Structural Biology in Cellular Environments Using Sensitivity Enhanced NMR

This dissertation describes the general applicability of DNP solid state NMR to mammalian cells. DNP NMR can detect proteins at their physiological concentrations within cellular environment in tractable times. However, these experiments were limited to cellular lysates, which did not recapitulate the intracellular environment completely. Studying proteins within their native cellular environment required in-cell NMR. This work focuses on developing methodology for in-cell DNP NMR and applying the technique to investigate the conformational ensemble of Tau. Chapter 1 provides an overview of in-cell NMR in biological systems and intrinsically disordered proteins. A brief primer of NMR, DNP and EPR is also provided in this chapter. Chapter 2 describes the development of methodology to maintain biological integrity of mammalian cells during DNP NMR. Description of a novel method to insert samples into a spectrometer is added as a supplementary chapter 2. Chapter 3 is based on a comparative study between different cryoprotectants to optimize biological integrity for in-cell DNP NMR. Chapter 4 focusses on the characterization of the reduction process of a polarizing agent, AMUPol inside intact and lysed HEK293 cells using electron paramagnetic resonance (EPR) spectroscopy. In the supplementary chapter 4, a novel DNP radical AsymPol-POK and its reduction kinetics within intact HEK293 cells is discussed. Chapter 5 deals with the application of in-cell DNP NMR to study the conformational ensemble of tau protein within cellular environment. The in-cell DNP NMR methodology developed in this work can be potentially applied to various biological systems. Additionally, the strategies and experiments described here to investigate the conformational ensemble of an intrinsically disordered protein, tau, within cellular environment, can be tailored to any other IDP or protein of interest within normal or pathological cellular milieu. Also, the EPR experiments described here will be a useful guide to test reduction kinetics of novel polarizing agents for in-cell DNP NMR. Overall, this dissertation should serve as a useful literature for technical advancement and biological application of in-cell DNP NMR

Kinetic and Thermodynamic Allostery in the Ras Protein Family

The general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.An understanding of allosteric communication is necessary for interpreting protein function and regulation. For proteins involved in disease, such knowledge can lead to discovery of new sites for therapeutic targeting. However allosteric mechanisms have proven to be diverse, and allosteric communication in many proteins cannot currently be explained by their structure or dynamics. Progress has been made in elucidating the ensemble nature of allosteric communication, especially using MD simulations to provide structural specificity of previously averaged conformations. Here I show how kinetic (i.e. temporal) correlations encode information that is orthogonal to existing work on thermodynamic correlations. I performed atomistic simulations on H, K, and NRas isoforms in various states in the Ras cycle, for a total of 0.5 milliseconds. I show that Ras' most important structural motifs, switch I and switch II, are the primary members of my calculated thermodynamic and kinetic allosteric networks, consistent with the known roles of these two motifs in Ras function. I also reveal how these communication networks are altered by the presence of the -phosphate, as well as binding of the downstream effector Raf kinase. Strikingly, these communication networks correspond to structural motifs that are functionally engaged in the Ras cycle step being simulated. I find that kinetics-based allosteric communication is not restricted to the boundaries of secondary structure elements and can occur across long distances. I show that known features of Ras regulation, such as activation of allostery upon Raf binding, necessitate kinetic allostery. These data explain experimentally observed allosteric relationships by revealing the kinetic and thermodynamic communication pathways, and show how both modes of communication are needed to relay information within proteins. This work suggests that kinetics-based communication is a key mechanism of protein function and regulation

