Pharmacology

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/4954

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AKAP2: a critical cytoskeletal scaffold for triple-negative breast cancer cell growth and metastasis
(2026-02-05) Rosenthal, Kacey; Scott, John D.
A Kinase Anchoring Proteins (AKAPs) that coordinate spatiotemporal signaling are increasingly implicated in cancer. Mutations and misregulation to AKAPs can facilitate aberrant signaling through amplified or mislocalization of the signaling enzymes anchored by these scaffolds. Accordingly, elevated AKAP2 is associated with triple-negative breast cancer. In addition, the gene expression of this cytoskeleton-localized scaffold is increased in basal-like breast cancer patient samples compared to other subtypes. A combination of biochemical, cellular, and omics approaches show that AKAP2 cytoskeleton and focal adhesion associated scaffolds contribute to the growth and motility of basal-like triple-negative breast cancer. Proximity proteomics identifies AKAP2 as an element of focal adhesions in MDA-MB-231 cells. In addition, AKAP2 is found to be localized to focal adhesions and constrains the signaling of focal adhesion kinase (FAK). As such, gene silencing of AKAP2 not only decreases FAK levels but also attenuates the phosphorylation of the cell motility adapter protein paxillin on Tyr118. Cell-derived xenograft studies in mice establish that AKAP2 is required for triple-negative breast cancer cell growth and metastasis, phenotypes that are linked to FAK action. These findings discover a new role for focal adhesion-associated AKAP2 in triple-negative breast cancer pathology.
Novel roles of the adaptor associated kinase 1 (AAK1)
(2025-08-01) Lius, Andrea; Ong, Shao-En
Epithelial-mesenchymal transition (EMT) is a reversible cellular program that is involved in normal biological processes, such as embryogenesis and wound healing, and pathological processes, such as fibrosis and cancer. In cancer, EMT can drive therapeutic resistance and metastasis, the hallmarks of malignancy. We recently developed a mass spectrometry-based proteomics approach to study protein complexes in native cells and tissues. Using this tool, we characterized protein complexes that are differentially enriched between epithelial-like and mesenchymal-like states in hepatocellular carcinoma (HCC). We found that the adaptor associated kinase 1 (AAK1) protein complex is highly enriched in mesenchymal-like HCC cells and tissues, and that the depletion of AAK1 resulted in EMT reversal. Before this, AAK1 was mostly known for its role in the regulation of Clathrin-mediated endocytosis through the phosphorylation of adaptor protein 2, and it was never directly linked to cancer or EMT. We also identified U3 small nucleolar RNA-associated protein 25 homolog (UTP25), an understudied nucleolar protein, as one of AAK1’s interacting partners. We showed that the depletion of UTP25, like AAK1, produced phenotypes that are consistent with EMT reversal. Finally, through our investigation of AAK1’s functional relationship with UTP25, we discovered that AAK1 can localize to the nucleus, and that this nuclear localization may have isoform-specific preference.
The Structural Basis of the Negative Regulation of NPR1 by Nimin-1 in Plant Immunity
(2025-08-01) Gish, Madeline; Zheng, Ning
Humans are dependent on plants for food, energy and materials, yet plants are constantly under threat from bacteria, viruses and insects. Plants defend themselves from pathogens primarily through the expression of specialized defense genes, initiated by the key defense hormone salicylic acid, which is structurally related to the drug aspirin. Defense gene transcription and translation is a massive metabolic commitment by the plant, requiring tight negative control for long-term plant health. The salicylic acid receptor, NPR1, interacts with transcription factors to initiate defense gene transcription through an unknown mechanism. The Nimin family of proteins, under the control of an SA responsive promoter, form a negative feedback loop by directly interacting with NPR1 to stop defense gene transcription. The mechanism of this interaction, although necessary for control of the plant immune response, is poorly defined. In this work, I present the first biophysical characterization of NPR1’s interaction with the Nimin family of proteins and report the x-ray crystal structure of NPR1 salicylic acid binding domain in complex with Nimin-1, showing separate Nimin and SA binding pockets. I then demonstrate that Nimin-1 proteins regulate NPR1 signaling by allosterically competing with salicylic acid to bind to NPR1’s C-terminal domain, and further identify key residues in the allosteric pathway between the two pockets.
