14-3-3 proteins facilitate the activation of MAP kinase cascades by upstream immunity-related kinases.

Activation of mitogen-activated protein kinase (MAP kinase) cascades is essential for plant immunity. Upon activation by surface-localized immune receptors, receptor-like cytoplasmic kinases (RLCKs) in the cytoplasm phosphorylate MAP kinase kinase kinases (MAPKKKs) to initiate MAP kinase activation. Surprisingly, we found that both the phosphorylation of Arabidopsis (Arabidopsis thaliana) MAPKKKs and the subsequent activation of MAP kinase cascades require the λ and κ isoforms of 14-3-3 proteins, which directly interact with multiple RLCKs and MAPKKKs. The N- and C-termini of MAPKKK5 interact intramolecularly to inhibit the access to the C terminus by RLCKs, whereas the 14-3-3 proteins relieve this inhibition and facilitate the interaction of RLCKs with the C-terminus of MAPKKK5. This enables the phosphorylation of MAPKK5 at Ser599 and Ser682, thus promoting MAP kinase activation and enhancing plant disease resistance. Our study reveals a role of 14-3-3 proteins as scaffolds and activators in the regulation of the RLCK-MAPKKK5 module and provides insight into the mechanism of plant immune signaling.

ASK1/ p38 axis inhibition blocks the release of mitochondrial "danger signals" from hepatocytes and suppresses progression to cirrhosis and liver cancer.

BACKGROUND AND AIMS: Apoptosis Signal-regulating Kinase 1 (ASK1) is activated by various pathological stimuli and induces cell apoptosis through downstream p38 activation. We studied the effect of pharmacological ASK1 inhibition on cirrhosis and its sequelae using comprehensive preclinical in vivo and in vitro systems. APPROACH AND RESULTS: Short-term (4-6 wk) and long-term (24-44 wk) ASK1 inhibition using small molecule GS-444217 was tested in thioacetamide-induced and BALB/c. Mdr2-/- murine models of cirrhosis and HCC, and in vitro using primary hepatocyte cell death assays. Short-term GS-444217 therapy in both models strongly reduced phosphorylated p38, hepatocyte death, and fibrosis by up to 50%. Profibrogenic release of mitochondrial DAMP mitochondrial deoxyribonucleic acid from dying hepatocytes was blocked by ASK1 or p38 inhibition. Long-term (24 wk) therapy in BALBc.Mdr2 - / - model resulted in a moderate 25% reduction in bridging fibrosis, but not in net collagen deposition. Despite this, the development of cirrhosis was effectively prevented, with strongly reduced p21 + hepatocyte staining (by 72%), serum ammonia levels (by 46%), and portal pressure (average 6.07 vs. 8.53 mm Hg in controls). Extended ASK1 inhibition for 44 wk in aged BALB/c. Mdr2-/- mice resulted in markedly reduced tumor number and size by ~50% compared to the control group. CONCLUSIONS: ASK1 inhibition suppresses the profibrogenic release of mitochondrial deoxyribonucleic acid from dying hepatocytes in a p38-dependent manner and protects from liver fibrosis. Long-term ASK1 targeting resulted in diminished net antifibrotic effect, but the progression to liver cirrhosis and cancer in BALBc/ Mdr2- / - mice was effectively inhibited. These data support the clinical evaluation of ASK1 inhibitors in fibrotic liver diseases.

ASK1 signaling regulates phase-specific glial interactions during neuroinflammation.

Neuroinflammation is well known to be associated with neurodegenerative diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that has been implicated in neuroinflammation, but its precise cellular and molecular mechanisms remain unknown. In this study, we generated conditional knockout (CKO) mice that lack ASK1 in T cells, dendritic cells, microglia/macrophages, microglia, or astrocytes, to assess the roles of ASK1 during experimental autoimmune encephalomyelitis (EAE). We found that neuroinflammation was reduced in both the early and later stages of EAE in microglia/macrophage-specific ASK1 knockout mice, whereas only the later-stage neuroinflammation was ameliorated in astrocyte-specific ASK1 knockout mice. ASK1 deficiency in T cells and dendritic cells had no significant effects on EAE severity. Further, we found that ASK1 in microglia/macrophages induces a proinflammatory environment, which subsequently activates astrocytes to exacerbate neuroinflammation. Microglia-specific ASK1 deletion was achieved using a CX3CR1CreER system, and we found that ASK1 signaling in microglia played a major role in generating and maintaining disease. Activated astrocytes produce key inflammatory mediators, including CCL2, that further activated and recruited microglia/macrophages, in an astrocytic ASK1-dependent manner. Astrocyte-specific analysis revealed CCL2 expression was higher in the later stage compared with the early stage, suggesting a greater proinflammatory role of astrocytes in the later stage. Our findings demonstrate cell-type-specific roles of ASK1 and suggest phase-specific ASK1-dependent glial cell interactions in EAE pathophysiology. We propose glial ASK1 as a promising therapeutic target for reducing neuroinflammation.

