Biology subjects

Hons, M.

Publications and source records attributed to Hons, M..

10 recordsLinked to original sources

bioRxiv2024

Multiple clustered centrosomes in antigen-presenting cells foster T cell activation without MTOC polarization

Cellular polarization plays a pivotal role in regulating immunological processes and is often associated with centrosome reorientation. During immune synapse (IS) formation centrosome repositioning in lymphocytes assists in T cell activation. While a single centrosome, consisting of two centrioles, is present in T cells, antigen-presenting cells (APCs) such as dendritic cells (DCs) amplify centrioles during maturation leading to increased centrosome numbers upon immune activation. How centrosome amplification in DCs affects IS formation and T cell activation is unclear. In this study, we combine experimental data with mathematical and computational modelling to provide evidence that centrosome amplification in DCs enhances antigen-specific T cell activation. Extra centrioles in DCs form active centrosomes, which cluster during DC-T cell interactions and unlike in T cells, localize close to the cell center. Perturbing either centriole numbers or centrosome configuration in DCs results in impaired T cell activation. Collectively, our results highlight a crucial role for centrosome amplification and optimal centrosome positioning in APCs for controlling T cell responses.

cell biology↗

bioRxiv2024

EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control

Imaging biological macromolecules in their native state with single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET) requires optimized approaches for the preparation and vitrification of biological samples. Here, we describe EasyGrid, a versatile technology enabling systematic, tailored and advanced sample preparation for cellular and structural biology. This automated, standalone platform combines in-line plasma treatment, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and on-the-fly grid quality control using light interferometry to streamline cryo-EM sample optimization. With EasyGrid, we optimized grid preparation for different purified macromolecular complexes and subsequently determined their structure with cryo-EM. We also demonstrated how the platform allows better vitrification of large, mammalian cells compared to standard plunge-freezing. Automated sample preparation with EasyGrid establishes an advanced, high-throughput platform for both single-particle cryo-EM and cellular cryo-ET sample preparation.

molecular biology↗

bioRxiv2022

Structural analysis shows that the BIR2 domain of E3 ligase XIAP binds across the RIP2 kinase dimer interface

NOD1 and NOD2 are innate immune system pattern recognition receptors that play a key role in the protection from bacterial infections and in the maintenance of gastro-intestinal homeostasis. Dysregulation of NOD signalling pathways promotes chronic inflammatory diseases such as inflammatory bowel disease. RIP2, which contains a kinase domain (RIP2K) and a CARD domain, is the immediate downstream signalling partner in the NOD pathway and inhibition of its kinase activity and ubiquitination are promising strategies to address these inflammatory diseases. However, recent work indicates that the phosphorylation activity of RIP2K is dispensable for signalling and that inhibitors of both RIP2K activity and RIP2 ubiquitination prevent the essential interaction between RIP2K and the BIR2 domain of XIAP, the key RIP2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined Native mass spectrometry, NMR, cryo-electron microscopy and Alphafold2 predictions to determine the structure of the RIP2K-XIAP BIR2 complex with 2:1 stoichiometry. The structure shows that complex formation requires that the kinase is in an active-like (i.e. C-helix IN) and dimeric conformation and explains both inhibitory mechanisms. It also shows why phosphorylation of the kinase activation loop is dispensable for signalling and reveals the structural role of RIP2K-K209 residue in the RIP2K-XIAP BIR2 interaction. Importantly, our results clarify the features of the RIP2K conformation essential for its role as a scaffold protein for ubiquitination.

biochemistry↗

bioRxiv2022

Structural basis and dynamics of Chikungunya alphavirus RNA capping by the nsP1 capping pores

Alphaviruses are emerging positive stranded RNA virus which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The non-structural protein 1 (nsP1) is responsible for RNA capping and the gating of replication organelles by assembling into monotopic membrane-associated dodecameric pores (Jones R. et al. Nature 2021). The capping path is unique for Alphavirus; beginning with the N7 methylation of a GTP molecule, followed by the covalent linkage of a m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA (Ahola T. et al. PNAS 1995). Here we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and SAM, how it reaches a metastable post-methylation state with SAH and m7GTP in the active site, the subsequent covalent transfer of m7GMP to nsP1 and post-reaction conformational changes triggering the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of intermediates of the reaction. Our data identify the molecular determinants allowing each pathway transition, provide explanation for the need for the SAM methyl donor all along the pathway and new clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together our results set new ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals. Significance statementHere we present biochemical and structural characterization of the capping pathway carried out by the Chikungunya virus nsP1 capping pores. We provide five Cryo-EM structures representative of the different steps of the reaction. These structures reveal the molecular determinants and dynamics associated with the alphavirus capping process. In addition, we biochemically show the RNA capping specificity and the reversibility of the reaction which allow nsP1 to cap and decap RNAs and to release intermediates of the reaction. These data provide a new biochemical clues on the enzymatic activity of nsP1 capping pores and a new structural landscape that will be instrumental for the design of effective antivirals targeting the viral RNA capping for blocking the infection.

molecular biology↗

bioRxiv2022

Structure of the human heparan sulfate polymerase complex EXT1-EXT2

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains with their catalytic sites facing in opposite directions. Along with in vitro activity assays using fluorescently labeled and chemically defined substrates, these findings provide a molecular insight into donor substrate recognition and demonstrate that the glycosyltransferase reactions are highly specific. A combination of in vitro and in cellulo mutational studies was used to dissect the functional role of the four catalytic sites. While EXT1 is able to catalyze both glycosyltransferase reactions, EXT2 harbors only N-acetylglucosamine transferase activity. Our results provide mechanistic insight into heparan sulfate chain elongation as a non processive process and lay the cornerstone for future studies on EXT1-EXT2 function in health and disease.

