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Chemotherapy drugs can cause pyroptotic cell death by activating caspase-3 to cleave gasdermin E, potentially contributing to their toxicity and adverse effects. Chemotherapy-induced cell death The negative side effects of chemotherapy are well documented but the mechanisms behind them are not always known. Feng Shao and colleagues show that chemotherapy drug treatment of gasdermin-E-expressing tumour cells results in a caspase-3-dependent switch from apoptotic to pyroptotic cell death. The majority of human tumours tested appeared to have lost expression of gasdermin E, whereas normal tissues express it. This observation has potential implications for the treatment of gasdermin-E-expressing tumours, and for chemotherapy-induced tissue damage. Pyroptosis is a form of cell death that is critical for immunity. It can be induced by the canonical caspase-1 inflammasomes or by activation of caspase-4, -5 and -11 by cytosolic lipopolysaccharide 1 , 2 , 3 . The caspases cleave gasdermin D (GSDMD) in its middle linker to release autoinhibition on its gasdermin-N domain, which executes pyroptosis via its pore-forming activity 4 , 5 , 6 , 7 , 8 , 9 . GSDMD belongs to a gasdermin family that shares the pore-forming domain 4 , 6 , 10 . The functions and mechanisms of activation of other gasdermins are unknown. Here we show that GSDME, which was originally identified as DFNA5 (deafness, autosomal dominant 5) 11 , can switch caspase-3-mediated apoptosis induced by TNF or chemotherapy drugs to pyroptosis. GSDME was specifically cleaved by caspase-3 in its linker, generating a GSDME-N fragment that perforates membranes and thereby induces pyroptosis. After chemotherapy, cleavage of GSDME by caspase-3 induced pyroptosis in certain GSDME-expressing cancer cells. GSDME was silenced in most cancer cells but expressed in many normal tissues. Human primary cells exhibited GSDME-dependent pyroptosis upon activation of caspase-3 by chemotherapy drugs. Gsdme −/− (also known as Dfna5 −/− ) mice were protected from chemotherapy-induced tissue damage and weight loss. These findings suggest that caspase-3 activation can trigger necrosis by cleaving GSDME and offer new insights into cancer chemotherapy.

Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1–activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity.

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. Liver diseases related to the human hepatitis B virus (HBV) kill about 1 million people every year, and more than 350 million people around the world are infected with the virus. Some 15 million of these people are also infected with the hepatitis D virus (HDV), which is a satellite virus of HBV, and this places them at an even higher risk of liver diseases, including cancer. The viruses are known to enter liver cells by binding to receptors on their surface before being engulfed. Both HBV and HDV have outer coats that consist of three kinds of envelope proteins, and a region called the pre-S1 domain in one of them is known to have a central role in the interaction between the viruses and the receptors and, therefore, in infecting the cells. However, the identity of the HBV receptor has remained a mystery. Now Yan et al. have identified this receptor to be sodium taurocholate cotransporting polypeptide. This protein, known as NTCP for short, is normally involved in the circulation of bile acids in the body. In addition to humans, only two species are known to be susceptible to infection by human HBV and HDV—chimpanzees and a small mammal known as the treeshrew. Yan et al. started by isolating primary liver cells from treeshrews, and then used a combination of advanced purification and mass spectrometry analysis to show that the NTCP on the surface of the cells interacts with the pre-S1 domain in HBV. The authors then performed a series of gene knockdown experiments on liver cells of both human and treeshrew origin: when the gene that codes for NTCP was silenced, HBV infection was greatly reduced. Moreover, they were able to transfect HepG2 cells—which are widely used in research into liver disease, but are not susceptible to HBV and HDV infection—with NTCP from humans and treeshrews to make them susceptible. Similarly, although monkeys are not susceptible to HBV, replacing just five amino acids in monkey NTCP with their human counterparts was enough to make the monkey NTCP a functional receptor for the viruses. In the past, basic research into HBV and the development of antiviral therapeutics have both been hindered by the lack of suitable in vitro infection systems and animal models. Now, the work of Yan et al. means that it will be possible to use NTCP-complemented HepG2 cells for challenges as diverse as fundamental studies of basic viral entry/replication mechanisms and large-scale drug screening. It is also possible that HBV and HDV infection might interfere with some of the important physiological functions carried out by NTCP, so the latest work could also be of interest to medical scientists working on other diseases related to these infections.