Walking on Weed: Predicting THC-induced motor impairment reveals disrupted cortical activity
(2025-08-01) English, Anthony; Stella, Nephi; Bruchas, Michael R
Cannabis has been used by humans for thousands of years for its multi-faceted properties as industrial tools, medicinal effects, and recreational value. And in the past several hundred years, international prohibition movements have been in a struggle with scientists investigating its effects on the human body. Despite this, scientists have persisted to isolate the primary psychoactive compound THC and use it as a basis to uncover an entire neurotransmitter system in humans: the endocannabinoid system. This system plays a critical role in modulating behavior, neurological development, and general brain activity. Further research into the endocannabinoid system and the Cannabis plant lags behind other similar fields despite its importance. The research and findings presented here show another step towards expanding our understanding and functional applications with THC and the endocannabinoid system.Cannabinoid compounds such as THC are notoriously difficult to work with due to their high lipophilicity and, in in vivo rodent studies, their high taste avoidance. Therefore, to counter the potent smells and flavors of raw THC, in an effort to uncover its in vivo behavioral effects. To address this gap, we developed a novel gelatin-based method for THC delivery that enhances palatability and voluntary intake in mice. By incorporating THC into a chocolate-flavored nutritional shake (Ensure™) and gelatinizing the mixture, we formulated an E-gel matrix. In mice, we enhanced voluntary consumption of high-dose THC compared to standard gelatin and succeeded in promoting the mice to consume enough to reach highly psychoactive behavioral effects. The use of Cannabis products containing high concentrations of THC is rapidly increasing, with a growing body of evidence links high-THC Cannabis use with increased psychotic and affective symptoms and Cannabis-associated vehicular accidents. The need for tools to investigate the mechanistic insight into how high-potency THC influences the brain is increasingly relevant. The E-gel model thus provides a translationally relevant, voluntary oral intake paradigm for characterizing THC's pharmacokinetics and behavioral impacts in rodents, offering an essential tool for investigating the consequences of high-dose THC exposure. The endogenous cannabinoid receptor CB1 plays a key role in brain development, but the long-term effects of adolescent THC exposure remain poorly understood. To explore its impact on addiction vulnerability, I exposed mice to THC during adolescence and assessed morphine-related behaviors in adulthood, finding no significant alterations in analgesia or drug-seeking. However, female mice showed THC-dependent impairments in memory recall, suggesting a sex-specific effect on learning. Preliminary behavioral analyses using pose estimation also revealed a unique exploratory behavior in THC-exposed animals, warranting further investigation into the neural circuits underlying these effects. Emerging technologies in other fields provide a pathway to overcome technical and political limitations to enable more sophisticated analyses. In recent years, computer vision tools like DeepLabCut and SLEAP have transformed behavioral research by allowing detailed tracking of individual points on animals during experiments, improving our ability to analyze animal behavior with greater precision than the human eye. Here, we utilized the SLEAP pose estimation algorithm within a linear track system that enables high-resolution and high-frame rate visualization of mice side and bottom-up profiles. Then, animal poses were used to calculate key features that to train dose prediction models capable of identifying the dose of THC animals were treated with solely based on their general or specifically locomotor behavior. These models were then utilized, along with the unsupervised identification of nuanced behaviors, to investigate the effects of THC on excitatory/inhibitory balance of cortical neurons in the mPFC. Inhibitory GABAergic neurons and excitatory glutamatergic neurons of the mPFC were observed to be modified in their activity during THC-impaired motor behavior. Manipulation of CB1R expression and activation of specific neurons time locked to movement with a closed-loop optogenetic model revealed the enhanced GABAergic activity correlates with worsened motor behavior, suggesting the E/I balances criticality in modifying THC-impairment. This effect also greatly modified native endocannabinoid signaling as identified by the novel GRABeCB2.0 senor. These findings marked a novel application of behavioral identification utilized to identify a novel THC-dependent modification in E/I balance of the cortical neurons during motor behavior. Together, the research displayed in this thesis combines advanced computational applications and cutting-edge biosensor technologies to address a pressing challenge in cannabinoid research: quantifying the multi-faceted signatures of THC’s effects in vivo.
