During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.

BACKGROUND: Autophagic defects are involved in Methamphetamine (Meth)-induced neurotoxicity. Syntaxin 17 (Stx17), a member of the SNARE protein family, participating in several stages of autophagy, including autophagosome-late endosome/lysosome fusion. However, the role of Stx17 and potential mechanisms in autophagic defects induced by Meth remain poorly understood.
METHODS: To address the mechanism of Meth-induced cognitive impairment, the adenovirus (AV) and adeno-associated virus (AAV) were injected into the hippocampus for stereotaxis to overexpress Stx17 in vivo to examine the cognitive ability via morris water maze and novel object recognition. In molecular level, the synaptic injury and autophagic defects were evaluated. To address the Meth induced neuronal damage, the epidermal growth factor receptor (EGFR) degradation assay was performed to evaluate the degradability of the \"cargos\" mediated by Meth, and mechanistically, the maturation of the vesicles, including autophagosomes and endosomes, were validated by the Co-IP and the GTP-agarose affinity isolation assays.
RESULTS: Overexpression of Stx17 in the hippocampus markedly rescued the Meth-induced cognitive impairment and synaptic loss. For endosomes, Meth exposure upregulated Rab5 expression and its guanine-nucleotide exchange factor (GEF) (immature endosome), with a commensurate decreased active form of Rab7 (Rab7-GTP) and impeded the binding of Rab7 to CCZ1 (mature endosome); for autophagosomes, Meth treatment elicited a dramatic reduction in the overlap between Stx17 and autophagosomes but increased the colocalization of ATG5 and autophagosomes (immature autophagosomes). After Stx17 overexpression, the Rab7-GTP levels in purified late endosomes were substantially increased in parallel with the elevated mature autophagosomes, facilitating cargo (Aβ42, p-tau, and EGFR) degradation in the vesicles, which finally ameliorated Meth-induced synaptic loss and memory deficits in mice.
CONCLUSIONS: Stx17 decrease mediated by Meth contributes to vesicle fusion defects which may ascribe to the immature autophagosomes and endosomes, leading to autophagic dysfunction and finalizes neuronal damage and cognitive impairments. Therefore, targeting Stx17 may be a novel therapeutic strategy for Meth-induced neuronal injury.

Bladder cancer (BC) cells exhibit a high basal level of autophagy activity, which contributes to the development of a protective mechanism for cellular survival against current treatments. Hsa‑microRNA‑34a (miR‑34a) presents anti‑tumor function in several types of cancer. However, the functional mechanism of miR‑34a in regulating tumor aggressiveness and protective autophagy of BC remains largely unknown. First, transfected BC cells with miR‑34a mimic exhibited LC3‑II and p62 accumulation through immunofluorescence staining. It was demonstrated that syntaxin 17 (STX17), which is required for autophagosome‑lysosome fusion, was downregulated upon miR‑34a mimic treatment. Mechanistically, miR‑34a reduced the expression of STX17 proteins that directly bind on STX17 3\'‑untranslated regions and thus suppressed STX17 mRNA translation to eventually inhibit protective autophagy in BC. Cell viability and colony formation assays revealed that overexpression of miR‑34a in BC cells enhances the chemosensitivity of cisplatin, doxorubicin, epirubicin and mitomycin C. Furthermore, miR‑34a inhibited cell proliferation and triggered G0/G1 cell cycle arrest by inhibiting cyclin D1 and cyclin E2 protein expression. Moreover, miR‑34a suppressed cell motility through the downregulation of epithelial‑mesenchymal transition. In summary, miR‑34a inhibits cell proliferation, motility and autophagy activity in BC, which can benefit BC treatment.

