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Kap-[beta]2 Recognition of Cargoes Involved in Neurodegenerative/Neurodevelopmental Diseases
No full textKaryopherin β2 is a nuclear-import receptor that transports many RNA-binding proteins into the nucleus by recognizing within the cargoes a unique nuclear-localization signal known as the PY-NLS. Several of Kapβ2 cargoes are mutated in neurological diseases. Two such proteins of interest to my research are the Fused in Sarcoma (FUS) and the HNRNPH2 proteins that are mutated in familial ALS and Bain disease, respectively.
I studied the interactions of Kapβ2 with HNRNPH2, which is mutated in the neurodevelopmental disease known as Bain disease. Mutations in HNRNPH2 were first found in 2016, and, since then, 14 mutations have been linked to this disorder, with several mutations found within its PY-NLS. We have observed that these mutations cause mislocalization of the cargo to stress granules in the cytoplasm. I performed biophysical and structural studies and found that the HNRNPH2 has a new type of epitope not observed before. Furthermore, I observed that this new epitope is essential for HNRNPH2 recognition. These findings are consistent with several mutations found within this epitope that are linked to Bain disease.
I also studied two FUS mutations, FUS(R495X) and FUS(P525L), that cause a severe type of ALS. First, I found that, in the absence of the PY-NLS like in FUS(R495X) mutation, Kap2 recognizes the arginine-glycine-glycine (RGG) regions of FUS as a secondary mechanism for nuclear transport. Lastly, together with the Shorter Lab, I characterized an engineered mutant Kap2 that can recognize FUS(P525L), transport it into the nucleus and decrease FUS aggregation more efficiently. I performed biophysical and structural studies in some of these engineered Kap2 to explain how these mutants improve interactions with the ALS-causing mutant FUS(P525L). We found that engineered Kap2 makes more contacts with FUS(P525L), rescuing binding activity
"Personalized medicine" wasn't personal; "precision medicine" isn't precise
Get PDFTuesday, April 8, 2025; noon to 1 p.m. (Central Time); Room NB2.100A or via Zoom. "'Personalized Medicine' Wasn't Personal; 'Precision Medicine' Isn't Precise". James Tabery, Ph.D., Professor of Philosophy at the University of Utah.Ever since the end of the Human Genome Project in 2003, geneticists have been forecasting a genomic revolution in healthcare. Initially, this revolution was packaged as "personalized medicine" -- the idea being that getting DNA from patients will allow for individualizing treatments. Eventually, though, geneticists became concerned that the language of "personalized medicine" misled the public about how medical genetics actually works, and so an effort was made to rebrand the revolution as "precision medicine". I'll tell the story of how this scientific marketing fiasco unfolded, explaining how it managed to only compound the confusion surrounding what genetics does and doesn't bring to medicine.UT Southwestern--Program in Ethic
HD-tDCS over Frontal Cortex and Neuropsychological Functioning in Cognitively Impaired Older Adults
Get PDFAlzheimer's Clinical Syndrome (ACS) is projected to triple in incidence over the next 30 years and is characterized by a gradual loss of cognitive and functional abilities. Mild cognitive impairment (MCI) is an earlier stage of cognitive impairment that often represents a prodromal stage of ACS. Individuals with MCI may have trouble completing complex instrumental activities of daily living such as managing medication regimens or multiple medical appointments, though they can maintain a level of independence with use of compensatory strategies. MCI and ACS commonly have an amnestic presentation (inability to learn and recall new information), though additional problems in executive functions, language, and/or visuospatial skills are typically present at the time of clinical diagnosis. As ACS progresses, the ability to function independently becomes compromised, and at this stage, assistance with complex activities of daily living (e.g., driving, financial management) is often necessary. Neuropsychiatric symptoms (NPS), such as depression, anxiety, and apathy, are common in MCI and ACS and are also associated with a faster rate of decline. Current treatments used to manage cognitive symptoms include acetylcholinesterase inhibitors (AChEIs) and NMDA-receptor blockers, while selective serotonin reuptake inhibitors (SSRI) and serotonin-norepinephrine reuptake inhibitors (SNRI) are common first-line medications for NPS, though all demonstrate short-lived efficacy in symptom control. Lifestyle changes such as dietary interventions, physical exercise, and therapeutic approaches (e.g., reality orientation training, cognitive training, rehabilitation) aim to reduce symptoms and some may stave off decline, though all are unproven to be a restorative treatment for ACS. As such, there exists an urgent need for investigation of novel treatment approaches.