The ascent of AKAPs: from architectural elements to isotype selective PKA scaffolds
(2025-05-12) Falcone, Jerome Ian; Scott, John D
The spatiotemporal organization of proteins within cells is essential for complex life. The scaffolding of protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) allows for the management of a multitude of simultaneous signal cascades within animal cells. AKAPs contain an 18-amino-acid-long amphipathic helix that anchors the docking and dimerization (d/d) domain of PKA regulatory subunits. There are four isoforms of the PKA regulatory subunit, RIα, RIβ, RIIα, and RIIβ, which fall into the two categories of type I and type II PKA (the α and β forms of each regulatory subunit share similar topology). There are >60 known AKAPs, and each one prefers to bind either type I or type II PKA. As PKA originated in the metazoan clade, the presence of 'AKAP' proteins in organisms that lack PKA suggests a PKA-independent function. These ancestral anchoring proteins localize to cilia and flagella across all eukaryotic kingdoms and bind docking and dimerization domain proteins that contain a d/d domain nearly identical to that of PKA, fused to other protein domains. In this work, we christen these PKA-independent scaffolds as docking and dimerization domain interacting proteins (DDIPs) and study the co-evolution of d/d proteins, PKA, AKAPs, and DDIPs. Phylogenetic analyses show that PKA and the d/d proteins exhibit telltale signs of divergent evolution. Driven by gene duplication and fusion events, both the structural folds and the primary sequences of these proteins have been highly conserved for over two billion years. The evolutionary path of AKAPs and DDIPs is strikingly different. Phylogenetic analyses reveal patterns of convergent evolution for these anchors. By tracing the evolution of anchoring domains on extant proteins, we reveal a paradigm of accretion of neutral mutations which eventually form a functional anchoring helix. Once this occurs, the mutation rate slows as this protein region becomes subject to selective pressures. Finally, I examine the features of AKAP helices which drive preference for either type I or type II PKA. Guided by bioinformatic analysis, we identified an F/Y, A motif at the N-terminus of all type I-selective AKAPs. We hypothesized that this "aromatic, alanine" motif controls AKAP preference. Using a total internal reflection fluorescence (TIRF)–fluorescence recovery after photobleaching (FRAP) assay, we measured the degree of R subunit binding of the characteristic type I and type II AKAPs, smAKAP and AKAP79. We then applied the "aromatic, alanine" motif to the helix of AKAP79, creating mutants with an "FA" on the N-terminus, an "AF" on the C-terminus, and a version with both terminal mutations known as "FAAF". Immunoprecipitation experiments with these mutants revealed that only the FAAF mutant gains the ability to pull down RI. Molecular dynamics simulations on AKAP79 and AKAP79FAAF reveal a rearrangement of contact points between the helix and the d/d domain which facilitate this preference switch. FRAP experiments confirm that AKAP79FAAF does switch its preference to RI in situ. The significance of this mutation on signaling was confirmed by a rescue of aberrant corticosterone release in an RI-biased Cushing's syndrome model. Finally, we performed a reciprocal experiment in which the 'aromatic, alanine' motif on smAKAP was replaced with two leucines to resemble AKAP79. This mutation was sufficient to impair RI binding by smAKAP in an immunoprecipitation, but did not confer RII binding measured by immunoprecipitation or RII overlay. Together these results clarify the evolutionary paths of these anchoring proteins and their d/d partners, and serve as a call to arms to identify and characterize new DDIPs. The analysis of AKAP preference will assist in the characterization of new AKAPs and reveals a new facet of what makes this binding interface such a powerful cellular tool.
High Throughput Identification of Mitochondrial Calcium Regulated Proteins
(2025-01-23) Locke, Timothy; Sancak, Yasemin
Mitochondrial calcium plays a well-known regulatory role in mitochondria. Disruption of mitochondrial Ca2+ uptake is implicated in diseases such as cancer, neurodegenerative, and metabolic diseases. Evaluating the relationship between the disease states and mitochondrial Ca2+ uptake has proven difficult due to limited knowledge of the mediators of mitochondrial Ca2+ signaling. Currently, there are 20 known mitochondrial Ca2+-binding proteins, which were identified using targeted biochemical assays or computational detection of EF-hand domains. It is unknown whether other calcium-regulated mitochondrial proteins with non-canonical Ca2+-binding domains, which are resistant to computational detection, exist. We set out to identify novel mitochondrial Ca2+-binding proteins in a high-throughput and unbiased manner and investigate how Ca2+ ions regulate these proteins and the mitochondrial pathways they control.To identify calcium-regulated proteins, I optimized a biochemical assay, PISA, that detects the conformational changes in proteins after they interact with calcium. I performed PISA on multiple samples - human cells, yeast cells, and mouse mitochondrial enriched samples from liver tissue– and showed the cross-species viability of this assay to find Ca2+-regulated proteins. The data of each PISA experiment, along with a table detailing data from the orthologous human and yeast proteins, are attached to this dissertation as supplemental tables. Focusing on the mitochondrial samples, I correctly identify known Ca2+-binding and non-Ca2+-binding proteins in an unbiased manner, as well as covering above 85% of the mouse liver mitochondrial proteome. Towards understanding the calcium-regulation of select hits, I used microscale thermophoresis (MST) to detect calcium-binding in vitro at physiologically relevant free calcium concentrations, successfully identifying a novel mitochondrial Ca2+-binding protein. My results fill a large hole in the field’s knowledge of mitochondrial Ca2+ signaling and provide multiple avenues for further research by highlighting new molecular players through which mitochondrial Ca2+ regulates mitochondrial functions.
