Targeting Autophagy for Acetaminophen-Induced Liver Injury: An Update.

Acetaminophen (APAP) overdose can induce hepatocyte necrosis and acute liver failure in experimental rodents and humans. APAP is mainly metabolized via hepatic cytochrome P450 enzymes to generate the highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which forms acetaminophen protein adducts (APAP-adducts) and damages mitochondria, triggering necrosis. APAP-adducts and damaged mitochondria can be selectively removed by autophagy. Increasing evidence implies that the activation of autophagy may be beneficial for APAP-induced liver injury (AILI). In this minireview, we briefly summarize recent progress on autophagy, in particular, the pharmacological targeting of SQSTM1/p62 and TFEB in AILI.

Oroxylin A attenuates psoriasiform skin inflammation by direct targeting p62 (sequestosome 1) via suppressing M1 macrophage polarization.

BACKGROUND AND PURPOSE: Psoriasis results from the interplay of innate and adaptive immunity in the skin. Oroxylin A (OA) has shown anti-inflammatory effects in various disorders. This study explores oroxylin A potential in treating psoriasis, particularly its impact on type I macrophage (Mφ1) polarization. EXPERIMENTAL APPROACH: Oroxylin A-mediated therapeutic effects were evaluated using imiquimod-induced or IL-23-injected psoriatic mice models, followed by proteomics assays to predict potential signalling and targeting proteins. Immunofluorescence and immunoblot assays verified that oroxylin A suppresses NF-kB signalling in M1 macrophages. Co-immunoprecipitation and microscale thermophoresis (MST) assays further demonstrated that p62 (sequestosome 1) is the target protein for oroxylin A in macrophages. Oroxylin A-p62-mediated suppression of psoriasis was validated in an imiquimod-induced p62 conditional knockout (cKO) mice model. KEY RESULTS: Oroxylin A demonstrated therapeutic efficacy in murine models induced by imiquimod or IL-23 by attenuating cutaneous inflammation and mitigating Mφ1 polarization via NF-κB signalling. Proteomics analysis suggested SQSTM1/p62 as a key target, confirmed to interact directly with oroxylin A. Oroxylin A disrupted the p62-PKCζ interaction by binding to PB1 domain of p62. Its anti-inflammatory effects were significantly reduced in macrophages from p62 cKO mice compared to the wild-type (WT) mice in psoriasis model, supporting oroxylin A role in suppressing Mφ1 polarization through its interaction with p62. CONCLUSION AND IMPLICATIONS: Our findings demonstrated oroxylin A suppressed psoriasiform skin inflammation in mouse models by blocking the PKCζ-p62 interaction, subsequently inhibiting the activation of NF-κB p65 phosphorylation in macrophages.

Knockdown of the Clock gene in the liver aggravates MASLD in mice via inhibiting lipophagy.

The increased global prevalence of metabolic dysfunction-associated steatohepatitis (MASLD) has been closely associated with chronic disorders of the circadian clock. Herein, we investigate the role of Clock, a core circadian gene, in the pathogenesis of MASLD. Wild-type (WT) and liver-specific Clock knockdown (Clock-KD) mice were fed a Western diet for 20 weeks to induce MASLD. A cellular MASLD model was established by treating AML12 cells with free fatty acids and the effects of Clock knockdown were examined following transfection with Clock siRNA. Increased lipid deposition and more severe steatohepatitis and fibrosis were observed in the livers of Western diet-fed but not normal chow diet-fed Clock-KD mice after 20 weeks compared to WT mice. Moreover, the Clock gene was found to be significantly downregulated in WT MASLD mice. The Clock gene was shown to regulate the expression of lipophagy-related proteins (LC3B, P62, RAB7, and PLIN2) in vivo and in vitro. Knockdown of Clock was found to inhibit lipophagy resulting in increased accumulation of lipid droplets in the mouse liver and AML12 cells. Interestingly, the CLOCK protein was shown to interact with P62. However, knockdown of the Clock gene did not promote transcription of the P62 gene but suppressed degradation of the P62 protein during lipophagy in AML12 cells. The hepatic Clock gene regulates lipophagy and affects lipid droplet deposition in liver cells, and thus plays a critical role in the development of MASLD induced by a Western diet.

