Biology subjects

Bernardes, G. J. L.

Publications and source records attributed to Bernardes, G. J. L..

6 recordsLinked to original sources

bioRxiv2025

LuxSit Pro and sPro: Next-Generation Designed Luciferases for Bioluminescent Reporting and Complementation-Based Detection

Bioluminescent systems are widely used as molecular reporters in life sciences due to their low background, broad dynamic range, and the key advantage of not requiring external excitation. However, natural luciferases often lack the brightness, stability, and long-lasting light emission needed to support a wide range of high-sensitivity applications. Here, we describe our next-generation luciferase solution: LuxSit Pro and its substrate, Luxterazine (LTZ). LuxSit Pro is a de novo luciferase optimized for steady brightness and structural stability through an iterative combination of computational and experimental methods. Composed of only 117 amino acids, LuxSit Pro is currently the smallest highest-performing known luciferase, exhibiting exceptional brightness and biochemical stability with robust recombinant expression in both mammalian and bacterial systems. Notably, LuxSit Pro was deliberately designed devoid of lysine and cysteine residues which enormously expands its potential applications. This property renders the reporter largely resistant to post-translational modifications such as ubiquitination and also enables site-directed chemical modifications, as lysines or cysteines can be reintroduced at specific sites to serve as chemical handles for augmenting or adapting the enzymes function. The novel synthetic substrate, Luxterazine (LTZ), is a compound that enables LuxSit Pro to deliver bright, steady, blue light emission ([~]490 nm) and offers improved solubility compared to existing Coelenterazine analogs. Finally, we also developed LuxSit sPro, a highly sensitive two-component complementation system for the quantitative detection of molecular interactions. The broad potential applications of LuxSit Pro and sPro, in combination with Luxterazine, are poised to drive the development of next-generation reporters and biosensor systems for the research and in vitro diagnostics fields.

molecular biology↗

bioRxiv2025

Bifunctional Phagocytic Synapse Enhancers for Cancer Immunotherapy

Immunotherapy profoundly impacted cancer treatments by harnessing the patients immune system. Phagocytosis, the process whereby immune cells engulf and destroy foreign particles or cells, plays a critical role in tumour cell clearance. Herein, we introduce a novel concept termed "ENPHASYS" - Enhancement of Phagocytic Synapses - designed to direct and amplify phagocytosis of cancer cells using heterobifunctional molecules named phagocytic synapse enhancers (PSEs). By engineering a de novo PD-L1 binder linked to a natural phagocytosis promoting peptide, tuftsin, the resulting PSE combines PD-L1 blockade with enhanced tumour cell phagocytosis; in addition, the PSEs induce macrophages to internalize a membrane or extracellular target. Intratumoural treatment of colorectal carcinoma- or glioblastoma-burdened immunocompetent animals resulted in beneficial overall survival, delayed tumour growth and a potent antitumor response driven by T-cell activation and TAM reprogramming, underpinning the translational relevance of ENPHASYS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/642658v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1673fd4org.highwire.dtl.DTLVardef@18fa319org.highwire.dtl.DTLVardef@11cf680org.highwire.dtl.DTLVardef@7b8784_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation and amplify signaling

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

synthetic biology↗

bioRxiv2023

Multiplexed Digital Characterisation of Misfolded Protein Oligomers via Solid-State Nanopores

Misfolded protein oligomers are of central importance in both the detection and treatment of Alzheimers and Parkinsons diseases. However, accurate high-throughput methods to identify and quantify oligomer populations are currently lacking. We present here a single-molecule approach for the detection of oligomeric species. The approach is based on the use of solid state nanopores and multiplexed DNA barcoding to identify and characterise oligomers from multiple samples. We study -synuclein oligomers in the presence of several small molecule inhibitors of -synuclein aggregation, as an illustration of the applicability of this method to assist the development of diagnostic and therapeutic methods for Parkinsons disease.

biophysics↗

bioRxiv2023

Targeted protein editing with an antibody-based system

The chemical modification of proteins is one of the major mechanisms used to regulate the properties and functions of these macromolecules in the cell. It is therefore of great interest to develop tools to exploit this type of modifications for applications in molecular biology, medicine and biotechnology. Here we present a method of using antibodies to perform post-translational covalent modifications of endogenous proteins in complex environments by exploiting proximity-driven chemistry. The method is based on the ability of antibodies to hold a weakly reactive group close to its intended site of reaction by binding the target protein on a nearby epitope. We characterise this approach by modifying the green fluorescent protein in increasingly complex environments, and illustrate its applicability by targeting the disease-associated protein beta-2 microglobulin.

biochemistry↗

bioRxiv2020

TRPV2 interaction with small molecules and lipids revealed by cryo-EM

Transient receptor potential vanilloid 2 (TRPV2) plays a critical role in a variety of physiological and pathophysiological processes, putting TRPV2 on the list of important drug targets. Yet, specific TRPV2 agonists and antagonists are currently unavailable. Their development requires a precise knowledge of how the currently known non-specific small molecules interact with TRPV2 at the molecular level. Here we present TRPV2 structures in ligand-bound states resolved by cryo-electron microscopy in the presence of 2-aminoethoxydiphenyl borate (2-ABP), 2-APB with doxorubicin (DOXO), and ruthenium red (RR). We identified a novel 2-APB drug binding site between the S5 helix and S4-S5 linker on two adjacent TRPV2 monomers and determined the mechanism of TRPV2 pore block by RR. We also showed that a large organic molecule like DOXO can enter the TRPV2 pore in the presence of 2-APB. Additionally, we discovered a structural lipid bound in a unique position in the "vanilloid pocket", which is absent in the 2-APB-bound state of the channel, allowing us to propose a model for TRPV2 channel gating. Together, this work provides a further understanding of TRPV2 function and a structural framework for the development of TRPV2-specific modulators.

biophysics↗