Blocking CHOP-dependent TXNIP shuttling to mitochondria attenuates albuminuria and mitigates kidney injury in nephrotic syndrome.

Albuminuria is a hallmark of glomerular disease of various etiologies. It is not only a symptom of glomerular disease but also a cause leading to glomerulosclerosis, interstitial fibrosis, and eventually, a decline in kidney function. The molecular mechanism underlying albuminuria-induced kidney injury remains poorly defined. In our genetic model of nephrotic syndrome (NS), we have identified CHOP (C/EBP homologous protein)-TXNIP (thioredoxin-interacting protein) as critical molecular linkers between albuminuria-induced ER dysfunction and mitochondria dyshomeostasis. TXNIP is a ubiquitously expressed redox protein that binds to and inhibits antioxidant enzyme, cytosolic thioredoxin 1 (Trx1), and mitochondrial Trx2. However, very little is known about the regulation and function of TXNIP in NS. By utilizing Chop-/- and Txnip-/- mice as well as 68Ga-Galuminox, our molecular imaging probe for detection of mitochondrial reactive oxygen species (ROS) in vivo, we demonstrate that CHOP up-regulation induced by albuminuria drives TXNIP shuttling from nucleus to mitochondria, where it is required for the induction of mitochondrial ROS. The increased ROS accumulation in mitochondria oxidizes Trx2, thus liberating TXNIP to associate with mitochondrial nod-like receptor protein 3 (NLRP3) to activate inflammasome, as well as releasing mitochondrial apoptosis signal-regulating kinase 1 (ASK1) to induce mitochondria-dependent apoptosis. Importantly, inhibition of TXNIP translocation and mitochondrial ROS overproduction by CHOP deletion suppresses NLRP3 inflammasome activation and p-ASK1-dependent mitochondria apoptosis in NS. Thus, targeting TXNIP represents a promising therapeutic strategy for the treatment of NS.

Reviving resilience: MEcPP-mediated ASK1-IMPα-9-TRP2 stress-responsive module.

Chloroplasts are not only critical photosynthesis sites in plants, but they also participate in plastidial retrograde signaling in response to developmental and environmental signals. MEcPP (2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate) is an intermediary in the methylerythritol phosphate (MEP) pathway in chloroplasts. It is a critical precursor for the synthesis of isoprenoids and terpenoid derivatives, which play crucial roles in plant growth and development, photosynthesis, reproduction, and defense against environmental constraints. Accumulation of MEcPP under stressful conditions triggers the expression of IMPα-9 and TPR2, contributing to the activation of abiotic stress-responsive genes. In this correspondence, we discuss plastidial retrograde signaling in support of a recently published paper in Molecular Plant (Zeng et al. 2024). We hope that it can shed more insight on the retrograde signaling cascade.

Co-targeting ASK1 and THRß synergistically improves steatohepatitis and fibrosis in a MASH animal model.

