CircRNA regulates the liquid-liquid phase separation of ATG4B, a novel strategy to inhibit cancer metastasis?

Anoikis is a common programmed death for most of detached cells, but cancer cells can obtain anoikis resistance to facilitate their distant metastasis through the circulation system. Researches have indicated that enhanced autophagic flux accounts for the survival of many cancer cells under detached conditions. Targeting ATG4B, the key factor of autophagy progress, can inhibit cancer metastasis in vitro, but ATG4B-deficient mice are susceptible to many serious diseases, which indicates the potential uncontrolled side effects of direct targeting of ATG4B. In our recent research, we confirmed that ATG4B is a novel RNA binding protein in the gastric cancer (GC) cell. It interacts with circSPECC1 which consequently facilitates the liquid-liquid phase separation and ubiquitination of ATG4B. Additionally, the m6A reader ELAVL1 inhibits the expression of circSPECC1 to enhance the expression of ATG4B and anoikis resistance of GC cells. Further, we screened out an FDA-approved compound, lopinavir, to restore circSPECC1 abundance and suppress GC metastasis. In conclusion, our research identified a novel signal pathway (ELAVL1-circSPECC1-ATG4B-autophagy) to facilitate anoikis resistance and metastasis of GC cells and screened out a compound with clinical application potential to block this pathway, providing a novel strategy for the prevention of GC metastasis.

Berberine increases the killing effect of pirarubicin on HCC cells by inhibiting ATG4B-autophagy pathway.

Pirarubicin (THP) is a new generation of cell cycle non-specific anthracycline-based anticancer drug. In the clinic, THP and THP combination therapies have been shown to be effective in hepatocellular carcinoma (HCC) patients with transcatheter arterial chemoembolization (TACE) without serious side effects. However, drug resistance limits its therapeutic efficacy. Berberine (BBR), an isoquinoline alkaloid, has been shown to possess antitumour properties against various malignancies. However, the synergistic effect of BBR and THP in the treatment of HCC is unknown. In the present study, we demonstrated for the first time that BBR sensitized HCC cells to THP, including enhancing THP-induced growth inhibition and apoptosis of HCC cells. Moreover, we found that BBR sensitized THP by reducing the expression of autophagy-related 4B (ATG4B). Mechanistically, the inhibition of HIF1α-mediated ATG4B transcription by BBR ultimately led to attenuation of THP-induced cytoprotective autophagy, accompanied by enhanced growth inhibition and apoptosis in THP-treated HCC cells. Tumor-bearing experiments in nude mice showed that the combination treatment with BBR and THP significantly suppressed the growth of HCC xenografts. These results reveal that BBR is able to strengthen the killing effect of THP on HCC cells by repressing the ATG4B-autophagy pathway, which may provide novel insights into the improvement of chemotherapeutic efficacy of THP, and may be conducive to the further clinical application of THP in HCC treatment.

Lopinavir enhances anoikis by remodeling autophagy in a circRNA-dependent manner.

Macroautophagy/autophagy-mediated anoikis resistance is crucial for tumor metastasis. As a key autophagy-related protein, ATG4B has been demonstrated to be a prospective anti-tumor target. However, the existing ATG4B inhibitors are still far from clinical application, especially for tumor metastasis. In this study, we identified a novel circRNA, circSPECC1, that interacted with ATG4B. CircSPECC1 facilitated liquid-liquid phase separation of ATG4B, which boosted the ubiquitination and degradation of ATG4B in gastric cancer (GC) cells. Thus, pharmacological addition of circSPECC1 may serve as an innovative approach to suppress autophagy by targeting ATG4B. Specifically, the circSPECC1 underwent significant m6A modification in GC cells and was subsequently recognized and suppressed by the m6A reader protein ELAVL1/HuR. The activation of the ELAVL1-circSPECC1-ATG4B pathway was demonstrated to mediate anoikis resistance in GC cells. Moreover, we also verified that the above pathway was closely related to metastasis in tissues from GC patients. Furthermore, we determined that the FDA-approved compound lopinavir efficiently enhanced anoikis and prevented metastasis by eliminating repression of ELAVL1 on circSPECC1. In summary, this study provides novel insights into ATG4B-mediated autophagy and introduces a viable clinical inhibitor of autophagy, which may be beneficial for the treatment of GC with metastasis.