PPAR-α Insufficiency Enhances Doxorubicin-Induced Nephropathy in PPAR-α Knockout Mice and a Murine Podocyte Cell Line.

Peroxisome proliferator-activated receptor-alpha (PPAR-α) and its exogenous activators (fibrates) promote autophagy. However, whether the deleterious effects of PPAR-α deficiency on doxorubicin (DOX)-induced podocytopathy are associated with reduced autophagy remains to be clarified. We investigated the mechanisms of PPAR-α in DOX-induced podocytopathy and tubular injury in PPAR-α knockout (PAKO) mice and in a murine podocyte cell line. DOX-treated PAKO mice showed higher serum levels of triglycerides and non-esterified fatty acids and more severe podocytopathy than DOX-treated wild-type mice, as evidenced by higher urinary levels of proteins and podocalyxin at 3 days to 2 weeks and higher blood urea nitrogen and serum creatinine levels at 4 weeks. Additionally, there was an increased accumulation of p62, a negative autophagy marker, in the glomerular and tubular regions in DOX-treated PAKO mice at Day 9. Moreover, DOX-treated PAKO mice showed more severe glomerulosclerosis and tubular damage and lower podocalyxin expression in the kidneys than DOX-treated control mice at 4 weeks. Furthermore, DOX treatment increased p-p53, an apoptosis marker, and cleaved the caspase-3 levels and induced apoptosis, which was ameliorated by fenofibrate, a PPAR-α activator. Fenofibrate further enhanced AMPK activation and autophagy under fed and fasting conditions. Conclusively, PPAR-α deficiency enhances DOX-induced podocytopathy, glomerulosclerosis, and tubular injury, possibly by reducing autophagic activity in mouse kidneys.

A robust evaluation of TDP-43, poly GP, cellular pathology and behavior in a AAV-C9ORF72 (G4C2)66 mouse model.

The G4C2 hexanucleotide repeat expansion in C9ORF72 is the major genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Despite considerable efforts, the development of mouse models of C9-ALS/FTD useful for therapeutic development has proven challenging due to the intricate interplay of genetic and molecular factors underlying this neurodegenerative disorder, in addition to species differences. This study presents a robust investigation of the cellular pathophysiology and behavioral outcomes in a previously described AAV mouse model of C9-ALS expressing 66 G4C2 hexanucleotide repeats. Despite displaying key molecular ALS pathological markers including RNA foci, dipeptide repeat (DPR) protein aggregation, p62 positive stress granule formation as well as mild gliosis, the AAV-(G4C2)66 mouse model in this study exhibits negligible neuronal loss, no motor deficits, and functionally unimpaired TAR DNA-binding protein-43 (TDP-43). While our findings indicate and support that this is a robust and pharmacologically tractable model for investigating the molecular mechanisms and cellular consequences of (G4C2) repeat driven DPR pathology, it is not suitable for investigating the development of disease associated neurodegeneration, TDP-43 dysfunction, gliosis, and motor performance. Our findings underscore the complexity of ALS pathogenesis involving genetic mutations and protein dysregulation and highlight the need for more comprehensive model systems that reliably replicate the multifaceted cellular and behavioral aspects of C9-ALS.

Study on the mechanism of activating SIRT1/Nrf2/p62 pathway to mediate autophagy-dependent ferroptosis to promote healing of diabetic foot ulcers.

