Targeting EGFR degradation by autophagosome degraders.

Several generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been developed for the treatment of non-small cell lung cancer (NSCLC) in clinic. However, emerging drug resistance mediated by new EGFR mutations or activations by pass, leads to malignant progression of NSCLC. Proteolysis targeting chimeras (PROTACs) have been utilized to overcome the drug resistance acquired by mutant EGFR, newly potent and selective degraders are still need to be developed for clinical applications. Herein, we developed autophagosome-tethering compounds (ATTECs) in which EGFR can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of the LC3 ligand GW5074. A series of EGFR-ATTECs have been designed and synthesized. Biological evaluations showed that these compounds could degrade EGFR and exhibited moderate inhibitory effects on certain NSCLC cell lines. The ATTEC 12c potently induced the degradation of EGFR with a DC50 value of 0.98 µM and a Dmax value of 81% in HCC827 cells. Mechanistic exploration revealed that the lysosomal pathway was mainly involved in this degradation. Compound 12c also exhibited promising inhibitory activity, as well as degradation efficiency in vivo. Our study highlights that EGFR-ATTECs could be developed as a new expandable EGFR degradation tool and also reveals a novel potential therapeutic strategy to prevent drug resistance acquired EGFR mutations.

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism.

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein-protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy.

Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter "delivery efficiency of autophagosome (DEAP)" to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.

Molecular mechanisms of secretory autophagy and its potential role in diseases.

Autophagy is a cellular degradation system that recycles or degrades damaged organelles, viral particles, and aggregated proteins through the lysosomal pathway. Autophagy plays an indispensable role in cellular homeostasis and communication processes. An interesting aspect is that autophagy also mediates the secretion of cellular contents, a process known as secretory autophagy. Secretory autophagy differs from macroautophagy, which sequesters recruited proteins, organelles, or viral particles into autophagosomes and degrades these sequesters in lysosomes, while the secretory autophagy pathway participates in the extracellular export of cellular contents sequestered by autophagosomes through autophagy and endosomal modulators. Recent evidence reveals that secretory autophagy is pivotal in the occurrence and progression of diseases. In this review, we summarize the molecular mechanisms of secretory autophagy. Furthermore, we review the impact of secretory autophagy on diseases, including cancer, viral infectious diseases, neurodegenerative diseases, and cardiovascular diseases. Considering the pleiotropic actions of secretory autophagy on diseases, studying the mechanism of secretory autophagy may help to understand the relevant pathophysiological processes.

SRT1720 inhibits bladder cancer cell progression by impairing autophagic flux.

Bladder cancer (BC) is the most common cancer of the urinary tract, with poor survival, high recurrence rates, and lacking of targeted drugs. In this study, we constructed a library to screen compounds inhibiting bladder cancer cells growth. Among them, SRT1720 was identified to inhibit bladder cancer cell proliferation in vitro and in vivo. SRT1720 treatment also suppressed bladder cancer cells migration, invasion and induced apoptosis. Mechanism studies shown that SRT1720 promoted autophagosomes accumulation by inducing early-stage autophagy but disturbed the late-stage of autophagy by blocking fusion of autophagosomes and lysosomes. SRT1720 appears to induce autophagy related proteins expression and alter autophagy-related proteins acetylation to impede the autophagy flux. LAMP2, an important lysosomal associated membrane protein, may mediate SRT1720-inhibited autophagy flux as SRT1720 treatment significantly deacetylated LAMP2 which may influence its activity. Taken together, our results demonstrated that SRT1720 mediated apoptosis and autophagy flux inhibition may be a novel therapeutic strategy for bladder cancer treatment.

Rim aperture of yeast autophagic membranes balances cargo inclusion with vesicle maturation.

Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle expansion. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy.

DNALI1 Promotes Neurodegeneration after Traumatic Brain Injury via Inhibition of Autophagosome-Lysosome Fusion.