ATG4B as a novel biomarker for abdominal aortic aneurysm: integrated evaluation through experimental and bioinformatics analyses.

Autophagy related gene 4B (ATG4B) plays a central role in autophagy machinery, but its clinical relevance to AAA remains unknown. In this study, 205 AAA patients and 205 age- and sex-matched controls were included to detect the serum ATG4B levels. Meanwhile, abdominal aortic specimens from 24 AAA patients and 6 human organ donors were collected to evaluate the mRNA and in situ protein expression of ATG4B. We observed significantly higher ATG4B mRNA and protein expression levels in AAA group compared to those in control group, with a positive correlation between mRNA levels and serum/in situ protein levels (serum, r = 0.518, P = 0.010; in situ, r = 0.453, P = 0.026). Serum ATG4B exhibited the diagnostic potential for AAA (AUC = 0.702, sensitivity = 75.6%) and intraluminal thrombus recognition (AUC = 0.602, sensitivity = 67.9%). Logistic regression revealed a significant association between elevated serum ATG4B and an increased risk of AAA and intraluminal thrombus formation. Deceased patients displayed higher baseline serum ATG4B levels, which could predict postoperative mortality (HR = 1.028, 95%CI = 1.007-1.049, P = 0.009, AUC = 0.612, sensitivity = 84.6%). The bioinformatics analysis suggested that ATG4B may modulate cellular autophagy and influence pathways associated with inflammation, lipid metabolism, or apoptosis, thereby contributing to the occurrence and development of AAA. The drug-gene interaction network identified 13 potential therapeutic drugs targeting ATG4B. In conclusion, ATG4B may serve as a promising biomarker for the diagnosis and prognostic assessment of AAA patients and play a key role in the pathogenesis of AAA.

Targeting GABARAPL1/HIF-2a axis to induce tumor cell apoptosis in nasopharyngeal carcinoma.

Objectives: The primary gene mutations associated with nasopharyngeal carcinoma (NPC) are located within the phosphoinositide 3-kinase-mammalian target of rapamycin signaling pathways, which have inhibitory effects on autophagy. Compounds that target autophagy could potentially be used to treat NPC. However, autophagy-related molecular targets in NPC remain to be elucidated. We aimed to examine levels of autophagy-related genes, including autophagy-related 4B cysteine peptidase (ATG4B) and gamma-aminobutyric acid (GABA) type A receptor-associated protein-like 1 (GABARAPL1), in NPC cells and explored their potential role as novel targets for the treatment of NPC. Materials and Methods: The mRNA and protein expression of autophagy-related genes were detected in several NPC cells. Levels of GABARAPL1 were modified by either overexpression or knockdown, followed by examining downstream targets using RT-qPCR and western blotting. The role of GABARAPL1 in NPC proliferation and apoptosis was examined by flow cytometry. Furthermore, the role of GABARAPL1 was assessed in vivo using a nude mouse xenograft tumor model. The underlying mechanism by which GABARAPL1 regulated nasopharyngeal tumor growth was investigated. Results: Autophagy-related 4B cysteine peptidase (ATG4B), GABARAPL1, and Unc-51-like kinase 1 (ULK1) were significantly down-regulated in multiple NPC cell lines. Overexpression of GABARAPL1 up-regulated the expression of autophagy-related proteins, decreased the level of hypoxia-inducible factor (HIF)-2α, and induced apoptosis in NPC cells. Importantly, overexpression of GABARAPL1 slowed tumor growth. Western blotting showed that autophagy was activated, and HIF-2α was down-regulated in tumor tissues. Conclusion: HIF-2α, as a substrate for autophagic degradation, may play an interesting role during NPC progression.

UBE3C tunes autophagy via ATG4B ubiquitination.

