In healthy cells, NRF2 is an essential transcription factor for regulating oxidative stress. However, when constitutively activated in cancer cells, it can lead to tumor cell proliferation and metabolic rewiring. When NRF2 is activated, it increases cysteine consumption in the cells through increased expression of the SLC7A11 cystine/glutamate antiporter. We discovered a dose dependent decrease in proliferation when exposed to higher cystine concentrations, unique to cells with NRF2 activation. To understand the kinetics of this proliferation defect, I am developing a tool to visualize and track cell proliferation using a live-cell imager. I will first genetically encode Nuclear Red Fluorescent Protein (NucRFP) into our NRF2-activated cells, using single-cell cloning and flow cytometry to isolate and establish clonal populations that stably express NucRFP. Then, I will use the live-cell imager to incubate cells with NucRFP expression, titrating different concentrations of cystine. Stable NucRFP expression will allow me to quantify cell growth overtime in different concentrations of cystine media to better understand cell growth. This research will generate insights into the consequences of cystine stress that inform the development of targeted treatments for NRF2 activation in cancer cells.

The external fertilization and transparent embryos of zebrafish make them an informative model of vertebrate embryonic development from the 1-cell stage. In this study, we examine the impact of de novo GTP synthesis on the formation of the embryonic somites, which are embryonic cells which develop into segmented blocks of muscle that run the length of the body. We hypothesize the de novo GTP synthesis is required for the correct patterning of somite borders in zebrafish embryos, and that this process facilitates the formation of a vertebrate body plan. Inosine monophosphate dehydrogenase 2 (IMPDH2) is the enzyme which catalyzes the conversion of inosine monophosphate (IMP) towards the de novo synthesis of GTP instead of ATP. To test the impact of de novo GTP synthesis on somite formation, we inhibited IMPDH2 function with mycophenolic acid (MPA) both before and after somite formation began. MPA caused stronger defects in the somite morphology and embryonic body shape when added to embryos before somite formation began, earlier in development. We performed in situ hybridization against xirp2a to assess the effect of inhibiting IMPDH2 function on the formation and patterning of the somite borders. MPA treatment decreased the definition of somite borders we could observe in the posterior tail. Inhibiting IMPDH2 with MPA produced somites with smooth, round borders instead of the chevron-shape typical of zebrafish. We next conducted immunohistochemistry against IMPDH2 to examine the expression and localization of this enzyme in embryonic cells when GTP conditions are low. In MPA-treated embryos, we observed increased expression of IMPDH2 across the entire embryo. We will next explore how GTP abundance affects activity of the clock, a mechanism which synchronizes gene expression of embryonic cells.

Carpenter syndrome is a congenital disorder mainly characterized by craniosynostosis – soft spots of the skull fuse prematurely resulting in skull elongation. MEGF8, a protein in the MEGF8-MGRN1-MOSMO (MMM) regulatory complex involved in Hedgehog (Hh) signaling, has been previously linked to Carpenter syndrome. Hh signaling, a core developmental pathway, plays many roles in skull development, including functioning as a guidance cue for cranial neural crest cells (foundational cells for skull bones) and regulating bone ossification (bone hardening). Despite the previous research linking MEGF8 and anomalous skull development, the role of the MMM complex and Hh signaling in skull development and the mechanism behind this abnormal skull development is still widely unknown. I’m exploring the mechanism by which elevated Hh signaling influences cell fates during the development of skull structures. I investigated a whole-embryo knockout mouse model to identify regions where overexpression of Hh correlates to abnormal skull phenotypes. I explored and measured these phenotypes via imaging a genetic Hedgehog reporter mouse line and skeletal stained embryos of various ages. These knockout mice were embryonic lethal because of a congenital heart defect associated with loss of Hh. We introduced an inducible Cre to conditionally knockout the MMM complex at a later age, avoiding heart defects. My findings identified the interparietal and parietal regions of the skull as areas of interest, visualized by over-ossification and lack of defined structural boundaries- nasal and palatal development was normally observed. These findings suggest that elevated Hh signals result in abnormal development of skull structures, similar to craniosynostosis, and are involved in intramembranous ossification (skull structures) rather than endochondral ossification (nasal/palate). From these findings, I’m investigating how Hh signaling plays a role in skull development and intramembranous ossification.

Dense-core vesicles are membrane-bound structures that carry neuromodulators such as insulin, dopamine, and serotonin. The peptides within dense-core vesicles are initially larger precursor proteins that undergo enzymatic processing to achieve their functional forms. During the defecation motor program in Caenorhabditis elegans, dense-core vesicles released from the intestine harbor neuropeptides that trigger neurons which activate enteric muscles, promoting the act of defecation. Failure of certain proneuropeptides to mature into neuropeptides results in decreased frequency of defecations. CPD-1, a conserved transmembrane carboxypeptidase, is a poorly understood processing enzyme that affects the defecation motor program. I built on our knowledge of EGL-21, another carboxypeptidase known to process neuropeptides and peptide hormones, to better understand CPD-1’s function. I show here that these two carboxypeptidases, EGL-21 and CPD-1, process neuropeptides necessary for successful defecation patterns. Mutants lacking egl-21 had decreased defecation frequency while worms lacking both egl-21 and cpd-1 had an even lower defecation frequency. Additionally, my results show that CPD-1 is expressed in intestinal cells and can compensate for EGL-21’s function. Finally, I am conducting experiments to determine whether one of CPD-1’s targets is NLP-40, an important neuro-like peptide released from the intestine that regulates defecation. These results contribute to our broader knowledge of peptide processing in dense-core vesicles.

Certain species exhibit the remarkable ability to regenerate their appendages, a process that requires complex metabolic pathways to facilitate the cellular proliferation needed to regrow tissue. Among these species, Xenopus tropicalis, the Tropical Clawed Frog, serves as a great model for regeneration studies because of its transient regenerative capacity. X. tropicalis tadpoles exhibit the ability to regenerate their tails, but this capability is gradually lost after metamorphosis. This unique characteristic allows for direct comparison of regenerative and non-regenerative processes within the same species. Previous work from the Wills lab indicates that genes encoding the enzymes of the pentose phosphate pathway (PPP), which generates precursors of biosynthetic molecules such as nucleotides and lipids, are highly expressed during tadpole tail regeneration. Although tail regeneration has been well studied, the variation in hind limb regenerative capacity across developmental stages and the metabolic pathways involved remains unclear. Hence, I performed a live imaging study to determine the developmental progression of hind limbs and assess their regenerative potential. This data suggests a decrease in success as the tadpole gets older. Immunohistochemistry staining of mitotic cells in developing limbs shows that proliferating cells decline as regeneration competency decreases. I hypothesized that genes for the PPP enzymes would also be expressed during successful limb regeneration, which was confirmed by in-situ hybridization. Together, these findings indicate that the regenerative capacity in limbs of X. tropicalis is stage-dependent and that PPP genes are expressed during all stages of regenerative capacity. This provides insights into the role of metabolic reprogramming in appendage regeneration, with the potential for translating it into non-regenerative species like mammals.

