Role of apoptosis repressor with caspase recruitment domain in human health and chronic diseases.

Apoptosis repressor with caspase recruitment domain (ARC) is a highly potent and multifunctional suppressor of various types of programmed cell death (PCD) (e.g. apoptosis, necroptosis, and pyroptosis) and plays a key role in determining cell fate. Under physiological conditions, ARC is predominantly expressed in terminally differentiated cells, such as cardiomyocytes and skeletal muscle cells. Its expression and activity are tightly controlled by a complicated system consisting of transcription factor (TF), non-coding RNA (ncRNA), and post-translational modification (PTM). ARC dysregulation has been shown to be closely associated with many chronic diseases, including cardiovascular disease, cancer, diabetes, and neurodegenerative disease. However, the detailed mechanisms of ARC involved in the progression of these diseases remain unclear to a large extent. In this review, we mainly focus on the regulatory mechanisms of ARC expression and activity and its role in PCD. We also discuss the underlying mechanisms of ARC in health and disease and highlight the potential implications of ARC in the clinical treatment of patients with chronic diseases. This information may assist in developing ARC-based therapeutic strategies for patients with chronic diseases and expand researchers' understanding of ARC.

ANKRD1 aggravates renal ischaemia‒reperfusion injury via promoting TRIM25-mediated ubiquitination of ACSL3.

BACKGROUND: Renal ischaemia‒reperfusion injury (IRI) is the primary cause of acute kidney injury (AKI). To date, effective therapies for delaying renal IRI and postponing patient survival remain absent. Ankyrin repeat domain 1 (ANKRD1) has been implicated in some pathophysiologic processes, but its role in renal IRI has not been explored. METHODS: The mouse model of IRI-AKI and in vitro model were utilised to investigate the role of ANKRD1. Immunoprecipitation-mass spectrometry was performed to identify potential ANKRD1-interacting proteins. Protein‒protein interactions and protein ubiquitination were examined using immunoprecipitation and proximity ligation assay and immunoblotting, respectively. Cell viability, damage and lipid peroxidation were evaluated using biochemical and cellular techniques. RESULTS: First, we unveiled that ANKRD1 were significantly elevated in renal IRI models. Global knockdown of ANKRD1 in all cell types of mouse kidney by recombinant adeno-associated virus (rAAV9)-mitigated ischaemia/reperfusion-induced renal damage and failure. Silencing ANKRD1 enhanced cell viability and alleviated cell damage in human renal proximal tubule cells exposed to hypoxia reoxygenation or hydrogen peroxide, while ANKRD1 overexpression had the opposite effect. Second, we discovered that ANKRD1's detrimental function during renal IRI involves promoting lipid peroxidation and ferroptosis by directly binding to and decreasing levels of acyl-coenzyme A synthetase long-chain family member 3 (ACSL3), a key protein in lipid metabolism. Furthermore, attenuating ACSL3 in vivo through pharmaceutical approach and in vitro via RNA interference mitigated the anti-ferroptotic effect of ANKRD1 knockdown. Finally, we showed ANKRD1 facilitated post-translational degradation of ACSL3 by modulating E3 ligase tripartite motif containing 25 (TRIM25) to catalyse K63-linked ubiquitination of ACSL3, thereby amplifying lipid peroxidation and ferroptosis, exacerbating renal injury. CONCLUSIONS: Our study revealed a previously unknown function of ANKRD1 in renal IRI. By driving ACSL3 ubiquitination and degradation, ANKRD1 aggravates ferroptosis and ultimately exacerbates IRI-AKI, underlining ANKRD1's potential as a therapeutic target for kidney IRI. KEY POINTS/HIGHLIGHTS: Ankyrin repeat domain 1 (ANKRD1) is rapidly activated in renal ischaemia‒reperfusion injury (IRI) models in vivo and in vitro. ANKRD1 knockdown mitigates kidney damage and preserves renal function. Ferroptosis contributes to the deteriorating function of ANKRD1 in renal IRI. ANKRD1 promotes acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) degradation via the ubiquitin‒proteasome pathway. The E3 ligase tripartite motif containing 25 (TRIM25) is responsible for ANKRD1-mediated ubiquitination of ACSL3.

