The potential importance of autophagy genes expression profile dysregulation and ATG polymorphisms in COVID-19 pathogenesis.

Autophagy is one of the important mechanisms in cell maintenance, which is considered associated with different pathological conditions such as viral infections. In this current study, the expression level and polymorphisms in some of the most important genes in the autophagy flux in COVID-19 patients were evaluated. This cross-sectional study was conducted among 50 confirmed COVID-19 patients and 20 healthy controls. The COVID-19 patients were divided into a severe group and a mild group according to their clinical features. The expression levels of ATG5, ATG16L1, LC3, and BECN1 were evaluated by the 2-∆∆CT method and beta-actin as the internal control. The polymorphisms of the ATG5 (rs506027, rs510432) and ATG16L1 (rs2241880 or T300A) were evaluated by the Sanger sequencing following the conventional PCR. The mean age of the included patients was 58.3 ± 17.9 and 22 (44%) were female. The expression levels of the LC3 were downregulated, while BECN1 and ATG16L1 genes represent an upregulation in COVID-19 patients. The polymorphism analysis revealed the ATG16L1 rs2241880 and AGT5 rs506027 polymorphism frequencies are statistically significantly different between COVID-19 and Healthy controls. The autophagy alteration represents an association with COVID-19 pathogenesis and severity. The current study is consistent with the alteration of autophagy elements in COVID-19 patients by mRNA expression-level evaluation. Furthermore, ATG16L1 rs2241880 and AGT5 rs506027 polymorphisms seem to be important in COVID-19 and are highly suggested for further investigations.

An approach combining bioinformatics and machine learning to identify eight autophagy-related biomarkers and construct molecular mechanisms underlying COVID-19 and major depressive disorders.

OBJECTIVE: A lack of objective biomarkers is preventing the screening and diagnosis of COVID-19 combined with major depression disorder (COVID-19-MDD). The purpose of this study was to identify diagnostic biomarkers and gene regulatory mechanisms associated with autophagy; a crucial process significantly involved in the pathogenesis of COVID-19-MDD. MATERIALS AND METHODS: In this study, differentially expressed genes (DEGs) were screened using GSE98793 from the GEO2R analysis (GEO) database, and intersected with the COVID-19-related gene (CRGs) and autophagy-related genes (ARGs) to obtain common genes involved in. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of these common genes were performed. Subsequently, the transcription factor (TF)-gene regulatory network and comorbidity network were constructed. In addition, 10 drug candidates were screened using the DSigDB database. To identify diagnostic markers, we used LASSO regression. RESULTS: In total, 13 common genes were screened, which were primarily enriched in lysosomes, endoplasmic reticulum membranes, and other endomembrane systems also associated with autophagy. Additionally, these genes were involved in neurological cell signaling and have a functional role in pathways related to vascular endothelial growth factor, tyrosine kinase, autophagy, inflammation, immunity, and carcinogenesis. Tumors and psychiatric disorders were the most highly linked diseases to COVID-19. Finally, ten drug candidates and eight diagnostic markers (STX17, NRG1, RRAGD, XPO1, HERC1, HSP90AB1, EPHB2, and S1PR3) were screened. CONCLUSIONS: This is the first study to screen eight diagnostic markers and construct a gene regulatory network for COVID-19-MDD from the perspective of autophagy. The findings of our study provide novel insights into the diagnosis and treatment of COVID-19-MDD.

Transcriptome analysis uncovers the autophagy-mediated regulatory patterns of the immune microenvironment in dilated cardiomyopathy.

The relationship between autophagy and immunity has been well studied. However, little is known about the role of autophagy in the immune microenvironment during the progression of dilated cardiomyopathy (DCM). Therefore, this study aims to uncover the effect of autophagy on the immune microenvironment in the context of DCM. By investigating the autophagy gene expression differences between healthy donors and DCM samples, 23 dysregulated autophagy genes were identified. Using a series of bioinformatics methods, 13 DCM-related autophagy genes were screened and used to construct a risk prediction model, which can well distinguish DCM and healthy samples. Then, the connections between autophagy and immune responses including infiltrated immunocytes, immune reaction gene-sets and human leukocyte antigen (HLA) genes were systematically evaluated. In addition, two autophagy-mediated expression patterns in DCM were determined via the unsupervised consensus clustering analysis, and the immune characteristics of different patterns were revealed. In conclusion, our study revealed the strong effect of autophagy on the DCM immune microenvironment and provided new insights to understand the pathogenesis and treatment of DCM.

VMP1 and TMEM41B are essential for DMV formation during ß-coronavirus infection.

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.

Cell deaths: Involvement in the pathogenesis and intervention therapy of COVID-19.

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.

Knockout of GGPPS1 restrains rab37-mediated autophagy in response to ventilator-induced lung injury.

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.

A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets.

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged-Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

SARS-CoV-2 ORF3a Induces Incomplete Autophagy via the Unfolded Protein Response.