Traumatic brain injury (TBI) leads to progressive neurodegeneration that may be caused by chronic traumatic encephalopathy (CTE). However, the precise mechanism remains unclear. Herein, the study identifies a crucial protein, axonemal dynein light intermediate polypeptide 1 (DNALI1), and elucidated its potential pathogenic role in post-TBI neurodegeneration. The DNALI1 gene is systematically screened through analyses of Aging, Dementia, and TBI studies, confirming its elevated expression both in vitro and in vivo. Moreover, it is observed that altered DNALI1 expression under normal conditions has no discernible effect. However, upon overexpression, DNALI1 inhibits autophagosome-lysosome fusion, reduces autophagic flux, and exacerbates cell death under pathological conditions. DNALI1 silencing significantly enhances autophagic flux and alleviates neurodegeneration in a CTE model. These findings highlight DNALI1 as a potential key target for preventing TBI-related neurodegeneration.

Rab32 family proteins regulate autophagosomal components recycling.

In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR.

Bortezomib modulated the autophagy-lysosomal pathway in a TFEB-dependent manner in multiple myeloma.

OBJECTIVE: To explore the involvement of TFEB-mediated autophagy-lysosomal mechanisms in multiple myeloma (MM) during bortezomib treatment. METHODS: MM cells were exposed to bortezomib or subjected to TFEB knockdown. CCK assay was used to assess the cell proliferation. Western blotting and fluorescent staining were conducted to examine autophagy and lysosomes. The TFEB expression pattern was analyzed, and whole transcriptome sequencing was carried out. Additionally, TFEB target genes were predicted using the GTRD(http://gtrd.biouml.org/) website, and pathway analysis was performed. RESULTS: Bortezomib demonstrated a dose-dependent and time dependent inhibition of cell proliferation. In MM cells treated with bortezomib, LC3B, Beclin-1, TFEB, and Lamp1 exhibited upregulation in a time- and concentration-dependent manner. LysoTracker dye labeling showed an increase in lysosomes in the bortezomib-treated group. Moreover, bortezomib elevated the expression of lysosome-associated factor Lamp1. Bortezomib promoted the nuclear translocation of TFEB, leading to decreased cytoplasmic TFEB and increased nuclear TFEB. TFEB gene silencing reversed bortezomib's inhibitory effect on MM cell lines, significantly reducing autophagosome expression and lysosome numbers. Furthermore, bioinformatic analysis identified the MAPK pathway as a potential downstream target of TFEB. CONCLUSION: Bortezomib effectively inhibits MM cell proliferation and induces autophagy, partly through TFEB-mediated mechanisms, with potential involvement of the MAPK pathway.

Key roles of autophagosome/endosome maturation mediated by Syntaxin17 in methamphetamine-induced neuronal damage in mice.

BACKGROUND: Autophagic defects are involved in Methamphetamine (Meth)-induced neurotoxicity. Syntaxin 17 (Stx17), a member of the SNARE protein family, participating in several stages of autophagy, including autophagosome-late endosome/lysosome fusion. However, the role of Stx17 and potential mechanisms in autophagic defects induced by Meth remain poorly understood. METHODS: To address the mechanism of Meth-induced cognitive impairment, the adenovirus (AV) and adeno-associated virus (AAV) were injected into the hippocampus for stereotaxis to overexpress Stx17 in vivo to examine the cognitive ability via morris water maze and novel object recognition. In molecular level, the synaptic injury and autophagic defects were evaluated. To address the Meth induced neuronal damage, the epidermal growth factor receptor (EGFR) degradation assay was performed to evaluate the degradability of the "cargos" mediated by Meth, and mechanistically, the maturation of the vesicles, including autophagosomes and endosomes, were validated by the Co-IP and the GTP-agarose affinity isolation assays. RESULTS: Overexpression of Stx17 in the hippocampus markedly rescued the Meth-induced cognitive impairment and synaptic loss. For endosomes, Meth exposure upregulated Rab5 expression and its guanine-nucleotide exchange factor (GEF) (immature endosome), with a commensurate decreased active form of Rab7 (Rab7-GTP) and impeded the binding of Rab7 to CCZ1 (mature endosome); for autophagosomes, Meth treatment elicited a dramatic reduction in the overlap between Stx17 and autophagosomes but increased the colocalization of ATG5 and autophagosomes (immature autophagosomes). After Stx17 overexpression, the Rab7-GTP levels in purified late endosomes were substantially increased in parallel with the elevated mature autophagosomes, facilitating cargo (Aß42, p-tau, and EGFR) degradation in the vesicles, which finally ameliorated Meth-induced synaptic loss and memory deficits in mice. CONCLUSION: Stx17 decrease mediated by Meth contributes to vesicle fusion defects which may ascribe to the immature autophagosomes and endosomes, leading to autophagic dysfunction and finalizes neuronal damage and cognitive impairments. Therefore, targeting Stx17 may be a novel therapeutic strategy for Meth-induced neuronal injury.