The angiopoietin-Tie2 signaling pathway is central to regulating vascular stability, remodeling, and permeability. Angiopoietin-1 (Ang1) promotes pAKT activation and vascular stability and regeneration, whereas Ang2 antagonizes these effects, leading to leaky vasculature. Although Tie2’s association with α5β1 integrin has been implicated in mediating these divergent outcomes, the requirement of direct F-domain ligand binding for integrin recruitment remains unclear. Here, we report the development and mechanistic evaluation of a de novo designed Tie2 mini binder (Tmb) that selectively targets the Tie2 receptor without engaging α5β1 integrin. Using an AI-based protein design pipeline, we designed Tmb with high affinity (KD ≈ 0.65 nM) for Tie2, as confirmed by CryoEM analysis, which demonstrated that Tmb accurately recapitulates its designed structure. When conjugated to multivalent scaffolds, Tmb effectively clusters Tie2 receptors, recapitulating the signaling profile of native Ang1. Notably, high valency Tmb constructs (e.g., H8T) robustly activated pAKT and induced nuclear FOXO1 exclusion, mirroring the pro-survival and vascular stabilizing effects of Ang1, despite lacking the capacity to bind α5β1 integrin directly. Detailed cellular assays revealed that Tie2 clustering leads to the formation of two distinct complexes: a Tie2–α5β1 integrin complex that facilitates focal adhesion assembly and cell migration via pCAS recruitment, and a Tie2–tight junction complex (comprising ZO1, claudin-5, and occludin) that underpins vascular barrier integrity. Importantly, competitive binding studies demonstrated that integrin recruitment to the Tie2 complex does not require direct F-domain engagement. In human iPSC-derived diabetic blood vessel organoids, treatment with Tmb-based Tie2 agonists ameliorated diabetic vasculopathy phenotypes by reducing pathogenic collagen IV deposition, restoring tight junction organization, and lowering nuclear FOXO1 levels. These findings provide novel insights into the mechanistic interplay between Tie2, integrin, and junctional proteins, and underscore the therapeutic potential of synthetic Tie2 agonists in vascular repair and diabetic vasculopathy.

Protein subunit vaccines are highly used today as an alternate vaccine platform to older vaccines such as live-attenuated viruses. They contain a protein antigen of the virus or bacterium that can be recognized and targeted by the immune system, and an adjuvant that amplifies the immune system response to this protein by widely putting the immune system on alert. The most commonly used adjuvants pose the risk of possible adverse reactions and are not created to target specific immune pathways, but rather stimulate general inflammation. To design a vaccine adjuvant that generates a more targeted immune response, we are using the self-assembling protein nanoparticle, I53-dn5, to display a CD40 binder that mimics the T Cell ligand, CD40L, by binding to B cell surface receptor CD40. We aim to create a particle that can replicate the binding interaction between B and T cells in the lymph node responsible for triggering antibody maturation, and B cell proliferation and differentiation. We hypothesize that this multivalent display of CD40 binder will generate potent B cell responses allowing us to respond to an antigen more effectively than current adjuvants. We are utilizing computational protein design methods like RFDiffusion, ProteinMPNN, and AlphaFold2 to optimize this display, and testing these designs in vitro for stability and ability to elicit downstream signaling effects of the CD40/CD40L interaction. This research holds two promising innovation potentials. The first is creating higher potency adjuvants by stimulating specific signaling pathways for use with protein subunit vaccines. Secondly, these materials can be used as a more stable and potent molecule in biochemical assays such as being an alternative to feeder cell lines in B cell support culture.

Neuronal and endocrine cells store and secrete molecular cargos like neurotransmitters and metabolic hormones through the regulated secretory pathway. Dense-core vesicles (DCVs) originate from the trans-Golgi network and undergo a maturation process involving peptide processing and cargo sorting before being stimulated to release their cargos outside the cell. Dysregulation of this process leads to a wide range of neurological and metabolic disorders; yet the molecular mechanisms underpinning it remain poorly understood. Vesicular traffic are largely coordinated by the Rab family of GTPase proteins.  Previous work identified the conserved proteins TBC8 and RUND1 as regulators of DCV maturation in Caenorhabditis elegans; and both proteins bind active GTP-bound RAB2. TBC8 is the putative RAB2 GTPase activating protein (GAP) which promotes conversion of GTP-RAB2 into GDP-RAB2, thereby inactivating the Rab. RUND1 interacts with both active GTP-RAB2 and TBC8, yet its precise function remains unknown. This study aims to characterize the biochemical function of TBC8 and RUND1 in regulating RAB2 activity. Using purified proteins, we demonstrate that TBC8 greatly promotes RAB2 GTP hydrolysis, indicating it is the bona fide RAB2 GAP. Additionally, we show that RUND1 strongly inhibits TBC8-stimulated RAB2 GTP hydrolysis, suggesting RUND1 may compete with TBC8 for RAB2 binding. Given this interplay between RUND1 and TBC8 in binding RAB2, we hypothesize that RAB2 exhibits exclusively pairwise interactions with its partners. To test this, we will use mass photometry to study whether RUND1 and TBC8 can bind RAB2 simultaneously or if one complex is preferentially formed. Based on our current findings, we propose a model where TBC8 promotes RAB2 inactivation by stimulating GTP hydrolysis and RUND1 blocks RAB2 inactivation by TBC8, prolonging the activate state of RAB2.

Organs maintain consistent shape, form, and volume through complex processes, one of which is cell-cell adhesion. E-Cadherin, a key cell-cell junction protein, is critical for cell shape, arrangement, and tissue structure. In this study, I investigate the role of E-Cadherin in the morphogenesis of the Drosophila Malpighian tubules, a model system where I can manipulate E-Cadherin expression and use fluorescence microscopy to observe the effects on organ growth. Previous work involved fixing and staining embryos to track E-Cadherin localization using fluorescent imaging to measure its intensity. I will further analyze E-Cadherin localization spatiotemporally by constructing a fluorescent fly line for live imaging during development. I expect E-Cadherin concentration to increase during elongation and to be enriched in looped regions of the tubules. To assess the requirement of E-Cadherin in organ formation, I will reduce its expression using RNAi and degradFP, expecting significant developmental defects due to the protein's vital role in morphogenesis. These defects will be quantified by comparing changes in cell and organ shape in control and E-Cadherin-reduced tubules. Additionally, I will help develop Python tools for 3D image analysis, including cell segmentation, creating a 3D model of E-Cadherin in tubular cells, and extracting protein intensity. Developing these tools not only enables our work in these tubular organs but also allows for comprehensive image analysis of other tubular 3D organ forms. Elucidating the precise mechanisms behind cell behavior, shape, and cell-cell interaction has important human health implications and will enable work in many other fields such as cancer, regenerative treatments, tissue growth, and organ synthesis. 

