USP25 regulates Wnt signaling by controlling the stability of tankyrases.

Aberrant activation of the Wnt signaling pathway plays an important role in human cancer development. Wnt signaling is negatively regulated by Axin, a scaffolding protein that controls a rate-limiting step in the destruction of ß-catenin, the central activator of the Wnt pathway. In Wnt-stimulated cells, Axin is rapidly modified by tankyrase-mediated poly(ADP-ribosyl)ation, which promotes the proteolysis of Axin and consequent stabilization of ß-catenin. Thus, regulation of the levels and activity of tankyrases is mechanistically important in controlling Wnt signaling. Here, we identify ubiquitin-specific protease 25 (USP25) as a positive regulator of Wnt/ß-catenin signaling. We found that USP25 directly interacted with tankyrases to promote their deubiquitination and stabilization. We demonstrated that USP25 deficiency could promote the degradation of tankyrases and consequent stabilization of Axin to antagonize Wnt signaling. We further characterized the interaction between TNKS1 and USP25 by X-ray crystal structure determination. Our results provide important new insights into the molecular mechanism that regulates the turnover of tankyrases and the possibility of targeting the stability of tankyrases by antagonizing their interaction with USP25 to modulate the Wnt/ß-catenin pathway.

SPATA2 regulates the activation of RIPK1 by modulating linear ubiquitination.

Stimulation of cells with TNFα leads to the formation of the TNF-R1 signaling complex (TNF-RSC) to mediate downstream cellular fate decision. Activation of the TNF-RSC is modulated by different types of ubiquitination and may lead to cell death, including apoptosis and necroptosis, in both RIPK1-dependent and RIPK1-independent manners. Spata2 (spermatogenesis-associated 2) is an adaptor protein recruited into the TNF-RSC to modulate the interaction between the linear ubiquitin chain assembly complex (LUBAC) and the deubiquitinase CYLD (cylindromatosis). However, the mechanism by which Spata2 regulates the activation of RIPK1 is unclear. Here, we report that Spata2-deficient cells show resistance to RIPK1-dependent apoptosis and necroptosis and are also partially protected against RIPK1-independent apoptosis. Spata2 deficiency promotes M1 ubiquitination of RIPK1 to inhibit RIPK1 kinase activity. Furthermore, we provide biochemical evidence for the USP domain of CYLD and the PUB domain of the SPATA2 complex preferentially deubiquitinating the M1 ubiquitin chain in vitro. Spata2 deficiency also promotes the activation of MKK4 and JNK and cytokine production independently of RIPK1 kinase activity. Spata2 deficiency sensitizes mice to systemic inflammatory response syndrome (SIRS) induced by TNFα, which can be suppressed by RIPK1 inhibitor Nec-1s. Thus, Spata2 can regulate inflammatory response and cell death in both RIPK1-dependent and RIPK1-independent manners.

Necroptosis activates UPR sensors without disrupting their binding with GRP78.

Necroptosis is a form of regulated necrosis mediated by the formation of the necrosome, composed of the RIPK1/RIPK3/MLKL complex. Here, we developed a proximity ligation assay (PLA) that allows in situ visualization of necrosomes in necroptotic cells and in vivo. Using PLA assay, we show that necrosomes can be found in close proximity to the endoplasmic reticulum (ER). Furthermore, we show that necroptosis activates ER stress sensors, PERK, IRE1α, and ATF6 in a RIPK1-RIPK3-MLKL axis-dependent manner. Activated MLKL can be translocated to the ER membrane to directly initiate the activation of ER stress signaling. The activation of IRE1α in necroptosis promotes the splicing of XBP1, and the subsequent incorporation of spliced XBP1 messenger RNA (mRNA) into extracellular vesicles (EVs). Finally, we show that unlike that of a conventional ER stress response, necroptosis promotes the activation of unfolded protein response (UPR) sensors without affecting their binding of GRP78. Our study reveals a signaling pathway that links MLKL activation in necroptosis to an unconventional ER stress response.

