This study investigated the protective effect of carnosine and its components (L-histidine and β-alanine [HA]) against dexamethasone (Dex)-induced muscle atrophy in C2C12 myotubes. Myotubes were treated with Dex (10 μM) to induce muscle atrophy manifested by decreased myotube diameter, low myosin heavy chain content, and increased expression of muscle atrophy-associated ubiquitin ligases (Atrogin-1, MuRF-1, and Cbl-b). Carnosine (20 mM) treatment significantly improved the myotube diameter and MyHC protein expression level in Dex-treated C2C12 myotubes. It also downregulated the expression of Atrogin-1, MuRF-1, and Cbl-b and suppressed the expression of forkhead box O3 (FoxO3a) mediated by Dex. Furthermore, reactive oxygen species production was increased by Dex but was ameliorated by carnosine treatment. However, HA (20 mM), the component of carnosine, treatment was found ineffective in preventing Dex-induced protein damage. Therefore, based on above results it can be suggested that carnosine could be a potential therapeutic agent to prevent Dex-induced muscle atrophy compared to its components HA.

FOXK2 is a crucial transcription factor implicated in a wide array of biological activities and yet understanding of its molecular regulation at the level of protein turnover is limited. Here, we identify that FOXK2 undergoes degradation in lung epithelia in the presence of the virulent pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae through ubiquitin-proteasomal processing. FOXK2 through its carboxyl terminus (aa 428-478) binds the Skp-Cullin-F-box ubiquitin E3 ligase subunit FBXO24 that mediates multisite polyubiquitylation of the transcription factor resulting in its nuclear degradation. FOXK2 was detected within the mitochondria and targeted depletion of the transcription factor or cellular expression of FOXK2 mutants devoid of key carboxy terminal domains significantly impaired mitochondrial function. In experimental bacterial pneumonia, Fbxo24 heterozygous mice exhibited preserved mitochondrial function and Foxk2 protein levels compared to WT littermates. The results suggest a new mode of regulatory control of mitochondrial energetics through modulation of FOXK2 cellular abundance.

Oleocanthal (OC) is a monophenol of extra-virgin olive oil (EVOO) endowed with antibiotic, cardioprotective and anticancer effects, among others, mainly in view of its antioxidant and anti-inflammatory properties. OC has been largely investigated in terms of its anticancer activity, in Alzheimer disease and in collagen-induced arthritis; however, the possibility that it can also affect muscle biology has been totally overlooked so far. This study is the first to describe that OC modulates alterations induced in C2C12 myotubes by stimuli known to induce muscle wasting in vivo, namely TNF-α, or in the medium conditioned by the C26 cachexia-inducing tumor (CM-C26). C2C12 myotubes were exposed to CM-C26 or TNF-α in the presence or absence of OC for 24 and 48 h and analyzed by immunofluorescence and Western blotting. In combination with TNF-α or CM-C26, OC was revealed to be able to restore both the myotube\'s original size and morphology and normal levels of both atrogin-1 and MuRF1. OC seems unable to impinge on the autophagic-lysosomal proteolytic system or protein synthesis. Modulations towards normal levels of the expression of molecules involved in myogenesis, such as Pax7, myogenin and MyHC, were also observed in the myotube cultures exposed to OC and TNF-α or CM-C26. In conclusion, the data presented here show that OC exerts a protective action in C2C12 myotubes exposed to TNF-α or CM-C26, with mechanisms likely involving the downregulation of ubiquitin-proteasome-dependent proteolysis and the partial relief of myogenic differentiation impairment.

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

There is an increasing tendency for sepsis patients to suffer from diaphragm atrophy as well as mortality. Therefore, reducing diaphragm atrophy could benefit sepsis patients\' prognoses. Studies have shown that Anisodamine (Anis) can exert antioxidant effects when blows occur. However, the role of Anisodamine in diaphragm atrophy in sepsis patients has not been reported. Therefore, this study investigated the antioxidant effect of Anisodamine in sepsis-induced diaphragm atrophy and its mechanism. We used cecal ligation aspiration (CLP) to establish a mouse septic mode and stimulated the C2C12 myotube model with lipopolysaccharide (LPS). After treatment with Anisodamine, we measured the mice\'s bodyweight, diaphragm weight, fiber cross-sectional area and the diameter of C2C12 myotubes. The malondialdehyde (MDA) levels in the diaphragm were detected using the oxidative stress kit. The expression of MuRF1, Atrogin1 and JAK2/STAT3 signaling pathway components in the diaphragm and C2C12 myotubes was measured by RT-qPCR and Western blot. The mean fluorescence intensity of ROS in C2C12 myotubes was measured by flow cytometry. Meanwhile, we also measured the levels of Drp1 and Cytochrome C (Cyt-C) in vivo and in vitro by Western blot. Our study revealed that Anisodamine alleviated the reduction in diaphragmatic mass and the loss of diaphragmatic fiber cross-sectional area and attenuated the atrophy of the C2C12 myotubes by inhibiting the expression of E3 ubiquitin ligases. In addition, we observed that Anisodamine inhibited the JAK2/STAT3 signaling pathway and protects mitochondrial function. In conclusion, Anisodamine alleviates sepsis-induced diaphragm atrophy, and the mechanism may be related to inhibiting the JAK2/STAT3 signaling pathway.