Identifying CDK8/19 as a Novel Regulator of EWS-FLI1 Function in Ewing Sarcoma

Ewing sarcoma, a pediatric bone cancer, is characterized by the translocation of the Low Complexity (LC) domain from an RNA binding protein (EWSR1, FUS, or TAF15) with the DNA binding domain of an ETS transcription factor. The translocation EWS-FLI1 accounts for 90% of Ewing sarcoma tumors and a large body of evidence suggests that EWS-FLI1, acting as an oncogenic transcription factor, is the main driver of proliferation in Ewing sarcoma. Targeted therapies for Ewing sarcoma that act through disruption of EWS-FLI1 function remain undiscovered. Using an engineered degron system I can deplete endogenous EWS-FLI1 and have identified transcripts that are activated or repressed by EWS-FLI1. From the list of EWS-FLI1 regulated transcripts I have generated a series of endogenous reporter alleles to monitor transcript levels in response to small molecules treatment. This system has been utilized to screen 380,000 compounds from the UTSW chemical library in high throughput. I have identified several independent scaffolds capable of modulating several EWS-FLI1 response genes in multiple Ewing sarcoma cell lines. Unexpectedly, our screen enriched for small molecules that resemble previously described CDK8/19 inhibitors. A role for CDK8/19 in EWS-FLI1 transcriptional activity has not been reported. To determine if the remaining small molecules capable of modulating EWS-FLI1 transcriptional activity were acting through CDK8/19 inhibition I utilized a previously described IFN-STAT1 phosphorylation assay used to test CDK8/19 activity. I identified a novel, potent small molecule capable of interrupting CDK8/19 activity. Through biochemical reconstitution of the full CDK8 module, I determined our novel CDK8/19 inhibitor is an ATP competitive active site inhibitor. Inhibition of CDK8/19 resulted in activation of genes that EWS-FLI1 represses, suggesting a role for CDK8/19 in EWS-FLI1 transcriptional repression. Chromatin profiling of CDK8 and EWS-FLI1 shows colocalization across the chromatin. In addition, treatment of Ewing sarcoma cells with either known CDK8/19 inhibitors or our novel CDK8/19 inhibitor causes growth inhibition

Investigation of Divergent Metabolic Programs in the Tumor-Immune Microenvironment

The current immune metabolism field has been using the term lactate and lactic acid interchangeably, with the notion that lactate and lactic acid are waste products of highly glycolytic cancer cells that negatively affects TIL function and viability to promote tumorigenesis. However, it is yet unclear whether lactate or its protonated counterpart lactic acid is responsible for the dampening of immune cell functions within the TIME. The in vitro cell culture studies aimed to delineate the impact of lactate and lactic acid in the context of cancer growth. Data showed that lactate alone significantly impairs cancer cell metabolism independent of the acidic pH environment. Additionally, therapeutic lactate treatment significantly increased the glycolytic activity and effector functions of CD8+ T-cells, contrary to the current understanding that lactate dampens immune cell functions. The comparison study between lactate and lactic acid further demonstrated that CD8+ T-cells undergo cell death that is dependent on pH levels, with approximately 50% of T-cell death occurring at pH level below 6.0, whereas cancer cells were able to metabolically adapt to the acidic environment and sustain growth. These findings provide a new perspective that challenges the current notion that lactate is considered a "pro-tumor" metabolite. In vivo metabolomics study of tumor cells and tumor-infiltrating CD8+ T-cells revealed that lactate initiates a divergent metabolic reprogramming in the two cell types. Systemic lactate treatment suppressed tumor glycolytic activities while boosting CD8+ T-cell glycolytic rate and effector function. Such divergent changes in two cell types provide a basis for developing potent antitumor responses in the complex heterogeneous tumor microenvironment. The impact of lactate on glycolytic flux modulation on both tumor cells and CD8+ T-cells, therefore, exposes a previously undefined difference in metabolic programs between the two cell types that can be exploited in the immune-suppressive tumor microenvironment

Development of the Multicultural Curriculum Appraisal (MCCA): A Process to Promote Multicultural Infusion into APA Accredited Doctoral Program Curriculum