In the past year and a half, SARS-CoV-2 has caused 240 million confirmed cases and 5 million deaths worldwide. Autophagy is a conserved process that either promotes or inhibits viral infections. Although coronaviruses are known to utilize the transport of autophagy-dependent vesicles for the viral life cycle, the underlying autophagy-inducing mechanisms remain largely unexplored. Using several autophagy-deficient cell lines and autophagy inhibitors, we demonstrated that SARS-CoV-2 ORF3a was able to induce incomplete autophagy in a FIP200/Beclin-1-dependent manner. Moreover, ORF3a was involved in the induction of the UPR (unfolded protein response), while the IRE1 and ATF6 pathways, but not the PERK pathway, were responsible for mediating the ORF3a-induced autophagy. These results identify the role of the UPR pathway in the ORF3a-induced classical autophagy process, which may provide us with a better understanding of SARS-CoV-2 and suggest new therapeutic modalities in the treatment of COVID-19.

Mitochondrial nanomedicine: Subcellular organelle-specific delivery of molecular medicines.

As mitochondria network together to act as the master sensors and effectors of apoptosis, ATP production, reactive oxygen species management, mitophagy/autophagy, and homeostasis; this organelle is an ideal target for pharmaceutical manipulation. Mitochondrial dysfunction contributes to many diseases, for example, ß-amyloid has been shown to interfere with mitochondrial protein import and induce apoptosis in Alzheimer's Disease while some forms of Parkinson's Disease are associated with dysfunctional mitochondrial PINK1 and Parkin proteins. Mitochondrial medicine has applications in the treatment of an array of pathologies from cancer to cardiovascular disease. A challenge of mitochondrial medicine is directing therapies to a subcellular target. Nanotechnology based approaches combined with mitochondrial targeting strategies can greatly improve the clinical translation and effectiveness of mitochondrial medicine. This review discusses mitochondrial drug delivery approaches and applications of mitochondrial nanomedicines. Nanomedicine approaches have the potential to drive the success of mitochondrial therapies into the clinic.

Construction of an autophagy interaction network based on competitive endogenous RNA reveals the key pathways and central genes of SARS-CoV-2 infection in vivo.

As of April 1, 2021, more than 2.8 million people have died of SARS-CoV-2 infection. In addition, the mutation of virus strains that have accompanied the pandemic has brought more severe challenges to pandemic control. Host microRNAs (miRNAs) are widely involved in a variety of biological processes of coronavirus infection, including autophagy in SARS-CoV-2 infection. However, the mechanisms underlying miRNAs involved in autophagy in SARS-CoV-2 infection have not been fully elucidated. In this study, the miRNA and messenger RNA (mRNA) expression profiles of patients with SARS-CoV-2 infection were investigated based on raw data from Gene Expression Omnibus (GEO) datasets, and potential novel biomarkers of autophagy were revealed by bioinformatics analyses. We identified 32 differentially expressed miRNAs and 332 differentially expressed mRNAs in patients with SARS-CoV-2 infection. Cytokine receptor related pathways were the most enriched pathways for differentially expressed miRNAs identified by pathway analysis. Most importantly, an autophagy interaction network, which was associated with the pathological processes of SARS-CoV-2 infection, especially with the cytokine storm, was constructed. In this network, hsa-miR-340-3p, hsa-miR-652-3p, hsa-miR-4772-5p, hsa-miR-192-5p, TP53INP2, and CCR2 may be biomarkers that predict changes in mild SARS-CoV-2 infection. Some molecules, including hsa-miR-1291 and CXCR4, were considered potential targets to predict the emergence of severe symptoms in SARS-CoV-2 infection. To our knowledge, this study provided the first profile analysis of an autophagy interaction network in SARS-CoV-2 infection and revealed several novel autophagy-related biomarkers for understanding the pathogenesis of SARS-CoV-2 infection in vivo.

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2-infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.

A Resource for the Network Representation of Cell Perturbations Caused by SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic has caused more than 2.3 million casualties worldwide and the lack of effective treatments is a major health concern. The development of targeted drugs is held back due to a limited understanding of the molecular mechanisms underlying the perturbation of cell physiology observed after viral infection. Recently, several approaches, aimed at identifying cellular proteins that may contribute to COVID-19 pathology, have been reported. Albeit valuable, this information offers limited mechanistic insight as these efforts have produced long lists of cellular proteins, the majority of which are not annotated to any cellular pathway. We have embarked in a project aimed at bridging this mechanistic gap by developing a new bioinformatic approach to estimate the functional distance between a subset of proteins and a list of pathways. A comprehensive literature search allowed us to annotate, in the SIGNOR 2.0 resource, causal information underlying the main molecular mechanisms through which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related coronaviruses affect the host-cell physiology. Next, we developed a new strategy that enabled us to link SARS-CoV-2 interacting proteins to cellular phenotypes via paths of causal relationships. Remarkably, the extensive information about inhibitors of signaling proteins annotated in SIGNOR 2.0 makes it possible to formulate new potential therapeutic strategies. The proposed approach, which is generally applicable, generated a literature-based causal network that can be used as a framework to formulate informed mechanistic hypotheses on COVID-19 etiology and pathology.