Uncorrected obesity is accompanied by unfavorable structural and functional changes in the heart, known as obesity cardiomyopathy. Recent evidence has revealed a crucial role for mitochondria-associated endoplasmic reticulum membranes (MAMs) in obesity-induced cardiac complication. Syntaxin 17 (STX17) serves as a scaffolding molecule localized on MAMs although its role in obesity heart complication remains elusive.
This study examined the role of STX17 in MAMs and mitochondrial Ca2+ homeostasis in HFD-induced obesity cardiomyopathy using tamoxifen-induced cardiac-specific STX17 knockout (STX17cko) and STX17 overexpression mice using intravenously delivered recombinant adeno-associated virus serotype-9 (AAV9-cTNT-STX17).
STX17 levels were significantly elevated in plasma from obese patients and heart tissues of HFD-fed mice. Our data revealed that cardiac STX17 knockout alleviated cardiac remodeling and dysfunction in obese hearts without eliciting any notable effect itself, while STX17 overexpression aggravated cardiac dysfunction in obese mice. STX17 deletion and STX17 overexpression annihilated and aggravated, respectively, HFD-induced oxidative stress (O2- production) and mitochondrial injury in the heart. Furthermore, STX17 transfection facilitated obesity-induced MAMs formation in cardiomyocytes and evoked excess mitochondrial Ca2+ influx, dependent upon interaction with mitochondrial Ca2+ uniporter dominant negative β (MCUb) through Habc domain. Our data also suggested that STX17 promoted ubiquitination and degradation of MCUb through the E3 ligase parkin in the face of palmitate challenging.
Taken together, our results identified a novel role for STX17 in facilitating obesity-induced MAMs formation, and subsequently mitochondrial Ca2+ overload, mitochondrial O2- accumulation, lipid peroxidation, resulting in cardiac impairment. Our findings denoted therapeutic promises of targeting STX17 in obesity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen that has devastated the worldwide swine industry for over 30 years. Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway, and previous studies have documented that PRRSV infection prompts autophagosome accumulation. However, whether PRRSV induces complete or incomplete autophagy remains controversial. Here, we demonstrated that overexpression of PRRSV nonstructural protein 5 (nsp5) induced the accumulation of autophagosomes, and a similar scenario was observed in PRRSV-infected cells. Moreover, both PRRSV infection and nsp5 overexpression activated incomplete autophagy, as evidenced by the blockage of autophagosome-lysosome fusion. Mechanistically, nsp5 overexpression, as well as PRRSV infection, inhibited the interaction of syntaxin 17 (STX17) with synaptosomal-associated protein 29 (SNAP29), two SNARE proteins that mediate autophagosome fusion with lysosomes, to impair the formation of autolysosomes. We further confirmed that nsp5 interacted with STX17, rather than SANP29, and the interacting domains of STX17 were the N-terminal motif and SNARE motif. Taken together, the findings of our study suggest a mechanism by which PRRSV induces incomplete autophagy by blocking autophagosome degradation and provide insights into the development of new therapeutics to combat PRRSV infection. IMPORTANCE A substantial number of viruses have been demonstrated to utilize or hijack autophagy to benefit their replication. In the case of porcine reproductive and respiratory syndrome virus (PRRSV), previous studies have demonstrated the proviral effects of autophagy on PRRSV proliferation. Thus, an investigation of the mechanism by which PRRSV regulates the autophagy processes can provide new insight into viral pathogenesis. Autophagic flux is a dynamic process that consists of autophagosome formation and subsequent lysosomal degradation. However, the exact effect of PRRSV infection on the autophagic flux remains disputed. In this study, we demonstrated that PRRSV infection, as well as PRRSV nsp5 overexpression, inhibited the interaction of STX17 with SNAP29 to impair the fusion of autophagosomes with lysosomes, thereby blocking autophagic flux. This information will help us to understand PRRSV-host interactions and unravel new targets for PRRS prevention and control.

Doxorubicin (Dox), an anthracycline antibiotic, is an anticancer drug that inhibits DNA replication and cellular metabolic processes in cancer cells with high proliferative potential. However, Dox causes severe side effects, including myocardial damage and heart failure, but the molecular mechanism underlying Dox-induced myocardial injury remains uncertain. In the present study, we evaluated the effects of Dox on the mitochondrial quality control system and regulation of mitochondrial respiration and autophagy in an in vitro rat myoblast H9c2 cell culture model using western blotting, immunohistochemistry, the Seahorse XF24 system, and flow cytometry. Our results showed that Dox did not impair the initiation of autophagic flux or the functions of lysosomes; however, Dox affected the mitochondrial quality control system, leading to a fission-dominant morphology and impaired regulation of mitochondrial respiration, thereby increasing oxidative stress and inhibited progression of autophagy, particularly the fusion of autophagosomes with lysosomes. This inhibition caused a significant decrease in the formation of autolysosomes and was responsible for the accumulation of dysfunctional mitochondria and subsequent increase in oxidative stress, eventually leading to increased myocardial cell death.

STX17 (syntaxin 17) mediates autophagosome-lysosome fusion, and the translocation of STX17 to autophagosomes is characteristic of this process. STX17 arrives at autophagosomes when they are closed, stays there for approximately 10 min to promote fusion with lysosomes, and leaves when the autolysosomes are mature. However, the mechanism of this transient visit remains largely unknown. Here, we summarize the current knowledge about this phenomenon, including a recently discovered retrieval mechanism, and discuss remaining questions.Abbreviations: MAM: mitochondria-associated membrane; SNX: sorting nexin; STX17: syntaxin 17.

The gram-negative bacterium, Legionella pneumophila is known to manipulate the host cellular functions. L. pneumophila secretes bacterial proteins called Legionella effectors into the host cytosol that are necessary for these manipulations. The Legionella effector Lpg1137 was identified as a serine protease responsible for the degradation of syntaxin 17 (Stx17). However, how Lpg1137 specifically recognizes and degrades Stx17 remained unknown. Given that Stx17 is localized in the ER, mitochondria-associated membrane (MAM), and mitochondria, Lpg1137 likely distributes to these compartments to recognize Stx17. Here, we show that the C-terminal region of Lpg1137 binds to phosphatidic acid (PA), a MAM and mitochondria-enriched phospholipid, and that this binding is required for the correct intracellular distribution of Lpg1137. Two basic residues in the C-terminal region of Lpg1137 are required for PA binding and their mutation causes mislocalization of Lpg1137. This mutant also fails to degrade Stx17 while retaining protease activity. Taken together, our data reveal that Lpg1137 utilizes PA for its distribution to the membranous compartments in which Stx17 is localized.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.