Synthetic protein circuits for programmable control of mammalian cell death.

Natural cell death pathways such as apoptosis and pyroptosis play dual roles: they eliminate harmful cells and modulate the immune system by dampening or stimulating inflammation. Synthetic protein circuits capable of triggering specific death programs in target cells could similarly remove harmful cells while appropriately modulating immune responses. However, cells actively influence their death modes in response to natural signals, making it challenging to control death modes. Here, we introduce naturally inspired "synpoptosis" circuits that proteolytically regulate engineered executioner proteins and mammalian cell death. These circuits direct cell death modes, respond to combinations of protease inputs, and selectively eliminate target cells. Furthermore, synpoptosis circuits can be transmitted intercellularly, offering a foundation for engineering synthetic killer cells that induce desired death programs in target cells without self-destruction. Together, these results lay the groundwork for programmable control of mammalian cell death.

FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis.

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

Structures and activation mechanism of the Gabija anti-phage system.

Prokaryotes have evolved intricate innate immune systems against phage infection1-7. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB8. GajA functions as a DNA endonuclease that is inactive in the presence of ATP9. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg2+. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.

Oligomerization-mediated activation of a short prokaryotic Argonaute.

Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P)+ to induce bacterial cell death1. Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR-APAZ, an associated protein2. SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA-DNA-bound state, two dimeric RNA-DNA-bound states and a tetrameric RNA-DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. The RNA-DNA-binding channel of apo inactive SPARTA is occupied by an auto-inhibitory motif in TIR-APAZ. After the binding of RNA-DNA, SPARTA transitions from a monomer to a symmetric dimer and then an asymmetric dimer, in which two TIR domains interact through charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolysing NAD(P)+. In addition, we observe unique features of interactions between SPARTA and RNA-DNA, including competition between the DNA 3' end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA-DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily.

The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages.

The interleukin-1 (IL-1) family cytokines are cytosolic proteins that exhibit inflammatory activity upon release into the extracellular space. These factors are released following various cell death processes, with pyroptosis being a common mechanism. Recently, it was recognized that phagocytes can achieve a state of hyperactivation, which is defined by their ability to secrete IL-1 while retaining viability, yet it is unclear how IL-1 can be secreted from living cells. Herein, we report that the pyroptosis regulator gasdermin D (GSDMD) was necessary for IL-1ß secretion from living macrophages that have been exposed to inflammasome activators, such as bacteria and their products or host-derived oxidized lipids. Cell- and liposome-based assays demonstrated that GSDMD pores were required for IL-1ß transport across an intact lipid bilayer. These findings identify a non-pyroptotic function for GSDMD, and raise the possibility that GSDMD pores represent conduits for the secretion of cytosolic cytokines under conditions of cell hyperactivation.

Gasdermin D pore structure reveals preferential release of mature interleukin-1.

As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1ß and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1ß and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1ß upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.

Gasdermin E suppresses tumour growth by activating anti-tumour immunity.

Cleavage of the gasdermin proteins to produce pore-forming amino-terminal fragments causes inflammatory cell death (pyroptosis)1. Gasdermin E (GSDME, also known as DFNA5)-mutated in familial ageing-related hearing loss2-can be cleaved by caspase 3, thereby converting noninflammatory apoptosis to pyroptosis in GSDME-expressing cells3-5. GSDME expression is suppressed in many cancers, and reduced GSDME levels are associated with decreased survival as a result of breast cancer2,6, suggesting that GSDME might be a tumour suppressor. Here we show that 20 of 22 tested cancer-associated GSDME mutations reduce GSDME function. In mice, knocking out Gsdme in GSDME-expressing tumours enhances, whereas ectopic expression in Gsdme-repressed tumours inhibits, tumour growth. This tumour suppression is mediated by killer cytotoxic lymphocytes: it is abrogated in perforin-deficient mice or mice depleted of killer lymphocytes. GSDME expression enhances the phagocytosis of tumour cells by tumour-associated macrophages, as well as the number and functions of tumour-infiltrating natural-killer and CD8+ T lymphocytes. Killer-cell granzyme B also activates caspase-independent pyroptosis in target cells by directly cleaving GSDME at the same site as caspase 3. Uncleavable or pore-defective GSDME proteins are not tumour suppressive. Thus, tumour GSDME acts as a tumour suppressor by activating pyroptosis, enhancing anti-tumour immunity.

Inflammation NODs to Antagonists of RIP2-XIAP Interaction.

While innate immunity is crucial for host defense, dysregulated signaling activation leads to pathological inflammation. In this issue of Molecular Cell, Goncharov et al. (2018) present a strategy to combat inflammatory diseases by disrupting RIP2-XIAP interaction in NOD2-mediated signaling.

Electrostatic influence on IL-1 transport through the GSDMD pore.

