De Novo Design of Integrin α5β1 Modulating Proteins for Regenerative Medicine
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PROVIDER: PRJNA1134765 | ENA |
REPOSITORIES: ENA
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Project description:Integrin α5β1 is crucial for cell attachment and migration in development and tissue regeneration, and α5β1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5β1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5β1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.
2025-08-01 | GSE272058 | GEO
Project description:Integrin α5β1 is crucial for cell attachment and migration in development and tissue regeneration, and α5β1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5β1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5β1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.
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Project description:Soluble VEGFR-1 (sVEGFR-1) acts both as a decoy receptor for VEGFs and as an extracellular matrix protein for α5β1 integrin. A sVEGFR-1-derived peptide that interacts with α5β1 integrin promotes angiogenesis. However, canonical signal downstream integrin activation is not induced, resulting into lack of focal adhesion maturation. We performed a gene expression profile of endothelial cells adhering on sVEGFR-1 compared to that of cells adhering on fibronectin, the principal α5β1 integrin ligand. Three protein kinase-C substrates, adducin, MARCKS, and radixin were differently modulated. Adducin and MARCKS were less phosphorylated whereas radixin was higher phosphorylated in sVEGFR-1 adhering cells, the latter leading to prolonged small GTPase Rac1 activation and induction of a pathway involving the heterotrimeric G protein α13. Altogether, our data indicated endothelial cell acquisition of an highly motile phenotype when adherent on sVEGFR-1. Finally, we indicated radixin as accountable for the angiogenic effect of α5β1 integrin interaction with sVEGFR-1 that in turn depends on an active VEGF-A/VEGFR-2 signaling.
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Project description:Cells treated with a specific antibody against α5β1 integrin exhibited cell spreading and scattering, over-expression of liver stem/progenitor cell markers and activation of the ERK1/2 and p38 MAPKs signaling cascades, in a similar manner to the process triggered by HGF/SF1 stimulation. Gene expression profiling revealed marked transcriptional changes of genes involved in cell adhesion and migration, as well as genes encoding chromatin remodeling factors. These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus. Untreated cells versus HGF/SF1 treated or α5β1 integrin-mediated cell migration
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Project description:Integrin α5β1 mediates cell adhesion to the extracellular matrix (ECM) by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type-III Fn domain (Fn10) and the synergy site in the 9th type-III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen deuterium exchange mass spectrometry (HDX MS) to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.
2022-08-09 | PXD031508 | Pride
Project description:Integrin α5β1 and αv crosstalk in chemotaxis and clonogenic survival of prostate cancer cells is abrogated by a bispecific α5β1/αv antibody (BsAbα5β1/αv), which uniquely induces internalization and lysosomal degradation of target integrins. We hypothesized that the BsAbα5β1/αv inactivates pathological mechanosignaling pathways that correlate with integrin expression from patient samples. Mechanistic studies indicate that the BsAbα5β1/αv uniquely reverses YAP, beta-catenin and FAK nuclear localization compared to monospecific integrin α5β1 and αv antibody controls in basal-type androgen-receptor negative prostate cancer cells. Dual integrin αv and α5 knockdown alone phenocopied the BsAbα5β1/αv effect. Following BsAbα5β1/αv treatment, ATAC-seq studies indicated the chromatin accessibility to TEAD and AP-1 family members was markedly reduced. In vitro and in vivo RNA-seq indicated down-regulation of Myc/E2F, TGF-beta and epithelial mesenchymal transition (EMT) and upregulation of Type I and II interferon transcriptomic pathways. The BsAbα5β1/αv induced CXCL10 and CCL5 cytokine secretion, immune-infiltration of tumors, and natural-killer cell-mediated elimination of the basal-type prostate cancer xenografts in nude mice. αv integrin was highly expressed and principally correlated with the Myc signaling pathway in rapid autopsy tissue microarrays, consistent with correlative data from the SU2C metastatic castration-resistant prostate cancer and DKFZ early-onset prostate cancer cohorts. These studies connect integrin signaling with the central biology of basal-type and castration-resistant prostate cancer and define a novel therapeutic strategy that controls critical immunosuppressive pathways.
