Student Award Details

Project Summary Mutations in genes encoding proteins that are essential for cell processes such as DNA replication can lead to cellular dysfunction and disease. Protein defects can perturb these cell processes by removing or surpassing cell cycle checkpoints, leading to errant growth and proliferation of cells, defining characteristics of cancer. As such, it is essential to investigate Proliferating Cell Nuclear Antigen (PCNA), the central player that coordinates DNA replication, DNA repair, and cell-cycle regulation. PCNA, also known as the sliding clamp, is a homotrimeric ring that slides along DNA to facilitate interactions of over 100 known proteins, many involved in cancer development and other important cellular processes. The sliding clamp is conserved across all life forms, providing insight into the evolution of DNA replication and cell-cycle machinery. Thus, PCNA is an ideal target to investigate mutational effects on protein function and the long-term impacts on the cell. In Aim 1, we will address an interesting paradox related to PCNA. Point mutations in PCNA that result in subtle biochemical effects cause severe disruption of organism fitness, suggesting that PCNA is especially sensitive to mutations. Conversely, PCNA genes across evolution are widely varying in sequence suggesting that PCNA is actually accepting of mutations. To investigate this contradiction, we will perform a mutational scan of all potential point mutations in the yeast PCNA protein. These mutants will then be exposed to DNA-damaging agents to assess the effects of the PCNA mutants on various PCNA functions. I predict that a mutational screen of PCNA will show mutational effects on cell viability and DNA damage response based on residue location in PCNA providing insight into the acceptability of point mutations in PCNA. This data will also provide insights into potential disease mutations that could impact human PCNA. The Kelch lab has previously investigated two disease-associated germline mutations in PCNA. These mutations lead to PCNA-associated DNA repair disorder (PARD), characterized by UV sensitivity, neurodegeneration, premature aging, and, most notably, the development of skin cancer. In Aim 2, we will investigate how patient-associated mutations in PCNA affect biochemical and cellular function. I selected variants based on association with cancer or PARD. I will establish mutant human retinal pigment epithelial (RPE1) cell lines using CRISPR/Cas9 techniques. Once these cell lines are established, I will assess the cellular impacts by using flow cytometry and DNA-damage assays. I will compare these results with tests of the biochemical functions using isothermal titration calorimetry and thermal shift assays. I predict that the mutations will exhibit defects in thermostability, cell regulation, and DNA repair. The impact of this study is two- fold. First, the study will enhance our understanding of how PCNA function and evolution are intertwined. Second, the study will investigate select human PCNA mutants that can inform cancer diagnosis and provide a framework for investigating other proteins.