ATG4B is a core protein and essential for cleaving precursor MAP1LC3/LC3 or deconjugating lipidated LC3-II to drive the formation of autophagosomes. The protein stability and activity of ATG4B regulated by post-translational modification (ubiquitination) will directly affect macroautophagy/autophagy. However, the mechanism involved in ATG4B ubiquitination is largely unclear. In this study, a new E3 ligase of ATG4B, UBE3C, was identified by mass spectra. UBE3C mainly assembles K33-branched ubiquitin chains on ATG4B at Lys119 without causing ATG4B degradation. In addition, the increased ubiquitination of ATG4B caused by UBE3C overexpression inhibits autophagy flux in both normal and starvation conditions, which might be due to the reduced activity of ATG4B and ATG4B-LC3 interaction. This reduction could be reversed once the lysine 119 of ATG4B was mutated to arginine. More important, under starvation conditions the interaction between ATG4B and UBE3C apparently decreased followed by the removal of the K33-branched ubiquitin chain of ATG4B. Thus, starvation-induced autophagy could be partially suppressed by an increased ubiquitination level of ATG4B. In conclusion, our research reveals a novel modification mode of ATG4B in which UBE3C can fine tune ATG4B activity by specific ubiquitination regulating autophagy without causing ATG4B degradation.Abbreviation: ATG: autophagy-related; Baf: bafilomycin A1; CBB: Coomassie Brilliant Blue; CM: complete medium; CQ: chloroquine; GFP: green fluorescent protein; HA-Ub: HA-tagged ubiquitin; IF: immunofluorescence; IP: immunoprecipitation; K: lysine; KO: knockout; K0: all K-to-R mutant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MS: mass spectrometry; NC: negative control; R: arginine; WCL: whole cell lysate; WT: wild-type.

FAT1 inhibits AML autophagy and proliferation via downregulating ATG4B expression.

BACKGROUND: Emerging studies have shown that FAT atypical cadherin 1 (FAT1) and autophagy separately inhibits and promotes acute myeloid leukemia (AML) proliferation. However, it is unknown whether FAT1 were associated with autophagy in regulating AML proliferation. METHODS: AML cell lines, 6-week-old male nude mice and AML patient samples were used in this study. qPCR/Western blot and cell viability/3H-TdR incorporation assays were separately used to detect mRNA/protein levels and cell activity/proliferation. Luciferase reporter assay was used to examine gene promoter activity. Co-IP analysis was used to detect the binding of proteins. RESULTS: In this study, we for the first time demonstrated that FAT1 inhibited AML proliferation by decreasing AML autophagy level. Moreover, FAT1 weakened AML autophagy level via decreasing autophagy related 4B (ATG4B) expression. Mechanistically, we found that FAT1 reduced the phosphorylated and intranuclear SMAD family member 2/3 (smad2/3) protein levels, thus decreasing the activity of ATG4B gene promoter. Furthermore, we found that FAT1 competitively bound to TGF-ßR II which decreased the binding of TGF-ßR II to TGF-ßR I and the subsequent phosphorylation of TGF-ßR I, thus reducing the phosphorylation and intranuclear smad2/3. The experiments in nude mice showed that knockdown of FAT1 promoted AML autophagy and proliferation in vivo. CONCLUSIONS: Collectively, these results revealed that FAT1 downregulates ATG4B expression via inhibiting TGFß-smad2/3 signaling activity, thus decreasing the autophagy level and proliferation activity of AML cells. GENERAL SIGNIFICANCE: Our study suggested that the "FAT1-TGFß-smad2/3-ATG4B-autophagy" pathway may be a novel target for developing new targeted drugs to AML treatment.

LncRNA AC125982.2 regulates apoptosis of cardiomyocytes through mir-450b-3p/ATG4B axis in a rat model with myocardial infarction.

Background: The occurrence and disability of myocardial infarction (MI) are on the rise globally, making it a significant contributor to cardiovascular mortality. Irreversible myocardial apoptosis plays a crucial role in causing MI. Long non-coding RNAs (LncRNAs) are key regulators of the cardiac remodeling process. Therefore, it is necessary to explore the effect of LncRNAs on cardiomyocyte apoptosis in MI. Methods: The rat-MI model was constructed, LncRNA-Seq and qPCR analyses were used to determine differentially expressed genes obtained from heart tissue of rats in the MI and sham groups. The miRanda software was used to predict the binding sites of LncRNA-miRNA and miRNA-mRNA, which were futhrer verified by dual luciferase assay. The LncRNA-miRNA-apoptosis pathway was further validated using hypoxia-exposed primary cardiomyocytes. Results: Compared to the sham group, 412 LncRNAs were upregulated and 501 LncRNAs were downregulated in MI-rat heart tissues. Among them, LncRNA AC125982.2 was most significantly upregulated in MI-rat heart tissues and hypoxic cardiomyocytes. Knockdown of AC125982.2 and ATG4B expression reversed hypoxia-induced apoptosis. In addition, transfection of mir-450b-3p inhibitor attenuated the protective effect of AC125982.2 knockdown. Moreover, we found that AC125982.2 modulated ATG4B expression by acting as a sponge for miR-450b-3p. Conclusion: Upregulated AC125982.2 expression regulates ATG4B by sponging miR-450b-3p, promoting cardiomyocyte apoptosis and contributing to rat MI development.

