Autophagy-lysosome Pathway Research Articles

Impairment of proteasome-associated deubiquitinating enzyme Uchl5/UBH-4 affects autophagy.

The autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) are the two major intracellular proteolytic systems that mediate protein turnover in eukaryotes. Although a crosstalk exists between these two systems, it is still unclear how UPS and ALP interact in vivo. Here, we investigated how impaired function of the proteasome-associated deubiquitinating enzyme (DUB) Uchl5/UBH-4 affects autophagy in human cells and in a multicellular organism. We show that downregulation of Uchl5 by siRNA reduces autophagy by partially blocking the fusion of autophagosomes with the lysosomes in HeLa cells, which is similar to a previously reported role of the proteasome-associated DUB Usp14 on autophagy. However, exposure of Caenorhabditis elegans to ubh-4 or usp-14 RNAi, or to their pharmacological inhibitors, results in diverse effects on numbers of autophagosomes and autolysosomes, without blocking the lysosomal fusion, in the intestine, hypodermal seam cells and the pharynx. Our results reveal that impairment of Uchl5/UBH-4 and Usp14 affects autophagy in a tissue context manner. A deeper insight into the interplay between UPS and ALP in various tissues in vivo has the potential to promote development of therapeutic approaches for disorders associated with proteostasis dysfunction.

Phytochemicals Targeting Mitophagy to Treat Heart Diseases: Retrospective Insights and Prospective Directions.

Mitophagy is a process by which cells selectively eliminate damaged or dysfunctional mitochondria through the autophagy-lysosome pathway, thereby maintaining mitochondrial quality and cellular homeostasis. This process is closely linked to the onset and progression of various heart diseases. Modern pharmacological research has demonstrated that phytochemicals can regulate mitochondrial homeostasis in cardiomyocytes through multiple mechanisms, influencing mitophagy and protecting cardiomyocytes, which in turn exerts anti-cardiovascular effects. However, the underlying mechanisms of these effects are not yet fully understood. This study summarizes the pharmacological effects and molecular mechanisms of mitophagy in heart diseases, aiming to provide reference for the research and treatment of phytochemicals targeting mitophagy against heart diseases. The results indicated that phytochemicals (such as Berberine, Ginsenoside Rg1, Quercetin, Resveratrol, Baicalein, and so on) can exert preventive and therapeutic effects on heart diseases (such as cardiac toxicity or damage, myocardial ischemia/reperfusion injury, heart failure, heart aging, cardiac hypertrophy, cardiomyopathy, and so on.) via regulating the PINK1/Parkin and FUNDC1-dependent mitophagy pathway. These compounds mainly exert their effects by regulating mitochondrial homeostasis, mitochondrial dynamics, mitochondrial oxidative stress, mitochondrial apoptosis, and mitochondrial energy metabolism. This study provides a reference that phytochemicals have effect on anti-cardiovascular effects by regulating mitophagy. However, further in-depth mechanistic and clinical research are needed in the future.

HBMSC-EVs alleviate weightlessness-induced skeletal muscle atrophy by suppressing oxidative stress and inflammation

