Designing conducting biomaterials capable of long-range electron transfer for the development of bioelectronics and biosensor remains a challenging and unsolved problem in protein design. The overarching goal of this project is to design protein fibers and template redox-active cofactors for long-range electron transfer. Recently our lab developed a method to design proteins that assemble into long fibers with a tunable diameter. We are using these fiber materials to assemble redox-active cofactors within 10-15 of each other. We are using a helical segment of a natural cytochrome-C protein as the electron transfer conduit. First, We docked this helical fragment inside the fiber molecules previously designed in our lab. Promising docks compatible with distance constraints for effective electron transfer (generally within 10-15Á of each other) were chosen for further design calculations to accommodate the cytochrome-C molecule. We ordered 11 proteins from the first round design calculations to characterize experimentally. Proteins were expressed using the usual E. coli. expression system and purified using Ni-NTA affinity chromatography. 9 out of 11 designs expressed and were soluble. For the helical cytochrome-C fragment, we expressed the native protein in E. coli. and obtained the heme-containing helical fragment by trypsin digestion. To ensure that the heme cofactors are incorporated in the intended positions of the fibers, high-resolution cryo-EM will be performed after cofactor incorporation. The redox potential of these fibers will then be measured using cyclic voltammetry (CV). If successful, this will lay the foundation for the development of a wide range of redox-active biomaterials capable of long-range electron transfer.

Self-assembling synthetic nucleocapsids have been at the center of increasing development as a medium for targeted drug delivery. By identifying certain cell markers of tumor cells, researchers at the Baker Lab have been evolving desired characteristics to this protein assembly by attaching different functional domains to specifically target cells. This screening of functional domains is possible because of the nucleocapsid’s ability to encapsulate its mRNA sequence, thereby linking genotype to phenotype. Moreover, this enables us to evolve desirable features in these functional domains through library selection. However, subsequent attachment of functional units to its exterior surface proved to decrease its mRNA packaging efficiency, thus limiting applications like mRNA delivery and evolution of functional units. This project aims to enhance the amount of encapsulated mRNA by mutating certain interior amino acids. Methods include cloning our I53-50-v4 protein assembly gene into Escherichia coli (E.coli) strains and over-expressing the proteins through IPTG induction. After the cells were harvested and lysed, proteins were purified through Immobilized Metal Affinity Chromatography (IMAC). RNA encapsulation levels were measured through Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) to compare between the original and mutated nucleocapsid. Promising results would demonstrate an increase in RNA concentration per nucleocapsid when compared to the original assembly across different constructs. The success of this project would allow for attaching multiple domains for evolution without compromising the nucleocapsid’s capacity to retain its biomolecular cargo. This research would greatly expand the utility of these nucleocapsids for evolving displayed proteins for desirable characteristics as well as enhance their efficacy for drug delivery applications.

Despite the yearly development of a vaccine, influenza (flu) still causes annual epidemics and is responsible for tens of thousands of deaths in the United States. Current flu vaccines are not fully effective in preventing viral infection because they protect people against a finite number of flu strains, which people may or may not be exposed to during a particular flu season. To address this challenge, we are engineering new protein therapeutics to increase the efficacy of flu vaccines. The two major proteins that flu viruses use to infect host cells, hemagglutinin (HA) and neuraminidase (NA), structurally parallel the two proteins that constitute a de novo designed nanocage. De novo protein design allows researchers to create proteins that are more stable than those produced in biological systems via evolution. The focus of this project is to use this designed nanocage to scaffold a more potent flu vaccine through a process called pseudo-symmetrization, which will allow the cage to display many different strains of HA and NA. To pseudo-symmetrize proteins, we used a modelling software to determine which protein components to modify to build a pseudo-symmetric protein. Then, we use molecular biology techniques to make modifications to regions of the protein that interact with other proteins when it self-assembles. As a result, the pseudo-symmetric protein is composed of building blocks that are all slightly different from one another, but can still assemble to create a nanocage with the same structure. So far, we have identified several variants of the modified cage component that can self-assemble, and we are continuing to test more interaction sites that make pseudo-symmetric proteins. These results, and continued experiments, will help us create new vaccines capable of displaying more flu virus antigens, thus protecting people from the flu using a single universal flu vaccine.