Amyotrophic lateral sclerosis (ALS) is a devastating degenerative motor neuron disease that is largely untreatable and leads to death within 5 years of diagnosis. ~10% of ALS cases are familial and caused by mutations in various ALS genes. Ultimately, the ideal treatment for genetic diseases such as ALS is somatic gene correction. Recently, advances in CRISPR/Cas systems have shown considerable promise for precise editing of disease loci using base and prime editing systems delivered by AAV. The second-most prevalent cause of familial ALS are mutations in the SOD1 gene. These mutations confer multiple toxic properties onto the protein. This project proposes to develop treatment to achieve somatic gene correction for common missense mutations in SOD1. The aims of this proposal are: (1) To develop AAV-mediated base editing gene correction strategies for the SOD1 A5V mutation in vitro. We will create next-generation base editors with a compact size, increased efficiency, and greater control over bystander editing. (2) To develop AAV-mediated prime editing gene correction strategies for the SOD1 A5V and G94A mutations in vitro. Different prime editor systems will be tested for optimal editing efficiencies and low off-target editing. (3) In in vivo studies, examine and optimize AAV- mediated base and prime editing gene correction strategies for the A5V and G94A mutations in A5V and SOD1G93A mouse models. Mice will receive AAV-mediated base and prime editors through an intracerebroventricular injection. Base and prime editor strategies will first be screened in mutation carrying HEK293T cells and then optimized in patient fibroblasts and mouse models. The effects of gene correction on gain- and loss-of-function molecular and motor phenotypes will next be evaluated. The fundamental hypothesis driving this proposal is that AAV-mediated somatic gene correction strategies, using base editing or prime editing to target the SOD1 mutations A5V and G94A, will decrease toxic GOF pathology and increase WT SOD1 protein levels in vivo, resulting in a balanced treatment for SOD1-ALS and a rescue of motor phenotype. In addition, with mentorship from experts in ALS and gene editing and the wealth of resources available at UMASS Chan, these studies will provide extensive training in gene editing for CNS diseases and project development that will be an essential foundation for a future career as an independent researcher developing gene therapies for a range of genetic CNS diseases.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a cytosolic double- stranded DNA (dsDNA) sensing pathway critical for regulating immune homeostasis. A series of gain-of-function (GOF) mutations result in constitutive activation of STING, causing an autoinflammatory disease called STING- Associated Vasculopathy with Onset in Infancy (SAVI). SAVI patients succumb to treatment resistant inflammatory lung disease and respiratory failure. There is little known about the mechanisms by which inflammation occurs. To address the urgent need to develop safe and effective therapies, we have developed a murine model for the most common STING gain-of-function mutation, STINGV154M/WT (VM). These mice recapitulate the lung inflammation exhibited by human SAVI patients. To identify the specific cell types involved in causing lung inflammation, we developed a novel VM conditional knock-in (CKI), allowing specific targeting of the VM mutation to different cell types. We demonstrated that endothelial cell (EC) STING GOF is sufficient in driving bronchus-associated lymphoid tissue (BALT) formation. However, the mechanism of action remains to be elucidated. Moreover, we have previously described SAVI lung disease as independent of type I interferon (IFN) and IRF3, signaling proteins downstream of STING activation. STING activation leads to downstream signaling of other pathways including NF-κB and autophagy. The signaling mechanism causing lung inflammation is also unknown. Additionally, STING GOF in ECs was insufficient to cause the extent of lung inflammation seen in VM mice, suggesting STING GOF in cells other than ECs is required for lung disease. Upon ubiquitous VM expression, we find evidence of fibroblast activation in the lung tissue. Fibroblastic reticular cells (FRCs) are a subset of fibroblasts that define the function and structure of lymphoid organs such as BALT. In addition to ECs, STING is highly expressed in FRCs, yet the role of STING in FRCs and contributions to lung disease is unknown. Thus, we hypothesize that coordinated interactions between ECs and FRCs exacerbate SAVI lung autoinflammation, which is dependent on NF-κB activation. In this proposal, Aim 1 will investigate how STING GOF mutation in ECs initiates immune cell recruitment. Aim 2 will determine the synergistic effects of STING GOF mutation in ECs and FRCs on lung autoinflammation. We propose to utilize in vivo, ex vivo, and in vitro techniques to test our hypothesis. The studies proposed in this application will provide critical insights that will enable us to design the best therapies. Furthermore, these studies will provide an opportunity to study the impact of STING activation on stromal cell types, an area of research that requires further exploration. Our findings will discern the role of ECs and FRCs in VM lung autoinflammation and will broadly provide insight into stromal cell-driven mechanisms of other lung disorders.

Tuberous sclerosis complex (TSC) is caused by Tsc1 or Tsc2 mutations and can affect several organs, including the kidneys, leading to benign tumors or cyst formation. Impaired TSC1/2 protein function promotes hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1), an important cellular nutrient sensor that controls cell growth and proliferation. However, a much deeper mechanistic understanding of mTORC1 regulation and function in kidney cells is required. The mTORC1 kinase complex is activated by the essential amino acid sensors called Rag-GTPases. There are four Rag GTPases isoforms (RagA, RagB, RagC, and RagD), which localize to lysosomes and function in a heterodimeric complex where RagA/RagB binds to RagC/RagD. The Rag-GTPase complex positively regulates mTORC1 by recruiting it to the lysosomal surface in the presence of amino acids, where mTORC1 is subsequently stimulated by another small GTPase called Rheb. The activated mTORC1 complex signals to several substrates that collectively regulate cell metabolism, growth, and proliferation, key factors for developing kidney cysts. Importantly, all current models indicate that RagA/B loss will inhibit mTORC1 signaling by preventing its localization to the lysosome. Unexpectedly, we discovered that deleting RagA and RagB in kidney tubular epithelial cells causes a striking and progressive cystic phenotype resembling TSC. Moreover, RagA/B deletion in the kidney is associated with increased, rather than decreased mTORC1 signaling, as in TSC. This phenotype is not observed when we delete the mTORC1 subunit Raptor in the kidney epithelium. Thus, we hypothesize that RagA/B loss triggers kidney cystogenesis via a non-canonical Rag-GTPase pathway, involving TFEB regulation and possible cellular crosstalk for mTORC1 hyperactivation, which can be a common mechanism of TSC cyst formation. Since aberrant mTORC1 activation is a hallmark of TSC and other kidney cystic diseases, resolving this unexpected mechanism and the Rag-GTPases role in kidney cyst development may have important translational implications for improving upon current mTOR-based therapeutic strategies for the management of TSC and other kidney cystic diseases. In this proposal, I aim to (1) identify the cellular origin and metabolic traits underlying RagA/B deletion-induced kidney cystogenesis, and (2) determine the mechanism linking RagA/B loss and mTORC1 activation, TFEB regulation, and the similarities to the pathophysiology of TSC.