Diabetic foot (DF), a prevalent and grave diabetes sequela, is considered as a notable clinical concern, with SIRT1 downregulation observed in DF patients' blood specimens. Nonetheless, the regulatory mechanisms of SIRT1 in diabetic foot ulcer (DFU) remain unclear. Thus, in the current study, we investigated the role and mechanisms of SIRT1 in alleviating DFU. Western blotting was used to detect the expression of autophagy and ferroptosis-related proteins, CCK8 assay was used to measure cell proliferation. Plate colony method was used to measure bacterial growth, and the inhibitory effect on intracellular and extracellular Staphylococcus aureus was observed after drug intervention. ELISA was used to detect inflammatory cytokines and oxidative stress markers levels. ROS, total iron, and Fe2+ levels were detected using corresponding assays. Additionally, HE staining detected the thickness of the epidermis and dermis of the rat wound tissue while the collagen deposition in the wound tissue was detected using Masson staining. In addition, Prussian blue staining was used to detect iron deposition, and C11 BODIPY 581/591 lipid peroxidation probe was used to detect lipid ROS. Our results suggested that the activation of SIRT1/Nrf2/p62 signaling affects cell proliferation, colony formation, ferroptosis, and the production of lipid ROS in DFU-infected cell model through autophagy. In vivo experiments indicated that activating SIRT1/Nrf2/p62 signaling affects oxidative stress, inflammation, and autophagy in wound tissue and promotes wound healing in DFU rats through mediating autophagy-dependent ferroptosis. Taken together, the activation of SIRT1/Nrf2/p62 pathway can promote DFU healing, which might be mediated by autophagy-dependent ferroptosis.

Abnormal expression of autophagy proteins in the duodenum of patients with functional dyspepsia: A preliminary study.

BACKGROUND AND STUDY AIMS: Functional dyspepsia (FD) is a common disease with an unclear pathology. Autophagy is associated with inflammation and has been proposed to play a role in the development of FD. This study aimed to evaluate expression of the autophagy proteins beclin1 and p62/SQSTM1 in patients with FD. PATIENTS AND METHODS: Duodenal mucosal tissues were collected from 10 patients with FD and 10 asymptomatic controls. The extent of autophagy was determined by examining expression levels of beclin1 and p62/SQSTM1 using quantitative polymerase chain reaction and immunohistochemistry techniques. RESULTS: Lower expression levels of beclin1 protein were detected in the duodenal bulb (D1) and the second portion of the duodenum (D2) in patients with FD compared with asymptomatic controls. Higher levels of p62 protein were expressed in D1 in patients with FD than in controls. No differences in mRNA expression of beclin1 and p62 were observed between patients with FD and controls. CONCLUSION: Abnormal autophagy was involved in FD, which may be associated with the pathogenesis of FD.

Zinc finger domain of p62/SQSTM1 is involved in the necroptosis of human cisplatin­resistant ovarian cancer cells treated with sulfasalazine.

Cisplatin resistance in ovarian cancer cells is mainly apoptosis resistant. Although other types of programmed cell death are highly involved in chemoresistance, which type can overcome cisplatin resistance remains unclear. The present study observed that cisplatin-sensitive SKOV3 cells and cisplatin-resistant SKOV3/DDP cells had different levels of sensitivity to sulfasalazine (SAS). The present study aimed to investigate the effect of SAS on necroptosis under the same inhibition rate in these two types of cells. Necroptosis inhibitor Necrostatin-1 (Nec-1) attenuated SAS-induced SKOV3/DDP cytotoxicity. SAS decreased SKOV3/DDP cells survival rate, accompanied by decreased cell adhesion and spreading. SAS treatment activated necrosome formation in SKOV3/DDP cells, suggesting the possibility of necroptosis. p62/sequestosome-1 (SQSTM1) protein expression levels were also increased over the same time period. The transfection of small interfering (si)-p62 could decrease the ratios of phosphorylated (p)-receptor-interacting serine/threonine kinase 1 (RIP1)/RIP1, p-receptor-interacting serine/threonine kinase 3 (RIP3)/RIP3 and p-mixed lineage kinase domain-like protein (MLKL)/MLKL proteins in SKOV3/DDP cells. Under the si-p62 condition, there was no increase in the rate of cell survival in Nec-1 and SAS combination group compared with SAS. The zinc finger domain deletion of p62/SQSTM1 effectively decreased the expression levels of necroptosis-related p-proteins. Collectively, certain drugs were able to induce necroptosis in SKOV3/DDP, while p62/RIP1/RIP3/MLKL was associated with the induction of necroptosis and with increasing the sensitivity of cisplatin-resistant ovarian cancer cells, which provided evidence for potential as a therapeutic target for overcoming resistance.

