (Sham). We observed that these infected mice had differential COVID-19 severity. Infected KO and 1A0 mice had more mortality and lung injury compared to other mouse lines, and disease severity correlated with enhanced upregulations of inflammatory genes that play vital roles in host immunity such as MyD88 and Stat3 in the lungs of KO and 1A0 mice. Furthermore, pathway analysis identified several important signaling pathways involved in lung defense, including pathogen-induced cytokine storm, NOD1/2, toll-like receptor, neuroinflammation, and Trem1 signaling pathways. Consistent with the transcriptomic data, expressions of inflammatory mediators such as G-CSF, IL-6, and IL-1β were comparatively higher in the lungs and sera of KO and 1A0 mice with the highest mortality rate. We further examined other organ injuries (kidney, intestine, and brain) in the infected mice; we found a more severe acute kidney injury (AKI) and intestinal damage in KO and 6A4 mice compared to other double-hTG mice. Viral titers were generally lower in the kidneys and brains of infected double-hTG mice relative to KO mice. Inflammatory mediators like TNF-α, IL-6, IL-1β, and MCP-1 were comparatively higher in KO and 6A4 mice with the most severe AKI. High virus presence and inflammatory markers were also observed in the brain and hippocampus of all infected mice. The results from in vivo studies suggest that SP-A variants differentially protect against severe COVID-19. Furthermore, the human COVID-19 patient studies revealed increased SP-A levels in the saliva of COVID-19 patients compared to healthy controls and highlighted the potential use of SP-A levels as a biomarker for COVID-19 severity. Collectively, these findings underscore the importance of host innate immune collectins and contribute to our understanding of the roles of host genetic variations in the observed population-level differences in COVID-19 susceptibility and severity.
  • Deciphering cellular dynamics and crosstalk of trabecular meshwork and Schlemm's canal cells in a bioengineered 3D extracellular matrix hydrogel microenvironment

    Singh, Ayushi (2024-11-14)
    In the conventional outflow pathway, Schlemm's canal (SC) inner wall endothelium interfaces with the trabecular meshwork (TM). Biomechanical changes in this microenvironment contribute to increased resistance to aqueous outflow, a characteristic of ocular hypertensive glaucoma. Notably, TM undergoes fibrotic-like remodeling and stiffening. Existing in vitro TM/SC models fail to accurately replicate native cell-cell and cell-extracellular matrix (ECM) interactions, limiting their use for studying glaucomatous outflow pathobiology. In this dissertation, we utilized a biomimetic ECM hydrogel system made from natural polymers resembling native tissue proteins. This ECM hydrogel can be (i) used to encapsulate donor-derived primary human TM cells or (ii) employed as a substrate for culturing donor-derived primary human SC cells on top. As ECM hydrogels gradually emerge as a preferred model in diverse research laboratories, a standardized fabrication method is essential to improve accessibility and consistency across experimental protocols. Thus, a detailed methodology for producing these ECM hydrogels is provided in Chapter 2. In Chapter 3, using the 3D TM hydrogel system, we demonstrated that simvastatin-mediated inactivation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) attenuates pathological changes in TM cells. YAP/TAZ are key mechanotransducers involved in glaucoma pathogenesis and are shown to be regulated by the mevalonate pathway. By inhibiting this pathway, we hypothesized that statins could potentially improve TM cell pathobiology by modulating YAP/TAZ activity. Thus, targeting the mevalonate pathway with statins may offer therapeutic potential for glaucoma. Despite significant progress in understanding TM and SC cells individually, the dynamic interactions between them and their role in glaucoma pathogenesis remain poorly understood. These interactions are crucial in the pathogenesis of glaucoma, yet no effective model exists to study them. Therefore, in Chapter 4, we developed a novel co-culture hydrogel system to explore TM-SC interactions and assess how glaucomatous TM cells affect SC behavior. Our findings show that glaucomatous TM cells alone can induce pathological changes in SC cells, underscoring the critical role of cell-cell and cell-ECM interactions in glaucoma progression. Collectively, these biomimetic ECM hydrogels provide a unique platform for investigating glaucomatous outflow mechanisms and offering insights into disease pathogenesis.
  • HIV-1 has a sweet tooth: glucose metabolism drives the multistep process of HIV-1 latency reversal

