Biology subjects

Courtney, K. C.

Publications and source records attributed to Courtney, K. C..

7 recordsLinked to original sources

bioRxiv2026

Strategies for Overcoming the Purification Challenges of Meis1 and HoxA9 and Their Functional Characterization

Transcription factors are critical regulators of homeostasis, and disruption of their function is a common underlying mechanism in cancer. The homeobox Myeloid ecotropic integration site-1 (Meis1) and its binding partner Homeobox A9 (HoxA9) are among the most highly dysregulated transcription factors in cancer, particularly in Acute Myeloid Leukemia (AML). Upregulation of HoxA9 is observed in more than 50% of AML cases and is a predictor of poor prognosis. Meis1 forms a cooperative DNA complex with HoxA9 and is required for HoxA9-mediated leukemogenesis. However, the mechanism by which HoxA9 and Meis1 interact and form a complex with DNA is unknown. To study the molecular mechanisms behind the HoxA9-Meis1-DNA complex and support future development of inhibitors, we describe optimized procedures for the recombinant expression and purification of full-length Meis1 and HoxA9. The constructs with an N-terminal His-tag showed degradation, but the addition of a small ubiquitin-like modifier (SUMO)-fusion tag improved solubility and stability for both proteins. Conditions were optimized for SUMO cleavage to obtain tag-free Meis1 and HoxA9 with high purity. We showed that both purified proteins bound to DNA with nanomolar affinities using a fluorescence polarization (FP) binding assay. The SUMO-fusion HoxA9 showed a distinct binding profile compared to the tag-free HoxA9 and formed a cooperative complex with Meis1. The tag-free HoxA9 appeared to oligomerize under the assay conditions. To the best of our knowledge, this is the first report to visualize the HoxA9-Meis1 cooperative interaction in a high-throughput assay. Our work establishes a foundation for future studies to decipher the protein-protein interaction of Meis1-HoxA9 and the development of inhibitors.

biochemistry↗

bioRxiv2026

Development of a Staining-based Electrophoretic Mobility Shift Assay for Analyzing Pbx1, DNA and HoxA9 Interactions

Transcription factors (TFs) are master regulators of gene expression and control a wide range of cellular functions including embryonic development, signaling pathways, immune response, and differentiation. A tight regulation of gene expression is crucial during all stages of life and changes in the TF function can lead to developmental abnormalities, diseases such as cancer, or resistance to treatment. Therefore, TFs are a promising class of drug targets, and the techniques that would contribute to the development of TF modulators are critical. Electrophoretic Mobility Shift Assay (EMSA) has been a primary tool to verify protein-DNA interactions, where a fluorescent, biotin, or isotope end-labelled DNA probe is used to quantify binding. Such labeling techniques, however, can be costly, time consuming, possess safety hazard risks and require capital equipment for imaging. Here, we optimized a label-free, staining-based EMSA to characterize a potential drug target, homeodomain (HD) TF Pre-B-cell leukemia homeobox-1 (Pbx1) and its binding partner Homeobox A9 (HoxA9). Staining the polyacrylamide EMSA gel with a DNA intercalating green cyanine dye - SYBR safe - allowed the visualization of Pbx1 homeodomain interactions with DNA at nanomolar concentrations and enabled quantitative determination of protein-DNA apparent binding affinity in the sub-micromolar range. Furthermore, a ternary complex of homeodomains of Pbx1 with HoxA9 and the DNA was also visible in the assay. We have shown that the staining-based EMSA can be used to evaluate inhibitors of Pbx1 that block the interaction with DNA. We have further validated the data from our assay with fluorophore labeling-based EMSA. Overall, using HD transcription factors Pbx1 and HoxA9 as a model, we have optimized a reliable and cost-effective staining-based EMSA that enables the high-sensitivity visualization and quantitative evaluation of transcription factor-DNA complexes without the need for end-labeled DNA probes. The streamlined workflow could be readily adapted to other DNA-binding proteins to study their interactions with the DNA, inhibitors, and other proteins.

biochemistry↗

bioRxiv2026

Structural Characterization of Calcium-Dependent Calmodulin-Calmidazolium Binding using Capillary Vibrating Sharp-Edge Spray-based Native Mass Spectrometry and In-Droplet Hydrogen Deuterium Exchange Mass Spectrometry