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) senses cytosolic DNA and activates an immune response. This signaling pathway is important for defending against viral infections and regulates cancer immunity. In order to study this signaling pathway, our goal was to develop an in vivo tool using the model organism Drosophila melanogaster to answer questions regarding the activity of STING signaling. To make this STING signaling reporter gene assay, we used molecular cloning to clone the promoter regions of three genes downstream of the STING pathway (Nazo, Srg1, Srg2). We used restriction enzymes to combine the promoter regions with a vector containing the reporter DsRed (sequence for red fluorescent protein). This DNA construct was injected into flies to create transgenic fly lines. When STING signaling is active, the reporter sequence is transcribed and translated along with the STING target genes. Thus, when the transgenic flies are dissected and stained for the reporter DsRed, the images show where and how much STING signaling is active in specific cells in tissue. We tested our reporter line with the antibiotic drug bleomycin which causes tissue and DNA damage that could activate STING signaling. We found that when flies from the reporter line were fed with sucrose containing bleomycin, images of their guts showed areas with high fluorescence that weren’t visible in flies fed with just sucrose. The fluorescent areas also aligned with areas appearing to have cells with broken nuclei, suggesting our reporter line was successful. This tool is useful for any project that needs to detect STING signaling, and is helpful for answering questions regarding the pathway. There are many unknowns regarding what regulates STING signaling and what STING signaling causes which can be further studied with this in vivo reporter.

Nucleotides are essential for diverse cellular functions, from DNA synthesis to signaling pathways. Inosine 5’ monophosphate dehydrogenase (IMPDH) is a highly conserved regulatory enzyme in the de novo pathway for guanine nucleotide synthesis. Humans have two isoforms of IMPDH, and both are highly regulated to maintain appropriate levels of purine nucleotides required by the cell. Mutations in IMPDH2 have recently been linked to dystonia, a neurological disorder. Through collaborations with clinicians, this work examines emerging mutations in IMPDH2 that have been identified in patients (ages 2-12) with neuromuscular symptoms such as hypotonia, developmental delay and impaired motor skills. All of the disease-causing mutations are located in or nearby the regulatory domain of the enzyme, desensitizing it to normal feedback inhibition by the downstream product GTP, and causing the enzyme to be hyperactive. In this research project, I am assaying these IMPDH2 mutants in the presence of potential IMPDH2 inhibitors to identify small molecules that will inhibit the hyperactive mutants. I am testing six small molecules, four of which are natural compounds derived from traditional Chinese medicine. The other two are mycophenolic acid (MPA) and ribavirin (RBV) which are previously established IMPDH inhibitors used for immunosuppression and hepatitis. I am also using electron microscopy to understand the effects of these inhibitors on the structure of IMPDH2. My preliminary data shows that MPA and RBV exhibit inhibitory activity on the disease mutants. I have also characterized new disease mutants as collaborators have connected with us. This led to the discovery of a new mutant that is hypersensitive to GTP inhibition, making it the only mutant that has behaved differently. I anticipate that the other small molecules will inhibit but not as strongly as RBV and MPA. The long-term goal of this work is to identify drug candidates for treating IMPDH2-related disorders.

Extracellular vesicles (EVs) are lipid-bilayer membrane-enclosed structures that cells produce and use for intercellular communication. Within the context of cancer, EVs have been shown to enhance cancer development by delivering cargo from malignant cells to recipient cells to promote survival, proliferation, and invasion. In a previous project, I conducted a chemical screen alongside my graudate mentor and other undergraduates to determine kinases that were important to EV biogenesis. One hit was the JNK pathway, which decreased EV production when inhibited. I studied the pathway in further detail utilizing a variety of experimental techniques to establish its importance for EV generation, and I was able to conclude that JNK regulates EV biogenesis. Another facet of cancer development is oxidative stress, caused by reactive oxygen species (ROS). When unregulated, these highly reactive free radicals and molecules derived from oxygen can damage DNA, facilitate metastasis, and aid in cancer progression. Given that surrounding literature revealed that JNK is activated by ROS, I hypothesized a connection between ROS and EV production. This project aims to more directly uncover the impact of ROS on EV generation by manipulating ROS-related genes in vivo. To do this, I knocked down ROS generator genes such as Dual Oxidase (Duox) in Drosophila melanogaster. I quantified ROS levels by staining the dissected tumor tissues with an ROS probe to ensure that the genes were functioning as expected. Then, I stained the tissues for phospho-JNK as a proxy for ROS quantification and to measure JNK activity. Finally, I conducted live imaging of the tumor tissues to quantify EV generation. I anticipate that impairing ROS generation will inhibit JNK activation, subsequently leading to a decrease in EV production. Understanding how factors involved in cancer development function in relation to each other is crucial for discovering novel cancer therapeutics.

Extracellular vesicles (EVs) are essential mediators in intercellular communication secreted by cells to transfer bioactive cargo that lead to biological effects. The crucial roles EVs have in maintaining biological homeostasis are similarly found within cancer cells in the tumor microenvironment, where they promote cell growth/survival, invasion, and metastasis. Investigating methods to reduce tumor-cell derived EVs could provide substantial remedies for cancer patients. One pathway of interest in cancer is the cellular response to reactive oxygen species (ROS)—highly reactive molecules which tumor cells use for oncogenic signaling, to damage macromolecules, and drive tumor progression. Modulation of ROS levels may yield anticancer effects, but research about the role of ROS in EV biogenesis has not been conducted. To assess their connection, I used MDA-MB-231 human breast cancer cells as an in vitro model for EV biogenesis. My interest in ROS and EVs began when I assisted my graduate mentor in an extensive chemical screen and found kinase inhibitors that altered EV production via an EV isolation protocol. From these hits, I identified ROS-activated pathways that promote cancer progression as important players in EV production. I then tested if chemicals known to directly affect ROS alter EV production by isolating and quantifying EVs and by imaging their production from MDA-MB-231 cells. To provide a comprehensive understanding of the pathway, I validated upstream interactions of EV biogenesis by measuring the production of ROS using a chemical marker that emits green fluorescence when oxidized. From this data, I can determine if there is a direct interaction between ROS and EV production. An understanding of EV biogenesis and its connection to ROS and cancer progression may unveil new opportunities for novel cancer therapeutics.