Mitochondria support multifaceted metabolic reactions in mammalian cells1. The vast majority of these metabolic pathways require the electron transport chain (ETC), a series of reactions in which ubiquinone (UQ) carries electrons to oxygen (O2) as the terminal electron acceptor (TEA). Recent studies have refuted this textbook model and have shown fumarate as a TEA2 through two mechanisms. First, under hypoxia, the reduced electron carrier ubiquinol accumulates driving complex II backwards to deliver electrons to fumarate. Second, in normoxic conditions, a novel mammalian metabolite rhodoquinone (RQ) can deliver electrons to fumarate3. Thus, the mammalian ETC is highly flexible and whether other electron acceptors beyond fumarate and O2 can be used has yet to be studied. The thermodynamic favorability of all reduction and oxidation (redox) reactions in the ETC are dictated by the Nernst equation. Electrons favorably transfer from metabolites with low to high reduction potential4. Thus, as RQ has a lower reduction potential than UQ, it can theoretically deliver electrons to other electron acceptors such as sulfites. Moreover, upon hypoxia exposure, UQH2 can build up enough to enable sulfites reduction as well. Oxidized sulfur-based molecules are known to act as TEA in certain sulfate-reducing prokaryotes5. The same molecules are known to interact with the mammalian ETC via enzymes sulfite oxidase (SUOX) and sulfide quinone oxidoreductase (SQOR) with cytochrome c and UQ, respectively. Importantly, SUOX functions in the cysteine catabolic pathway to oxidize sulfite (SO3-2) into sulfate (SO4-2), subsequently transferring electrons to cytochrome c6. Similarly, SQOR oxidizes hydrogen sulfide (H2S) to glutathione persulfide (S2O3-2) by transferring electrons to UQ6. Although these electron donor mechanisms (Fig.1) are well established, the reversibility of these reactions, enabling reduction of sulfate and glutathione persulfide as electron acceptors, have never been examined in mammals. We hypothesize that sulfate via SUOX and glutathione persulfide via SQOR can serve as TEA for UQ-dependent ETC circuits in hypoxic (low oxygen) environments, and RQ-dependent ETC circuits at any oxygen tension (Fig. 2). The proposed research will test the propensity for mammalian cells using the UQ- and RQ-directed ETC circuits to employ oxidized sulfur species as TEA. We will leverage directing UQ vs RQ circuits in vitro to measure reversibility of SUOX and SQOR activities in varying oxygen availabilities (Aim 1) and an established conditional mouse model to examine the tissue specificity of these enzymes and circuits (Aim 2). This work will add to the growing body of literature on novel mechanisms of flexibility in the mammalian ETC and will provide meaningful insights to mitochondrial function. [1]Monzel, Nat Met 2023 [2]Spinelli, Science 2021 [3]Valeros, Cell 2025 [4]Alberts, Mol Bio of the Cell., 2002 [5]Muyzer, Nat Rev Microbiol 2008 [6]Kohl, Br J Pharmacol 2019

