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1.
J Cell Biol ; 221(6)2022 06 06.
Article in English | MEDLINE | ID: covidwho-1960887

ABSTRACT

ß-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3-nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.


Subject(s)
COVID-19 , Membrane Proteins , SARS-CoV-2 , Viral Replication Compartments , Autophagy/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Organelles/metabolism , Phosphatidylserines , SARS-CoV-2/physiology , Viral Nonstructural Proteins/genetics , Virus Replication
2.
Cells ; 11(15)2022 Jul 22.
Article in English | MEDLINE | ID: covidwho-1957235

ABSTRACT

Autophagy is a highly conserved lysosomal degradation pathway active at basal levels in all cells. However, under stress conditions, such as a lack of nutrients or trophic factors, it works as a survival mechanism that allows the generation of metabolic precursors for the proper functioning of the cells until the nutrients are available. Neurons, as post-mitotic cells, depend largely on autophagy to maintain cell homeostasis to get rid of damaged and/or old organelles and misfolded or aggregated proteins. Therefore, the dysfunction of this process contributes to the pathologies of many human diseases. Furthermore, autophagy is highly active during differentiation and development. In this review, we describe the current knowledge of the different pathways, molecular mechanisms, factors that induce it, and the regulation of mammalian autophagy. We also discuss its relevant role in development and disease. Finally, here we summarize several investigations demonstrating that autophagic abnormalities have been considered the underlying reasons for many human diseases, including liver disease, cardiovascular, cerebrovascular diseases, neurodegenerative diseases, neoplastic diseases, cancers, and, more recently, infectious diseases, such as SARS-CoV-2 caused COVID-19 disease.


Subject(s)
COVID-19 , Animals , Autophagy/physiology , Homeostasis , Humans , Lysosomes/metabolism , Mammals , SARS-CoV-2
3.
J Cell Biol ; 221(7)2022 Jul 04.
Article in English | MEDLINE | ID: covidwho-1956550

ABSTRACT

The process of membrane atg8ylation, defined herein as the conjugation of the ATG8 family of ubiquitin-like proteins to membrane lipids, is beginning to be appreciated in its broader manifestations, mechanisms, and functions. Classically, membrane atg8ylation with LC3B, one of six mammalian ATG8 family proteins, has been viewed as the hallmark of canonical autophagy, entailing the formation of characteristic double membranes in the cytoplasm. However, ATG8s are now well described as being conjugated to single membranes and, most recently, proteins. Here we propose that the atg8ylation is coopted by multiple downstream processes, one of which is canonical autophagy. We elaborate on these biological outputs, which impact metabolism, quality control, and immunity, emphasizing the context of inflammation and immunological effects. In conclusion, we propose that atg8ylation is a modification akin to ubiquitylation, and that it is utilized by different systems participating in membrane stress responses and membrane remodeling activities encompassing autophagy and beyond.


Subject(s)
Autophagy , Ubiquitins , Animals , Autophagy/physiology , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Mammals/metabolism , Microtubule-Associated Proteins/metabolism , Ubiquitination , Ubiquitins/genetics
4.
PLoS Pathog ; 18(7): e1010736, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1951573

ABSTRACT

Intracellular pathogens cause membrane distortion and damage as they enter host cells. Cells perceive these membrane alterations as danger signals and respond by activating autophagy. This response has primarily been studied during bacterial invasion, and only rarely in viral infections. Here, we investigate the cellular response to membrane damage during adenoviral entry. Adenoviruses and their vector derivatives, that are an important vaccine platform against SARS-CoV-2, enter the host cell by endocytosis followed by lysis of the endosomal membrane. We previously showed that cells mount a locally confined autophagy response at the site of endosomal membrane lysis. Here we describe the mechanism of autophagy induction: endosomal membrane damage activates the kinase TBK1 that accumulates in its phosphorylated form at the penetration site. Activation and recruitment of TBK1 require detection of membrane damage by galectin 8 but occur independently of classical autophagy receptors or functional autophagy. Instead, TBK1 itself promotes subsequent autophagy that adenoviruses need to take control of. Deletion of TBK1 reduces LC3 lipidation during adenovirus infection and restores the infectivity of an adenovirus mutant that is restricted by autophagy. By comparing adenovirus-induced membrane damage to sterile lysosomal damage, we implicate TBK1 in the response to a broader range of types of membrane damage. Our study thus highlights an important role for TBK1 in the cellular response to adenoviral endosome penetration and places TBK1 early in the pathway leading to autophagy in response to membrane damage.


