A recent study of the D614G variant spike protein of SARS-CoV-2 sheds light on a mechanism underlying the relatively increased infectivity of virions expressing it and has implications for vaccine ...design.
Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human ...fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost ...advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas 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 noninfectious.
ER-associated degradation (ERAD) is essential for protein quality control in the ER, not only when the ER is stressed, but also at steady state. We report a new layer of homeostatic control, in which ...ERAD activity itself is regulated posttranscriptionally and independently of the unfolded protein response by adjusting the endogenous levels of EDEM1, OS-9, and SEL1L (ERAD enhancers). Functional UBC6e requires its precise location in the ER to form a supramolecular complex with Derlin2. This complex targets ERAD enhancers for degradation, a function that depends on UBC6e’s enzymatic activity. Ablation of UBC6e causes upregulation of active ERAD enhancers and so increases clearance not only of terminally misfolded substrates, but also of wild-type glycoproteins that fold comparatively slowly in vitro and in vivo. The levels of proteins that comprise the ERAD machinery are thus carefully tuned and adjusted to prevailing needs.
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•Deletion of UBC6e upregulates selective ERAD enhancers without activation of the UPR•Levels of ERAD enhancers are controlled by UBC6e-containing complexes•UBC6e deficiency causes hyperactive ERAD through functional ERAD enhancers•Slow folding glycoprotein is more rapidly degraded in UBC6e−/− cells and mice
Hagiwara et al. describe a new layer of ERAD control exerted by the ER-resident E2 ubiquitin-conjugating enzyme, UBC6e. Functional EDEM1, OS-9, and SEL1L accumulate in UBC6e-deficient cells and not only accelerate degradation of misfolded ERAD substrates, but also lead to premature degradation of normal glycoproteins that fold comparatively slowly.
Tissue-resident memory CD8
T cells (T
) constitute a major component of the immune-surveillance system in nonlymphoid organs. Local, noncognate factors are both necessary and sufficient to support ...the programming of T
cell fate in tissue-infiltrating T cells. Recent evidence suggests that TCR signals received in infected nonlymphoid tissues additionally contribute to T
cell formation. Here, we asked how antigen-dependent pathways influence the generation of skin-resident memory T cells that arise from a polyclonal repertoire of cells induced by infection with an antigenically complex virus and recombinant vaccine vector. We found that CD8
T cells of different specificities underwent antigen-dependent competition in the infected tissue, which shaped the composition of the local pool of T
cells. This local cross-competition was active for T cells recognizing antigens that are coexpressed by infected cells. In contrast, T
cell development remained largely undisturbed by the presence of potential competitors when antigens expressed in the same tissue were segregated through infection with antigenically distinct viral quasispecies. Functionally, local cross-competition might serve as a gatekeeping mechanism to regulate access to the resident memory niche and to fine-tune the local repertoire of antiviral T
cells.
Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are ...mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.
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•NLRP3 inflammasome activation by imiquimod and CL097 is K+ efflux-independent•Imiquimod and CL097 inhibit NQO2 and Complex I•Imiquimod and CL097 trigger ROS and cysteine oxidation•NLRP3 activation by these molecules requires ROS, Complex I dysfunction, and NEK7
Potassium (K+) efflux is widely accepted as a universal requirement for NLRP3 inflammasome activation. Groß et al. report that the topical immunomodulator imiquimod and the related molecule CL097 trigger K+ efflux-independent NLRP3 activation by targeting the ROS-producing quinone oxidoreductases NQO2 and mitochondrial Complex I.
Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two ...transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.
ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated ...in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1−/− mice have reduced viability and fail to thrive early after birth. Male Ube2j1−/− mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1−/− elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control.
Background: The physiological roles of many ER quality control components are unknown.
Results: Male Ube2j1−/− mice are sterile with defects in flagella and acrosome function, and cytoplasm removal in sperm cells.
Conclusion: Spermatogenesis is a previously unknown process requiring ER quality control.
Significance: ER quality control components might serve as diagnostic or therapeutic targets for male infertility in the future.
Fungal infections are major causes of morbidity and mortality, especially in immunocompromised individuals. The innate immune system senses fungal pathogens through Syk-coupled C-type lectin ...receptors (CLRs), which signal through the conserved immune adaptor Card9. Although Card9 is essential for antifungal defense, the mechanisms that couple CLR-proximal events to Card9 control are not well defined. Here, we identify Vav proteins as key activators of the Card9 pathway. Vav1, Vav2, and Vav3 cooperate downstream of Dectin-1, Dectin-2, and Mincle to engage Card9 for NF-κB control and proinflammatory gene transcription. Although Vav family members show functional redundancy, Vav1/2/3−/− mice phenocopy Card9−/− animals with extreme susceptibility to fungi. In this context, Vav3 is the single most important Vav in mice, and a polymorphism in human VAV3 is associated with susceptibility to candidemia in patients. Our results reveal a molecular mechanism for CLR-mediated Card9 regulation that controls innate immunity to fungal infections.
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•Vav proteins control CLR-mediated inflammatory responses•CLR-induced NF-κB activation is regulated by Vav proteins•Vav/Card9 signaling is critical for antifungal host defense
CLR/Card9 signaling is essential for antifungal immunity. Roth et al. have identified the Vav family of proteins as key activators of the Card9/NF-kB pathway, essential for CLR-induced inflammatory responses and host defense against fungi.
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