PURPOSE: Metabolic dysfunction-associated steatohepatitis (MASH) is a liver disease that has gained widespread attention globally. Unfortunately, there is no approved treatment for this condition yet. However, recent research has identified Apoptosis signal-regulating kinase 1 (ASK1) and thyroid hormone receptor-ß (THR-ß) as potential targets for treating MASH. Although the individual effects of these two targets have been studied, their combinatory effect has not been well defined. Therefore, further research is needed to investigate the potential benefits of targeting both ASK1 and THR-ß for treating MASH. METHODS: We established a MASH model using the HFHFrC diet (high fat, high fructose, and cholesterol) and carbon tetrachloride (CCL4). Forty mice were evenly assigned to four groups: vehicle, GS4997 (an ASK1 inhibitor), MGL3196 (a THRß agonist), GS4997+ MGL3196 combination (combo). The drugs were administered for 8 weeks, after which the mice were sacrificed for serum biochemical tests, liver TG and TC evaluation, liver histopathological study, and gene expression validation. RESULTS: GS4997 and MGL3196, when used in combination, have been shown to have synergistic effects on various parameters. Firstly, they synergistically reduced body weight and liver body weight ratio. Secondly, this combination also synergistically lowered AST and TC. Thirdly, synergistic effects were also observed in liver TG and TC reduction. Fourthly, we further confirmed that GS4997 mildly improved liver inflammation, ballooning, and fibrosis, but exhibited incredible histopathological efficacy when combined with MGL3196. Finally, this combinatory effect can be interpreted by synergistically regulating lipid-related genes such as Dio1, Ctp1-α, and Cat, inflammation-related genes such as Il-6, Il-8, and Mcp-1, and fibrosis-related genes such as Tgf-ß, Col1α1, and Col6α3. CONCLUSION: GS4997 and MGL3196, when used in combination, have been shown to have a comprehensive effect on MASH by synergistically regulating lipid, inflammation, and fibrosis-related gene expression through co-targeting ASK1 and THRß.

MITOGEN-ACTIVATED PROTEIN KINASE3 enhances disease resistance of edr1 mutants by phosphorylating MAPKKK5.

Mitogen-activated protein kinase (MAPK/MPK) cascades are key signaling modules that regulate plant immunity. ENHANCED DISEASE RESISTANCE1 (EDR1) encodes a Raf-like MAPK kinase kinase (MAPKKK) that negatively regulates plant defense in Arabidopsis (Arabidopsis thaliana). The enhanced resistance of edr1 requires MAPK KINASE4 (MKK4), MKK5, and MPK3. Although the edr1 mutant displays higher MPK3/6 activation, the mechanism by which plants increase MAPK cascade activation remains elusive. Our previous study showed that MAPKKK5 is phosphorylated at the Ser-90 residue in edr1 mutants. In this study, we demonstrated that the enhanced disease resistance of edr1 required MAPKKK5. Phospho-dead MAPKKK5S90A partially impaired the resistance of edr1, and the expression of phospho-mimetic MAPKKK5S90D in mapkkk5-2 resulted in enhanced resistance to the powdery mildew Golovinomyces cichoracearum strain UCSC1 and the bacterial pathogen Pseudomonas syringae pv. tomato (Pto) strain DC3000. Thus, Ser-90 phosphorylation in MAPKKK5 appears to play a crucial role in disease resistance. However, MAPKKK5-triggered cell death was not suppressed by EDR1. Furthermore, activated MPK3 phosphorylated the N terminus of MAPKKK5, and Ser-90 was one of the phosphorylated sites. Ser-90 phosphorylation increased MAPKKK5 stability, and EDR1 might negatively regulate MAPK cascade activation by suppressing the MPK3-mediated feedback regulation of MAPKKK5. Taken together, these results indicate that MPK3 phosphorylates MAPKKK5 to enhance MAPK cascade activation and disease resistance in edr1 mutants.

Reactions of Cisplatin with Thioredoxin-1 Regulate Intracellular Redox Homeostasis.

Cisplatin is a widely used anticancer drug. In addition to inducing DNA damage, increased levels of reactive oxygen species (ROS) play a significant role in cisplatin-induced cell death. Thioredoxin-1 (Trx1), a redox regulatory protein that can scavenge ROS, has been found to eliminate cisplatin-induced ROS, while elevated Trx1 levels are associated with cisplatin resistance. However, it is unknown whether the effect of Trx1 on the cellular response to cisplatin is due to its direct reaction and how this reaction influences the activity of Trx1. In this work, we performed detailed studies of the reaction between Trx1 and cisplatin. Trx1 is highly reactive to cisplatin, and the catalytic motif of Trx1 (CGPC) is the primary binding site of cisplatin. Trx1 can bind up to 6 platinum moieties, resulting in the structural alteration and oligomerization of Trx1 depending on the degree of platination. Platination of Trx1 inhibits its interaction with ASK1, a Trx1-binding protein that regulates cell apoptosis. Furthermore, the reaction with cisplatin suppresses drug-induced ROS generation, which could be associated with drug resistance. This study provides more insight into the mechanism of action of cisplatin.