Gynogenesis is an asexual reproduction strategy where sperm is necessary for fertilization, but the resultant offspring have no paternal DNA and two maternal sets of chromosomes. This strange reproductive strategy has never been observed before in nematodes (round worms), until a few years ago when a previous student at Ailion Lab observed the phenomenon when investigating the hybrid offspring of two species of Caenorhabditis roundworms; C.Becei and C. Nouraguensis. On their own, neither of these species exhibit asexual reproduction. Furthermore, C. Nouraguensis females normally produce haploid eggs, but when cross bred with C. Becei, they began to produce almost only diploid eggs. It is known that asexuality has arisen from previously sexually reproducing species, but the exact mechanisms of this evolution are unknown. This research project uses CRISPR techniques to attach fluorescent proteins to key structures involved in meiosis, which can then be imaged to reveal any irregularities which could explain the production of diploid eggs instead of haploid. The main goal is to understand the cellular mechanisms which facilitate such a dramatic change in reproductive strategy. 

Through the tagging and cleaving of DNA sequences in Saccharomyces cerevisiae, we observe changes in MCM (minichromosome maintenance) protein recruitment, loading, and activation. The functions of MCM2-7 are critical to separate and unwind DNA in preparation for replication. In the G1 phase, MCMs are recruited and loaded to replication origins in an inactive state, within G1 cells. S phase follows, in which the CDC7/DBF4 kinase phosphorylates the MCM, allowing it to fire and initiate DNA unraveling for replication. The regulation of licensing and activation through these phases is crucial to ensure appropriate replication timing (early vs. late) in the genome. By tagging either a histone or one of the MCMs with micrococcal nuclease (MNase), I implement ChEC (chromatin endogenous cleavage) sequencing to cleave the DNA specifically where it surrounds the nucleosome or the MCM complexes. This method allows for precise mapping of the location of MCM binding sites and nucleosomes. We expect to see an increase in MCM helicase complex licensing and firing in regions occupied by less nucleosomes, resulting in regions of earlier DNA replication timing.

Congenital hydrocephalus is a condition that is characterized by an accumulation of cerebrospinal fluid in the brain. This increases pressure in the brain, leading to neurological deficits. Surgery is the only current intervention however, even after surgery, patients experience lifelong complications. The underlying genetic mechanisms that cause human hydrocephalus are still unknown. To investigate the mechanisms behind hydrocephalus, the lab developed a mouse model by selectively ablating Notch signaling in specific brain regions. These mice developed obstructive hydrocephalus due to a loss of cell adhesion in the Sylvian aqueduct, a thin channel connecting the 3rd and 4th ventricles of the brain. Interestingly, brain regions exposed to high levels of Hedgehog signaling retained cell adhesion, indicating a possible protective role. Based on this, I hypothesized that Hedgehog signaling plays an unexpected role in supporting Notch-mediated cell adhesion. To test this hypothesis, I utilized small molecule Notch inhibitors to suppress Notch signaling activity in cortical spheroids. Cortical spheroids are precursors to cortical organoids derived from mouse embryonic stem cells. Preliminary data showed that various concentrations of different inhibitors were able to reduce cell adhesion and disrupt neural rosette formation in the spheroids. Neural rosettes are structures that recapitulate early cortical formation. After treatment with Hedgehog agonists, there was an increase in the number of neural rosettes in both untreated and inhibitor-treated spheroids, indicating that Hedgehog signaling can compensate for a loss of Notch signaling and preserve cell adhesion in the developing brain. These results seem to suggest that Hedgehog signaling can compensate for a loss of Notch through cell adhesion maintenance and prevent premature neural progenitor cell differentiation. The goal of the project is to establish cortical spheroids as a model system to screen potential genes associated with human hydrocephalus along with future drug therapies.
 

The Hedgehog signaling pathway is essential for embryonic development. Errors in the Hedgehog pathway can cause limb, heart, and left-right patterning defects. However, Hedgehog signaling also plays a crucial role in the regeneration and maintenance of adult tissues and cells. Mutations in key components can lead to the constitutive activation of the pathway, leading to uncontrolled cell proliferation and cancer. Dysregulated Hedgehog signaling is associated with two major cancer types: basal cell carcinoma (skin cancer) and medulloblastoma (a pediatric brain tumor). To counteract this, small molecule inhibitors like Vismodegib have been developed to directly bind to and suppress the activity of the Hedgehog transducer, Smoothened (SMO). While Vismodegib is a potent inhibitor of Hedgehog signaling, mutations in SMO eventually lead to drug resistance and tumor relapse.The mechanisms underlying Vismodegib drug resistance and how the Hedgehog signaling pathway is reactivated in its presence remains unknown. To investigate these mechanisms, a constitutively active fluorescent Hedgehog reporter was knocked into the mouse skin cells, and a genome-wide CRISPR knockout (KO) library approach was used to generate a pool of gene-edited cells. Following treatment with the Hedgehog ligand Sonic Hedgehog (SHH) to activate the pathway and Vismodegib to inhibit it, fluorescence-activated cell sorting (FACS) was performed to sort the cells with high fluorescence to identify the KO cells that retained Hedgehog pathway activity after treatment with the Hedgehog inhibitor. This screen identified 10 novel genes associated with Vismodegib resistance. For further studies, I used a dual guide approach to generate knockouts of each gene respectively and clone them into CRISPR/Cas9 gene-editing vectors. My goal is to evaluate the expression of different Hedgehog genes using biochemical approaches. This would allow us to understand how each gene affects downstream pathway activity and identify the mechanism through which these genes could potentially impart drug resistance.

Downregulation of the mTOR complex has been shown to increase lifespan and delay development of multiple organisms, including Drosophila melanogaster. Rapamycin, an inhibitor of this complex, is undergoing FDA-approved clinical trials as a promising anti-aging drug. However the impact of genetic variation on rapamycin's response is unknown. Our study of 140+ genetically diverse Drosophila strains revealed significant variation in pupation time after rapamycin exposure, however, the underlying mechanisms of this variation remain poorly understood. Surprisingly, this sensitivity does not correlate with genetic variation in or around the mTOR gene. We therefore hypothesize that differences in phosphorylation of downstream mTOR targets may explain this variation. Currently, we are using multiple approaches to investigate how activation of downstream targets differs between highly resistant and sensitive strains. We aim to characterize the phosphoproteome of first instar Drosophila larvae from highly sensitive and resistant strains. First instar larvae were treated with rapamycin for 12 hours, followed by mass spectrometry analysis to identify phosphorylation changes in mTOR pathway targets. To validate that 12 hours of treatment induces a rapamycin response, we monitored the growth of a parallel group of larvae until 72 hours and measured their size. Sensitive DGRP strains, 348 and 517, showed a twofold reduction in length when treated with 20uM rapamycin compared to control (p-value <0.0001), while the resistant strain, 441, showed no significant decrease. Comparing the phosphoproteome of multiple resistant and sensitive lines will uncover molecular factors associated with resistance or sensitivity. Additionally, whole-larvae RNA-seq will assess the expression profile of these factors, revealing whether gene expression of tor pathway-related genes contributes to sensitivity. Understanding the mechanisms behind rapamycin resistance or sensitivity is critical for its clinical application. This project highlights the value of accounting for genetic variation in drug development, guiding future approaches for developing new drugs.