Migrasomal autophagosomes relieve endoplasmic reticulum stress in glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative. Migrasomes are newly discovered membrane structures observed in migrating cells. Thus, they can be generated from GBM cells that have the ability to migrate along the brain parenchyma. However, the function of migrasomes has not yet been elucidated in GBM cells. RESULTS: Here, we describe the composition and function of migrasomes generated along with GBM cell migration. Proteomic analysis revealed that LC3B-positive autophagosomes were abundant in the migrasomes of GBM cells. An increased number of migrasomes was observed following treatment with chloroquine (CQ) or inhibition of the expression of STX17 and SNAP29, which are involved in autophagosome/lysosome fusion. Furthermore, depletion of ITGA5 or TSPAN4 did not relieve endoplasmic reticulum (ER) stress in cells, resulting in cell death. CONCLUSIONS: Taken together, our study suggests that increasing the number of autophagosomes, through inhibition of autophagosome/lysosome fusion, generates migrasomes that have the capacity to alleviate cellular stress.

Targeting PKD2 aggravates ferritinophagy-mediated ferroptosis via promoting autophagosome-lysosome fusion and enhances efficacy of carboplatin in lung adenocarcinoma.

Ferroptosis is an iron-dependent cell death and affects efficacies of multiple antitumor regimens, showing a great potential in cancer therapy. Protein kinase D2 (PKD2) plays a crucial role in regulating necrosis and apoptosis. However, the relationship of PKD2 and ferroptosis is still elusive. In this study, we mainly analyzed the roles of PKD2 on ferroptosis and chemotherapy in lung adenocarcinoma (LUAD). We found PKD2 was highly expressed in LUAD and silencing PKD2 could promote erastin-induced reactive oxygen species (ROS), malondialdehyde (MDA) accumulation, intracellular iron content and LUAD cells death. Mechanistically, augmenting PKD2 could prevent autophagic degradation of ferritin, which could be impaired by bafilomycin A1. We further found that PKD2 overexpression would promote LC3B-II, p62/SQSTM1 accumulation and block autophagosome-lysosome fusion in a TFEB-independent manner, which could be impaired by bafilomycin A1. Bafilomycin A1 stimulation could weaken ferroptosis promotion by PKD2 abrogation. Silencing ferritin heavy chain-1 (FTH1) could reverse the resistance to ferroptosis by PKD2 overexpression. Additionally, in vitro and vivo experiments validated PKD2 promoted proliferation, migration and invasion of LUAD cells. PKD2 knockdown or pharmacological inhibition by CRT0066101 could enhance efficacy of carboplatin in LUAD via ferroptosis and apoptosis. Collectively, our study revealed that abrogation of PKD2 could aggravate ferritinophagy-mediated ferroptosis by promoting autophagosome-lysosome fusion and enhance efficacy of carboplatin in LUAD. Targeting PKD2 to induce ferroptosis may be a promising strategy for LUAD therapy.

S670, an amide derivative of 3-O-acetyl-11-keto-ß-boswellic acid, induces ferroptosis in human glioblastoma cells by generating ROS and inhibiting STX17-mediated fusion of autophagosome and lysosome.