Mitochondrial Calcium Signaling Regulates Branched-Chain Amino Acid Catabolism in Fibrolamellar Carcinoma
(2024-10-16) Marsh, Nicole; Sancak, Yasemin S
Metabolic adaptations in response to changes in energy supply and demand are essential for survival. The mitochondrial calcium uniporter coordinates metabolic homeostasis by regulating TCA cycle activation, mitochondrial fatty acid oxidation and cellular calcium signaling. However, a comprehensive analysis of uniporter-regulated mitochondrial metabolic pathways has remained unexplored. Here, we investigate the metabolic consequences of uniporter loss- and gain-of-function and find that mitochondrial calcium signaling regulates the branched-chain amino acid (BCAA) catabolism pathway. Loss of uniporter function activates the pathway through two mechanisms: increased expression of pathway proteins and stimulation of the enzyme that catalyzes the committed step of the pathway by dephosphorylation. Conversely, in the liver cancer fibrolamellar carcinoma (FLC) – which we demonstrate to have increased mitochondrial calcium levels – expression of BCAA catabolism enzymes is suppressed. The transcription factor KLF15, a master regulator of liver metabolic gene expression, is also regulated by mitochondrial calcium signaling. KLF15 is downregulated in FLC patient tumors and cellular models. The KLF15 target gene ornithine transcarbamylase (OTC) is downregulated in FLC, which is thought to contribute to urea cycle defects and hyperammonemia observed in some FLC patients, suggesting a role for mitochondrial calcium in hyperammonemia. Inhibition of uniporter activity in FLC cellular models upregulates KLF15, BCAA catabolism genes, and OTC. Collectively, we identify an important role for mitochondrial calcium signaling in metabolic adaptation through transcriptional and post-translational regulation of metabolism and elucidate its importance for BCAA and ammonia regulation in FLC.
The impact of repeated opioid withdrawal on mouse behavior and microglia
(2024-10-16) Bergkamp, David; Neumaier, John F
Microglia are specialized cells of the central nervous system (CNS), often mentioned as the resident myeloid population or the resident immune cells of the CNS. Microglia, as well as astrocytes and oligodendrocytes, contribute to drug induced changes in CNS function, which can be investigated using powerful new omics techniques. Microglia have been shown to play a role in opioid withdrawal by modulating the actions of various signaling molecules and by releasing inflammatory agents, leading to alterations in animal behavior. We hypothesized that microglia may contribute to the behavioral signs of opioid withdrawal when an individual experiences withdrawal several times in succession. Here we present work studying the impact of multiple withdrawal experiences on mouse behavior, as well as changes to microglia morphology and microglia RNA translation, specific to the mouse striatum. We find that five withdrawal experiences lead to more intense and protracted withdrawal signs in mice. Additionally, the impact of multiple cycles of withdrawal changes microglia cells to adopt a more ameboid and inflammatory-like state, perhaps proliferating specifically in the striatum of female mice. Our RNA sequencing results also indicate that multiple cycles of withdrawal induce an inflammatory signature in the microglia translatome, and support our finding that microglia are shifted to a proliferative state in the mouse striatum.
Leveraging Molecular Pharmacological Principles for the Design of Novel Treatments for Drug Addiction and Mood Disorders
(2024-09-09) Neiswanger, Carlie; Chavkin, Charles
The dynorphin/Kappa opioid receptor (KOR) system has been implicated in the regulation of stress, anxiety, depression, and substance use disorders. KOR antagonists have great potential as a therapeutic for these disorders, but the culmination of preclinical work over the last two decades has made for very little success in the clinic. The work here aims to identify and design a repeated dosing regimen to use with partial and G-biased KOR agonists. These agonists, specifically nalfurafine and nalmefene, activate the G-protein mediated JNK-ROS pathway that leads to long-term inactivation seen with norBNI and JDTic. Using a new tool, oROS-Gr, ROS generation was detected in ex vivo slice and in vivo fiber photometry with the treatment of these agonists. Repeated dosing caused significant KOR inactivation and blocked stress-induced aversion and aversion during opioid-withdrawal mediated by the dyn/KOR system. Low dosing combined withpreviously demonstrated high degree of safety profiles for nalfurafine and nalmefene make them promising new options for the treatment of substance use and mood disorders in humans.
Development of Human Pancreatic Ductal Epithelial Cells as a model of Cystic Fibrosis Pancreatic Disease
(2024-09-09) Malik, Sarah; Hull-Meichle, Rebecca L
Impaired insulin release underlies the development of cystic-fibrosis-related diabetes (CFRD) which affects 40-50% of adults with CF and is associated with significantly increased morbidity and mortality. The initiating site of CF pancreas pathology is the pancreatic duct as CFTR expression is highest in pancreatic ductal epithelial cells (PDECs). While loss-of-function mutations in CFTR result in CF, these mutations lead to the destruction of pancreatic exocrine (acinar) tissue which is characterized by the clinical manifestation of pancreatic exocrine insufficiency in 85% of people with CF. Despite this destruction of the pancreas, there is only modest loss of islets. While the pathophysiology of CF pancreas disease and CFRD are not well understood, it is appreciated that paracrine signals in the PDEC-islet axis could be one mechanism contributing to the deficit in insulin release in CFRD. The data supporting the existence of crosstalk between PDECs and islets are limited in translational human models, despite the observation of their close proximity within the pancreas. The studies presented in this thesis represent an advancement in our tools to study this PDEC-islet axis. Collectively, the experiments enclosed in this thesis provides a better foundational understanding of ex vivo and cultured PDEC CFTR, SOX9, AQP1, KRT7 and KRT19 mRNA expression. Additionally, these findings represent advancement in developing a model to modulate CFTR expression in primary human PDECs. Ultimately, these discoveries set a path for downstream analysis that will prove valuable in the discovery and development of future therapeutics to treat CFRD.