BackgroundMuscle disuse and offloading in microgravity are likely the primary factors mediating spaceflight-induced muscle atrophy, for which there is currently no effective treatment other than exercise. Extracellular vesicles derived from bone marrow mesenchymal stem cells (BMSC-EVs) possess anti-inflammatory and antioxidant properties, offering a potential strategy for combating weightless muscular atrophy.MethodsIn this study, human BMSCs-EVs (hBMSC-EVs) were isolated using super-centrifugation and characterized. C2C12 myotube nutrition-deprivation and mice tail suspension models were established. Subsequently, the diameter of C2C12 myotubes, Soleus mass, cross-sectional area (CSA) of muscle fibers, and grip strength in mice were assessed to investigate the impact of hBMSC-EVs on muscle atrophy. Immunostaining, transmission electron microscopy observation, and western blot analysis were employed to assess the impact of hBMSC-EVs on muscle fiber types, ROS levels, inflammation, ubiquitin–proteasome system activity, and autophagy lysosome pathway activation in skeletal muscle atrophy.ResultsThe active hBMSC-EVs can be internalized by C2C12 myotubes and skeletal muscle. hBMSC-EVs can effectively reduce C2C12 myotube atrophy caused by nutritional deprivation, with a concentration of 10 × 108 particles/mL showing the best effect (P < 0.001). Additionally, hBMSC-EVs can down-regulate the protein levels associated with UPS and oxidative stress. Moreover, intravenous administration of hBMSC-EVs at a concentration of 1 × 1010 particles/mL can effectively reverse the reduction in soleus mass (P < 0.001), CSA (P < 0.01), and grip strength (P < 0.001) in mice caused by weightlessness. They demonstrate the ability to inhibit protein degradation mediated by UPS and autophagy lysosome pathway, along with the suppression of oxidative stress and inflammatory responses. Furthermore, hBMSC-EVs impede the transition of slow muscle fibers to fast muscle fibers via upregulation of Sirt1 and PGC-1α protein levels.ConclusionsOur findings indicate that hBMSC-EVs are capable of inhibiting excessive activation of the UPS and autophagy lysosome pathway, suppressing oxidative stress and inflammatory response, reversing muscle fiber type transformation, effectively delaying hindlimb unloading-induced muscle atrophy and enhancing muscle function. Our study has further advanced the understanding of the molecular mechanism underlying muscle atrophy in weightlessness and has demonstrated the protective effect of hBMSC-EVs on muscle atrophy.

UBE2T/CDC42/CD276 signaling axis mediates brain metastasis of triple-negative breast cancer via lysosomal autophagy

BackgroundAdvanced triple-negative breast cancer (TNBC) is prone to brain metastasis (BrM). The precise molecular mechanism responsible for this phenomenon has not yet been completely established, so it is vital to comprehend the molecular mechanism behind it.MethodsThe protein chip analysis was conducted to identify any abnormal UBE2T protein expression in TNBC, especially BrM. Here, we used public databases and bioinformatics analysis as well as clinical samples from different cohorts to investigate the interrelationship between UBE2T/CDC42/CD276. This predicted relationship was then repeatedly validated using different in vivo and in vitro experimental methods. Additionally, multiple experimental approaches were implemented, encompassing western blotting, Co-IP, GST pull-down, flow cytometry, mass spectrometry, immunofluorescence, immunohistochemistry, and qRT-PCR to reveal the molecular mechanism of UBE2T-mediated immune escape and BrM.ResultsOur results indicate that expressed at elevated levels in breast cancer, UBE2T is negatively linked to patient prognosis, especially in BrM of TNBC. Data from clinical samples from our different cohorts and TCGA indicate a significant correlation between UBE2T and immunosuppression. Mechanistically, UBE2T directly interacts with CDC42, promoting its K48-linked polyubiquitination and proteasomal degradation, thereby inhibiting CDC42 from degrading CD276 via the autophagy-lysosomal pathway, indirectly upregulating CD276 and thereby impairing the CD8+T cells function, ultimately mediating tumor immune escape and BrM. Finally, animal experimental results also showed that inhibition of UBE2T elevated the TNBC sensitivity to immune checkpoint CD276 blockade and inhibited BrM of TNBC.ConclusionsIn conclusion, our results indicate a new mechanism whereby UBE2T-mediated ubiquitination positively controls the UBE2T/CDC42/CD276 axis to upregulate tumor cell expression of CD276 and thereby impair CD8+T cells function, ultimately leading to tumor cell immune escape and BrM.

The TFEB activator clomiphene citrate ameliorates lipid metabolic syndrome pathology by activating lipophagy and lipolysis

The balance between lipid synthesis and lipid catabolism is critical to maintain energy homeostasis. Lipophagy and lipolysis are two important pathways for lipid selective catabolism. Defects in lipophagy and lipolysis are linked to lipid metabolic diseases. Transcription factor EB (TFEB) is a master regulator of autophagy and lysosome biogenesis, as well as lipid metabolism by promoting expression of genes encoding fat catabolic lipases. Therefore, targeting TFEB provides a novel potential strategy for the treatment of lipid metabolic diseases. In this study, we showed that the TFEB activator clomiphene citrate (CC) activated the autophagy-lysosome and lipolysis pathways, and promoted degradation of lipid droplets induced by the free fatty acids oleate and palmitate in HepG2 cells. Moreover, CC treatment promoted lipid catabolism and attenuated obesity, restored lipid levels, blood glucose levels and insulin resistance, hepatocellular injury and hepatic steatosis, as well as liver inflammation in the HFD fed mice. In addition, we found that En-CC, a trans-isomer of CC, displayed less toxicity and more efficient activation of TFEB. Consistent with CC, En-CC treatment improved lipid metabolic syndrome pathology. These findings demonstrate that CC promotes clearance of lipids and ameliorates HFD-induced lipid metabolic syndrome pathology through activating TFEB-mediated lipophagy and lipolysis, indicating that CC has the potential to be used to treat lipid metabolic diseases.

