Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.

Nucleophagy in Aspergillus oryzae is Mediated by Autophagosome Formation and Vacuole-Mediated Degradation.

We previously reported autophagy-mediated degradation of nuclei, nucleophagy, in the filamentous fungus Aspergillus oryzae. In this study, we examined whether nuclei are degraded as a whole. We generated A. oryzae mutants deleted for orthologs of Saccharomyces cerevisiae YPT7 and ATG15 which are required, respectively, for autophagosome-vacuole fusion and vacuolar degradation of autophagic bodies. Degradation of histone H2B-EGFP under starvation conditions was greatly decreased in the ΔAoypt7 and ΔAoatg15 mutants. Fluorescence and electron microscopic observations showed that autophagosomes and autophagic bodies surrounding the entire nuclei were accumulated in the cytoplasm of ΔAoypt7 and the vacuole of ΔAoatg15, respectively. These results indicate that nuclei are engulfed in the autophagosomes as a whole and transported/released into the vacuolar lumen where they are degraded.

Sigma-1 receptor recruits LC3 mRNA to ER-associated omegasomes to promote localized LC3 translation enabling functional autophagy.

Autophagosome formation initiated on the endoplasmic reticulum (ER)-associated omegasome requires LC3. Translational regulation of LC3 biosynthesis is unexplored. Here we demonstrate that LC3 mRNA is recruited to omegasomes by directly binding to the ER transmembrane Sigma-1 receptor (S1R). Cell-based and in vitro reconstitution experiments show that S1R interacts with the 3' UTR of LC3 mRNA and ribosomes to promote LC3 translation. Strikingly, the 3' UTR of LC3 is also required for LC3 protein lipidation, thereby linking the mRNA-3' UTR to LC3 function. An autophagy-defective S1R mutant responsible for amyotrophic lateral sclerosis cannot bind LC3 mRNA or induce LC3 translation. We propose a model wherein S1R de-represses LC3 mRNA via its 3' UTR at the ER, enabling LC3 biosynthesis and lipidation. Because several other LC3-related proteins use the same mechanism, our data reveal a conserved pathway for localized translation essential for autophagosome biogenesis with insights illuminating the molecular basis of a neurodegenerative disease.

Spatial Activation of Autophagy in Human Placenta-Related Tissue During Labor: A Possible Mechanism for Labor Onset.

INTRODUCTION: To explore the mechanisms of labor by investigating the autophagy of placental and fetal membranes tissue in normal pregnant women. METHODS: Placenta and fetal membranes were collected from women with singleton pregnancies without any medical complications and from women who delivered vaginally (labor-initiated group; L group) or by caesarean section (labor-noninitiated group; NL group). Autophagosomes were observed by transmission electron microscopy (TEM). Immunofluorescence and western blotting (WB) were used to detect protein levels of the autophagy markers LC3A and LC3B. TEM, immunohistochemistry (IHC), and WB were used to compare autophagy in different parts of the placenta and fetal membranes in the L and NL groups. The expression of LC3B/LC3A, ROCK1, and ROCK2 in the placenta of nonpregnant and pregnant rats was detected by WB and IHC. RESULTS: TEM and IHC results showed an increase in the number of autophagosomes and autophagic lysosomes in the L group, and WB results indicated an increase in the LC3B/A ratio between the placenta and fetal membranes in the L group. Autophagy was significantly increased on the maternal side of the placenta in the L group, and the level of autophagy became higher near rupture in the fetal membranes and near the point where the umbilical cord joins the placenta in the L group. The LC3B/A ratio increased and ROCK1 and ROCK2 levels decreased in postnatal rats. DISCUSSION: Autophagy can occur in the placenta and fetal membranes and its activity is higher at the onset of labor, suggesting a role in labor.

Aloperine Suppresses Cancer Progression by Interacting with VPS4A to Inhibit Autophagosome-lysosome Fusion in NSCLC.

Aloperine (ALO), a quinolizidine-type alkaloid isolated from a natural Chinese herb, has shown promising antitumor effects. Nevertheless, its common mechanism of action and specific target remain elusive. Here, it is demonstrated that ALO inhibits the proliferation and migration of non-small cell lung cancer cell lines in vitro and the tumor development in several mouse tumor models in vivo. Mechanistically, ALO inhibits the fusion of autophagosomes with lysosomes and the autophagic flux, leading to the accumulation of sequestosome-1 (SQSTM1) and production of reactive oxygen species (ROS), thereby inducing tumor cell apoptosis and preventing tumor growth. Knockdown of SQSTM1 in cells inhibits ROS production and reverses ALO-induced cell apoptosis. Furthermore, VPS4A is identified as a direct target of ALO, and the amino acids F153 and D263 of VPS4A are confirmed as the binding sites for ALO. Knockout of VPS4A in H1299 cells demonstrates a similar biological effect as ALO treatment. Additionally, ALO enhances the efficacy of the anti-PD-L1/TGF-ß bispecific antibody in inhibiting LLC-derived subcutaneous tumor models. Thus, ALO is first identified as a novel late-stage autophagy inhibitor that triggers tumor cell death by targeting VPS4A.