Molecular interactions and signaling mechanisms of the oncogenic fusion protein DNAJ-PKAc in fibrolamellar carcinoma
(2023-09-27) Lauer, Sophia My-Linh; Scott, John D
The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperone binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ-PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and live-cell photoactivation microscopy we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the co-chaperone Bcl-2 associated athanogene 2 (BAG2), a regulator of Hsp70-mediated protein refolding and inhibitor of CHIP-mediated ubiquitination. Immunoprecipitation experiments in AML12 hepatocytes and FLC patient tissue showed co-precipitation of BAG2 with DNAJ-PKAc but not wild-type PKAc. Use of a kinase-dead mutant (K72H) in AML12 cells demonstrated that this interaction is independent of fusion kinase activity and reconstitution of FLC complexes using the SpyCatcher/SpyTag heterodimerization system also confirmed this interaction. Furthermore, immunoblot and immunohistochemical analyses of FLC patient samples demonstrate increased levels of BAG2 in primary tumors and metastatic recurrence. BAG2 is linked to the anti-apoptotic factor Bcl-2. Pharmacological approaches using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax show that the DNAJ-PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in a cellular model of FLC. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.
Stimulus-Dependent Regulation of 2-Arachidonoyl Glycerol Signaling and the Mechanism of its Hydrolysis by ABHD6
(2023-09-27) Singh, Simar; Stella, Nephi
Cannabis has been used for thousands of years for recreational and therapeutic effects. The efficacy of medicinal cannabis for conditions such as epilepsy and pain has resulted in the discovery of the molecular targets of the bioactive compounds, or phyto-cannabinoids, found in cannabis. These targets are part of a signaling system called the endocannabinoid (eCB) system, which is a neuromodulatory system consisting of the receptors targeted by phyto-cannabinoids, the endogenously produced lipid neuromodulators, or eCBs, that act at these targets, and the machinery that regulates eCB levels, including biosynthetic and degradative enzymes. Enhancing eCB signaling in the brain can provide therapeutic benefit for neurological diseases like epilepsy and chronic pain.A strategy for enhancing eCB signaling is by inhibiting its enzymatic hydrolysis. One of the eCB hydrolyzing enzymes expressed in the brain is ɑ/β hydrolase domain containing 6 (ABHD6). ABHD6 hydrolyzes the eCB 2-arachidonoyl glycerol (2-AG), which is the most abundant eCB in the central nervous system. Production of neuronal 2-AG is activity-dependent, resulting in localized and tightly regulated signaling at targets such as the cannabinoid receptors (CBRs). Therefore, inhibiting ABHD6 can increase intrinsic 2-AG levels and resulting CBR activation. Thus, inhibition will have an effect only when and where activated cells produce 2-AG. Recent studies have demonstrated efficacy of ABHD6 inhibition in preclinical models of epilepsy and neuropathic pain, which are pathological conditions that share key elements: hyperexcitability of neurons and neuroinflammation. While 2-AG’s role in reducing neuronal activity has been well-described, the mechanism of neuronal 2-AG production following inflammation and the effect of ABHD6 inhibition on enhancing these 2-AG levels remains unclear. Here, we characterize a genetically encoded sensor, the GRABeCB2.0, and then use the sensor to show that two inflammatory mediators, ATP and bradykinin (BK), can stimulate 2-AG production in neuro2a mouse neuroblastoma cells through separate receptor-dependent mechanisms. I discovered that, in a co-culture model system, ATP and BK-stimulated 2-AG can participate in paracrine activation of GRABeCB2.0. These findings describe a potential molecular mechanism by which inflammatory mediators may act directly on sensory CB1R-expressing neurons and engage the eCB system, which can function in a negative feedback loop to decrease hyperactivity of peripheral nociceptors that underlies hyperalgesia. Since ABHD6 inhibition has the potential to enhance 2-AG signaling and produces analgesia in neuropathic pain models, we further described the mechanism of ABHD6 hydrolysis activity and discovered that an undescribed membrane factor increases in vitro ABHD6 activity and are now testing a first-in-class ABHD6 inhibitor. This work expands our understanding of the pathophysiological role of 2-AG and describes the function of ABHD6, a potential therapeutic target which can allow for the development selective inhibitors with a clinical benefit.
