Glutathione S-transferase (GST) is an enzyme that is present in most organisms. Its primary function is the detoxification of xenobiotic compounds. In mammals, some GST isoforms have a role in modulating the MAPK signaling pathway. The GST protein from Schistosoma japonicum (26 kDa) is also an important tool used in biochemistry labs as a tag for the isolation of proteins via affinity chromatography. Reduced glutathione (GSH) is a substrate for GST that binds to its G-site with a high affinity, and this interaction can be exploited to purify GST-fusion proteins on agarose beads containing a GSH moiety. In the research presented, GST was expressed in E. coli, purified, and dialyzed to remove GSH ligand completely. The protein concentration of the purified, dialyzed GST sample was quantified using Qubit™ fluorescence spectroscopy. Isothermal titration calorimetry (ITC) was utilized to measure the dissociation constant and thermodynamic properties of the binding of GSH to GST. ITC is a highly sensitive analytical method that monitors changes in heat produced from noncovalent chemical associations. In order to obtain accurate ITC measurements, various concentration ratios of GSH/GST were explored. Quantifying the binding affinity and thermodynamic properties of GSH binding with GST provides useful information for investigators who use GST as a fusion tag or who are interested in reactions between the GSH-GST complex and its substrates.

During the formation of the early embryonic zebrafish body, bipotential neuromesodermal progenitor cells (NMPs) that are located at the posterior end begin to differentiate via regulation of the Wnt pathway. Relatively higher Wnt-signaling activates the expression of the tbx16 gene, which in turn inhibits the neural fate and commits cells to a mesodermal (muscle) fate. The aim of this study is to understand the downstream effects of tbx16 on the rate and mechanism of anterior migratory transitions of mesodermal cells as they leave the progenitor region and travel to the somite-forming mesoderm region. We have previously shown that successful anterior migration and differentiation of mesodermal cells is dependent upon tbx16 downregulating the expression level of two Arhgaps, a Rho GTPase-activating protein. Hence, we heat shock hsp70: Arhgap29 and hsp70: Arhgap35 zebrafish transgenic lines at early somite stages in order to compare and contrast the effects of sustained Arhgap activation during the critical period of mesodermal cell anterior migration. At 24 hours post-heat shock, embryos are sorted by wild-type and transgenic, with transgenics having severe defects in their posterior somite formation. Followed by immunocytochemistry for a muscle antibody, confocal imaging on whole-mount embryos captures the shape and number of somites from each transgenic line. My results show that while Arhgap29 affects both the number and morphology of somites, Arhgap35 only affects their morphology. This suggests that these two Arhgaps do not have overlapping roles in the control of mesodermal cell migration. I am currently performing a separate in-situ hybridization experiment to help me understand the role of these Arhgaps on the essential genes involved in muscle cell differentiation. This study contributes to understanding early vertebrate development as a model for human embryogenesis.

Hydrogen Sulfide (H₂S) has potent physiological effects. In large doses, it is a deadly toxin, but in small doses, it acts as a signalling molecule with a wide range of physiological effects. The worm C. elegans is a good model organism for the study of H₂S because of its short lifespan, ease of genetic manipulation, and ease of maintenance. In C. elegans, H₂S induces a stress response pathway, coordinated by the HIF-1 and SKN-1 transcription factors, that plays a role in extending lifespan and improves resistance to various stresses. A previous screen in our lab identified several mutations that suppress the requirement of hif-1 for survival in H₂S by increasing SKN-1 activity. One SKN-1-upregulated transcript, rhy-1, was sufficient to rescue hif-1 lethality in H₂S. RHY-1 is an integral membrane protein that is localized in the hypodermis, intestine, and some head neurons of C. elegans. It has predicted acyl transferase activity, but its specific molecular function is unknown. The role of RHY-1 as a hif-1 lethality suppressor indicates that it acts in a hif-1 independent H₂S response pathway. The goal of this study is to better understand the role of RHY-1 in this hif-1 independent response pathway. In order to identify its binding partners and elucidate its role in cellular signalling, we performed immunoprecipitation of RHY-1 for use in tandem mass spectrometry. This approach allows for the identification of its binding partners under baseline and H₂S stress conditions, which may further define the role of RHY-1 in the stress response to H₂S. Defining the role of RHY-1 will enhance our understanding of how the H₂S stress response leads to increased lifespan in C. elegans. Since several members of this pathway are well conserved in humans, this work may provide a mechanism for the therapeutic use of H₂S in human medicine.