    Kayode, Yetunde (2024-11-04)
    The major barrier to a cure for HIV-1 is the establishment of latency in long-lived CD4+ T cells within lymphoid tissues which readily fuel viral rebound upon antiretroviral therapy (ART) interruption. Therapeutic approaches aimed at eliminating these HIV reservoirs with latency reversal agents (LRAs) have hitherto yielded underwhelming results in clinical trials owing to our incomplete understanding of the exact determinants of meaningful latency reversal in vivo. While previous studies have associated glycolysis with HIV productive replication and latency reversal, the exact role and mechanistic link of glycolysis to HIV latency reversal remains undefined. Furthermore, few studies have investigated HIV latency under physiologically relevant metabolic conditions found in the anatomical reservoirs of HIV in vivo. The studies in this thesis reveal that glycolysis is a metabolic determinant of HIV latency reversal, particularly during physiological hypoxia. We show that the capacity of LRAs to modulate glycolysis determines their efficacies over a physiological range of glucose and oxygen availabilities as found across tissues in vivo. Mechanistically, glycolysis fuels histone lactylation, a novel post-translational modification (PTM) which we show is a stronger predictor xviii of latency reversal than the canonically recognized acetylation marks, and promotes chromatin accessibility at the HIV LTR. Beyond histone PTM modulation, glycolysis also modulates HIV RNA splicing, a critical post-transcriptional step in HIV latency reversal. Specifically, multiple splicing of rev, an HIV regulatory gene, is significantly downmodulated by glycolytic restriction in a hypoxia-dependent fashion. Finally, we show that glucose and oxygen availability impact the phosphorylation and lactylation of splicing factor 3B subunit 1 (SF3B1), a core component of the U2 spliceosome complex and HIV dependency factor which provides preliminary mechanistic insight to how glycolysis and hypoxia modulate HIV RNA splicing. Collectively, our findings uncover glucose and oxygen availability as critical metabolic determinants of HIV-1 latency reversal and support the rationale that physiologically relevant experimental conditions should be utilized in studies aimed at identifying therapeutic agents that effectively target the latent reservoir in vivo.
  • Kohlschütter-Tönz protein ROGDI is the homolog of yeast Rav2 and a novel Rabconnectin-3 subunit

    Winkley, Samuel (2024-10-10)
    V-ATPases are rotary proton pumps that are extraordinarily well-conserved among eukaryotes. V-ATPases function primarily to acidify intracellular compartments, critical to maintaining cellular homeostasis. The V-ATPase-generated proton gradient provides the optimal environment for lysosomal catabolism and drives intracellular protein trafficking. V-ATPases serve important functions throughout the human body. For example, V-ATPase activity energizes the active transport of neurotransmitters into synaptic vesicles, regulates the acid/base balance in the kidney, and helps the immune system recognize invading pathogens. However, when V-ATPase activity is inappropriately increased or decreased, these processes are affected, and disease can result. V-ATPases are composed of peripheral V₁ and integral membrane V₀ subcomplexes; V₁ hydrolyzes ATP and transmits rotation to V₀, which moves protons across a membrane. V-ATPase activity is regulated in part through the reversible association of the V₁ subcomplex and V₁C subunit from V₀. Upon disassembly, both V₁ and V₀ are catalytically inactivated. In yeast, the RAVE complex catalyzes the efficient reassembly of V-ATPases. Rabconnectin-3 is the human homolog of the RAVE complex and functions similarly. Mutations in the Rabconnectin-3 complex can reduce V-ATPase activity through decreased assembly, which leads to disease. Both Rabconnectin-3 subunits share substantial homology with the RAVE subunit Rav1. We have identified the poorly characterized protein ROGDI as the mammalian homolog of the yeast RAVE subunit, Rav2. ROGDI shares strong functional and structural homology with yeast Rav2. Expression of ROGDI in a rav2Δ yeast strain partially rescues the growth phenotype characteristic of RAVE mutants. ROGDI binds to the structurally conserved N-terminal β-sheet rich domain. AlphaFold3 modeling predicts that ROGDI binds between the Rabconnectin-3 subunits. ROGDI coimmunoprecipitates with Rabconnectin-3 and V-ATPase subunits. Additionally, ROGDI is present alongside V-ATPase and Rabconnectin-3 subunits on lysosomal membranes. This indicates that, like RAVE and Rav2, Rabconnectin-3 and ROGDI localize intracellular regions rich in V-ATPases. Identifying ROGDI as a novel Rabconnectin-3 subunit is a substantial step forward in our understanding of Rabconnectin-3 and how it influences V-ATPase activity.
  • Human cytomegalovirus (HCMV) exploits heat-shock transcription factor 1 (HSF1) to promote viral replication: a potential novel antiviral target to combat HCMV infection