Subject(s)
Adenoviridae Infections , Autophagy , Endosomes , Protein Serine-Threonine Kinases , Adenoviridae/metabolism , Adenoviridae Infections/metabolism , Endosomes/metabolism , Galectins/metabolism , Humans , Protein Serine-Threonine Kinases/genetics
5.
Int J Biol Sci ; 18(12): 4690-4703, 2022.
Article in English | MEDLINE | ID: covidwho-1954689

ABSTRACT

There is increasing amount of evidence indicating the close interplays between the replication cycle of SARS-CoV-2 and the autophagy-lysosome pathway in the host cells. While autophagy machinery is known to either assist or inhibit the viral replication process, the reciprocal effects of the SARS-CoV-2 on the autophagy-lysosome pathway have also been increasingly appreciated. More importantly, despite the disappointing results from the clinical trials of chloroquine and hydroxychloroquine in treatment of COVID-19, there is still ongoing effort in discovering new therapeutics targeting the autophagy-lysosome pathway. In this review, we provide an update-to-date summary of the interplays between the autophagy-lysosome pathway in the host cells and the pathogen SARS-CoV-2 at the molecular level, to highlight the prognostic value of autophagy markers in COVID-19 patients and to discuss the potential of developing novel therapeutic strategies for COVID-19 by targeting the autophagy-lysosome pathway. Thus, understanding the nature of such interactions between SARS-CoV-2 and the autophagy-lysosome pathway in the host cells is expected to provide novel strategies in battling against this global pandemic.


Subject(s)
COVID-19 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Autophagy , COVID-19/drug therapy , Humans , Lysosomes , Pandemics , SARS-CoV-2
6.
Front Cell Infect Microbiol ; 12: 856711, 2022.
Article in English | MEDLINE | ID: covidwho-1924078

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) could cause lethal diarrhea and dehydration in suckling piglets, which can adversely affect the development of the global swine industry. The lack of effective therapeutical and prophylactic treatment especially for PEDV variant strains underlines the importance of effective antiviral strategies, such as identification of novel antiviral agents. In the present study, the antiviral activity of cinchonine against PEDV was investigated in Vero CCL81 and LLC-PK1 cells at a non-cytotoxic concentration determined by Cell Counting Kit-8 assay in vitro. We found that cinchonine exhibited a significant suppression effect against PEDV infection and its inhibitory action was primarily focused on the early stage of PEDV replication. Moreover, we also observed that cinchonine could significantly induce autophagy by detecting the conversion of LC3-I to LC3-II by using western blot analysis. Cinchonine treatment could inhibit PEDV replication in a dose-dependent manner in Vero CCL81 cells, while this phenomenon disappeared when autophagy was attenuated by pre-treatment with autophagy inhibitor 3MA. Consequently, this study indicated that cinchonine can inhibit PEDV replication via inducing cellular autophagy and thus from the basis for successful antiviral strategies which potentially suggest the possibility of exploiting cinchonine as a novel antiviral agent.


Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Autophagy , Chlorocebus aethiops , Cinchona Alkaloids , Coronavirus Infections/drug therapy , Coronavirus Infections/veterinary , Porcine epidemic diarrhea virus/physiology , Swine , Swine Diseases/drug therapy , Vero Cells , Virus Replication
8.
Front Immunol ; 13: 903498, 2022.
Article in English | MEDLINE | ID: covidwho-1903026

ABSTRACT

Autophagy is a homeostatic process responsible for the self-digestion of intracellular components and antimicrobial defense by inducing the degradation of pathogens into autophagolysosomes. Recent findings suggest an involvement of this process in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the role of autophagy in the immunological mechanisms of coronavirus disease 2019 (COVID-19) pathogenesis remains largely unexplored. This study reveals the presence of autophagy defects in peripheral immune cells from COVID-19 patients. The impairment of the autophagy process resulted in a higher percentage of lymphocytes undergoing apoptosis in COVID-19 patients. Moreover, the inverse correlation between autophagy markers levels and peripheral lymphocyte counts in COVID-19 patients confirms how a defect in autophagy might contribute to lymphopenia, causing a reduction in the activation of viral defense. These results provided intriguing data that could help in understanding the cellular underlying mechanisms in COVID-19 infection, especially in severe forms.