Humans have limited ability to regenerate many tissues. In instances such as limb loss or spinal cord injuries there is a dramatic decrease in quality of life and there are few therapeutic options to help recover from these serious injuries. My research characterizes how epigenetic changes affect tail regeneration in the frog, Xenopus tropicalis. Xenopus tadpoles have significant regenerative abilities when they are developing but that ability is lost as they age and undergo metamorphosis. This transition in regenerative ability makes them a great model for understanding the molecular differences between tissue that can regenerate and tissue that can not. Other advantages of Xenopus for epigenetic studies of regeneration include a well-characterized diploid genome and many well-established molecular tools. Previous work by our group has shown that the accessibility of chromatin undergoes drastic remodeling in which many of the genes are made inaccessible shortly after injury in the Xenopus tail. These genes are then reopened during regeneration. These changes are likely due in large part to histone deacetylases (HDACs) and histone acetyltransferases (HATs). We hypothesize that HDACs are necessary for causing the initial inaccessibility of the chromatin and that HATs are necessary for the reopening of chromatin for regeneration. We used an Assay for Transposase-Accessible Chromatin and sequencing (ATAC-Seq) to show that the chromatin does not undergo its normal shift toward inaccessibility over the course of regeneration when tadpoles are treated with HDAC inhibitors, coupled with a reduction in regenerative ability. This study will help us identify how histone acetylation directs changes in chromatin accessibility. Additionally, with immunohistochemistry we can study specific sites of histone acetylation and validate these antibodies for future epigenetic research. Our study of large-scale epigenetic changes during regeneration will help characterize how epigenetic gene regulation enables regeneration.

For more than a century, Drosophila melanogaster (fruit flies) have been an invaluable and versatile tool to further our understanding of cell signaling and survival mechanisms. To this day, they continue to shed light on the endogenous pathways that cancer cells can hijack in order to proliferate, metastasize, and recur following remission. The molecular conservation of these pathways invites parallels between the germline stem cells in D. melanogaster and the cancer stem-like cells in human carcinoma. In the same way that a tumor can relapse following a period of dormancy, Drosophila germline stem cells are capable of repopulating their niche after insult from Ionizing Radiation (IR). Utilizing this powerful model, we have conducted a small molecule drug screen of 512 compounds that we have narrowed down to eight candidate drugs that appear to increase cell death in Drosophila germline stem cells. Having already characterized the wild type Drosophila germline stem cell response to IR-induced DNA damage, we  probed how drug treatment and gene knockdown affected the germline stem cells' ability to recover from insult. Previous work in the Ruohola-Baker Lab has demonstrated the critical importance of the mechanistic target of rapamycin (mTOR) and the Tie receptor pathways in regulating regeneration after insult in the Drosophila germline. The Tuberous Sclerosis Complex (TSC), a heterodimer comprised of Tsc1 and Tsc2, is a known negative regulator of mTOR. Additionally, the Tie receptor is central to anti-apoptotic signal transduction in the Drosophila ovary. We have screened four candidate drugs to see if they effectively increase stem cell death in Tsc1-knockdown and Tie-null flies, in order to ascertain whether our drugs affect stem cell survival mechanisms through mTOR and/or Tie signaling. Our findings may shed light onto how to mitigate the quiescent threat of tumor relapse.