Hydronidone induces apoptosis in activated hepatic stellate cells through endoplasmic reticulum stress-associated mitochondrial apoptotic pathway.

BACKGROUND AND AIM: Hydronidone (HDD) is a novel pirfenidone derivative developed initially to reduce hepatotoxicity. Our previous studies in animals and humans have demonstrated that HDD treatment effectively attenuates liver fibrosis, yet the underlying mechanism remains unclear. This study aimed to investigate whether HDD exerts its anti-fibrotic effect by inducing apoptosis in activated hepatic stellate cells (aHSCs) through the endoplasmic reticulum stress (ERS)-associated mitochondrial apoptotic pathway. METHODS: The carbon tetrachloride (CCl4)- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver fibrosis models were used for in vivo studies. In vitro studies were conducted using the human hepatic stellate cell line LX-2. The apoptotic effect of HDD on aHSCs was examined using TUNEL and flow cytometry assays. The small interfering RNA (siRNA) technique was employed to downregulate the expression of interest genes. RESULTS: HDD treatment significantly promoted apoptosis in aHSCs in both the CCl4- and DDC-induced liver fibrosis in mice and LX-2 cells. Mechanistic studies revealed that HDD triggered ERS and subsequently activated the IRE1α-ASK1-JNK pathway. Furthermore, the influx of cytochrome c from the mitochondria into the cytoplasm was increased, leading to mitochondrial dysfunction and ultimately triggering apoptosis in aHSCs. Notably, inhibition of IRE1α or ASK1 by siRNA partially abrogated the pro-apoptotic effect of HDD in aHSCs. CONCLUSIONS: The findings of both in vivo and in vitro studies suggest that HDD induces apoptosis in aHSCs via the ERS-associated mitochondrial apoptotic pathway, potentially contributing to the amelioration of liver fibrosis.

CLDN6 inhibited cellular biological function of nonsmall cell lung cancer cells through suppressing aerobic glycolysis via the RIP1/ASK1/JNK axis.

Claudin-6 (CLDN6) has been extensively studied in different tumors to date. However, in the case of nonsmall cell lung cancer (NSCLC), CLDN6 has a largely unknown role and molecular mechanism. We detected the expression of CLDN6 in NSCLC tissues and cells using reverse transcription-quantitative polymerase chain reaction (PCR) and western blot assays. A gain-of-function experiment was performed to evaluate the biological effects of CLDN6 on NSCLC cell behaviors. Methylation-specific PCR was utilized to detect the DNA methylation of CLDN6 gene promoter region. The interaction of CLDN6 and receptor interacting protein 1 (RIP1) was determined by coimmunoprecipitation assay. Furthermore, the modulation of CLDN6 on RIP1/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) axis was confirmed. The results showed that in NSCLC tissues and cells, CLDN6 expression level was declined, and was associated with a high level of DNA methylation. CLDN6 overexpression suppressed the viability, invasion, migration, and promoted cell apoptosis. Besides, the enhanced expression of CLDN6 reduced the glycolysis and the dysfunction of mitochondrial respiration of NSCLC cells. Mechanistic investigation confirmed that CLDN6 interacted with RIP1 and inhibited cellular biological function of NSCLC cells via RIP1/ASK1/JNK axis. Besides, CLDN6 overexpression inhibited tumor growth in vivo. In conclusion, CLDN6 inhibited NSCLC cell proliferation through inactivating aerobic glycolysis via the RIP1/ASK1/JNK axis.

RING finger protein 5 protects against acute myocardial infarction by inhibiting ASK1.