Current methods of drug delivery include the use of antibody drug conjugates (ADCs), lipid and polymeric nanoparticles, and top-down modified viruses. While successful in certain applications, these delivery platforms involve laborious production methods and have limited engineering opportunities. Synthetically designed proteins are viable drug delivery candidates that can be precisely modified to overcome the engineering challenges associated with other delivery platforms. Specifically, synthetic nucleocapsids (synNCs) are computationally designed icosahedral protein assemblies evolved to have virus-like genome packaging. The linkage between the encapsulated mRNA (genotype) and exterior proteins (phenotype) allows for directed evolution to be achieved through library selection. The synNC was evolved to increase the in vivo circulation half-life from less than 5 minutes to over 4.5 hours. The next step is to optimize the targeting ability of synNCs toward specific organs. We performed site-directed mutagenesis to introduce several mutations on the exterior of the synNC, performed two rounds of in vivo library selection, and identified several mutations that favored specific organs. Our current work involves producing five promising synNC mutants, biochemically characterizing the mutants, and examining their biodistribution in mice. The ability to target specific organs will be a successful step towards utilizing nucleocapsids in drug delivery and other biomedical applications.

With regards to most tissues, humans lack the ability to regenerate, instead scarring in response to injury. This often leads to poor patient outcomes, especially in the event of spinal cord damage. Xenopus tropicalis are capable of avoiding this scarring response as tadpoles but not as adults. They instead fully regenerate tail tissue and are thus an excellent model system for the investigation of how regenerative and non-regenerative organisms differ in their response to injury. Due to the complexity of this process, many transcription factors have been implicated to have a role in regeneration, though the precise roles of many such transcription factors remain unknown. Here, we focus on the transcription factor Hif1α, which is canonically involved in responses to hypoxia and oxidative stress. Using an Assay for Transposase-Accessible Chromatin (ATAC-Seq) we have found that over the course of regeneration, Hif1α response elements (HREs) increase in accessibility. To understand the role of Hif1α during regeneration, I have used echinomycin, a small molecule known to inhibit binding of Hif1α to HREs. Tadpoles treated with echinomycin fail to regenerate, indicating the necessity of Hif1α in regeneration. In order to determine the effects of Hif1α on gene expression, I have queried several genes known to be differentially expressed during regeneration through the use of quantitative polymerase chain reaction. I have shown that inhibition of Hif1α transcriptional activity via echinomycin significantly alters Wnt target gene expression, indicating that Hif1α regulates Wnt target genes. This provides an improved understanding of the regulatory processes that enable regeneration.

The blood brain barrier (BBB) is an almost impenetrable obstacle for therapeutic delivery, hampering the treatment of many neurological diseases. By exploiting natural transport mechanisms utilized by the brain such as iron import via transferrin receptor (TfR), researchers have been able to transport therapeutic molecules into the brain, albeit with a low efficiency. TfR is a transmembrane protein that is highly expressed on the BBB where it binds its ligand, Transferrin. Transferrin-bound iron binds on the blood side of the BBB, is subsequently endocytosed and trafficked through the cell before being exocytosed on the brain side. We have computationally designed a protein that, like the transferrin ligand, can bind TfR and pass the BBB in in vitro BBB models. The goal of my project is to attach existing protein nanocages to this binder which have previously been shown to package therapeutic molecules. I have generated constructs by fusing the binder to these cages, purifying the cages, analyzing their stability, and testing their binding affinity to transferrin receptor. We currently have one cage fusion that successfully binds the transferrin receptor and is being tested for BBB traversal. We are continuing our work to create more variants that can successfully cross this barrier. Using computationally designed de novo proteins has many advantages over traditional protein engineering approaches such as hyperstability and a high degree of customizability. In the future, this project could provide new opportunities for treatment of many neurological diseases.

Hydrogen sulfide (H₂S) is a common cause of workplace injuries and deaths for industrial workers. In our project, we use Caenorhabditis elegans (C. elegans) as a model organism for investigating how cells behave under an environmental stressor and the long-lasting effects of that behavior. Previous work in our lab has shown that early exposure to low H₂S (50 ppm) enable C. elegans adults to survive a much higher subsequent exposure by forming a cellular memory known as a “bookmark.” Bookmarked animals survive at high H₂S (150 ppm), while animals without previous exposure do not. In a genetic screen, we identified various epigenetic factors that are involved in this process; however, it is still unclear when in the “life” of the bookmark and where in the animal these factors are required. The required bookmarking gene swsn-4 is part of the SWI/SNF complex, a group of proteins that regulate compaction of DNA and thus the accessibility of genes. We are interested in assessing the spatial requirements for swsn-4 by rescuing mutant animals that lack this chromatin-remodeling factor. For the first part of the project, we use Gateway recombination cloning technology to enable tissue-specific expression of swsn-4. In the next part of the project, we test whether introducing swsn-4 in specific tissues rescues bookmark retention. A recent study identified hif-1, a transcription factor, to be broadly needed to rescue animals exposed to both low and high H₂S, suggesting that the response is needed in most cells to ensure survival of the animal. Because swsn-4 is also present broadly in the body of C. elegans, we predict it will be needed in a similar way to hif-1. We hope that our investigation would lead us to discovering methods in which we can utilize the properties of H₂S as a chemical messenger to help patients.