A variety of signals, including inflammasome activation, trigger the formation of large transmembrane pores by gasdermin D (GSDMD). There are primarily two functions of the GSDMD pore, to drive lytic cell death, known as pyroptosis, and to permit the release of leaderless interleukin-1 (IL-1) family cytokines, a process that does not require pyroptosis. We are interested in the mechanism by which the GSDMD pore channels IL-1 release from living cells. Recent studies revealed that electrostatic interaction, in addition to cargo size, plays a critical role in GSDMD-dependent protein release. Here, we determined computationally that to enable electrostatic filtering against pro-IL-1ß, acidic lipids in the membrane need to effectively neutralize positive charges in the membrane-facing patches of the GSDMD pore. In addition, we predicted that salt has an attenuating effect on electrostatic filtering and then validated this prediction using a liposome leakage assay. A calibrated electrostatic screening factor is necessary to account for the experimental observations, suggesting that ion distribution within the pore may be different from the bulk solution. Our findings corroborate the electrostatic influence of IL-1 transport exerted by the GSDMD pore and reveal extrinsic factors, including lipid and salt, that affect the electrostatic environment.

Highly Sensitive Diethylamine Detection at Room Temperature Using g-C3N4 Nanosheets Decorated with CuO Hollow Polyhedral Structures.

Chemiresistive-based metal oxide semiconductor (MOS) gas sensors are widely used in gas sensing due to their advantageous properties. Graphitic carbon nitride (g-C3N4) and metal oxide heterostructure materials can improve charge transport properties, selectivity, and sensitivity in MOS gas sensor materials. Herein, for the first time, CuO hollow polyhedral structures (HPSs) were synthesized via a hydrothermal technique and annealed at different temperatures, with the 400 °C annealed (CuO-400 HPSs) demonstrating remarkable sensing capabilities for diethylamine (DEA) gas at room temperature (RT). The x-g-C3N4 nanosheets were decorated with CuO HPSs in varying amounts (x = 0.8, 1.8, 2.1, and 3.1 wt %) and then annealed at 400 °C for x-g-C3N4-CuO-400 hollow polyhedral heterostructures (HPHSs). Indeed, among the synthesized samples, the 1.8%-g-C3N4-CuO-400 HPHSs have a higher sensitivity to DEA (resistance change in gas (Rg) and air (Ra); Rg/Ra= 65 @ 20 ppm), a low detection limit (Rg/Ra= 6 @ 500 ppb), wide dynamic response (Rg/Ra= 190 @ 80 ppm), strong stability (30 days), and 21.6 times higher sensitivity than pure CuO at RT toward 20 ppm of DEA. The exceptional gas-sensing behavior can be attributed to various factors, including controlled annealing conditions that result in the formation of well-defined structures and greater porosity, efficient charge transfer properties resulting from an optimized ratio of g-C3N4 to CuO in HPHSs, an abundance of defects, unsaturated Cu sites, and synergistic effects. The study presents a universal strategy for generating sensitive and selective g-C3N4-based composite materials for low-temperature gas sensors.

Deciphering the Mechanisms of Photo-Enhanced Catalytic Activities in Plasmonic Pd-Au Heteromeric Nanozymes for Colorimetric Analysis.

The growing demand for highly active nanozymes in various fields has led to the development of several strategies to enhance their activity. Plasmonic enhancement, a strategy used in heterogenous catalysis, represents a promising strategy to boost the activity of nanozymes. Herein, Pd-Au heteromeric nanoparticles (Pd-Au dimers) with well-defined heterointerfaces have been explored as plasmonic nanozymes. As a model system, the Pd-Au dimers with integrated peroxidase (POD)-like activity and plasmonic activity are used to investigate the effect of plasmons on enhancing the activity of nanozymes under visible light irradiation. Mechanistic studies revealed that the generation of hot electron-hole pairs plays a dominant role in plasmonic effect, and it greatly enhances the decomposition of H2 O2 to the reactive oxygen species (ROS) intermediates (•OH, •O2 - and 1 O2 ), leading to elevated POD-like activity of the Pd-Au dimers. Finally, the Pd-Au dimers are applied in the plasmon-enhanced colorimetric method for the detection of alkaline phosphatase, exhibiting broad linear range and low detection limit. This study not only provides a straightforward approach for regulating nanozyme activity through plasmonic heterostructures but also sheds light on the mechanism of plasmon-enhanced catalysis of nanozymes.

Steering the Selective Production of Glycolic Acid by Electrocatalytic Oxidation of Ethylene Glycol with Nanoengineered PdBi-Based Heterodimers.