Sodium pump subunit NKAα1 protects against diabetic endothelial dysfunction by inhibiting ferroptosis through the autophagy-lysosome degradation of ACSL4.

The sodium pump Na+/K+-ATPase (NKA), an enzyme ubiquitously expressed in various tissues and cells, is a critical player in maintaining cellular ion homeostasis. Dysregulation of α1 subunit of NKA (NKAα1) has been associated with cardiovascular and metabolic disorders, yet the exact role of NKAα1 in diabetes-induced endothelial malfunction remains incompletely understood. The NKAα1 expression and NKA activity were examined in high-glucose (HG)-exposed endothelial cells (ECs) and mouse aortae, as well as in high-fat-diet (HFD)-fed mice. Acetylcholine (Ach) was utilised to assess endothelium-dependent relaxation (EDR) in isolated mouse aortae. We found that both NKAα1 protein and mRNA levels were significantly downregulated in the aortae of HFD-fed mice, and HG-incubated mouse aortae and ECs. Gain- andloss-of-functionexperiments revealed that NKAα1 preserves EDR by mitigating oxidative/nitrative stresses in ECs. Overexpression of NKAα1 facilitated EC viability, migration, and angiogenesis by inhibiting the overproduction of superoxide and peroxynitrite. Mechanistically, dysfunctional NKAα1 impaired autophagy process, and prevented the transfer of acyl-CoA synthetase long-chain family member 4 (ACSL4) to the lysosome for degradation, thereby resulting in lipid peroxidation and ferroptosis in ECs. Induction of ferroptosis and inhibition of the autophagy-lysosome pathway blocked the protective effects of NKAα1 on EDR. Eventually, we identified Hamaudol as a potent activator of NKAα1 by restraining the phosphorylation and endocytosis of NKAα1, restoring EDR in obese diabetic mice. Overall, NKAα1 facilitates the autophagic degradation of ACSL4 via the lysosomal pathway, preventing ferroptosis and oxidative/nitrative stress in ECs. NKAα1 may serve as an attractive candidate for the management of vascular disorders associated with diabetes. KEY POINTS: NKAα1 downregulation impairs endothelial function in diabetes by promoting oxidative/nitrative stress and ferroptosis. NKAα1 supports lysosomal degradation of ACSL4 via autophagy, preventing lipid peroxidation and ferroptosis. Hamaudol, an activator of NKAα1, restores endothelial relaxation in diabetic mice by inhibiting NKAα1 phosphorylation and endocytosis.

TFEB-dependent autophagy-lysosomal pathway is required for NRF2-driven antioxidative action in obstructive sleep apnea-induced neuronal injury.

Nearly one billion individuals worldwide suffer from obstructive sleep apnea (OSA) and are potentially impacted by related neurodegeneration. TFEB is considered a master regulator of autophagy and lysosomal biogenesis, but little is known about its role in neuronal oxidative stress and resultant injury induced by OSA. This study aimed to investigate these issues. Here, we demonstrated that neuronal TFEB induction is repressed in OSA mouse models. Activation of a TFEB-dependent autophagy-lysosomal pathway (ALP) reduces hippocampal neuronal cell death and mitigates OSA-related cognitive impairment. Neuronal NRF2 induction was also found to be defective in OSA mouse models. A series of staining assays for HO1, SOD3, ROS, GSH, 8-OHdG, MDA and PI revealed that enhancement of NRF2 expression restores neuronal redox balance and protects hippocampal neurons. We then identified a novel interplay between TFEB-dependent ALP and NRF2-mediated relief of oxidative stress. Inhibition of NRF2 hinders TFEB expression and lysosomal biogenesis. Conversely, knockdown of TFEB or blocking autophagy dampens the antioxidative effect of NRF2. Our findings highlight the unexpected and crucial role of TFEB-dependent ALP as a downstream event of NRF2 in NRF2-promoted redox balance. This study provides novel insights into the mechanism behind NRF2-driven antioxidative action and the regulation of TFEB-dependent ALP.