USP14 regulates autophagy by suppressing K63 ubiquitination of Beclin 1.

The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular degradative mechanisms that mediate the turnover of complementary repertoires of intracellular proteomes. Simultaneously activating both UPS and autophagy might provide a powerful strategy for the clearance of misfolded proteins. However, it is not clear whether UPS and autophagy can be controlled by a common regulatory mechanism. K48 deubiquitination by USP14 is known to inhibit UPS. Here we show that USP14 regulates autophagy by negatively controlling K63 ubiquitination of Beclin 1. Furthermore, we show that activation of USP14 by Akt-mediated phosphorylation provides a mechanism for Akt to negatively regulate autophagy by promoting K63 deubiquitination. Our study suggests that Akt-regulated USP14 activity modulates both proteasomal degradation and autophagy through controlling K48 and K63 ubiquitination, respectively. Therefore, regulation of USP14 provides a mechanism for Akt to control both proteasomal and autophagic degradation. We propose that inhibition of USP14 may provide a strategy to promote both UPS and autophagy for developing novel therapeutics targeting neurodegenerative diseases.

Thromboelastography (TEG) parameters as potential predictors of malignancy and tumor progression in colorectal cancer.

PURPOSE: The purpose of this study was to investigate the use of thromboelastography (TEG) in patients with colorectal cancer and to examine whether the TEG parameters can be used as potential markers for disease screening and prediction of disease severity. METHODS: One-hundred fifteen healthy controls (HC), 43 patients with benign adenoma (BA), and 387 patients with colorectal cancers (CRC) were included in the study. TEG parameters (reaction time, R; clot kinetics, K; alpha angle, α-angle; maximum amplitude, MA), conventional laboratory parameters, and clinical information were collected and analyzed among the HC, BA, and CRC groups. Receiver operating characteristics (ROC) were used for differential analysis. The correlation between TEG parameters and pathological information of CRC (differentiation degree, vaso-nerve infiltration, TNM stage) was analyzed. The differences in TEG parameters at different stages of disease and pre-/post operation were compared. RESULTS: Shorter K and higher α-angle/MA were found in patients with CRC compared with HC and BA (P < 0.001). TEG parameters demonstrated moderate diagnostic value (distinguish CRC from HC + BA: K-AUC = 0.693, α-angle-AUC = 0.687, MA-AUC = 0.700) in CRC but did not outperform traditional laboratory parameters. TEG hypercoagulability was closely associated with tumor markers (carcinoma embryonic antigen and carbohydrate antigen 19-9) and pathological information (differentiation degree, vaso-nerve infiltration, and TNM stage) (P < 0.05). Trend analysis showed that K decreased, but α-angle/MA increased gradually as the tumor progressed (P < 0.001). K- and α-angle showed slightly better sensitivity in predicting advanced tumors compared to traditional laboratory parameters. In CRC patients, 3-6 months after tumor resection, K [from 1.8 (1.5, 2.3) to 1.9 (1.6, 2.6)], α-angle [from 65.3 (59.0, 68.6) to 63.7 (56.6, 68.5)], and MA [from 61.0 (58.2, 66.0) to 58.9 (55.8, 61.3)] exhibited modest improvements compared to their preoperative values (P < 0.05). CONCLUSION: TEG parameters possess moderate diagnostic value in CRC diagnosis and predicting advanced tumors, and they are closely linked to surgical interventions. Although TEG parameters do not significantly outperform traditional laboratory parameters, they still hold promise as potential alternative indicators in CRC patients.

Hepatocyte-specific TAK1 deficiency drives RIPK1 kinase-dependent inflammation to promote liver fibrosis and hepatocellular carcinoma.