Heterodimers of metal nanocrystals (NCs) with tailored elemental distribution have emerged as promising candidates in the field of electrocatalysis, owing to their unique structures featuring heterogeneous interfaces with distinct components. Despite this, the rational synthesis of heterodimer NCs with similar elemental composition remains a formidable challenge, and their impact on electrocatalysis has remained largely elusive. In this study, Pd@Bi-PdBi heterodimer NCs are synthesized through an underpotential deposition (UPD)-directed growth pathway. In this pathway, the UPD of Bi promotes a Volmer-Weber growth mode, allowing for judicious modulation of core-satellite to heterodimer structures through careful control of supersaturation and growth kinetics. Significantly, the heterodimer NCs are employed in the electrocatalytic process of ethylene glycol (EG) with high activity and selectivity. Compared with pristine Pd octahedra and common PdBi alloy NC, the unique heterodimer structure of the Pd@Bi-PdBi heterodimer NCs endows them with the highest electrocatalytic performance of EG and the best selectivity (≈93%) in oxidizing EG to glycolic acid (GA). Taken together, this work not only heralds a new strategy for UPD-directed synthesis of bimetallic NCs, but also provides a new design paradigm for steering the selectivity of electrocatalysts.

Probing local charge transfer processes of Pt-Au heterodimers in plasmon-enhanced electrochemistry by CO stripping techniques.

CO-stripping experiments are employed as a highly structure-sensitive and in situ strategy to explore the mechanisms of plasmon-enhanced electrooxidation reactions. By using Pt-Au heterodimers as a model catalyst, the plasmon-induced current and potential changes on Pt and Au sites can be identified and explained.

Cryo-EM structure of the gasdermin A3 membrane pore.

Gasdermins mediate inflammatory cell death after cleavage by caspases or other, unknown enzymes. The cleaved N-terminal fragments bind to acidic membrane lipids to form pores, but the mechanism of pore formation remains unresolved. Here we present the cryo-electron microscopy structures of the 27-fold and 28-fold single-ring pores formed by the N-terminal fragment of mouse GSDMA3 (GSDMA3-NT) at 3.8 and 4.2 Å resolutions, and of a double-ring pore at 4.6 Å resolution. In the 27-fold pore, a 108-stranded anti-parallel ß-barrel is formed by two ß-hairpins from each subunit capped by a globular domain. We identify a positively charged helix that interacts with the acidic lipid cardiolipin. GSDMA3-NT undergoes radical conformational changes upon membrane insertion to form long, membrane-spanning ß-strands. We also observe an unexpected additional symmetric ring of GSDMA3-NT subunits that does not insert into the membrane in the double-ring pore, which may represent a pre-pore state of GSDMA3-NT. These structures provide a basis that explains the activities of several mutant gasdermins, including defective mutants that are associated with cancer.

Enhanced Luminol Chemiluminescence with Oxidase-like Properties of FeOOH Nanorods for the Sensitive Detection of Uric Acid.

FeOOH nanorods, as one-dimensional nanomaterials, have been widely used in many fields due to their stable properties, low cost, and easy synthesis, but their application in the field of chemiluminescence (CL) is rarely reported. In this work, FeOOH nanorods were synthesized by a simple and environmentally friendly one-pot hydrothermal method and used for the first time as a catalyst for generating strong CL with luminol without additional oxidant. Remarkably, luminol-FeOOH exhibits about 250 times stronger CL than the luminol-H2O2 system. Its CL intensity was significantly quenched by uric acid. We established a simple, rapid, sensitive, and selective CL method for the detection of uric acid with a linear range of 20-1000 nM and a detection limit of 6.3 nM (S/N = 3). In addition, we successfully applied this method to the detection of uric acid in human serum, and the standard recoveries were 95.6-106.4%.

Ultrafast Response and High Selectivity of Diethylamine Gas Sensors at Room Temperature Using MOF-Derived 1D CuO Nano-Ellipsoids.

Environmental and health monitoring requires low-cost, high-performance diethylamine (DEA) sensors. Materials based on metal-organic frameworks (MOFs) can detect hazardous gases due to their large specific surface area, many metal sites, unsaturated sites, functional connectivity, and easy calcination to remove the scaffold. However, developing facile materials with high sensitivity and selectivity in harsh environments for accurate DEA detection at a low detection limit (LOD) at room temperature (RT) is challenging. In this study, p-type semiconducting porous CuOx sensing materials were synthesized using a simple solvothermal process and annealed in an argon atmosphere at three different temperatures (x = 400, 600, and 800 °C). Significant variations in particle size, specific area, crystallite size, and shape were noticed when the annealing temperature was elevated. Cu-MIL-53 annealed at 400 °C (CuO-400) has a typical nanoellipsoid (NEs) shape with a length of 61.5 nm and a diameter of 33.2 nm. Surprisingly, CuO-400 NEs showed an excellent response to DEA with an ultra-LOD (Rg/Ra = 7.3 @ 100 ppb, 55% relative humidity), excellent selectivity and sensitivity (Rg/Ra = 236 @ 15 ppm), exceptional long-term stability and repeatability, and a fast response/recovery period at RT, outperforming most previously reported materials. CuO-400 NEs have outstanding gas-sensing characteristics due to their high porosity, 1D nanostructure, unsaturated Cu sites (Cu+ and Cu2+), large specific surface area, and numerous oxygen vacancies. This study presents a generic approach to produce future CuO derived from Cu-MOFs-sensitive materials, revealing new insights into the design of effective sensors for environmental monitoring at RT.