The current American Psychological Association's standard for Diversity Education and Training in doctoral-level accredited Health Service Psychology programs is sparce and lacks a measurable expectation of programs. There is no published research on broad application or outcomes of this standard across Health Service Psychology curricula. To address any variation in the interpretation of the diversity education and training standard, we created an audit tool, the Multicultural Curriculum Appraisal (MCCA) Tool, to capture the amount of multicultural education and training present in today's psychology programs. One important step is to understand the how much multicultural content psychology instructors are incorporating into their course syllabi. Another essential step is to identify the areas, within a syllabus, that contain multicultural content. Lastly, it is critical to understand any potential barriers to multicultural infusion identified by psychology instructors. Our study surveyed doctoral level instructors (n=55) and audited their course syllabi (n=92). The primary aim was to explore the influence of instructor identity on the amount of multicultural content in a course. We expected instructors who identified with historically marginalized groups to have more multicultural infusion into their courses. This studies secondary aim examined barriers to multicultural infusion using three types of barriers: institutional, instructor and student. We anticipated participants who identified instructor barriers to have lower scores than participants who selected other barrier types. Contrary to expectations, participants with marginalized identities and those who selected instructor barriers did not have significantly different scores than their peers. Participants who were trained as counseling psychologists, taught at counseling psychology programs, were younger, ranked themselves lower on a national subjective socioeconomic scale, and in the early career stage demonstrated higher scores on the Multicultural Curriculum Appraisal. Additionally, participants who identified institutional barriers also demonstrated higher scores on the Multicultural Curriculum Appraisal. This study lays the groundwork for future research designed to better understand and correct for the variation between instructors, academic programs, and psychology disciplines and understand the potential influences of instructor identity and barriers in multicultural education and training in psychology curricula

RNA Exonuclease Xrn1 Regulates TORC1 and Autophagy in Response to SAM Availability

During methionine deprivation, yeast cells experience loss of S-adenosyl methionine (SAM), resulting in globally reduced histone methylation levels. Investigations into the location of the enduring histone methylation initiated our studies into Y' elements, subtelomeric noncoding RNAs that we have found are highly expressed under this condition. Their accumulation following loss of methionine is caused by reduced degradation by the conserved 5'-3' exonuclease Xrn1, rather than increased transcription during this condition. The finding that Xrn1 has altered activity under methionine deprivation led to investigate how Xrn1 may sense the change in nutrient availability. Autophagy is a conserved process of cellular self-digestion that promotes survival during nutrient stress. In yeast, methionine starvation is sufficient to induce autophagy. One pathway of autophagy induction is governed by the SEACIT complex, which regulates TORC1 activity in response to amino acids through the Rag GTPases Gtr1 and Gtr2. However, the precise mechanism by which SEACIT senses amino acids and regulates TORC1 signaling remains incompletely understood. We identified Xrn1 as a surprising and novel regulator of TORC1 activity in response to methionine starvation. This role of Xrn1 is dependent on its catalytic activity, but not on degradation of any specific class of mRNAs. Instead, Xrn1 modulates the nucleotide-binding state of the Gtr1/2 complex, which is key for its interaction with and activation of TORC1. This work identifies a critical role for Xrn1 in nutrient sensing and growth control that extends beyond its canonical housekeeping function in RNA degradation and indicates an avenue for RNA metabolism to function in amino acid signaling into TORC1

Improving Awareness of Skin Cancer in Organ Transplant Recipients of Color

Organ transplant recipients are at high risk for skin cancer. Currently, more than half of the transplant waiting list is composed of skin of color patients. Skin cancer in skin of color is associated with higher morbidity and mortality and has a different clinical presentation and risk factors. Yet, skin cancer prevention resources and efforts are primarily focused on non-skin of color patients. A cross-sectional pilot survey was administered to assess and compare skin cancer attitudes, behaviors, and knowledge especially risk factors and features specific to skin of color between skin of color and non-skin of color organ transplant recipients. Patients from a patient list obtained from the University of Texas Southwestern Medical Center organ transplant center were randomized on Excel and contacted by phone with the choice to participate by phone or online. 219 of 403 patients completed the survey. Skin of color organ transplant recipients was significantly more likely to never practice recommended skin cancer preventative behaviors (p = 0.002, 0.006, 0.02), to hold a lower perceived self-risk (p = 0.02), to worry less about getting skin cancer (p = 0.003), and to have false perceptions about risk factors (p = 0.001, 0.005) in either univariable or multivariable analysis. However, they were more likely (38%, p = 0.02) to recognize human papillomavirus as a risk factor. The knowledge gaps identified can guide the development of skin cancer educational resources that are more comprehensive and relevant to skin of color recipients. This can lead to improved skin cancer awareness and better outcomes and reduce racial health disparities

