Biology subjects

Teixeira, A. I.

Publications and source records attributed to Teixeira, A. I..

5 recordsLinked to original sources

bioRxiv2024

Engineering multivalent Fc display for FcγR blockade

Autoimmune diseases, driven by Fc{gamma} receptor (Fc{gamma}R) activation through autoantibody immune complexes (IC), present a complex therapeutic challenge of achieving pharmacological blockade of Fc{gamma}R without triggering receptor activation. The assembly of ICs into polydisperse, higher-order structures is required for Fc{gamma}R activation. However, engineered multimeric, monodisperse Fc assemblies have been reported to prevent Fc{gamma}R activation, suggesting that Fc spatial organization determines Fc{gamma}R activation. In this study, we engineered a functional single-chain Fc domain protein (scFc) for unidirectional, multivalent presentation by virus-like particles (VLPs), used as a display platform. We found that the multivalent display of scFc on the VLPs elicited distinct cellular responses compared with monovalent scFc, highlighting the importance of the structural context of scFc on its function. scFc-VLPs had minimal impact on the nanoscale spatial organization of Fc{gamma}R at the cell membrane and caused limited receptor activation and internalization. In contrast, the monovalent scFc acted as an Fc{gamma}R agonist, inducing receptor clustering, activation, and internalization. Increasing scFc valency in scFc-VLPs was associated with increased binding to monocytes, reaching a plateau at high valencies. Notably, the ability of scFc-VLPs to block IC-mediated phagocytosis in vitro increased with scFc valency. In a murine model of passive immune thrombocytopenia (ITP), a high valency scFc-VLP variant with a desirable immunogenicity profile induced attenuation of thrombocytopenia. Here we show that multivalent presentation of an engineered scFc on a display platform can be tailored to promote suppression of IC-mediated phagocytosis while preventing Fc{gamma}R activation. This work introduces a new paradigm that can contribute to the development of therapies for autoimmune diseases.

bioengineering↗

bioRxiv2023

Biodistribution of DNA-origami nanostructures in live zebrafish embryos with single-cell resolution

DNA origami-based nanotechnology is a versatile tool for exploring fundamental biological questions and holds significant promise for future biomedical applications. However, the development of DNA origami-based therapeutic agents is hindered by the challenge of translating in vitro performance into effective applications in vivo. Here, we exploit the optical transparency of the embryonic zebrafish to track intravenously injected, fluorescently labelled wireframe DNA origami nanostructures. Our approach integrated long-term, high-resolution imaging of transgenic live embryos with single-cell RNA sequencing, to elucidate the biodistribution of DNA nanostructures over time, up to 3 days post-injection (dpi). Notably, we observed rapid accumulation of nanostructures in the caudal hematopoietic tissue (CHT), akin to the fetal liver in mammals. We tested the effects of coating the nanostructures with an oligolysine PEG copolymer (K-PEG), a widely used strategy to enhance their stability. The K-PEG coating mitigated the accumulation rate in CHT, enabling higher percentages of the nanostructures to engage with other tissues. Additionally, our findings highlighted the pivotal role of scavenger endothelial cells in DNA origami clearance, with K-PEG offering sustained protection for the nanostructures at the CHT. Furthermore, by monitoring DNA origami in a transgenic zebrafish line designed for targeted macrophage ablation, we found that macrophages contribute to nanostructure clearance at later time points. This study introduces a framework for the analyses of the biodistribution and clearance of DNA origami nanostructures in vivo with single cell resolution and establishes a foundation for the investigation of DNA origami-based nanomedicines in animal models.

bioengineering↗

bioRxiv2022

Notch engagement by Jag1 nanoscale clusters indicates a force-independent mode of activation

The Notch signaling pathway is a cell-cell communication system with fundamental roles in embryonic development and the nervous system. The model of Notch receptor activation that is currently most accepted, involves a force-induced conformation change at the negative regulatory region of the receptor, the subsequent recruitment of ADAM metalloproteases and a cleavage cascade that releases the Notch intracellular domain. Here, we define conditions that enable force-independent Notch activation through the formation of soluble, long-lived, multivalent ligand-receptor complexes. To investigate how ligand valency affects activation of Notch receptors, we treated iPSc-derived neuroepithelial stem-like (lt-NES) cells with different spatially defined, molecularly precise ligand nanopatterns on DNA origami nanostructures. Our data indicate that Notch signaling is activated via stimulation with multivalent clusters of the ligand Jag1, and even multivalent chimeric structures where some Jag1 proteins are replaced by other binders that do not target Notch. The findings are corroborated by systematic elimination, through experimental control, of several confounding factors that potentially could generate forces, including electrostatic interactions, endocytosis and non-specific binding. Taken together, our data suggest a model where Jag1 ligands are able to activate Notch receptors upon prolonged binding, which subsequently triggers downstream signaling in a force independent manner. These findings reveal a distinct mode of activation of Notch and could lay the foundation for the development of soluble Notch agonists that currently remain elusive.

cell biology↗

bioRxiv2019

A DNA nanoassembly-based approach to map membrane protein nanoenvironments

Super-resolution imaging has revealed that most proteins at the plasma membrane are not uniformly distributed but localize to dynamic domains of nanoscale dimensions. To investigate their functional relevance, there is a need for methods that enable comprehensive mapping of the compositions and spatial organizations of membrane protein nanodomains in cell populations. However, current superresolution methods are limited to analysing small, preselected subsets of proteins, at very low sampling fractions. Here we describe the development of a non-microscopy based super-resolution method for unbiased ensemble analysis of membrane protein nanodomains. The method, termed NANOscale DEciphEring of membrane Protein nanodomains (NanoDeep), is based on the use of DNA nanoassemblies to translate membrane protein organization information into a DNA sequencing readout. Using NanoDeep, we characterized the nanoenvironments of Her2, a membrane receptor of critical relevance in cancer. We found that the occupancies of Her2, Her3 and EGFR in the nanoenvironments surrounding Her2 were similar in two cell lines with vastly different expression levels of Her2. Further, we found that adding Heregulin-{beta}1 to cancer cells led to increased occupancy of Her2 and Her3, and to a lesser extent EGFR, in Her2 nanoenvironments. NanoDeep has the potential to provide new insights into the roles of the composition and spatial organization of protein nanoenvironments in the regulation of membrane protein function.

bioengineering↗

bioRxiv2019

Live detection of neural stem and glioblastoma cells by an oligothiophene derivative

Here we report a luminescent conjugated oligothiophene (LCO), named p-HTMI, for non-invasive and non-amplified real-time detection of live human patient-derived glioblastoma (GBM) cells and embryonic neural stem cells (NSCs). While p-HTMI stained only a small fraction of other cell types investigated, the mere addition of p-HTMI to the cell culture resulted in efficient detection of NSCs or GBM cells from rodents and humans within minutes. p-HTMI is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. p-HTMI were readily able to detect a subpopulation of human cells in vivo in mouse brain sections with tumors developed from orthotopically transplanted patient-derived GBM cells. Cell sorting experiments of human GBM cells demonstrated that p-HTMI labeled the same population as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO p-HTMI is a versatile tool for immediate and selective detection of subpopulations of neural stem and glioma cells.

bioengineering↗