The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization.

Autophagy initiation is regulated by the ULK1 kinase complex. To gain insights into functions of the holo-complex, we generated a deep interactome by combining affinity purification- and proximity labeling-mass spectrometry of all four complex members: ULK1, ATG13, ATG101, and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases, implying the formation of a signalosome. Interestingly, several selective autophagy receptors also interact with ULK1, indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, which supports the recruitment of AMBRA1 to the ER membrane, positively affecting autophagy.

Role of AMBRA1 in mitophagy regulation: emerging evidence in aging-related diseases.

Aging is a gradual and irreversible physiological process that significantly increases the risks of developing a variety of pathologies, including neurodegenerative, cardiovascular, metabolic, musculoskeletal, and immune system diseases. Mitochondria are the energy-producing organelles, and their proper functioning is crucial for overall cellular health. Over time, mitochondrial function declines causing an increased release of harmful reactive oxygen species (ROS) and DNA, which leads to oxidative stress, inflammation and cellular damage, common features associated with various age-related pathologies. The impairment of mitophagy, the selective removal of damaged or dysfunctional mitochondria by autophagy, is relevant to the development and progression of age-related diseases. The molecular mechanisms that regulates mitophagy levels in aging remain largely uncharacterized. AMBRA1 is an intrinsically disordered scaffold protein with a unique property of regulating the activity of both proliferation and autophagy core machineries. While the role of AMBRA1 during embryonic development and neoplastic transformation has been extensively investigated, its functions in post-mitotic cells of adult tissues have been limited due to the embryonic lethality caused by AMBRA1 deficiency. Recently, a key role of AMBRA1 in selectively regulating mitophagy in post-mitotic cells has emerged. Here we summarize and discuss these results with the aim of providing a comprehensive view of the mitochondrial roles of AMBRA1, and how defective activity of AMBRA1 has been functionally linked to mitophagy alterations observed in age-related degenerative disorders, including muscular dystrophy/sarcopenia, Parkinson diseases, Alzheimer diseases and age-related macular degeneration.Abbreviations: AD: Alzheimer disease; AMD: age-related macular degeneration; AMBRA1: autophagy and beclin 1 regulator 1; APOE4: apolipoprotein E4; ATAD3A: ATPase family AAA domain containing 3A; ATG: autophagy related; BCL2: BCL2 apoptosis regulator; BH3: BCL2-homology-3; BNIP3L/NIX: BCL2 interacting protein 3 like; CDK: cyclin dependent kinase; CHUK/IKKα: component of inhibitor of nuclear factor kappa B kinase complex; CRL2: CUL2-RING ubiquitin ligase; DDB1: damage specific DNA binding protein 1; ER: endoplasmic reticulum; FOXO: forkhead box O; FUNDC1: FUN14 domain containing 1; GBA/ß-glucocerebrosidase: glucosylceramidase beta; HUWE1: HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1; IDR: intrinsically disordered region; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MCL1: MCL1 apoptosis regulator, BCL2 family member; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MSA: multiple system atrophy; MYC: MYC proto-oncogene, bHLH transcription factor; NUMA1: nuclear mitotic apparatus protein 1; OMM; mitochondria outer membrane; PD: Parkinson disease; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PTK2/FAK: protein tyrosine kinase 2; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SAD: sporadic AD; SOCS3: suppressor of cytokine signaling 3; SRC, SRC proto-oncogene, non-receptor tyrosine kinase; STAT3: signal transducer and activator of transcription 3; STING1: stimulator of interferon response cGAMP interactor 1; SQSTM1/p62: sequestosome 1; TBK1: TANK binding kinase 1; TGFB/TGFß: transforming growth factor beta; TOMM: translocase of outer mitochondrial membrane; TRAF6: TNF receptor associated factor 6; TRIM32: tripartite motif containing 32; ULK1: unc-51 like autophagy activating kinase 1.

AMBRA1 phosphorylation by CDK1 and PLK1 regulates mitotic spindle orientation.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.

The ubiquitin ligase TRIM32 promotes the autophagic response to Mycobacterium tuberculosis infection in macrophages.