Circ-0000953 deficiency exacerbates podocyte injury and autophagy disorder by targeting Mir665-3p-Atg4b in diabetic nephropathy.

Circular RNAs (circRNAs) are special non-coding RNA (ncRNA) molecules that play a significant role in many diseases. However, the biogenesis and regulation of circRNAs in diabetic nephropathy (DN) are largely unknown. Here, we investigated the expression profile of circRNAs in kidney of DN mice through circular RNA sequencing (circRNA-seq). The renal biopsy samples of patients with DN had low circ -0,000,953 expression, which was significantly associated with renal function. Furthermore, loss-of-function and gain-of-function experiments were carried out to prove the role of circ -0,000,953 in DN. Podocyte conditional knockin (cKI) or systemic overexpression of circ -0,000,953 alleviated albuminuria and restored macroautophagy/autophagy in kidney of diabetic mice. However, circ -0,000,953 knockdown exacerbated albuminuria and podocyte injury. Mechanistically, we found circ -0,000,953 directly binds to Mir665-3p-Atg4b to perform its function. Silencing of Mir665-3p or overexpression of Atg4b recovered podocyte autophagy both in vitro and in vivo. To examine the cause of circ -0,000,953 downregulation in DN, bioinformatics prediction found that circ -0,000,953 sequence has a high possibility of containing an m6A methylation site. Additionally, METTL3 was proved to regulate the expression and methylation level of circ -0,000,953 through YTHDF2 (YTH N6-methyladenosine RNA binding protein 2). In conclusion, this study revealed that circ -0,000,953 regulates podocyte autophagy by targeting Mir665-3p-Atg4b in DN. Therefore, circ -0,000,953 is a potential biomarker for prevention and cure of DN.Abbreviation: CCL2/MCP-1: C-C motif chemokine ligand 2; ceRNA: competing endogenous RNA; circRNA: circular RNA; cKI: conditional knockin; cKO: conditional knockout; CRE: creatinine; DM: diabetes mellitus; DN: diabetic nephropathy; ESRD: end-stage renal disease; HG: high glucose; IF: immunofluorescence; MAP1LC3/LC3B: microtubule-associated protein 1 light chain 3 beta; MPC5: mouse podocyte clone 5; MTECs: mouse tubular epithelial cells; MTOR: mechanistic target of rapamycin kinase; NC: normal control; ncRNA: non-coding RNA; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PAS: periodic acid-Schiff; RELA/p65: v-rel reticuloendotheliosis viral oncogene homolog A (avian); SDs: slit diaphragm proteins; Seq: sequencing; STZ: streptozotocin; SV40: SV40-MES13-cells, mouse mesangial cell line; T1D: type 1 diabetes mellitus; T2D: type 2 diabetes mellitus; TEM: transmission electron microscopy; TNF/TNF-α: tumor necrosis factor; VECs: vascular endothelial cells; WT1: WT1 transcription factor; YTHDF2: YTH N6-methyladenosine RNA binding protein 2.