Transforming growth factor ß-activated kinase1 (TAK1) encoded by the gene MAP3K7 regulates multiple important downstream effectors involved in immune response, cell death, and carcinogenesis. Hepatocyte-specific deletion of TAK1 in Tak1ΔHEP mice promotes liver fibrosis and hepatocellular carcinoma (HCC) formation. Here, we report that genetic inactivation of RIPK1 kinase using a kinase dead knockin D138N mutation in Tak1ΔHEP mice inhibits the expression of liver tumor biomarkers, liver fibrosis, and HCC formation. Inhibition of RIPK1, however, has no or minimum effect on hepatocyte loss and compensatory proliferation, which are the recognized factors important for liver fibrosis and HCC development. Using single-cell RNA sequencing, we discovered that inhibition of RIPK1 strongly suppresses inflammation induced by hepatocyte-specific loss of TAK1. Activation of RIPK1 promotes the transcription of key proinflammatory cytokines, such as CCL2, and CCR2+ macrophage infiltration. Our study demonstrates the role and mechanism of RIPK1 kinase in promoting inflammation, both cell-autonomously and cell-nonautonomously, in the development of liver fibrosis and HCC, independent of cell death, and compensatory proliferation. We suggest the possibility of inhibiting RIPK1 kinase as a therapeutic strategy for reducing liver fibrosis and HCC development by inhibiting inflammation.

Tissue-specific roles of Axin2 in the inhibition and activation of Wnt signaling in the mouse embryo.

Axin proteins are key negative regulators of the canonical Wnt signal transduction pathway. Although Axin2 null mice are viable, we identified an unusual ENU-induced recessive allele of Axin2, canp, that causes midgestation lethality in homozygotes. We show that the Axin2(canp) mutation is a V26D substitution in an invariant N-terminal sequence motif and that the Axin2(canp) protein is more stable than wild type. As predicted for an increased level of a negative regulator, the Axin2(canp) mutation leads to decreased Wnt signaling in most tissues, and this can account for most of the morphological phenotypes of Axin2(canp) mutants. In contrast, there is a paradoxical increase in canonical Wnt activity in the late primitive streak of all Axin2(canp) mutant embryos that is associated with the formation of an ectopic tail in some mutants. Treatment of wild-type embryos with an inhibitor of Tankyrase that stabilizes Axin proteins also causes inhibition of Wnt signaling in anterior regions of the embryo and a gain of Wnt signaling in the primitive streak. The results indicate that although increased stability of Axin2 leads to a loss of canonical Wnt signaling in most tissues, stabilized Axin2 enhances Wnt pathway activity in a specific progenitor population in the late primitive streak.

Morroniside improves the symptoms of post-stroke depression in mice through the BDNF signaling pathway mediated by MiR-409-3p.

BACKGROUND: Post-stroke depression (PSD) is a common psychiatric symptom after a stroke. Morroniside, an iridoid glycoside found in Cornus officinalis, has garnered significant attention for its potential to alleviate symptoms associated with depression. PURPOSE: This study aims to highlight the potential use of morroniside in the treatment of PSD and elucidate the underlying molecular mechanisms. METHODS: To establish a reliable PSD model, male C57BL/6 mice were subjected to brief MCAO in conjunction with CUMS. Post-morroniside administration, neuronal viability, and hippocampal cell apoptosis were evaluated by Nissl staining and TUNEL detection, respectively. Depression-like behaviors were evaluated using SPT, TST, and FST. The Longa score and cylinder test were used to evaluate the effect of morroniside on motor function. Furthermore, to investigate the underlying molecular mechanisms, bioinformatic analysis and the dual luciferase assay were performed to investigate the MiR-409-3p-BDNF interaction. In addition, subsequent to MiR-409-3p overexpression via AAV virus, we assessed mRNA expression and protein levels of key components within the BDNF/TrkB signaling pathway using RT-qPCR, immunohistochemistry, and western blot analysis. RESULTS: The observed decrease in apoptosis and amelioration of depression-like behaviors strongly indicate the potential of morroniside as a therapeutic agent for PSD. Furthermore, the upregulation of key proteins within the BDNF/TrkB signaling pathway in the cortex suggests that morroniside activates this pathway. Through bioinformatics analysis, MiR-409-3p was identified and found to bind to the BDNF gene, resulting in the inhibition of BDNF expression. Importantly, we demonstrate that morroniside mitigates this inhibitory effect of MiR-409-3p on BDNF, thereby facilitating the activation of the BDNF/TrkB signaling pathway. CONCLUSION: The findings suggest that morroniside demonstrates the ability to improve PSD symptoms through the BDNF/TrkB signaling pathway mediated by MiR-409-3p. These results emphasize the importance of the BDNF signaling pathway in improving PSD symptoms and provide a possible mechanism for morroniside to treat PSD.