Directly converting fibroblasts (that make up scar tissue) into skeletal or heart muscle without a pluripotent intermediate (direct skeletal muscle or cardiac reprogramming) is one of the most promising methods for regenerating lost muscle tissue, but its low efficiency in human cells remains a significant obstacle toward clinical application. In collaboration with the Institute of Protein Design, UW, we have designed several synthetic minibinders against receptor kinases which are highly specific to their cognate receptor. Utilizing these minibinders we have created a new class of designed protein, called heterofusions, that fuse two unrelated minibinders together to force the two cognate receptor kinases together in an unnatural pairing, which could elicit novel signaling responses not achievable using natural ligands. However, which heterofusions elicit novel signaling is unknown. We aim to use direct skeletal muscle and cardiac reprogramming systems, which would benefit from this novel signaling, to screen which heterofusions elicit novel signaling to increase efficiency. To do this I developed an inducible direct cardiac reprogramming system and we also used a previously established inducible direct skeletal muscle reprogramming system to be backgrounds for screening heterofusions, with efficiency determined by imaging cardiac and skeletal muscle development makers. We found a few heterofusions, including that which brings together TrkA and BMPRII (TAB2), increased the efficiency of skeletal muscle reprogramming. I found in signaling experiments using Chinese hamster ovary cells modified to express human TrkA and BMPRII that TAB2 upregulates pERK and pCREB. Interestingly, pCREB is not part of native TrkA or BMPRII signaling, meaning novel signaling is occuring. Additionally, I have shown pCREB inhibition with a small molecule impairs direct skeletal reprogramming and TAB2’s ability to increase efficiency, showing pCREB is TAB2’s mechanism of increasing efficiency. These results show heterofusions novel signaling abilities and its applications in revolutionizing regenerative therapies.

The Type 4 pilus (T4P) in Neisseria gonorrhoeae, and other bacterial species, is a protein system responsible for host-cell adhesion of the pathogen. Insight into the structure of this system necessary for N. gonorrhoeae pathogenesis can aid the development of novel therapeutic avenues. PilC, the adhesin located at the tip of the T4P, is essential for the initiation of pilus assembly, DNA transformation, and host-cell adhesion. It is believed to interact with a complex of minor pilin proteins to initiate pilus assembly, but the mechanisms of this process are unclear. My project aims to develop an amber-codon suppression system to investigate the function of PilC and its interactions with minor pilins and host cells. Based on computational modeling, the last 12 amino acids of PilC form a beta-strand that binds to the minor pilin PilK to initiate piliation. I designed a mutated version of the PilC gene by inserting an amber stop codon (sequence “TAG”) before the genetic code for this beta-strand. When expressed in gonorrhoeae, the mutated gene leads to a loss of T4P. Next, I aim to genetically modify an existing tRNA to read an amber stop codon. I hypothesize that such a tRNA, known as an “amber suppressor,” when expressed in the non-piliated cell, should rescue the defect in PilC by reading the amber stop codon, thus enabling translation of the complete, functional protein. The resulting cell should change from non-piliated to piliated, confirming that the final beta-strand of PilC is essential for T4P formation. Once I develop a functional amber-suppressor system in N. gonorrhoeae, I intend to study other domains of PilC and the minor pilins essential to T4P biogenesis, by extending the system to enable site-specific incorporation of non-canonical amino acids with useful properties.

G proteins play a vital role in regulating neuronal activity by acting as key intermediaries that relay extracellular signals inside the cell, triggering a cascade of further signaling events that impact cellular function. This signaling can modulate the activity of ion channels in the neuronal membrane, which control membrane excitability by opening or closing in response to signals, thereby affecting the cell's electrical potential. We are studying the signal transduction pathway that acts downstream of the heterotrimeric G protein Gq to regulate the NCA cation channel in Caenorhabditis elegans. My project focuses on characterizing an unidentified mutant yak133, which has a distinct phenotype defined by deep body bends, also referred to as "loopy." This phenotype suggests that yak133 could be connected to Gq signaling, as activating the Gq pathway leads to a loopy phenotype. The goal of my project is to identify the gene affected by yak133 and understand how it functions to modulate the NCA channel. I narrowed down a list of candidate genes from whole genome sequencing of yak133 by performing a genetic cross to deficiency strains that lack a specific segment of DNA. I then carried out a forward genetic screen and identified a new recessive mutant, yak193, which appears to affect the same gene. I am currently preparing this strain for genome sequencing, and by analyzing both mutants, I expect to identify the gene affected by yak133 and yak193, as they should share mutations in one gene in common. This work will provide relevant insights into the molecular mechanisms regulating neuronal activity and how disruptions in this pathway affect motor and behavioral function. Since many of the genes in C. elegans are conserved in humans, these findings could have broader implications, potentially advancing our understanding of human neuronal function and related disorders. 

Upon nerve injury and neurodegeneration, neuron regeneration is crucial to maintain proper function. However, this natural process happens infrequently and slowly. Neuron regeneration is known to be mediated by the activity of nerve growth factor (NGF) in neurons, which binds to two receptors: tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR). Previous studies have shown that engaging the receptor p75NTR activates a signaling pathway that also triggers a pain response, thus it would be ideal to have a ligand that only activates TrkA for neuron regeneration without initiating the pain response. In collaboration with the Institute for Protein Design (IPD), this study investigated an AI-designed TrkA agonist that specifically binds to and activates only the TrkA receptor. We used fibroblasts transdifferentiated into neurons as a model to study the efficiency of this TrkA agonist. Western blotting was used to study the phosphorylation of the proteins downstream of TrkA in the signaling pathway, such as pPLCγ, pAkt, and pErk, and the activity of transient receptor potential vanilloid 1 (TRPV1), a calcium channel that indicates the sensitivity of a neuron. Immunofluorescence staining was used to examine the expression of calcitonin gene-related peptide (CGRP), a neuropeptide involved in pain perception. We found that the designed TrkA agonist generates a similar level of activation of downstream proteins as NGF while successfully preventing the expression of pain response markers. Directly injecting NGF as a treatment for neurodegenerative diseases is generally not considered viable as it often induces significant pain, therefore this TrkA agonist has the potential for therapeutic use.