During embryonic development, a dormancy-like state known as diapause arises during the transition from pre to post implantation. This state of suspended development is a reproductive strategy which favors newborn survival in mammals during nutritional deprivation or stress. Studies from the Ruohola-Baker lab found potential candidate regulators of diapause by establishing an in-vitro diapause model using pluripotent mouse embryonic stem cells (mESC). One of the genes is Activating Transcription Factor 5 (ATF5) which encodes a protein capable of survival-mediated functions such as maintaining mitochondrial activity during stress, modulating cell differentiation, preventing apoptosis and regulating cancer pathway. ATF5 has been known to transcriptionally target mTOR, a mechanistic target of rapamycin. Energy stress in the form of starvation and pharmacological inhibition of mTOR has shown to induce diapause-like state in mESCs in vitro. Our hypothesis is that upregulation of ATF5 under energy stress will reestablish diapause-like state in naïve mouse embryonic stem cells in vitro. We will test our hypothesis by loss-of-function and overexpression experiments. We test if ATF5 gene knockout using CRISPR-Cas9 prevents the mutant lines from entering diapause-like state from energy stress. Using western blots, we will quantify phospho-mTOR levels and its downstream targets in the ATF5 KO lines and compare them with the wildtype lines. For the overexpression of ATF5, we will make rescue lines for the ATF5 KO cells. We predict that overexpressed ATF5 in rescue lines will enter diapause-like state, and have reduced mTOR and its downstream target signals compared to KO lines. Our discoveries of ATF5 function in diapause can be useful in understanding how early-staged cancer stem cells enter a diapause-like state or quiescent state which enables them to escape chemotherapy detection. We can potentially contribute to the development of therapies to target ATF5 mechanism so that these undetected cancer stem cells can be detected.

Amyloid diseases are characterized by the aggregation and buildup of proteins in vital tissues and organs. Insoluble β-sheet amyloid fibrils were previously thought to be the major underlying cause of tissue degeneration and cell death. However, recent experimental evidence suggests that soluble oligomers, which form during protein aggregation and before polymerization into fibrils, are the principal cause of toxicity in mammalian cells. These toxic oligomeric protein assemblies are believed to share a common sequence-independent secondary protein backbone structure known as α-sheet. This project proposes the investigation of a synthetic peptide known as AP3 that is capable of forming toxic oligomers and β-sheet amyloid fibrils. This peptide was de novo designed with a completely randomized sequence which preserves the underlying chirality that produces α-sheet character leading to its exhibition of amyloidogenic properties under acidic conditions. Furthermore, AP3 aggregation was shown to be inhibited by three naturally occuring amyloid proteins implicated in their respective dieseases: Amyloid Beta (Alzheimer’s), IAPP (Type II Diabetes), and Transthyretin (Cardiac Amyloidosis). Analysis using dot-blot assays, soluble oligomer binding assays (SOBA), and BLITz assays will provide additional insight into the behavioral, binding, and kinetic properties of AP3. Upon further evaluation, we aim to demonstrate the ability of AP3 to serve as a synthetic model for naturally occuring amyloids and provide a better understanding of amyloidogenesis as well as the interactions between amyloidogenic species. This research will prove useful in the creation of more effective amyloid inhibitors and treatments for amyloid diseases.

Membrane trafficking in the eukaryotic cell is a highly controlled and significant process that is related to various inherited disorders and cancers. Among numerous regulatory proteins, Uso1 protein – an essential, long, coiled-coil protein – plays a key role in the tethering process, capturing and pulling the vesicle toward the target membrane. Despite many years of work, the tethering mechanism has not been fully understood. Although several tethering models have been proposed, none of them were tested with Uso1 protein. Hence, to test these models, we engineered a mechanically deficient version of Uso1 protein, which lacks the critical tethering region for functioning. After the protein characterization, in cell survival tests and in test tube tests in chemically defined fusion system have been performed. The failure of the trafficking event, which would support our hypothesis, along with other functional test data, would provide a promising demonstration for the tethering mechanism of the long coiled-coil tether and the role of Uso1 protein.