Conjugation of ATG8s to single membranes at a glance.

Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.

Methods to Monitor Nucleophagy in Yeast.

The selective degradation of nuclear components via autophagy, termed nucleophagy, is an essential process observed from yeasts to mammals and crucial for maintaining nucleus homeostasis and regulating nucleus functions. In the budding yeast Saccharomyces cerevisiae, nucleophagy occurs in two different manners: one involves autophagosome formation for the sequestration and vacuolar transport of nucleus-derived vesicles (NDVs), and the other proceeds with the invagination of the vacuolar membrane for the uptake of NDVs into the vacuole, termed macronucleophagy and micronucleophagy, respectively. This chapter describes methods to analyze and quantify activities of these nucleophagy pathways in yeast.

Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.

Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.

Bafilomycin 1A Affects p62/SQSTM1 Autophagy Marker Protein Level and Autophagosome Puncta Formation Oppositely under Various Inflammatory Conditions in Cultured Rat Microglial Cells.

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings.

Microscopy Analysis of Autophagosomal Structures in Plant Cells.

Macroautophagy, hereafter autophagy, plays a crucial role in the degradation of harmful or unwanted cellular components through a double-membrane autophagosome. Upon autophagosome fusion with the vacuole, the degraded materials are subsequently recycled to generate macromolecules, contributing to cellular homeostasis, metabolism, and stress tolerance in plants. A hallmark during autophagy is the formation of isolation membrane structure named as phagophore, which undergoes multiple steps to become as a complete double-membrane autophagosome. Methodologies have been developed in recent years to observe and quantify the autophagic process, which greatly advance knowledge of autophagosome biogenesis in plant cells. In this chapter, we will introduce two methods to dissect the autophagosome-related structures in the Arabidopsis plant cells, including the correlative light and electron microscopy, to map the ultrastructural feature of autophagosomal structures, and time-lapse imaging to monitor the temporal recruitment of autophagy machinery during autophagosome formation.

Determination of the Cryo-EM Structure of ATG9 from Arabidopsis thaliana.

Macroautophagy/autophagy is a highly conserved process for the degradation of cellular components and plays an essential role in cellular homeostasis maintenance. During autophagy, specialized double-membrane vesicles known as autophagosomes are formed and sequester cytoplasmic cargoes and deliver them to lysosomes or vacuoles for breakdown. Central to this process are autophagy-related (ATG) proteins, with the ATG9-the only integral membrane protein in this core machinery-playing a central role in mediating autophagosome formation. Recent years have witnessed the maturation of cryo-electron microscopy (cryo-EM) and single-particle analysis into powerful tools for high-resolution structural determination of protein complexes. These advancements have significantly deepened our understanding of the intricate molecular mechanisms underlying autophagosome biogenesis. In this study, we present a protocol detailing the acquisition of the three-dimensional structure of ATG9 from Arabidopsis thaliana. The structural resolution achieved 7.8 Å determined by single-particle cryo-electron microscopy (cryo-EM).

Ac-HSP20 regulates autophagy and promotes the encystation of Acanthamoeba castellanii by inhibiting the PI3K/AKT/mTOR signaling pathway.