Chronic pain is a public health crisis that has been clinically demonstrated to disrupt reward learning and motivation in affected individuals. Previous literature has indicated that Calca neurons in the parabrachial nucleus (PBN) play a key role in the sensory and emotional processing of pain and become hyperactive in chronic pain models. Despite this, how PBN Calca signalling impacts adaptive decision-making in a positive-reinforcement context remains unclear. This study aims to explore how chronic PBN Calca hyperactivity impacts learning and motivation. Using chemogenetics, a technique that selectively modulates neuronal activity, we chronically activated PBN Calca neurons in transgenic mice. These mice were then tested in a two-phase positive-reinforcement operant conditioning paradigm to assess how chronic PBN Calca activation altered learning rates and motivation compared to controlled animals. In phase one, mice underwent a fixed ratio schedule in which they learned to press a lever during a distinct cue to obtain a food reward. In phase two, mice underwent a progressive ratio schedule in which they had to press a lever an increasing number of times to obtain a food reward. We hypothesized that chronic activation of PBN Calca neurons would impair both learning rate and motivation. With this work, we hope to clarify the impact of centrally-mediated chronic pain on motivational and cognitive processes, which could inform the development of future therapeutic strategies.

Natural growth factors like fibroblast growth factor (FGF) are essential for maintaining pluripotency in induced pluripotent stem cells (iPSCs). However, current limitations of native growth factors include signal instability, off-target pathway activation, and dependence of xenogenic components for production. To address these issues, we developed a synthetic protein, C6-79C, which consists of six scaffolded subunits of a de novo designed FGFR1/2c binder, mb7. While mb7 functions as an FGF pathway inhibitor, the hexameric C6-79C acts as a receptor tyrosine kinase (RTK) agonist, providing more isoform-specific and prolonged signaling compared to native FGF. We formulated SynGrow, replacing FGF with C6-79C in minimal E8 media, and compared its performance against commercial media. Our study focused on three objectives: (1) comparing the expression of pluripotency markers (Oct4, NANOG, SOX2, and TRA1-60) in cells grown in SynGrow versus commercial media, and (2) evaluating morphology and viability under different media change regimens (daily, every other day, or no change). iPSCs grown in SynGrow exhibited superior morphology compared to those in mTeSR (commercial media). Pluripotency markers (Oct4, NANOG, and SOX2) were expressed at similar levels in both media, with SynGrow also showing higher expression of TRA1-60 across passages, confirmed by flow cytometry. Future evaluations will assess germ layer marker expression following directed differentiation. Our findings demonstrate that synthetic protein-based media formulations, like SynGrow, can effectively replace native growth factor-based media. This approach offers stable, prolonged, and xeno-free alternatives for stem cell culture, with broad implications for improving reproducibility and safety in regenerative medicine and cell-based therapies. 

Genetic mutations in Caenorhabditis elegans (C. elegans) worms can be studied to understand disruptions in pathways relevant to those in humans, due to ortholog between worm genes and human counterparts. These mutations can manifest as an unmotivated phenotype where the worm displays decreased motivation to move. To explore this phenotype, we performed a series of crosses on a strain of mutated worms to map and identify which gene the mutation is on and to gain a better understanding of the underlying reasons behind the unmotivated phenotype. Our work thus far has led to the potential uncovering of a new gene correlating with this phenotype that has never been associated together before. The worm mutation named yak187 was first generated through random mutagenesis. I performed crosses between yak187 worms and various other strains that each contained a fluorescent marker on a different chromosome. Results yielded little correlations between yak187 and any of the chromosomes we tried. We continued crossing with more strains that contained markers near the ends of chromosomes of suspect and eventually narrowed our highest probable linkage to the right arm of the X chromosome. There are no mutants with this phenotype known in this region yet so our next steps are to sequence the whole genome to pinpoint the location. Furthermore, we have reason to believe that this mutation impacts the dense core vesicle (DCV) pathway impacting neuropeptide release. This pathway is important for regulating body functions, development, and emotions. Disruptions to DCV processes can result in diminished abilities for organisms to operate correctly, resulting in similar consequences as those seen in the mutated worms. The overall pathway involving the production and maturation of DCVs and the secretion of neuropeptides is similar to that in humans, making the study of this system in C. elegans further more exciting.

Salivary glands are organs in the mouth which produce and secrete saliva, a multifunctional fluid crucial for processes including oral cavity lubrication, digestion, and antimicrobial functions. Diabetes mellitus has been associated with salivary gland dysfunction and harmful oral consequences including severe tooth decay and disrupted wound healing, yet it is not currently known what cell populations are affected in salivary glands and how this disease affects cell organization, function, and metabolic response. One model for diseases in human tissues are organoids, three-dimensional multicellular systems derived from stem cells which self-organize to mimic the structure and function of tissues in vivo when given the right cues. Dr. Devon Ehnes in the Ruohola-Baker Lab recently created a protocol to develop salivary gland organoids from induced pluripotent stem cells (iPSCs), and through additional culture in a high-glucose media along with inflammatory cytokines, this organoid has been used to study how diabetes affects salivary glands. Preliminary analysis has suggested acinar and ductal cell dysfunction and mitochondrial stress as causes of salivary gland dysfunction, but further work is necessary to understand how this diabetic environment leads to changes in cell function and mitochondrial activity. Here, I use a human iPSC-derived organoid model to assess how diabetic conditions affect the expression and localization of the acinar marker AMY1A, the ductal marker KRT19, the cell stress marker FOXO1, and the mitochondrial marker ATPB to determine the mechanisms for salivary gland dysfunction in diabetes.

Human tooth development is a complex and tightly regulated process that involves multiple signaling pathways and specialized proteins coordinating enamel formation. Enamel, the hardest tissue in the human body, is secreted by ameloblasts, which follow a distinct developmental process. Disruptions in these processes can lead to enamel-related disorders, such as amelogenesis imperfecta, a genetic condition characterized by defective enamel formation. A key factor in this disorder is WDR72, a gene that encodes the tryptophan-aspartate repeat domain 72 (WDR72) protein, which is critical for intracellular trafficking during enamel maturation. Although WDR72 has been studied in animal models, its precise localization and function in human fetal tooth buds remain incompletely understood. To address this question, I cryosectioned human fetal tooth samples at 19 and 22 gestational weeks and performed immunochemistry staining to visualize WDR72 alongside key enamel proteins. I performed cryosectioning to prepare thin tissue sections of each tooth bud sample, followed by immunohistochemical staining with antibodies specific to WDR72. I then imaged selected sections under a fluorescence microscope. Preliminary results suggest distinct WDR72 distribution in regions corresponding to secretory ameloblasts. These findings offer insights into the localization of WDR72 during tooth formation and lay the groundwork for future studies on the mechanisms of tooth regeneration. 