Loss of ATG4B and ATG4A results in two-stage cell cycle defects in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) exhibits elevated levels of autophagy, which promote tumor progression and treatment resistance. ATG4B is an autophagy-related cysteine protease under consideration as a potential therapeutic target, but it is largely unexplored in PDAC. Here, we investigated the clinical and functional relevance of ATG4B expression in PDAC. Using two PDAC patient cohorts, we found that low ATG4B mRNA or protein expression is associated with worse patient survival outcomes, poorly differentiated PDAC tumors and a lack of survival benefit from adjuvant chemotherapy. In PDAC cell lines, ATG4B knockout reduced proliferation, abolished processing of LC3B (also known as MAP1LC3B), and reduced GABARAP and GABARAPL1 levels, but increased ATG4A levels. ATG4B and ATG4A double knockout lines displayed a further reduction in proliferation, characterized by delays in G1-S phase transition and mitosis. Pro-LC3B accumulated aberrantly at the centrosome with a concomitant increase in centrosomal proteins PCM1 and CEP131, which was rescued by exogenous ATG4B. The two-stage cell cycle defects following ATG4B and ATG4A loss have important therapeutic implications for PDAC.

YY1 is regulated by ALKBH5-mediated m6A modification and promotes autophagy and cancer progression through targeting ATG4B.

YY1 affects tumorigenesis and metastasis in multiple ways. However, the function of YY1 and the potential mechanisms through which it operates in gastric cancer (GC) progression by regulating autophagy remains poorly understood. This study aimed to assess the essential transcription factors (TFs) involved in autophagy regulation in GC. Western blot, RFP-GFP-LC3 double fluorescence and transmission electron microscopy (TEM) assays were used to probe autophagy activity in GC cells. Methylated RNA immunoprecipitation (MeRIP) was utilized to evaluate the ALKBH5-regulated m6A levels of YY1. Gain- and loss-of-function assays were employed in the scrutiny of the biological effects of the ALKBH5/YY1/ATG4B axis on cancer cell proliferation and invasion abilities in vitro. Per the findings, YY1 was identified as a crucial transcriptional activator of cancer autophagy-related genes and promoted the proliferation and aggressiveness of cancer cells associated with enhanced ATG4B-mediated autophagy. However, ectopic ALKBH5 expression abolished the YY1-induced effect via m6A modification. Importantly, YTHDF1 facilitated the mRNA stability of YY1 through m6A recognition. Collectively, this study found that YY1 was regulated by ALKBH5 and YTHDF1-mediated m6A modification and served as an autophagy-dependent tumor driver to accelerate cancer progression through ATG4B transactivation, providing an exploitable therapeutic target for GC.

In vitro production of N-degron fused proteins and its application.

The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.

ATG4B antagonizes antiviral immunity by GABARAP-directed autophagic degradation of TBK1.

ABBREVIATIONS: Baf A1: bafilomycin A1; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IFN: interferon; IKBKE/IKKi: inhibitor of nuclear factor kappa B kinase subunit epsilon; IRF3: interferon regulatory factor 3; ISG: interferon-stimulated gene; ISRE: IFN-stimulated response element; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMPs: pathogen-associated molecule patterns; RIGI/DDX58: RNA sensor RIG-I; SeV: Sendai virus; siRNA: small interfering RNA; TBK1: TANK binding kinase 1; WT: wild-type; VSV: vesicular stomatitis virus.

Ethanol inhibits pancreatic acinar cell autophagy through upregulation of ATG4B, mediating pathological responses of alcoholic pancreatitis.