My research project involves the AP-3 (adaptor protein) complex, which plays a key role in membrane trafficking cells. Within all eukaryotic cells, there are membrane-bound sections of the cell that interact with each other in various ways to drive the cell's function. Vesicles mediate the transport of proteins and lipids among cellular organelles. These vesicles are created in various ways by proteins throughout the cell that form a "coat" around the vesicle as it travels to its destination. AP-3 is a protein complex that mediates vesicular transport from the Golgi apparatus to the lysosome. The current aim of my project is to analyze evolutionarily conserved features of the AP-3 complex by mutating subunits of the complex and observing resultant phenotypes in our model organism, Saccharomyces cerevisiae (baker’s yeast). In doing so, we utilize a gene reporter system called GNSI to analyze AP-3 function in different genetic strains. This reporter system is mainly analyzed through a colorimetric assay (chemical test to determine components), using qualitative observations of the intensity of colored halos around yeast colonies on a gel plate. We also use fluorescence microscopy by using signals from the Green Fluorescent Protein (GFP) to determine whether AP-3 was successful in trafficking to the lysosomal vacuole. So far, our results have shown that targeted truncations of proteins within the AP-3 subunit Apl6 yielded loss of function in trafficking. We are continuing our analyses by focusing on other proteins within AP-3. These basic studies will further our understanding of membrane trafficking and may provide insight into diseases linked to AP-3 function, including HIV-1 particle assembly and human genetic disorders.

Ribonucleotides are essential components of the cell: they are the building blocks of RNA and DNA, key signaling molecules, and metabolic intermediates. Maintaining proper ribonucleotide balance is critical for cell survival. An essential, highly-conserved enzyme that regulates purine biosynthesis is IMP dehydrogenase (IMPDH), which catalyzes the first committed step in the production of GTP. In humans, there are two IMPDH isoforms: IMPDH1 is present in low quantities in most cells, and IMPDH2 is upregulated during proliferation. Both IMPDH isoforms form higher order structures in cells and assemble into filaments and bundles of filaments in vitro. Nine point mutations in IMPDH1 have been discovered that cause retinal degeneration and ultimately blindness in humans. Our current findings show that these point mutations in IMPDH differentially impact the protein’s ability to assemble into filaments in vitro. We hypothesize that the ability of IMPDH to polymerize will affect the protein’s activity because of the interplay between protein structure and substrate binding. We use negative stain electron microscopy to first characterize the polymerization behavior of IMPDH in the presence of different ligands: the substrates IMP & NAD+, as well as allosteric effectors ATP and GTP that promote polymerization. In addition, we employ kinetics assays that vary levels of substrate and GTP to quantitatively assess enzyme activity by measuring NADH output. Characterizing each mutant’s behavior will be the first step to understanding the molecular mechanisms that cause retinal disease in IMPDH. In a future study, we can use cryo electron microscopy to solve the structures of mutant protein.

Neonatal mammalian heart tissues possess regenerative capabilities after injuries like myocardial infarctions that are mostly lost in adult mammalian tissues but conserved through adulthood in other vertebrates like zebrafish. Previous studies have shown that regeneration in ventricular cardiomyocytes (CM) occurs through de-differentiation and proliferation, but the underlying mechanisms that cause cardiomyocytes to enter the primed cell-cycle are unknown. Here we show that amino acid and metabolite levels in injured cardiomyocytes result in a primed state for regenerating cells. In chemically ablated zebrafish, it is shown that the amino acid profile activates the mTOR pathway to drive regeneration. Amino acid activation of mTOR is a result of high glutamine and leucine levels post-injury and in early heart regeneration in adult zebrafish, which is lost in adut mammals. Inhibition of the Wnt/β-catenin signaling pathway upstream of mTOR shows down regulation of mTORC1, showing that mTOR is necessary for CM proliferation in regenerating heart tissue. How Wnt signaling gets activated upon injury is unknown, and this study aims to understand the pathways upstream of Wnt signaling for activation. It is known that scarring needs to occur before regeneration occurs in heart tissue. This study also investigates why macrophages are essential for scar formation in ablated heart tissue and its underlying mechanisms. Further, single cell RNA sequencing one-week post injury is used to determine cell fates of the heart tissue. Cardiac cell types like CMs, endocardial and epicardial cells, and bulbus arteriosus (BA) cells were activated post-injury, with epicaridal cells promoting CM regeneration and BA cells activating signaling pathways during heart regeneration. This study demonstrates the signaling and metabolic pathways that activate cardiomyocyte regeneration in zebrafish hearts.