Diterpenoid tanshinones inhibit gastric cancer angiogenesis through the PI3K/Akt/mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge is a kind of Chinese herbal medicine known for activating blood circulation and removing blood stasis, with the effect of cooling blood and eliminating carbuncles, and has been proven to have the effect of treating tumors. However, the inhibitory effect of Salvia miltiorrhiza Bunge extracts (Diterpenoid tanshinones) on tumors by inhibiting angiogenesis has not been studied in detail. AIM OF THE STUDY: This study aimed to investigate the anti-gastric cancer effect of diterpenoid tanshinones (DT) on angiogenesis, including the therapeutic effects and pathways. MATERIALS AND METHODS: This experiment utilized network pharmacology was used to identify relevant targets and pathways of Salvia miltiorrhiza Bunge-related components in the treatment of gastric cancer. The effects of DT on the proliferation and migration of human gastric cancer cell line SGC-7901 and human umbilical vein endothelial cell line HUVECs were evaluated, and changes in the expression of angiogenesis-related factors were measured. In vivo, experiments were conducted on nude mice to determine tumor activity, size, immunohistochemistry, and related proteins. RESULTS: The findings showed that DT could inhibit the development of gastric cancer by suppressing the proliferation of gastric cancer cells, inducing apoptosis, and inhibiting invasion and metastasis. In addition, the content of angiogenesis-related factors and proteins was significantly altered in DT-affected cells and animals. CONCLUSIONS: Results suggest that DT has potential as a therapeutic agent for the treatment of gastric cancer, as it can inhibit tumor growth and angiogenesis. It was also found that DT may affect the expression of the angiogenic factor VEGF through the PI3K/Akt/mTOR pathway, leading to the regulation of tumor angiogenesis. This study provides a new approach to the development of anti-tumor agents and has significant theoretical and clinical implications for the treatment of gastric cancer.

Rutaecarpine Alleviates Early Brain Injury-Induced Inflammatory Response Following Subarachnoid Hemorrhage via SIRT6/NF-[Formula: see text]B Pathway.

Subarachnoid hemorrhage (SAH), a specific subtype of cerebrovascular accident, is characterized by the extravasation of blood into the interstice between the brain and its enveloping delicate tissues. This pathophysiological phenomenon can precipitate an early brain injury (EBI), which is characterized by inflammation and neuronal death. Rutaecarpine (Rut), a flavonoid compound discovered in various plants, has been shown to have protective effects against SAH-induced cerebral insult in rodent models. In our study, we used a rodent SAH model to evaluate the effect of Rut on EBI and investigated the effect of Rut on the inflammatory response and its regulation of SIRT6 expression in vitro. We found that Rut exerts a protective effect on EBI in SAH rats, which is partly due to its ability to inhibit the inflammatory response. Notably, Rut up-regulated Sirtuin 6 (SIRT6) expression, leading to an increase in H3K9 deacetylation and inhibition of nuclear factor-kappa B (NF-[Formula: see text]B) transcriptional activation, thereby mediating the inflammatory response. In addition, further data showed that SIRT6 was proven to mediate the regulation of Rut on the microglial inflammatory response. These findings highlight the importance of SIRT6 in the regulation of inflammation and suggest a potential mechanism for the protective effect of Rut on EBI. In summary, Rut may have the potential to prevent and treat SAH-induced brain injury by interacting with SIRT6. Our findings may provide a new therapeutic strategy for the treatment of SAH-induced EBI.