Subject(s)
COVID-19 , Lymphopenia , Autophagy , Humans , Leukocytes, Mononuclear , SARS-CoV-2
9.
Int J Mol Sci ; 23(11)2022 May 24.
Article in English | MEDLINE | ID: covidwho-1892892

ABSTRACT

A major cause of cancer cell resistance to chemotherapeutics is the blocking of apoptosis and induction of autophagy in the context of cell adaptation and survival. Therefore, new compounds are being sought, also among drugs that are commonly used in other therapies. Due to the involvement of histamine in the regulation of processes occurring during the development of many types of cancer, antihistamines are now receiving special attention. Our study concerned the identification of new mechanisms of action of azelastine hydrochloride, used in antiallergic treatment. The study was performed on HeLa cells treated with different concentrations of azelastine (15-90 µM). Cell cycle, level of autophagy (LC3 protein activity) and apoptosis (annexin V assay), activity of caspase 3/7, anti-apoptotic protein of Bcl-2 family, ROS concentration, measurement of mitochondrial membrane potential (Δψm), and level of phosphorylated H2A.X in response to DSB were evaluated by cytometric method. Cellular changes were also demonstrated at the level of transmission electron microscopy and optical and fluorescence microscopy. Lysosomal enzyme activities-cathepsin D and L and cell viability (MTT assay) were assessed spectrophotometrically. Results: Azelastine in concentrations of 15-25 µM induced degradation processes, vacuolization, increase in cathepsin D and L activity, and LC3 protein activation. By increasing ROS, it also caused DNA damage and blocked cells in the S phase of the cell cycle. At the concentrations of 45-90 µM, azelastine clearly promoted apoptosis by activation of caspase 3/7 and inactivation of Bcl-2 protein. Fragmentation of cell nucleus was confirmed by DAPI staining. Changes were also found in the endoplasmic reticulum and mitochondria, whose damage was confirmed by staining with rhodamine 123 and in the MTT test. Azelastine decreased the mitotic index and induced mitotic catastrophe. Studies demonstrated the multidirectional effects of azelastine on HeLa cells, including anti-proliferative, cytotoxic, autophagic, and apoptotic properties, which were the predominant mechanism of death. The revealed novel properties of azelastine may be practically used in anti-cancer therapy in the future.


Subject(s)
Cathepsin D , Uterine Cervical Neoplasms , Apoptosis , Autophagy , Caspase 3/metabolism , Cell Line, Tumor , Female , HeLa Cells , Humans , Phthalazines , Proto-Oncogene Proteins c-bcl-2 , Reactive Oxygen Species/metabolism , Uterine Cervical Neoplasms/drug therapy
10.
Signal Transduct Target Ther ; 7(1): 186, 2022 06 13.
Article in English | MEDLINE | ID: covidwho-1890154

ABSTRACT

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has dramatically influenced various aspects of the world. It is urgent to thoroughly study pathology and underlying mechanisms for developing effective strategies to prevent and treat this threatening disease. It is universally acknowledged that cell death and cell autophagy are essential and crucial to maintaining host homeostasis and participating in disease pathogenesis. At present, more than twenty different types of cell death have been discovered, some parts of which have been fully understood, whereas some of which need more investigation. Increasing studies have indicated that cell death and cell autophagy caused by coronavirus might play an important role in virus infection and pathogenicity. However, the knowledge of the interactions and related mechanisms of SARS-CoV-2 between cell death and cell autophagy lacks systematic elucidation. Therefore, in this review, we comprehensively delineate how SARS-CoV-2 manipulates diverse cell death (including apoptosis, necroptosis, pyroptosis, ferroptosis, and NETosis) and cell autophagy for itself benefits, which is simultaneously involved in the occurrence and progression of COVID-19, aiming to provide a reasonable basis for the existing interventions and further development of novel therapies.