Breast Cancer type 1 gene (BRCA1) was first linked to a high risk for breast and ovarian cancer over two decades ago, yet much remains elusive about its functional mechanism and how inheritance of a variant of the ubiquitously-expressed gene can lead to markedly increased risk for tissue-specific cancers. A gene found in the nematode C. elegans was demonstrated to be homologous to human BRCA1 including at the protein product level. Its protein product was shown to have the same type of enzymatic activity as the human protein product of BRCA1, and to be involved in the same DNA repair pathway. Using purified protein products and both structural analysis via Nuclear Magnetic Resonance and in vitro biochemical enzyme activity assays, I have shown that the C. elegans and human BRCA1 share some structural similarities and that the enzymatic activity is conserved. These results suggest that C. elegans could be developed into a model system that would allow for experimentation to directly move between in vitro biochemical studies and in vivo genomic/genetic studies. My current work has focused on furthering the development of the C. elegans model system by using it to identify other protein-protein interactions, including the identification of new protein targets. Additionally, my work explores the effect that cancer associated variants have on the enzymatic activity of the C. elegans BRCA1. The development of C. elegans as a model system could lead to new insights into conserved functions of BRCA1 and a fuller understanding of its biological and developmental importance.

Cancer stem cells are thought to play a role in relapses and metastasis in numerous cancers. The inability of traditional cancer therapies, such as chemotherapy, to eradicate these cancer stem cells prompted a search for small molecules that induced apoptosis in cancer stem cells. Drosophila germline stem cells can be used as a model system to emulate cancer stem cells. Upon irradiation, Drosophila germline stem cells are able to survive apoptosis through a molecular signal released by the apoptotic daughter cells via the TIE receptor. We have conducted an in vivo drug screen of 512 compounds in Drosophila melanogaster to find drugs that would disrupt this protective mechanism and induce apoptosis. In particular, Camptothecin, NSC 125197, and NSC 127458 were effective in killing germline stem cells. To test the effect of the small molecules we fed female flies the compound for 3 days, dissected and fixed the ovaries and stained for an apoptosis marker, activated caspase, and the GSC marker, adducin. We quantified the effect of the drug by analyzing the number of germline stem cells and caspase signaling on a confocal microscope. Through this system we aim to find potentially new, and more powerful anti-cancer drugs that can affect cancer stem cells. Future directions include attempting to understand the mechanism of action of these apoptotic compounds in hopes that they might be useful in the fight against cancer stem cells.

It is not fully known what controls signal preference in branched signal transduction pathways. One hypothesis is that the configuration and orientation of receptors determines signal preference. We are investigating this hypothesis in the context of the Tie2 receptor pathway which has downstream signaling involved in cell survival via Akt, migration via Dok-R, and sprouting via FAK, among other branches. We are using computer designed protein tools, short-armed claw trimers and nanocages, conjugated to F domains of the canonical Tie2 ligand angiopoietin 1 in our investigation. We have shown that short-armed claw trimers with three conjugated F domains do not yield increased phosphorylation of Akt, suggesting that more than three receptors must cluster together for signal activation. The nanocages are multivalent, for they have multiple F domains conjugated to them. We have different valency nanocages with different percentages of F domain conjugation. We are using these different valency nanocages to investigate which valency is optimal for receptor activation. We have shown that higher valency nanocages yield increased phosphorylation of Akt. We predict that higher valency nanocages cluster more Tie2 receptors together in a specific conformation. Thus, we show a positive correlation between Tie2 clustering and the phosphorylation of Akt due to Tie2 activation. Now we will investigate combination conjugations of F domain and integrin binders to the nanocages as well as other branches of the Tie2 pathway: Dok-R corresponding to wound healing ability and FAK corresponding to tube formation ability. This work has potential to help generate therapeutic compounds for wound healing.