Enhancing hepatocellular carcinoma therapy with DOX-loaded SiO2 nanoparticles via mTOR-TFEB pathway autophagic flux inhibition

Chemotherapeutic drugs often fail to provide long-term efficacy due to their lack of specificity and high toxicity. To enhance the biosafety and reduce the side effects of these drugs, various nanocarrier delivery systems have been developed. In this study, we loaded the anticancer drug doxorubicin (DOX) and an MRI contrast agent into silica nanoparticles, coating them with pH-responsive and tumor cell-targeting polymers. These polymers enable the carrier to achieve targeted delivery and controlled drug release in acidic environments. This integrated diagnostic and therapeutic strategy successfully achieved both the diagnosis and treatment of liver cancer. Additionally, we demonstrated that the nanocarrier inhibits autophagic flux in liver cancer cells by targeting the autophagy-lysosome pathway and regulating the nuclear translocation of TFEB, thereby promoting tumor cell death. This novel diagnostic-integrated nanocarrier is expected to be a promising tool for targeted liver cancer treatment.

AlphaFold-Based AI Docking Reveals AMPK/SIRT1-TFEB Pathway Modulation by Traditional Chinese Medicine in Metabolic-Associated Fatty Liver Disease.

Metabolic-associated fatty liver disease (MAFLD) is a chronic, progressive disorder characterized by hepatic steatosis and excessive lipid accumulation. Its high global adult prevalence (approximately 50.7%), however, FDA-approved therapeutic drugs remains lacking. Qigui Jiangzhi Formula (QGJZF) shows promise in treating MAFLD by effectively decreasing lipid levels and improving hepatic steatosis, however its mechanisms remain unclear. This study investigated QGJZF's effects in high-fat diet-induced zebrafish and golden hamsters, and in palmitate (PA) and oleic acid (OA) - induced HepG2 cells, using the SymMap database to identify potential targets and pathways of QGJZF in MAFLD and AlphaFold algorithms to predict protein structures. In vivo, QGJZF significantly alleviatied hepatic lipid deposition. Intriguingly, QGJZF decreased lipid droplets and its levels are negative correlation with the numbers of autolysosomes, indicating that QGJZF's mechanism of meliorating liver lipid deposition may be relate to the regulation of autophagy. QGJZF upregulated the expressions of phosphorylated -Adenosine 5'-monophosphate (AMP) - activated protein kinase (p-AMPK), Sirtuin deacetylase 1 (SIRT1) and Transcription factor EB (TFEB), accompanied by the changes in autophagy-related proteins. In vitro, QGJZF inhibited the lipid deposition in, and its effect was blocked by Baf-A1, which was mediated through upregulation of TFEB and its mediated autophagy-lysosomal pathway. Moreover, cotreatment with AMPK inhibitor Compound C, the regulation of QGJZF on TFEB, SIRT1, autophagy-related protein levels, and lipid deposition were reversed. Network pharmacology identified the PRKAA2 (AMPK) and SIRT1 as key hub targets. Futher analysis of their structures using AlphaFold3 algorithms, yielded high-ranking scores of 0.97 and 0.93, respectively. Liquid chromatography-mass spectrometry combined with molecular docking expounded its five compounds in QGJZF binding to AMPK protein. These findings suggest that QGJZF as a therapeutic agent in augmenting autophagy-facilitated lipid clearance for the management of MAFLD via AMPK/SIRT1-TFEB axis.

Engineering Covalent Aptamer Chimeras for Enhanced Autophagic Degradation of Membrane Proteins.