PLK2-mediated phosphorylation of SQSTM1 S349 promotes aggregation of polyubiquitinated proteins upon proteasomal dysfunction.

Dysregulation in protein homeostasis results in accumulation of protein aggregates, which are sequestered into dedicated insoluble compartments so-called inclusion bodies or aggresomes, where they are scavenged through different mechanisms to reduce proteotoxicity. The protein aggregates can be selectively scavenged by macroautophagy/autophagy called aggrephagy, which is mediated by the autophagic receptor SQSTM1. In this study, we have identified PLK2 as an important regulator of SQSTM1-mediated aggregation of polyubiquitinated proteins. PLK2 is upregulated following proteasome inhibition, and then associates with and phosphorylates SQSTM1 at S349. The phosphorylation of SQSTM1 S349 strengthens its binding to KEAP1, which is required for formation of large SQSTM1 aggregates/bodies upon proteasome inhibition. Our findings suggest that PLK2-mediated phosphorylation of SQSTM1 S349 represents a critical regulatory mechanism in SQSTM1-mediated aggregation of polyubiquitinated proteins.

Proximity labeling reveals dynamic changes in the SQSTM1 protein network.

Sequestosome1 (SQSTM1) is an autophagy receptor that mediates the degradation of intracellular cargo, including protein aggregates, through multiple protein interactions. These interactions form the SQSTM1 protein network, and these interactions are mediated by SQSTM1 functional interaction domains, which include LIR, PB1, UBA, and KIR. Technological advances in cell biology continue to expand our knowledge of the SQSTM1 protein network and the relationship between the actions of the SQSTM1 protein network in cellular physiology and disease states. Here we apply proximity profile labeling to investigate the SQSTM1 protein interaction network by fusing TurboID with the human protein SQSTM1 (TurboID::SQSTM1). This chimeric protein displayed well-established SQSTM1 features including production of SQSTM1 intracellular bodies, binding to known SQSTM1 interacting partners, and capture of novel SQSTM1 protein interactors. Strikingly, aggregated tau protein altered the protein interaction network of SQSTM1 to include many stress-associated proteins. We demonstrate the importance of the PB1 and/or UBA domains for binding network members, including the K18 domain of tau. Overall, our work reveals the dynamic landscape of the SQSTM1 protein network and offers a resource to study SQSTM1 function in cellular physiology and disease state.

P62-autophagic pathway degrades SLC7A11 to regulate ferroptosis in doxorubicin-induced cardiotoxicity.

Doxorubicin-induced cardiotoxicity (DIC) poses a significant challenge, impeding its widespread application. Emerging evidence suggests the involvement of ferroptosis in the DIC. While the downregulation of SLC7A11 expression has been linked to the promotion of ferroptosis, the precise regulatory mechanism remains unclear. Recent studies, including our own, have highlighted abnormal levels of autophagy adapter protein P62 and autophagy in DIC development. Thus, our study aimed to further investigate the role of autophagy and ferroptosis in DIC, elucidating underlying molecular mechanisms across molecular, cellular, and whole-organ levels utilizing gene knockdown, immunoprecipitation, and mass spectrometry techniques. The results of our findings unveiled cardiomyocyte damage, heightened autophagy levels, and ferroptosis in DOX-treated mouse hearts. Notably, inhibition of autophagy levels attenuated DOX-induced ferroptosis. Mechanistically, we discovered that the autophagy adaptor protein P62 mediates the entry of SLC7A11 into the autophagic pathway for degradation. Furthermore, the addition of autophagy inhibitors (CQ or BAF) could elevate SLC7A11 and GPX4 protein expression, reduce the accumulation of Fe2+ and ROS in cardiomyocytes, and thus mitigate DOX-induced ferroptosis. In summary, our findings underscore the pivotal role of the P62-autophagy pathway in SLC7A11 degradation, modulating ferroptosis to exacerbate DIC. This finding offers significant insights into the underlying molecular mechanisms of DOX-induced ferroptosis and identifies new targets for reversing DIC.