Enteric diseases, or diseases of the Gastrointestinal (GI) tract, remain one of the most prevalent killers of children in sub-Saharan Africa. The most practical way to prevent such diseases is through vaccination, but antigens for enteric diseases need to be delivered directly to the GI tract to be most efficient, making vaccination difficult. Recent studies by the von Adrian group at Harvard University have found that both T and B cells are reprogrammed to home to the GI tract when they encounter retinoic acid, a metabolite of vitamin A. The King Lab at the University of Washington is working to develop a novel vaccine candidate using recently developed self-assembling protein nanoparticles, that can simultaneously package all-trans retinoic acid (ATRA) and multivalently display enteric antigens. Recent data indicate that preliminary versions of these nanoparticles can successfully elicit a mucosal immune response when delivered with free ovalbumin, an avian egg protein that is frequently used as a model antigen. We expect to elicit more potent immune responses when the antigen is multivalently displayed at high density on the nanoparticle. To accomplish this, I created a library of plasmid constructs combining ovalbumin and several nanoparticle subunits using a variety of different linkers. I expressed, purified, and evaluated these nanoparticles for their expression of the antigen using enzyme-linked immunosorbent assays, or ELISAs.

East Coast Fever (ECF) is a tick-transmitted disease caused by Theileria parva in cattle that is detrimental to the economic well-being of Eastern and Southern Africa. Current disease control involves pesticides, antibiotics, and a commercial live parasite vaccine, but they are insufficient and thus developing an effective, affordable, and protective vaccine for ECF is a pressing need. Studies by the Nene group at International Livestock Research Institute have demonstrated that the T. parva sporozoite stage-specific surface coat protein p67 provides partial protective immunity to cattle when used as a subunit vaccine. Within the King group at the University of Washington, I generated novel p67C nanoparticle immunogens intended to induce more potent and durable immune responses in immunized cattle. I genetically fused the p67C epitope to a variety of self-assembling protein nanoparticle subunits and screened for stability and expression levels. Based on these data, I selected three best-performing p67C nanoparticles for larger-scale expression and purification: I32-19, I32-28, and I53-50. Each of them, with 60 copies of the p67C, was expressed, purified, and extensively quality-controlled to confirm monodispersity, purity, and low endotoxin levels for immunization studies. Immunogenicity data from the ILRI show that the nanoparticles with p67C induce a similar level of p67C-specific antibodies as a combination of HepB core antigen and mesoporous silica nanoparticles containing more than twice as much p67C antigen, and far higher antibody levels than p67C alone. It is an outstanding vaccine candidate to help those suffering from ECF. These preliminary results will be confirmed by parasite challenge studies in 2018.

Posttranslational modification with the small protein ubiquitin (Ub) is an essential process in various regulatory pathways of eukaryotes, such as protein degradation, DNA repair, and the cell cycle. Misregulation of ubiquitylation has been associated with diseases such as cancer and neurodegenerative disorders. Ubiquitylation is accomplished by an enzymatic cascade that involves a family of ~40 ubiquitin conjugating enzymes (E2), which form thioester intermediates with Ub, called E2~Ub conjugates. In most ubiquitylation reactions, it is the E2 that covalently attaches Ub to substrate proteins. While the overwhelming majority of E2s ubiquitylate the ε-amino group of substrates’ lysine residues, important exceptions have recently been discovered. For example, Ube2W has been identified as the only human E2 that exclusively ubiquitylates the α-amino group of substrates’ unstructured N-termini. Knockout of Ube2W in mice leads to severe developmental abnormalities, suggesting its importance for early postnatal survival. Little is known about the molecular mechanism of Ube2W’s unusual specificity and structural insights to the catalytically-relevant species, the Ube2W~Ub conjugate, are lacking. Here, I report the formation and purification of the first stable Ube2W~Ub conjugate mimic. Using a combination of biochemical techniques and nuclear magnetic resonance (NMR), I characterized atomic-level changes within the Ube2W~Ub conjugate during substrate recognition and catalysis. This investigation will expand our mechanistic understanding of E2s and establish insights to a biochemically fascinating and biologically relevant enzyme.