Role of Krox20 Transcription Factor and Krox20+ Stem Cells in Epithelial Tissue Development and Homeostasis

Resident stem cells (SCs) within tissues are important for normal homeostasis maintenance and wound repair. This is mediated by the ability of SCs to properly self-renew, maintain their identity, and differentiate. Epidermal SCs occupy exclusive niches, priming them for giving rise to specific portions of the of the interfollicular epidermis (IFE) and hair follicle (HF). During homeostasis, the bulge SCs generate the lower portion of the HF, while isthmus, junctional zone, and infundibular SCs give rise to the upper HF and IFE. Beyond their unique cell fate, these cells are also capable of generating other portions of the epidermis, but only during pathological conditions (e.g., wounding). In this study, we report a newly identified infundibular SCs, marked by transcription factor Krox20. While Krox20-expessing cells are limited to the infundibular niche, lineage tracing analysis shows that these cells differentiate to generate both the entire HF and the IFE during homeostasis. Notably, previously identified epidermal SCs are derived and continually replenished from Krox20 lineage cells. The ablation of K14-derived Krox20-expressing cells results in deformed HF structure and the lack of a hair shaft, as well as abnormal differentiation and stratification of the IFE. Taken together, these results highlight Krox20-expressing cells as epidermal SCs, crucial for HF and IFE homeostasis and maintenance. Notably, this study represents the first description of a SC population which give rise to both the HF and the IFE during homeostasis, representing a paradigm shift in the current understanding of epidermal SCs differentiation. To evaluate the role of the Krox20 protein in these cells, we ablated Krox20 expression in K14-derived epithelial cells, and found that this resulted in spontaneous hair loss, correlated with aberrant HF structure and epidermal differentiation. Interestingly, these mice also serendipitously developed squamous metaplasia of the ocular surface, resulting from the loss of Meibomian glands (MGs). Expression analyses showed that Krox20-expressing cells constitute stem/progenitor cells that are restricted to the ductal region of the MG, but differentiate to give rise to the entire MG structure. Overall, this study identifies Krox20 as a marker of resident SCs that are essential to continually replenish epithelial-derived tissues (e.g., the epidermis and the MG), with Krox20 expression playing an important role in the function of these cells

Physician Burden and Time Delays in Initiating Immunomodulatory Therapy for Non-Infectious Uveitis and Inflammatory Eye Diseases

