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The murine caspase-11 non-canonical inflammasome responds to various bacterial infections. Caspase-11 activation-induced pyroptosis, in response to cytoplasmic lipopolysaccharide (LPS), is critical for endotoxic shock in mice. The mechanism underlying cytosolic LPS sensing and the responsible pattern recognition receptor are unknown. Here we show that human monocytes, epithelial cells and keratinocytes undergo necrosis upon cytoplasmic delivery of LPS. LPS-induced cytotoxicity was mediated by human caspase-4 that could functionally complement murine caspase-11. Human caspase-4 and the mouse homologue caspase-11 (hereafter referred to as caspase-4/11) and also human caspase-5, directly bound to LPS and lipid A with high specificity and affinity. LPS associated with endogenous caspase-11 in pyroptotic cells. Insect-cell purified caspase-4/11 underwent oligomerization upon LPS binding, resulting in activation of the caspases. Underacylated lipid IVa and lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) could bind to caspase-4/11 but failed to induce their oligomerization and activation. LPS binding was mediated by the CARD domain of the caspase. Binding-deficient CARD-domain point mutants did not respond to LPS with oligomerization or activation and failed to induce pyroptosis upon LPS electroporation or bacterial infections. The function of caspase-4/5/11 represents a new mode of pattern recognition in immunity and also an unprecedented means of caspase activation. Caspase-4 and caspase-11 are shown to be the direct sensors for cytoplasmic lipopolysaccharide in humans and mice, respectively, mediating inflammatory cell death in intracellular bacterial infection. Sensing role for caspases in innate immunity A 'non-canonical' innate immune pathway, independent of Toll-like receptor 4 but involving caspase-11, was recently discovered in mice, where it acts to recognize lipopolysaccharide (LPS) from pathogenic bacteria. Here Feng Shao and colleagues investigate this pathway and a similar one in humans. They find that caspase-11 and caspase-4 are the direct sensors for cytoplasmic LPS in mice and humans, respectively, mediating inflammatory cell death in intracellular bacterial infection.

The kinase Mst1, which acts in the Hippo pathway, controls cell proliferation, differentiation and apoptosis. Junichi Sadoshima and his colleagues show that Mst1 in cardiomyocytes phosphorylates the protein Beclin1 to coordinately suppress autophagy and promote apoptosis, thereby having deleterious effects on the heart. Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.

Innate immunity to viral infection involves induction of the type I IFN response; however, dysfunctional regulation of this pathway leads to inappropriate inflammation. Here, we evaluated a nonconsanguineous family of mixed European descent, with 4 members affected by systemic inflammatory and autoimmune conditions, including lupus, with variable clinical expression. We identified a germline dominant gain-of-function mutation in TMEM173, which encodes stimulator of type I IFN gene (STING), in the affected individuals. STING is a key signaling molecule in cytosolic DNA-sensing pathways, and STING activation normally requires dimerization, which is induced by 2'3' cyclic GMP-AMP (cGAMP) produced by the cGAMP synthase in response to cytosolic DNA. Structural modeling supported constitutive activation of the mutant STING protein based on stabilized dimerization. In agreement with the model predictions, we found that the STING mutant spontaneously localizes in the Golgi of patient fibroblasts and is constitutively active in the absence of exogenous 2'3'-cGAMP in vitro. Accordingly, we observed elevated serum IFN activity and a type I IFN signature in peripheral blood from affected family members. These findings highlight the key role of STING in activating both the innate and adaptive immune responses and implicate aberrant STING activation in features of human lupus.

Chronic infections can result in harmful production of the proinflammatory cytokine IL-1 generated via the NLRP3 inflammasome. Sassetti et al . demonstrate that IL-1 activated by Mycobacteria results in nitrosylation and consequent regulation of the NLRP3 inflammasome. Interleukin 1 (IL-1) is an important mediator of innate immunity but can also promote inflammatory tissue damage. During chronic infections such as tuberculosis, the beneficial antimicrobial role of IL-1 must be balanced with the need to prevent immunopathology. By exogenously controlling the replication of Mycobacterium tuberculosis in vivo , we obviated the requirement for antimicrobial immunity and discovered that both IL-1 production and infection-induced immunopathology were suppressed by lymphocyte-derived interferon-γ (IFN-γ). This effect was mediated by nitric oxide (NO), which we found specifically inhibited assembly of the NLRP3 inflammasome via thiol nitrosylation. Our data indicate that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses elicited during persistent infections.

Although clinical trials have shown that RAF inhibitors prolong the survival of patients with BRAF-mutant melanoma, resistance inevitably develops; resistance is shown here to be frequently mediated by the expression of splicing variants of mutant BRAF. Mechanism of RAF inhibitor resistance Although recent clinical trials have shown the efficacy of B-RAF inhibitors in the treatment of melanomas with activating B-RAF mutations, the patients inevitably develop resistance. David Solit and colleagues now identify a mechanism of acquired resistance conferred by a structural change in B-RAF itself. The expression of a 61-kilodalton splice variant of mutant B-RAF leads to enhanced B-RAF dimerization, rendering it resistant to kinase inhibitors. This variant was found to be expressed in 6 of 19 patients who had developed resistance to the B-RAF inhibitor PLX4032. Activated RAS promotes dimerization of members of the RAF kinase family 1 , 2 , 3 . ATP-competitive RAF inhibitors activate ERK signalling 4 , 5 , 6 , 7 by transactivating RAF dimers 4 . In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E) 8 . However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4–8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS–GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Cas12a is a bacterial RNA-guided nuclease used widely for genome editing and, more recently, as a molecular diagnostic. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three such inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity via functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit recognition of double-stranded DNA (dsDNA), with AcrVA4 driving dimerization of Cas12a. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophages with tools to evade CRISPR-Cas12a and support biotechnological applications for which multiple-turnover enzymatic inhibition of Cas12a is desirable.Doudna and colleagues determine the mechanisms used by type V anti-CRISPR proteins. AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the Cas12a-bound guide RNA, while AcrVA4 and AcrVA5 inhibit recognition of dsDNA.

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.

Detection of more than 1,000 different T-cell specificities in parallel facilitates identification of cancer and autoimmune antigens. Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10–100 distinct T-cell specificities in one sample. Here we use peptide–major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ54-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ’s AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP–complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.

Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H2O2, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.