The Pyrin inflammasome detects the presence of a pathogen not through recognition of a microbial molecule but by the activity of a bacterial toxin that modifies host Rho activity. A novel angle on innate immunity The inflammasome complex, mediated by NOD-like receptor (NLR) proteins or other Pyrin-domain pattern recognition receptors (PRRs), plays a critical role in innate immune defence against various microbial infections. Feng Shao and colleagues show that the Pyrin inflammasome detects the presence of the pathogen Burkholderia cenocepacia not by recognizing a microbial molecule, the mechanism commonly adopted by mammalian PRRs, but by sensing the response of host Rho GTPase to a bacterial toxin. Infection with B. cenocepacia is of clinical importance as it can often be fatal in cystic fibrosis and chronic granulomatous disease, therefore the findings reported here could be of relevance to vaccine development and immunotherapy prevention against this type of pathogen. Cytosolic inflammasome complexes mediated by a pattern recognition receptor (PRR) defend against pathogen infection by activating caspase 1. Pyrin, a candidate PRR, can bind to the inflammasome adaptor ASC to form a caspase 1-activating complex 1 , 2 . Mutations in the Pyrin-encoding gene, MEFV , cause a human autoinflammatory disease known as familial Mediterranean fever 3 , 4 , 5 . Despite important roles in immunity and disease, the physiological function of Pyrin remains unknown. Here we show that Pyrin mediates caspase 1 inflammasome activation in response to Rho-glucosylation activity of cytotoxin TcdB 6 , 7 , 8 , a major virulence factor of Clostridium difficile , which causes most cases of nosocomial diarrhoea. The glucosyltransferase-inactive TcdB mutant loses the inflammasome-stimulating activity. Other Rho-inactivating toxins, including FIC-domain adenylyltransferases ( Vibrio parahaemolyticus VopS and Histophilus somni IbpA) and Clostridium botulinum ADP-ribosylating C3 toxin, can also biochemically activate the Pyrin inflammasome in their enzymatic activity-dependent manner. These toxins all target the Rho subfamily and modify a switch-I residue. We further demonstrate that Burkholderia cenocepacia inactivates RHOA by deamidating Asn 41, also in the switch-I region, and thereby triggers Pyrin inflammasome activation, both of which require the bacterial type VI secretion system (T6SS). Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. Thus, Pyrin functions to sense pathogen modification and inactivation of Rho GTPases, representing a new paradigm in mammalian innate immunity.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling 1 . Recent studies indicate that the bacterial metabolite d -glycero-β- d -manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol 2 – 4 , but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-β- d -manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR–Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)–TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand–receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1 -dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively. The bacterial metabolite ADP-heptose activates NF-κB in host cells via alpha-kinase 1 and the TIFA–TRAF signalling pathway.

Background The healthcare system is currently facing challenges in transitioning to a more integrated, patient-centered care. China has implemented various practical exploration and pilot programs in developing an integrated healthcare service system. However, there remains an absence of appropriate instrument to assess integrated care from the patient’s perspective, particularly one that aligns with China's national conditions. Methods Using a literature review, Delphi methods, a pilot study involving 171 chronic patients in Guangdong Province, and the analytic hierarchy process, we developed and validated the Chinese Patient-Centered Integrated Care (CPCIC) with a weighting system. Results The results of exploratory factor analysis indicated CPCIC scale had robust structural validity. The overall Cronbach’s α coefficient was 0.868, signifying excellent internal consistency. The final scale included four first-level indicators (dimensions): services responsiveness, services convenience, services continuity and services coordination and 14 s-level indicators (items). The assigned weights of these dimensions were 0.1070, 0.2926, 0.4155 and 0.1849, respectively. Conclusions The four-dimension, 14-item CPCIC scale with a weighting system was successfully developed and validated. The scale provides a reliable and valid instrument for evaluating the degree of integrated care from the patient’s perspective, and has potential applications in informing the reform of China’s healthcare system to enhance patient-centered care.

