Biology subjects

Woodruff, T.

Publications and source records attributed to Woodruff, T..

6 recordsLinked to original sources

bioRxiv2025

Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

The conceptual framework of biased signaling has revolutionized our understanding of GPCR signaling and regulatory paradigms, and greatly impacted the efforts focused on the discovery of GPCR-targeted therapeutics. However, the mechanistic basis of biased signaling remains primarily defined based on synthetic ligands and receptor mutants with relatively limited progress in understanding naturally-encoded signaling-bias. Here, we present fundamental molecular and structural insights into naturally-encoded signaling-bias at the complement anaphylatoxin C5a receptors namely, C5aR1 and C5aR2. We first discover that C5a-d-Arg, the naturally-occurring version of C5a lacking the terminal arginine, exhibits robust G-protein signaling-bias at C5aR1, characterised by attenuated {beta}arr recruitment. This signaling-bias manifests in both cytokine release from primary human immune cells, and in vivo, during neutrophil mobilization. We combine the cryo-EM structures of C5a/C5a-d-Arg-C5aR1 complexes with MD simulation, site-directed mutagenesis, and cellular experiments to elucidate that the G-protein-bias exhibited by C5a-d-Arg results from a distinct orientation of TM7 and helix 8 in C5aR1 leading to inefficient GRK recruitment and receptor phosphorylation. Next, we determine the first cryo-EM structures of C5aR2, a naturally-encoded {beta}-arrestin-biased receptor, in an apo state, complexed with the natural agonists C5a and C5a-d-Arg, and three peptide agonists including a first-in-class, newly discovered C5aR2-selective agonist, R8Y. These structural snapshots reveal key differences between the binding of C5a and C5a- d-Arg to C5aR1 and C5aR2, and provide a molecular basis of functional specialization at these two receptors. Moreover, the structural insights also allow us to decipher the molecular basis of naturally-encoded signaling-bias at C5aR2 originating from a shallower cytoplasmic interface with hydrophobic interior pocket that is not permissive to efficient G-protein-coupling and activation. Finally, we also engineer and characterize loss-of-function and gain-of-function variants of C5aR1 and C5aR2, which in turn corroborate and validate the structural observations presented here. Collectively, our findings offer crucial insights into previously lacking molecular mechanisms of the naturally-encoded signaling-bias at GPCRs, which have broad implications not only for the general framework of biased-signaling, but also for novel therapeutic design.

biochemistry↗

bioRxiv2024

Analyzing high-throughput assay data to advance the rapid screening of environmental chemicals for human reproductive toxicity

While high-throughput (HTP) assays have been proposed as platforms to rapidly assess reproductive toxicity, there is currently a lack of established assays that specifically address germline development/function and fertility. We assessed the applicability domains of yeast (S. cerevisiae) and nematode (C. elegans) HTP assays in toxicity screening of 124 environmental chemicals, determining their agreement in identifying toxicants and their concordance with reproductive toxicity in vivo. We integrated data generated in the two models and compared results using a streamlined, semi-automated benchmark dose (BMD) modeling approach. We then extracted and modeled relevant mammalian in vivo data available for the matching chemicals included in the Toxicological Reference Database (ToxRefDB). We ranked potencies of common compounds using the BMD and evaluated correlation between the datasets using Pearson and Spearman correlation coefficients. We found moderate to good correlation across the three data sets, with r = 0.48 (95% CI: 0.28-1.00, p<0.001) and rs = 0.40 (p=0.002) for the parametric and rank order correlations between the HTP BMDs; r = 0.95 (95% CI: 0.76-1.00, p=0.0005) and rs = 0.89 (p=0.006) between the yeast assay and ToxRefDB BMDs; and r = 0.81 (95% CI: 0.28-1.00, p=0.014) and rs = 0.75 (p=0.033) between the worm assay and ToxRefDB BMDs. Our findings underscore the potential of these HTP assays to identify environmental chemicals that exhibit reproductive toxicity. Integrating these HTP datasets into mammalian in vivo prediction models using machine learning methods could further enhance the predictive value of these assays in future rapid screening efforts.

developmental biology↗

bioRxiv2022

A high-throughput approach to identify reproductive toxicants among environmental chemicals using an in vivo evaluation of gametogenesis in budding yeast Saccharomyces cerevisiae

BackgroundEnvironmental chemical exposures are likely making important contributions to current levels of infertility and its increasing incidence. Yet the US produces high volumes of industrial chemicals for which there is limited data on their potential human reproduction toxicity. Current assays typically used in policy and regulatory settings involve costly and timeconsuming whole-animal rodent tests which limit the rapidity with which one can assess the thousands of chemicals yet to be tested. ObjectiveOur aim was to develop a fast and reliable strategy to evaluate a large number of chemicals for reproductive toxicity by developing a high-throughput toxicity assessment using the yeast S. cerevisiae. MethodsYeast are chronically exposed to each environmental chemical at two doses, 30 M and 100 M, in a 96-well plate-based format throughout gametogenesis. Non-gametes are removed and chemicals are washed away before gamete viability is measured using absorbance at 600 nm to produce growth curves. The difference in time at half-maximal saturation with and without exposure is used to determine the extent of reproductive toxicity. ResultsWe validated our assay using bisphenol A (BPA), a well-established mammalian reproductive toxicant. We find that BPA in yeast has similar detrimental effects in meiosis as shown in worms and mammals. Competition assays with BPA analogs reveal that two of out of 19 BPA analogs examined (bisphenol E and 17{beta}-estradiol) show synergistic effects with BPA at doses tested and none show antagonistic effects. Out of 179 additional environmental chemicals, we designated 57 chemicals as reproductively toxic. Finally, by comparing chemicals in our cohort that have been evaluated for reproductive toxicity in mammalian studies, we find a statistically significant association between toxic chemicals in yeast and mammals. ConclusionWe show that a high-throughput assay using yeast may be a useful approach for rapidly and reliably identifying chemicals that pose a reproductive risk.