Targeted degradation of membrane proteins represents an attractive strategy for eliminating pathogenesis-related proteins. Aptamer-based chimeras hold great promise as membrane protein degraders, however, their degradation efficacy is often hindered by the limited structural stability and the risk of off-target effects due to the non-covalent interaction with target proteins. We here report the first design of a covalent aptamer-based autophagosome-tethering chimera (CApTEC) for the enhanced autophagic degradation of cell-surface proteins, including transferrin receptor 1 (TfR1) and nucleolin (NCL). This strategy relies on the site-specific incorporation of sulfonyl fluoride groups onto aptamers to enable the cross-linking with target proteins, coupled with the conjugation of an LC3 ligand to hijack the autophagy-lysosomal pathway for targeted protein degradation. The chemically engineered CApTECs exhibit enhanced on-target retention and improved structural stability. Our results also demonstrate that CApTECs achieve remarkably enhanced and prolonged degradation of membrane proteins compared to the non-covalent designs. Furthermore, the CApTEC targeting TfR1 is combined with 5-fluorouracil (5-FU) for synergistic tumor therapy in a mouse model, leading to substantial suppression of tumor growth. Our strategy may provide deep insights into the LC3-mdiated autophagic degradation, affording a modular and effective strategy for membrane protein degradation and precise therapeutic applications.

Hypoxia-triggered ERRα acetylation enhanced its oncogenic role and promoted progression of renal cell carcinoma by coordinating autophagosome-lysosome fusion

Estrogen-related receptor α (ERRα) is dysregulated in many types of cancer and exhibits oncogenic activity by promoting tumorigenesis and metastasis of cancer cells. However, its defined role in renal cell carcinoma (RCC) has not been fully elucidated. To reveal the biological function of ERRα and determine the underlying regulatory mechanism in RCC, the quantitative proteomics analysis and mechanism investigation were conducted. The results demonstrated that ERRα promoted the proliferation and tumorigenesis of RCC cells by maintaining lysosome-dependent autophagy flux. ERRα inhibition impaired the transcriptional expression of LAMP2 and VAMP8 and blocked the fusion of autophagosomes with lysosomes, causing the impairment of the autophagy-lysosome pathway and tumor repression in RCC. Moreover, VHL mutant-induced hyperactive hypoxia signaling in RCC triggered p300/CBP-mediated acetylation at the DNA-binding domain of ERRα, and this acetylation promoted its affinity toward targeting DNA and Parkin-mediated ubiquitination and proteasome-dependent degradation. This regulatory model enhanced ERRα transactivation on the expression of LAMP2 and VAMP8, which then maintained autophagy flux and RCC progression. Pharmaceutical inhibition on ERRα acetylation-mediated autophagy-lysosome pathway led to growth repression and sunitinib sensitivity of RCC cells. Taken together, this study uncovered a novel regulatory mechanism of acetylation contributing to the transcriptional performance and the oncogenic role of ERRα in RCC progression by modulating the autophagy-lysosome pathway. These findings might provide a novel approach for the clinical diagnosis and resolution of sunitinib resistance of RCC.

Actions of dexmedetomidine in regulating NLRP3 in postoperative cognitive dysfunction in aged mice via the autophagy-lysosome pathway.

Autophagy-lysosomal pathway dysfunction leads to postoperative cognitive dysfunction (POCD). Dexmedetomidine (Dex) improves POCD, and we probed the effects of Dex on autophagy-lysosomal pathway dysfunction in a POCD model. A POCD mouse model was established and intraperitoneally injected with Dex. Cognitive function was evaluated by Morris water maze/open field test/novel object recognition assay. Levels of neurotransmitters/inflammatory cytokines in hippocampus, and NLRP3/ASC/Cleaved Caspase-1 proteins were determined by ELISA/Western blot. NLRP3 inflammasome-mediated microglial activation/astrocyte A1 differentiation in the hippocampal CA1 region were assessed by immunofluorescence assay. BV-2 cells were treated with lipopolysaccharide (LPS) and Dex and/or the NLRP3 inflammasome activator Nigericin, and transfected with si-TFEB for co-culture with primary reactive astrocytes (RAs) to verify the function of Dex in vitro. Dex alleviated cognitive dysfunction in POCD mice and repressed NLRP3 inflammasome-mediated microglial activation and astrocyte A1 differentiation. NLRP3 inflammasome activation partially reversed the protective effect of Dex on the POCD condition. In vitro experiments verified the inhibitory properties of Dex on microglial activation and astrocyte A1 differentiation. Dex induces TFEB nuclear translocation, microglial autophagy and lysosomal biogenesis. By activating the autophagy-lysosome pathway, Dex regulated NLRP3 inflammasome-mediated microglial activation, inhibited astrocyte A1 differentiation and alleviated POCD in vivo. Dex regulates NLRP3 inflammasome-mediated hippocampal microglial activation by promoting TFEB nuclear translocation and activating the autophagy-lysosome pathway and inhibits astrocyte A1 differentiation, thereby alleviating POCD.