    Dilruba, Akter (2024)
    Human cytomegalovirus (HCMV) is a highly prevalent beta-Herpesviridae virus infecting almost 80-90 % of the world population. Though HCMV infection is typically asymptomatic, it can cause significant morbidity and mortality among immunocompromised individuals. Because of its obligate intracellular nature, HCMV modulates the cellular environment to promote infection. HCMV activates different cellular responses and signaling pathways to facilitate a favorable state for viral replication. During the lytic cycle of HCMV infection, viral entry, and replication inside the cell initiate stress response due to nutrient deficiency, energy depletion, hypoxia, and proteotoxic stress. Stress responses are designed to sense the damage, initiating a cascade of events to survive the stress. Several studies showed that HCMV usurps components of heat shock-stress response (HSR) to mitigate stress-associated damage and promote viral gene expression and replication. In this study, we found that HCMV infection in fibroblast cells induces a unique biphasic activation of heat shock transcription factor 1 (HSF1), a master transcription factor that is activated in response to heat-induced proteotoxic stress. HCMV binding to the integrin-ᵝ receptor activates HSF1 through Src- kinases. Importantly, HCMV infection drives the translocation of HSF1 into the infected cell nucleus. During canonical activation of HSF1, nuclear HSF1 binds to the specific sequence on the genome called heat shock element (HSE) and initiates transcription of a wide variety of stress-related genes. Interestingly, HCMV also utilizes this master transcription factor by harboring HSEs on major immediate early promoter (MIEP) to regulate viral immediate early (IE) gene expression. We found inhibition of HSF1 with a novel anti-HSF1 targeting drug SISU102 (Direct Targeted HSF1 InhiBitor) attenuated IE protein expression, indicating that the HSF1 regulates HCMV lytic replication. Additionally, inhibition of HSF1 reduced late (L) gene expression and subsequent viral progeny production. To explore HSF1 as a potential in vivo anti-HCMV target, we employed a murine model involving the subcutaneous transplantation of human skin into athymic nude mice. Treatment with SISU102 significantly diminishes viral replication in skin xenografts compared to the vehicle-treated group, indicating HSF1 as a possible cellular protein target for HCMV antiviral therapy. Overall, our data suggest that HCMV infection rapidly activates HSF1 during viral binding and entry, driving nuclear localization to promote lytic replication, which can be exploited as an antiviral strategy.
  • Microbiota Colonization Dynamics Dictate Systemic IgA

    Harris, Joshua (2024-08-22)
    Evolution of the mammalian gut is intimately linked with the microbes that inhabit this space. Immunological development of gastrointestinal and systemic tissues is fundamentally dependent on stimulation by symbiotic microorganisms. In some cases, the same species that are critical for host immunity display pathogenic qualities when homeostasis is disrupted. Bacteroides fragilis is one such species with numerous symbiotic and pathogenic characteristics. This thesis explores the generation of B. fragilis-specific systemic IgA and the role of this response in protecting the host from B. fragilis pathogenicity. Induction of systemic IgA specific to B. fragilis requires exposure of this bacterium to small intestinal Peyer's patches and results in migration of newly generated IgA plasma cells to systemic tissues. Colonization dynamics of B. fragilis in mouse models with endogenous gut microbiota revealed that the magnitude of systemic IgA responses occurs in a dose-dependent fashion. Finally, a framework for establishing B. fragilis colonization and subsequent immune modulation within a highly diverse intestinal ecosystem was developed.
  • Investigating the role of formin FHOD3 during myofibrillogenesis in embryonic chick cardiomyocytes