Protein homooligomers, multibodied assemblies built from identical polypeptide chains, comprise a large fraction of known cellular proteins. Homooligomers prove to be particularly amenable for many biological applications; they hold the potential as oligomerization domains, often have enzymatic functionality as a byproduct of their oligomeric configuration, and can serve as structural scaffolds for bionanomaterials. While there exists a multitude of protein homooligomers in the Protein Data Bank, the finite number of existing homooligomers limits the potential for custom applications. Our current work involves designing novel cyclic protein homooligomers from a set of de novo designed repeat proteins that bind the Fc region of antibodies. Using the Rosetta software suite, we generated a set of de novo homooligomer models by designing the oligomeric interface to direct self-assembly into a target configuration with three to six identical chains. After a round of refinement, we expressed the designs in Escherichia coli and purified them by immobilized metal affinity chromatography. Their oligomerization state was validated by measuring the molecular weight in solution by size exclusion chromatography paired with multi-angled light scattering and comparing it to the predicted molecular weight of the design. Designs that exhibited the desired molecular weight were submitted to collaborators for small angle X-ray scattering data and X-ray crystallography. The exclusive use of de novo proteins in homooligomer design granted a greater control over the shape and stability by nature of the repeats, thus making one successful interface design useful for a multitude of shapes and sizes. This variability opens up a wide scope of scaffolds for using these homooligomers for near atomic scale structural characterization by cryo-EM.

Metalloproteins account for at least one-third of all known proteins and take part in a myriad of redox chemistry. In spite of the long-standing interest, designing metalloproteins from scratch with structural precision and tunable redox properties remains an elusive goal. The underlying hypothesis for this project is to design redox active, iron- sulfur [Fe-S] proteins using protein designing software called Rosetta. Iron-sulfur proteins are nature's modular unit for electron transfer and redox catalysis. We used Rosetta to design classes of iron sulfur proteins including [4Fe-4S] and [2Fe-2S] clusters. We have expressed these designed proteins in E.coli and tested their binding properties to their respective inorganic clusters using biophysical techniques such as ultraviolet–visible spectroscopy (UV-Vis), circular dichroism (CD), and electron paramagnetic resonance spectroscopy (EPR). Initial results show that designed proteins can be expressed in E.coli and bind to [4Fe-4S] clusters as designed. The Cluster incorporation was verified by UV-Vis spectroscopy, which shows absorption centered around 410nm, characteristics of a [4Fe-4S] cluster. We also verified that the holo protein is folded and thermostable using CD. We are currently focusing on crystallizing these holoproteins to verify the accuracy of the design process and investigate their redox properties. These designed proteins will help in explaining the fundamental factors controlling the redox properties of metalloproteins and lay the foundation for designing a new class of metalloenzyme relevant to metabolic engineering.

Previous research on JNK-mediated stress signals demonstrated that stem cells in the posterior midgut of Drosophila Melanogaster only undergo compensatory proliferation or apoptosis. However, our group discovered that the stem cells can also undergo the process of basal extrusion and dissemination, resulting in the cells being eliminated from the tissue into the hemocoel, the blood containing intertissue body cavity. The JNK signal promotes the cells to exit the epithelium of the gut, move through the muscle layer, and be released to the hemocoel, which resembles the process of metastasis in human cancer. In order to understand the mechanism of this extrusion process, we carried out an RNA Interference (RNAi) screen and sought to find genes that are necessary for the stem cell extrusion in the JNK activator, HepCA, expressed flies. We used the ESG-GAL4, UAS-GFP, TUB-GAL80TS(EGT) genetic system to study the knockdown effect from the RNAi of each gene. We selected 215 lines of kinases and phosphatases of flies to test if the knocked-down gene results in suppression of cell elimination by the JNK stress signal. After 4 days of inducement, which is the sufficient time for the JNK signal can promote complete extrusion of the intestinal stem cells, the intestines were dissected, fixed, mounted, and examined with a fluorescent microscope for any presence of stem cells. Through this screen, we could find 33 lines that had strong suppression of cell elimination, 39 lines that had a moderate effect. Our results suggest that the knocked down genes with strong suppression of cell elimination are involved in the mechanism of basal cell extrusion, and future research dictates an investigation into the molecular function of each of these genes.

