Biology subjects

Egan, E.

Publications and source records attributed to Egan, E..

3 recordsLinked to original sources

bioRxiv2024

Design of a Soluble Multivalent Notch Agonist

Designed protein agonists can enhance the efficiency of endogenous signaling pathways, and provide a powerful means to control cellular functions and develop disease therapeutics. Designing a soluble cytokine-like agonist for Notch signaling, an evolutionarily conserved pathway that regulates cell fate in embryonic and adult development, is especially challenging because Notch receptor activation requires a mechanical force that is typically mediated by cell-associated transmembrane ligands at sites of cell-cell contact. Moreover, free soluble Notch ligand is signal inhibitory. Here, we exploit computationally designed protein oligomers with precise geometries and valencies to generate cytokine-like, protein only, multivalent soluble Notch agonists. These tools promote cell-cell contact, cluster Notch proteins in synapses at the cell surface, and activate Notch signaling in reporter cell lines and cells expressing endogenous receptors. We demonstrate the utility of these soluble Notch agonists in T cell differentiation from cord blood (CB) and human induced pluripotent stem cells (iPSCs), and in bioreactor production of T cells in liquid suspension. Soluble multivalent Notch agonists can be applied broadly to in vitro cellular differentiation methods to generate clinical cell products and to develop immunotherapies.

synthetic biology↗

bioRxiv2021

SGLT2 inhibitors therapy protects glucotoxicity-induced β-cell failure in a mouse model of human KATP-induced diabetes trough mitigation of oxidative and ER stress

Progressive loss of pancreatic {beta}-cell functional mass and anti-diabetic drug responsivity are classic findings in diabetes, frequently attributed to compensatory insulin hypersecretion and {beta}-cell exhaustion. However, loss of {beta}-cell mass and identity still occurs in mouse models of human KATP-gain-of-function induced Neonatal Diabetes Mellitus (NDM), in the absence of insulin secretion. Here we studied the mechanisms underlying and temporal progression of glucotoxicity-induced loss of functional {beta}-cell mass in NDM mice, and the effects of sodium-glucose transporter 2 inhibitors (SGLT2i) therapy. Upon tamoxifen induction of transgene expression, NDM mice developed severe diabetes followed by an unexpected loss of insulin content, decreased proinsulin processing and proinsulin accumulation at 2-weeks of diabetes. This was accompanied by a marked increase in {beta}-cell oxidative and ER stress, without changes in islet cell identity. Strikingly, early treatment with the SGLT2 inhibitor dapagliflozin restored insulin content, decreased proinsulin:insulin ratio and reduced oxidative and ER stress. However, despite reduction of blood glucose, dapagliflozin therapy was ineffective in restoring {beta}-cell function in NDM mice when tit was initiated at >40 days of diabetes, when loss of {beta}-cell mass and identity had already occurred. These results have important clinical implications as they demonstrate that: i) hyperglycemia per se, and not insulin hypersecretion, drives {beta}-cell failure in diabetes, ii) recovery of {beta}-cell function by SGLT2 inhibitors is through reduction of oxidative and ER stress, iii) SGLT2 inhibitors revert/prevent {beta}-cell failure when used in early stages of diabetes, but not when loss of {beta}-cell mass/identity already occurred, iv) common execution pathways underlie loss and recovery of {beta}-cell function in different forms of diabetes.

cell biology↗

bioRxiv2020

Varying co-factor requirements for MAML1-dependent transcription at different Notch-responsive target genes

Mastermind proteins are required for transcription of Notch target genes, yet the molecular basis for mastermind function remains incompletely understood. Previous work has shown that Notch can induce transcriptional responses by binding to promoters, but more often by binding to enhancers, with HES4 and DTX1 as representative mammalian examples of promoter and enhancer responsiveness, respectively. Here, we show that mastermind dependence of the Notch response at these loci is differentially encoded in Jurkat T-ALL cells. Knockout of Mastermind-like1 (MAML1) eliminates Notch responsive activation of both these genes, and reduced target gene expression is accompanied by a decrease in H3K27 acetylation, consistent with the importance of MAML1 for p300 activity. Add-back of defined MAML1 constructs in knockout cells identifies residues 151-350 of MAML1 as essential for expression of either Notch-responsive gene. Fusion of the Notch-binding region of MAML1 to the HAT domain of p300 rescues expression of HES4 but not DTX1, suggesting that an additional activity of MAML1 is needed for gene induction at a distance. Together, these studies establish the functional importance of the MAML1151-350 region for Notch-dependent transcriptional induction, and reveal differential requirements for MAML1-dependent recruitment activities at different Notch responsive loci, highlighting the molecular complexity of NTC-stimulated transcription.

molecular biology↗