Given the unmet treatment needs for MCI and ACS, it is important to evaluate new potential treatments that might provide symptom improvement. One line of possible treatment emerging in the literature is transcranial direct current stimulation (tDCS), which is a non-invasive brain stimulation technique aimed at altering neuronal activity in the brain. The frontal cortex serves as a brain circuitry hub connected to a variety of cortical and subcortical structures. Within the frontal cortex lies the dorsal anterior cingulate cortex (dACC) and pre-supplementary motor area (preSMA), which are differentially important areas for cognitive and emotional functioning. Some preliminary evidence also suggests that both areas are amenable to treatment in some patients with neurologic conditions.
This investigation comprised two parts. Part One focused on understanding the current state of knowledge and findings of neuropsychological effects of tDCS over the frontal cortex in individuals with MCI and ACS through a comprehensive narrative review. This review revealed limited evidence to date (9 randomized controlled trials meeting criteria) for the utility of tDCS in treating cognitive deficits and NPS, likely due to a lack of thorough neuropsychological investigation and heterogeneity of study design and methods. However, the technology showed some early promise, as clinically meaningful change on global cognitive screeners was detected in a single trial with an MCI sample. Part Two of the investigation sought to add new knowledge and fill critical gaps currently in the literature by leveraging two mirrored randomized controlled clinical trials recently conducted at UT Southwestern Medical Center. The trials evaluated neuropsychological effects of High Definition tDCS over the dACC/preSMA in individuals with MCI and ACS before and after 10 treatments. Secondary analysis of the combined samples revealed no statistically significant treatment effects on a depressive symptom measure or on composite scores of episodic memory, executive function, language, and working memory. Individuals with MCI and ACS were also evaluated for clinically meaningful changes, and while this revealed several treatment responders across outcome measures in the MCI and ACS active treatment groups, no significant differences were detected between the proportion of responders to non-responders. The individual clinical improvements detected may suggest that certain characteristics (e.g., disease severity) in MCI and ACS may be associated with differential effects from this technology that are worth future investigation. Future studies would likely benefit from investigating different dosage parameters in these populations and use of innovative methods for targeted stimulation over cortical regions important for cognitive and emotional functioning (e.g., dACC/preSMA)
Delayed Graft Function after Kidney Transplant: Complication or Quality Metric?
Get PDFThe 63rd Annual Medical Student Research Forum at UT Southwestern Medical Center (Tuesday, January 28, 2025; 3-6 p.m.; D1.700 Lecture Hall)Each year the Medical Student Research Program awards students for the best oral presentation and the best poster presentation as judged by faculty across campus. This author received an award as one of the best poster presentations at this forum.INTRODUCTION: With the general shortage of deceased donor kidneys in the US, providers and centers must consider all kidney offers, including those traditionally considered to be of higher risk (e.g. kidneys from older donors or with longer cold ischemic times). This study aims to clarify the role of delayed graft function (DGF), defined as the need for dialysis within a week after renal transplantation, as a center-level metric for appropriate deceased donor kidney acceptance practices.
HYPOTHESIS: We hypothesize that DGF rates can be used as a method to assess a center's aggressiveness in acceptance practices (i.e. accepting higher risk kidneys) to maximize transplantation rates. Secondarily, DGF rates will not significantly impact center-level patient and renal graft survival outcomes.
METHODS: We used program specific reports from the Scientific Registry of Transplant Recipients (SRTR) to obtain center-level waitlist and transplant outcomes, updated through 12/31/2023. All active U.S. adult kidney transplant centers were included (N=196) and centers were stratified into quartiles based on DGF rates (Q1 with lowest rates and Q4 with highest). Offer acceptance practices, patient and graft survival rates, and waitlist (WL) outcomes were compared.