Regulatory Mechanisms and Therapeutic Implications of Lysosomal Dysfunction in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-beta (Aβ) plaques, neurofibrillary tangles (NFTs) formed from hyperphosphorylated Tau, and widespread neuronal loss. The autophagy-lysosomal pathway plays a crucial role in maintaining cellular homeostasis by degrading and recycling of damaged organelles and aggregate amyloid proteins implicated in AD. Lysosomes are key effectors of autophagic process, responsible for the breakdown of a variety of damaged organelles and aggregate or dysfunctional proteins. This review examines the role of lysosomal dysfunction in AD pathophysiology, focusing on genetic factors, acidification abnormalities, and other contributing factors. We also explore the involvement of lysosomal dysfunction of microglia in AD pathology, and cover the role of lysosomal stress response (LSR) in cellular response to neuronal injury associated with AD. Furthermore, we discuss potential therapeutic strategies targeting lysosomal proteolysis pathway and addressing lysosomal dysfunction for AD treatment, including the pharmacologically activating lysosomal activity, regulating TFEB, and considering other emerging approaches.

Itaconic acid ameliorates necrotizing enterocolitis through the TFEB-mediated autophagy-lysosomal pathway.

Excessive autophagy has been implicated in the pathogenesis of necrotizing enterocolitis (NEC), yet the molecular underpinnings of the autophagy-lysosomal pathway (ALP) in NEC are not well characterized. This study aimed to elucidate alterations within the ALP in NEC by employing RNA sequencing on intestinal tissues obtained from affected infants. Concurrently, we established animal and cellular models of NEC to assess the therapeutic efficacy of itaconic acid (ITA). Our results indicate that the ALP is significantly disrupted in NEC. Notably, ITA was found to modulate the ALP, enhancing autophagic flux and lysosomal function, which consequently alleviated NEC symptoms. Further analysis revealed that ITA's beneficial effects are mediated through the promotion of TFEB nuclear translocation, thereby augmenting the ALP. These findings suggest that targeting the ALP with ITA to modulate TFEB activity may represent a viable therapeutic approach for NEC.

Serum starvation induces cytosolic DNA trafficking via exosome and autophagy-lysosome pathway in microglia.

The imbalance of microglial homeostasis is highly associated with age-related neurological diseases, where cytosolic endogenous DNA is also likely to be found. As the main medium for storing biological information, endogenous DNA could be localized to cellular compartments normally free of DNA when cells are stimulated. However, the intracellular trafficking of endogenous DNA remains unidentified. In this study, we demonstrated that nuclear DNA (nDNA) and mitochondrial DNA (mtDNA), as the components of endogenous DNA, undergo different intracellular trafficking under conditions of microglial homeostasis imbalance induced by serum starvation. Upon detecting various components of endogenous DNA in the cytoplasmic and extracellular microglia, we found that cytosolic nDNA primarily exists in a free form and undergoes degradation through the autophagy-lysosome pathway. In contrast, cytosolic mtDNA predominantly exists in a membrane-wrapped form and is trafficked through both exosome and autophagy-lysosome pathways, with the exosome pathway serving as the primary one. When the autophagy-lysosome pathway was inhibited, there was an increase in exosomes. More importantly, the inhibition of the autophagy-lysosome pathway resulted in enhanced trafficking of mtDNA through the exosome pathway. These findings unveiled the crosstalk between these two pathways in the trafficking of microglial cytosolic DNA and thus provide new insights into intervening in age-related neurological diseases.

FOXS1, frequently inactivated by promoter methylation, inhibited colorectal cancer cell growth by promoting TGFBI degradation through autophagy-lysosome pathway.