Mycobacterium tuberculosis (Mtb) is known to evade host immune responses and persist in macrophages for long periods. A mechanism that the host uses to combat Mtb is xenophagy, a selective form of autophagy that targets intracellular pathogens for degradation. Ubiquitination of Mtb or Mtb-containing compartments is a key event to recruit the autophagy machinery and mediate the bacterial delivery to the lysosome. This event relies on the coordinated and complementary activity of different ubiquitin ligases, including PARKIN, SMURF1, and TRIM16. Because each of these factors is responsible for the ubiquitination of a subset of the Mtb population, it is likely that additional ubiquitin ligases are employed by macrophages to trigger a full xenophagic response during Mtb infection. In this study, we investigated the role TRIM proteins whose expression is modulated in response to Mtb or BCG infection of primary macrophages. These TRIMs were ectopically expressed in THP1 macrophage cell line to assess their impact on Mtb replication. This screening identified TRIM32 as a novel player involved in the intracellular response to Mtb infection, which promotes autophagy-mediated Mtb degradation. The role of TRIM32 in xenophagy was further confirmed by silencing TRIM32 expression in THP1 cells, which causes increased intracellular growth of Mtb associated to impaired Mtb ubiquitination, reduced recruitment of the autophagy proteins NDP52/CALCOCO2 and BECLIN 1/BECN1 to Mtb and autophagosome formation. Overall, these findings suggest that TRIM32 plays an important role in the host response to Mtb infection through the induction of autophagy, representing a promising target for host-directed tuberculosis therapies.

A microRNA Arising from the Negative Strand of SARS-CoV-2 Genome Targets FOS to Reduce AP-1 Activity.

Virus-encoded microRNAs were first reported in the Epstein-Barr virus in 2004. Subsequently, a few hundred viral miRNAs have been identified, mainly in DNA viruses belonging to the herpesviridae family. To date, only 30 viral miRNAs encoded by RNA viruses are reported by miRBase. Since the outbreak of the SARS-CoV-2 pandemic, several studies have predicted and, in some cases, experimentally validated miRNAs originating from the positive strand of the SARS-CoV-2 genome. By integrating NGS data analysis and qRT-PCR approaches, we found that SARS-CoV-2 also encodes for a viral miRNA arising from the minus (antisense) strand of the viral genome, in the region encoding for ORF1ab, herein referred to as SARS-CoV-2-miR-AS1. Our data show that the expression of this microRNA increases in a time course analysis of SARS-CoV-2 infected cells. Furthermore, enoxacin treatment enhances the accumulation of the mature SARS-CoV-2-miR-AS1 in SARS-CoV-2 infected cells, arguing for a Dicer-dependent processing of this small RNA. In silico analysis suggests that SARS-CoV-2-miR-AS1 targets a set of genes which are translationally repressed during SARS-CoV-2 infection. We experimentally validated that SARS-CoV-2-miR-AS1 targets FOS, thus repressing the AP-1 transcription factor activity in human cells.

NAADP-Evoked Ca2+ Signaling Leads to Mutant Huntingtin Aggregation and Autophagy Impairment in Murine Astrocytes.

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by mutations in the huntingtin gene (mHtt), causing an unstable repeat of the CAG trinucleotide, leading to abnormal long repeats of polyglutamine (poly-Q) in the N-terminal region of the huntingtin, which form abnormal conformations and aggregates. Alterations in Ca2+ signaling are involved in HD models and the accumulation of mutated huntingtin interferes with Ca2+ homeostasis. Lysosomes are intracellular Ca2+ storages that participate in endocytic and lysosomal degradation processes, including autophagy. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an intracellular second messenger that promotes Ca2+ release from the endo-lysosomal system via Two-Pore Channels (TPCs) activation. Herein, we show the impact of lysosomal Ca2+ signals on mHtt aggregation and autophagy blockade in murine astrocytes overexpressing mHtt-Q74. We observed that mHtt-Q74 overexpression causes an increase in NAADP-evoked Ca2+ signals and mHtt aggregation, which was inhibited in the presence of Ned-19, a TPC antagonist, or BAPTA-AM, a Ca2+ chelator. Additionally, TPC2 silencing revert the mHtt aggregation. Furthermore, mHtt has been shown co-localized with TPC2 which may contribute to its effects on lysosomal homeostasis. Moreover, NAADP-mediated autophagy was also blocked since its function is dependent on lysosomal functionality. Taken together, our data show that increased levels of cytosolic Ca2+ mediated by NAADP causes mHtt aggregation. Additionally, mHtt co-localizes with the lysosomes, where it possibly affects organelle functions and impairs autophagy.

The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis.