Chemically induced dimerization (CID) systems, which are systems that involve the dimerization of two proteins only in the presence of a specific molecule, have shown a great potential in studying and controlling cellular signaling pathways. However, current methods for identifying CID systems for any given target based on small molecule-protein interactions remains a challenge in the area of protein design. Our research team proposes a general approach for identifying new CID systems for which may have clinical and therapeutic applications. To do this, we aim to create nanobody-based CID binders. Using a technique known as “Trinucleotide-Directed Mutagenesis,” our team has been able to create a diverse nanobody library of over 109. Phage display allows us to screen this library for specific nanobodies that can bind with high specificity and affinity to a specific molecule. Currently, we are using cannabidiol (CBD) as a target molecule. After performing several rounds of selection, in order to validate our system’s specificity and selectivity, we use ELISA assay tests as well as Bio-Layer Interferometry to measure the binding affinity and binding rate of our system. In order to ensure the specificity of our CID system we tested it against, tetrahydrocannabinol (THC), a structural homolog. To date, we have successfully found three different nanobodies that can bind with high affinity and specificity to our small molecule of choice. As we move into phase two of our project, we are currently working on identifying the second nanobody in order to form a complete CID system. Designed CID systems will not only provide personalized choices for patients showing different drug responses, but also serve as orthogonal chemical control of different cell subpopulations and cell behaviors to improve therapeutic efficacy and safety.

Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Smoking is the major cause of lung cancer. While tobacco smoke is well-known to cause mutations in DNA that drive the cancerous phenotype, it also induces epigenetic changes - changes in the regulatory information overlaid on the genome. Smoking has been shown in numerous epidemiological studies of subjects’ whole blood to affect an epigenetic mark called DNA methylation. DNA methylation commonly refers to the addition of a methyl group to cytosine in the context of a Cytosine-Guanine (CpG) dinucleotide. Hypomethylation at several CpGs in the blood has been shown to predict lung cancer risk, but the mechanisms by which DNA methylation changes in blood are linked to cancer in the lung are poorly understood. To investigate this, I used R to analyze 18 published peer-reviewed articles and collected 20,946 unique CpGs that have been found to be statistically significantly differentially methylated in the blood of smokers compared to non-smokers. I then utilized the UCSC Genome Browser and The Cancer Genome Atlas (TCGA) data to compare smoking-induced methylation changes in whole blood with those of non-tumor lung tissue and lung adenocarcinoma. Understanding the relationship between whole blood methylation and lung methylation can reveal if blood DNA methylation patterns are good surrogate markers for DNA methylation that occurs in the lung. Further studies of select differentially methylated lung CpGs can help unravel the mechanism by which smoking-induced epigenetic changes contribute to lung cancer development.

Hydrogen sulfide (H₂S) is a gas most commonly known for its pungent scent and toxicity. However, administered in low doses, H₂S can be tolerated, and it can even facilitate the acclimation to higher doses in the nematode Caenorhabditis elegans, a model organism with relatively short generation times and extensive tools for genetic manipulation. Furthermore, H₂S could have important medical uses due to its function as a vasodilator and chemical signal in cellular signaling, such as conserving donor organs, extending the time prior to an ischemic response, and decreasing nociception in severe injuries (Miller, and Roth). Data from our lab suggest that a low dose exposure to H₂S during early stages of development forms a “bookmark” which permits the survival of the animal at high doses of H₂S during adulthood. Through a genetic screen, epigenetic factors including spr-5, set-2, swsn-4, and swsn-1 were discovered to be involved in the bookmark. The ease of bookmark formation and the short time between formation and manifestation of phenotype present a unique opportunity to dissect a complete epigenetic pathway in addition to understanding cellular responses to H₂S. Our project aims to develop tools that will help understand temporal and spatial requirements for the protein products of these bookmarking genes. To achieve this, we intend to use CRISPR to attach a tag to the endogenous loci of target genes that allows for auxin-induced degradation (AID) (Zhang, L. et al.). Our targets will include both bookmarking genes and previously identified H₂S response elements such as hif-1. Thus far, we have tested the efficiency of the tag on Green Fluorescent Protein (GFP). Presently, we are working to create the transgenic lines of nematodes that carry the AID tag alongside proteins involved in the formation or maintenance of the H₂S bookmark.