Subject(s)
COVID-19 , Apoptosis , Autophagy/genetics , Humans , Pandemics , SARS-CoV-2
11.
Molecules ; 27(11)2022 May 26.
Article in English | MEDLINE | ID: covidwho-1869715

ABSTRACT

Impaired autophagy, responsible for increased inflammation, constitutes a risk factor for the more severe COVID-19 outcomes. Spermidine (SPD) is a known autophagy modulator and supplementation for COVID-19 risk groups (including the elderly) is recommended. However, information on the modulatory effects of eugenol (EUG) is scarce. Therefore, the effects of SPD and EUG, both singularly and in combination, on autophagy were investigated using different cell lines (HBEpiC, SHSY5Y, HUVEC, Caco-2, L929 and U937). SPD (0.3 mM), EUG (0.2 mM) and 0.3 mM SPD + 0.2 mM EUG, significantly increased autophagy using the hallmark measure of LC3-II protein accumulation in the cell lines without cytotoxic effects. Using Caco-2 cells as a model, several crucial autophagy proteins were upregulated at all stages of autophagic flux in response to the treatments. This effect was verified by the activation/differentiation and migration of U937 monocytes in a three-dimensional reconstituted intestinal model (Caco-2, L929 and U937 cells). Comparable benefits of SPD, EUG and SPD + EUG in inducing autophagy were shown by the protection of Caco-2 and L929 cells against lipopolysaccharide-induced inflammation. SPD + EUG is an innovative dual therapy capable of stimulating autophagy and reducing inflammation in vitro and could show promise for COVID-19 risk groups.


Subject(s)
COVID-19 , Syzygium , Aged , Autophagy , COVID-19/drug therapy , Caco-2 Cells , Eugenol/pharmacology , Humans , Inflammation , Monocytes , Plant Oils , Spermidine/pharmacology , Triticum
12.
Aging (Albany NY) ; 14(10): 4195-4210, 2022 05 23.
Article in English | MEDLINE | ID: covidwho-1863474

ABSTRACT

Previous studies have shown that the polyamine spermidine increased the maximum life span in C. elegans and the median life span in mice. Since spermidine increases autophagy, we asked if treatment with chloroquine, an inhibitor of autophagy, would shorten the lifespan of mice. Recently, chloroquine has intensively been discussed as a treatment option for COVID-19 patients. To rule out unfavorable long-term effects on longevity, we examined the effect of chronic treatment with chloroquine given in the drinking water on the lifespan and organ pathology of male middle-aged NMRI mice. We report that, surprisingly, daily treatment with chloroquine extended the median life span by 11.4% and the maximum life span of the middle-aged male NMRI mice by 11.8%. Subsequent experiments show that the chloroquine-induced lifespan elevation is associated with dose-dependent increase in LC3B-II, a marker of autophagosomes, in the liver and heart that was confirmed by transmission electron microscopy. Quite intriguingly, chloroquine treatment was also associated with a decrease in glycogenolysis in the liver suggesting a compensatory mechanism to provide energy to the cell. Accumulation of autophagosomes was paralleled by an inhibition of proteasome-dependent proteolysis in the liver and the heart as well as with decreased serum levels of insulin growth factor binding protein-3 (IGFBP3), a protein associated with longevity. We propose that inhibition of proteasome activity in conjunction with an increased number of autophagosomes and decreased levels of IGFBP3 might play a central role in lifespan extension by chloroquine in male NMRI mice.