    Sausville, Damien (2024-08-05)
    Formins are major actin polymerizing proteins which act via the FH2 domain to promote actin nucleation and polymerization, as well as the FH1 domain to accelerate FH2 mediated actin elongation. FHOD3 is a formin that has been shown to be expressed predominantly in the heart and is critical for myofibril maturation during development in mice. FHOD3 has been shown to localize where actin filaments overlap myosin filaments within the sarcomeres of mice, rat, and human induced pluripotent stem-cell derived cardiomyocytes, flanking both sides of the M-line in the sarcomere. However, the role of FHOD3 in the myofibrillogenesis and the timing of FHOD3's activity in myofibrils has yet to be determined. Using RT-PCR, I successfully identified expression of at least two different isoforms of FHOD3 within heart tissue, matching to predicted isoforms X5 and X6. I also identified two chemically conserved regions within the FHOD3 amino acid sequence that are related to the cardiac FHOD3 isoform's localization to myofibrils. Using immunofluorescence microscopy and western blotting I found that FHOD3 is present within embryonic chick cardiomyocytes and that the localization of FHOD3 matches prior reports. FHOD3 was determined to be transiently expressed at significantly higher rates on Days 3 and 4 of culture in cardiomyocyte myofibrils. 90% of measured sarcomeres containing FHOD3 had a Z-line to Z-line length ranging from 1.4-1.9 µm, suggesting not only a length-dependent role of FHOD3, but a myofibril maturity dependent localization of FHOD3. These observations illustrate that FHOD3 likely does not have a function in the initiation of myofibrillogenesis but may instead have a role in the maturation and elongation of sarcomeres. The transient nature observed also suggests that FHOD3 may be localized within the sarcomere only as needed. Knockdowns of FHOD3 performed with shRNAs showed no indication of knockdown causing myofibrillar disruption. Knockdowns of FHOD3 using DsiRNAs were statistically inconclusive for knockdown occurring but did have an upwards nonsignificant trend in the percentage of myofibril disruption in cardiomyocytes.
  • HSV-1 targets a novel antiviral response of the STING pathway

    Szemere, Zsuzsa (2024-07-08)
    In order to establish a successful infection, herpes simplex virus-1 (HSV-1), a ubiquitous virus with high seropositivity in the human population, must undermine a multitude of host innate and intrinsic immune defense mechanisms, including key players of the stimulator of interferon genes (STING) pathway. Recently it was discovered that not only de novo produced intracellular 2'-3'cGAMP, but also extracellular 2'-3'cGAMP can activate the STING pathway by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this critical signaling molecule in cancer cells. We hypothesized that the import of exogenous 2'-3'cGAMP would function to establish an antiviral state similar to that seen with the paracrine antiviral activities of interferon. Further, to establish a successful infection, viruses, such as HSV-1, must undermine this induction of the STING pathway by inhibiting the biological functions of SLC19A1. Herein, we report that treatment of the monocytic cell line, THP-1 cells and SH-SY5Y neuronal cell line with exogenous 2'-3'cGAMP induces interferon production and establishes an antiviral state. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2'-3'cGAMP-induced inhibition of viral replication. Additionally, HSV-1 infection results in the reduction of SLC19A1 transcription, translation, and importantly, the rapid removal of SLC19A1 from the cell surface of infected cells. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2'-3'cGAMP which is undermined by HSV-1 protein ICP27. This work presents novel and important findings about how HSV-1 manipulates the host's immune environment for viral replication and discovers details about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future.
  • Structural insights of the histone H3 tail and its role in the mechanism of histone H3 lysine-4 methylation