 Respiratory Syncytial Virus (RSV) is a common respiratory virus for which there is no vaccine. Over 60% of children are infected with the virus during their first season (winter months) and nearly everyone gets the virus before age 2, with reinfection occurring numerous times throughout life. In healthy individuals, symptoms are usually mild, but in young children, especially premature babies, and elderly (older than 65years old) adults, RSV can cause serious bronchiolitis and lead to hospitalization and death. While no prophylactic RSV vaccines are currently on the market, many are in development including the UW IPD Icosavax’s protein nanoparticle vaccine which shows a strong humoral response with 8x better neutralizing antibody titers than antigen alone. A business case assessment and asset valuation for commercial development of the RSV nanoparticle vaccine for adults 65 years of age or older was performed. We calculated the current expected net present value for the asset based on RSV prevalence, the burden it creates, current vaccination practices, and industry standards. Gathering this information required extensive review of published literature and market reports, and the data was used to create a series of discounted cash flow valuation models. These models indicate that the vaccine asset is worth somewhere between $8M and $38M today, depending heavily on competition, public acceptance and vaccine coverage rates, and cost of goods. The valuation models can be used to facilitate conversation with potential investors and acquirers and plan future decision making to profitably operate Icosavax.

Ca²⁺, or Calcium ions, are very important signaling molecules that are essential to normal eyesight. A light-sensitive layer in the eyes, called the retina, contain photoreceptors which require proper concentrations of Ca²⁺ for correct function. Mitochondria in the retina may help store Ca²⁺. To transport Ca²⁺, the mitochondria rely on Mitochondrial Calcium Uniporter proteins (MCU) and Mitochondrial Calcium Uptake 1 regulators (Micu1). This study hypothesizes that the Micu1 to MCU ratio influences the levels of Ca²⁺ in the photoreceptors and that an abnormal Micu1 to MCU ratio may compromise photoreceptor health. To analyze the Micu1 to MCU ratio, quantitative polymerase chain reactions (qPCR) are conducted. Retina cells are compared to other cell types, such as brain and heart cells, to determine the ratio of Micu1 to MCU relative to other tissue types. qPCR is also used to determine the Micu1 to MCU ratio in cells designed to express Micu1 or MCU abnormally. Current results suggest that the retina and brain express higher amounts of Micu1 than the heart, but the retina expresses the least MCU. It is hypothesized that most of the MCU channels in the retina are regulated by Micu1. On the other hand, heart tissue has more MCU and less Micu1, suggesting that the MCU channels are not regulated to the same extent. This makes sense because the retina and brain both contain large amounts of neurons, while the heart largely contains muscle. Therefore, different cell types with different functions express unique Micu1 to MCU ratios. Cells which are designed to express Micu1 or MCU abnormally have different ratios that may be insightful in addressing visual disorders. Further work may include manipulating the Micu1 to MCU ratio and observing the consequent effects on the photoreceptors.

Synthetic nucleocapsids have the potential to be a novel class of drug delivery vehicle that represent a happy medium between viral and polymeric delivery systems. They were recently engineered by the Baker lab to encapsulate their own RNA. RNA encapsulation provided the genotype-phenotype linkage required for evolution. Nucleocapsids were then evolved to have relatively long circulation times (4.5 hours), which gives them the potential to deliver nucleic acids to many cell types in vivo. My project focuses on attaching pH-responsive proteins to the surface of nuclecapsids to mediate RNA delivery. Since the endosome is typically the first compartment the nucleocapsids enter, it is vital that the nucleocapsids escape the endosome before being trafficked to the lysosome and degraded. The pH-responsive proteins are proven to rupture membranes upon pH changes similar to those found in the transition from endosome to lysosome. This would allow the cages to break out of the endosomal pathway and deposit their RNA cargo into the cell cytoplasm. Once the RNA is deposited, native cellular machinery can transcribe the desired proteins. We aim to program the packaged RNA to either induce apoptosis or slow cell growth in tumor-related cells. This revolutionary delivery vehicle could be utilized for more precise and effective biologics delivery. 