Cystic fibrosis (CF) is a rare autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation is F508del-CFTR (ΔF) which leads the encoded ion channel towards misfolding and premature degradation. The disease is characterized by chronic bronchopulmonary obstruction, inflammation and airways colonization by bacteria, which are the major cause of morbidity and mortality. The STING pathway is the main signaling route activated in the presence of both self and pathogen DNA, leading to Type I Interferon (IFN I) production and the innate immune response. In this study, we show for the first time the relationship existing in CF between resistant and recurrent opportunistic infections by Pseudomonas aeruginosa and the innate immunity impairment. We demonstrate through ex vivo and in vivo experiments that the pathway is inadequately activated in ΔF condition and the use of direct STING agonists, as 2',3'-cyclic GMP-AMP (2', 3' cGAMP), is able to restore the immune response against bacterial colonization. Indeed, upon treatment with the STING pathway agonists, we found a reduction of colony forming units (CFUs) consequent to IFN-ß enhanced production in Pseudomonas aeruginosa infected bone marrow derived macrophages and lung tissues from mice affected by Cystic Fibrosis. Importantly, we also verified that the impairment detected in the primary PBMCs obtained from ΔF patients can be corrected by 2', 3' cGAMP. Our work indicates that the cGAS/STING pathway integrity is crucial in the Cystic Fibrosis response against pathogens and that the restoration of the pathway by 2', 3' cGAMP could be exploited as a possible new target for the symptomatic treatment of the disease.

Type 2 transglutaminase in the nucleus: the new epigenetic face of a cytoplasmic enzyme.

One of the major mysteries in science is how it is possible to pack the cellular chromatin with a total length of over 1 m, into a small sphere with a diameter of 5 mm "the nucleus", and even more difficult to envisage how to make it functional. Although we know that compaction is achieved through the histones, however, the DNA needs to be accessible to the transcription machinery and this is allowed thanks to a variety of very complex epigenetic mechanisms. Either DNA (methylation) or post-translational modifications of histone proteins (acetylation, methylation, ubiquitination and sumoylation) play a crucial role in chromatin remodelling and consequently on gene expression. Recently the serotonylation and dopaminylation of the histone 3, catalyzed by the Transglutaminase type 2 (TG2), has been reported. These novel post-translational modifications catalyzed by a predominantly cytoplasmic enzyme opens a new avenue for future investigations on the enzyme function itself and for the possibility that other biological amines, substrate of TG2, can influence the genome regulation under peculiar cellular conditions. In this review we analyzed the nuclear TG2's biology by discussing both its post-translational modification of various transcription factors and the implications of its epigenetic new face. Finally, we will focus on the potential impact of these events in human diseases.

Proteomic analysis identifies a signature of disease severity in the plasma of COVID-19 pneumonia patients associated to neutrophil, platelet and complement activation.

Most patients infected with SARS-CoV-2 display mild symptoms with good prognosis, while 20% of patients suffer from severe viral pneumonia and up to 5% may require intensive care unit (ICU) admission due to severe acute respiratory syndrome, which could be accompanied by multiorgan failure.Plasma proteomics provide valuable and unbiased information about disease progression and therapeutic candidates. Recent proteomic studies have identified molecular changes in plasma of COVID-19 patients that implied significant dysregulation of several aspects of the inflammatory response accompanied by a general metabolic suppression. However, which of these plasma alterations are associated with disease severity remains only partly characterized.A known limitation of proteomic studies of plasma samples is the large difference in the macromolecule abundance, with concentration spanning at least 10 orders of magnitude. To improve the coverage of plasma contents, we performed a deep proteomic analysis of plasma from 10 COVID-19 patients with severe/fatal pneumonia compared to 10 COVID-19 patients with pneumonia who did not require ICU admission (non-ICU). To this aim, plasma samples were first depleted of the most abundant proteins, trypsin digested and peptides subjected to a high pH reversed-phase peptide fractionation before LC-MS analysis.These results highlighted an increase of proteins involved in neutrophil and platelet activity and acute phase response, which is significantly higher in severe/fatal COVID-19 patients when compared to non-ICU ones. Importantly, these changes are associated with a selective induction of complement cascade factors in severe/fatal COVID-19 patients. Data are available via ProteomeXchange with identifier PXD036491. Among these alterations, we confirmed by ELISA that higher levels of the neutrophil granule proteins DEFA3 and LCN2 are present in COVID-19 patients requiring ICU admission when compared to non-ICU and healthy donors.Altogether, our study provided an in-depth view of plasma proteome changes that occur in COVID-19 patients in relation to disease severity, which can be helpful to identify therapeutic strategies to improve the disease outcome.

Analysis of Secreted Proteins from Prepubertal Ovarian Tissues Exposed In Vitro to Cisplatin and LH.