    Connelly, Michael (2024-07-26)
    Structural insights of the histone H3 tail and its role in the mechanism of H3 lysine-4 methylation Gene expression relies on the proper chromatin structure to provide the necessary access to the DNA for the large transcription complexes to carry out their tasks. If the chromatin is tightly condensed, transcription is unable to occur. To regulate and initiate access to the DNA, an elaborate network of histone modifying enzymes, chromatin remodeling complexes, and other supporting proteins must coordinate the writing, reading, and erasing of histone post-translational modifications (PTMs). One such PTM, methylation of histone H3 on the lysine-4 (H3K4) residue, is critically important for maintenance of gene expression states. This is done in a spatiotemporal manner, which is influenced by the number of methyl groups that are present. However, an understanding of how the degree of H3K4 methylation is regulated remains elusive. In this dissertation, we demonstrate the remarkable conservation of length and composition in the flexible N-terminal tails of histone proteins across evolution. Recent structural studies indicate several methyltransferase complexes bind to the nucleosome core, often leaving the N-terminal tails unbound. Research from our lab has also demonstrated that non-processive buildup of lysine-4 methyl groups takes place at multiple active sites. Based on these observations, we propose a hypothesis whereby the histone H3 tail acts as a swinging arm substrate, delivering residue side chains to different active sites to facilitate the progressive establishment of these epigenetic states. To investigate this hypothesis, we employed the CRISPR/Cas9 system in Saccharomyces cerevisiae to systematically modify the length of the H3 tail. We monitored histone H3 lysine 4 (H3K4) methylation, mediated by SET1, the primary H3K4 methyltransferase in budding yeast. Our findings demonstrate that altering the length of the H3 tail has varying effects on the extent of H3K4 methylation, in accordance with the swinging arm model. We also demonstrate that three proline residues are responsible for providing a segmented, tripartite structure with hinge-like joints that likely influence the tail's range of motion. Furthermore, the results support the proposed multiple active-site model, where mono-, di-, and trimethylation occur at distinct active sites within the COMPASS or MLL Core Complexes.
  • Role of TLDc Proteins Oxr1 and Rtc5 in Yeast V-ATPase Reversible Disassembly

    Khan, Md. Murad (2024-06)
    The vacuolar H+-ATPase (V-ATPase; V1Vo-ATPase) is a highly conserved, ATP hydrolysis-driven dedicated proton pump found on the membranes of intracellular organelles in virtually all eukaryotic cells and on the plasma membrane of specialized cell types. Regulation of V-ATPase activity is key to maintaining normal physiological functions, as aberrations in its activity are associated with several pathophysiological conditions. V-ATPase activity is mainly regulated by a mechanism called reversible disassembly, in which the assembly state - and hence the activity - of the enzyme is controlled by nutrient availability and extracellular cues. During the process, V-ATPase activity becomes either turned off by dissociation of the V1-ATPase from the Vo proton channel, or turned on by reassembling the two subcomplexes into an active enzyme. While the process is well-characterized at the cellular level, the molecular mechanism at the level of the enzyme remains elusive. Here, we show that two TLDc proteins, Oxr1p and Rtc5p, control the assembly state of yeast V-ATPase, with the former promoting disassembly, and the latter (re)assembly of the enzyme. Based on cryoEM analysis and in vitro and in vivo approaches, we discovered that Oxr1p is a V-ATPase disassembly factor. Oxr1p binding to V-ATPase results in autoinhibited V1 in two steps - first producing a disassembly intermediate, which, upon ATP hydrolysis, gets converted into autoinhibited V1. From in vitro experiments, we find that the second TLDc protein, Rtc5p, primes autoinhibited V1 for (re)assembly with Vo. CryoEM structures of Rtc5p bound V1 show Rtc5p's C-terminal ⍺ helix inserted into the catalytic core of the enzyme, thereby opening a second catalytic site, a conformational change that may facilitate (re)assembly of V1Vo. In vivo experiments, however, suggest that Rtc5p is not essential for V-ATPase reassembly in the cell, suggesting redundancy and/or alternative pathways. Overall, this study enhances our understanding of the molecular basis for the regulation of V-ATPase activity by reversible disassembly.
  • Exploring the role of single nucleotide polymorphisms in varicella zoster virus vaccine attenuation in skin

