Recent progresses in the late stages of autophagy.

Autophagy, a lysosome-dependent degradation process, plays a crucial role in maintaining cell homeostasis. It serves as a vital mechanism for adapting to stress and ensuring intracellular quality control. Autophagy deficiencies or defects are linked to numerous human disorders, especially those associated with neuronal degeneration or metabolic diseases. Yoshinori Ohsumi was honored with the Nobel Prize in Physiology or Medicine in 2016 for his groundbreaking discoveries regarding autophagy mechanisms. Over the past few decades, autophagy research has predominantly concentrated on the early stages of autophagy, with relatively limited attention given to the late stages. Nevertheless, recent studies have witnessed substantial advancements in understanding the molecular intricacies of the late stages, which follows autophagosome formation. This review provides a comprehensive summary of the recent progresses in comprehending the molecular mechanisms of the late stages of autophagy.

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.

The STX17-SNAP47-VAMP7/VAMP8 complex is the default SNARE complex mediating autophagosome-lysosome fusion.

Autophagosome-lysosome fusion mediated by SNARE complexes is an essential step in autophagy. Two SNAP29-containing SNARE complexes have been extensively studied in starvation-induced bulk autophagy, while the relevant SNARE complexes in other types of autophagy occurring under non-starvation conditions have been overlooked. Here, we found that autophagosome-lysosome fusion in selective autophagy under non-starvation conditions does not require SNAP29-containing SNARE complexes, but requires the STX17-SNAP47-VAMP7/VAMP8 SNARE complex. Further, the STX17-SNAP47-VAMP7/VAMP8 SNARE complex also functions in starvation-induced autophagy. SNAP47 is recruited to autophagosomes following concurrent detection of ATG8s and PI(4,5)P2 via its Pleckstrin homology domain. By contrast, SNAP29-containing SNAREs are excluded from selective autophagy due to inactivation by O-GlcNAcylation under non-starvation conditions. These findings depict a previously unknown, default SNARE complex responsible for autophagosome-lysosome fusion in both selective and bulk autophagy, which could guide research and therapeutic development in autophagy-related diseases.

ULK phosphorylation of STX17 controls autophagosome maturation via FLNA.

Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also participates in the late stages of autophagy remains unknown. Here, we found that the autophagosomal SNARE protein, STX17, is phosphorylated by ULK at residue S289, beyond which it localizes specifically to autophagosomes. Inhibition of STX17 phosphorylation prevents such autophagosome localization. FLNA was then identified as a linker between ATG8 family proteins (ATG8s) and STX17 with essential involvement in STX17 recruitment to autophagosomes. Phosphorylation of STX17 S289 promotes its interaction with FLNA, activating its recruitment to autophagosomes and facilitating autophagosome-lysosome fusion. Disease-causative mutations around the ATG8s- and STX17-binding regions of FLNA disrupt its interactions with ATG8s and STX17, inhibiting STX17 recruitment and autophagosome-lysosome fusion. Cumulatively, our study reveals an unexpected role of ULK in autophagosome maturation, uncovers its regulatory mechanism in STX17 recruitment, and highlights a potential association between autophagy and FLNA.

The fate of autophagosomal membrane components.

During macroautophagy/autophagy, autophagosomes fuse with lysosomes to form autolysosomes. After fusion, the autophagosome inner membrane and enclosed substrates are degraded and transported out of lysosomes for recycling. The lysosomal membrane components are recycled by autophagic lysosome reformation (ALR) to generate new lysosomes. However, the fate of autophagosome outer membrane components on autolysosomes remains unknown. Our recent work discovered that autophagosome outer membrane components are not degraded but are recycled through an unidentified process which we named autophagosomal components recycling (ACR). Further investigation revealed the recycler complex (SNX4-SNX5-SNX17) responsible for ACR. The discovery of ACR not only fills a missing part in autophagy, but also reveals a new recycling pathway on autolysosomes.

Bifendate inhibits autophagy at multiple steps and attenuates oleic acid-induced lipid accumulation.

Some traditional Chinese medicines exert roles in the therapy of liver diseases by modulating autophagy. Bifendate (DDB), a synthetic intermediate of Schisandrin C extracted from Schisandrae chinensis, is clinically used to treat hepatitis in China. While DDB is a positive control to research some potential hepatoprotective agents, its related molecular mechanisms are unknown. In this study, we show that DDB inhibited autophagosome-lysosome fusion, lysosome acidification and autophagic lysosome reformation. Moreover, DDB attenuated oleic acid-induced lipid droplet accumulation. These findings reveal the effects of DDB on the autophagy-related processes and lysosomal function, and also provide a possibility to understand the bioactivity mechanism of DDB in the future.

Autophagosomal components recycling on autolysosomes.

Autophagy is a multistage, intracellular process. Here, we highlight a recently identified autophagosomal components recycling (ACR) stage and the recycler complex (SNX4-SNX5-SNX17), which mediates recycling of autophagosomal outer membrane proteins on the autolysosome surface immediately following autophagosome-lysosome fusion. This discovery opens numerous research directions into the postfusion fate of autophagosomes.

STING controls energy stress-induced autophagy and energy metabolism via STX17.

The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. Here, we report that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, we demonstrate a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17.

Recycling of autophagosomal components from autolysosomes by the recycler complex.

Autolysosomes contain components from autophagosomes and lysosomes. The contents inside the autolysosomal lumen are degraded during autophagy, while the fate of autophagosomal components on the autolysosomal membrane remains unknown. Here we report that the autophagosomal membrane components are not degraded, but recycled from autolysosomes through a process coined in this study as autophagosomal components recycling (ACR). We further identified a multiprotein complex composed of SNX4, SNX5 and SNX17 essential for ACR, which we termed 'recycler'. In this, SNX4 and SNX5 form a heterodimer that recognizes autophagosomal membrane proteins and is required for generating membrane curvature on autolysosomes, both via their BAR domains, to mediate the cargo sorting process. SNX17 interacts with both the dynein-dynactin complex and the SNX4-SNX5 dimer to facilitate the retrieval of autophagosomal membrane components. Our discovery of ACR and identification of the recycler reveal an important retrieval and recycling pathway on autolysosomes.

Gene Manipulation Protocols in Autophagy.

Macroautophagy (referred to as autophagy hereafter) is a highly conserved catabolic process in eukaryotic cells. Autophagy is essential for cellular homeostasis through elimination and recycling of large cytoplasmic components, such as abnormal protein aggregates and damaged organelles, via lysosomal degradation. Since being originally identified by genetic screening in yeast, autophagy-related (ATG) genes have played a central role in autophagy research in different organisms, including plants, worms, flies, and mammals. Mouse models for monitoring autophagic activity or clarifying its biological functions have also been established. These mice are powerful tools to investigate roles of autophagy in vivo. Owing to the rapid technological advances in molecular biology, it is ever more efficient and simpler to manipulate autophagy-associated genes. Herein, we will introduce some commonly used approaches of gene silencing in mammalian cells, including CRIPSR/Cas9-mediated gene knockout and siRNA- and shRNA-mediated gene knockdown. We also summarized the common mouse models used for assessing autophagy. We hope to bring the researchers some useful information as they study autophagy.

Analysis of Ovarian Injury Associated With COVID-19 Disease in Reproductive-Aged Women in Wuhan, China: An Observational Study.

Objective: This study was intended to investigate the relationship between COVID-19 disease and ovarian function in reproductive-aged women. Methods: Female COVID-19 patients of reproductive age were recruited between January 28 and March 8, 2020 from Tongji Hospital in Wuhan. Their baseline and clinical characteristics, as well as menstrual conditions, were recorded. Differentials in ovarian reserve markers and sex hormones (including anti-Müllerian hormone [AMH], follicle-stimulating hormone [FSH], the ratio of FSH to luteinizing hormone [LH], estradiol [E2], progesterone [P], testosterone [T], and prolactin [PRL] were compared to those of healthy women who were randomly selected and individually matched for age, region, and menstrual status. Uni- and multi-variable hierarchical linear regression analyses were performed to identify risk factors associated with ovarian function in COVID-19 women. Results: Seventy eight patients agreed to be tested for serum hormone, of whom 17 (21.79%) were diagnosed as the severe group and 39 (50%) were in the basal level group. Menstrual status (P = 0.55), menstrual volumes (P = 0.066), phase of menstrual cycle (P = 0.58), and dysmenorrhea history (P = 0.12) were similar without significant differences between non-severe and severe COVID-19 women. Significant lower serum AMH level/proportion (0.19/0.28 vs. 1.12 ng/ml, P = 0.003/0.027; AMH ≤ 1.1 ng/ml: 75/70.4 vs. 49.7%, P = 0.009/0.004), higher serum T (0.38/0.39 vs. 0.22 ng/ml, P < 0.001/0.001) and PRL (25.43/24.10 vs. 12.12 ng/ml, P < 0.001/0.001) levels were observed in basal level and the all-COVID-19 group compared with healthy age-matched control. When adjusted for age, menstrual status and parity variations in multivariate hierarchical linear regression analysis, COVID-19 disease was significantly associated with serum AMH (ß = -0.191; 95% CI: -1.177-0.327; P = 0.001), T (ß = 0.411; 95% CI: 11.154-22.709; P < 0.001), and PRL (ß = 0.497; 95% CI: 10.787-20.266; P < 0.001), suggesting an independent risk factor for ovarian function, which accounted for 3.2% of the decline in AMH, 14.3% of the increase in T, and 20.7% of the increase in PRL. Conclusion: Ovarian injury, including declined ovarian reserve and reproductive endocrine disorder, can be observed in women with COVID-19. More attention should be paid to their ovarian function under this pandemic, especially regarding reproductive-aged women. Clinical Trial Number: ChiCTR2000030015.

Potential Influence of Menstrual Status and Sex Hormones on Female Severe Acute Respiratory Syndrome Coronavirus 2 Infection: A Cross-sectional Multicenter Study in Wuhan, China.

BACKGROUND: Recent studies have indicated that females with coronavirus disease 2019 (COVID-19) have a lower morbidity, severe case rate, and mortality and better outcome than those of male individuals. However, the reasons remained to be addressed. METHODS: To find the factors that potentially protect females from COVID-19, we recruited all confirmed patients hospitalized at 3 branches of Tongji Hospital (N = 1902), and analyzed the correlation between menstrual status (n = 509, including 68 from Mobile Cabin Hospital), female hormones (n = 78), and cytokines related to immunity and inflammation (n = 263), and the severity/clinical outcomes in female patients <60 years of age. RESULTS: Nonmenopausal female patients had milder severity and better outcome compared with age-matched men (P < .01 for both). Menopausal patients had longer hospitalization times than nonmenopausal patients (hazard ratio [HR], 1.91 [95% confidence interval {CI}, 1.06-3.46]; P = .033). Both anti-Müllerian hormone (AMH) and estradiol (E2) showed a negative correlation with severity of infection (adjusted HR, 0.146 [95% CI, .026-.824], P = .029 and 0.304 [95% CI, .092-1.001], P = .05, respectively). E2 levels were negatively correlated with interleukin (IL) 2R, IL-6, IL-8, and tumor necrosis factor alpha in the luteal phase (P = .033, P = .048, P = .054, and P = .023) and C3 in the follicular phase (P = .030). CONCLUSIONS: Menopause is an independent risk factor for female COVID-19 patients. AMH and E2 are potential protective factors, negatively correlated with COVID-19 severity, among which E2 is attributed to its regulation of cytokines related to immunity and inflammation.

RNF186 regulates EFNB1 (ephrin B1)-EPHB2-induced autophagy in the colonic epithelial cells for the maintenance of intestinal homeostasis.

Although genome-wide association studies have identified the gene RNF186 encoding an E3 ubiquitin-protein ligase as conferring susceptibility to ulcerative colitis, the exact function of this protein remains unclear. In the present study, we demonstrate an important role for RNF186 in macroautophagy/autophagy activation in colonic epithelial cells and intestinal homeostasis. Mechanistically, RNF186 acts as an E3 ubiquitin-protein ligase for EPHB2 and regulates the ubiquitination of EPHB2. Upon stimulation by ligand EFNB1 (ephrin B1), EPHB2 is ubiquitinated by RNF186 at Lys892, and further recruits MAP1LC3B for autophagy. Compared to control mice, rnf186-/- and ephb2-/- mice have a more severe phenotype in the DSS-induced colitis model, which is due to a defect in autophagy in colon epithelial cells. More importantly, treatment with ephrin-B1-Fc recombinant protein effectively relieves DSS-induced mouse colitis, which suggests that ephrin-B1-Fc may be a potential therapy for human inflammatory bowel diseases.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG16L1: autophagy related 16 like 1; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; CD: Crohn disease; CQ: chloroquine; Csf2: colony stimulating factor 2; Cxcl1: c-x-c motif chemokine ligand 1; DMSO: dimethyl sulfoxide; DSS: dextran sodium sulfate; EFNB1: ephrin B1; EPHB2: EPH receptor B2; EPHB3: EPH receptor B3; EPHB2K788R: lysine 788 mutated to arginine in EPHB2; EPHB2K892R: lysine 892 mutated to arginine in EPHB2; ER: endoplasmic reticulum; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; GWAS: genome-wide association studies; HRP: horseradish peroxidase; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IBD: inflammatory bowel diseases; Il1b: interleukin 1 beta; Il6: interleukin 6; IRGM:immunity related GTPase M; i.p.: intraperitoneally; IPP: inorganic pyrophosphatase; KD: knockdown; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; NOD2: nucleotide binding oligomerization domain containing 2; PI3K: phosphoinositide 3-kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; RNF186: ring finger protein 186; RNF186A64T: alanine 64 mutated to threonine in RNF186; RNF186R179X: arginine 179 mutated to X in RNF186; RPS6: ribosomal protein S6; Tnf: tumor necrosis factor; SQSTM1: sequestosome 1; Ub: ubiquitin; UBE2D2: ubiquitin conjugating enzyme E2 D2; UBE2H: ubiquitin conjugating enzyme E2 H; UBE2K: ubiquitin conjugating enzyme E2 K; UBE2N: ubiquitin conjugating enzyme E2 N; UC: ulcerative colitis; ULK1:unc-51 like autophagy activating kinase 1; WT: wild type.

CAV1-CAVIN1-LC3B-mediated autophagy regulates high glucose-stimulated LDL transcytosis.

Diabetes is a recognized high-risk factor for the development of atherosclerosis, in which macroautophagy/autophagy is emerging to play essential roles. The retention of low-density lipoprotein (LDL) particles in subendothelial space following transcytosis across the endothelium is the initial step of atherosclerosis. Here, we identified that high glucose could promote atherosclerosis by stimulating transcytosis of LDL. By inhibiting AMPK-MTOR-PIK3C3 pathway, high glucose suppresses the CAV-CAVIN-LC3B-mediated autophagic degradation of CAV1; therefore, more CAV1 is accumulated in the cytosol and utilized to form more caveolae in the cell membrane and facilitates the LDL transcytosis across endothelial cells. For a proof of concept, higher levels of lipids were accumulated in the subendothelial space of umbilical venous walls from pregnant women with gestational diabetes mellitus (GDM), compared to those of pregnant women without GDM. Our results reveal that high glucose stimulates LDL transcytosis by a novel CAV1-CAVIN1-LC3B signaling-mediated autophagic degradation pathway. ABBREVIATIONS: 3-MA: 3-methyladenine; ACTB: actin beta; AMPK: AMP-activated protein kinase; Bafi: bafilomycin A1; CAV1: caveolin-1; CAVIN1: caveolae associated protein 1; CSD: the CAV1 scaffolding domain; GDM: gestational diabetes mellitus; IMD: intramembrane domain; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule- associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTOR: mechanistic target of rapamycin kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; SQSTM1/p62: sequestosome 1.

Anatomy of autophagy: from the beginning to the end.

Autophagy is a highly regulated process in eukaryotes to maintain homeostasis and manage stress responses. Understanding the regulatory mechanisms and key players involved in autophagy will provide critical insights into disease-related pathogenesis and potential clinical treatments. In this review, we describe the hallmark events involved in autophagy, from its initiation, to the final destruction of engulfed targets. Furthermore, based on structural and biochemical data, we evaluate the roles of key players in these processes and provide rationale as to how they control autophagic events in a highly ordered manner.

MTORC1-mediated NRBF2 phosphorylation functions as a switch for the class III PtdIns3K and autophagy.

NRBF2/Atg38 has been identified as the fifth subunit of the macroautophagic/autophagic class III phosphatidylinositol 3-kinase (PtdIns3K) complex, along with ATG14/Barkor, BECN1/Vps30, PIK3R4/p150/Vps15 and PIK3C3/Vps34. However, its functional mechanism and regulation are not fully understood. Here, we report that NRBF2 is a fine tuning regulator of PtdIns3K controlled by phosphorylation. Human NRBF2 is phosphorylated by MTORC1 at S113 and S120. Upon nutrient starvation or MTORC1 inhibition, NRBF2 phosphorylation is diminished. Phosphorylated NRBF2 preferentially interacts with PIK3C3/PIK3R4. Suppression of NRBF2 phosphorylation by MTORC1 inhibition alters its binding preference from PIK3C3/PIK3R4 to ATG14/BECN1, leading to increased autophagic PtdIns3K complex assembly, as well as enhancement of ULK1 protein complex association. Consequently, NRBF2 in its unphosphorylated form promotes PtdIns3K lipid kinase activity and autophagy flux, whereas its phosphorylated form blocks them. This study reveals NRBF2 as a critical molecular switch of PtdIns3K and autophagy activation, and its on/off state is precisely controlled by MTORC1 through phosphorylation.

Kinesin 1 Drives Autolysosome Tubulation.

Autophagic lysosome reformation (ALR) plays an important role in maintaining lysosome homeostasis. During ALR, lysosomes are reformed by recycling lysosomal components from autolysosomes. The most noticeable step of ALR is autolysosome tubulation, but it is currently unknown how the process is regulated. Here, using an approach combining in vivo studies and in vitro reconstitution, we found that the kinesin motor protein KIF5B is required for autolysosome tubulation and that KIF5B drives autolysosome tubulation by pulling on the autolysosomal membrane. Furthermore, we show that KIF5B directly interacts with PtdIns(4,5)P2. Kinesin motors are recruited and clustered on autolysosomes via interaction with PtdIns(4,5)P2 in a clathrin-dependent manner. Finally, we demonstrate that clathrin promotes formation of PtdIns(4,5)P2-enriched microdomains, which are required for clustering of KIF5B. Our study reveals a mechanism by which autolysosome tubulation was generated.

ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes.

Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.