molecular biology↗

bioRxiv2022

A Duplex Structure of SARM1 Octamers Induced by a New Inhibitor

In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1 by stabilizing the ARM-dependent inactive, compact octamer ring conformation, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a Calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also promotes the formation of a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 [A] resolution. In the duplex, each ARM domain protomer is not only engaged in lateral interactions with neighboring protomers but is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.

biochemistry↗

bioRxiv2021

ADAPs intrinsically disordered region is an actin sponge regulating T cell motility

Intrinsically disordered proteins (IDPs) play a vital role in biological processes that rely on transient molecular compartmentation1. In T cells, the dynamic switching between migration and adhesion mandates a high degree of plasticity in the interplay of adhesion and signaling molecules with the actin cytoskeleton2,3. Here, we show that the N-terminal intrinsically disordered region (IDR) of adhesion- and degranulation-promoting adapter protein (ADAP) acts as a multipronged scaffold for G- and F-actin, thereby promoting actin polymerization and bundling. Positively charged motifs, along a sequence of at least 200 amino acids, interact with both longitudinal sides of G-actin in a promiscuous manner. These polymorphic interactions with ADAP become constrained to one side once F-actin is formed. Actin polymerization by ADAP acts in synergy with a capping protein but competes with cofilin. In T cells, ablation of ADAP impairs adhesion and migration with a time-dependent reduction of the F-actin content in response to chemokine or T cell receptor (TCR) engagement. Our data suggest that IDR-assisted molecular crowding of actin above the critical concentration defines a new mechanism to regulate cytoskeletal dynamics. The principle of IDRs serving as molecular sponges to facilitate regulated self-assembly of filament-forming proteins might be a general phenomenon.

biochemistry↗

bioRxiv2021

Multi-tier mechanics control stromal adaptations in swelling lymph nodes

Lymph nodes (LNs) comprise two main structural elements: Fibroblastic reticular cells (FRCs) that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. While LNs are fairly stable in size during homeostatic conditions, immunological challenge causes more than 10-fold increase in size within only a few days. How a solid organ can accommodate such extreme volumetric changes is poorly understood. Here, we characterize the biomechanics of LN swelling on the cellular and organ scale. We identify lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing FRCs and their associated conduits to stretch. After an initial phase of relaxation, FRCs sense the resulting strain via cell matrix adhesions, which coordinates local growth and remodeling of the stromal network. While the expanded FRC network adopts its typical configuration, a massive fibrotic reaction of the organ capsule sets in and counters further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multi-tier fashion.

immunology↗

bioRxiv2021

Mechanosensitivity of amoeboid cells crawling in 3D

Efficient immune-responses require migrating leukocytes to be in the right place at the right time. When crawling through the body amoeboid leukocytes must traverse complex three-dimensional tissue-landscapes obstructed by extracellular matrix and other cells, raising the question how motile cells adapt to mechanical loads to overcome these obstacles. Here we reveal the spatio-temporal configuration of cortical actin-networks rendering amoeboid cells mechanosensitive in three-dimensions, independent of adhesive interactions with the microenvironment. In response to compression, Wiskott-Aldrich syndrom protein (WASp) assembles into dot-like structures acting as nucleation sites for actin spikes that in turn push against the external load. High precision targeting of WASp to objects as delicate as collagen fibers allows the cell to locally and instantaneously deform its viscoelastic surrounding in order to generate space for forward locomotion. Such pushing forces are essential for fast and directed leukocyte migration in fibrous and cell-packed tissues such as skin and lymph nodes. In BriefWASp-driven actin spikes counter compressive loads of crowded tissue-landscapes.

cell biology↗

bioRxiv2020

The Structural Basis for SARM1 Inhibition, and Activation Under Energetic Stress

SARM1 is a central executor of axonal degeneration (1). Mechanistically, SARM1 contains NADase activity, which, in response to nerve injury, depletes the key cellular metabolite, NAD+ (2-5). Interestingly, SARM1 knockout mouse models do not present any apparent physiological impairment. Yet, the lack of SARM1 protects against various neuropathies (6, 7), thereby highlighting SARM1 as a likely safe and effective drug target (8). However, the absence of a SARM1 structure, in its active or inhibited form, makes it impossible to understand the molecular basis of SARM1 inhibition, and its activation under stress conditions. In this study we present two cryo-EM maps of SARM1 (at 2.6 [A] and 2.9 [A] resolution). We show that the inhibited SARM1 homo-octamer assumes a packed conformation with well-ordered inner and peripheral rings. Here the catalytic TIR domains are held apart from each other and are unable to form dimers, which is a prerequisite for NADase activity. More importantly, after screening several cellular metabolites we discovered that the inactive conformation is stabilized by the binding of SARM1s own substrate: NAD+. The NAD+ inhibitory allosteric site is located away from the NAD+ catalytic site of the TIR domain. Site-directed mutagenesis of the allosteric site leads to constitutive active SARM1. Based on our data we propose that a reduction of cellular NAD+ concentrations (an early indication of disease-associated and age-related neurodegeneration (9)) disassemble SARM1s peripheral ring, which allows NADase activity. This leads to an energetic catastrophe and eventually cell death. The discovery of the allosteric inhibitory site opens the door for the development of effective drugs that will prevent SARM1 activation, rather than compete for binding to the NADase catalytic site. Brief descriptionIt is not known how NAD+ depletion brings about neurodegeneration. Here, we show that the intrinsic NADase activity of SARM1 is allosterically inhibited by physiological concentrations of NAD+. NAD+ stabilizes a compact, auto-inhibited conformation of the SARM1 octamer. Once NAD+ levels are depleted, the allosteric inhibition is released, enabling SARM1s NADase activity, which eventually leads to energetic catastrophe and cell death.

biochemistry↗