Mitochondrial fusion is essential for cellular function, metabolism, apoptosis, and stress responses. Mitochondrial outer membrane fusion is mediated by two mitofusin paralogs, Mfn1 andMfn2, which are large GTPases that remodel cellular membranes. Membrane fusion likely proceeds through two distinct steps, first tethering two organelles and second lipid mixing; however, much of the mechanism is poorly defined. Previous studies have solved crystal structures of a partial construct of the mitofusins, revealing a GTP dependent conformational change ; however, this is not a complete analysis as at least two states in the catalytic cycle are missing. Our project aims to quantify the conformational changes of Mfn2 throughout the entire mechanism of GTP hydrolysis. To achieve this, we are utilizing a novel transition metal Förster Resonance Energy Transfer (tmFRET) developed by Dr. Gordon and Dr. Zagotta. This system utilizes a noncanonical amino acid as the donor and a transition metal as the acceptor to measure changes as small as 3Å. Currently, we’re mutating the cystines to develop a single donor-acceptor pair, while keeping the stability and GTPase function of Mfn2. Our main approach is to introduce targeted mutations in key cysteine residues and analyze their effects on the protein’s enzymatic activity. Using molecular biology, we design DNA plasmids encoding the mutations,and express and purify the mutant proteins.  Finally we measure the GTPase activity using malachite green assays. Our current findings suggest some mutations have trivial impact on MFN2’s GTP hydrolysis, suggesting that it’s viable. The further goal of our project is to keep only one solvent accessible cysteine while maintaining protein function. This research will further elucidate the mechanism of mitochondrial fusion and its role in disease pathogenesis. Explanding the biophysical understanding of membrane remodeling.

Per-Arnt-Sim (PAS) domains are ubiquitous protein modules that enable cells to detect and respond to environmental signals. For instance, circadian rhythm regulators leverage PAS domains to sense stimuli and initiate protein-protein interactions critical for maintaining biological oscillations. Structurally, the sensory region of PAS domains detects environmental cues—such as fluctuations in phosphorylation levels—while the effector domain converts these signals into cellular responses, including altered gene expression or protein interactions. Inspired by this natural framework, our project aims to design de novo sensory domains that selectively recognize tyrosine phosphorylation, a key post-translational modification in cellular signaling, through association/dissociation between bound and unbound states regulated by the phosphorylation/dephosphorylation cycles. During the design phase, we prioritized synthetic peptide targets for initial proof of principle and systematically deployed computational pipelines: (1) Rosetta introduced phosphotyrosine modifications into pre-designed protein-peptide heterodimer scaffolds; (2) iterative LigandMPNN with Rosetta FastRelax optimized binding interfaces to accommodate the phosphotyrosine modifications; (3) RFdiffusion Partial Diffusion enhanced the structural diversity around promising designs with the aim of improving affinity and specificity; and (4) Chai-1 and AlphaFold enabled in silico folding and structure-based filtering of final candidates. High-confidence designs will be expressed and purified from E. coli, and then undergo in vivo characterization via size exclusion chromatography (SEC) binding assays and enzyme-linked immunosorbent arrays (ELISA) to quantify their binding affinity, specificity, and the function of phosphorylation-dependent switching. Validated scaffolds will then be integrated with pre-designed effector domains to assemble fully de novo PAS domains. This modular platform establishes a foundation for designing phosphorylation-sensitive biosensors. Future adaptation to natural phosphorylation sites could yield programmable tools for interrogating signaling networks, advancing synthetic biology, and enabling precise manipulation of cellular communication pathways.

In nature, Per-Arnt-Sim (PAS) domains comprise a sensor that undergoes conformational changes upon signal recognition which either activates or deactivates an effector domain. Natural PAS domains detect environmental cues, such as oxygen, light, and small ligands; however, they do not sense phosphorylation, a key post-translational modification. Here, we present a designed de novo phosphorylation-inducible heterodimer that serves as a sensor domain. This system toggles between association and dissociation states in response to phosphorylation and dephosphorylation events. To engineer reversible association and dissociation, we designed phosphorylated peptides and their corresponding binders. Starting from a library of previously designed peptide-binder complexes, mutations were introduced into the peptide sidechains, replacing selected residues with phosphorylated tyrosine, serine, or threonine. Next, we ran iterative cycles of LigandMPNN-FastRelax to generate binder sequence candidates. Finally, we used AlphaFold2 and Chai1 to predict the folded structures of our input sequences and selected those that were predicted with high confidence. For experimental validation, the designed proteins will be overexpressed in Escherichia coli and purified using affinity and size exclusion chromatography. Phosphorylation-dependent binding specificity and affinity will be assessed through enzyme-linked immunosorbent assays (ELISA), surface plasmon resonance (SPR), and fluorescence polarization (FP). Subsequently, we will fuse these sensor domain designs to a collection of previously designed hinge proteins—which can bind/release an effector protein—to produce de novo PAS domains, thereby linking the sensing event to downstream functional responses. This adaptable system offers broad applications in biomaterials and synthetic biology, including the development of responsive scaffolds for biosensors and synthetic protein motors with controlled conformational cycles. 

Under acute genotoxic stress, such as chemoradiation, stem cells can undergo cell cycle arrest at the G1/S phase to avoid apoptosis. This protective state, called quiescence, is reversible once stress-free conditions allow re-entry into the cell cycle to regenerate daughter cells. We have previously demonstrated a common mechanism by which two types of stem cells—Drosophila germline stem cells (GSCs) and human-induced pluripotent stem cells (hiPSCs)—enter quiescence. Recently, we found Cyclin E (CycE) associated with the outer mitochondrial membrane (OMM) in both GSCs and hiPSCs. We are interested in studying the interaction between CycE mitochondrial localization domains and mitochondrial proteins responsible for CycE localization.To map the CycE mitochondrial localization domain, I have generated four CycE truncations tagged with GFP: ΔN-terminus, ΔCyclin Box_N terminus, ΔCyclin Box_C terminus, and ΔC-terminus. I have tested these constructs in various cell lines, including Rcc4, HCT116, MCF10A, HEK, and HeLa, and found that HCT116 exhibits mitochondrial localization of CycE. I will compare the localization of wild-type CycE-GFP versus mutant CycE using immunofluorescent staining of CycE and mitochondria in HCT116, as this cell line is well-suited for transfection studies. We have shown that mitochondrial CycE is degraded in quiescent stem cells through PINK1/PARKIN-mediated mitophagy. We propose that CycE degradation is necessary for quiescence entry. In Drosophila GSCs, we observe that upon irradiation, cells overexpressing non-degradable CycE continue cell division, whereas control cells undergo quiescence. Understanding the mechanism by which Cyclin E localizes to the OMM will enhance our knowledge of how it prevents quiescence entry, thereby contributing to the development of anti-cancer treatments.

