Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

Nanodecoy systems based on analogues of viral cellular receptors assembled onto fluid lipid-based membranes of nano/extravescicles are potential new tools to complement classic therapeutic or preventive antiviral approaches. The need for lipid-based membranes for transmembrane receptor anchorage may pose technical challenges along industrial translation, calling for alternative geometries for receptor multimerization. Here we developed a semisynthetic self-assembling SARS-CoV-2 nanodecoy by multimerizing the biotin labelled virus cell receptor -ACE2- ectodomain onto a poly-avidin nanoparticle (NP) based on the Avidin-Nucleic-Acid-NanoASsembly-ANANAS. The ability of the assembly to prevent SARS-CoV-2 infection in human lung cells and the affinity of the ACE2:viral receptor-binding domain (RBD) interaction were measured at different ACE2:NP ratios. At ACE2:NP = 30, 90 % SARS-CoV-2 infection inhibition at ACE2 nanomolar concentration was registered on both Wuhan and Omicron variants, with ten-fold higher potency than the monomeric protein. Lower and higher ACE2 densities were less efficient suggesting that functional recognition between multi-ligand NPs and multi-receptor virus surfaces requires optimal geometrical relationships. In vivo studies in mice showed that the biodistribution and safety profiles of the nanodecoy are potentially suitable for preventing viral infection upon nasal instillation. Viral receptor multimerization using ANANAS is a convenient process which, in principle, could be rapidly adapted to counteract also other viral infections.

Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability.

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called "NT-mini" and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT's heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension.

Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In this regard, human embryonic kidney 293 (HEK293) cells constitute one of the main standard lab-scale mammalian hosts for recombinant protein production since these cells are relatively easy to handle, scale-up, and transfect. Here, we present a detailed protocol for the cost-effective, reproducible, and scalable implementation of HEK293 cell cultures in suspension (suitable for commercially available HEK293 cells, HEK293-F) for high-quantity recombinant production of secreted soluble multi-domain proteins. In addition, the protocol is optimized for a Monday-to-Friday maintenance schedule, thus simplifying and streamlining the work of operators responsible for cell culture maintenance. Graphic abstract: Schematic overview of the workflow described in this protocol.

Dissecting the Extracellular Complexity of Neuromuscular Junction Organizers.

Synapse formation is a very elaborate process dependent upon accurate coordination of pre and post-synaptic specialization, requiring multiple steps and a variety of receptors and signaling molecules. Due to its relative structural simplicity and the ease in manipulation and observation, the neuromuscular synapse or neuromuscular junction (NMJ)-the connection between motor neurons and skeletal muscle-represents the archetype junction system for studying synapse formation and conservation. This junction is essential for survival, as it controls our ability to move and breath. NMJ formation requires coordinated interactions between motor neurons and muscle fibers, which ultimately result in the formation of a highly specialized post-synaptic architecture and a highly differentiated nerve terminal. Furthermore, to ensure a fast and reliable synaptic transmission following neurotransmitter release, ligand-gated channels (acetylcholine receptors, AChRs) are clustered on the post-synaptic muscle cell at high concentrations in sites opposite the presynaptic active zone, supporting a direct role for nerves in the organization of the post-synaptic membrane architecture. This organized clustering process, essential for NMJ formation and for life, relies on key signaling molecules and receptors and is regulated by soluble extracellular molecules localized within the synaptic cleft. Notably, several mutations as well as auto-antibodies against components of these signaling complexes have been related to neuromuscular disorders. The recent years have witnessed strong progress in the understanding of molecular identities, architectures, and functions of NMJ macromolecules. Among these, prominent roles have been proposed for neural variants of the proteoglycan agrin, its receptor at NMJs composed of the lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific kinase (MuSK), as well as the regulatory soluble synapse-specific protease Neurotrypsin. In this review we summarize the current state of the art regarding molecular structures and (agrin-dependent) canonical, as well as (agrin-independent) non-canonical, MuSK signaling mechanisms that underscore the formation of neuromuscular junctions, with the aim of providing a broad perspective to further stimulate molecular, cellular and tissue biology investigations on this fundamental intercellular contact.

Structural characterization of the third scavenger receptor cysteine-rich domain of murine neurotrypsin.

Neurotrypsin (NT) is a multi-domain serine protease of the nervous system with only one known substrate: the large proteoglycan Agrin. NT has seen to be involved in the maintenance/turnover of neuromuscular junctions and in processes of synaptic plasticity in the central nervous system. Roles which have been tied to its enzymatic activity, localized in the C-terminal serine-protease (SP) domain. However the purpose of NT's remaining 3-4 scavenger receptor cysteine-rich (SRCR) domains is still unclear. We have determined the crystal structure of the third SRCR domain of murine NT (mmNT-SRCR3), immediately preceding the SP domain and performed a comparative structural analysis using homologous SRCR structures. Our data and the elevated degree of structural conservation with homologous domains highlight possible functional roles for NT SRCRs. Computational and experimental analyses suggest the identification of a putative binding region for Ca2+ ions, known to regulate NT enzymatic activity. Furthermore, sequence and structure comparisons allow to single out regions of interest that, in future studies, might be implicated in Agrin recognition/binding or in interactions with as of yet undiscovered NT partners.

Identifying and Visualizing Macromolecular Flexibility in Structural Biology.

Structural biology comprises a variety of tools to obtain atomic resolution data for the investigation of macromolecules. Conventional structural methodologies including crystallography, NMR and electron microscopy often do not provide sufficient details concerning flexibility and dynamics, even though these aspects are critical for the physiological functions of the systems under investigation. However, the increasing complexity of the molecules studied by structural biology (including large macromolecular assemblies, integral membrane proteins, intrinsically disordered systems, and folding intermediates) continuously demands in-depth analyses of the roles of flexibility and conformational specificity involved in interactions with ligands and inhibitors. The intrinsic difficulties in capturing often subtle but critical molecular motions in biological systems have restrained the investigation of flexible molecules into a small niche of structural biology. Introduction of massive technological developments over the recent years, which include time-resolved studies, solution X-ray scattering, and new detectors for cryo-electron microscopy, have pushed the limits of structural investigation of flexible systems far beyond traditional approaches of NMR analysis. By integrating these modern methods with powerful biophysical and computational approaches such as generation of ensembles of molecular models and selective particle picking in electron microscopy, more feasible investigations of dynamic systems are now possible. Using some prominent examples from recent literature, we review how current structural biology methods can contribute useful data to accurately visualize flexibility in macromolecular structures and understand its important roles in regulation of biological processes.