The papain-like protease of coronaviruses cleaves ULK1 to disrupt host autophagy.

The ongoing pandemic of COVID-19 alongside the outbreaks of SARS in 2003 and MERS in 2012 underscore the significance to understand betacoronaviruses as a global health challenge. SARS-CoV-2, the etiological agent for COVID-19, has infected over 50 million individuals' worldwide with more than ∼1 million fatalities. Autophagy modulators have emerged as potential therapeutic candidates against SARS-CoV-2 but recent clinical setbacks urge for better understanding of viral subversion of autophagy. Using MHV-A59 as a model betacoronavirus, time-course infections revealed significant loss in the protein level of ULK1, a canonical autophagy-regulating kinase, and the concomitant appearance of a possible cleavage fragment. To investigate whether virus-encoded proteases target ULK1, we conducted in-vitro and cellular cleavage assays and identified ULK1 as a novel bona fide substrate of SARS-CoV-2 papain-like protease (PLpro). Mutagenesis studies discovered that ULK1 is cleaved at a conserved PLpro recognition sequence (LGGG) after G499, separating its N-terminal kinase domain from a C-terminal substrate recognition region. Over-expression of SARS-CoV-2 PLpro is sufficient to impair starvation-induced autophagy and disrupt formation of ULK1-ATG13 complex. Finally, we demonstrated a dual role for ULK1 in MHV-A59 replication, serving a pro-viral functions during early replication that is inactivated at late stages of infection. In conclusion, our study identified a new mechanism by which PLpro of betacoronaviruses induces viral pathogenesis by targeting cellular autophagy.

A multifactorial score including autophagy for prognosis and care of COVID-19 patients.

In less than eleven months, the world was brought to a halt by the COVID-19 outbreak. With hospitals becoming overwhelmed, one of the highest priorities concerned critical care triage to ration the scarce resources of intensive care units. Which patient should be treated first? Based on what clinical and biological criteria? A global joint effort rapidly led to sequencing the genomes of tens of thousands of COVID-19 patients to determine the patients' genetic signature that causes them to be at risk of suddenly developing severe disease. In this commentary, we would like to consider some points concerning the use of a multifactorial risk score for COVID-19 severity. This score includes macroautophagy (hereafter referred to as autophagy), a critical host process that controls all steps harnessed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Abbreviation list: ATG5: autophagy related 5; BECN1: beclin 1; COVID-19: coronavirus infectious disease-2019; EGR1: early growth response 1; ER: endoplasmic reticulum; DMVs: double-membrane vesicles; IBV: infectious bronchitis virus; MAP1LC3: microtubule associated protein 1 light chain 3; LC3-I: proteolytically processed, non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; MEFs: mouse embryonic fibroblasts; MERS-CoV: Middle East respiratory syndrome-coronavirus; MHV: mouse hepatitis virus; NSP: non-structural protein; PEDV: porcine epidemic diarrhea virus; PLP2-TM: membrane-associated papain-like protease 2; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TGEV: transmissible gastroenteritis virus.

Modeling Potential Autophagy Pathways in COVID-19 and Sarcoidosis.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mainly affects the lungs. Sarcoidosis is an autoinflammatory disease characterized by the diffusion of granulomas in the lungs and other organs. Here, we discuss how the two diseases might involve some common mechanistic cellular pathways around the regulation of autophagy.

A pharmacological perspective of chloroquine in SARS-CoV-2 infection: An old drug for the fight against a new coronavirus?

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having serious consequences on health and the economy worldwide. All evidence-based treatment strategies need to be considered to combat this new virus. Drugs need to be considered on scientific grounds of efficacy, safety and cost. Chloroquine (CQ) and hydroxychloroquine (HCQ) are old drugs used in the treatment of malaria. Moreover, their antiviral properties have been previously studied, including against coronaviruses, where evidence of efficacy has been found. In the current race against time triggered by the COVID-19 pandemic, the search for new antivirals is very important. However, consideration should be given to old drugs with known anti-coronavirus activity, such as CQ and HCQ. These could be integrated into current treatment strategies while novel treatments are awaited, also in light of the fact that they display an anticoagulant effect that facilitates the activity of low-molecular-weight heparin, aimed at preventing acute respiratory distress syndrome (ARDS)-associated thrombotic events. The safety of CQ and HCQ has been studied for over 50 years, however recently published data raise concerns for cardiac toxicity of CQ/HCQ in patients with COVID-19. This review also re-examines the real information provided by some of the published alarming reports, although concluding that cardiac toxicity should in any case be stringently monitored in patients receiving CQ/HCQ.