Biology subjects

Andreeva, L.

Publications and source records attributed to Andreeva, L..

6 recordsLinked to original sources

bioRxiv2026

Human NLRC4 can act as a direct sensor for cytosolic flagellin

Innate immune cells sense pathogenic bacteria like Legionella pneumophila through patterns such as the protein flagellin, a critical component of the bacterial motility apparatus. Recognition of cytosolic flagellin in mouse immune cells is well understood and mediated by the receptors, neuronal apoptosis inhibitory protein (Naip) 5 or Naip6, which activate the Nlrc4 inflammasome multi-protein complex for initiating cell death or interleukin-1 family cytokine release. However, the role of human NAIP as a cytosolic flagellin sensor remains controversial. Using a multipronged approach, we demonstrate that in a reconstituted cell system human NLRC4 engaged Legionella FlaA flagellin directly (i.e. without the need for hNAIP), whereas human NAIP did not interact with FlaA. Ectopic cytosolic FlaA expression also induced NLRC4 oligomerization, a prerequisite for inflammasome activation, in the absence of NAIP. Unexpectedly, the presence of NAIP diminished the binding of NLRC4 to flagellins and subsequent interleukin-1{beta} release. Interestingly, in resting THP-1 cells, NAIP stably interacted with NLRC4, and during infection or stimulation with FlaA pro-inflammatory responses in THP-1 cells were predominantly NLRC4-dependent. Our data highlight NLRC4 as a putative direct sensor of cytosolic flagellins in the human system and NAIP as a potential negative regulator of flagellin sensing.

immunology↗

bioRxiv2026

Structure of zebrafish NLRP3 reveals a novel mode of inflammasome activation

NLRP3 is an innate immune sensor of a broad range of stimuli, which upon activation forms a multiprotein inflammasome complex triggering caspase-1 activation, IL-1{beta} and IL-18 maturation, and inflammatory cell death. The canonical NLRP3 activation pathway has been well characterized from a structural perspective. It involves the association of NLRP3 with membranes in the form of inactive oligomeric "cage" complexes, which, upon activation, convert to an active oligomeric NLRP3 disc. NLRP3 structural rearrangements during non-classical NLRP3 activation pathways, however, remain unknown. Here, we report a novel mode of NLRP3 activation utilized by the NLRP3 homolog from zebrafish. The cryo-EM structure of zebrafish NLRP3 shows that, unlike human NLRP3, it forms disc-shaped heptamers that undergo further trimerization, resulting in a 21-mer oligomeric arrangement. Surprisingly, a single zebrafish NLRP3 heptamer cannot arrange its PYD domains into a PYD helix and therefore requires a trimer of heptamers to form a PYD filament that enables ASC oligomerization. Furthermore, zebrafish NLRP3 does not associate with the Golgi network, nor does it form inactive "cage" oligomers or interact with NEK7. Thus, our data demonstrate an ancestral non-canonical structural mechanism of NLRP3 activation, which may shed light on alternative NLRP3 activation pathways present in humans.

immunology↗

bioRxiv2026

Crystal structure of E. coli Nissle 1917 flagellin reveals novel features that modulate bacterial motility but not TLR5 recognition

The probiotic E. coli Nissle 1917 (EcN) strain is known to promote intestinal homeostasis via flagellin, the protomer of its motility apparatus, the flagellum. The flagellin of EcN shows atypical features, namely a hypervariable region (HVR), whose structure and significance have remained elusive. We therefore determined the crystal structure of the E. coli Nissle 1917 flagellin FliC at a resolution of 1.2 [A] which revealed an unusual domain architecture: the canonical D1 domain was found connected by an extended linker to an extensive HVR whose D2, D3 and D4 domains form an outer domain (OD) which surrounds the filament core comprised of conserved domains D0-D1. Using both recombinant proteins and gene-edited EcN strains expressing mutant flagellins, the functional requirement for these unique features was subsequently studied for effects on immune recognition on intestinal epithelial and immune cells, as well as on flagellar protein expression, assembly and bacterial motility. While human and mouse TLR5 immune recognition of flagellar proteins or intact bacteria was only moderately affected by removal of linker or D4, especially linker removal reduced protein stability and bacterial motility in both soft agar and liquid media swimming assays. Interestingly, depending on the environment, D4 or HVR removal had different effects on motility and surface structure. Finally, a site-directed mutagenesis approach highlighted that loss of TLR5 recognition strictly entails loss of motility but not vice versa. Our data indicate that specific HVRs/OD might be relevant for motility of E. coli Nissle 1917 in specialized environments, but not for immune recognition. Moreover, we find mutational tolerance is greater for immune recognition than for motility, providing new insights into bacterial adaptation to the host environment.