BACKGROUND: The encystation of Acanthamoeba castellanii has important ecological and medical significance. Blocking encystation is the key to preventing transmission and curing infections caused by A. castellanii. The formation of autophagosomes is one of the most important changes that occur during the encystation of Acanthamoeba. Our previous studies have shown that the heat shock protein 20 of A. castellanii (Ac-HSP20) is involved in its encystation. This study aimed to determine the role and mechanism of Ac-HSP20 in regulating autophagy involved in the encystation of A. castellanii. METHODS: Immunofluorescence assay, western blotting and transmission electron microscopy were used to analyze the dynamic changes in autophagy during the initiation and continuation of encystation. The knockdown of Ac-HSP20 was performed to clarify its regulation of encystation and autophagy and to elucidate the molecular mechanism by which Ac-HSP20 participates in autophagy to promote cyst maturation. RESULTS: The encystation rates and autophagosomes were significantly decreased by treatment with the autophagy inhibitor 3-MA. The autophagy marker LC3B and autophagic lysosomes increased with the induced duration of encystation and reached the maximum at 48 h. The encystation rate, LC3B expression and autophagosomes decreased when Ac-HSP20 was knocked down by siRNA transfection. In addition, the expression levels of Ac-HSP20 and LC3B increased and the expressions of p-AKT and p-mTOR decreased after 48 h of encystation without knockdown. However, the expressions of p-AKT and p-mTOR increased while the expression of LC3B decreased under the knockdown of Ac-HSP20. Furthermore, the protein expression of LC3B increased when the PI3K/AKT/mTOR signaling pathway was inhibited but decreased when the pathway was activated. CONCLUSIONS: The results demonstrated that autophagy is positively correlated with the encystation of A. castellanii, and Ac-HSP20 regulates autophagy to maintain the homeostasis of A. castellanii by inhibiting the PI3K /AKT /mTOR signaling pathway, thus promoting the maturation and stability of encystation.

An Intrinsic Host Defense against HSV-1 Relies on the Activation of Xenophagy with the Active Clearance of Autophagic Receptors.

Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy.

The actin binding protein profilin 1 localizes inside mitochondria and is critical for their function.

The monomer-binding protein profilin 1 (PFN1) plays a crucial role in actin polymerization. However, mutations in PFN1 are also linked to hereditary amyotrophic lateral sclerosis, resulting in a broad range of cellular pathologies which cannot be explained by its primary function as a cytosolic actin assembly factor. This implies that there are important, undiscovered roles for PFN1 in cellular physiology. Here we screened knockout cells for novel phenotypes associated with PFN1 loss of function and discovered that mitophagy was significantly upregulated. Indeed, despite successful autophagosome formation, fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells accumulate depolarized, dysmorphic mitochondria with altered metabolic properties. Surprisingly, we also discovered that PFN1 is present inside mitochondria and provide evidence that mitochondrial defects associated with PFN1 loss are not caused by reduced actin polymerization in the cytosol. These findings suggest a previously unrecognized role for PFN1 in maintaining mitochondrial integrity and highlight new pathogenic mechanisms that can result from PFN1 dysregulation.

TMEM9 activates Rab9-dependent alternative autophagy through interaction with Beclin1.

Transmembrane protein 9 (TMEM9) is a transmembrane protein that regulates lysosomal acidification by interacting with the v-type ATPase complex. However, the role of TMEM9 in the lysosome-dependent autophagy machinery has yet to be identified. In this study, we demonstrate that the lysosomal protein TMEM9, which is involved in vesicle acidification, regulates Rab9-dependent alternative autophagy through its interaction with Beclin1. The cytosolic domain of TMEM9 interacts with Beclin1 via its Bcl-2-binding domain. This interaction between TMEM9 and Beclin1 dissociates Bcl-2, an autophagy-inhibiting partner, from Beclin1, thereby activating LC3-independent and Rab9-dependent alternative autophagy. Late endosomal and lysosomal TMEM9 apparently colocalizes with Rab9 but not with LC3. Furthermore, we show that multiple glycosylation of TMEM9, essential for lysosomal localization, is essential for its interaction with Beclin1 and the activation of Rab9-dependent alternative autophagy. These findings reveal that TMEM9 recruits and activates the Beclin1 complex at the site of Rab9-dependent autophagosome to induce alternative autophagy.

HaloTag as a substrate-based macroautophagy reporter.

Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation.

FUNDC1 alleviates doxorubicin-induced cardiotoxicity by restoring mitochondrial-endoplasmic reticulum contacts and blocked autophagic flux.

Rationale: Autophagy dysregulation is known to be a mechanism of doxorubicin (DOX)-induced cardiotoxicity (DIC). Mitochondrial-Endoplasmic Reticulum Contacts (MERCs) are where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a tethering protein of MERCs. We aim to investigate the effect of DOX on MERCs in cardiomyocytes and explore whether it is involved in the dysregulated autophagy in DIC. Methods: We employed confocal microscopy and transmission electron microscopy to assess MERCs structure. Autophagic flux was analyzed using the mCherry-EGFP-LC3B fluorescence assay and western blotting for LC3BII. Mitophagy was studied through the mCherry-EGFP-FIS1 fluorescence assay and colocalization analysis between LC3B and mitochondria. A total dose of 18 mg/kg of doxorubicin was administrated in mice to construct a DIC model in vivo. Additionally, we used adeno-associated virus (AAV) to cardiac-specifically overexpress FUNDC1. Cardiac function and remodeling were evaluated by echocardiography and Masson's trichrome staining, respectively. Results: DOX blocked autophagic flux by inhibiting autophagosome biogenesis, which could be attributed to the downregulation of FUNDC1 and disruption of MERCs structures. FUNDC1 overexpression restored the blocked autophagosome biogenesis by maintaining MERCs structure and facilitating ATG5-ATG12/ATG16L1 complex formation without altering mitophagy. Furthermore, FUNDC1 alleviated DOX-induced oxidative stress and cardiomyocytes deaths in an autophagy-dependent manner. Notably, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. Conclusions: In summary, our study identified that FUNDC1-meditated MERCs exerted a cardioprotective effect against DIC by restoring the blocked autophagosome biogenesis. Importantly, this research reveals a novel role of FUNDC1 in enhancing macroautophagy via restoring MERCs structure and autophagosome biogenesis in the DIC model, beyond its previously known regulatory role as an mitophagy receptor.