Novel tools for multi-omic characterization of subnuclear RNA-scaffolded structures in development and disease
(2023-09-27) Kania, Evan Erik; Shechner, David M; Scott, John
RNA molecules are increasingly recognized as fundamental regulators of subcellular organization. In the nucleus, RNAs scaffold numerous structures that mediate diverse and essential genomic functions. These range from small-scale chromatin interactions that co-regulate handfuls of genes, to subnuclear organelles that collectively control cellular gene-expression, epigenetic, and metabolic programs. RNA-scaffolded structures are characteristically dysregulated in, and thought to be causally linked to, a plethora of human diseases, including developmental and neurodegenerative disorders, retroviral infections, and cancer. Nuclear architectural RNAs may therefore represent a large, untapped pool of novel therapeutic targets. However, elucidating and characterizing these targets—identifying the proteins, RNAs, and genomic loci with which a given RNA interacts—remains challenging.The studies presented in this thesis represent groundbreaking advancements in technologies I generated to capture and characterize nuclear RNA-scaffolded structures and their associated genomic loci, proteins, and RNAs, even those with low abundance. Employing these innovative techniques on gene silencing long non-coding RNAs (lncRNAs), I identified the biomolecular constituents of the nuclear structures they scaffold. Notably, this work shed light on the molecular constituents of the X-chromosome inactivation center, scaffolded by the nascent Xist transcript, and provided a better understanding of gene silencing lncRNA function. Moreover, I also applied the suite of technologies I developed to investigate a severe form of dilated cardiomyopathy caused by RBM20 mutations (RBM20-DCM), focusing on the nascent TTN transcript that naturally scaffolds nuclear RBM20 foci. These analyses revealed key biomolecules crucial for the native function of RBM20 foci and unveiled aberrations in alternative splicing and mitochondrial reactive oxygen species handling upon loss of RBM20, offering insight into the pathogenesis of RBM20-DCM. Collectively, this thesis lays the foundation for exploring nuclear RNA interactomes in various contexts, facilitating a deeper understanding of these structures in both native biological and aberrant disease states. The newly developed technologies hold significant potential for broader applications in understanding the composition and localization of other RNA-scaffolded structures, providing invaluable information for future research and potential therapeutic interventions. By delving into the complexities of nuclear RNA-scaffolded structures, these studies not only advance our understanding of cellular regulation but also open up exciting possibilities for targeted therapies against numerous human diseases. These findings represent a significant advancement in the field of RNA biology and lay the foundation for further investigations in the realm of RNA-based therapeutics and precision medicine. Ultimately, the discoveries made here pave the way for future breakthroughs in deciphering the mechanisms governing RNA-mediated subcellular organization and the promising utilization of RNA scaffolded structures and their constituents as therapeutic targets.
Development of Oligonucleotide-directed proximity-interactome MAPping (O-MAP), for characterizing RNA-protein interactions and higher order subnuclear architecture in situ
(2023-08-14) Tsue, Ashley Frances; Shechner, David M
RNA-protein interactions underscore a broad array of regulatory mechanisms throughout biology, and dysregulation of these interactions contributes to a broad range of human pathologies such as neurodegenerative disorders and many cancers. Recent studies have discovered thousands of RNA-binding proteins that lack canonical RNA binding domains. Additionally, the human transcriptome harbors tens of thousands of novel RNAs that can be differentially regulated in disease. RNA-protein complexes, termed ribonucleoproteins (RNPs), can also serve as structural scaffolds for reorganizing local subcellular environments and assembling subnuclear bodies. A classic example of RNA-scaffolded structures is the nucleolus, a subnuclear body that serves as the site of ribosome biogenesis, cell cycle regulation, and stress responses. Nucleolar assembly is initiated de novo after each cell division—nucleated by pre-ribosomal RNA (pre-rRNA) and other nucleolar factors that are preserved during the disassembly of the nucleolus at the start of mitosis. This formation is thought to be driven by RNA-protein interactions that phase separate into the nucleolar tripartite structure. Other RNAs, such as X inactive specific transcript (XIST) and MALAT1, are thought to have an architectural role in organizing subnuclear Barr bodies and nuclear speckles for gene regulation. However, studying specific RNA and their protein interactomes is challenging by conventional methods. Most approaches for analyzing the composition of a subcellular structure rely on the biochemical purification of that structure or enriching for interacting proteins using antisense oligonucleotides of the target RNA bound to a resin. These approaches are either impossible for smaller structures or are plagued with nonspecific binding and low specificity after exposure to crude lysate. To address these problems, recent work has utilized proximity labeling as a way to study specific RNA-protein interactomes within the subcellular context; however, few methods exist to specifically target and enrich for RNA scaffolded subnuclear bodies. Utilizing existing RNA-fluorescence in situ hybridization (FISH) technologies and proximity labeling, this thesis focuses on the development of Oligonucleotide-directed proximity-interactome (O-MAP) to target specific RNAs within their subcellular context and probe for interacting proteins. This technique targets antisense oligonucleotides to a RNA of interest in fixed cells and recruits a proximity labeling enzyme, horse radish peroxidase (HRP), to the target RNA enabling the promiscuous biotinylation of nearby proteins. These biotinylated proteins can be isolated by streptavidin enrichment and analyzed by downstream omics such as mass spectrometry (MS). Using the 47S pre-ribosomal RNA, long noncoding RNA XIST and 7SK small nuclear RNA as models, O-MAP induces precise biotinylation of target RNA and nearby proteins that can then be systematically analyzed by mass spectrometry. O-MAP-MS at these RNA targets characterized these RNA scaffolded compartments and interacting proteomes, highlighting classes of proteins involved in nucleolar biology and nuclear speckles. Without the need for genetic manipulation, O-MAP is both easily portable to other cell lines, organoids, tissues as well as RNA targets of varying expression and lengths. Additionally, O-MAP can probe differentially-regulated proteomes in disease-relevant states including pancreatic cancer and cellular stress. These studies demonstrate the versatility and specificity of O-MAP as well as its potential to provide new characterizations in RNA interactome biology.