As of now, mRNA vaccines have been deemed as a potent replacement for current vaccine models against infectious diseases for their improvements in B-cell and T-cell immune responses. Usually, when soluble, subunit antigens are delivered, they are scattered and randomly bind to B-cell receptors, often loosely. However, with a nanoparticle carrier for antigens, there would be more effective crosslinking with B-cell surface immunoglobins as there is a higher density of structurally ordered antigen arrays presented by the nanoparticle. As a result, the B-cell creates a stronger immune response. Additionally, the multivalent particles also favors the creation of long-lasting immunity against a given virus. My team and I are currently developing a self-assembling protein platform using dn5A and dn5B protein components as a carrier for an mRNA vaccine against the flu. My project mainly focused on optimizing the co-secretion of the two particles by exploring different models and combinations of both. This is important as the translated cage not only has to be able to self-assemble but also be capable of doing so without producing excess protein in order achieve its purpose. To do so, I investigated 12 different constructs of dn5A and dn5B through transfections and analysis with western blots and electron microscopy. We used the data collected to improve the dn5A/dn5b protein platform utilized alongside flu mRNA vaccines, helping them better achieve potency. Overall, if effective, the new vaccination model can be utilized for other infectious diseases, including HIV and meningococcus.

Larval tadpoles of the frog Xenopus tropicalis exhibit a natural ability to regenerate multiple tissue types in response to injury. Unlike tadpoles, humans are incapable of regenerating a majority of their major organs and tissues following traumatic injury, often resulting in an irreversible loss of function of the affected tissues. While both non-regenerative and regenerative organisms undergo a period of wound healing in response to injury, the former then undergo scarring, whereas regenerative systems forgo scarring and ultimately regenerate the lost or damaged tissue. After tail amputation, reactivation of the cell cycle in the remaining tissue is required to promote cell proliferation in order to create the cells that will populate the regenerated tail. However, the molecular mechanisms that enable naturally occurring regeneration are not entirely understood. In order to better understand how wound healing promotes regeneration in tadpoles, I used immunofluorescent microscopy of Phospho-histone 3 (PH3) to assess the mitotic activity of Xenopus tropicalis tails during early regeneration. Over the first two hours post tail amputation, image analysis of PH3-positive cells shows that the amount and localization of mitotic activity varies greatly in the remaining tail tissue. Specifically, tissues adjacent to the amputation site transiently experience a dramatic decrease in mitotic activity beginning at 45 minutes post amputation (mpa), followed by the return of mitotic activity to these areas after 75mpa. I hypothesize that cell cycle inhibition during this 30 minute window is an important point of regulation during the regenerative process and may be a critical component of setting up a regenerative response to traumatic injury. Identifying mechanisms that enable regeneration will be critical for the development of clinical therapies that promote regeneration in humans

The Polycomb Repressive Complex 2 (PRC2) complex is known to be important in the development of zebrafish and Human Embryonic Stem Cells (hESC’s) by aiding in the transition from naïve to primed stem cells. H3K27me3 is a PCR2 dependent methylation of histone 3. The catalytic subunit responsible for trimethylation is the methyltransferase EZH2 and is required for correct zebrafish embryogenesis. The EZH2 interacts with the PRC2 complex’s EED (Embryonic Ectoderm Development) which is critical for EZH2 activity presumably because EED binds EZH2 to its specific substrate. A computational protein design was utilized to engineer a synthetic, novel protein that is a competitive EZH2 inhibitor with 300 times more affinity to the EED binding site than endogenous EZH2. This synthetic protein is referred to as EED binder (EB). Our previous results show that EB allow hESC exit pluripotency. EB has now been injected into zebrafish embryos in combination with a heat shock protein that will activate the EB, and a GFP indicator. A founder animal has been found that allows us to create an F1 generation of fish that are all positive carriers of the EB protein. These F1 fish can be used to create an F2 generation that receive a heat shock treatment at different times of development in order to observe the affects of inhibiting PRC2. It is assumed that if PRC2 is inhibited in a developing fish it will not survive as this is what was found in hESC’s. This is novel research in that we are now able to control temporal aspects of development of PRC2. This research lead to testing tissue regeneration and embryogenesis in zebrafish which will impact new research of regenerative medicine.

