Biology subjects

Reyes-Robles, T.

Publications and source records attributed to Reyes-Robles, T..

4 recordsLinked to original sources

bioRxiv2022

Proteomic Mapping of Intercellular Synaptic Environments via Flavin-Dependent Photoredox Catalysis

Receptor-ligand interactions play essential signaling roles within intercellular contact regions. This is particularly important within the context of the immune synapse where protein communication at the surface of physically interacting T cells and antigen-presenting cells regulate downstream immune signaling responses. To identify protein microenvironments within immunological synapses, we combined a flavin-dependent photocatalytic labeling strategy with quantitative mass spectrometry-based proteomics. Using -PD-L1 or -PD-1 single-domain antibody (VHH)-based photocatalyst targeting modalities, we profiled protein microenvironments within the intercellular region of an immune synapse-forming co-culture system. In addition to enrichment of both PD-L1 and PD-1 with either targeting modality, we also observed enrichment of both known immune synapse residing receptor-ligand pairs and surface proteins, as well as previously unknown synapse residing proteins.

biochemistry↗

bioRxiv2022

High resolution photocatalytic mapping of SARS-CoV-2 Spike protein-host cell membrane interactions

Identifying protein environments at the virus-host cell interface can improve our understanding of viral entry and pathogenesis. SARS-CoV-2, the virus behind the ongoing COVID-19 pandemic, uses the cell surface ACE2 protein as a major receptor, but the contribution of other cellular proteins in the entry process is unknown. To probe the microenvironment of SARS-CoV-2 Spike-ACE2 protein interactomes on human cells, we developed a photocatalyst-based viral-host protein microenvironment mapping platform (ViraMap) employing iridium photocatalysts conjugated to Spike for visible-light driven proximity labelling on host cells. Application of ViraMap on ACE2-expressing cells captured ACE2, the established co-receptor NRP1, as well as other proteins implicated in host cell entry and immunomodulation. We further investigated these enriched proteins via loss-of-function and over-expression in pseudotype and authentic infection models and observed that the Ig receptor PTGFRN and tyrosine kinase ligand EFNB1 can serve as SARS-CoV-2 entry factors. Our results highlight additional host targets that participate infection and showcase ViraMap for interrogating virus-host cell surface interactomes.

biochemistry↗

bioRxiv2021

Detection of Cell-Cell Interactions via Photocatalytic Cell Tagging

Cell-cell interactions drive essential biological processes critical to cell and tissue development, function, pathology, and disease outcome. The growing appreciation of immune cell interactions within disease environments has led to significant efforts to develop protein- and cell-based therapeutic strategies. A better understanding of these cell-cell interactions will enable the development of effective immunotherapies. However, characterizing these complex cellular interactions at molecular resolution in their native biological contexts remains challenging. To address this, we introduce photocatalytic cell tagging (PhoTag), a modality agnostic platform for profiling cell-cell interactions. Using photoactivatable flavin-based cofactors, we generate phenoxy radical tags for targeted labeling at the cell surface. Through various targeting modalities (e.g. MHC-Multimer, antibody, single domain antibody (VHH)) we deliver a flavin photocatalyst for cell tagging within monoculture, co-culture, and peripheral blood mononuclear cells. PhoTag enables highly selective tagging of the immune synapse between an immune cell and an antigen-presenting cell through targeted labeling at the cell-cell junction. This allowed for the ability to profile gene expression-level differences between interacting and bystander cell populations. Given the modality agnostic and spatio-temporal nature of PhoTag, we envision its broad utilization to detect and profile intercellular interactions within an immune synapse and other confined cellular regions for any biological system.

immunology↗

bioRxiv2021

Small molecule photocatalysis enables drug target identification via energy transfer

The identification of cellular targets that can be exploited for therapeutic benefit, broadly known as target ID, remains a fundamental goal in drug discovery. In recent years, the application of new chemical and biological technologies that accelerate target ID has become commonplace within drug discovery programs, as a complete understanding of how molecules react in a cellular environment can lead to increased binding selectivity, improved safety profiles, and clinical efficacy. Established approaches using photoaffinity labelling (PAL) are often costly and time-consuming due to poor signal-to-noise coupled with extensive probe optimization. Such challenges are exacerbated when dealing with low abundance membrane proteins or multiple protein target engagement, typically rendering target ID unfeasible. Herein, we describe a general platform for photocatalytic small molecule target ID, which hinges upon the generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead from the drug, catalytic signal amplification results in multiple labelling events per drug, leading to unprecedented levels of target enrichment. Through the development of cell permeable photo-catalyst conjugates, this method has enabled the quantitative target and off target identification of several drugs including (+)-JQ1, paclitaxel, and dasatinib. Moreover, this methodology has led to the target ID of two GPCRs - ADORA2A and GPR40 - a class of drug target seldom successfully uncovered in small molecule PAL campaigns.

molecular biology↗