microbiology↗

bioRxiv2026

NLRP3 acts as a direct sensor of intracellular potassium ions

The NLRP3 inflammasome is a sentinel of cellular homeostasis, and its activation triggers the assembly of a molecular machinery that drives inflammation in infection, cardiovascular, metabolic, and neurodegenerative diseases. The majority of the many triggers known to activate NLRP3 are believed to induce potassium ion (K+) efflux from the cell as a fundamental danger signal for compromised cellular integrity. However, it has remained unclear how a reduction in intracellular K+ concentration is mechanistically translated into conformational changes in NLRP3 that promote inflammasome assembly, interleukin (IL)-1 release, and cell death. Here, we provide evidence that alterations in K+ levels directly regulate the conformation of the NLRP3 protein. In cell-free lysates derived from cell lines and primary blood immune cells high K+ concentrations stabilized a compact, protease-resistant structure resembling inhibitor-bound NLRP3, whereas low K+ conditions or the presence of a K+ chelator favored an open, more flexible and protease-accessible conformation. Notably, human NLRP3 remained responsive to K+ even when exogenously expressed in macrophage-like Drosophila cells or purified as recombinant protein. This indicates that K+ sensing occurs independently of cellular co-factors and is consistent with direct ion coordination. Of note, stimulation with the K+-independent NLRP3 agonist CL097 failed to recapitulate the conformational transition caused by K+ efflux inducer, nigericin. Moreover, pathogenic gain-of-function mutant variants of NLRP3 constitutively resembled the open and flexible protease-accessible conformation. Mapping K+-interactions by high-performance computation suggested that K+ ions populate the nucleotide binding pocket of the FISNA-NACHT module of individual NLRP3 chains but also stabilize face-to-face interactions within inactive oligomeric cage assemblies via the NACHT-adjacent acidic loop. Collectively, our findings enable us to propose a mechanistic model of how intracellular K+ ions preclude NLRP3 activation prior to efflux and thus how NLRP3 responds to cellular danger as a direct K+ sensing protein.

immunology↗

bioRxiv2021

Full-length NLRP3 forms oligomeric cages to mediate NLRP3 sensing and activation

The nucleotide-binding domain and leucine-rich-repeat (LRR) containing protein 3 with a pyrin domain (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here we report that the endogenous, stimulus-responsive form of full-length NLRP3 is a 12-16 mer double ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for all NLRP3 activating stimuli, requires the double ring cages of NLRP3. Double ring-defective NLRP3 mutants further abolish inflammasome punctum formation, caspase-1 processing and cell death. Thus, unlike other inflammasome sensors that are monomeric when inactive, our data uncover a unique NLRP3 oligomer on membrane that is poised to sense diverse signals to induce inflammasome activation.

immunology↗

bioRxiv2019

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity

Activity of the NLRP3 inflammasome, a critical mediator of inflammation (1), is controlled by accessory proteins (2, 3), post-translational modifications (4, 5), cellular localization (6, 7) and oligomerization (8). How these factors relate, is unclear. We show that the established drug target, Brutons Tyrosine Kinase (BTK) (2, 9), integrates several levels of NLRP3 regulation: BTK phosphorylation of four conserved tyrosine residues, by neutralizing the charge of a polybasic linker region, weakens the interaction of NLRP3 with Golgi phospholipids and may thus guide NLRP3 cytosolic localization. BTK activity also promotes NLRP3 oligomerization and subsequent formation of inflammasomes. As NLRP3 tyrosine modification ultimately also impacts on IL-1{beta} release, we propose BTK-mediated, charge-switch-based NLRP3 regulation as a novel and therapeutically tractable step in the control of inflammation. One Sentence SummaryMulti-phosphorylation of NLRP3 by Brutons tyrosine kinase modulates NLRP3 cellular localization, inflammasome assembly, and IL-1{beta} release.

immunology↗