Chemically induced dimerization (CID), in which two proteins dimerize only in the presence of a small molecule, has been widely used to control cell signaling, regulatory, and metabolic pathways, and used as logic gates for biological computation in living mammalian cells. However, few naturally occuring CID systems and their derivatives are currently available. Creating a CID system with desired affinity and specificity for any given small molecule remains an unsolved problem for computational design and other protein engineering approaches. To address this challenge, we have used a novel strategy to select CID binders from a vastly diverse combinatorial nanobody library. We have created new CID systems that can sense cholecalciferol and artemisinin. We are validating CID biosensors by a yeast three-hybrid system and built structural models to understand the small molecule-induced dimerization. Our work is a proof-of-concept that can be generalized to create CID systems for many applications.

The identification and characterization of natural and artificial protein-protein interactions are fundamental to science and engineering. Although many technologies have been established to expedite research in this area, all can hardly meet the need to analyze vastly diverse protein-interactome networks, dynamics, evolution, and druggability. To develop a high-throughput quantitative interaction profiling technology, multimeric protein interaction sequencing (mPI-seq), we performed a 20×20-plex binding assay in a single aqueous solution using a set of de novo designed heterodimeric pairs. First, we barcoded the proteins with DNAs by ribosome and mRNA display. Then, barcoded proteins were assayed en masse in aqueous solution and immobilized into an ultrathin polyacrylamide gel layer attached to glass surface for in situ sequencing. DNA barcodes were amplified into discrete DNA clusters (polymerase colonies or polonies) and then sequenced using sequencing by synthesis. Finally, the protein-protein interactions were measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Through this project, we aim to establish a robust quantitative method to measure protein binding affinity massively in parallel and to use the large-scale functional data set to guide computational protein design.

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 a RNA Interference (RNAi) screen and sought to find genes that are necessary for the stem cell extrusion in the JNK activators, rasv12 and 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. After 4 days of inducement, which is the sufficient time for the JNK signal to 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. Our previous research from last year focused on the first phase of screening of possible RNAi lines involved in cell extrusion. Our results from this year focuses on our second phase of further identifying characteristics of cell movement in rasv12 flies.

Loss of heterozygosity (LOH) is a form of genomic instability that is common in cancer and can result in loss of function of tumor suppressor genes. LOH can occur as a result of interhomolog recombination (IHR). We are studying the mechanism of IHR using a human cell line engineered to report on the frequency of IHR at the gene encoding the cell surface protein, CD44. In this line, one allele of CD44 carries an inactivating mutation in exon 1 and the other in exon 17, so no surface CD44 protein is produced and the cells are sCD44- by flow cytometry. Our laboratory has shown that double strand breaks (DSBs) targeted by CRISPR/Cas9 to sites between the two mutations will stimulate IHR and generate sCD44+ cells, which are readily quantified by flow cytometry. We are working to identify the factors that regulate IHR. This will enable us to control the frequency of IHR and may help to reduce the frequency of IHR that results in loss of tumor suppressor gene activity in tumors.

In humans, limb amputation and recovery post-amputation is characterized by inflammation and scarring that lead to poor clinical outcomes. In contrast, amphibians such as the frog Xenopus tropicalis are capable of healing scarlessly and can fully regenerate previously amputated appendages. Successful limb regeneration depends on precisely choreographed expression of genes, directed in part by the deposition and removal of epigenetic markers. The broad aim of this research is to identify the spatiotemporal dynamics of epigenetic modifications and how they play a role in regulating gene expression during regeneration. It is known that histone deacetylases (HDACs) and H3K27-specific methyltransferase EZH2 enzymes limit chromatin accessibility and are necessary for regeneration to occur properly. However, the precise mechanisms and genomic targets of these enzymes remain unknown. We hypothesize that inhibiting these enzymes will leave chromatin in a constitutively accessible state, disrupting the gene expression required for successful regeneration. I am utilizing the drugs Trichostatin A (TSA) and DZNep to inhibit HDACs and EZH2 respectively at differing sequential time points throughout tail regeneration. In addition to characterizing the morphological outcome of regenerating tails that have been treated with these drugs at varying intervals post-amputation, I  also use immunofluorescence to identify the targeted location relative to the injury site and tissue types as they are affected across time. For humans and other mammals with limited regenerative capability, studying these epigenetic changes and their impact on Xenopus tropicalis tadpole tail regeneration is especially significant: it has the potential to determine how changes in gene regulation may enable and facilitate a broader capacity for limb regeneration by informing future therapeutic possibilities.