Glioblastoma (GBM) is the most common malignant tumor in the brain with temozolomide (TMZ) as the only approved chemotherapy agent. GBM is characterized by susceptibility to radiation and chemotherapy resistance and recurrence as well as low immunological response. There is an urgent need for new therapy to improve the outcome of GBM patients. We previously reported that 3-O-acetyl-11-keto-ß-boswellic acid (AKBA) inhibited the growth of GBM. In this study we characterized the anti-GBM effect of S670, a synthesized amide derivative of AKBA, and investigated the underlying mechanisms. We showed that S670 dose-dependently inhibited the proliferation of human GBM cell lines U87 and U251 with IC50 values of around 6 µM. Furthermore, we found that S670 (6 µM) markedly stimulated mitochondrial ROS generation and induced ferroptosis in the GBM cells. Moreover, S670 treatment induced ROS-mediated Nrf2 activation and TFEB nuclear translocation, promoting protective autophagosome and lysosome biogenesis in the GBM cells. On the other hand, S670 treatment significantly inhibited the expression of SXT17, thus impairing autophagosome-lysosome fusion and blocking autophagy flux, which exacerbated ROS accumulation and enhanced ferroptosis in the GBM cells. Administration of S670 (50 mg·kg-1·d-1, i.g.) for 12 days in a U87 mouse xenograft model significantly inhibited tumor growth with reduced Ki67 expression and increased LC3 and LAMP2 expression in the tumor tissues. Taken together, S670 induces ferroptosis by generating ROS and inhibiting STX17-mediated fusion of autophagosome and lysosome in GBM cells. S670 could serve as a drug candidate for the treatment of GBM.

Inhibition of PRKAA/AMPK (Ser485/491) phosphorylation by crizotinib induces cardiotoxicity via perturbing autophagosome-lysosome fusion.

Crizotinib, a small-molecule tyrosine kinase inhibitor targeting ALK, MET and ROS1, is the first-line drug for ALK-positive metastatic non-small cell lung cancer and is associated with severe, sometimes fatal, cases of cardiac failure, which increases the risk of mortality. However, the underlying mechanism remains unclear, which causes the lack of therapeutic strategy. We established in vitro and in vivo models for crizotinib-induced cardiotoxicity and found that crizotinib caused left ventricular dysfunction, myocardial injury and pathological remodeling in mice and induced cardiomyocyte apoptosis and mitochondrial injury. In addition, we found that crizotinib prevented the degradation of MET protein by interrupting autophagosome-lysosome fusion and silence of MET or re-activating macroautophagy/autophagy flux rescued the cardiomyocytes death and mitochondrial injury caused by crizotinib, suggesting that impaired autophagy activity is the key reason for crizotinib-induced cardiotoxicity. We further confirmed that recovering the phosphorylation of PRKAA/AMPK (Ser485/491) by metformin re-activated autophagy flux in cardiomyocytes and metformin rescued crizotinib-induced cardiomyocyte injury and cardiac complications. In summary, we revealed a novel mechanism for crizotinib-induced cardiotoxicity, wherein the crizotinib-impaired autophagy process causes cardiomyocyte death and cardiac injury by inhibiting the degradation of MET protein, demonstrated a new function of impeded autophagosome-lysosome fusion in drugs-induced cardiotoxicity, pointed out the essential role of the phosphorylation of PRKAA (Ser485/491) in autophagosome-lysosome fusion and confirmed metformin as a potential therapeutic strategy for crizotinib-induced cardiotoxicity.Abbreviations and Acronyms: AAV: adeno-associated virus; ACAC/ACC: acetyl-Co A carboxylase; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATG7: autophagy related 7; CHX: cycloheximide; CKMB: creatine kinase myocardial band; CQ: chloroquine; c-PARP: cleaved poly (ADP-ribose) polymerase; DAPI: 4'6-diamidino-2-phenylindole; EF: ejection fraction; FOXO: forkhead box O; FS: fractional shortening; GSEA: gene set enrichment analysis; H&E: hematoxylin and eosin; HF: heart failure; HW: TL: ratio of heart weight to tibia length; IR: ischemia-reperfusion; KEGG: Kyoto encyclopedia of genes and genomes; LAMP2: lysosomal-associated membrane protein 2; LDH: lactate dehydrogenase; MCMs: mouse cardiomyocytes; MMP: mitochondrial membrane potential; mtDNA: mitochondrial DNA; MYH6: myosin, heavy peptide 6, cardiac muscle, alpha; MYH7: myosin, heavy peptide 7, cardiac muscle, beta; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; PI: propidium iodide; PI3K: phosphoinositide 3-kinase; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; qPCR: quantitative real-time PCR; SD: standard deviation; SRB: sulforhodamine B; TKI: tyrosine kinase inhibitor; WGA: wheat germ agglutinin.