Daidzein Inhibits Muscle Atrophy by Suppressing Inflammatory Cytokine- and Muscle Atrophy-Related Gene Expression.

BACKGROUND: Sarcopenic obesity, which is associated with a poorer prognosis than that of sarcopenia alone, may be positively affected by soy isoflavones, known inhibitors of muscle atrophy. Herein, we hypothesize that these compounds may prevent sarcopenic obesity by upregulating the gut metabolites with anti-inflammatory effects. METHODS: To explore the effects of soy isoflavones on sarcopenic obesity and its mechanisms, we employed both in vivo and in vitro experiments. Mice were fed a high-fat, high-sucrose diet with or without soy isoflavone supplementation. Additionally, the mouse C2C12 myotube cells were treated with palmitic acid and daidzein in vitro. RESULTS: The isoflavone considerably reduced muscle atrophy and the expression of the muscle atrophy genes in the treated group compared to the control group (Fbxo32, p = 0.0012; Trim63, p < 0.0001; Foxo1, p < 0.0001; Tnfa, p = 0.1343). Elevated levels of daidzein were found in the muscles and feces of the experimental group compared to the control group (feces, p = 0.0122; muscle, p = 0.0020). The real-time PCR results demonstrated that the daidzein decreased the expression of the palmitate-induced inflammation and muscle atrophy genes in the C2C12 myotube cells (Tnfa, p = 0.0201; Il6, p = 0.0008; Fbxo32, p < 0.0001; Hdac4, p = 0.0002; Trim63, p = 0.0114; Foxo1, p < 0.0001). Additionally, it reduced the palmitate-induced protein expression related to the muscle atrophy in the C2C12 myotube cells (Foxo1, p = 0.0078; MuRF1, p = 0.0119). CONCLUSIONS: The daidzein suppressed inflammatory cytokine- and muscle atrophy-related gene expression in the C2C12 myotubes, thereby inhibiting muscle atrophy.

Blockade of forkhead box protein O1 signaling alleviates primary sclerosing cholangitis-induced sarcopenia in mice model.

AIMS: Primary sclerosing cholangitis (PSC) is a cholestatic liver disease that affects the hepatic bile ducts, leading to hepatic inflammation and fibrosis. PSC can also impact skeletal muscle through the muscle-liver axis, resulting in sarcopenia, a complication characterized by a generalized loss of muscle mass and strength. The underlying mechanisms and therapy of PSC-induced sarcopenia are not well understood, but one potential regulator is the transcription factor forkhead box protein O1 (FOXO1), which is involved in the ubiquitin proteasome system. Thus, the aim of this study is to assess the pharmacological potential of FOXO1 inhibition for treating PSC-induced sarcopenia. MATERIALS AND METHODS: To establish diet-induced PSC model, we provided mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 4 weeks. Mice were intramuscularly injected with AS1842856 (AS), a FOXO1 inhibitor, at a dose of 3.5 mg/kg twice a week for last two weeks. C2C12 myotubes with cholic acid (CA) or deoxycholic acid (DCA) were treated with AS. KEY FINDINGS: We observed a decrease in muscle size and performance in DDC-fed mice with upregulated expression of FOXO1 and E3 ligases such as ATROGIN1 and MuRF1. We found that myotube diameter and MyHC protein level were decreased by CA or DCA in C2C12 myotubes, but treatment of AS reversed these reductions. We observed that intramuscular injection of AS effectively mitigates DDC diet-induced sarcopenia in a rodent PSC model. SIGNIFICANCE: Our study suggests that a FOXO1 inhibitor could be a potential leading therapeutic drug for relieving PSC-induced sarcopenia.

Exploring the Effects of S-Nitrosylation on Caspase-3 Modification and Myofibril Degradation of Beef In Vitro.