Mechanisms and physiological implications of mitochondrial and cellular calcium signaling
(2023-04-17) MacEwen, Melissa Jane; Sancak, Yasemin
Calcium signaling is central to fields as disparate as memory formation, protein structure, and cellular migration. The five chapters of this dissertation each examine calcium signaling in a different context. First, an introduction contextualizes this dissertation in the broader history of calcium signaling and mitochondrial biology. Second, a published review details the most salient contributions to the field of mitochondrial calcium signaling in recent years. Third, a research publication clarifies why the metazoan mitochondrial calcium uniporter (MCU) requires another protein, EMRE, to conduct calcium ions. The fourth chapter provides an overview of an ongoing research project examining potential metabolic “rewiring” that enables mitochondria to produce energy and metabolites even when mitochondrial calcium signaling is compromised. Finally, the fifth chapter showcases a research publication that used experimental biochemistry to determine which of three proposed mathematical models most accurately reflects the binding between calmodulin (CaM) and myosin light-chain kinase (MLCK).
Uncovering the Role of Protein Kinase TAOK2 as an Endoplasmic Reticulum-Microtubule Tether
(2022-01-26) Nourbakhsh, Kimya; Yadav, Smita
Kinase signaling drives a multitude of components of neuronal development such as establishment of polarity, dendritic growth and arborization, dendritic spine formation, and synaptogenesis. Thousand And One amino acid Kinases (TAOKs) are a subfamily of sterile-20 (STE20) kinases possessing an N-terminal kinase domain and diverging C-terminal extended regulatory domains. As evolutionarily conserved kinases, TAO kinases have been shown to play important roles in neuronal development in rodents and invertebrates. Recent studies have linked de novo variants of human TAOK1/2 to neurodevelopmental diseases, thus making it imperative to understand their role in neuronal development. Despite its disease association, the molecular functions of TAO kinases remain unknown. Here, I focus on understanding the molecular and cellular function of the largest member of the TAO family, TAOK2 kinase. We find that TAOK2 is an endoplasmic reticulum (ER) resident multi-pass membrane-spanning kinase that localizes to distinct junctures of the ER through six transmembrane helices and an amphipathic helix. Additionally, we found that TAOK2 associates to microtubules directly and with high affinity through its cytoplasmic facing C-terminal tail. Furthermore, TAOK2 acts as a functional ER-microtubule tether, knockout of which leads to increased ER motility, increased microtubule growth but decreased ER tip attachment-complex movement. We determine that TAOK2 interacts with microtubule plus-end binding (EB) proteins through a distinctive SxIP motif. Finally, we determine that TAOK2 microtubule binding is regulated by its catalytic activity, loss of which leads to perturbations of ER and microtubule motility. This work identifies TAOK2 as an ER-microtubule tether and reveals a kinase-regulated mechanism for control of ER dynamics critical for cell growth and division. To further understand the context of TAOK2 signaling and determine TAOK2 interactors between the ER and microtubules, I propose the use of proximity labeling utilizing BioID2 and mass spectrometry. I lay out the blueprint for these experiments and the preliminary data from pilot experiments. Finally, I provide implications of these findings on how TAOK2 functions as an ER-microtubule tether might contribute to neurodevelopment.