It is well known that secreted and exosomal proteins are associated with a broad range of physiological processes involving tissue homeostasis and differentiation. In the present paper, our purpose was to characterize the proteome of the culture medium in which the oocytes within the primordial/primary follicles underwent apoptosis induced by cisplatin (CIS) or were, for the most part, protected by LH against the drug. To this aim, prepubertal ovarian tissues were cultured under control and in the presence of CIS, LH, and CIS + LH. The culture media were harvested after 2, 12, and 24 h from chemotherapeutic drug treatment and analyzed by liquid chromatography-mass spectrometry (LC-MS). We found that apoptotic conditions generated by CIS in the cultured ovarian tissues and/or oocytes are reflected in distinct changes in the extracellular microenvironment in which they were cultured. These changes became evident mainly from 12 h onwards and were characterized by the inhibition or decreased release of a variety of compounds, such as the proteases Htra1 and Prss23, the antioxidants Prdx2 and Hbat1, the metabolic regulators Ldha and Pkm, and regulators of apoptotic pathways such as Tmsb4x. Altogether, these results confirm the biological relevance of the LH action on prepuberal ovaries and provide novel information about the proteins released by the ovarian tissues exposed to CIS and LH in the surrounding microenvironment. These data might represent a valuable resource for future studies aimed to clarify the effects and identify biomarkers of these compounds' action on the developing ovary.

Peroxiredoxin 6 Modulates Insulin Secretion and Beta Cell Death via a Mitochondrial Dynamic Network.

In pancreatic beta cells, mitochondrial metabolism controls glucose-stimulated insulin secretion (GSIS) by ATP production, redox signaling, and calcium (Ca2+) handling. Previously, we demonstrated that knockout mice for peroxiredoxin 6 (Prdx6-/- ), an antioxidant enzyme with both peroxidase and phospholipase A2 activity, develop a mild form of diabetes mellitus with a reduction in GSIS and in peripheral insulin sensitivity. However, whether the defect of GSIS present in these mice is directly modulated by Prdx6 is unknown. Therefore, the main goal of the present study was to evaluate if depletion of Prdx6 affects directly GSIS and pancreatic beta ß-cell function. Murine pancreatic ß-cell line (ßTC6) knockdown for Prdx6 (Prdx6KD) was employed, and insulin secretion, ATP, and intracellular Ca2+ content were assessed in response to glucose stimulation. Mitochondrial morphology and function were also evaluated through electron microscopy, and by testing mitochondrial membrane potential, oxygen consumption, and mitochondrial mass. Prdx6KD cells showed a significant reduction in GSIS as confirmed by decrease in both ATP release and Ca2+ influx. GSIS alteration was also demonstrated by a marked impairment of mitochondrial morphology and function. These latest are mainly linked to mitofusin downregulation, which are, in turn, strictly related to mitochondrial homeostasis (by regulating autophagy) and cell fate (by modulating apoptosis). Following a pro-inflammatory stimulus (typical of diabetic subjects), and in agreement with the deregulation of mitofusin steady-state levels, we also observed an enhancement in apoptotic death in Prdx6KD compared to control cells. We analyzed molecular mechanisms leading to apoptosis, and we further demonstrated that Prdx6 suppression activates both intrinsic and extrinsic apoptotic pathways, ultimately leading to caspase 3 and PARP-1 activation. In conclusion, Prdx6 is the first antioxidant enzyme, in pancreatic ß-cells, that by controlling mitochondrial homeostasis plays a pivotal role in GSIS modulation.

The interplay between SARS-CoV-2 infected airway epithelium and immune cells modulates regulatory/inflammatory signals.

To assess the cross-talk between immune cells and respiratory tract during SARS-CoV-2 infection, we analyzed the relationships between the inflammatory response induced by SARS-CoV-2 replication and immune cells phenotype in a reconstituted organotypic human airway epithelium (HAE). The results indicated that immune cells failed to inhibit SARS-CoV-2 replication in the HAE model. In contrast, immune cells strongly affected the inflammatory profile induced by SARS-CoV-2 infection, dampening the production of several immunoregulatory/inflammatory signals (e.g., IL-35, IL-27, and IL-34). Moreover, these mediators were found inversely correlated with innate immune cell frequency (NK and γδ T cells) and directly with CD8 T cells. The enriched signals associated with NK and CD8 T cells highlighted the modulation of pathways induced by SARS-CoV-2 infected HAE. These findings are useful to depict the cell-cell communication mechanisms necessary to develop novel therapeutic strategies aimed to promote an effective immune response.

