The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to spread with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. Here, we generated four neutralizing nanobodies that target the receptor-binding domain of the SARS-CoV-2 spike protein. We defined two distinct binding epitopes using x-ray crystallography and cryo-electron microscopy. Based on the structures, we engineered multivalent nanobodies with more than 100-fold improved neutralizing activity than monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor-binding competition, while other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion, and rendered the virions non-infectious.

Inflammasomes integrate cytosolic evidence of infection or damage to mount inflammatory responses. The inflammasome sensor NLRP1 is expressed in human keratinocytes and coordinates inflammation in the skin. We found that diverse stress signals induce human NLRP1 inflammasome assembly by activating MAP kinase p38: While the ribotoxic stress response to UV and microbial molecules exclusively activates p38 through MAP3K ZAKα, infection with arthropod-borne alphaviruses, including Semliki Forest and Chikungunya virus, activates p38 through ZAKα and potentially other MAP3K. We demonstrate that p38 directly phosphorylates NLRP1 and that serine 107 in the linker region is critical for activation. NLRP1 phosphorylation is followed by ubiquitination of NLRP1PYD, N-terminal degradation of NLRP1, and nucleation of inflammasomes by NLRP1UPA-CARD. In contrast, activation of NLRP1 by nanobody-mediated ubiquitination, viral proteases, or inhibition of DPP9 was independent of p38 activity. Taken together, we define p38 activation as a unifying signaling hub that controls NLRP1 inflammasome activation by integrating a variety of cellular stress signals relevant to the skin.

Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron-sized "specks" to maximize caspase-1 activation and the maturation of IL-1 cytokines. Caspase-1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid-derived nanobodies against ASC (VHH ASC ) target and disassemble post-pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis-driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHH ASC to target inflammasomes while preserving pre-pyroptotic IL-1b release, essential to host defense. Systemically administrated mouse-specific VHH ASC attenuated inflammation and clinical gout, and antigen-induced arthritis disease. Hence, VHH ASC neutralized post-pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHH ASC are the first biologicals that disassemble pre-formed inflammasomes while preserving their functions in host defense.

To disentangle the elusive lipid–protein interactions in T‐cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T‐cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity‐sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid‐mediated interactions in membrane‐hosted signalling events.

Inflammasomes integrate cytosolic evidence of infection or damage to mount inflammatory responses. The inflammasome sensor NLRP1 is expressed in human keratinocytes and coordinates inflammation in the skin. We found that diverse stress signals converge on the activation of p38 kinases to initiate human NLRP1 inflammasome assembly: UV irradiation and microbial molecules that initiate the ribotoxic stress response critically relied on the MAP3 kinase ZAKα to activate p38 and ultimately human NLRP1. Infection with insect-transmitted alphaviruses, including Semliki Forest, Ross River, and Chikungunya virus, also activated NLRP1 in a p38-dependent manner. In the absence on ZAKα, inflammasome assembly was maintained, although at reduced levels, indicating contribution of other upstream kinases. NLRP1 activation by direct nanobody-mediated ubiquitination was independent of p38 activity. Stimulation of p38 by overexpression of MAP2 kinases MKK3 or MKK6 is sufficient for NLRP1 activation, and NLRP1 is directly phosphorylated by p38. Taken together, we define p38 activation as a unifying signaling hub that controls NLRP1 inflammasome activation by integrating a variety of cellular stress signals relevant to the skin.

Activation of various inflammasomes converges on the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases, followed by oligomerization of the N-terminal domain (GSDMDNT) and the assembly of pores penetrating target membranes. Yet, it remained unclear what triggers the conformational changes that allow membrane insertion, as methods to study pore formation in living cells were limited. We raised nanobodies specific for human GSDMD and found two nanobodies that prevent pyroptosis and IL-1β release when expressed in the cytosol of human macrophages. Nanobody binding to GSDMDNTblocked its oligomerization, while inflammasome assembly and GSDMD processing itself were not affected. The nanobody-stabilized monomers of GSDMDNTpartitioned into the plasma membrane, suggesting that pore formation is initiated by insertion of monomers, followed by oligomerization in the target membrane. When GSDMD pore formation was inhibited, cells still underwent caspase-1-dependent apoptosis, likely due to the substantially augmented caspase-1 activity. This hints at a novel layer of regulation of caspase-1 activity by GSDMD pores. Moreover, we revealed the unexpected therapeutic potential of antagonistic GSDMD nanobodies, as recombinant nanobodies added to the medium prevented cell death by pyroptosis, likely by entering through GSDMD pores and curtailing the assembly of additional pores. GSDMD nanobodies may thus be suitable to treat the ever-growing list of diseases caused by activation of the (non-) canonical inflammasomes.

Gasdermin D (GSDMD) is a key mediator of pyroptosis, a pro-inflammatory form of cell death occurring downstream of inflammasome activation as part of the innate immune defence. Upon cleavage by inflammatory caspases, the N-terminal domain of GSDMD forms pores in the plasma membrane resulting in cytokine release and eventually cell death. Targeting GSDMD is an attractive way to dampen inflammation. In this study, six GSDMD targeting nanobodies were characterized in terms of their binding affinity, stability, and effect on GSDMD pore formation. Three of the nanobodies inhibited GSDMD pore formation in a liposome leakage assay, although caspase cleavage was not perturbed. We determined the crystal structure of human GSDMD in complex with two nanobodies at 1.9 Ang resolution, providing detailed insights into the GSDMD-nanobody interactions and epitope binding. The pore formation is sterically blocked by one of the nanobodies that binds to the oligomerization interface of the N-terminal domain in the multi-subunit assembly. Our biochemical and structural findings provide new tools for studying inflammasome biology and build a framework for the design of novel GSDMD targeting drugs.

The quest for understanding of numerous vital membrane-associated cellular processes, such as signalling, has largely focussed on the spatiotemporal orchestration and reorganisation of the identified key proteins, including their binding and aggregation. Despite strong indications of the involvement of membrane lipid heterogeneities, historically often termed lipid rafts, their roles in many processes remain controversial and mechanisms elusive. Taking activation of T lymphocytes as an example, we here investigate membrane properties around the key proteins − in particular the T cell receptor (TCR), its main kinase Lck, and phosphatase CD45. We determine their partitioning and co-localisation in passive cell-derived model membranes (i.e. giant plasma-membrane vesicles, GPMVs), and explore their mobility and local lipid order in live Jurkat T cells using fluorescence correlation spectroscopy and spectral imaging with polarity-sensitive membrane probes. We find that upon aggregation and partial immobilisation, the TCR changes its preference towards more ordered lipid environments, which can in turn passively recruit Lck. We observe similar aggregation-induced local membrane ordering and recruitment of Lck also by CD45, as well as by a membrane protein of antigen-presenting cells, CD86, which is not supposed to interact with Lck directly. This highlights the involvement of lipid-mediated interactions and suggests that the cellular membrane is poised to modulate the frequency of protein encounters according to their aggregation state and alterations of their mobility, e.g. upon ligand binding.