Subject(s)
Autophagy , Chloroquine , Glycogenolysis , Longevity , Proteasome Endopeptidase Complex , Proteasome Inhibitors , Animals , Autophagy/drug effects , COVID-19/drug therapy , Chloroquine/pharmacology , Glycogen , Glycogenolysis/drug effects , Longevity/drug effects , Male , Mice , Proteasome Inhibitors/pharmacology , Spermidine/pharmacology
13.
Mol Immunol ; 140: 175-185, 2021 12.
Article in English | MEDLINE | ID: covidwho-1851816

ABSTRACT

SFN, a dietary phytochemical, is a significant member of isothiocyanates present in cruciferous vegetables at high levels in broccoli. It is a well-known activator of the Nrf2/ARE antioxidant pathway. Long since, the therapeutic effects of SFN have been widely studied in several different diseases. Other than the antioxidant effect, SFN also exhibits an anti-inflammatory effect through suppression of various mechanisms, including inflammasome activation. Considerably, SFN has been demonstrated to inhibit multiple inflammasomes, including NLRP3 inflammasome. NLRP3 inflammasome induces secretion of pro-inflammatory cytokines and promotes inflammatory cell death. The release of pro-inflammatory cytokines enhances the inflammatory response, in turn leading to tissue damage. These self-propelling inflammatory responses would need modulation with exogenous therapeutic agents to suppress them. SFN is a promising candidate molecule for the mitigation of NLRP3 inflammasome activation, which has been related to the pathogenesis of numerous disorders. In this review, we have provided fundamental knowledge about Sulforaphane, elaborated its characteristics, and evidentially focused on its mechanisms of action with regard to its anti-inflammatory, anti-oxidative, and neuroprotective features. Thereafter, we have summarized both in vitro and in vivo studies regarding SFN effect on NLRP3 inflammasome activation.


Subject(s)
Inflammasomes/metabolism , Isothiocyanates/pharmacology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Sulfoxides/pharmacology , Animals , Autophagy/drug effects , Epigenesis, Genetic/drug effects , Humans , Neurogenesis/drug effects
14.
PLoS One ; 17(4): e0266337, 2022.
Article in English | MEDLINE | ID: covidwho-1846925

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has resulted in an urgent need for identifying potential therapeutic drugs. In the first half of 2020 tropic antimalarial drugs, such as chloroquine (CQ) or hydroxochloroquine (HCQ) were the focus of tremendous public attention. In the initial periods of the pandemic, many scientific results pointed out that CQ/HCQ could be very effective for patients with severe COVID. While CQ and HCQ have successfully been used against several diseases (such as malaria, autoimmune disease and rheumatic illnesses); long term use of these agents are associated with serious adverse effects (i.e. inducing acute kidney injury, among many others) due to their role in blocking autophagy-dependent self-degradation. Recent experimental and clinical trial data also confirmed that there is no sufficient evidence about the efficient usage of CQ/HCQ against COVID-19. By using systems biology techniques, here we show that the cellular effect of CQ/HCQ on autophagy during endoplasmic reticulum (ER) stress or following SARS-CoV-2 infection results in upregulation of ER stress. By presenting a simple mathematical model, we claim that although CQ/HCQ might be able to ameliorate virus infection, the permanent inhibition of autophagy by CQ/HCQ has serious negative effects on the cell. Since CQ/HCQ promotes apoptotic cell death, here we confirm that addition of CQ/HCQ cannot be really effective even in severe cases. Only a transient treatment seemed to be able to avoid apoptotic cell death, but this type of therapy could not limit virus replication in the infected host. The presented theoretical analysis clearly points out the utility and applicability of systems biology modelling to test the cellular effect of a drug targeting key major processes, such as autophagy and apoptosis. Applying these approaches could decrease the cost of pre-clinical studies and facilitate the selection of promising clinical trials in a timely fashion.


Subject(s)
COVID-19 , Autophagy , COVID-19/drug therapy , Chloroquine/pharmacology , Chloroquine/therapeutic use , Humans , Hydroxychloroquine/adverse effects , Pandemics , SARS-CoV-2 , Systems Biology
15.
Ann N Y Acad Sci ; 1510(1): 79-99, 2022 04.
Article in English | MEDLINE | ID: covidwho-1822055

ABSTRACT

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.