RESULTS: There was a large spread of DGF rates, with Q1 <20% and Q4>41.78%. Higher DGF centers (Q2, Q3, and Q4) had higher median yearly transplant volumes than those in Q1 (p=0.005). Overall offer-to-acceptance ratios (OAR) did not differ across quartiles. However, Q4 and Q3 had significantly higher OARs for both high risk kidneys (Q4- 1.27 vs. Q3- 1.06 vs. Q2- 1.04 vs. Q1- 0.78; p=0.028) as well as difficult-to-place kidneys with over 100 offers (Q4: 1.09 vs. Q3: 0.98 vs. Q2: 0.83 vs. Q1: 0.52; p = 0.019). Despite these practices, there was no difference in percentage of center waitlist transplanted across groups (p=0.43). Importantly, 30-day and 1-year graft and patient survival hazard ratios did not differ across quartiles. At 3 years, higher DGF centers had a lower and more favorable 3-year patient survival hazard ratio (Q1: 1.15 vs. Q2: 1.10 vs. Q3: 0.93 vs. Q4: 0.95; p = 0.001).
CONCLUSIONS: Higher DGF centers were found to accept more high-risk kidneys with similar long-term outcomes, indicating that DGF is not disadvantageous when managed well. However, this metric was not concordant with waitlist removal for transplant or median time to transplant. Center DGF rates may act as an adjunctive metric to assess acceptance practices, but should not be used in isolation as a determination of center aggressiveness.Southwestern Medical Foundatio
The Role of Pelvic Tilt in Femoroacetabular Impingement Syndrome, Hip Dysplasia, and Osteoarthritis
No full textThe 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.BACKGROUND: In femoroacetabular impingement syndrome (FAIS), pelvic tilt is believed to impact disease progression and symptomology by influencing impingement free range of motion, with similar associations observed in hip dysplasia through modification of acetabular coverage and osteoarthritis via alterations in joint movements. While the apparent role of pelvic tilt in hip pathology has been reported, the exact effects of many therapeutic interventions on pelvic tilt are unknown.
OBJECTIVE: The primary purpose of these studies are two-fold: first, to investigate the relationship between preoperative pelvic tilt and change in postoperative outcomes in patients undergoing surgery for either FAIS or hip dysplasia. Second, to investigate the role of surgical intervention on pelvic tilt in FAIS, dysplasia, and osteoarthritis patients.
METHODS: A prospective hip preservation registry of demographic, radiographic, and outcomes data for all patients operated on by the senior author between October 2016 and January 2020 were queried, and all patients who underwent surgery with a primary diagnosis of FAIS, hip dysplasia, or osteoarthritis were considered for inclusion. Pelvic tilt was assessed in the standing position on the AP radiograph with the PS-SI distance both pre and postoperatively, and the outcomes were assessed with the HOS, iHOT-12, UCLA Activity Score, EQ-VAS, and HHS.
RESULTS: Regarding the first study question, the average PS-SI distance was 86.4±18.3 mm for the FAIS group and 96.2±15.1 mm for the dysplasia group. Analysis demonstrated a positive relationship between pelvic tilt and change in iHOT12 (rs = 0.262, p=0.019) for all 89 patients with hip pathology. No other significant relationships were observed. Regarding the second research question, the FAIS + dysplasia + OA cohort had an average preoperative PS-SI distance of 73.9±22.9 mm and an average postoperative PS-SI distance of 64.9±25.5 mm on the standing AP radiograph. The results from the linear regression revealed a significant negative predictive association between standing preoperative PS-SI distance and standing postoperative PS-SI distance for all 3 cohorts of patients (p<0.0001 for all groups).