The Structure of Sperm Autoantigenic Protein (SPA17): An R2D2 Protein Critical to Cilia and Implicated in Oncogenesis
(2021-08-26) Dahlin, Heather Raquel; Scott, John DW
A-Kinase Anchoring proteins (AKAPs) localize the activity of cyclic AMP (cAMP)-Dependent Protein Kinase (PKA) through interaction of an amphipathic helix that binds to a conserved RIIα docking and dimerization (R2D2) domain on the N-terminus of PKA. Genome analysis indicates that at least thirteen other RIIα superfamily proteins exist in humans, which are not coupled to cyclic nucleotide binding domains and are largely localized to cilia and flagella. The newly reported R2D2 proteins exist in two lineages differing by their similarity to Type I or Type II PKA. Moreover, R2D2 domains bind to AKAPs and can contain extra regulatory sequences conferring novel functions and binding specificity. Here we detail the structure of one such domain comprising the N-terminus of Sperm Autoantigenic Protein 17 (SPA17) resolved to 1.72 Ã . The structure of core hydrophobic sites for dimerization and AKAP binding are highly conserved between PKA and SPA17. Additional flanking sequences outside of the core R2D2 domain occlude the AKAP binding site and reduce the affinity for AKAP helices in the absence of heterodimerization with another R2D2 protein, ROPN1L.
Adrenergic Regulation of Cardiac CaV1.2 via Direct Phosphorylation and the GTPase RAD
(2021-08-26) Hovey, Liam; Catterall, William A
Dysregulation of the cardiac fight-or-flight response has been linked to chronic heart failure and arrhythmic disorders. The L-type calcium channel CaV1.2 has a central role in mediating the cardiac fight-or-flight response, but the precise molecular mechanisms that transduce adrenergic stimulation to increased cardiac output are not completely characterized. This work seeks to address fundamental mechanistic questions related to the regulation of CaV1.2 during the cardiac fight-or-flight response, and thus lay the groundwork for next-generation therapies targeting the ?-adrenergic-CaV1.2 signaling axis. I have specifically focused on a critical question in the field: is CaV1.2 activity primarily regulated by direct phosphorylation during the fight-or-flight response, or through other molecular mediators? I report here that direct phosphorylation of the C-Terminal Domain of CaV1.2 has an important role in cardiac function and CaV1.2 activity, and that the small GTPase RAD co-regulates the channel alongside direct phosphorylation. These findings indicate that the molecular mechanism of the cardiac fight-or-flight response is characterized by convergence of the direct phosphorylation and RAD pathways to co-regulate CaV1.2 activity.
Serotonergic Circuits Mediating Stress Potentiation of Addiction Risk
(2021-08-26) Fontaine, Harrison; Chavkin, Charles
Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in monoaminergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. We found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT+ neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3+ neurons blocked subsequent cocaine CPP. SERT+/VGluT3- expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not present in mNAc. Optical inhibition of SERT+ neurons produced place aversion, whereas optical stimulation of SERT+ terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.
Identifying the Mechanisms Behind Radiation Resistance in Treatment NaÃ¯ve Diffuse Intrinsic Pontine Glioma Models
(2021-07-07) Ferguson, Eric; Olson, Jim
Diffuse Intrinsic Pontine Glioma (DIPG) is a near universally fatal pediatric brain tumor that occurs in the pons and other midline structures. These tumors are characterized by the presence of a unique histone mutation: H3 K27M. This mutation inhibits the polycomb repressive complex 2 (PRC2) and reduces the global H3 K27 trimethylation mark. DIPG is resistant to conventional therapies such as radiation and chemotherapy which contributes to the high mortality rate in DIPG patients. A major challenge to researching DIPG is a lack of tissue available for research models. Due to the sensitive location of DIPG tumors, these tumors cannot be removed by surgery. All current DIPG models are either generated from autopsies or genetically engineered from mice or human stem cells. Currently no drug developed from these research models have translated into a successful clinical therapy. In order to develop a model that more accurately represents to disease state, we developed a protocol to collect biopsies from DIPG patients prior to treatment. Using these treatment naive DIPG biopsy models, we observed similar resistance to radiation therapy compared to how the tumors behaved in the clinic. A key component of the resistance to radiation was the inability of DIPG cells to undergo apoptosis. We found that the pro-apoptotic protein BCL-2 associated X (BAX) was significantly reduced in DIPG compared to other pediatric brain tumors. When radiation sensitive cells were irradiated, they upregulated BAX but in DIPG this response was significantly reduced. In order to overcome the reduced levels of BAX, we inhibited the pro survival BCL-2 family proteins and found that the pan BCL-2, BCL-xl inhibitor navitoclax was able to sensitize DIPG cells to radiation. We measured the percent of pro-apoptotic cells and found the combination of navitoclax and radiation significantly increase the percent of pro-apoptotic cells in the combination treatment compared to either treatment individually. Future work will focus on why BAX is downregulated in DIPG compared to other pediatric brain tumors. We have identified several transcription factors and epigenetic regulators of BAX that are dysregulated in DIPG. This research has contributed to our understanding of how DIPG resists conventional therapy and may lead to targeted therapies which sensitize DIPG and improve the overall patient survival.

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