BACKGROUND: Myocardial infarction (MI) is a major disease with high morbidity and mortality worldwide. However, existing treatments are far from satisfactory, making the exploration of potent molecular targets more imperative. The E3 ubiquitin ligase RING finger protein 5 (RNF5) has been previously reported to be involved in several diseases by regulating ubiquitination-mediated protein degradation. Nevertheless, few reports have focused on its function in cardiovascular diseases, including MI. METHODS: In this study, we established RNF5 knockout mice through precise CRISPR-mediated genome editing and utilized left anterior descending coronary artery ligation in 9-11-week-old male C57BL/6 mice. Subsequently, serum biochemical analysis and histopathological examination of heart tissues were performed. Furthermore, we engineered adenoviruses for modulating RNF5 expression and subjected neonatal rat cardiomyocytes to oxygen-glucose deprivation (OGD) to mimic ischemic conditions, demonstrating the impact of RNF5 manipulation on cellular viability. Gene and protein expression analysis provided insights into the molecular mechanisms. Statistical methods were rigorously employed to assess the significance of experimental findings. RESULTS: We found RNF5 was downregulated in infarcted heart tissue of mice and NRCMs subjected to OGD treatment. RNF5 knockout in mice resulted in exacerbated heart dysfunction, more severe inflammatory responses, and increased apoptosis after MI surgery. In vitro, RNF5 knockdown exacerbated the OGD-induced decline in cell activity, increased apoptosis, while RNF5 overexpression had the opposite effect. Mechanistically, it was proven that the kinase cascade initiated by apoptosis signal-regulating kinase 1 (ASK1) activation was closely regulated by RNF5 and mediated RNF5's protective function during MI. CONCLUSIONS: We demonstrated the protective effect of RNF5 on myocardial infarction and its function was dependent on inhibiting the activation of ASK1, which adds a new regulatory component to the myocardial infarction associated network and promises to enable new therapeutic strategy.

Discovery of a quinoline-containing compound JT21-25 as a potent and selective inhibitor of apoptosis signal-regulating kinase 1 (ASK1).

ASK1 kinase inhibition has become a promising strategy for treating inflammatory diseases, such as non-alcoholic steatohepatitis and multiple sclerosis. Here, we reported the discovery of a promising compound 9h (JT21-25) containing quinoline structures as a potent small molecule inhibitor of ASK1. The compound JT21-25 was selective against MAP3K kinases TAK1 (>1960.8-fold), and much higher than the selectivity of GS-4997 for TAK1 (312.3-fold). In addition, different concentrations of JT21-25 did not show significant toxicity in normal LO2 liver cells, and the cell survival rate was greater than 80 %. The Oil Red O staining experiment showed that at the 4 µM and 8 µM concentrations of JT21-25, only slight cytoplasmic fat droplets were observed in LO2 cells, and there was no significant fusion between fat droplets. In the biochemical analysis experiment, JT21-25 significantly reduced the content of CHOL, LDL, TG, ALT, and AST. In summary, these findings suggested that compound JT21-25 might be valuable for further investigation as a potential candidate in the treatment of associated diseases.

Exploring novel indazole derivatives as ASK1 inhibitors: Design, synthesis, biological evaluation and docking studies.

Apoptosis signal regulated kinase 1 (ASK1, MAP3K5) is a member of the mitogen activated protein kinase (MAPK) signaling pathway, involved in cell survival, differentiation, stress response, and apoptosis. ASK1 kinase inhibition has become a promising strategy for the treatment of Non-alcoholic steatohepatitis (NASH) disease. A series of novel ASK1 inhibitors with indazole scaffolds were designed and synthesized, and their ASK1 kinase activities were evaluated. The System Structure Activity Relationship (SAR) study discovered a promising compound 33c, which has a strong inhibitory effect on ASK1. Noteworthy observations included a discernible reduction in lipid droplets within LO2 cells stained with Oil Red O, coupled with a decrease in LDL, CHO, and TG content within the NASH model cell group. Mechanistic inquiries revealed that compound 33c could inhibit the protein expression levels of the upregulated ASK1-p38/JNK signaling pathway in TNF-α treated HGC-27 cells and regulate apoptotic proteins. In summary, these findings suggest that compound 33c may be valuable for further research as a potential candidate compound against NASH.

Oxidative Stress-Induced Platelet Apoptosis/Activation: Alleviation by Purified Curcumin via ASK1-JNK/p-38 Pathway.