Subject(s)
Proteasome Endopeptidase Complex , Ubiquitin , Autophagy/physiology , Humans , Organelles , Proteasome Endopeptidase Complex/metabolism , Proteins/metabolism , Proteolysis , Ubiquitin/metabolism
16.
Oxid Med Cell Longev ; 2022: 9366494, 2022.
Article in English | MEDLINE | ID: covidwho-1807713

ABSTRACT

Trehalose, a natural disaccharide, is synthesized by many organisms when cells are exposed to stressful stimuli. On the basis of its ability to modulate autophagy, trehalose has been considered an innovative drug for ameliorating many diseases, but its molecular mechanism is not well described. Previous findings demonstrated that trehalose plays a photoprotective role against ultraviolet (UV) B-induced damage through autophagy induction in keratinocytes. In this study, coimmunoprecipitation, label-free quantitative proteomic and parallel reaction monitoring, and western blot analysis demonstrated that trehalose promotes the interaction between tissue inhibitor of metalloproteinase (TIMP) 3 and Beclin1. Western blot and immunofluorescence staining analysis suggested that trehalose increases ATG9A localization in lysosomes and decreases its localization in the endoplasmic reticulum. Furthermore, in the presence or absence of UVB radiation, we evaluated the influence of TIMP3 and ATG9A small interfering RNA (siRNA) on the effect of trehalose on autophagy, cell death, migration, or interleukin-8 expression in keratinocytes, including HaCaT, A431, and human epidermal keratinocytes. The results revealed that in HaCaT cells, TIMP3 and ATG9A siRNA resulted in attenuation of trehalose-induced autophagy and inhibited cell death. In A431 cells, TIMP3 and ATG9A siRNA led to attenuation of trehalose-induced autophagy and cell death and inhibited migration. In human epidermal keratinocytes, trehalose-induced autophagy and inhibition of the interleukin-8 expression were blocked by ATG9A but not TIMP3 siRNA. In addition, the results of quantitative real-time PCR and immunohistochemistry analysis demonstrated the abnormal expression of TIMP3 and ATG9A in actinic keratosis and cutaneous squamous cell carcinoma skin tissues. These findings suggest the protective effects of trehalose in normal keratinocytes and its inhibitory effects on cancerous keratinocytes, possibly mediated by activation of autophagy and regulation of TIMP3 and ATG9A, providing the mechanistic basis for the potential use of trehalose in the prevention or treatment of UVB-induced skin diseases.


Subject(s)
Carcinoma, Squamous Cell , Skin Neoplasms , Autophagy , Autophagy-Related Proteins/metabolism , Carcinoma, Squamous Cell/pathology , Humans , Interleukin-8/metabolism , Keratinocytes/metabolism , Membrane Proteins/metabolism , Proteomics , RNA, Small Interfering/metabolism , Skin Neoplasms/metabolism , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/metabolism , Tissue Inhibitor of Metalloproteinase-3/pharmacology , Trehalose/pharmacology , Ultraviolet Rays/adverse effects , Vesicular Transport Proteins/metabolism
17.
J Cell Biol ; 221(6)2022 06 06.
Article in English | MEDLINE | ID: covidwho-1806200

ABSTRACT

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.


Subject(s)
Autophagy , Extracellular Vesicles , Lysosomes , Proteostasis , Autophagosomes/metabolism , Extracellular Vesicles/metabolism , Humans , Lysosomes/metabolism , SARS-CoV-2 , Sequestosome-1 Protein , Viroporin Proteins , rab27 GTP-Binding Proteins
18.
Cells ; 11(8)2022 04 13.
Article in English | MEDLINE | ID: covidwho-1798904

ABSTRACT

Autophagy plays a key role in eliminating and recycling cellular components in response to stress, including starvation. Dysregulation of autophagy is observed in various diseases, including neurodegenerative diseases, cancer, and diabetes. Autophagy is tightly regulated by autophagy-related (ATG) proteins. Autophagy-related 4 (ATG4) is the sole cysteine protease, and four homologs (ATG4A-D) have been identified in mammals. These proteins have two domains: catalytic and short fingers. ATG4 facilitates autophagy by promoting autophagosome maturation through reversible lipidation and delipidation of seven autophagy-related 8 (ATG8) homologs, including microtubule-associated protein 1-light chain 3 (LC3) and GABA type A receptor-associated protein (GABARAP). Each ATG4 homolog shows a preference for a specific ATG8 homolog. Post-translational modifications of ATG4, including phosphorylation/dephosphorylation, O-GlcNAcylation, oxidation, S-nitrosylation, ubiquitination, and proteolytic cleavage, regulate its activity and ATG8 processing, thus modulating its autophagic activity. We reviewed recent advances in our understanding of the effect of post-translational modification on the regulation, activity, and function of ATG4, the main protease that controls autophagy.


