Biology subjects

Hons, M.

Publications and source records attributed to Hons, M..

5 recordsLinked to original sources

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↗