Platelets are known for their indispensable role in hemostasis and thrombosis. However, alteration in platelet function due to oxidative stress is known to mediate various health complications, including cardiovascular diseases and other health complications. To date, several synthetic molecules have displayed antiplatelet activity; however, their uses are associated with bleeding and other adverse effects. The commercially available curcumin is generally a mixture of three curcuminoids: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Although crude curcumin is known to inhibit platelet aggregation, the effect of purified curcumin on platelet apoptosis, activation, and aggregation remains unclear. Therefore, in this study, curcumin was purified from a crude curcumin mixture and the effects of this preparation on the oxidative stress-induced platelet apoptosis and activation was evaluated. 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) compound was used as an inducer of oxidative stress. Purified curcumin restored AAPH-induced platelet apoptotic markers like reactive oxygen species, intracellular calcium level, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release from mitochondria to the cytosol, and phosphatidyl serine externalization. Further, it inhibited the agonist-induced platelet activation and aggregation, demonstrating its antiplatelet activity. Western blot analysis confirms protective effect of the purified curcumin against oxidative stress-induced platelet apoptosis and activation via downregulation of MAPKs protein activation, including ASK1, JNK, and p-38. Together, these results suggest that the purified curcumin could be a potential therapeutic bioactive molecule to treat the oxidative stress-induced platelet activation, apoptosis, and associated complications.

Thioredoxin 1 overexpression attenuated diabetes-induced endoplasmic reticulum stress in Müller cells via apoptosis signal-regulating kinase 1.

As one of the common and serious chronic complications of diabetes mellitus (DM), the related mechanism of diabetic retinopathy (DR) has not been fully understood. Müller cell reactive gliosis is one of the early pathophysiological features of DR. Therefore, exploring the manner to reduce diabetes-induced Müller cell damage is essential to delay DR. Thioredoxin 1 (Trx1), one of the ubiquitous redox enzymes, plays a vital role in redox homeostasis via protein-protein interactions, including apoptosis signal-regulating kinase 1 (ASK1). Previous studies have shown that upregulation of Trx by some drugs can attenuate endoplasmic reticulum stress (ERS) in DR, but the related mechanism was unclear. In this study, we used DM mouse and high glucose (HG)-cultured human Müller cells as models to clarify the effect of Trx1 on ERS and the underlying mechanism. The data showed that the diabetes-induced Müller cell damage was increased significantly. Moreover, the expression of ERS and reactive gliosis was also upregulated in diabetes in vivo and in vitro. However, it was reversed after Trx1 overexpression. Besides, ERS-related protein expression, reactive gliosis, and apoptosis were decreased after transfection with ASK1 small-interfering RNA in stable Trx1 overexpression Müller cells after HG treatment. Taken together, Trx1 could protect Müller cells from diabetes-induced damage, and the underlying mechanism was related to inhibited ERS via ASK1.

Protein Phosphatase 5-Recruiting Chimeras for Accelerating Apoptosis-Signal-Regulated Kinase 1 Dephosphorylation with Antiproliferative Activity.

A normal phosphorylation state is essential for the function of proteins. Biased regulation frequently results in morbidity, especially for the hyperphosphorylation of oncoproteins. The hyperphosphorylation of ASK1 at Thr838 leads to a persistently high activity state, which accelerates the course of gastric cancer. Under normal conditions, PP5 specifically dephosphorylates p-ASK1T838 in cells, thereby weakening ASK1 to a low-basal activity state. However, in tumor types, PP5 shows low activity with a self-inhibition mechanism, making p-ASK1T838 remain at a high level. Thus, we aim to design phosphatase recruitment chimeras (PHORCs) through a proximity-mediated effect for specifically accelerating the dephosphorylation of p-ASK1T838. Herein, we describe DDO3711 as the first PP5-recruiting PHORC, which is formed by connecting a small molecular ASK1 inhibitor to a PP5 activator through a chemical linker, to effectively decrease the level of p-ASK1T838 in vitro and in vivo. DDO3711 shows preferable antiproliferative activity (IC50 = 0.5 µM) against MKN45 cells through a direct binding and proximity-mediated mechanism, while the ASK1 inhibitor and the PP5 activator, used alone or in combination, exhibit no effect on MKN45 cells. Using DDO3711, PHORCs are identified as effective tools to accelerate the dephosphorylation of POIs and provide important evidence to achieve precise phosphorylation regulation, which will promote confidence in the further regulation of abnormally phosphorylated oncoproteins.

Bone marrow mesenchymal stem cell-derived exosomes carrying E3 ubiquitin ligase ITCH attenuated cardiomyocyte apoptosis by mediating apoptosis signal-regulated kinase-1.