Subject(s)
Autophagy , Microtubule-Associated Proteins , Animals , Autophagy/physiology , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/metabolism , Mammals/metabolism , Microtubule-Associated Proteins/metabolism , Peptide Hydrolases/metabolism , Protein Processing, Post-Translational
19.
Front Cell Infect Microbiol ; 12: 845368, 2022.
Article in English | MEDLINE | ID: covidwho-1793038

ABSTRACT

Coronaviruses are the etiologic agents of several diseases. Coronaviruses of critical medical importance are characterized by highly inflammatory pathophysiology, involving severe pulmonary impairment and infection of multiple cell types within the body. Here, we discuss the interplay between coronaviruses and autophagy regarding virus life cycle, cell resistance, and inflammation, highlighting distinct mechanisms by which autophagy restrains inflammatory responses, especially those involved in coronavirus pathogenesis. We also address different autophagy modulators available and the rationale for drug repurposing as an attractive adjunctive therapy. We focused on pharmaceuticals being tested in clinical trials with distinct mechanisms but with autophagy as a common target. These autophagy modulators act in cell resistance to virus infection and immunomodulation, providing a double-strike to prevent or treat severe disease development and death from coronaviruses diseases.


Subject(s)
Coronavirus Infections , Coronavirus , Autophagy/physiology , Coronavirus/physiology , Coronavirus Infections/pathology , Humans , Inflammation , Viral Load , Virus Replication/physiology
20.
Hum Cell ; 35(3): 871-884, 2022 May.
Article in English | MEDLINE | ID: covidwho-1762779

ABSTRACT

Mechanical ventilation may cause ventilator-induced lung injury (VILI) in patients requiring ventilator support. Inhibition of autophagy is an important approach to ameliorate VILI as it always enhances lung injury after exposure to various stress agents. This study aimed to further reveal the potential mechanisms underlying the effects of geranylgeranyl diphosphate synthase large subunit 1 (GGPPS1) knockout and autophagy in VILI using C57BL/6 mice with lung-specific GGPPS1 knockout that were subjected to mechanical ventilation. The results demonstrate that GGPPS1 knockout mice exhibit significantly attenuated VILI based on the histologic score, the lung wet-to-dry ratio, total protein levels, neutrophils in bronchoalveolar lavage fluid, and reduced levels of inflammatory cytokines. Importantly, the expression levels of autophagy markers were obviously decreased in GGPPS1 knockout mice compared with wild-type mice. The inhibitory effects of GGPPS1 knockout on autophagy were further confirmed by measuring the ultrastructural change of lung tissues under transmission electron microscopy. In addition, knockdown of GGPPS1 in RAW264.7 cells reduced cyclic stretch-induced inflammation and autophagy. The benefits of GGPPS1 knockout for VILI can be partially eliminated through treatment with rapamycin. Further analysis revealed that Rab37 was significantly downregulated in GGPPS1 knockout mice after mechanical ventilation, while it was highly expressed in the control group. Simultaneously, Rab37 overexpression significantly enhances autophagy in cells that are treated with cyclin stretch, including GGPPS1 knockout cells. Collectively, our results indicate that GGPPS1 knockout results in reduced expression of Rab37 proteins, further restraining autophagy and VILI.


Subject(s)
Ventilator-Induced Lung Injury , Animals , Autophagy/genetics , Dimethylallyltranstransferase , Farnesyltranstransferase , Geranyltranstransferase , Humans , Lung/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Ventilator-Induced Lung Injury/genetics , Ventilator-Induced Lung Injury/metabolism , Ventilator-Induced Lung Injury/pathology , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism
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