This study aimed to explore the effects of S-nitrosylation on caspase-3 modification and its subsequent effects on beef myofibril degradation in vitro. Recombinant caspase-3 was reacted with different concentrations of S-nitrosoglutathione (GSNO, nitric oxide donor) at 37 °C for 30 min and subsequently incubated with purified myofibrillar protein from bovine semimembranosus muscle. Results indicated that the activity of caspase-3 was significantly reduced after GSNO treatments (P < 0.05) and showed a dose-dependent inhibitory effect, which was attributed to the increased S-nitrosylation extent of caspase-3. LC-MS/MS analysis revealed that caspase-3 was S-nitrosylated at cysteine sites 116, 170, 184, 220, and 264. Moreover, the degradation of desmin and troponin-T was notably suppressed by S-nitrosylated caspase-3 (P < 0.05). To conclude, protein S-nitrosylation could modify the cysteine residues of caspase-3, which accounts for the reduced caspase-3 activity and further represses its proteolytic ability on beef myofibrillar protein.

Antioxidative, Glucose Management, and Muscle Protein Synthesis Properties of Fish Protein Hydrolysates and Peptides.

The marine environment is an excellent source for many physiologically active compounds due to its extensive biodiversity. Among these, fish proteins stand out for their unique qualities, making them valuable in a variety of applications due to their diverse compositional and functional properties. Utilizing fish and fish coproducts for the production of protein hydrolysates and bioactive peptides not only enhances their economic value but also reduces their potential environmental harm, if left unutilized. Fish protein hydrolysates (FPHs), known for their excellent nutritional value, favorable amino acid profiles, and beneficial biological activities, have generated significant interest for their potential health benefits. These hydrolysates contain bioactive peptides which are peptide sequences known for their beneficial physiological effects. These biologically active peptides play a role in metabolic regulation/modulation and are increasingly seen as promising ingredients in functional foods, nutraceuticals and pharmaceuticals, with potential to improve human health and prevent disease. This review aims to summarize the current in vitro, cell model (in situ) and in vivo research on the antioxidant, glycaemic management and muscle health enhancement properties of FPHs and their peptides.

Methods for production and assaying catalysis of isolated recombinant human aspartate/asparagine-ß-hydroxylase.

Aspartate/asparagine-ß-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer. This chapter describes methods for the production and purification of soluble constructs of recombinant human AspH suitable for biochemical and crystallographic studies. The chapter also describes efficient methods for performing turnover and inhibition assays which monitor catalysis of isolated recombinant human AspH in vitro using solid phase extraction coupled to mass spectrometry (SPE-MS). The SPE-MS assays employ synthetic disulfide- or thioether-bridged macrocyclic oligopeptides as substrates; a macrocycle is an apparently essential requirement for productive AspH catalysis and mimics an EGFD disulfide isomer that is not typically observed in crystal and NMR structures. SPE-MS assays can be used to monitor catalysis of 2OG oxygenases other than AspH; the methods described herein are representative for 2OG oxygenase SPE-MS assays useful for performing kinetic and/or inhibition studies.

Buzhong Yiqi decoction attenuates acquired myasthenia by regulating the JAK2/STAT3/AKT signaling pathway, inhibiting inflammation, and improving mitochondrial function.

Acquired myasthenia (AM), a debilitating autoimmune disease, is typically characterized by skeletal muscle fatigue and weakness. Despite advances in myasthenia gravis treatment, current approaches remain unsatisfactory and many result in unexpected side effects. Traditional Chinese medicine has shown great potential in the treatment of myasthenia gravis, including relieving myasthenic symptoms, improving patients' quality of life, and reducing Western medicine side effects. This study investigates the protective effects and mechanism of BZYQD in mice with acquired myasthenia. BZYQD alleviates the reduced grip strength and increased expression of MAFbx and MuRF-1 in mice with acquired myasthenia. It also reduces levels of pro-inflammatory factors IL-1ß, IL-6, and TNF-α in the mouse serum. In addition, BZYQD reduces ROS accumulation and the mitochondrial ROS production rate, while increasing ATP levels and mitochondrial membrane potential in mice with acquired myasthenia. Moreover, BZYQD decreases the expression of p-JAK2, p-STAT3, and p-AKT in the skeletal muscle of mice with acquired myasthenia. In summary, BZYQD reduces inflammation, enhances mitochondrial function, and regulates the JAK2/STAT3/AKT signaling pathway to treat acquired myasthenia.