AMBRA1 regulates mitophagy by interacting with ATAD3A and promoting PINK1 stability.

PINK1 accumulation at the outer mitochondrial membrane (OMM) is a key event required to signal depolarized mitochondria to the autophagy machinery. How this early step is, in turn, modulated by autophagy proteins remains less characterized. Here, we show that, upon mitochondrial depolarization, the proautophagic protein AMBRA1 is recruited to the OMM and interacts with PINK1 and ATAD3A, a transmembrane protein that mediates mitochondrial import and degradation of PINK1. Downregulation of AMBRA1 expression results in reduced levels of PINK1 due to its enhanced degradation by the mitochondrial protease LONP1, which leads to a decrease in PINK1-mediated ubiquitin phosphorylation and mitochondrial PRKN/PARKIN recruitment. Notably, ATAD3A silencing rescues defective PINK1 accumulation in AMBRA1-deficient cells upon mitochondrial damage. Overall, our findings underline an upstream contribution of AMBRA1 in the control of PINK1-PRKN mitophagy by interacting with ATAD3A and promoting PINK1 stability. This novel regulatory element may account for changes of PINK1 levels in neuropathological conditions.Abbreviations: ACTB/ß-actin: actin beta; AMBRA1: autophagy and beclin 1 regulator 1; ATAD3A: ATPase family AAA domain containing 3A; BCL2L1/BCL-xL: BCL2 like 1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMA1: OMA1 zinc metallopeptidase; OMM: outer mitochondrial membrane; PARL: presenilin associated rhomboid like; PARP: poly(ADP-ribose) polymerase; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; SDHA: succinate dehydrogenase complex flavoprotein subunit A; TOMM70: translocase of outer mitochondrial membrane 70.

Multi-omics approach to COVID-19: a domain-based literature review.

BACKGROUND: Omics data, driven by rapid advances in laboratory techniques, have been generated very quickly during the COVID-19 pandemic. Our aim is to use omics data to highlight the involvement of specific pathways, as well as that of cell types and organs, in the pathophysiology of COVID-19, and to highlight their links with clinical phenotypes of SARS-CoV-2 infection. METHODS: The analysis was based on the domain model, where for domain it is intended a conceptual repository, useful to summarize multiple biological pathways involved at different levels. The relevant domains considered in the analysis were: virus, pathways and phenotypes. An interdisciplinary expert working group was defined for each domain, to carry out an independent literature scoping review. RESULTS: The analysis revealed that dysregulated pathways of innate immune responses, (i.e., complement activation, inflammatory responses, neutrophil activation and degranulation, platelet degranulation) can affect COVID-19 progression and outcomes. These results are consistent with several clinical studies. CONCLUSIONS: Multi-omics approach may help to further investigate unknown aspects of the disease. However, the disease mechanisms are too complex to be explained by a single molecular signature and it is necessary to consider an integrated approach to identify hallmarks of severity.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Rationale and Criteria for a COVID-19 Model Framework.

Complex systems are inherently multilevel and multiscale systems. The infectious disease system is considered a complex system resulting from the interaction between three sub-systems (host, pathogen, and environment) organized into a hierarchical structure, ranging from the cellular to the macro-ecosystem level, with multiscales. Therefore, to describe infectious disease phenomena that change through time and space and at different scales, we built a model framework where infectious disease must be considered the set of biological responses of human hosts to pathogens, with biological pathways shared with other pathologies in an ecological interaction context. In this paper, we aimed to design a framework for building a disease model for COVID-19 based on current literature evidence. The model was set up by identifying the molecular pathophysiology related to the COVID-19 phenotypes, collecting the mechanistic knowledge scattered across scientific literature and bioinformatic databases, and integrating it using a logical/conceptual model systems biology. The model framework building process began from the results of a domain-based literature review regarding a multiomics approach to COVID-19. This evidence allowed us to define a framework of COVID-19 conceptual model and to report all concepts in a multilevel and multiscale structure. The same interdisciplinary working groups that carried out the scoping review were involved. The conclusive result is a conceptual method to design multiscale models of infectious diseases. The methodology, applied in this paper, is a set of partially ordered research and development activities that result in a COVID-19 multiscale model.

High Levels of TRIM5α Are Associated with Xenophagy in HIV-1-Infected Long-Term Nonprogressors.