Capillary vibrating sharp-edge spray ionization (cVSSI)-based native mass spectrometry (nMS) and in-droplet hydrogen deuterium exchange (HDX-MS) were used to evaluate calcium-dependent interactions between calmodulin and calmidazolium (CDZ). cVSSI was found to produce a narrow charge-state distribution (CSD) with low average charge states, indicating that this method preserved native-like states. cVSSI was also able to resolve stepwise Ca2+ binding for species containing one to four Ca2+ ions. In the absence of Ca2+, no detectable CDZ binding was observed. However, CDZ binding was observed when calmodulin was fully loaded with Ca2+. CDZ binding to the protein caused marked redistribution of the CSD toward lower charge states, consistent with ligand-induced stabilization of the protein into a more compact conformational ensemble. The apparent dissociation constant (Kd) of the interaction was determined to be 261 {+/-} 29 nM and 126 {+/-} 17 nM from Langmuir and quadratic binding models, respectively. Complementary in-droplet HDX-MS revealed that deuterium uptake was correlated with charge state across the CSD, corroborating the nMS evidence for stepwise conformational compaction of CaM. Within the individual charge states, two distinct populations representing CDZ-bound and unbound CaM were resolved by HDX-MS, which were not detectable by nMS alone. Comparison of these populations within each of the 6+, 5+ and 4+ charge states revealed reductions in deuterium uptake by approximately 37%, 22%, and 11% upon CDZ binding, respectively, consistent with ligand occlusion and local structural tightening at the binding interface. The gradient in protection across charge states further reflects differences in binding interface accessibility among CaM conformers spanning a range of CDZ-induced conformational states. Together, these results demonstrate that cVSSI-based nMS coupled with in-droplet HDX-MS provides an integrated platform for simultaneously resolving metal loading, ligand binding, binding affinity, and ligand-induced conformational changes. This approach complements traditional structural methods by enabling direct interrogation of dynamic, metal-dependent protein-ligand interactions in their native states.

biochemistry↗

bioRxiv2022

Synaptotagmin 7 outperforms synaptotagmin 1 to open and stabilize nascent fusion pores via robust membrane penetration

Synaptotagmin-1 and synaptotagmin-7 are two prominent Ca2+ sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding Ca2+, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examined the biophysical properties of these two synaptotagmin isoforms and compared their interactions with phospholipid membranes. We discovered that synaptotagmin-1*membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a "super-penetrator". We exploited these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to Ca2+.

neuroscience↗

bioRxiv2021

Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release

Synaptotagmin-1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer, however biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures ([~]180 nm) were reduced to smaller rings ([~]30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in: a) clamping spontaneous release, and b) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.

neuroscience↗

bioRxiv2021

Synaptotagmin 7 is enriched at the plasma membrane to promote vesicle docking and control synaptic plasticity

Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. Zap-and-freeze electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via {gamma}-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity. O_LISYT7 mediated asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment was observed optically at individual hippocampal synapses C_LIO_LILocalization, trafficking, and stability of SYT7 is dependent on processing by {gamma}-secretase C_LIO_LIShort term plasticity defects arise in SYT7KOs due to decreased docking of synaptic vesicles after stimulation C_LIO_LISYT7 promotes paired-pulse facilitation and asynchronous release via distinct mechanisms C_LI

neuroscience↗

bioRxiv2017

C24 sphingolipids play a surprising and central role in governing cholesterol and lateral organization of the live cell plasma membrane

Mammalian cell sphingolipids, primarily with C24 and C16 acyl chains, reside in the outer leaflet of the plasma membrane. Curiously, little is known how C24 sphingolipids impact cholesterol and membrane microdomains. Here, we generated giant unilamellar vesicles and live mammalian cells with C24 or C16 sphingomyelin exclusively in the outer leaflet and compared microdomain formation. In giant unilamellar vesicles, we observed that asymmetrically placed C24 sphingomyelin suppresses microdomains. Conversely, C16 sphingomyelin facilitates microdomains. Replacing endogenous sphingolipids with C24 or C16 sphingomyelin in live HeLa cells has a similar impact on microdomains, characterized by FRET between GPI-anchored proteins: C24, but not C16, sphingomyelin suppresses submicron domains in the plasma membrane. Molecular dynamics simulations indicated that, when in the outer leaflet, the acyl chain of C24 sphingomyelin interdigitates into the opposing leaflet, thereby favouring cholesterol in the inner leaflet. We indeed found that cholesterol prefers the inner over the outer leaflet of asymmetric unilamellar vesicles (80/20) when C24 sphingomyelin is in the outer leaflet. However, when C16 sphingomyelin is in the outer leaflet, cholesterol is evenly partitioned between leaflets (50/50). Interestingly, when a mixture of C24/C16 sphingomyelin is in the outer leaflet of unilamellar vesicles, cholesterol still prefers the inner leaflet (80/20). Indeed, in human erythrocyte plasma membrane, where a mixture of C24 and C16 sphingolipids are naturally in the outer leaflet, cholesterol prefers the cytoplasmic leaflet (80/20). Therefore, C24 sphingomyelin uniquely interacts with cholesterol and governs the lateral organization in asymmetric membranes, including the plasma membrane, potentially by generating cholesterol asymmetry.\n\nStatement of SignificanceThe plasma membrane bilayer of mammalian cells has distinct phospholipids between the outer and inner leaflet, with sphingolipids exclusively in the outer leaflet. A large portion of mammalian sphingolipids have very long acyl chains (C24). Little is known how C24 sphingolipids function in the outer leaflet. Mutations in the ceramide synthase 2 gene is found to decrease C24. This severely perturbs homeostasis in mice and humans. Here, we investigated unilamellar vesicles and mammalian cells with C24 sphingomyelin exclusively in the outer leaflet. We provide evidence that outer leaflet C24 sphingomyelin suppresses microdomains in model membranes and live cells by partitioning cholesterol into the inner leaflet. We propose that C24 sphingolipids are critical to the function of the plasma membrane.

cell biology↗