Transcription factors (TFs) capable of binding specific DNA sequences are integral to targeted genetic regulation in both natural and synthetic contexts. However, the design of de novo TFs has proven challenging despite major advancements in computational tools for protein design. Preliminary efforts to design de novo TFs yielded a small library of dimers composed of two protein subunits in complex with one another, mimicking a common structural conformation of native TFs. Although these de novo TFs induced genetic repression, the magnitude of repression was relatively modest compared to natural repressors. Furthermore, the design characteristics indicative of the highest performing de novo TFs were unclear, suggesting that TF-induced repression was more complex than just allosteric inhibition of the RNA polymerase. To create TFs capable of higher levels of repression, machine learning tools including RFdiffusion, ProteinMPNN, AlphaFold3, and RoseTTAFoldNA were used to design de novo homodimeric TFs able to bend the DNA upon binding. In doing so, designed TFs could further inhibit the function of the cellular machinery involved in transcription by altering the structure of the DNA promoter region. The top designs were selected, synthesized, and tested for efficacy as genetic inhibitors in Escherichia coli, with preliminary results suggesting that these DNA-bending TFs successfully magnified repression of the target gene. These TFs represent a major advancement in engineering protein-DNA interactions and could have a variety of applications across synthetic biology and genetic engineering. In particular, successful designs could have applications in synthetic gene circuits, as biosensors for various cellular processes, and even therapeutics for a wide range of genetic diseases.

Humans have limited regenerative capabilities, providing incentive to study other natural models of regeneration to make advances in the field of regenerative medicine. In response to injury, species including Xenopus tropicalis employ cellular mechanisms to replenish lost tissue, a process that has high metabolic demands. Depending on their developmental stage, X. tropicalis tadpoles exhibit different regenerative capabilities after tail amputation, posing them as a unique model system. Three-day-old tadpoles (NF stage 41) are able to regenerate their tails completely after injury, but transiently lose this ability during what is known as the refractory period. However, they soon regain regenerative capabilities in the tail and in the developing hind limb before permanently losing them during metamorphosis. Previous work by the Wills lab has determined that the pentose phosphate pathway (PPP) is required for successful tail regeneration in stage 41 tadpoles, but leaves open the question of whether the PPP remains significant at subsequent regenerative stages and structures. Here I test the hypothesis that the PPP continues to facilitate appendage regeneration in post-refractory tadpoles. To functionally test the requirement of the PPP in post-refractory tail regeneration, I performed pharmacological inhibition of g6pd, a key enzyme in the PPP, during tail regeneration. To assess regeneration quality, I developed a pipeline using FIJI ImageJ and R to quantify metrics of regenerative success such as tail area and length. Using this framework, I found that post-refractory tadpoles had diminished regenerative success under PPP inhibition similar to stage 41 tadpoles. These results suggest that the PPP is required at all stages of tadpole tail regeneration and will provide a more comprehensive understanding of metabolism during regeneration, a potentially beneficial insight for research in wound-healing initiatives in mammals. 

According to the Centers for Disease Control and Prevention, the U.S. has more than 2.8 million antibiotic-resistant infections each year. The rise of multidrug resistance in bacteria poses an urgent clinical threat contributing to these various infections. UPAB1 is a specific strain of a notoriously drug-resistant bacteria Acinetobacter baumannii associated with catheter-associated urinary tract infections (CAUTI). UPAB1 infects the urinary tract through the introduction of a foreign object, such as a catheter. In response, the immune system coats the catheter with fibrinogen, a glycoprotein complex that assists in wound healing. UPAB1 uses its bacterial adhesin proteins, such as Abp2D, to bind to fibrinogen, deplete essential nutrients, and infect the urinary tract. By designing Abp2D inhibitors as de novo miniproteins, we hypothesize that A. baumannii will be prevented from establishing a bacterial infection and allow us to offer a potential alternative in combating antibiotic resistance in CAUTIs. Targeting UPAB1 Abp2D, we first developed designs of Abp2D inhibitors utilizing computational software like RoseTTAFold Diffusion (RFdiffusion) for miniprotein backbone design, ProteinMPNN for sequence design, and AlphaFold2 (AF2) for structure prediction of the sequences to validate and filter. Afterwards, in the laboratory, we expressed and purified the miniprotein designs. We are currently testing these designs as Abp2D inhibitors via E. coli cultures to determine their success in binding to UPAB1 Abp2D.

Eukaryotic cells contain many membrane-bound organelles and rely on precise vesicle trafficking to transport cargo between them and maintain organelle function and identity. Functional defects in Adaptor Protein complex 3 (AP-3) disrupt vesicle trafficking, leading to disorders such as albinism, seizures, and neutropenia. In Saccharomyces cerevisiae, AP-3 carries cargo from the late Golgi to the lysosomal vacuole, but how it dissociates from the carrier vesicle is not clear. Adenosine diphosphate (ADP)-ribosylation factor 1 (ARF1) regulates AP-3 recruitment and shedding, relying on GTPase-activating proteins (GAPs) for proper function. AGE2, an ARF1 GAP, functions redundantly with GCS1 to regulate ARF1 (Schoppe, 2020), thus AP-3 trafficking. This study aims to identify the interaction site between AP-3 and AGE2 to better understand AP-3 shedding molecularly. Using AlphaFold3, the Merz lab predicted a conserved alpha-helix region in the AP-3 subunit Apl5 C-terminal domain (CTD) as a potential interaction site. To test this hypothesis, I introduced substitution mutations in Apl5 CTD and conducted spinning disc confocal microscope experiments to assess AP-3 pathway defects with a GNSI reporter, which enables to quantify AP-3 function via fluorescence distribution. My results show no statistically significant difference in trafficking defects between wild-type and mutant strains, suggesting that the predicted site is either not a binding site, or not necessary for AP-3 and AGE2 function. Although this study yielded a negative result, it refines our understanding of AP-3 shedding. Future studies will explore alternative regions on Apl5 subunit of AP-3 to identify the true interaction site and uncover the molecular mechanism of AP-3 shedding.

The root cause of neurodegenerative diseases such as Alzheimer’s, Parkinson's and dementia is protein misfolding which leads to toxic aggregations in the brain, causing neuron death. At the molecular level, these diseases are offset by chaperone proteins, which have the task of stopping toxic aggregation events which directly causes onset of many neurodegenerative diseases. Understanding interactions between small heat shock proteins (sHSP), which are a class of chaperone proteins, and their client proteins, such as those involved in neurodegeneration is key for preventing these diseases. The sHSPs are a class of chaperone proteins which have the purpose of preventing other proteins from misfolding. The formation of toxic aggregates plays a factor in the first steps to pathology. Prevention of these aggregates and thus the toxic events that follow means understanding the protective mechanism that exists to stop aggregation. The challenge of these mechanisms is their immense complexity and there are not many methods in which small changes in the proteins can be detected. One possible technique that allows these small changes to be detected is Fluorescence Resonance Energy Transfer (FRET), which is a highly sensitive distance-dependent physical process. Fundamentally, energy is transferred non-radiatively via an excited molecular fluorophore (the donor) to another fluorophore (the acceptor). The goal of my work is to incorporate the FRET pairs into sHSP oligomers to probe changes in these oligomers. These changes could be the binding of another protein, such as a client protein, or another sHSP. These changes in the FRET signal will be indicative of how the probes are orientated relative to each other, allowing us to gauge what interactions are happening. My work validates the use of FRET to gauge how sHSP are interacting on a molecular level.