Excessive alcohol intake is a major risk factor for pancreatitis, sensitizing the exocrine pancreas to stressors by mechanisms that remain obscure. Impaired autophagy drives nonalcoholic pancreatitis, but the effects of ethanol (EtOH) and alcoholic pancreatitis on autophagy are poorly understood. Here, we find that ethanol reduces autophagosome formation in pancreatic acinar cells, both in a mouse model of alcoholic pancreatitis induced by a combination of EtOH diet and cerulein (a CCK ortholog) and in EtOH+CCK-treated acinar cells (ex vivo model). Ethanol treatments decreased pancreatic level of LC3-II, a key mediator of autophagosome formation. This was caused by ethanol-induced upregulation of ATG4B, a cysteine protease that, cell dependently, regulates the balance between cytosolic LC3-I and membrane-bound LC3-II. We show that ATG4B negatively regulates LC3-II in acinar cells subjected to EtOH treatments. Ethanol raised ATG4B level by inhibiting its degradation, enhanced ATG4B enzymatic activity, and strengthened its interaction with LC3-II. We also found an increase in ATG4B and impaired autophagy in a dissimilar, nonsecretagogue model of alcoholic pancreatitis induced by EtOH plus palmitoleic acid. Adenoviral ATG4B overexpression in acinar cells greatly reduced LC3-II and inhibited autophagy. Furthermore, it aggravated trypsinogen activation and necrosis, mimicking key responses of ex vivo alcoholic pancreatitis. Conversely, shRNA Atg4B knockdown enhanced autophagosome formation and alleviated ethanol-induced acinar cell damage. The results reveal a novel mechanism, whereby ethanol inhibits autophagosome formation and thus sensitizes pancreatitis, and a key role of ATG4B in ethanol's effects on autophagy. Enhancing pancreatic autophagy, particularly by downregulating ATG4B, could be beneficial in mitigating the severity of alcoholic pancreatitis.NEW & NOTEWORTHY Ethanol sensitizes mice and humans to pancreatitis, but the underlying mechanisms remain obscure. Autophagy is important for maintaining pancreatic acinar cell homeostasis, and its impairment drives pancreatitis. This study reveals a novel mechanism, whereby ethanol inhibits autophagosome formation through upregulating ATG4B, a key cysteine protease. ATG4B upregulation inhibits autophagy in acinar cells and aggravates pathological responses of experimental alcoholic pancreatitis. Enhancing pancreatic autophagy, particularly by down-regulating ATG4B, could be beneficial for treatment of alcoholic pancreatitis.

Atg4b Overexpression Extends Lifespan and Healthspan in Drosophila melanogaster.

Autophagy plays important but complex roles in aging, affecting health and longevity. We found that, in the general population, the levels of ATG4B and ATG4D decreased during aging, yet they are upregulated in centenarians, suggesting that overexpression of ATG4 members could be positive for healthspan and lifespan. We therefore analyzed the effect of overexpressing Atg4b (a homolog of human ATG4D) in Drosophila, and found that, indeed, Atg4b overexpression increased resistance to oxidative stress, desiccation stress and fitness as measured by climbing ability. The overexpression induced since mid-life increased lifespan. Transcriptome analysis of Drosophila subjected to desiccation stress revealed that Atg4b overexpression increased stress response pathways. In addition, overexpression of ATG4B delayed cellular senescence, and improved cell proliferation. These results suggest that ATG4B have contributed to a slowdown in cellular senescence, and in Drosophila, Atg4b overexpression may have led to improved healthspan and lifespan by promoting a stronger stress response. Overall, our study suggests that ATG4D and ATG4B have the potential to become targets for health and lifespan interventions.

ATG4B and pS383/392-ATG4B serve as potential biomarkers and therapeutic targets of colorectal cancer.

BACKGROUND: Autophagy related protease 4B (ATG4B) is a protease required for autophagy processing, which is strongly implicated in cancer progression.  Phosphorylation of ATG4B is crucial for activation of its protease activity.  However, little is known about the relationship of ATG4B and its phosphorylated form at Ser 383 and 392 sites (pS383/392-ATG4B), with clinical outcomes, particularly in colorectal cancer (CRC). METHODS: The ATG4B gene expression in CRC patients was obtained from The Cancer Genome Atlas (TCGA) database to analyze its clinical relevance. Tissue microarrays composed of 118 CRC patient specimens were used to determine the associations of ATG4B and pS383/392-ATG4B protein levels with prognosis. The biological functions of ATG4B in CRC cells were inspected with cell proliferation, mobility and spheroid culture assays. RESULTS: ATG4B gene expression was elevated in tumor tissues of CRC patients compared to that in adjacent normal tissues and high level of ATG4B expression was associated with poor survival. Similarly, protein levels of ATG4B and pS383/392-ATG4B were highly correlated with worse overall survival and disease-free survival. Stratification analysis results showed that high level of ATG4B had significantly higher risk of mortality in males and elderly patients compared to those female patients and patients 60 years or younger. In contrast, multivariate Cox's regression analysis indicated that high level of pS383/392-ATG4B was significantly linked to unfavorable overall survival and disease-free survival of males and elderly patients, whereas, it had no correlation with female patients and patients 60 years or younger. Moreover, high level of ATG4B was positively associated with increased mortality risk in patients with advanced AJCC stages (III and IV) and lymph node invasion (N1 and N2) for both overall survival and disease-free survival. Nevertheless, high level of pS383/392-ATG4B was positively correlated with increased mortality risk in patients with early AJCC stages (I and II) and without lymph node invasion (N0). In addition, silencing ATG4B attenuated migration, invasion, and further enhanced the cytotoxic effects of chemotherapeutic drugs in two and three-dimensional cultures of CRC cells. CONCLUSIONS: Our results suggest that ATG4B and pS383/392-ATG4B might be suitable biomarkers and therapeutic targets for CRC.