Studying zebrafish embryos allows us to understand features of vertebrate embryonic development. Neuro-mesodermal progenitor cells at the very posterior end, or tailbud, of an embryo are bipotential. This is because the presence or absence of Wnt signaling commits them to either neural or mesodermal fate. Directed by environmental cues, mesodermal cells exit the tailbud, migrate anteriorward, and become somites, structural segments from which muscles differentiate. The Kimelman lab has found that Tbx16/Spadetail, a major driver of mesodermal morphogenesis, downregulates Arhgap29 and Arhgap35, members of Rho family GTPase activating proteins. This suggests Arhgap29 and Arhgap35 may be involved in mesodermal cell movement. My work in the lab is focused on finding out what roles these two genes play. I used heat shock promoter hsp70 to overexpress Arhgap29 and Arhgap35 in transgenic fish lines. Previously, our lab showed that sustained Arhgap35 affected somite morphology, and that sustained Arhgap29 also decreased the number of somites. In my experiments, I carried out in-situ hybridization in wild-type, Arhgap29- and Arhgap35-expressing embryos to examine genes regulating specification/differentiation of muscle cells and genes involved in transmembrane cell adhesion. I will present data on cell tracking and cell protrusions collected from Arhgap29- and Arhgap35-expressing embryos. These results will help me compare cell migration between Arhgap-expressing and wild-type embryos. The purpose of these analyses is to understand how these two proteins control cell movement in the embryo. In the future, I will continue to investigate cellular mechanisms underlying vertebrate posterior elongation.

During the early stages of vertebrate embryonic development, blocks of muscle tissue called somites form progressively along the anterior-posterior (head to tail) body axis. As the embryo grows in length, new somites are continuously added at the posterior end until the tail reaches its final length. Our work focuses on a subset of genes called the hox genes. These genes encode transcriptional regulatory proteins that are involved in controlling the formation of the body plan along the anterior-posterior (AP) axis. In vertebrates, these genes are present in four major clusters (A, B, C and D) and within a cluster they are expressed temporally from 3’ to 5’ of DNA, with hox13 being at the very 5’ end and thus the most posteriorly expressed hox genes. In this study we use zebrafish as a model organism. Zebrafish embryos are excellent for investigation because: 1) the genome has been fully sequenced to a very high quality allowing the use of CRISPR mutagenesis; 2) the zebrafish embryos are transparent and so very easy to study using live microscope imaging; 3) much of the early development is similar among all vertebrates including humans. The role of the hox genes during the somite-forming states has almost entirely been characterized based on the overexpression of individual hox genes in previous research. We developed a zebrafish loss-of-function hoxa13 CRISPR mutant and are investigating the roles of this gene on the formation of the body plan. My research focuses on understanding how a loss of hoxa13 gene affects cell movement as the AP axis forms using spinning desk confocal microscopy to capture cells and Imaris software to track them, and I will present the results of this analysis. We expect to observe abnormal or reversal of cell movement in the prognitor area of the tailbud.

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. Previous work has suggested that two cystine mutations to Cellular Retinoic Acid Binding Protein I (CRABP-I) create a disulfide bond as a result of the conformational change that CRABP-I undergoes when it binds ATRA. This disulfide bond would essentially lock ATRA into CRABP-I, reducing its dissociation constant in vivo and maintaining the gut-homing properties of the nanoparticle post-injection. In order to assess the efficacy of these cysteine mutations, I expressed two versions of CRABP-I, the wildtype protein with no cysteine residues, and a version with no cystine residues except for the two that create the disulfide bond. After establishing that these new CRABP-I mutants folded into the approximate shape of wildtype CRABP-I via circular dichroism, I designed and tested new assays that measured free thiol concentrations of each protein after binding ATRA, as well as free ATRA concentration overtime. This data will help us determine whether these two cystine mutations make a significant difference in the ATRA binding quality of CRABP-I, which could improve the immune response generated by our vaccine candidate.