The Importance of Marital Status in the Morbidity and Prognosis of Lung Metastasis in Newly Diagnosed Ovarian Cancer.

Objective: The study aimed to evaluate the risk factors for the morbidity and prognosis of lung metastases (LM) in patients with newly diagnosed ovarian cancer (OC), and further explore the important role of marital status. Materials and methods: Based on the Surveillance, Epidemiology, and End Results (SEER) dataset, OC patients from 2010 and 2019 were retrospectively analyzed. Logistic regression analysis and Kaplan-Meier method were applied to evaluate the vital factors of incidence and survival outcome in LM population. Cox regression analysis was performed to identify risk factors for the prognosis of OC patients with LM. The predictive potential was showed by two established nomograms and examined by the concordance index (C-index), calibration curves, the area under the curve (AUC), decision curve analyses (DCAs) and clinical impact curves (CICs). Results: There are 25,202 eligible OC patients were enrolled in the study, the morbidity of LM at 5.61%. Multivariable logistic regression models illustrated that chemotherapy (P<0.01), surgical treatment of bilateral or more areas (P<0.01), T stage (P<0.01), N1 stage (P<0.01), bone metastasis (P<0.01), brain metastasis (P<0.01) and liver metastasis (P<0.01) were all significantly connected with LM in OC. Multivariable Cox regression analyses illustrated that unmarried, radiotherapy, elder people and positive cancer antigen 125 (CA-125) were significantly associated with shorter survival time, while chemotherapy made contributions to improve survival. Our study found that marital relationships promoted LM and was associated with the better prognosis, while unmarried patients had the opposite results. With the further development of our research, the cross-action of social, economic and psychological factors together determined the great impact of marital status on the morbidity and prognosis of OC patients combined with LM. Finally, the stability of the models was proved by internal verification. Conclusion: The population-based cohort study provides references for guiding clinical screening and individualized treatment of OC patients with LM. Under the influence of society and economy, marital status is closely related to the morbidity and prognosis of OC, which can be an important direction to explore the risk of OC lung metastasis in the future.

Exploring the Pharmacological Mechanism of the Effective Chinese Medicines Against Gynecological Cancer Based on Meta-Analysis Combined With Network Pharmacology Analysis.

This meta-analysis plus network pharmacology aimed to investigate whether traditional Chinese medicine (TCM) combined with chemotherapy is associated with more beneficial efficacy data in the treatment of gynecological cancer (GC). A total of 11 randomized controlled trials (RCTs) consisting of 863 GC patients were included. Results showed a better ORR (RR: 1.42, 95% CI: 1.18-1.71; I 2 = 21.4%; p = 0.282), DCR (RR: 1.13, 95% CI: 1.03-1.25; I 2 = 0.0%; p = 0.492), PD (RR: 0.27, 95% CI: 0.11-0.65, p = 0.003; I 2 = 0.0%, p = 0.930), and QOL (SMD: 0.85, 95% CI: 0.38-1.33, p = 0.005) and higher proportions of CD3+ T (WMD: 5.65, 95% CI: 4.23-7.08, p = 0.000; I 2 = 68.3%, p = 0.004), CD4+ T (WMD: 6.97, 95% CI: 5.35-8.59, p = 0.000; I 2 = 83.4%, p = 0.000), and the CD4+/CD8+ T ratio (WMD: 0.32, 95% CI: 0.23-0.42, p = 0.000; I 2 = 78.0%, p = 0.000). The number of adverse events (AEs) was significantly lower in the TCM + chemotherapy group. The active components and targets of 19 high-frequency Chinese medicines obtained from the meta-analysis were screened and explored in network pharmacology analysis. Also, a regulatory network of active components and targets, a core network and key genes, a diagram of protein interaction, network topology analysis, and gene body GO function and KEGG pathway enrichment analysis were performed. A total of 120 active components were identified. NPM1 and HSPA8 are the most critical target proteins in the core network of protein interaction. HSP90AA1 is the most important target protein in the TCM group. KEGG enrichment analysis showed that it was highly significant in the lipid and atherosclerotic pathways. Therefore, moderate evidence revealed that TCM plus chemotherapy has obvious advantages over chemotherapy alone in terms of tumor responses, QOL, peripheral blood lymphocyte levels, and fewer AEs in the treatment of GC. The potential important targets and core genes were displayed. Systematic Review Registration: www.crd.york.ac.uk/PROSPERO/, identifier CRD42021252500.