BACKGROUND: Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been verified to perform an effective role in treating acute myocardial infarction (MI). Herein, we aimed to investigate the role of BMSC-derived exosomes carrying itchy E3 ubiquitin ligase (ITCH) in MI and the underlying mechanism involved. METHODS: BMSCs were isolated from rat bone marrow and exosomes were extracted using ultra-high speed centrifugation. Exosomes uptake by cardiomyoblasts was determined by PKH-67 staining. Rat cardiomyoblast cell line H9C2 was stimulated by hypoxia, as in vitro model. H9C2 cell apoptosis was determined by flow cytometry. Cell viability was examined by cell counting kit-8 assay. Western blotting was performed to determine the expression of ITCH, apoptosis signal-regulated kinase-1 (ASK1), and apoptotic-related protein cleaved-caspase 3 and Bcl-2. Ubiquitination assay was employed to measure the levels of ASK1 ubiquitination. RESULTS: Exosomes derived from BMSCs were endocytosed by H9C2 cardiomyoblasts. BMSC-Exo downregulated cleaved-caspase 3 expression, upregulated Bcl-2 expression, further suppressed H9C2 cell apoptosis under hypoxia treatment, meanwhile the expression of ASK1 was downregulated, and similar effects were observed in BMSC-cultured supernatant (BMSC-S). However, these effects were reversed by exosome inhibitor GW4869. BMSC-derived exosomes enhanced ASK1 ubiquitination and degradation. Mechanically, exosomes of ITCH-knockdown BMSCs promoted H9C2 cell apoptosis and upregulated ASK1 expression. Overexpression of ITCH enhanced ASK1 ubiquitination and degradation. Further, the protein expression of ASK1 and cleaved-caspase 3 was upregulated and Bcl-2 protein expression was downregulated. ITCH-knockdown BMSC exosomes increased cardiomyoblast apoptosis. CONCLUSION: BMSC-derived exosomes carrying ITCH suppressed cardiomyoblast apoptosis, promoted cardiomyoblast viability, and improved myocardial injury in AMI by mediating ASK1 ubiquitination.

ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood-brain barrier integrity after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a significant worldwide public health concern that necessitates attention. Apoptosis signal-regulating kinase 1 (ASK1), a key player in various central nervous system (CNS) diseases, has garnered interest for its potential neuroprotective effects against ischemic stroke and epilepsy when deleted. Nonetheless, the specific impact of ASK1 on TBI and its underlying mechanisms remain elusive. Notably, mutation of ATP-binding sites, such as lysine residues, can lead to catalytic inactivation of ASK1. To address these knowledge gaps, we generated transgenic mice harboring a site-specific mutant ASK1 Map3k5-e (K716R), enabling us to assess its effects and elucidate potential underlying mechanisms following TBI. METHODS: We employed the CRIPR/Cas9 system to generate a transgenic mouse model carrying the ASK1-K716R mutation, aming to investigate the functional implications of this specific mutant. The controlled cortical impact method was utilized to induce TBI. Expression and distribution of ASK1 were detected through Western blotting and immunofluorescence staining, respectively. The ASK1 kinase activity after TBI was detected by a specific ASK1 kinase activity kit. Cerebral microvessels were isolated by gradient centrifugation using dextran. Immunofluorescence staining was performed to evaluate blood-brain barrier (BBB) damage. BBB ultrastructure was visualized using transmission electron microscopy, while the expression levels of endothelial tight junction proteins and ASK1 signaling pathway proteins was detected by Western blotting. To investigate TBI-induced neuroinflammation, we conducted immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analyses. Additionally, immunofluorescence staining and electrophysiological compound action potentials were conducted to evaluate gray and white matter injury. Finally, sensorimotor function and cognitive function were assessed by a battery of behavioral tests. RESULTS: The activity of ASK1-K716R was significantly decreased following TBI. Western blotting confirmed that ASK1-K716R effectively inhibited the phosphorylation of ASK1, JNKs, and p38 in response to TBI. Additionally, ASK1-K716R demonstrated a protective function in maintaining BBB integrity by suppressing ASK1/JNKs activity in endothelial cells, thereby reducing the degradation of tight junction proteins following TBI. Besides, ASK1-K716R effectively suppressed the infiltration of peripheral immune cells into the brain parenchyma, decreased the number of proinflammatory-like microglia/macrophages, increased the number of anti-inflammatory-like microglia/macrophages, and downregulated expression of several proinflammatory factors. Furthermore, ASK1-K716R attenuated white matter injury and improved the nerve conduction function of both myelinated and unmyelinated fibers after TBI. Finally, our findings demonstrated that ASK1-K716R exhibited favorable long-term functional and histological outcomes in the aftermath of TBI. CONCLUSION: ASK1-K716R preserves BBB integrity by inhibiting ASK1/JNKs pathway in endothelial cells, consequently reducing the degradation of tight junction proteins. Additionally, it alleviates early neuroinflammation by inhibiting the infiltration of peripheral immune cells into the brain parenchyma and modulating the polarization of microglia/macrophages. These beneficial effects of ASK1-K716R subsequently result in a reduction in white matter injury and promote the long-term recovery of neurological function following TBI.