ATPase-regulated autophagosome biogenesis.

Omega-shaped domains of the endoplasmic reticulum, known as omegasomes, have been suggested to contribute to autophagosome biogenesis, although their exact function is not known. Omegasomes are characterized by the presence of the double FYVE domain containing protein ZFYVE1/DFCP1, but it has remained a paradox that depletion of ZFYVE1 does not prevent bulk macroautophagy/autophagy. We recently showed that ZFYVE1 contains an N-terminal ATPase domain which dimerizes upon ATP binding. Mutations in the ATPase domain that inhibit ATP binding or hydrolysis do not prevent omegasome expansion and maturation. However, omegasome constriction is inhibited by these mutations, which results in an increased lifetime and thereby higher number of omegasomes. Interestingly, whereas ZFYVE1 knockout or mutations do not significantly affect bulk autophagy, selective autophagy of mitochondria, protein aggregates and micronuclei is inhibited. We propose that ATP binding and hydrolysis control the di- or multimerization state of ZFYVE1 which could provide the mechanochemical energy to drive large omegasome constriction and autophagosome completion.

Tumor-derived autophagosome vaccines combined with immune adjuvants mediate antitumor immune responses via the neoantigen pathway.

Vaccines composed of autophagosomes derived from tumor cells called DRibbles (DRiPs-containing blebs) are involved in the cross-presentation of tumor antigens, thus inducing cross-reactive T-cell responses against the tumor. Compared with traditional tumor lysate vaccines, autophagosome vaccines were found to be better sources of multiple tumor-associated antigens (TAAs) that activate antigen-specific T-cells. However, the involvement of tumor neoantigens in the immune responses of autophagosome vaccines remains unclear. The present study showed that exogenous autophagosome vaccines (DRibbles) combined with immune adjuvants (anti-OX40 antibody and ATP) can effectively activate functional T cells in vitro. Importantly, the combination of exogenous tumor-derived autophagosome vaccines and immune adjuvants was found to induce tumor regression in B16F10 and 4T1 tumor-bearing mice. The combination of autophagosome-enriched DRibbles with anti-OX40 antibody and ATP also exhibited optimal immune stimulation and antitumor efficiency in vivo. The effectiveness of exogenous DRibble vaccines was mainly due to their enhancement of tumor immunogenicity by increasing the presentation and release of tumor neoantigens. These findings suggest that this immunotherapeutic method may be effective in the treatment of cancer.

The Molecular Mechanism and Therapeutic Application of Autophagy for Urological Disease.

Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It comprises microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Macroautophagy consists of three stages: induction, autophagosome formation, and autolysosome formation. Atg8-family proteins are valuable for tracking autophagic structures and have been widely utilized for monitoring autophagy. The conversion of LC3 to its lipidated form, LC3-II, served as an indicator of autophagy. Autophagy is implicated in human pathophysiology, such as neurodegeneration, cancer, and immune disorders. Moreover, autophagy impacts urological diseases, such as interstitial cystitis /bladder pain syndrome (IC/BPS), ketamine-induced ulcerative cystitis (KIC), chemotherapy-induced cystitis (CIC), radiation cystitis (RC), erectile dysfunction (ED), bladder outlet obstruction (BOO), prostate cancer, bladder cancer, renal cancer, testicular cancer, and penile cancer. Autophagy plays a dual role in the management of urologic diseases, and the identification of potential biomarkers associated with autophagy is a crucial step towards a deeper understanding of its role in these diseases. Methods for monitoring autophagy include TEM, Western blot, immunofluorescence, flow cytometry, and genetic tools. Autophagosome and autolysosome structures are discerned via TEM. Western blot, immunofluorescence, northern blot, and RT-PCR assess protein/mRNA levels. Luciferase assay tracks flux; GFP-LC3 transgenic mice aid study. Knockdown methods (miRNA and RNAi) offer insights. This article extensively examines autophagy's molecular mechanism, pharmacological regulation, and therapeutic application involvement in urological diseases.