Tumor suppressor gene (TSG) inactivation by epigenetic modifications contributes to the carcinogenesis and progression of colorectal cancer (CRC). Expression profiling and CpG methylomics revealed that a forkhead-box transcriptional factor, FOXS1, is downregulated and methylated in CRC. To assess the biological functions and underlying mechanisms of FOXS1 in colorectal cancer. Public databases, semi-quantitative RT-PCR, immunohistochemistry, MSP, and BGS were used to analyze FOXS1 expression and promoter methylation in CRC. Stable FOXS1-overexpressing or knockdown cell lines were established. Cell growth, colony formation, flow cytometry, GFP-LC3 puncta detection, Ad-mCherry-GFP-LC3B, qPCR, in vivo subcutaneous tumor model, RNA-seq, western blotting, immunofluorescence, Co-IP assays, and protein stability analysis were performed to investigate the underlying molecular mechanisms of FOXS1. In CRC, FOXS1 was frequently downregulated due to promoter CpG methylation, acting as an independent prognostic marker. Moreover, FOXS1 exerts inhibitory effects on the growth of CRC cells in vitro and in vivo, while concurrently promoting CRC cell autophagy. Intriguingly, we found that FOXS1 interacted with transforming growth factor beta induced (TGFBI) and FOXS1 promoted TGFBI degradation through the autophagy-lysosome pathway rather than the ubiquitin-proteasome system. FOXS1 was also found to facilitate the interaction between TGFBI and lysosomal associated membrane protein 2A (LAMP2A), leading to the translocation of TGFBI into lysosomes for degradation. Additionally, FOXS1 regulates AKT phosphorylation and FOXO3a nuclear translocation, promoting the transcription of autophagy-related genes downstream of FOXO3a. Restoration of TGFBI expression reversed the suppressive effect exerted by FOXS1 on the growth of colorectal cancer cells. FOXS1 functions as a tumor suppressor that is methylated in CRC and promotes the lysosomal degradation of TGFBI, regulates cell growth and promotes autophagy in CRC through the TGFBI/AKT/FOXO3a signaling pathway. These findings indicate that FOXS1 exhibits potential as a promising biomarker and therapeutic target for colorectal cancer.

GDF15 inhibits early-stage adipocyte differentiation by enhancing HOP2 expression and suppressing C/EBPα expression.

Excessive adipocyte differentiation and accumulation contribute to the development of metabolic disorders. Growth differentiation factor 15 (GDF15) plays an essential role in energy homeostasis and is considered an anti-obesity factor; however, elevated serum levels of endogenous GDF15 have been reported in certain individuals with obesity. In this study, to gain a better understanding of this complex relationship between GDF15 levels and obesity, we investigated GDF15 expression and function during adipogenesis. Compared with mice fed a normal diet, those fed a short-term high-fat diet exhibited a reduction in epididymal white adipose tissue and serum GDF15 expression. These results were confirmed in human adipose-derived stem cells that showed reduced GDF15 expression during adipogenesis differentiation. During adipogenesis, GDF15 was primarily degraded via the autophagy lysosomal pathway, and GDF15 overexpression in pre-adipocytes inhibited adipogenesis by suppressing CCAAT enhancer binding protein alpha (C/EBPα). Furthermore, whereas we detected a reduction in homologous-pairing protein 2 (HOP2) expression during adipogenesis, expression increased in response to an overexpression of GDF15. Furthermore, following knockdown of HOP2 during GDF15 overexpression, there was no suppression of C/EBPα expression. These findings indicate that GDF15 undergoes lysosomal degradation via an autophagic pathway and suppresses adipocyte differentiation via the HOP2-mediated inhibition of C/EBPα expression. Collectively, our findings indicate that GDF15 could serve as a potential therapeutic target for the treatment of metabolic disorders.

Histone Lysine Crotonylation Accelerates ACSL4-Mediated Ferroptosis of Keratinocytes via Modulating Autophagy in Diabetic Wound Healing.