Gasdermin-D (GSDMD), the executioner of pyroptotic cell death when it is cleaved by inflammatory caspases, plays a crucial role in host defense and the response to danger signals. So far, there are no known mechanisms, other than cleavage, for regulating GSDMD. Here, we show that tripartite motif protein TRIM21 acts as a positive regulator of GSDMD-dependent pyroptosis. TRIM21 interacted with GSDMD via its PRY-SPRY domain, maintaining GSDMD stable expression in resting cells yet inducing the N-terminus of GSDMD (GSDMD-N) aggregation during pyroptosis. TRIM21-deficient cells displayed a reduced cell death in response to NLRP3 or NLRC4 inflammasome activation. Genetic ablation of TRIM21 in mice conferred protection from LPS-induced inflammation and dextran sulfate sodium-induced colitis. Therefore, TRIM21 plays an essential role in GSDMD-mediated pyroptosis and may be a viable target for controlling and treating inflammation-associated diseases.

Receptor interacting protein kinase 3 (RIP3 or RIPK3), the critical executor of cell programmed necrosis, plays essential roles in maintaining immune responses and appropriate tissue homeostasis. Although the E3 ligases CHIP and PELI1 are reported to promote RIP3 degradation, however, how post-translational modification regulates RIP3 activity and stability is poorly understood. Here, we identify the tripartite motif protein TRIM25 as a negative regulator of RIP3-dependent necrosis. TRIM25 directly interacts with RIP3 through its SPRY domain and mediates the K48-linked polyubiquitination of RIP3 on residue K501. The RING domain of TRIM25 facilitates the polyubiquitination chain on RIP3, thereby promoting proteasomal degradation of RIP3. Also, TRIM25 deficiency inhibited the ubiquitination of RIP3, thus promoting TNF-induced cell necrosis. Our current finding reveals the regulating mechanism of polyubiquitination on RIP3, which might be a potential therapeutic target for the intervention of RIP3-dependent necrosis-related diseases.

A novel hydrophilic porous biocomposite was fabricated by incorporating graphene oxide (GO) @chitosan (CS) foam substrate (GO@CS@ZIF-8 foam) with ZIF-8 crystals in situ via a facile stirring method for simultaneous enrichment of glycopeptides and phosphopeptides from complex biological samples. The experimental results demonstrated that GO@CS@ZIF-8 foam exhibited favorable specificity for simultaneous enrichment of N-glycopeptides and phosphopeptides under the same condition for HRP and β-casein tryptic digest mixtures. The novel material was further applied to enriching both glycopeptides and phosphopeptides simultaneously from 4 μL complex human serum, and 423 N-glycopeptides and 40 phosphopeptides corresponding to 133 glycoproteins and 29 phosphoproteins were identified, respectively.

Gasdermin-E (GSDME), the executioner of pyroptosis when cleaved by caspase 3, plays a crucial role in tumor defense and the response to chemotherapy drugs in cells. So far, there are poorly known mechanisms for the expression regulation of GSDME during cell death. Here, we identify the transcription factor Sp1 (Specificity protein 1) as a positive regulator of GSDME-mediated pyroptosis. Sp1 directly interacts with the GSDME promoter at −36 ~ −28 site and promotes GSDME gene transcription. Further, Sp1 knockdown or inhibition suppresses GSDME expression, thus reducing chemotherapy drugs (topotecan, etoposide, doxorubicin, sorafinib and cisplatin) induced cell pyroptosis. The regulation process synergizes with STAT3 (Signal transducer and activator of transcription 3) activity and antagonizes with DNA methylation but barely affects GSDMD-mediated pyroptosis or TNF-induced necroptosis. Our current finding reveals a new regulating mechanism of GSDME expression, which may be a viable target for the intervention of GSDME-dependent inflammatory diseases and cancer therapy.