The 63rd Annual Medical Student Research Forum at UT Southwestern Medical Center (Tuesday, January 28, 2025; 3-6 p.m.; D1.700 Lecture Hall)PURPOSE: To assess the additional time and effort requirements and resultant time delays necessitated by insurance-mandated prior authorization for medications used in the treatment of uveitis and ocular inflammatory disease. DESIGN: Retrospective observational study PARTICIPANTS: Patients with uveitis and ocular inflammatory disease at the University of Texas Southwestern Medical Center in Dallas, TX for whom the uveitis specialist attempted approval for initiation of a new systemic immunomodulatory therapy between 02/01/2023 - 01/31/2024. METHODS: Electronic medical records were reviewed to determine prior authorization requests, appeals, and relevant data related to the process required to initiate new immunomodulatory therapy. Infusion related medications were excluded for analysis. MAIN OUTCOME MEASURES: Primary outcome measures were the success rate of obtaining the requested medication and time from medication request to initiation. Secondary measures included the number of additional steps required (e.g. appeals, peer-to-peer consultations), factors influencing medication approval (e.g. insurance subtype, medication class), and rates of reliance on patient assistance programs. RESULTS: A total of 83 patients underwent 107 attempts for initiation of a new immunomodulatory therapy. Underlying uveitis disease entities were pemphigoid (30, 38%), anterior uveitis (10, 13%), intermediate uveitis (6, 8%), posterior uveitis (1, 1%), panuveitis (28, 35%), and scleritis (4, 5%). Medications attempted included methotrexate (22, 20.6%), mycophenolate mofetil (13, 12.1%), mycophenolic acid (4, 3.7%), adalimumab (37, 34.6%), baricitinib (18, 16.8%), tofacitinib (10, 9.3%), and tocilizumab (2, 1.9%). The majority (71%) of medications were considered "off-label". Approval rates were 100% for on-label and 52.31% for off-label medications (p=0.033). Patients requesting off-label use medications were twice as likely to be denied and require an appeals process (1.07 vs 0.50; p=0.01). Average time to initiation was 28 days for on-label and 41 days for off-label medications (p=0.01). For off-label medications, private insurance approval rates were significantly higher than Medicare (59.52% vs 36.36%; p=0.05). Patient assistance programs were utilized to cover some or all medication costs in 63% of cases. CONCLUSION: Prior authorization requirements present an under-recognized and considerable administrative burden on healthcare providers in obtaining vision-saving therapy for their patients. Denials result in substantial delays in initiating crucial vision-preserving treatment.Southwestern Medical Foundatio

Intracellular Lipid Surveillance Through Interplay Between a Nuclear Receptor and Rab GTPases

The general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.Cells continuously experience fluctuations in resource availability and therefore require adaptive mechanisms to recognize these changes and respond accordingly to ensure metabolic health. Signaling mechanisms like the AMPK and mTOR pathways enable cells to monitor energy and amino acid availability, respectively, and cholesterol sensing by SREBP provides a means for cells to maintain homeostatic lipid saturation. Lipids comprise a vast class of energy-rich molecules that, altogether, are essential for most every cellular process. Yet independent of sterol sensing, it remains unclear how cells sense and respond to global lipid depletion. While starving cells initiate fatty acid β-oxidation via mammalian nuclear receptors, PPAR and HNF4, it is unknown how cells couple this catabolic process with mechanisms to increase nutrient intake and prevent further starvation. Comprised predominantly of lipid droplets, the C. elegans intestine is an excellent model for elucidating the relationship between lipid depletion and nutrient absorption in the context of lipid sensing. The work presented herein defines a novel signaling mechanism through which the transcriptional regulator of β-oxidation in C. elegans, Nuclear Hormone Receptor 49 (NHR-49), senses intracellular lipid availability and restores lipid homeostasis following starvation-induced lipid depletion by monitoring abundance of a single de novo-synthesized lipid, geranylgeranyl pyrophosphate. In this pathway, lipid availability inactivates NHR-49 through its sequestration to cytosolic transport vesicles by the geranylgeranyl-conjugated RAB-11.1 GTPase. Lipid depletion prevents NHR-49 vesicular association, thereby promoting its nuclear translocation and activation of genes involved in nutrient absorption and catabolism. Importantly, activation of endocytic recycling regulator, rab-11.2, enhances nutrient transporter residency on the cell surface. Upon investigation of how this intracellular lipid surveillance pathway relates to other homeostatic mechanisms, the findings described in the succeeding chapter establish a cooperative relationship between NHR-49 and the regulator of cytosolic protein folding, Heat Shock Factor (HSF-1), in mediating metabolic health and age progression. Through stabilizing the intestinal actin network, HSF-1 ensures proper vesicle trafficking and acts as an upstream regulator of NHR-49 in promoting lipid homeostasis. Overall, this work expands our understanding of lipid sensing and details a novel mechanism by which cells increase nutrient intake in times of metabolic demand