Prion diseases occur from the misfolding of the Prion Protein cellular form (PrP^C) under low pH conditions to the infectious scrapie species (PrP^Sc), which can aggregate further into insoluble fibrils. Previous studies have demonstrated that along with other amyloid oligomers, the prion scrapie oligomers cause neurotoxicity by disrupting the membrane, increasing its permeability and affecting calcium ion influx; however, the molecular mechanism for this effect is unknown. Molecular Dynamics simulations were performed to gain insight into the molecular mechanism of PrP^Sc-induced misfolding of PrP^C and oligomer toxicity in a membrane environment. The system was composed of the hexameric bovine PrP^Sc spiral model oligomer and the di-glycosylated human PrP^C attached to a POPC membrane via a glycophosphatidylinositol (GPI) anchor. Prior unpublished membrane simulations of this system have suggested that PrP^Sc induced PrP^C conformational changes as well as significant membrane disruption from oligomer-binding. Here we confirm and build upon these earlier studies demonstrating the reproducibility and robustness of oligomer binding affinity by varying the proximity of the oligomer to the membrane, providing key insight into infectious scrapie propagation and PrP^Sc cellular toxicity.

Natural proteins often assemble into various complex geometric structures based on their interactions with each other. These structures can hold and transport "cargo" as well as display antigens, making them extremely useful in vaccine design. The King Lab at the University of Washington uses the way these proteins assemble to develop computational models that help them design novel self-assembling protein cages, or nanoparticles. These nanoparticles are then used to develop vaccines or treatments for diseases. Components of the designed protein cage can be modified and expressed individually before being assembled together into the nanoparticle. I am working on stabilizing one of these protein cages known as T33DN2, so it can be used towards creating a vaccine. T33DN2 is a tetrahedral cage comprised of two trimeric proteins known as T33DN2A and T33DN2B. When expressed individually through E.coli, DN2A is produced in a soluble form while DN2B is produced in a mostly insoluble form. T33DN2 is currently an unstable cage, as only the A component is expressed solubly. Soluble proteins are generally more stable and thus easier to work with than their insoluble counterparts. To increase the solubility of DN2B, I have been making mutations to specific amino acids in the DNA that produces this protein, as well as expressing and purifying this component to determine its stability.

Thermoregulation, the maintenance of core body temperature in a constantly changing enviroment, is a critical aspect of homeostasis. Despite its importance, the neural mechanism by which thermoregulatory processes occur is not very well understood at the circuit level. Afferent skin temperature information travels through the spinal cord to the parabrachial nucleus (PBN), where it passes on to the preoptic area of the hypothalamus (POA). A subset of prodynorphin (Pdyn)-expressing neurons in the PBN (PdynPBN neurons) are activated when mice are exposed to warm environments, and 80% of these neurons project to the POA. The exact role of PdynPBN neurons has not been characterized, however, and their full projection profile is not established. Using genetic and viral techniques, we inserted a Cre-dependent designer receptor exclusively activated by designer drugs (DREADD) into mouse PdynPBN neurons and labeled their synaptic projections with GFP-bound synaptophysin, an abundant synaptic vesicle protein used for neurotransmitter trafficking. The use of Cre-dependent DREADD and synaptophysin-GFP allowed us to specifically label and activate PdynPBN neurons. We found that activation of these cells increases tail-skin temperature with a concurrent drop in core-body temperature. These data suggest that PdynPBN neurons may convey environmental temperature information that is sufficient to activate heat-defense responses. Establishing the genetic identity of neurons in a circuit that helps to maintain constant core body temperature will allow for the elucidation of downstream nodes in this circuit.

For many globular proteins, the sequence and native structure are known. However, less is understood about how a string of amino acids folds into a functional protein. Experimental study of folding presents challenges due to the transience and variability of folding/unfolding transition states and intermediates. Alternatively, computational study of unfolding can provide significant insight into folding. Here, molecular dynamics simulations have been used to study the unfolding pathways of the SH3 domain structural family and to investigate the factors that determine the path and outcome. To separate folding determinants from amino acid sequence, 17 SH3 proteins were chosen with an average sequence identity of only 27%. Six unfolding simulations were performed for each protein, and the unfolding transition state ensemble was identified by locating the large, rapid conformational changes that signal the start of unfolding. Contact analysis was used to characterize the structure of the transition states ensembles. Two general pathways at the transition state were identified, distinguished based on the specific β-sheet structure lost at the transition state. In the first, more populated pathway contacts in the β-sheet containing the N- and C- terminal β-strands were lost while the second pathway was defined by structure loss in the other β-sheet. Though many of the investigated proteins went through both pathways in different simulations, most showed a clear bias towards one pathway. This work demonstrates that similar protein structures can fold through different pathways. The bias of many SH3 proteins towards one folding pathway also suggests the presence of some elements of primary structure that direct folding. Further investigation of the SH3 domain may yield ‘rules’ that determine the structure and folding pathway of the domain, and these rules may inform the study of other, similar proteins.