N-acetylgalactosaminyltransferase-4 protects against hepatic ischemia/reperfusion injury by blocking apoptosis signal-regulating kinase 1 N-terminal dimerization.

BACKGROUND AND AIMS: Ischemia-reperfusion (I/R) injury is an inevitable complication of liver transplantation (LT) and compromises its prognosis. Glycosyltransferases have been recognized as promising targets for disease therapy, but their roles remain open for study in hepatic I/R (HIR) injury. Here, we aim to demonstrate the exact function and molecular mechanism of a glycosyltransferase, N-acetylgalactosaminyltransferase-4 (GALNT4), in HIR injury. APPROACH AND RESULTS: By an RNA-sequencing data-based correlation analysis, we found a close correlation between GALNT4 expression and HIR-related molecular events in a murine model. mRNA and protein expression of GALNT4 were markedly up-regulated upon reperfusion surgery in both clinical samples from subjects who underwent LT and in a mouse model. We found that GALNT4 deficiency significantly exacerbated I/R-induced liver damage, inflammation, and cell death, whereas GALNT4 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration clarified that GALNT4 directly binds to apoptosis signal-regulating kinase 1 (ASK1) to inhibit its N-terminal dimerization and subsequent phosphorylation, leading to a robust inactivation of downstream c-Jun N-terminal kinase (JNK)/p38 and NF-κB signaling. Intriguingly, the inhibitory capacity of GALNT4 on ASK1 activation is independent of its glycosyltransferase activity. CONCLUSIONS: GALNT4 represents a promising therapeutic target for liver I/R injury and improves liver surgery prognosis by inactivating the ASK1-JNK/p38 signaling pathway.

Activation of osteoblast ferroptosis via the METTL3/ASK1-p38 signaling pathway in high glucose and high fat (HGHF)-induced diabetic bone loss.

Diabetes mellitus (DM) and osteoporosis are two common diseases that may develop as a cause-and-effect relationship since the incidence of osteoporotic fractures is significantly increased in DM patients. However, the pathophysiology of diabetic osteoporosis is yet to be clearly understood. Iron overload has been reported to lead to bone loss and closely related to osteoporosis. In this study, we hypothesized that high glucose and high fat (HGHF) may induce osteoblastic ferroptosis for the pathogenesis of diabetic osteoporosis and explored the possible molecular mechanisms behind. Using the diabetic rat model established by HGHF feeding with a subsequent intraperitoneal injection of a single low dose of streptozocin, we found that the serum ferritin level (a biomarker for body iron store) was significantly elevated in HGHF-fed rats and the expression of SLC7A11 and GPX4 (inhibitory marker proteins for ferroptosis) was markedly attenuated in the bone tissue of the rats with diabetic bone loss as compared to the normal rats. In an osteoblast cell model, treatment of pre-osteoblastic MC3T3-E1 cells with high glucose and palmitic acid (HGPA) not only suppressed osteoblast differentiation and mineralization but also triggered ferroptosis-related osteoblastic cell death. m6 A-seq revealed that m6 A methylation on ASK1 was 80.9-fold higher in HGPA-treated cells. The expression of p-ASK1 and p-p38 was also significantly elevated in the HGPA-treated cells. Knockout of METTL3 (methyltransferase-like 3), one of the major m6 A methyltransferases, in MC3T3-E1 cells not only abrogated HGPA-induced activation of ASK1-p38 signaling pathway but also attenuated the level of ferroptosis. Therefore, HGHF-induced ferroptosis in osteoblasts may be the main cause of osteoporosis in DM via activation of METTL3/ASK1-p38 signaling pathway, and inhibition of ferroptosis in osteoblasts may provide a potential therapeutic strategy for diabetic osteoporosis.