Dysfunction of keratinocytes affects diabetic wound healing, but underlying mechanisms have not been understood. This study examines crotonylation's role in ferroptosis and autophagy in keratinocytes, particularly regarding ACSL4, using STZ-induced diabetic rats and high glucose-exposed keratinocytes to assess these processes. The ACSL4 knockdown was achieved using adenovirus in wounds to examine the impact of ferroptosis modulation on healing diabetic wounds. MB-3 was utilized to block the H3K27 crotonylation (H3K27cr) in order to clarify the regulatory function of crotonylation in both autophagy and ferroptosis. In STZ-induced diabetic skin and high glucose-exposed keratinocytes, ferroptosis mediated by ACSL4 and suppression of autophagic flux were demonstrated. Moreover, the downregulation of ACSL4 triggered ferroptosis in adjacent wounds of diabetic rats and improved wound healing. The degradation of ACSL4 may be observed via the autophagy-lysosome pathway in keratinocytes. Downregulation of SQSTM1 in diabetic keratinocytes leads to autophagy inhibition and modulates the protein level of ACSL4. Mechanistically, total crotonylation levels and H3K27cr were remarkably elevated in the skin and keratinocytes of diabetic rats; blocking high glucose-induced H3K27cr with MB-3 can enhance SQSTM1 transcription and expression while promoting autophagy and reducing ACSL4-induced ferroptosis in keratinocytes. Therefore, H3K27cr influences autophagy by adjusting SQSTM1 to facilitate ACSL4-triggered ferroptosis in diabetic keratinocytes. This study clarifies the relationships between acylation modifications, autophagy, and ferroptosis, while also offering mechanistic insights and potential therapeutic targets for issues associated with diabetic wound healing.

Astaxanthin promotes the longevity of Caenorhabditis elegans via modulation of the intracellular redox status and PHA-4-mediated autophagy.

Astaxanthin is a xanthophyll carotenoid which has been associated with a number of health-promoting effects, including anti-aging; however, the underlying mechanisms are not fully understood. In the present study, it was found that astaxanthin promoted the longevity of wild-type (N2) Caenorhabditis elegans (C. elegans). The lifespan-extending effect of astaxanthin was associated with a significant decrease of lipofuscin accumulation and the reduction of the age-related decline in spontaneous motility. Meanwhile, astaxanthin enhanced the oxidative stress resistance in C. elegans, preventing the elevation of the reactive oxygen species and alleviating juglone-induced toxicity. Further studies revealed that astaxanthin treatment induced the expression of the skn-1 gene; besides, the lifespan-extending effect of astaxanthin relied on SKN-1. Additionally, the expression of age-1, a PI3K homolog gene, and let-363, a target of the rapamycin (TOR) homolog gene, was decreased, while the expression of PHA-4, a transcription factor negatively regulated by TOR signaling, was increased by astaxanthin treatment. PHA-4 has been demonstrated to regulate the expression of genes playing critical roles in the autophagy-lysosome pathway (ALP). Consistently, several key genes related to ALP, including lgg-1, atg-5, vps-34, ncr-1 and asm-1 were upregulated in C. elegans treated with astaxanthin. Knockdown of pha-4 expression by siRNA prevented the elevation of the above ALP-related genes, while diminishing the lifespan-extension effect of astaxanthin. Overall, these results indicated that astaxanthin prolonged the lifespan of C. elegans via modulating the intracellular redox status and promoting PHA-4-mediated autophagy.

BMSCs-derived small extracellular vesicles antagonize cerebral endothelial Caveolin-1 driven autophagic degradation of tight-junction proteins to protect blood-brain barrier post-stroke.

Bone marrow mesenchymal stem cells (BMSCs) -derived extracellular vesicles (EVs), especially small EVs (sEVs), were vastly reported to enable multiple restorative effects on ischemic stroke, yet the protective mechanism of blood-brain barrier (BBB) has not been fully illustrated. In the present study, we investigated the therapeutic effects and mechanism of BMSCs-derived sEVs on BBB injury after ischemic stroke. In-vivo, administering sEVs to transient middle cerebral artery occlusion (tMCAo) mice mitigated the brain infarct volume, BBB permeability and neural apoptosis, and improved the cerebral blood flow perfusion and neurological function. Simultaneously, cerebral vascular endothelial overexpressed Caveolin-1 (Cav-1) together with its strong co-localization with autophagosome protein LC3B were suppressed, and ZO-1 and Occludin expressions were enhanced, whose results were consistent with those of oxygen-glucose-deprivation/reperfusion (OGD/R)-insulted brain endothelial cells (BECs) in vitro. Furthermore, by employing Cav-1 siRNA and pcDNA3.1 transfection, Co-immunoprecipitation, cycloheximide assay, and molecular docking, it proved that brain endothelial Cav-1 was an essential upstream of autophagy activation, contributing to tight-junction proteins delegation via the autophagy-lysosomal pathway. Altogether, our study demonstrates the novel mechanism of Cav-1-dependent tight-junction proteins autophagic disruption on BBB integrity after ischemic stroke, and BMSC-sEVs treatment can reverse such hazard cascades.