An ATG4B inhibitor blocks autophagy and sensitizes Sorafenib inhibition activities in HCC tumor cells.

Autophagy related 4B (ATG4B) which regulates autophagy by promoting the formation of autophagosome through reversible modification of LC3, is closely related to cancer cell growth and drug resistance, and therefore is an attractive therapeutic target. Recently, ATG4B inhibitors have been reported, yet with drawbacks including weak potency. To discover more promising ATG4B inhibitors, we developed a high-throughput screening (HTS) assay and identified a new ATG4B inhibitor named DC-ATG4in. DC-ATG4in directly binds to ATG4B and inhibits its enzyme activity with an IC50 of 3.08 ± 0.47 µM. We further confirmed that DC-ATG4in is an autophagy inhibitor and blocks autophagy induced by Sorafenib in Hepatocellular Carcinoma (HCC) cells. More importantly, combination of DC-ATG4in with Sorafenib synergized the cancer cell killing effect and proliferation inhibition activities on HCC cells. Our data suggested that inactivation of autophagy via ATG4B inhibition may be a viable strategy to sensitize existing targeted therapy such as Sorafenib in the future.

The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells.

Although several mechanisms of macroautophagy/autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to its activation, the ATG4B protease primes the key autophagy player MAP1LC3B/LC3B. Given the lack of reporters to follow this event in living cells, we developed a Förster's resonance energy transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine-tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image makes it possible to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are pools of unprimed LC3B upon ATG4B downregulation, and the priming of the biosensor is abolished in ATG4B knockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Moreover, we screened for commercially-available ATG4B inhibitors, and illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Finally, we uncovered the CDK1-dependent regulation of the ATG4B-LC3B axis at mitosis. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution.Abbreviations: Aqua: aquamarine; ATG: autophagy related; AURKA: aurora kinase A; BafA1: bafilomycin A1; CDK1: cyclin dependent kinase 1; DKO: double knockout; FLIM: fluorescence lifetime imaging microscopy; FP: fluorescence protein; FRET: Förster's resonance energy transfer; GABARAP: GABA type A receptor-associated protein; HBSS: Hanks' balanced salt solution; KO: knockout; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NSC: NSC 185058; PE: phosphatidylethanolamine; SKO: single knockout; TKO: triple knockout; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type; ZPCK: Z-L-phe chloromethyl ketone.

Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD.

In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1ß: interleukin 1 beta; IS: Inner segment; ITGB1/integrin ß1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.

Involvement of acetylation of ATG4B in controlling autophagy induction.

ATG4B, a cysteine protease promoting autophagosome formation by reversibly modifying Atg8-family proteins, plays a vital role in controlling macroautophagy/autophagy initiation in response to stress. However, the molecular mechanism underlying the regulation of ATG4B activity is far from well elucidated. In the current study, we firstly revealed that the acetylation level of ATG4B at lysine residue 39 (K39) is strongly involved in regulating its activity and autophagy. Specifically, SIRT2 deacetylates ATG4B K39, enhancing ATG4B activity and autophagic flux, which can be antagonized by EP300/p300. Starvation treatment contributes to EP300 suppression and SIRT2 activation, promoting the deacetylation of ATG4B K39, which leads to the elevation of ATG4B activity and finally autophagy initiation. Mechanistic investigation showed that starvation reduces CCNE (cyclin E), resulting in the downregulation of the CCNE-CDK2 protein complex, decreasing the phosphorylation of SIRT2 Ser331 and finally activating SIRT2. In addition, we confirmed that SIRT2 promotes autophagy via suppressing acetylation of ATG4B at K39 using sirt2 gene knockout (sirt2-/-) mice. Collectively, our results have revealed the acetylation-mediated regulation of ATG4B cysteine protease activity in autophagy initiation in response to nutritional deficiency.