The new exploration of pure total flavonoids extracted from Citrus maxima (Burm.) Merr. as a new therapeutic agent to bring health benefits for people.

The peel and fruit of Citrus varieties have been a raw material for some traditional Chinese medicine (TCM). Pure total flavonoids from Citrus maxima (Burm.) Merr. (PTFC), including naringin, hesperidin, narirutin, and neohesperidin, have been attracted increasing attention for their multiple clinical efficacies. Based on existing in vitro and in vivo research, this study systematically reviewed the biological functions of PTFC and its components in preventing or treating liver metabolic diseases, cardiovascular diseases, intestinal barrier dysfunction, as well as malignancies. PTFC and its components are capable of regulating glycolipid metabolism, blocking peroxidation and persistent inflammation, inhibiting tumor progression, protecting the integrity of intestinal barrier and positively regulating intestinal microbiota, while the differences in fruit cultivation system, picking standard, manufacturing methods, delivery system and individual intestinal microecology will have impact on the specific therapeutic effect. Thus, PTFC is a promising drug for the treatment of some chronic diseases, as well as continuous elaborate investigations are necessary to improve its effectiveness and bioavailability.

Caspase inhibition prolongs inflammation by promoting a signaling complex with activated RIPK1.

Activation of inflammation by lipopolysaccharide (LPS) is an important innate immune response. Here we investigated the contribution of caspases to the LPS-mediated inflammatory response and discovered distinctive temporal roles of RIPK1 in mediating proinflammatory cytokine production when caspases are inhibited. We propose a biphasic model that differentiates the role of RIPK1 in early versus late phase. The early production of proinflammation cytokines stimulated by LPS with caspase inhibition is mediated by the NF-κB pathway that requires the scaffold function of RIPK1 but is kinase independent. Autocrine production of TNFα in the late phase promotes the formation of a novel TNFR1-associated complex with activated RIPK1, FADD, caspase-8, and key mediators of NF-κB signaling. The production of proinflammatory cytokines in the late phase can be blocked by RIPK1 kinase inhibitor Nec-1s. Our study demonstrates a mechanism by which the activation of RIPK1 promotes its own scaffold function to regulate the NF-κB-mediated proinflammatory cytokine production that is negatively regulated by caspases to restrain inflammatory signaling.

Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners.

RIPK1 is a death-domain (DD) containing kinase involved in regulating apoptosis, necroptosis and inflammation. RIPK1 activation is known to be regulated by its DD-mediated interaction and ubiquitination, though underlying mechanisms remain incompletely understood. Here we show that K627 in human RIPK1-DD and its equivalent K612 in murine RIPK1-DD is a key ubiquitination site that regulates the overall ubiquitination pattern of RIPK1 and its DD-mediated interactions with other DD-containing proteins. K627R/K612R mutation inhibits the activation of RIPK1 and blocks both apoptosis and necroptosis mediated by TNFR1 signaling. However, Ripk1K612R/K612R mutation sensitizes cells to necroptosis and caspase-1 activation in response to TLRs signaling. Ripk1K612R/K612R mice are viable, but develop age-dependent reduction of RIPK1 expression, spontaneous intestinal inflammation and splenomegaly, which can be rescued by antibiotic treatment and partially by Ripk3 deficiency. Furthermore, we show that the interaction of RIPK1 with FADD contributes to suppressing the activation of RIPK3 mediated by TLRs signaling. Our study demonstrates the distinct roles of K612 ubiquitination in mRIPK1/K627 ubiquitination in hRIPK1 in regulating its pro-death kinase activity in response to TNFα and pro-survival activity in response to TLRs signaling.