2025-06-10 | GSE292297 | GEO
Project description:Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin a5b1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin a5b1 or depletion of the a5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5β1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5β1 integrin inhibition as a promising therapy for pulmonary hypertension
2024-12-31 | GSE277556 | GEO
Project description:Primary tumor growth and metastasis in triple-negative breast cancer (TNBC) require supporting vasculature, which develop through a combination of endothelial angiogenesis and vasculogenic mimicry (VM), a process associated with aggressive metastatic behavior in which vascular-like structures are lined by tumor cells. We developed αEGFR-E-P125A, an antibody-endostatin fusion protein that delivers a dimeric, mutant endostatin (E-P125A) payload that inhibits TNBC angiogenesis and VM in vitro and in vivo. To characterize the mechanisms associated with induction and inhibition of VM, RNA-seq of MDA-MB-231-4175 TNBC cells grown in a monolayer (2D) was compared to cells plated on Matrigel undergoing VM (3D). We then compared RNA-seq between TNBC cells in 3D and cells in 3D with VM inhibited by αEGFR-E-P125A (αEGFR-E-P125A). Gene set enrichment analysis (GSEA) demonstrated that VM induction activated the IL6-JAK-STAT3 and angiogenesis pathways, which were downregulated by αEGFR-E-P125A treatment. Correlative analysis of the phospho-proteome demonstrated decreased EGFR phosphorylation at Y1069, along with decreased phosphorylation of focal adhesion kinase (FAK) Y397 and STAT3 Y705 sites downstream of α5β1 integrin. Suppression of phosphorylation events downstream of EGFR and α5β1 integrin demonstrated that αEGFR-E-P125A interferes with ligand-receptor activation, inhibits VM, and overcomes oncogenic signaling associated with EGFR and α5β1 integrin crosstalk. In vivo, αEGFR-E-P125A treatment decreased primary tumor growth and VM, reduced lung metastasis, and confirmed the inhibition of signaling events observed in vitro. Simultaneous inhibition of EGFR and α5β1 integrin signaling by αEGFR-E-P125A is a promising strategy for the inhibition of VM, tumor growth, motility, and metastasis in TNBC and other EGFR-overexpressing tumors.
2024-01-29 | GSE253789 | GEO
Project description:Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2 and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of bFGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4 and NANOG, while the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state, showing high similarity to their embryonic stem (ES) cell counterparts we generated recently. However, their global gene expression analysis revealed a slight, but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and ES cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans. Comparison of rabbit embryonic stem cells, rabbit somatic cells, and rabbit induced pluripotent stem cells at several timing (passage number 6, 7, 22, 23) of cultures. Eight samples were used for assessment.
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Project description:Fibronectin (FN)-binding integrins control a variety of cellular responses through Rho GTPases. The FN-binding integrins, αvβ3 and α5β1, are known to induce different effects on cell morphology and motility. Here we report that FN-bound αvβ3 integrin, but not FN-bound α5β1 integrin, triggers the dissociation of the RhoA GEF Lfc (GEF-H1 in humans) from microtubules (MT), leading to the activation of RhoA, formation of stress fibres and maturation of focal adhesions (FAs). Conversely, loss of Lfc expression decreases RhoA activity, stress fibre formation and FA size, suggesting that Lfc is the major GEF downstream of FN-bound αvβ3 that controls RhoA activity. Mechanistically, FN-engaged αvβ3 integrin activates a kinase cascade involving MARK2/3, which in turn leads to phosphorylation of several phospho-sites on Lfc. In particular, S151 was identified as the main site involved in the regulation of Lfc localization and activity. Our findings indicate that activation of Lfc/RhoA is orchestrated in FN-adherent cells in an integrin-specific manner.
2023-04-17 | PXD037596 | Pride