Remarkably Enhanced Luminol/H2O2 Chemiluminescence with Excellent Peroxidase-like Activity of FeCoNi-based Metal-Organic Xerogels for the Sensitive Detection of Dopamine.

Metal-organic gels (MOGs) are a category of metal-organic smart soft materials with large specific surface areas, loose porous structures, and open metal active sites. In this work, trimetallic Fe(III)Co(II)Ni(II)-based MOGs (FeCoNi-MOGs) were synthesized at room temperature via a simple and mild one-step procedure. Fe3+, Co2+, and Ni2+ were the three central metal ions in it, while 1,3,5-benzenetricarboxylic acid (H3BTC) served as the ligand. The solvent enclosed in it was then removed by freeze-drying to get the corresponding metal-organic xerogels (MOXs). The as-prepared FeCoNi-MOXs have excellent peroxidase-like activity and can significantly enhance luminol/H2O2 chemiluminescence (CL) by more than 3000 times, which is very effective compared with other reported MOXs. Based on the inhibitory effect of dopamine on the CL of the FeCoNi-MOXs/luminol/H2O2 system, a simple, rapid, sensitive, and selective CL method for dopamine detection was established with a linear range of 5-1000 nM and a limit of detection of 2.9 nM (LOD, S/N = 3). Furthermore, it has been effectively used for the quantitative measurement of dopamine in dopamine injections and human serum samples, with a recovery rate of 99.5-109.1%. This research brings up prospects for the application of MOXs with peroxidase-like activity in CL.

Maneuvering the Peroxidase-Like Activity of Palladium-Based Nanozymes by Alloying with Oxophilic Bismuth for Biosensing.

Engineering the catalytic performance of nanozymes is of vital importance for their broad applications in biological analysis, cancer treatment, and environmental management. Herein, a strategy to boost the peroxidase-like activity of Pd-based nanozymes with oxophilic metallic bismuth (Bi) is demonstrated, which is based on the incorporation of oxophilic Bi in the Pd-based alloy nanocrystals (NCs). To synthesize PdBi alloy NCs, a seed-mediated method is employed with the assistance of underpotential deposition (UPD) of Bi on Pd. The strong interaction of Bi atoms with Pd surfaces favors the formation of alloy structures with controllable shapes and excellent monodispersity. More importantly, the PdBi NCs show excellent peroxidase-like activities compared with pristine Pd NCs. The structure-function correlations for the PdBi nanozymes are elucidated, and an indirect colorimetric method based on cascade reactions to determine alkaline phosphatase (ALP) is established. This method has good linear range, low detection limit, excellent selectivity, and anti-interference. Collectively, this work not only provides new insights for the design of high-efficiency nanozymes, expands the colorimetric sensing platform based on enzyme cascade reactions, but also represents a new example for UPD-directed synthesis of alloy NCs.

Clinical and pathological features of omicron variant of SARS-CoV-2-associated kidney injury.

Kidney injury is common in patients with Coronavirus Disease-19 (COVID-19), which is related to poor prognosis. We aim to summarize the clinical features, athological types, and prognosis of COVID-19 associated kidney injury caused by the Omicron strain. In this study, 46 patients with Omicron-associated kidney injury were included, 38 of whom performed renal biopsy. Patients were divided into two groups: group A for patients with onset of kidney injury after SARS-CoV-2 infection; group B for patients with pre-existing kidney disease who experienced aggravation of renal insufficiency after SARS-CoV-2 infection. The clinical, pathological, and prognostic characteristics of the patients were observed. Acute kidney injury (AKI) (35%) was the most common clinical manifestation in group A. Patients in group B mainly presented with chronic kidney disease (CKD) (55%) and nephrotic syndrome (NS) (40%). The pathological type was mainly IgA nephropathy (IgAN) (39% in group A and 45% in group B). Among all of them, one case presenting with thrombotic microangiopathy had worse kidney function at biopsy time. Mean serum C3 levels were 1.2 ± 0.5 and 1.0 ± 0.2 g/L in group A and group B, respectively. In renal tissues, C3 deposits were observed in 71.1% of patients. 11.8% (n = 2) patients experienced deterioration of renal function after treatment, but no patients developed to end-stage renal disease. In our single-center study in China, the main clinical manifestations were AKI, CKD, and NS, while the main pathological type was IgAN. Compared with previous strains of SARS-CoV-2, patients with the Omicron infection had a favorable prognosis.