RBM8A, a new target of TEAD4, promotes breast cancer progression by regulating IGF1R and IRS-2.

BACKGROUND: Breast cancer (BC) is the most common malignant tumor in women worldwide, and further elucidation of the molecular mechanisms involved in BC pathogenesis is essential to improve the prognosis of BC patients. RNA Binding Motif Protein 8 A (RBM8A), with high affinity to a myriad of RNA transcripts, has been shown to play a crucial role in genesis and progression of multiple cancers. We attempted to explore its functional significance and molecular mechanisms in BC. METHODS: Bioinformatics analysis was performed on publicly available BC datasets. qRT-PCR was used to determine the expression of RBM8A in BC tissues. MTT assay, clone formation assay and flow cytometry were employed to examine BC cell proliferation and apoptosis in vitro. RNA immunoprecipitation (RIP) and RIP-seq were used to investigate the binding of RBM8A/EIF4A3 to the mRNA of IGF1R/IRS-2. RBM8A and EIF4A3 interactions were determined by co-immunoprecipitation (Co-IP) and immunofluorescence. Chromatin immunoprecipitation (Ch-IP) and dual-luciferase reporter assay were carried out to investigate the transcriptional regulation of RBM8A by TEAD4. Xenograft model was used to explore the effects of RBM8A and TEAD4 on BC cell growth in vivo. RESULTS: In this study, we showed that RBM8A is abnormally highly expressed in BC and knockdown of RBM8A inhibits BC cell proliferation and induces apoptosis in vitro. EIF4A3, which phenocopy RBM8A in BC, forms a complex with RBM8A in BC. Moreover, EIF4A3 and RBM8A complex regulate the expression of IGF1R and IRS-2 to activate the PI3K/AKT signaling pathway, thereby promoting BC progression. In addition, we identified TEAD4 as a transcriptional activator of RBM8A by Ch-IP, dual luciferase reporter gene and a series of functional rescue assays. Furthermore, we demonstrated the in vivo pro-carcinogenic effects of TEAD4 and RBM8A by xenograft tumor experiments in nude mice. CONCLUSION: Collectively, these findings suggest that TEAD4 novel transcriptional target RBM8A interacts with EIF4A3 to increase IGF1R and IRS-2 expression and activate PI3K/AKT signaling pathway, thereby further promoting the malignant phenotype of BC cells.

Filbertone Reduces Senescence in C2C12 Myotubes Treated with Doxorubicin or H2O2 through MuRF1 and Myogenin.

It has been demonstrated that filbertone, the principal flavor compound of hazelnuts, exhibits preventive effects against hypothalamic inflammation, obesity, neurodegenerative diseases, and muscle lipid accumulation. However, its influence on muscle aging has yet to be elucidated. The objective of this study was to investigate the effects of filbertone on muscle aging in C2C12 myotubes subjected to senescence induction by either doxorubicin or hydrogen peroxide. To ascertain the mechanisms by which filbertone exerts its effects, we conducted a series of experiments, including Western blot analysis, reverse transcription quantitative polymerase chain reaction (qRT-PCR), and senescence-associated ß-galactosidase (SA-ß-gal) staining. Filbertone was markedly observed to decrease not only the protein levels of p53 (p < 0.01) in senescence-induced skeletal muscle cells, but also the gene expression levels of p21 (p < 0.05), a direct target of p53. The expression of muscle-related genes, including myogenin and muscle RING-finger protein-1 (MuRF1), was found to be significantly enhanced in senescent muscle cells following treatment with filbertone (p < 0.05). In addition, the number of senescent skeletal muscle cells exhibiting ß-galactosidase activity was found to be markedly reduced in the presence of filbertone (p < 0.01). Collectively, these findings suggest that filbertone plays a pivotal role in the regulation of muscle aging.