FBXO32, a member of the F-box protein family, is known to play both oncogenic and tumor-suppressive roles in different cancers. However, the functions and the molecular mechanisms regulated by FBXO32 in lung adenocarcinoma (LUAD) remain unclear. Here, we report that FBXO32 is overexpressed in LUAD compared with normal lung tissues, and high expression of FBXO32 correlates with poor prognosis in LUAD patients. Firstly, we observed with a series of functional experiments that FBXO32 alters the cell cycle and promotes the invasion and metastasis of LUAD cells. We further corroborate our findings using in vivo mouse models of metastasis and confirmed that FBXO32 positively regulates LUAD tumor metastasis. Using a proteomic-based approach combined with computational analyses, we found a positive correlation between FBXO32 and the PI3K/AKT/mTOR pathway, and identified PTEN as a FBXO32 interactor. More important, FBXO32 binds PTEN via its C-terminal substrate binding domain and we also validated PTEN as a bona fide FBXO32 substrate. Finally, we demonstrated that FBXO32 promotes EMT and regulates the cell cycle by targeting PTEN for proteasomal-dependent degradation. In summary, our study highlights the role of FBXO32 in promoting the PI3K/AKT/mTOR pathway via PTEN degradation, thereby fostering lung adenocarcinoma progression.

This study aimed to investigate the effects and mechanism of Lactobacillus gasseri BNR17, a probiotic strain isolated from human breast milk, on dexamethasone-induced muscle loss in mice and cultured myotubes. BALB/c mice were intraperitoneally injected with dexamethasone, and orally administered L. gasseri BNR17 for 21 days. L. gasseri BNR17 treatment ameliorated dexamethasone-induced decline in muscle function, as evidenced by an increase in forelimb grip strength, treadmill running time, and rotarod retention time in both female and male mice. In addition, L. gasseri BNR17 treatment significantly increased the mass of the gastrocnemius and quadriceps muscles. Dual-energy X-ray absorptiometry showed a significant increase in lean body mass and a decrease in fat mass in both whole body and hind limb after treatment with L. gasseri BNR17. It was found that L. gasseri BNR17 treatment downregulated serum myostatin level and the protein degradation pathway composed of muscle-specific ubiquitin E3 ligases, MuRF1 and MAFbx, and their transcription factor FoxO3. In contrast, L. gasseri BNR17 treatment upregulated serum insulin-like growth factor-1 level and Akt-mTOR-p70S6K signaling pathway involved in protein synthesis in muscle. As a result, L. gasseri BNR17 treatment significantly increased the levels of major muscular proteins such as myosin heavy chain and myoblast determination protein 1. Consistent with in vivo results, L. gasseri BNR17 culture supernatant significantly ameliorated dexamethasone-induced C2C12 myotube atrophy in vitro. In conclusion, L. gasseri BNR17 ameliorates muscle loss by downregulating the protein degradation pathway and upregulating the protein synthesis pathway.

Astaxanthin (AST) is a natural marine carotenoid with a variety of biological activities. This study aimed to demonstrate the possible mechanisms by which AST improves skeletal muscle atrophy in cancer cachexia. In this study, the effects of different doses of AST (30 mg/kg b.w., 60 mg/kg b.w. and 120 mg/kg b.w.) on skeletal muscle functions were explored in mice with cancer cachexia. The results showed that AST (30, 60 and 120 mg/kg b.w.) could effectively protect cachexia mice from body weight and skeletal muscle loss. AST dose-dependently ameliorated the decrease in myofibres cross-sectional area and increased the expression of myosin heavy chain (MHC). AST treatment decreased both the serum and muscle level of IL-6 but not TNF-α in C26 tumor-bearing cachexia mice. Moreover, AST alleviated skeletal muscle atrophy by decreasing the expression of two muscle-specific E3 ligases MAFBx and MuRF-1. AST improved mitochondrial function by downregulating the levels of muscle Fis1, LC3B and Bax, upregulating the levels of muscle Mfn2 and Bcl-2. In conclusion, our study show that AST might be expected to be a nutritional supplement for cancer cachexia patients.

Skeletal muscle wasting results from numerous pathological conditions affecting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role of E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss-of-function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 resulted in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fiber integrity. We further implicated a disruption in atrogin-1-mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We revealed that BiP was not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorated pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicate atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy and highlight atrogin-1\'s essential role in maintaining muscle homeostasis.

Understanding the mechanisms that govern the stability of functionally crucial proteins is essential for various cellular processes, development, and overall cell viability. Disturbances in protein homeostasis are linked to the pathogenesis of neurodegenerative diseases. PTEN-induced kinase 1 (PINK1), a protein kinase, plays a significant role in mitochondrial quality control and cellular stress response, and its mutated forms lead to early-onset Parkinson\'s disease. Despite its importance, the specific mechanisms regulating PINK1 protein stability have remained unclear. This study reveals a cytoplasmic interaction between PINK1 and F-box and WD repeat domain-containing 7β (FBW7β) in mammalian cells. FBW7β, a component of the Skp1-Cullin-1-F-box protein complex-type ubiquitin ligase, is instrumental in recognizing substrates. Our findings demonstrate that FBW7β regulates PINK1 stability through the Skp1-Cullin-1-F-box protein complex and the proteasome pathway. It facilitates the K48-linked polyubiquitination of PINK1, marking it for degradation. When FBW7 is absent, PINK1 accumulates, leading to heightened mitophagy triggered by carbonyl cyanide 3-chlorophenylhydrazone treatment. Moreover, exposure to the toxic compound staurosporine accelerates PINK1 degradation via FBW7β, correlating with increased cell death. This study unravels the intricate mechanisms controlling PINK1 protein stability and sheds light on the novel role of FBW7β. These findings deepen our understanding of PINK1-related pathologies and potentially pave the way for therapeutic interventions.