ABIN-1 heterozygosity sensitizes to innate immune response in both RIPK1-dependent and RIPK1-independent manner.

ABIN-1 (encoded by the gene Tnip1) is a ubiquitin-binding protein that can interact with ubiquitin-editing enzyme A20 (encoded by the gene TNFAIP3) to restrain the activation of necroptosis and NF-κB activation. Genetic variants in the genes Tnip1 and TNFAIP3 are both strongly associated with susceptibility to autoimmune chronic inflammatory diseases such as psoriasis vulgaris and systemic lupus erythematosus (SLE) in humans. Here we investigated the mechanism by which ABIN-1 regulated innate immune responses. We show that ABIN-1 heterozygosity sensitizes cells to antiviral response by mediating NF-κB-dependent and RIPK1-independent expression of pattern recognition molecules, including TLR3, RIG-I, and MDA5, in MEFs. Furthermore, we demonstrate that increased interaction of ABIN-1 and A20 with prolonged poly(I:C) stimulation of WT cells leads to A20-dependent reduction of ABIN-1 protein. Finally, we show that ABIN-1 heterozygosity sensitizes innate immune response of Abin-1+/- mice in vivo by promoting the production of proinflammatory cytokines, which can be blocked upon inhibition of RIPK1 kinase. Inhibition of RIPK1 kinase activity in vivo partially reduces the expression of MDA5, RIG-I, and caspase-11 in Abin-1+/- mice but not in WT mice. Thus, we conclude that ABIN-1 is a suppressor of innate immune response and the interaction of ABIN-1 with A20 controls innate immunity response through the NF-κB pathway and in both RIPK1 kinase activity-independent and dependent manner.

Necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression.

Necroptosis, a form of regulated necrotic cell death, is mediated by receptor interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). However, the mechanism by which necroptosis promotes inflammation is still unclear. Here we report that the expression of cytokines is robustly upregulated in a cell-autonomous manner during necroptosis induced by tumor necrosis factor alpha (TNFα). We demonstrate that TNFα-induced necroptosis leads to two waves of cytokine production. The first wave, more transient and weaker than the second, is in response to TNFα alone; whereas the second wave depends upon the necroptotic signaling. We show that necroptosis promotes the transcription of TNFα-target genes in a cell-intrinsic manner. The activation of both NF-κB and p38 by the necroptotic machinery, RIPK1, RIPK3, and MLKL, is involved in mediating the robust induction of cytokine expression in the second wave. In contrast, necroptosis induced by direct oligomerization of MLKL promotes cytokine production at much lower levels than that of necroptosis induced with TNFα. Thus, we conclude that TNFα-induced necroptosis signaling events mediated by RIPK1 and RIPK3 activation, in addition to the MLKL oligomerization, promotes the expression of cytokines involving multiple intracellular signaling mechanisms including NF-κB pathway and p38. These findings reveal that the necroptotic cell death machinery mounts an immune response by promoting cell-autonomous production of cytokines. Our study provides insights into the mechanism by which necroptosis promotes inflammation in human diseases.

Small molecule probes for cellular death machines.

The past decade has witnessed a significant expansion of our understanding about the regulated cell death mechanisms beyond apoptosis. The application of chemical biological approaches had played a major role in driving these exciting discoveries. The discovery and use of small molecule probes in cell death research has not only revealed significant insights into the regulatory mechanism of cell death but also provided new drug targets and lead drug candidates for developing therapeutics of human diseases with huge unmet need. Here, we provide an overview of small molecule modulators for necroptosis and ferroptosis, two non-apoptotic cell death mechanisms, and discuss the molecular pathways and relevant pathophysiological mechanisms revealed by the judicial applications of such small molecule probes. We suggest that the development and applications of small molecule probes for non-apoptotic cell death mechanisms provide an outstanding example showcasing the power of chemical biology in exploring novel biological mechanisms.

Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system.

Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells.