Biology subjects

Richardson, W.

Publications and source records attributed to Richardson, W..

7 recordsLinked to original sources

bioRxiv2024

A BTB extension and ion-binding domain contribute to the pentameric structure and TFAP2A binding of KCTD1

KCTD family proteins typically assemble into Cullin-RING E3 ligases. KCTD1 is an atypical member that functions instead as a transcriptional repressor. Mutations in KCTD1 cause developmental abnormalities and kidney fibrosis in scalp-ear-nipple syndrome. Here, we present unexpected mechanistic insights from the structure of full-length KCTD1. Disease-causing mutation P20S maps to an unrecognized extension of the BTB domain that contributes both to its pentameric structure and TFAP2A binding. The C-terminal domain (CTD) shares its fold and pentameric assembly with the protein GFRP despite lacking discernible sequence similarity. Most surprisingly, the KCTD1 CTD establishes a central channel occupied by alternating sodium and iodide ions that restrict TFAP2A dissociation. The elucidation of the structure redefines the KCTD family BTB domain fold and identifies an unexpected ion pore for future study of KCTD1s function in the ectoderm, neural crest and kidney.

molecular biology↗

bioRxiv2023

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD have indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3{beta}, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3{beta} activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3{beta} activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3{beta}. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces post-synaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated, key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity and human neuropathology.

neuroscience↗

bioRxiv2023

Inclusive pedagogy strategies to introduce high schoolers to systems biology

Women and non-white racial and ethnic groups remain underrepresented in science, technology, engineering, and math (STEM). To achieve a more diverse and equitable STEM workforce, the recruitment and retention of these historically marginalized communities in postsecondary education will also need to increase. Recently, the lens has turned to pedagogy and how creating a more inclusive classroom environment can foster STEM identity in previously marginalized communities. This work focuses on developing and piloting systems biology education modules designed to promote an inclusive learning environment in a summer outreach program for high school students by (1) utilizing a hybrid of unplugged activities with coded simulation and (2) a female-oriented problem statement. Based on initial findings in our pilot program, we anticipate that these techniques will enable students with limited prior computational experience to feel more comfortable and able to complete tasks, thereby increasing their self-efficacy and STEM identity. These modules could be a valuable tool for practitioners teaching high school or early college-level computational courses who instruct students with varied coding experience. Additionally, our analysis of the use of a female-oriented problem statement on female-identifying students self-efficacy provides potential evidence of the usefulness of representative problem statements in engaging underrepresented student populations. We believe these techniques could be adapted to address a variety of contexts across STEM disciplines and other fields to foster inclusive learning environments.

scientific communication and education↗

bioRxiv2023

Vascular Smooth Muscle Cell Mechanotransduction Through Serum and Glucocorticoid Inducible Kinase-1 Promotes Interleukin-6 Production and Macrophage Accumulation in Murine Hypertension