Cholesterol-rich lysosomes induced by respiratory syncytial virus promote viral replication by blocking autophagy flux.

Respiratory syncytial virus (RSV) hijacks cholesterol or autophagy pathways to facilitate optimal replication. However, our understanding of the associated molecular mechanisms remains limited. Here, we show that RSV infection blocks cholesterol transport from lysosomes to the endoplasmic reticulum by downregulating the activity of lysosomal acid lipase, activates the SREBP2-LDLR axis, and promotes uptake and accumulation of exogenous cholesterol in lysosomes. High cholesterol levels impair the VAP-A-binding activity of ORP1L and promote the recruitment of dynein-dynactin, PLEKHM1, or HOPS VPS39 to Rab7-RILP, thereby facilitating minus-end transport of autophagosomes and autolysosome formation. Acidification inhibition and dysfunction of cholesterol-rich lysosomes impair autophagy flux by inhibiting autolysosome degradation, which promotes the accumulation of RSV fusion protein. RSV-F storage is nearly abolished after cholesterol depletion or knockdown of LDLR. Most importantly, the knockout of LDLR effectively inhibits RSV infection in vivo. These findings elucidate the molecular mechanism of how RSV co-regulates lysosomal cholesterol reprogramming and autophagy and reveal LDLR as a novel target for anti-RSV drug development.

Tumor cell-released autophagosomes (TRAPs) induce PD-L1-decorated NETs that suppress T-cell function to promote breast cancer pulmonary metastasis.

BACKGROUND: Lung metastasis is the primary cause of breast cancer-related mortality. Neutrophil extracellular traps (NETs) are involved in the progression of breast cancer. However, the mechanism of NET formation is not fully understood. This study posits that tumor cell-released autophagosomes (TRAPs) play a crucial role in this process. METHODS: TRAPs were isolated from breast cancer cell lines to analyze their impact on NET formation in both human and mouse neutrophils. The study used both in vitro and in vivo models, including Toll-like receptor 4 (TLR4-/-) mice and engineered breast cancer cell lines. Immunofluorescence, ELISA, Western blotting, RNA sequencing, and flow cytometry were employed to dissect the signaling pathways leading to NET production and to explore their immunosuppressive effects, particularly focusing on the impact of NETs on T-cell function. The therapeutic potential of targeting TRAP-induced NETs and their immunosuppressive functions was evaluated using DNase I and αPD-L1 antibodies. Clinical relevance was assessed by correlating circulating levels of TRAPs and NETs with lung metastasis in patients with breast cancer. RESULTS: This study showed that TRAPs induced the formation of NETs in both human and mouse neutrophils by using the high mobility group box 1 and activating the TLR4-Myd88-ERK/p38 signaling axis. More importantly, PD-L1 carried by TRAP-induced NETs inhibited T-cell function in vitro and in vivo, thereby contributing to the formation of lung premetastatic niche (PMN) immunosuppression. In contrast, Becn1 KD-4T1 breast tumors with decreased circulating TRAPs in vivo reduced the formation of NETs, which in turn attenuated the immunosuppressive effects in PMN and resulted in a reduction of breast cancer pulmonary metastasis in murine models. Moreover, treatment with αPD-L1 in combination with DNase I that degraded NETs restored T-cell function and significantly reduced tumor metastasis. TRAP levels in the peripheral blood positively correlated with NET levels and lung metastasis in patients with breast cancer. CONCLUSIONS: Our results demonstrate a novel role of TRAPs in the formation of PD-L1-decorated NETs, which may provide a new strategy for early detection and treatment of pulmonary metastasis in patients with breast cancer.

Altered autophagic flux in GNE mutant cells of Indian origin: Potential drug target for GNE myopathy.

Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of ß-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.