Cardiovascular disease is the leading cause of death in the United States. Despite decades of research, it is unclear how the RNase A family nuclease angiogenin stimulates blood vessel formation, and why angiogenin dysfunction is associated with heart failure and poor cardiovascular health. Recent work revealed that angiogenin’s nuclease activity, which is required for its angiogenic function, is stimulated by binding to the ribosome, but it remains unclear whether angiogenin’s ribosome-dependent mechanism is involved in angiogenesis. A bacterial nuclease named ribocin with a strikingly angiogenin-like structure and ribosome- dependent activity holds similar potential for understanding lung health. Nearly all Cystic Fibrosis patients experience Pseudomonas aeruginosa bacterial pneumonia and subsequently suffer from lung tissue inflammation and lasting damage long after the infection has cleared. Ribocin encoded by P. aeruginosa damages human ribosomes specifically at central helix 69 of the 28S rRNA and inhibits translation. We hypothesize that like other ribosome-inactivating proteins, ribocin induces a ribotoxic stress response that causes inflammation and cell death in human lung tissues. To inform future therapeutic studies aimed at treating cardiovascular disease and post-infection pulmonary damage, the mechanisms of translation control by these RNase A-family nucleases must be elucidated in the context of their cellular functions. The goal of this project is to determine the structural basis of translation control by angiogenin during angiogenesis and determine the impact of translation control by ribocin on ribotoxic stress response and cell death. With guidance from the sponsor, an expert in biochemical and structural basis of translation, and collaborators, who are experts in the RNA developmental biology and cryo-EM method development, the trainee will apply cutting edge methods for in-cell cryogenic electron microscopy (cryo-EM) complemented by cell assay and biochemical approaches to visualize structural changes to actively translating ribosomes during angiogenin- stimulated vascularization and ribocin-mediated tissue damage. Aim 1 will determine the contribution of angiogenin’s ribosome-specific activity on tube formation (angiogenesis) in human umbilical vascular endothelial cells (HUVEC). Aim 2 will elucidate the structural mechanism of translation inhibition by ribocin and investigate the impact of selective ribosome damage by ribocin on ribotoxic stress response in human lung cells (IB3). The results of this study will reveal details of mechanisms underlying fundamental cardiovascular function and novel components of pulmonary disfunction, necessary for future work in developing therapeutics for cardiovascular and lung disease.

As we age, cells accumulate damage, become less efficient at converting energy and produce more toxic byproducts leading to an overall decline of the human body. This decline is linked to mitochondria, the cells energy supplier. Normally mitochondria rely on oxygen to provide energy, which comes at the cost of producing toxic molecules in the process. However, it was recently found that a previously unknown mammalian molecule can reprogram the mitochondrial energy supply route to become independent of oxygen reducing toxic byproduct levels. This project will explore how this alternative energy pathway changes with age and whether activating it can restore mitochondrial health in older mice. By tracing metabolic changes in reprogrammed mitochondria, the goal is to find out if this “low-damage” energy mode can slow or even reverse some of the effects of aging. This could lead to new ways to support healthy aging at the cellular level.

Zoonotic transmission of avian Influenza A virus (IAV) to humans poses a pandemic threat. Recent transmission of avian IAV to dairy cattle, and to dairy farm employees emphasizes the importance of identifying molecular mechanisms that regulate zoonotic events. It is thought that the IAV envelope glycoprotein, hemagglutinin (HA), must adapt its receptor specificity to bind α2,6-linked SA that predominates the human upper airway to initiate infection. However, recognition of α2,6-linked SA by HA is insufficient for avian H5N1 IAVs to enter human cells, indicating that other adaptations are necessary for zoonotic transmission to occur. Although evidence suggests that the pH and temperature sensitivity of HA are important factors that govern host tropism of IAV, a molecular mechanism that explains the temperature and pH dependence is missing. To define the pH and temperature dependence of HAs pre- to post-fusion conformational change, we first focused on visualizing conformational dynamics of HA with single-molecule FRET (smFRET). Preliminary data reveal that under neutral pH and room temperature conditions, the head domains of A/Vietnam/1194/2004 (H5N1) (VN04) HA, HA1, undergo a breathing motion where the heads are either caged, or uncaged. At pH8.0 HA spends more time in the fully caged conformation and less time in the uncaged conformation. Decreasing the pH to 6.5 shifts the equilibrium to where HA spends more time in the caged conformation and 80% of the time in the uncaged conformation. These data allow us to establish a conformational phenotype for HA where can determine the pH and temperature dependence of a given conformational phenotype. Thus, the central hypotheses of this proposal are that (1) the regulation of the pre- to post-fusion conformational change of avian HA is differentially regulated by pH and temperature compared to human adapted HA and (2) the membrane fusion phenotypes of HA are different. Experiments performed in Aim 1 will characterize the pH and temperature dependence of HAs conformational phenotype by monitoring conformational dynamics of HA at pH 8.0, pH 6.5, and pH 5.5 as well as at room temperature, 32°C and 37°C. I will implement the smFRET for three HA serotypes: A/Vietnam/1194/2004 (H5N1) (VN04), A/California/07/2009 (H1N1) (CA09) and A/Hong Kong/1/1968 (H3N2) (HK68). Furthermore, mutations that alter pH and temperature stability will be characterized to evaluate a molecular mechanism that can explain the differences in the conformational phenotype between the different HA serotypes. Aim 2 will characterize the pH and temperature dependence of HA-mediated membrane fusion by monitoring the extent and rates of fusion of HA pseudo-typed virus with the three previously mentioned HA serotypes. Additionally, the same pH and temperature stability mutations will be used to assess their impact on membrane fusion. Collectively, these data will reveal a mechanism that explains the zoonotic transmission potential of IAV and help improve genetic surveillance efforts.

