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International Journal of Molecular... Jun 2024In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing...
The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.
In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.
Topics: Humans; Viral Nonstructural Proteins; Virus Replication; Autophagosomes; HeLa Cells; Phlebovirus; Autophagy; Tyrosine; Tryptophan; TOR Serine-Threonine Kinases; Mutation; Amino Acid Substitution; Severe Fever with Thrombocytopenia Syndrome; Lysosomes; Nucleoproteins
PubMed: 38928101
DOI: 10.3390/ijms25126394 -
Journal For Immunotherapy of Cancer Jun 2024Lung metastasis is the primary cause of breast cancer-related mortality. Neutrophil extracellular traps (NETs) are involved in the progression of breast cancer. However,...
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, 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.
Topics: Animals; Humans; Mice; Female; Breast Neoplasms; Lung Neoplasms; Extracellular Traps; B7-H1 Antigen; Autophagosomes; T-Lymphocytes; Cell Line, Tumor
PubMed: 38926151
DOI: 10.1136/jitc-2024-009082 -
Journal of Fungi (Basel, Switzerland) Jun 2024is a native woody oil plant in southern China and is infected with anthracnose wherever it is grown. We previously identified as the major causal agent of anthracnose...
is a native woody oil plant in southern China and is infected with anthracnose wherever it is grown. We previously identified as the major causal agent of anthracnose on and found that CfAtg8 regulates the pathogenicity and development of . Here, we revealed that CfAtg4 interacts with CfAtg8, contributing to the formation of autophagosomes. The CfAtg8 allele, which only contains 1-160 amino acids of the CfAtg8, partially recovered the autophagosome numbers and autophagy defects of the Δ mutant. Consequently, these recoveries resulted in the restoration of the defects of the Δ mutant in growth and responses to different external stresses, albeit to an extent. Importantly, we illustrated the critical roles of CfAtg8 in appressoria formation, and pathogenicity. Collectively, our findings provide new insights into the importance of the interaction between CfAtg8 and CfAtg4 in the growth, autophagy and pathogenicity of the phytopathogenic fungi.
PubMed: 38921417
DOI: 10.3390/jof10060431 -
Microbiology Spectrum Jun 2024() as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug...
UNLABELLED
() as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategies for mycobacterial infections. In this study, we identified amiodarone as a potential HDT candidate that inhibited both intracellular and in primary human macrophages without directly impairing bacterial growth, thereby confirming that amiodarone acts in a host-mediated manner. Moreover, amiodarone induced the formation of (auto)phagosomes and enhanced autophagic targeting of mycobacteria in macrophages. The induction of autophagy by amiodarone is likely due to enhanced transcriptional regulation, as the nuclear intensity of the transcription factor EB, the master regulator of autophagy and lysosomal biogenesis, was strongly increased. Furthermore, blocking lysosomal degradation with bafilomycin impaired the host-beneficial effect of amiodarone. Finally, amiodarone induced autophagy and reduced bacterial burden in a zebrafish embryo model of tuberculosis, thereby confirming the HDT activity of amiodarone . In conclusion, we have identified amiodarone as an autophagy-inducing antimycobacterial HDT that improves host control of mycobacterial infections.
IMPORTANCE
Due to the global rise in antibiotic resistance, there is a strong need for alternative treatment strategies against intracellular bacterial infections, including () and non-tuberculous mycobacteria. Stimulating host defense mechanisms by host-directed therapy (HDT) is a promising approach for treating mycobacterial infections. This study identified amiodarone, an antiarrhythmic agent, as a potential HDT candidate that inhibits the survival of and in primary human macrophages. The antimycobacterial effect of amiodarone was confirmed in an tuberculosis model based on infection of zebrafish embryos. Furthermore, amiodarone induced autophagy and inhibition of the autophagic flux effectively impaired the host-protective effect of amiodarone, supporting that activation of the host (auto)phagolysosomal pathway is essential for the mechanism of action of amiodarone. In conclusion, we have identified amiodarone as an autophagy-inducing HDT that improves host control of a wide range of mycobacteria.
PubMed: 38916320
DOI: 10.1128/spectrum.00167-24 -
Frontiers in Microbiology 2024Herpes Simplex Virus type 1 (HSV-1) 1 is a neurotropic virus that has been associated with neurodegenerative disorders. The dysregulation of autophagy by HSV-1 has been...
Herpes Simplex Virus type 1 (HSV-1) 1 is a neurotropic virus that has been associated with neurodegenerative disorders. The dysregulation of autophagy by HSV-1 has been proposed as a potential cause of neurodegeneration. While studies have extensively tackled the interaction between autophagy and HSV-1 in neurons, research in glial cells is currently limited. Our studies demonstrate that HSV-1 inhibits, but not completely blocks, the formation of autophagosomes in human oligodendroglioma- and astrocytoma- derived cell lines. These findings have been confirmed in murine oligodendrocyte precursor cells (OPCs). Finally, this study investigates the impact of autophagy on HSV-1 infection in glial cells. While the lack of basal autophagy in LC3B knockout glial cells does not have a significant effect on viral infection, cells without the autophagy-related protein ATG5 exhibit reduced viral production. The absence of ATG5 leads to a decrease in the transcription and replication of viral genes, as well as a delay in the initial stages of the formation of HSV-1 replication compartments. These findings indicate that while autophagy may not play a significant role in antiviral defense in glial cells, HSV-1 may be inhibiting autophagy to exploit non-canonical functions of certain components of the autophagic machinery, such as ATG5, to benefit its lifecycle.