Moschus ameliorates glutamate-induced cellular damage by regulating autophagy and apoptosis pathway.

Alzheimer's disease (AD), a neurodegenerative disorder, causes short-term memory and cognition declines. It is estimated that one in three elderly people die from AD or other dementias. Chinese herbal medicine as a potential drug for treating AD has gained growing interest from many researchers. Moschus, a rare and valuable traditional Chinese animal medicine, was originally documented in Shennong Ben Cao Jing and recognized for its properties of reviving consciousness/resuscitation. Additionally, Moschus has the efficacy of "regulation of menstruation with blood activation, relief of swelling and pain" and is used for treating unconsciousness, stroke, coma, and cerebrovascular diseases. However, it is uncertain whether Moschus has any protective effect on AD patients. We explored whether Moschus could protect glutamate (Glu)-induced PC12 cells from cellular injury and preliminarily explored their related action mechanisms. The chemical compounds of Moschus were analyzed and identified by GC-MS. The Glu-induced differentiated PC12 cell model was thought to be the common AD cellular model. The study aims to preliminarily investigate the intervention effect of Moschus on Glu-induced PC12 cell damage as well as their related action mechanisms. Cell viability, lactate dehydrogenase (LDH), mitochondrial reactive oxygen species, mitochondrial membrane potential (MMP), cell apoptosis, autophagic vacuoles, autolysosomes or autophagosomes, proteins related to apoptosis, and the proteins related to autophagy were examined and analyzed. Seventeen active compounds of the Moschus sample were identified based on GC-MS analysis. In comparison to the control group, Glu stimulation increased cell viability loss, LDH release, mitochondrial damage, loss of MMP, apoptosis rate, and the number of cells containing autophagic vacuoles, and autolysosomes or autophagosomes, while these results were decreased after the pretreatment with Moschus and 3-methyladenine (3-MA). Furthermore, Glu stimulation significantly increased cleaved caspase-3, Beclin1, and LC3II protein expression, and reduced B-cell lymphoma 2/BAX ratio and p62 protein expression, but these results were reversed after pretreatment of Moschus and 3-MA. Moschus has protective activity in Glu-induced PC12 cell injury, and the potential mechanism might involve the regulation of autophagy and apoptosis. Our study may promote research on Moschus in the field of neurodegenerative diseases, and Moschus may be considered as a potential therapeutic agent for AD.

Dual Implications of Nanosilver-Induced Autophagy: Nanotoxicity and Anti-Cancer Effects.

In recent years, efforts have been made to identify new anti-cancer therapies. Various types of nanomaterials, including silver nanoparticles (AgNPs), are being considered as an option. In addition to its well-known antibacterial activity, AgNPs exhibit cytotoxic potential in both physiological and cancer cells by inducing stress-mediated autophagy and apoptotic cell death. A rapidly growing collection of data suggests that the proper regulation of autophagic machinery may provide an efficient tool for suppressing the development of cancer. In this light, AgNPs have emerged as a potential anti-cancer agent to support therapy of the disease. This review summarizes current data indicating the dual role of AgNP-induced autophagy and highlights factors that may influence its protective vs. its toxic potential. It also stresses that our understanding of the cellular and molecular mechanisms of autophagy machinery in cancer cells, as well as AgNP-triggered autophagy in both normal and diseased cells, remains insufficient.

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin.

BACKGROUND: The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. METHODS: We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. RESULTS: We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. CONCLUSION: Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.