[Effect of Peitu Yifei Granules on autophagy mechanism in rats with idiopathic pulmonary fibrosis based on PI3K/Akt/mTOR signaling pathway].

To investigate the mechanism by which Peitu Yifei Granules inhibit idiopathic pulmonary fibrosis(IPF) in rats, fifty specific-pathogen-free(SPF) grade male Wistar rats were randomly divided into blank group and modeling group. IPF was induced in the modeling group rats by tracheal infusion of 5 mg·kg~(-1) bleomycin(BLM) and then randomly divided into model group, pirfenidone group, and high-dose, medium-dose, and low-dose groups treated with Peitu Yifei Granules. After 24 hours of modeling, the treatment groups received intragastric administration of either Peitu Yifei Granules or pirfenidone as a positive control drug; meanwhile, the model group received an equal volume of normal saline. After 21 days of treatment administration, lung tissue samples were collected for analysis. Pathological changes in lung tissues were assessed using hematoxylin-eosin(HE) staining and Masson's trichrome staining. The expression levels of protein kinase B(Akt), mammalian target of rapamycin(mTOR), their phosphorylated forms, and sequestosome 1(p62) were determined through Western blot(WB). Fluorescent quantitative real-time polymerase chain reaction(RT-qPCR) was used to measure messenger ribonucleic acid(mRNA) expression levels of Beclin-1, microtubule-associated proteins 1A/1B light chain 3B(LC3B), and p62. Immunohistochemistry was performed to assess protein expression levels of Beclin-1 and LC3B in lung tissue samples. RESULTS:: demonstrated that lung tissue structure appeared normal without significant collagen deposition in the blank group rats. In contrast, rats from the model group exhibited thickened alveolar septa along with evident inflammatory changes and collagen deposition. Compared to the model group rats, those treated with Peitu Yifei Granules or pirfenidone showed significantly improved lung tissue structure with reduced inflammation and collagen deposition observed histologically. Furthermore, compared with those of the blank group, the expressions of p62 and its mRNA, p-Akt and p-mTOR protein in lung tissues of the model group were significantly increased, while Beclin-1, LC3B and their mRNA levels were significantly decreased. Compared with those of the model group, the expressions of p62 and its mRNA, p-Akt and p-mTOR in lung tissues of the pirfenidone group and Peitu Yifei Granules high-dose and medium-dose groups were significantly decreased, while Beclin-1, LC3B and their mRNA expressions were significantly increased. The above results indicate that Peitu Yifei Granules can improve autophagy levels in lung tissues by inhibiting the phosphoinositide 3-kinase(PI3K)/Akt/mTOR signaling pathway and delay the development of IPF disease.

BNIP3-mediated mitophagy attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting ferroptosis through P62-KEAP1-NRF2 pathway activation to maintain iron and redox homeostasis.

As a major contributor to neonatal death and neurological sequelae, hypoxic-ischemic encephalopathy (HIE) lacks a viable medication for treatment. Oxidative stress induced by hypoxic-ischemic brain damage (HIBD) predisposes neurons to ferroptosis due to the fact that neonates accumulate high levels of polyunsaturated fatty acids for their brain developmental needs but their antioxidant capacity is immature. Ferroptosis is a form of cell death caused by excessive accumulation of iron-dependent lipid peroxidation and is closely associated with mitochondria. Mitophagy is a type of mitochondrial quality control mechanism that degrades damaged mitochondria and maintains cellular homeostasis. In this study we employed mitophagy agonists and inhibitors to explore the mechanisms by which mitophagy exerted ferroptosis resistance in a neonatal rat HIE model. Seven-days-old neonatal rats were subjected to ligation of the right common carotid artery, followed by exposure to hypoxia for 2 h. The neonatal rats were treated with a mitophagy activator Tat-SPK2 peptide (0.5, 1 mg/kg, i.p.) 1 h before hypoxia, or in combination with mitochondrial division inhibitor-1 (Mdivi-1, 20 mg/kg, i.p.), and ferroptosis inhibitor Ferrostatin-1 (Fer-1) (2 mg/kg, i.p.) at the end of the hypoxia period. The regulation of ferroptosis by mitophagy was also investigated in primary cortical neurons or PC12 cells in vitro subjected to 4 or 6 h of OGD followed by 24 h of reperfusion. We showed that HIBD induced mitochondrial damage, ROS overproduction, intracellular iron accumulation, lipid peroxidation and ferroptosis, which were significantly reduced by the pretreatment with Tat-SPK2 peptide, and aggravated by the treatment with Mdivi-1 or BNIP3 knockdown. Ferroptosis inhibitors Fer-1 and deferoxamine B (DFO) reversed the accumulation of iron and lipid peroxides caused by Mdivi-1, hence reducing ferroptosis triggered by HI. We demonstrated that Tat-SPK2 peptide-activated BNIP3-mediated mitophagy did not alleviate neuronal ferroptosis through the GPX4-GSH pathway. BNIP3-mediated mitophagy drove the P62-KEAP1-NRF2 pathway, which conferred ferroptosis resistance by maintaining iron and redox homeostasis via the regulation of FTH1, HO-1, and DHODH/FSP1-CoQ10-NADH. This study may provide a new perspective and a therapeutic drug for the treatment of neonatal HIE.