Autoimmune diseases are characterized by one’s own immune system attacking healthy cells in the body. Current treatments for autoimmune diseases require regular intravenous injections. The objective of this project is to develop a new generation of oral protein therapeutics. As a proof of concept, we computationally designed inhibitors against the inflammatory cytokine IL-17 in order to inhibit its activity in autoimmune conditions. IL-17 elicits its effect by triggering a signal cascade through heterodimerization of its cognate receptors. Our goal is to selectively occlude this dimerization to prevent the progression of disease. We have designed our inhibitors to bind in the same location as the native receptor, preventing downstream signaling that leads to autoimmune diseases. Historically, binders have been created by using existing protein interfaces grafted onto native protein shapes. However, we succeeded in designing IL-17 binders without using any prior protein interface information through de novo protein scaffolds. This project could revolutionize the way protein therapeutics are developed. Our goal is to analyze the biophysical characterization of our binders. In order to create a new standard in protein design our binders should have accurate size and folding in relation to the designed model which we are analyzing through mass spectrometry and circular dichroism spectroscopy. In order to become an effective drug candidate the binders must have successful binding to the IL-17 target which we are analyzing using octet binding analysis. In the end we hope to test our successful IL-17 binders in mouse models of multiple sclerosis, eventually transitioning to human clinical trials.

The microbial community residing in the gastrointestinal tract, called the gut microbiota, plays an important role in host energy homeostasis. Since energy homeostasis is essential for survival, it is plausible that neural circuits involved in maintaining host energy homeostasis would evolve to shape the composition of the gut microbiota in alignment with the energy needs of the host. In humans and mice, neurons in the arcuate nucleus of the hypothalamus that express agouti-related protein (AgRP neurons) play an important role in stimulating food intake and maintaining energy balance. AgRP neurons are maximally active during starvation conditions; thus, we are investigating whether they help conserve energy under these conditions by altering the composition of the gut microbiota to a state with a greater capacity for energy harvest. To test this hypothesis, we expressed an excitatory designer receptor, the hM3Dq receptor, in the AgRP neurons of mice and then analyzed the bacterial composition and energy harvest capacity of the gut microbiota of these mice following pharmacological activation of the hM3Dq receptor relative to unstimulated control mice. We assessed the bacterial composition of the gut microbiota using 16S rRNA amplification, sequencing, and characterization, and we assessed the energy harvest capacity of the gut microbiota by determining the energy content of the mouse fecal matter using bomb calorimetry. Our results show how AgRP neuronal activity influences the composition and energy harvest capacity of the gut microbiota. These results reveal that the central nervous system can shape the gut microbiota to promote energy balance. This finding contributes to a better understanding of why and how gut microbiota changes occur, which may be relevant to understanding metabolic diseases such as obesity.

This project is to structurally verify computationally designed genetic fusions between helical bundle and repeat protein sets. With the use of this computational method, Rosetta, combining two discrete numbers of proteins can generate a much larger library of protein scaffolds. By biochemically characterizing and solving X-ray crystal structures of the designs, my work aims to experimentally validate this new computational protocol so that the designed proteins can be used with confidence for downstream applications. The wet lab protocol I will be performing follows common standard protein purification procedure. First, I will transform plasmid of genes containing the designed fusion proteins into an Escherichia coli strain, which is capable of over-expressing proteins of interest. After growing large cultures of these bacteria, the cell will be harvested and lysed. Following with the technique of immobilized metal affinity chromatography to purify the hexahistidine- tagged proteins- that binds to the nickel- charged resin—out of the soluble fraction of the cell lysate. The method of size exclusion chromatography (SEC) will be performed after purification takes place. This is going to further purify as well as giving information of the protein designs. I will perform this protocol on close to one hundred designs to calculate a “success rate” for the computational protocol. In greater picture, the overall goal of this project will be researching self- assembled protein design for crystallization from the computational design. Aiming for the result of producing various proteins have the ability of forming crystal in various solution conditions that confirm the structural characterization of designed protein and also validate both experimental and computational protocols. This research provides an avenue for greatly expanding the database of proteins can be utilized in fields such as materials engineering and creating binders of chemical molecules.