pharmacology and toxicology↗

bioRxiv2020

Stage-specific muscle wasting mechanisms in a novel cancer cachexia mouse model for ovarian granulosa cell tumor

Cachexia is a progressive muscle wasting syndrome that increases mortality risk in cancer patients, while there are still no effective treatment due to the complexity of syndrome and the lack of preclinical models. We identified a transgenic mice model with ovarian granulosa cell tumors mimic the progression of cachexia seen in humans, including drastic weight loss, skeletal muscle wasting and increased serum cachexia biomarker activin A and GDF15. Hypercatabolism was detected in skeletal muscle, having upregulation of E3 ligases Atrogin-1 and Murf-1. Our cachexia model exhibited stage-specific muscle wasting mechanisms. At precachexia stage, elevation of activin A activates p38 MAPK. Inhibition of activin A with Follistatin reversed weight loss at precachexia stage. At cachexia stage, energy stress in skeletal muscle activates AMPK and leads to upregulation of FoxO3. Our results indicate this novel preclinical cancer cachexia model is exploitable for studying pathophysiological mechanisms and testing therapeutic agents of cachexia.

cancer biology↗

bioRxiv2019

Ligands, receptors and transcription factors that mediate inter-cellular and intra-cellular communication during ovarian follicle development

Reliably producing a competent oocyte entails a deeper comprehension of ovarian follicle maturation, a very complex process that includes meiotic maturation of the female gamete, the oocyte, together with the mitotic divisions of the hormone-producing somatic cells. In this report, we investigate mice ovarian folliculogenesis in vivo using publically available time-series microarrays from primordial to antral stage follicles. Manually curated protein interaction networks were employed to identify autocrine and paracrine signaling between the oocyte and the somatic cells (granulosa and theca cells) and the oocyte and cumulus and mural cells at multiple stages of follicle development. We established protein binding interactions between expressed genes that encoded secreted factors and expressed genes that encoded cellular receptors. Some of computationally identified signaling interactions are well established, such as the paracrine signaling from the oocyte to the somatic cells through the secreted oocyte growth factor Gdf9; while others are novel connections in term of ovarian folliculogenesis, such as the possible paracrine connection from somatic secreted factor Ntn3 to the oocyte receptor Neo1. Additionally, we identify several of the likely transcription factors that might control the dynamic transcriptome during ovarian follicle development, noting that the YAP/TAP signaling is very active in vivo. This novel dynamic model of signaling and regulation can be employed to generate testable hypotheses regarding follicle development, guide the improvement of culture media to enhance in vitro ovarian follicle maturation and possibly as novel therapeutic targets for reproductive diseases.

systems biology↗

bioRxiv2019

Partial ligand-receptor engagement yields functional bias at the human complement receptor, C5aR1

The human complement component, C5a, binds two different seven transmembrane receptors termed as C5aR1 and C5aR2. C5aR1 is a prototypical G protein-coupled receptor that couples to Gi sub-family of heterotrimeric G proteins and {beta}-arrestins ({beta}arr) following C5a stimulation. Peptide fragments derived from the carboxyl-terminus of C5a can still interact with the receptor, albeit with lower affinity, and can act as agonists or antagonists. However, whether such fragments might display ligand bias at C5aR1 remains unexplored. Here, we compare C5a and a modified C-terminal fragment of C5a, C5apep, in terms of G protein coupling, {beta}arr recruitment, endocytosis and ERK1/2 MAP kinase activation at the human C5aR1. We discover that C5apep acts as a full-agonist for G protein coupling, while only displaying partial agonism for {beta}arr recruitment. We also observe that whilst C5apep is significantly less efficient in inducing C5aR1 endocytosis compared to C5a, it exhibits robust activation of ERK1/2 phosphorylation at levels similar to C5a. Interestingly, C5apep displays full-agonist efficacy with respect to inhibiting LPS induced IL-6 secretion in human macrophages, but its ability to induce human neutrophil migration is substantially lower compared to C5a. Taken together, our findings reveal ligand-bias at C5aR1, not only with respect to transducer-coupling and receptor trafficking but also in terms of cellular responses. Our findings therefore establish a framework to explore additional levels of biased signaling and biased ligands at C5aR1 with therapeutic potential. More generally, our findings may be extended to discover biased ligands for the broad sub-family of chemokine GPCRs which also interact with chemokine ligands through a biphasic mechanism.

biochemistry↗