The Chorioallantoic Membrane (CAM) Assay for the Analysis of Starvation-Induced Autophagy.

During avian development, the chorioallantoic membrane (CAM) is generated around 4 days after fertilization following the fusion of the allantois and the chorion. The CAM develops rapidly over the next several days and gets heavily vascularized and therefore has been explored widely as a tool for the study of angiogenesis. Additionally, being immunodeficient, the CAM can be used for tumor growth of human origin and its metastasis. Of note, the CAM assay is minimally invasive for the chicken embryo and lacks innervation, which gives this in vivo model a low ethical burden. Here, we describe the protocol for the generation of microtumors from human colorectal cancer cell lines on the CAM, incubated in a nutrient-deficient medium for the activation of autophagy. We show that pre-inoculation markers of autophagy induced through nutrient deficiency are retained in the microtumors generated on the CAM.

Muscle-derived IL-1ß regulates EcSOD expression via the NBR1-p62-Nrf2 pathway in muscle during cancer cachexia.

Oxidative stress contributes to the loss of skeletal muscle mass and function in cancer cachexia. However, this outcome may be mitigated by an improved endogenous antioxidant defence system. Here, using the well-established oxidative stress-inducing muscle atrophy model of Lewis lung carcinoma (LLC) in 13-week-old male C57BL/6J mice, we demonstrate that extracellular superoxide dismutase (EcSOD) levels increase in the cachexia-prone extensor digitorum longus muscle. LLC transplantation significantly increased interleukin-1ß (IL-1ß) expression and release from extensor digitorum longus muscle fibres. Moreover, IL-1ß treatment of C2C12 myotubes increased NBR1, p62 phosphorylation at Ser351, Nrf2 nuclear translocation and EcSOD protein expression. Additional studies in vivo indicated that intramuscular IL-1ß injection is sufficient to stimulate EcSOD expression, which is prevented by muscle-specific knockout of p62 and Nrf2 (i.e. in p62 skmKO and Nrf2 skmKO mice, respectively). Finally, since an increase in circulating IL-1ß may lead to unwanted outcomes, we demonstrate that targeting this pathway at p62 is sufficient to drive muscle EcSOD expression in an Nrf2-dependent manner. In summary, cancer cachexia increases EcSOD expression in extensor digitorum longus muscle via muscle-derived IL-1ß-induced upregulation of p62 phosphorylation and Nrf2 activation. These findings provide further mechanistic evidence for the therapeutic potential of p62 and Nrf2 to mitigate cancer cachexia-induced muscle atrophy. KEY POINTS: Oxidative stress plays an important role in muscle atrophy during cancer cachexia. EcSOD, which mitigates muscle loss during oxidative stress, is upregulated in 13-week-old male C57BL/6J mice of extensor digitorum longus muscles during cancer cachexia. Using mouse and cellular models, we demonstrate that cancer cachexia promotes muscle EcSOD protein expression via muscle-derived IL-1ß-dependent stimulation of the NBR1-p62-Nrf2 signalling pathway. These results provide further evidence for the potential therapeutic targeting of the NBR1-p62-Nrf2 signalling pathway downstream of IL-1ß to mitigate cancer cachexia-induced muscle atrophy.