PubMed: 38915300
DOI: 10.3389/fmicb.2024.1411655 -
Hepatobiliary Surgery and Nutrition Jun 2024Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. Emerging evidence suggests that autophagy plays a critical role in HCC...
BACKGROUND
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. Emerging evidence suggests that autophagy plays a critical role in HCC tumorigenesis, metastasis, and prognosis. Choline is an essential nutrient related to prolonged survival and reduced risk of HCC. However, it remains unclear whether this phenomenon is mediated by autophagy.
METHODS
Two HCC cell lines (HUH-7 and Hep3B) were used in the present study. Cell growth was evaluated by cell counting kit 8 (CCK-8), colony formation, and mouse xenografts assays. Cell motility was calculated by wound healing and transwell assays. Autophagosomes were measured by transmission electron microscope (TEM), and autophagy flux was detected by mRFP-GFP-labeled LC3 protein. The mRNA level of genes was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels were detected by Western blotting (WB).
RESULTS
We found that choline inhibited the proliferation, migration, and invasion of HCC cells by downregulating autophagy and . Upregulated expression of the solute carrier family 5 member 7 (SLC5A7), a specific choline transporter, correlated with better HCC prognosis. We further discovered that choline could promote SLC5A7 expression, upregulate cytoplasm p53 expression to impair the AMPK/mTOR pathway, and attenuate autophagy. Finally, we found that choline acted synergistically with sorafenib to attenuate HCC development and .
CONCLUSIONS
Our findings provide novel insights into choline-mediated autophagy in HCC, providing the foothold for its future application in HCC treatment.
PubMed: 38911213
DOI: 10.21037/hbsn-22-476 -
Ferroptosis inhibitor improves outcome after early and delayed treatment in mild spinal cord injury.Acta Neuropathologica Jun 2024We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and...
We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and functional loss in the acute and chronic periods after spinal cord injury (SCI) in young, adult, female mice. Phagocytosis of red blood cells at sites of hemorrhage is the main source of iron derived from hemoglobin after SCI. Expression of hemeoxygenase-1 that induces release of iron from heme, is increased in spinal cord macrophages 7 days after injury. While iron is stored safely in ferritin in the injured spinal cord, it can, however, be released by NCOA4-mediated shuttling of ferritin to autophagosomes for degradation (ferritinophagy). This leads to the release of redox active iron that can cause free radical damage. Expression of NCOA4 is increased after SCI, mainly in macrophages. Increase in the ratio of redox active ferrous (Fe) to ferric iron (Fe) is also detected after SCI by capillary electrophoresis inductively coupled mass spectrometry. These changes are accompanied by other hallmarks of ferroptosis, i.e., deficiency in various elements of the antioxidant glutathione (GSH) pathway. We also detect increases in enzymes that repair membrane lipids (ACSL4 and LPCAT3) and thus promote on-going ferroptosis. These changes are associated with increased levels of 4-hydroxynonenal (4-HNE), a toxic lipid peroxidation product. Mice with mild SCI (30 kdyne force) treated with the ferroptosis inhibitor (UAMC-3203-HCL) either early or delayed times after injury showed improvement in locomotor recovery and secondary damage. Cerebrospinal fluid and serum samples from human SCI cases show evidence of increased iron storage (ferritin), and other iron related molecules, and reduction in GSH. Collectively, these data suggest that ferroptosis contributes to secondary damage after SCI and highlights the possible use of ferroptosis inhibitors to treat SCI.
Topics: Ferroptosis; Animals; Spinal Cord Injuries; Mice; Female; Mice, Inbred C57BL; Iron; Treatment Delay
PubMed: 38907771
DOI: 10.1007/s00401-024-02758-2 -
Life Science Alliance Sep 2024Apolipoprotein E4, the most important genetic risk factor for Alzheimer's disease, is shown to internalize into neurons and intersect with amyloid-β in...
Apolipoprotein E4, the most important genetic risk factor for Alzheimer's disease, is shown to internalize into neurons and intersect with amyloid-β in endosomes-autophagosomes of neurites and modulate intraneuronal amyloid-β-42.
PubMed: 38906679
DOI: 10.26508/lsa.202402875 -
International Journal of Biological... 2024Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is...
Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.
Topics: Pulmonary Artery; Animals; Cell Proliferation; Humans; Zinc; Mice; Rats; Myocytes, Smooth Muscle; Metallothionein 3; Male; Autophagosomes; Autophagy-Related Protein 5; Rats, Sprague-Dawley; Transcription Factors; Autophagy; Hypertension, Pulmonary; Mice, Inbred C57BL; DNA-Binding Proteins; Transcription Factor MTF-1; Metallothionein
PubMed: 38904023
DOI: 10.7150/ijbs.92992 -
Nature Communications Jun 2024Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require...
Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.
Topics: Arabidopsis; Autophagosomes; Endosomal Sorting Complexes Required for Transport; Arabidopsis Proteins; Calcium-Binding Proteins; Autophagy; Phosphatidylinositol Phosphates; Autophagy-Related Protein 8 Family; Vacuoles; Phase Separation
PubMed: 38898014
DOI: 10.1038/s41467-024-49485-6