ObjectiveThe objective of this investigation was to demonstrate that in vivo induction of hypertension (HTN) and in vitro cyclic stretch of aortic VSMCs can cause SGK-1-dependent production of cytokines to promote macrophage accumulation as agents of vascular remodeling. MethodsHTN was induced in C57Bl/6 mice with AngiotensinII (AngII) infusion (1.46mg/kg/day x 21 days) with or without systemic infusion of EMD638683 (2.5mg/kg/day x 21 days), a selective SGK-1 inhibitor. Systolic blood pressure (SBP) was recorded on days 0 and 21. At terminal study, abdominal aortas were harvested to quantify SGK-1 activity (pSGK-1:SGK-1) by immunoblot. Additional replicates were digested and analyzed by flow cytometry for abundance of CD11b+/F4-80+ cells (macrophages). Plasma was analyzed by ELISA to quantify IL-6 and MCP-1. Aortic VSMCs from wild-type (WT) mice were subjected to 12% biaxial cyclic stretch for 3 or 12 hours +/- EMD638683 (10M) and +/- SGK-1siRNA with subsequent QPCR for IL-6 and MCP-1 expression. Culture media was analyzed by ELISA for IL-6 and MCP-1. Aortic VSMCs from SGK-1flox+/+ mice were transfected with Cre-Adenovirus to knockout SGK-1 (SGK-1KO VSMCs) and underwent parallel tension experimentation. Computational modeling was employed to simulate VSMC signaling due to mechanical strain and AngII. Statistical analysis included ANOVA with significance at p<0.05. ResultsSGK-1 activity (pSGK-1:SGK-1) was upregulated in the abdominal aorta of mice with HTN and significantly reduced by treatment with EMD638683. Concurrently, increased CD11b+/F4-80+ cells and plasma IL-6 levels in the HTN group and reduction with EMD638683 was observed. This mirrored the increased abundance of IL-6 in media from Stretch WT VSMCs, and attenuation of the effect with EMD638683. Treating WT VSMCs with SGK-1siRNA likewise inhibited IL-6 expression. MCP-1 also demonstrated increased expression and secretion into the media in WT VSMCs with Stretch. Further supporting the integral role of mechanical signaling through SGK-1, target gene expression and cytokine secretion was unchanged in SGK-1KO VSMCs with Stretch, and computer modeling confirmed SGK-1 as an intersecting node of signaling due to mechanical strain and AngII. In summation, this data suggests a biomechanical link between aortic VSMC mechanotransduction and cytokine production to promote macrophage accumulation, mediated in-part by SGK-1 activation. ConclusionMechanotransduction through SGK-1 is instrumental in pro-inflammatory cytokine production and aortic macrophage accumulation in systemic HTN, therefore further investigation into targeting this kinase may present opportunities to modulate hypertensive vascular remodeling.

physiology↗

bioRxiv2023

A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3/{beta} affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3/{beta} signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3/{beta}. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3/{beta} promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

cell biology↗

bioRxiv2021

Fabrication and Characterization Methods for Investigating Cell-Matrix Interactions in Environments Possessing Spatial Orientation Heterogeneity

Fibrillar collagen is a ubiquitous structural protein that plays a significant role in determining the mechanical properties of various tissues. The constituent collagen architecture can give direct insight into the respective functional role of the tissue due to the strong structure-function relationship that is exhibited. In such tissues, matrix structure can vary across local subregions contributing to mechanical heterogeneity which can be implicated in tissue function or failure. The post-myocardial infarction scar environment is an example of note where mechanically insufficient collagen can result in impaired cardiac function and possibly tissue rupture due to post-MI cellular response and matrix interactions. In order to further develop the understanding of cell-matrix and cell-cell interactions within heterogeneous environments, we developed a method of heterogeneous collagen gel fabrication which produces a region of randomly oriented fibers directly adjacent to an interconnected region of anisotropic alignment. To fully capture and evaluate the degree of alignment and spatial orientation heterogeneity, several image processing and automated analysis methods were employed. Our analysis revealed the successful fabrication of an interconnected spatially heterogeneous collagen gel possessing distinct regions of random or preferential alignment. Additionally, embedded cell populations were observed to recognize and reorient with their underlying and surrounding architectures through our cell-centric analysis techniques.

bioengineering↗

bioRxiv2020

Fibroblast Mechanotransduction Network Predicts Targets for Mechano-Adaptive Infarct Therapies

Regional control of fibrosis after myocardial infarction is critical for maintaining structural integrity in the infarct while preventing collagen accumulation in non-infarcted areas. Cardiac fibroblasts modulate matrix turnover in response to biochemical and biomechanical cues, but the complex interactions between signaling pathways confounds efforts to develop therapies for regional scar formation. Here, we employed a logic-based ordinary differential equation model of fibroblast mechano-chemo signal transduction to predict matrix protein expression in response to canonical biochemical stimuli and mechanical tension. Functional analysis of mechano-chemo interactions showed extensive pathway crosstalk with tension amplifying, dampening, or reversing responses to biochemical stimuli. Comprehensive drug target screens in low- and high-tension contexts identified 13 mechano-adaptive therapies that promote matrix accumulation in regions where it is needed and reduce matrix levels in regions where it is not needed. Our predictions demonstrate this approachs utility for discovering context-specific mechanisms mediating fibrosis and druggable targets for spatially resolved therapies.

systems biology↗