In the context of human immunodeficiency virus (HIV), both CD4+ T cells and macrophages contribute to the viral reservoir within people living with HIV on combination antiretroviral therapy. Natural killer (NK) cells are the major cytolytic cells of the innate immune system, and their failure to effectively eliminate infected cells allows propagation and persistence of infection. In in vitro models of infection, HIV-infected macrophages are more resistant to NK cell-mediated killing than their CD4+ T cell counterparts, implicating macrophage-specific mechanisms of resistance. However, these mechanisms have yet to be determined. The overall objective of this proposal is to define macrophage-specific mechanisms that facilitate increased resistance to NK cellmediated killing. The central hypothesis is that HIV-infected macrophages resist NK cell-mediated killing by reducing immediate NK cell lytic function, and antagonizing death receptor signaling. To address this hypothesis, the following aims will be pursued: Specific Aim #1 will characterize release of HIV-infected macrophage lysosomal granules towards NK cells. Published work shows that melanoma cells being targeted by CD8+ cytolytic T lymphocytes release their lysosomes at the immunological synapse to degrade perforin, protecting them from elimination. To measure lysosome release, the investigators used CD107a, which lines the membrane of lysosomes and lytic granules and is surface exposed following degranulation. My preliminary data shows that HIV-infected macrophages increase surface CD107a expression upon co-culture with autologous NK cells. Therefore, I will investigate whether this mechanism is also being used by HIV-infected macrophages to neutralize NK cell degranulation. Specific Aim #2 will define mechanisms of HIV-infected macrophage resistance to NK cell FasL-mediated killing. Preliminary data shows that HIV-infected macrophages, but not CD4+ T cells, are not susceptible to apoptosis induced by incubation with recombinant FasL, despite both cells expressing the Fas receptor. I will investigate whether this resistance is due to increased anti-apoptotic activity of the protein cFLIP, which regulates caspase-8 activity. To complement these in vitro experiments, I will also analyze published single-cell RNA-sequencing data sets to determine tissue resident macrophage expression of cFLIP and other anti-apoptotic proteins. Results from these studies will elucidate how HIV-infected macrophages escape NK cell-mediated killing and will ultimately inform clinicals strategies utilizing NK cells to control pathogenesis. The work outlined above will be conducted at the University of Massachusetts Chan Medical School, within the Immunology and Microbiology Ph.D. program. The training plan, which will help me achieve my goal of becoming an independent investigator, includes coursework on basic/advanced principles of immunology and ethics of research. Finally, I will attend national/international conferences where I will be able to receive feedback from external scientists on my research and engage with others performing cutting edge research.

Despite progress in pharmacological advances, the rate of obesity incidence rises every year with an increased risk of developing insulin resistance, cardiovascular disease, and other cardiovascular risk factors including type 2 diabetes (T2D). Current treatments are not curative and typically require compliance with life-long, daily treatments. Thus, there is a critical need for novel therapeutic advancements to combat obesity and its comorbidities. We plan to generate metabolically active, thermogenic adipocytes from human white adipose tissue (WAT) to advance a cell therapy strategy for alleviating insulin resistance and cardiovascular risk in T2D. Using a CRISPR-based system to target a thermogenic suppressor gene, Nrip1/RIP140, we were able to induce "browning" in mouse and human white adipocytes with high efficiency. The browning of WAT is favorable as the brown-like human adipocytes express a brown fat-specific marker, uncoupling protein 1 (UCP1), and other beneficial secreted factors that can improve metabolism in obese mice. Although our published method is effective in vivo, we only manage to achieve ~10% of the brown fat UCP1 expression, implying that there is room for more browning to generate a more potent thermogenic fat. PPAR and cAMP signaling are key in the activation of brown fat. Thus, our goal is to generate fully brown adipocytes from human white adipocytes by combining Nrip1 disruption with PPAR and cAMP activation. We predict that this combination will result in maximally induced brown human adipocytes that can improve systemic metabolism in our mouse model. The long-term goal in our lab is to genetically modify human adipocytes ex vivo to enhance therapeutic activity and implant the metabolically active and thermogenic adipocytes into obese patients.