    Lee, Elizabeth Da-Yong (2024-05)
    Varicella zoster virus (VZV) is a disease that can be detrimental to the health of children in its primary form, chicken pox, and later in the elderly as its reactivated form, shingles. Before the advent of the vaccine, Varivax, VZV was endemic in the United States as it is highly contagious and can be spread through both direct contact and aerosol particles. Varivax, or vOka, is a live attenuated vaccine, and while effective, has side effects ranging from rashes to possible VZV reactivation. While the vaccine has reduced the incidence and severity of VZV, there is still little known about the mechanism of its attenuation in skin. vOka is genetically heterogeneous with hundreds of single nucleotide polymorphisms (SNPs) that are a mixture of wild-type and vOka nucleotides. Previous studies have demonstrated the key to attenuation may be through five SNPs in the open reading frame (ORF) 62 region found to be fixed and stable across different licensed vOka preparations around the world. ORF62 contains the gene for IE62, a transactivator protein responsible for regulating the expression of viral genes and the host gene for KRT15, a cytokeratin protein. This project focused on if two SNPs, located in the loci positions 106262 and 107252, that are found to be almost 100% conserved across all variations of vOka are responsible for the attenuation in human skin and induction of KRT15. We evaluated four mutant viruses with SNPs found in vOka and discovered that a double SNP mutation stunted virus growth in HFF cells. In addition, we found no significant difference in the growth of our viruses in skin but variability in successful infection. Furthermore, in infected skin, we found that VZV-ORF57-Luc and single mutant virus, 68-958, upregulate KRT15 expression with VZV infection while single mutant virus, 68-62S-A, may downregulate KRT15 expression with VZV infection. This project is important because it may reveal the molecular basis of attenuation of the licensed varicella vaccine. This information could be used to make a vaccine that contains only the attenuated genotype.
  • From bud scars to molecular insights: investigating V-ATPase function and assembly in yeast replicative aging

    Hashmi, Fiza (2024-05-28)
    Aging is a complex process that involves the progressive decline of physiological functions over time. As the global population continues to age, understanding the mechanisms underlying aging has become an important area of study. Lysosomes play a crucial role in maintaining protein quality control and degrading unneeded or damaged proteins through proteolysis. Therefore, lysosomes play a prominent role in theories of aging due to their significant role in cellular homeostasis. Interestingly, many of the hallmarks of aging are conserved between yeast and humans, highlighting the relevance of yeast as a model organism to study aging processes. One key enzyme responsible for acidifying lysosomes and lysosome-like vacuoles in yeast is the vacuolar-type H+-ATPase (V-ATPase). Despite evidence showing that lysosomes alkalinize with age, compromising their proteolytic function, little is known about the regulation of V-ATPase in aging cells. Yeast cells divide asymmetrically with each division leaving a "bud scar" that can be stained to determine replicative age. In comparing cells of distinct replicative age, we find significant decreases in V-ATPase assembly, accompanied by poor vacuolar acidification, in older cells. Remarkably, partial disassembly of the V-ATPase occurs at a relatively early age, indicating its potential as a phenotypic driver in the aging process. Reversible disassembly is controlled in part by the activity of two opposing and conserved factors, the RAVE complex and Oxr1. The RAVE complex promotes V-ATPase assembly and a rav1∆ mutant has a significantly shorter lifespan than wild-type cells; Oxr1 promotes disassembly and an oxr1∆ mutation significantly extends lifespan. These data indicate that reduced V-ATPase assembly may drive the loss of lysosome acidification with age and place the balance of V-ATPase assembly factors at the center of this process. Caloric restriction, defined as reduced calories with adequate nutrition, has been shown to extend lifespan in multiple organisms including yeast. We find that caloric restriction reverses the age-related decreases in V-ATPase assembly and vacuolar acidification in yeast as well as restoring balance of assembly factors. We investigated three conserved metabolic signaling pathways that have been linked to acidification, caloric restriction, and aging: PKA, mTORC1/S6K (TORC1/Sch9 in yeast), and AMPK (Snf1 in yeast). By utilizing non-essential nutrient mutations in these signaling pathways, we determined the impact on V-ATPase assembly during replicative aging. Mutations compromising TORC1 function were known to extend lifespan and preserved V-ATPase assembly even in older cells. In contrast, a mutation that prevents recruitment of Snf1/AMPK to vacuoles prevented V-ATPase assembly even in young cells and shortened lifespan. This study provides novel insights into the importance of V-ATPase assembly and function in the aging process and suggests novel interventions to promote health aging.

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