CONCLUSION: The improvement in iHOT12 was greater for patients with more anterior tilt and less for patients with posterior pelvic tilt regardless of underlying hip etiology. There is a statistically significant decrease in pelvic tilt from pre to postoperatively in FAIS, dysplasia, and OA patients undergoing hip surgery. These results confirm that surgical intervention alters the pelvic orientation and indicate that impact of hip surgery on pelvic tilt should be considered within the therapeutic plan in order to optimize pelvic orientation
HF, 2025: a spectacular journey in science, discovery, and implementation
Get PDFDetailed formal protocol with illustrations and extensive bibliography.A recording of the protocol presentation is available on UT Southwestern’s Mediasite. Note: Access to the video is restricted to authorized UT Southwestern users only.UT Southwestern--Internal Medicin
Shield and Shuttle: Importins as Protective Chaperones and the Nuclear Transport Mechanism of H2A-H2B
Get PDFThe active transport of macromolecules from the cytoplasm to the nucleus is carried out by a sub-class of Karyopherins called Importins. It was hypothesized back in the early 2000s that importins could have dual functions as 1) nuclear importers and 2) molecular chaperones. This thought stems back to characteristics of importins: they are negatively charged and shuttle positively charged DNA and RNA binding proteins prone to aggregation such as histones, ribosomal proteins, and transcription factors.
Nuclear import begins in the cytoplasm where Importins typically recognize a linear pattern of sequences in disordered regions of their cargoes called Nuclear Localization Signals. However, there are growing cases where Importins recognize the cargo's protein fold. Once in the nucleus, cargo release from the Importin is facilitated by a small GTPase, Ran, bound to GTP. Interestingly, there have been many biochemical experiments showing that RanGTP binding is not enough for cargo release of histones H2A-H2B and transcription factors TBP and TFIIB for yeast Importin Kap114. It has been shown that these cargos need both RanGTP and DNA to be fully released from the importin, suggesting these cargos are chaperoned for targeted delivery.
Here, I will further discuss how I played a part in unraveling the structural mechanism of the evolutionary conserved H2A-H2B core recognition, chaperoning, and release by Kap114 and its human homolog Imp9. I will also expand upon another importin, Kapβ2, and its mechanism in preventing RNA binding protein FUS, a NLS containing cargo, from phase separating and aggregating, providing strong evidence that importins can function as both protective chaperones and nuclear importers
Lamellipodia as Proliferation Machines: How Branched Actin Based Feedback Loops Allow Melanoma to Evade Growth Inhibition
No full textThe 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.Spatial regulation of Rac1 activity is well-characterized and important for several cellular functions, including building and sustaining protrusive branched actin networks at the cell edge. High Rac1 activity sits atop a dense cytoskeletal scaffold that drives the extension of a flat lamellipodium, whose tightly packed volume forms a microdomain of enhanced signaling activity with elevated transduction efficiency. Cancer cells can coopt this mechanism to drive proliferation and survival signaling. For example, the hyperactive Rac1P29S mutation found in 10% of melanoma patients is associated with advanced disease, increased resistance to MAPK pathway inhibitors, and decreased patient survival. Previous work demonstrated that these clinical phenotypes correlate at a cellular level with sustained proliferation under drug challenge and is linked to a cell's ability to build extended lamellipodia. Using FRET- and localization-based biosensors for Rac1 activity, we observe larger and more potent microdomains in melanoma cells expressing Rac1P29S. The resultant branched actin network provides abundant binding sites for the tumor suppressor NF2/merlin. Superimposition of heightened Rac1 activity poises the Rac1 effector kinase PAK to inactivate merlin through phosphorylation. Displacement of merlin from the lamellipodia by a competitive actin-binding peptide interrupts this phospho-inactivation, re- sensitizing melanoma cells to MAPK inhibition. However, merlin plays an additional role upstream of the Rac1 signaling domain. Merlin recruitment to the cell edge limits protrusion formation. Knockout of merlin recapitulates the Rac1P29S-induced phenotype, producing enhanced lamellipodia with elevated wildtype Rac1 activity upon MAPK-inhibition. Breaking merlin's inhibition of the branched actin network similarly allows for enhanced lamellipodia formation, and ultimately merlin phospho-inactivation. Together these data support a double-negative feedback--Rac1 microdomains inactivate merlin localized to the dense branched-actin networks; active merlin, in turn, attenuates microdomain formation through inhibition of branched actin polymerization. This localized signaling feedback is thus prone, when perturbed, to produce self-sustaining patterns of signaling activity, allowing for MAPK-inhibited melanoma to escape merlin-mediated growth control
Identifying CDK8/19 as a Novel Regulator of EWS-FLI1 Function in Ewing Sarcoma
Get PDFEwing 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