Autophagy is a lysosomal-dependent degradative mechanism essential in maintaining cellular homeostasis, but it is also considered an ancient form of innate eukaryotic fighting against invading microorganisms. Mounting evidence has shown that HIV-1 is a critical target of autophagy that plays a role in HIV-1 replication and disease progression. In a special subset of HIV-1-infected patients that spontaneously and durably maintain extremely low viral replication, namely, long-term nonprogressors (LTNP), the resistance to HIV-1-induced pathogenesis is accompanied, in vivo, by a significant increase in the autophagic activity in peripheral blood mononuclear cells. Recently, a new player in the battle of autophagy against HIV-1 has been identified, namely, tripartite motif protein 5α (TRIM5α). In vitro data demonstrated that TRIM5α directly recognizes HIV-1 and targets it for autophagic destruction, thus protecting cells against HIV-1 infection. In this paper, we analyzed the involvement of this factor in the control of HIV-1 infection through autophagy, in vivo, in LTNP. The results obtained showed significantly higher levels of TRIM5α expression in cells from LTNP with respect to HIV-1-infected normal progressor patients. Interestingly, the colocalization of TRIM5α and HIV-1 proteins in autophagic vacuoles in LTNP cells suggested the participation of TRIM5α in the autophagy containment of HIV-1 in LTNP. Altogether, our results point to a protective role of TRIM5α in the successful control of the chronic viral infection in HIV-1-controllers through the autophagy mechanism. In our opinion, these findings could be relevant in fighting against HIV-1 disease, because autophagy inducers might be employed in combination with antiretroviral drugs.

Transglutaminase 2 Regulates Innate Immunity by Modulating the STING/TBK1/IRF3 Axis.

We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-ß production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNß production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-ß production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.

Pharmacological Modulators of Autophagy as a Potential Strategy for the Treatment of COVID-19.

The family of coronaviruses (CoVs) uses the autophagy machinery of host cells to promote their growth and replication; thus, this process stands out as a potential target to combat COVID-19. Considering the different roles of autophagy during viral infection, including SARS-CoV-2 infection, in this review, we discuss several clinically used drugs that have effects at different stages of autophagy. Among them, we mention (1) lysosomotropic agents, which can prevent CoVs infection by alkalinizing the acid pH in the endolysosomal system, such as chloroquine and hydroxychloroquine, azithromycin, artemisinins, two-pore channel modulators and imatinib; (2) protease inhibitors that can inhibit the proteolytic cleavage of the spike CoVs protein, which is necessary for viral entry into host cells, such as camostat mesylate, lopinavir, umifenovir and teicoplanin and (3) modulators of PI3K/AKT/mTOR signaling pathways, such as rapamycin, heparin, glucocorticoids, angiotensin-converting enzyme inhibitors (IECAs) and cannabidiol. Thus, this review aims to highlight and discuss autophagy-related drugs for COVID-19, from in vitro to in vivo studies. We identified specific compounds that may modulate autophagy and exhibit antiviral properties. We hope that research initiatives and efforts will identify novel or "off-label" drugs that can be used to effectively treat patients infected with SARS-CoV-2, reducing the risk of mortality.

Proteomic analysis identifies the RNA helicase DDX3X as a host target against SARS-CoV-2 infection.

COVID-19 is currently a highly pressing health threat and therapeutic strategies to mitigate the infection impact are urgently needed. Characterization of the SARS-CoV-2 interactome in infected cells may represent a powerful tool to identify cellular proteins hijacked by viruses for their life cycle and develop host-oriented antiviral therapeutics. Here we report the proteomic characterization of host proteins interacting with SARS-CoV-2 Nucleoprotein in infected Vero E6 cells. We identified 24 high-confidence proteins mainly playing a role in RNA metabolism and translation, including RNA helicases and scaffold proteins involved in the formation of stress granules, cytoplasmic aggregates of messenger ribonucleoproteins that accumulate as a result of stress-induced translation arrest. Analysis of stress granules upon SARS-CoV-2 infection showed that these structures are not induced in infected cells, neither eIF2α phosphorylation, an upstream event leading to stress-induced translation inhibition. Notably, we found that G3BP1, a stress granule component that associates with the Nucleoprotein, is required for efficient SARS-CoV-2 replication. Moreover, we showed that the Nucleoprotein-interacting RNA helicase DDX3X colocalizes with viral RNA foci and its inhibition by small molecules or small interfering RNAs significantly reduces viral replication. Altogether, these results indicate that SARS-CoV-2 subverts the stress granule machinery and exploits G3BP1 and DDX3X for its replication cycle, offering groundwork for future development of host-directed therapies.