Autophagic degradation of CDK4 is responsible for G0/G1 cell cycle arrest in NVP-BEZ235-treated neuroblastoma.

BACKGROUND: CDK4 is highly expressed and associated with poor prognosis and decreased survival in advanced neuroblastoma (NB). Targeting CDK4 degradation presents a potentially promising therapeutic strategy compared to conventional CDK4 inhibitors. However, the autophagic degradation of the CDK4 protein and its anti-proliferation effect in NB cells has not been mentioned. RESULTS: We identified autophagy as a new pathway for the degradation of CDK4. Firstly, autophagic degradation of CDK4 is critical for NVP-BEZ235-induced G0/G1 arrest, as demonstrated by the overexpression of CDK4, autophagy inhibition, and blockade of autophagy-related genes. Secondly, we present the first evidence that p62 binds to CDK4 and then enters the autophagy-lysosome to degrade CDK4 in a CTSB-dependent manner in NVP-BEZ235 treated NB cells. Similar results regarding the interaction between p62 and CDK4 were observed in the NVP-BEZ235 treated NB xenograft mouse model. CONCLUSIONS: Autophagic degradation of CDK4 plays a pivotal role in G0/G1 cell cycle arrest in NB cells treated with NVP-BEZ235.

The Potential Association Between microRNA 135-5P and p62 and Their Effect on NRF2 Pathway in Multiple Sclerosis.

Background: Multiple Sclerosis (MS) is a prevalent non-traumatic disabling disease affecting young adults, characterized by complexity in its pathogenesis. Nuclear factor erythroid 2-Related Factor 2 (NRF2) serves as a crucial transcriptional regulator of anti-inflammatory and antioxidant enzymes, influenced by the ubiquitous protein p62. It acts as a scaffold directing substrates to autophagosomes. This study aims to explore the potential association between microRNA 135-5p and p62 and their impact on inflammation and oxidative stress through the NRF2 pathway in MS. Methods: The study included 30 healthy controls and 60 MS patients (relapsing-remitting and secondary progressive). Real-time PCR was employed for the detection of Nrf2, p62, miRNA135-5P, and NF-κB in serum, while p53 levels were determined using ELISA. Results: Nrf2 and p62 expression was significantly downregulated in the MS group compared to controls. Conversely, miRNA135-5P, NF-κB expression, and P53 levels were significantly elevated in the MS group. Conclusions: This study reveals a potential association between miRNA 135-5p and p62, indicating their role in the pathogenesis of MS. Results suggest that miRNA 135-5p and p62 may influence inflammation and oxidative stress in MS through the NRF2 pathway, potentially mediated by NF-κB and p53.

In Silico Assessment of LIR Motif-Containing Proteins Binding to ATG8-Family Proteins.

Selective autophagic degradation of cellular components has been shown to be mediated by the interaction of LIR motif-containing proteins with ATG8-family proteins. Here, we present a detailed methodology for the in silico evaluation of potential binding between LIR motif-containing proteins and ATG8-family proteins. We visualize AlphaFold-predicted protein complexes using PyMOL to assess potential interactions, providing an effective computational tool for this purpose.

Method to Purify Phase-Separated p62 Bodies Using Fluorescence-Activated Particle Sorting.

p62 bodies are ubiquitin-positive cytoplasmic condensates formed by liquid-liquid phase separation. They are targeted by selective autophagy and play important roles in intracellular quality control and stress responses. However, little is known about their constituents. In this chapter, we describe a method for purifying p62 bodies using fluorescence-activated particle sorting. This method contributes to the identification of novel components of p62 bodies under various physiological and stress conditions.

Bafilomycin 1A Affects p62/SQSTM1 Autophagy Marker Protein Level and Autophagosome Puncta Formation Oppositely under Various Inflammatory Conditions in Cultured Rat Microglial Cells.

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings.