MK8722 initiates early-stage autophagy while inhibiting late-stage autophagy via FASN-dependent reprogramming of lipid metabolism.

Background and objective: Epithelial ovarian cancer (EOC) is associated with latent onset and poor prognosis, with drug resistance being a main concern in improving the prognosis of these patients. The resistance of cancer cells to most chemotherapeutic agents can be related to autophagy mechanisms. This study aimed to assess the therapeutic effect of MK8722, a small-molecule compound that activates AMP-activated protein kinase (AMPK), on EOC cells and to propose a novel strategy for the treatment of EOC. Purpose: To explore the therapeutic effects of MK8722 on EOC cells, and to elucidate the underlying mechanism. Methods and results: It was found that MK8722 effectively inhibited the malignant biological behaviors of EOC cells. In vitro experiments showed that MK8722 targeted and decreased the lipid metabolic pathway-related fatty acid synthase (FASN) expression levels, causing the accumulation of lipid droplets. In addition, transmission electron microscopy revealed the presence of autophagosome-affected mitochondria. Western blotting confirmed that MK8722 plays a role in activating autophagy upstream (PI3K/AKT/mTOR) and inhibiting autophagy downstream via FASN-dependent reprogramming of lipid metabolism. Plasmid transient transfection demonstrated that MK8722 suppressed late-stage autophagy by blocking autophagosome-lysosome fusion. Immunofluorescence and gene silencing revealed that this effect was achieved by inhibiting the interaction of FASN with the SNARE complexes STX17-SNP29-VAMP8. Furthermore, the antitumor effect of MK8722 was verified using a subcutaneous xenograft mouse model. Conclusion: The findings suggest that using MK8722 may be a new strategy for treating EOC, as it has the potential to be a new autophagy/mitophagy inhibitor. Its target of action, FASN, is a molecular crosstalk between lipid metabolism and autophagy, and exploration of the underlying mechanism of FASN may provide a new research direction.

RUNDC1 negatively mediates the fusion of autophagosomes with lysosomes via regulating SNARE complex assembly.

Macroautophagy/autophagy is an essential pro-survival mechanism activated in response to nutrient deficiency. The proper fusion between autophagosomes and lysosomes is a critical step for autophagic degradation. We recently reported that RUNDC1 (RUN domain containing 1) inhibits autolysosome formation via clasping the ATG14-STX17-SNAP29 complex to hinder VAMP8 binding. We showed that RUNDC1 colocalizes with LC3 and associates with mature autophagosomes in cell lines and the zebrafish model. We utilized liposome fusion and in vitro autophagosome-lysosome fusion assays to demonstrate that RUNDC1 inhibits autolysosome formation. Moreover, we found that RUNDC1 clasps the ATG14-STX17-SNAP29 complex via stimulating ATG14 homo-oligomerization to inhibit ATG14 dissociation, which in turn prevents VAMP8 from binding to STX17-SNAP29. Our results demonstrate that RUNDC1 is a negative regulator of autophagy that restricts autophagosome fusion with lysosomes and is crucial for zebrafish survival in nutrient-deficient conditions. Here, we summarize our findings and discuss their implications for our understanding of autophagy regulation.

Syntaxin-5's flexibility in SNARE pairing supports Golgi functions.

Deficiency in the conserved oligomeric Golgi (COG) complex that orchestrates SNARE-mediated tethering/fusion of vesicles that recycle the Golgi's glycosylation machinery results in severe glycosylation defects. Although two major Golgi v-SNAREs, GS28/GOSR1, and GS15/BET1L, are depleted in COG-deficient cells, the complete knockout of GS28 and GS15 only modestly affects Golgi glycosylation, indicating the existence of an adaptation mechanism in Golgi SNARE. Indeed, quantitative mass-spectrometry analysis of STX5-interacting proteins revealed two novel Golgi SNARE complexes-STX5/SNAP29/VAMP7 and STX5/VTI1B/STX8/YKT6. These complexes are present in wild-type cells, but their usage is significantly increased in both GS28- and COG-deficient cells. Upon GS28 deletion, SNAP29 increased its Golgi residency in a STX5-dependent manner. While STX5 depletion and Retro2-induced diversion from the Golgi severely affect protein glycosylation, GS28/SNAP29 and GS28/VTI1B double knockouts alter glycosylation similarly to GS28 KO, indicating that a single STX5-based SNARE complex is sufficient to support Golgi glycosylation. Importantly, co-depletion of three Golgi SNARE complexes in GS28/SNAP29/VTI1B TKO cells resulted in severe glycosylation defects and a reduced capacity for glycosylation enzyme retention at the Golgi. This study demonstrates the remarkable plasticity in SXT5-mediated membrane trafficking, uncovering a novel adaptive response to the failure of canonical intra-Golgi vesicle tethering/fusion machinery.

siRNA screening reveals that SNAP29 contributes to exosome release.

Cells release extracellular vesicles (EVs) of different sizes. Small EVs (< 200 nm) can originate from the fusion of multivesicular bodies with the plasma membrane, i.e. exosomes, and from budding of the plasma membrane, i.e. small ectosomes. To investigate the molecular machinery required for the release of small EVs, we developed a sensitive assay based on incorporation of radioactive cholesterol in EV membranes and used it in a siRNA screening. The screening showed that depletion of several SNARE proteins affected the release of small EVs. We focused on SNAP29, VAMP8, syntaxin 2, syntaxin 3 and syntaxin 18, the depletion of which reduced the release of small EVs. Importantly, this result was verified using gold standard techniques. SNAP29 depletion resulted in the largest effect and was further investigated. Immunoblotting analysis of small EVs showed that the release of several proteins considered to be associated with exosomes like syntenin, CD63 and Tsg101 was reduced, while the level of several proteins that have been shown to be released in ectosomes (annexins) or by secretory autophagy (LC3B and p62) was not affected by SNAP29 depletion. Moreover, these proteins appeared in different fractions when the EV samples were further separated by a density gradient. These results suggest that SNAP29 depletion mainly affects the secretion of exosomes. To investigate how SNAP29 affects exosome release, we used microscopy to study the distribution of MBVs using CD63 labelling and CD63-pHluorin to detect fusion events of MVBs with the plasma membrane. SNAP29 depletion caused a redistribution of CD63-labelled compartments but did not change the number of fusion events. Further experiments are therefore needed to fully understand the function of SNAP29. To conclude, we have developed a novel screening assay that has allowed us to identify several SNAREs involved in the release of small EVs.

Lipid phosphatase SAC1 suppresses hepatitis B virus replication through promoting autophagic degradation of virions.

Phosphatidylinositol lipids play vital roles in lipid signal transduction, membrane recognition, vesicle transport, and viral replication. Previous studies have revealed that SAC1-like phosphatidylinositol phosphatase (SACM1L/SAC1), which uses phosphatidylinositol-4-phosphate (PI4P) as its substrate, greatly affects the replication of certain bacteria and viruses in vitro. However, it remains unclear whether and how SAC1 modulates hepatitis B virus (HBV) replication in vitro and in vivo. In the present study, we observed that SAC1 silencing significantly increased HBV DNA replication, subviral particle (SVP) expression, and secretion of HBV virions, whereas SAC1 overexpression exerted the opposite effects. Moreover, SAC1 overexpression inhibited HBV DNA replication and SVP expression in a hydrodynamic injection-based HBV-persistent replicating mouse model. Mechanistically, SAC1 silencing increased the number of HBV-containing autophagosomes as well as PI4P levels on the autophagosome membrane. Moreover, SAC1 silencing blocked autophagosome-lysosome fusion by inhibiting the interaction between synaptosomal-associated protein 29 and vesicle-associated membrane protein 8. Collectively, our data indicate that SAC1 significantly inhibits HBV replication by promoting the autophagic degradation of HBV virions. Our findings support that SAC1-mediated phospholipid metabolism greatly modulates certain steps of the HBV life-cycle and provide a new theoretical basis for antiviral therapy.

Snap29 Is Dispensable for Self-Renewal Maintenance but Required for Proper Differentiation of Mouse Embryonic Stem Cells.

Pluripotent embryonic stem cells (ESCs) can self-renew indefinitely and are able to differentiate into all three embryonic germ layers. Synaptosomal-associated protein 29 (Snap29) is implicated in numerous intracellular membrane trafficking pathways, including autophagy, which is involved in the maintenance of ESC pluripotency. However, the function of Snap29 in the self-renewal and differentiation of ESCs remains elusive. Here, we show that Snap29 depletion via CRISPR/Cas does not impair the self-renewal and expression of pluripotency-associated factors in mouse ESCs. However, Snap29 deficiency enhances the differentiation of ESCs into cardiomyocytes, as indicated by heart-like beating cells. Furthermore, transcriptome analysis reveals that Snap29 depletion significantly decreased the expression of numerous genes required for germ layer differentiation. Interestingly, Snap29 deficiency does not cause autophagy blockage in ESCs, which might be rescued by the SNAP family member Snap47. Our data show that Snap29 is dispensable for self-renewal maintenance, but required for the proper differentiation of mouse ESCs.

Blockage of autophagosome-lysosome fusion through SNAP29 O-GlcNAcylation promotes apoptosis via ROS production.

Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer stage. Therefore, development of potent autophagy modulators, with a clear mechanistic understanding of their target action, has paramount importance in both mechanistic and clinical studies. In the process of exploring the mechanism of action of a previously identified cytotoxic small molecule (SM15) designed to target microtubules and the interaction domain of microtubules and the kinetochore component NDC80/HEC1, we discovered that the molecule acts as a potent autophagy inhibitor. By using several biochemical and cell biology assays we demonstrated that SM15 blocks basal autophagic flux by inhibiting the fusion of correctly formed autophagosomes with lysosomes. SM15-induced autophagic flux blockage promoted apoptosis-mediated cell death associated with ROS production. Interestingly, autophagic flux blockage, apoptosis induction and ROS production were rescued by genetic or pharmacological inhibition of OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) or by expressing an O-GlcNAcylation-defective mutant of the SNARE fusion complex component SNAP29, pointing to SNAP29 as the molecular target of SM15 in autophagy. Accordingly, SM15 was found to enhance SNAP29 O-GlcNAcylation and, thereby, inhibit the formation of the SNARE fusion complex. In conclusion, these findings identify a new pathway in autophagy connecting O-GlcNAcylated SNAP29 to autophagic flux blockage and autophagosome accumulation, that, in turn, drives ROS production and apoptotic cell death. Consequently, modulation of SNAP29 activity may represent a new opportunity for therapeutic intervention in cancer and other autophagy-associated diseases.

Snapshots from within the cell: Novel trafficking and non trafficking functions of Snap29 during tissue morphogenesis.

Membrane trafficking is a core cellular process that supports diversification of cell shapes and behaviors relevant to morphogenesis during development and in adult organisms. However, how precisely trafficking components regulate specific differentiation programs is incompletely understood. Snap29 is a multifaceted Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptor, involved in a wide range of trafficking and non-trafficking processes in most cells. A body of knowledge, accrued over more than two decades since its discovery, reveals that Snap29 is essential for establishing and maintaining the operation of a number of cellular events that support cell polarity and signaling. In this review, we first summarize established functions of Snap29 and then we focus on novel ones in the context of autophagy, Golgi trafficking and vesicle fusion at the plasma membrane, as well as on non-trafficking activities of Snap29. We further describe emerging evidence regarding the compartmentalisation and regulation of Snap29. Finally, we explore how the loss of distinct functions of human Snap29 may lead to the clinical manifestations of congenital disorders such as CEDNIK syndrome and how altered SNAP29 activity may contribute to the pathogenesis of cancer, viral infection and neurodegenerative diseases.

The ORF7a protein of SARS-CoV-2 initiates autophagy and limits autophagosome-lysosome fusion via degradation of SNAP29 to promote virus replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is closely related to various cellular aspects associated with autophagy. However, how SARS-CoV-2 mediates the subversion of the macroautophagy/autophagy pathway remains largely unclear. In this study, we demonstrate that overexpression of the SARS-CoV-2 ORF7a protein activates LC3-II and leads to the accumulation of autophagosomes in multiple cell lines, while knockdown of the viral ORF7a gene via shRNAs targeting ORF7a sgRNA during SARS-CoV-2 infection decreased autophagy levels. Mechanistically, the ORF7a protein initiates autophagy via the AKT-MTOR-ULK1-mediated pathway, but ORF7a limits the progression of autophagic flux by activating CASP3 (caspase 3) to cleave the SNAP29 protein at aspartic acid residue 30 (D30), ultimately impairing complete autophagy. Importantly, SARS-CoV-2 infection-induced accumulated autophagosomes promote progeny virus production, whereby ORF7a downregulates SNAP29, ultimately resulting in failure of autophagosome fusion with lysosomes to promote viral replication. Taken together, our study reveals a mechanism by which SARS-CoV-2 utilizes the autophagic machinery to facilitate its own propagation via ORF7a.Abbreviations: 3-MA: 3-methyladenine; ACE2: angiotensin converting enzyme 2; ACTB/ß-actin: actin beta; ATG7: autophagy related 7; Baf A1: bafilomycin A1; BECN1: beclin 1; CASP3: caspase 3; COVID-19: coronavirus disease 2019; GFP: green fluorescent protein; hpi: hour post-infection; hpt: hour post-transfection; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERS: Middle East respiratory syndrome; MTOR: mechanistic target of rapamycin kinase; ORF: open reading frame; PARP: poly(ADP-ribose) polymerase; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; shRNAs: short hairpin RNAs; siRNA: small interfering RNA; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TCID50: tissue culture infectious dose; TEM: transmission electron microscopy; TUBB, tubulin, beta; ULK1: unc-51 like autophagy activating kinase 1.

SNARE-binding protein synaptosomal-associated protein of 29 kDa (SNAP29) regulates the intracellular sequestration of glucose transporter 4 (GLUT4) vesicles in adipocytes.

AIMS/INTRODUCTION: Insulin stimulates translocation of glucose transporter 4 (GLUT4) from the perinuclear location to the plasma membrane. In the unstimulated state, intracellular vesicles containing GLUT4 are sequestered into specialized storage vesicles that have come to be known as the insulin-responsive compartment (IRC). The IRC is a functional compartment in the perinuclear region that is a target of the insulin signaling cascade, although its precise nature is unclear. Here, we report a novel molecular mechanism facilitating formation of the IRC. MATERIALS AND METHODS: We determined synaptosomal-associated protein of 29 kDa (SNAP29) by mass spectrometry to be an EH domain-containing protein 1 (EHD1)-binding protein. Then, its expression was confirmed by western blotting. Subcellular localization of SNAP29 was determined by immunofluorescent microscopy. Interactions between SNAP29 and syntaxins were determined by immunoprecipitation. We measured glucose uptake and GLUT4 translocation in 3T3-L1 adipocyte expressing SNAP29 or silencing SNAP29. RESULTS: We found SNAP29 to be localized in the perinuclear region and to show partial co-localization with GLUT4 under basal conditions. We also found that SNAP29 binds to syntaxin6, a Qc-SNARE, in adipocytes. In SNAP29-expressing cells, vesicles containing GLUT4 were observed to aggregate around the perinuclear region. In contrast, when SNAP29 was silenced, perinuclear GLUT4 vesicles were dispersed throughout the cytosol. Insulin-stimulated glucose uptake was inhibited in both SNAP29-expressing and SNAP29-silenced cells. CONCLUSIONS: These data suggest that SNAP29 sequesters and anchors GLUT4-containing vesicles in the perinuclear region, and might have a role in the biogenesis of the perinuclear IRC.

Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis.

The endocytic protein EHD1 controls primary ciliogenesis by facilitating fusion of the ciliary vesicle and by removal of CP110 from the mother centriole. EHD3, the closest EHD1 paralog, has a similar regulatory role, but initial evidence suggested that the other two more distal paralogs, EHD2 and EHD4 may be dispensable for ciliogenesis. Herein, we define a novel role for EHD4, but not EHD2, in regulating primary ciliogenesis. To better understand the mechanisms and differential functions of the EHD proteins in ciliogenesis, we first demonstrated a requirement for EHD1 ATP-binding to promote ciliogenesis. We then identified two sequence motifs that are entirely conserved between EH domains of EHD1, EHD3 and EHD4, but display key amino acid differences within the EHD2 EH domain. Substitution of either P446 or E470 in EHD1 with the aligning S451 or W475 residues from EHD2 was sufficient to prevent rescue of ciliogenesis in EHD1-depleted cells upon reintroduction of EHD1. Overall, our data enhance the current understanding of the EHD paralogs in ciliogenesis, demonstrate a need for ATP-binding and identify conserved sequences in the EH domains of EHD1, EHD3 and EHD4 that regulate EHD1 binding to proteins and its ability to rescue ciliogenesis in EHD1-depleted cells.

Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts.

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.

Activity of the SNARE Protein SNAP29 at the Endoplasmic Reticulum and Golgi Apparatus.

Snap29 is a conserved regulator of membrane fusion essential to complete autophagy and to support other cellular processes, including cell division. In humans, inactivating SNAP29 mutations causes CEDNIK syndrome, a rare multi-systemic disorder characterized by congenital neuro-cutaneous alterations. The fibroblasts of CEDNIK patients show alterations of the Golgi apparatus (GA). However, whether and how Snap29 acts at the GA is unclear. Here we investigate SNAP29 function at the GA and endoplasmic reticulum (ER). As part of the elongated structures in proximity to these membrane compartments, a pool of SNAP29 forms a complex with Syntaxin18, or with Syntaxin5, which we find is required to engage SEC22B-loaded vesicles. Consistent with this, in HeLa cells, in neuroepithelial stem cells, and in vivo, decreased SNAP29 activity alters GA architecture and reduces ER to GA trafficking. Our data reveal a new regulatory function of Snap29 in promoting secretory trafficking.

Decreased neuronal synaptosome associated protein 29 contributes to poststroke cognitive impairment by disrupting presynaptic maintenance.

Background: Poststroke cognitive impairments are common in stroke survivors, and pose a high risk of incident dementia. However, the cause of these cognitive impairments is obscure and required an investigation. Methods: Oxygen-glucose deprivation (OGD) model and middle cerebral artery occlusion (MCAO) model were used to imitate in vitro or in vivo acute cerebral ischemia, respectively. The differentially expressed synaptosome associated protein 29 (SNAP29)-interacting proteins upon ischemia and reperfusion were analyzed with bioinformatics analysis and the results indicated that the changes of SNAP29 after acute ischemia were mainly involved in the synaptic functions. The outcomes of SNAP29 reduction were assessed with SNAP29 knockdown, which mimicked the distribution of SNAP29 along neuronal processes after acute ischemia. Using the whole-cell patch clamp recording method and transmission electron microscope, the pre-synaptic function and readily releasable pool (RRP) were observed after SNAP29 knock down. Using photogenetic manipulations and behavioral tests, the neuronal projection and cognitive functions of mice with SNAP29 knock down in hippocampus CA1 region were evaluated. Results: It was found that SNAP29 protein levels decreased in both in vitro and in vivo ischemic models. Further, the SNAP29 reduction wasn't associated with impaired autophagy flux and neuronal survival. When SNAP29 was knocked down in primary cortical neurons, the frequency of AMPARs-mediated mEPSCs, but not the amplitude, significantly decreased. Meanwhile, the mice with SNAP29 knockdown at CA1 region of hippocampus developed an impairment in hippocampus-mPFC (middle prefrontal cortex) circuit and behavioral dysfunctions. Moreover, the size of RRP at presynaptic sites was diminished. Conclusion: Since SNAP29 protein levels didn't significantly influence the neuronal survival and its decrease was sufficient to disturb the neural circuit via a presynaptic manner, the SNAP29-associated strategies may be an efficient target against poststroke synaptic dysfunction and cognitive deficits.

Tumor Necrosis Factor α Reduces SNAP29 Dependent Autolysosome Formation to Increase Prion Protein Level and Promote Tumor Cell Migration.

Tumor Necrosis Factor α (TNFα) is best known as a mediator of inflammation and immunity, and also plays important roles in tumor biology. However, the role of TNFα in tumor biology is complex and not completely understood. In a human melanoma cell line, M2, and a lung carcinoma cell line, A549, TNFα up-regulates prion protein (PrP) level, and promotes tumor cell migration in a PrP dependent manner. Silencing PRNP abrogates TNFα induced tumor cell migration; this phenotype is reversed when PRNP is re-introduced. Treatment with TNFα activates nuclear factor kappa B (NF-κB) signaling, which then mitigates autophagy by reducing the expression of Forkhead Box P3 (FOXP3). Down regulation of FOXP3 reduces the transcription of synaptosome associated protein 29 (SNAP29), which is essential in the fusion of autophagosome and lysosome creating autolysosome. FOXP3 being a bona fide transcription factor for SNAP29 is confirmed in a promoter binding assay. Accordingly, silencing SNAP29 in these cell lines also up-regulates PrP, and promotes tumor cell migration without TNFα treatment. But, when SNAP29 or FOXP3 is silenced in these cells, they are no longer respond to TNFα. Thus, a reduction in autophagy is the underlying mechanism by which expression of PrP is up-regulated, and tumor cell migration is enhanced upon TNFα treatment. Disrupting the TNFα-NF-κB-FOXP3-SNAP29 signaling axis may provide a therapeutic approach to mitigate tumor cell migration.

SNAP29 mediates the assembly of histidine-induced CTP synthase filaments in proximity to the cytokeratin network.

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.

Silencing DAPK3 blocks the autophagosome-lysosome fusion by mediating SNAP29 in trophoblast cells under high glucose treatment.

Autophagy plays an essential role in gestational diabetes mellitus (GDM). Death-associated protein kinase-3 (DAPK3) regulates a variety of cellular functions; however, the relationship between DAPK3 and autophagy is unknown. In this study, we aim to investigate whether DAPK3 is associated with autophagy in GDM, and we found that DAPK3 was upregulated in the placenta of GDM patients and extravillous trophoblast cells under high-glucose conditions. Silencing DAPK3 decreased the assembly of the STX17-SNAP29-VAMP8 complex, leading to the blockade of autophagosome-lysosome fusion by mediating synaptosomal-associated protein 29 (SNAP29). Moreover, knockdown of DAPK3 ameliorates cell invasion and mediates autophagy in high glucose, and does not alter the expression of autophagy-related genes in normal glucose. Our study demonstrates the significance of DAPK3 in autophagy and GDM, which may provide new insights into the molecular mechanisms regulating trophoblast invasion.

Regulation of Autophagic Flux by the 20S Proteasome.

The proteolytic arm of the protein homeostasis network is maintained by both the ubiquitin-proteasome system (UPS) and autophagy. A well-balanced crosstalk between the two catabolic pathways ensures energy-efficient maintenance of cellular function. Our current understanding of the crosstalk between the UPS and autophagy is centered around substrate ubiquitination. Herein we report an additional method of crosstalk involving ubiquitin-independent 20S proteasome regulation of autophagosome-lysosome fusion. We found that enhancement of 20S proteasome activity increased the degradation of the disordered soluble N-ethylmaleimide-sensitive factor activating protein receptor proteins, synaptosomal-associated protein 29 (SNAP29), and syntaxin 17 (STX17), but not vesicle-associated membrane protein 8. This resulted in a reduction of autophagosome-lysosome fusion, which was ameliorated upon overexpression of both SNAP29 and STX17. In all, we herein present a mechanism of crosstalk between the proteasome and autophagy pathway that is regulated by ubiquitin-independent 20S proteasome-mediated degradation of SNAP29 and STX17.

The Glycoprotein and Nucleocapsid Protein of Hantaviruses Manipulate Autophagy Flux to Restrain Host Innate Immune Responses.

Hantavirus infection, which causes severe zoonotic diseases with high mortality in humans, has become a global public health concern. Here, we demonstrate that Hantaan virus (HTNV), the prevalent prototype of the hantavirus in Asia, can restrain innate immune responses by manipulating host autophagy flux. HTNV induces complete mitophagy at the early stage of infection but incomplete autophagy at the late stage, and these responses involve the viral glycoprotein (Gn) and nucleocapsid protein (NP), respectively. Gn translocates to mitochondria and interacts with TUFM, recruiting LC3B and promoting mitophagy. Gn-induced mitophagy inhibits type I interferon (IFN) responses by degrading MAVS. Additionally, we found that NP competes with Gn for binding to LC3B, which inhibits Gn-mediated autophagosome formation, and interacts with SNAP29, which prevents autophagosome-lysosome fusion. Thus, NP disturbs the autophagic degradation of Gn. These findings highlight how hantaviruses repurpose host autophagy and evade innate immune responses for their life cycle and pathogenesis.

CEDNIK syndrome in an Indian patient with a novel mutation of the SNAP29 gene.

CEDNIK (CErebral Dysgenesis, Neuropathy, Ichthyosis, and Keratoderma) syndrome is a neuroichthyotic syndrome characterized by a constellation of clinical features including severe developmental retardation, microcephaly, and facial dysmorphism. Here, we report the first case of CEDNIK syndrome from India presenting with characteristic clinical features and harboring a novel mutation of SNAP29 gene.