Better understanding on interactions between SARS‐CoV‐2 and host cells should help to identify host factors that may be targetable to combat infection and COVID‐19 pathology. To this end, we have ...conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen in human lung cancer cells infected with SARS‐CoV‐2‐pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS‐CoV‐2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS‐CoV‐2 viruses confirm that loss‐of‐function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single‐cell RNA‐Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS‐CoV‐2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.
Synopsis
Identifying targetable host factors to fight COVID‐19 requires full understanding of virus‐host cell interactions. Here, a genome‐wide loss‐of‐function screen identifies new regulators of the intracellular fate of endocytosed SARS‐CoV‐2 virions.
A genome‐wide CRISPR/Cas9‐based screen in human lung cancer cells identifies lysosomal membrane proteins PLAC8 and SPNS1 as required for viral entry.
PLAC8 loss reduces SARS‐CoV‐2 infection efficiency, while overexpression boosts it in several lung cancer lines
Single‐cell RNA‐Seq analyses reveal a strong enrichment of PLAC8 expression in lung and colon SARS‐CoV‐2 target cells
PLAC8 colocalizes with ACE2, but its loss‐of‐function does not affect binding or endocytosis in experiments with recombinant SPIKE receptor binding domain
PLAC8 loss‐of‐function causes an expansion of the autophagolysosomal compartment
Genome‐wide CRISPR screens identify lysosomal efflux transporter SPNS1 and transmembrane protein PLAC8 as regulators of the intracellular fate of endocytosed SARS‐CoV‐2 virions.
This study was designed to investigate the controversy on the potential role of sKlotho as an early biomarker in Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD), to assess whether sKlotho is a ...reliable marker of kidney α-Klotho, to deepen the effects of sKlotho on vascular smooth muscle cells (VSMCs) osteogenic differentiation and to evaluate the role of autophagy in this process. Experimental studies were conducted in CKD mice fed a normal phosphorus (CKD+NP) or high phosphorus (CKD+HP) diet for 14 weeks. The patients' study was performed in CKD stages 2-5 and in vitro studies which used VSMCs exposed to non-calcifying medium or calcifying medium with or without sKlotho. The CKD experimental model showed that the CKD+HP group reached the highest serum PTH, P and FGF23 levels, but the lowest serum and urinary sKlotho levels. In addition, a positive correlation between serum sKlotho and kidney α-Klotho was found. CKD mice showed aortic osteogenic differentiation, together with increased autophagy. The human CKD study showed that the decline in serum sKlotho is previous to the rise in FGF23. In addition, both serum sKlotho and FGF23 levels correlated with kidney function. Finally, in VSMCs, the addition of sKlotho prevented osteogenic differentiation and induced autophagy. It can be concluded that serum sKlotho was the earliest CKD-MBD biomarker, a reliable indicator of kidney α-Klotho and that might protect against osteogenic differentiation by increasing autophagy. Nevertheless, further studies are needed to investigate the mechanisms of this possible protective effect.
Eukaryotic cells have developed mechanisms that allow them to link growth and proliferation to the availability of energy and biomolecules. AMPK (adenosine monophosphate-activated protein kinase) is ...one of the most important molecular energy sensors in eukaryotic cells. AMPK activity is able to control a wide variety of metabolic processes connecting cellular metabolism with energy availability. Autophagy is an evolutionarily conserved catabolic pathway whose activity provides energy and basic building blocks for the synthesis of new biomolecules. Given the importance of autophagic degradation for energy production in situations of nutrient scarcity, it seems logical that eukaryotic cells have developed multiple molecular links between AMPK signaling and autophagy regulation. In this review, we will discuss the importance of AMPK activity for diverse aspects of cellular metabolism, and how AMPK modulates autophagic degradation and adapts it to cellular energetic status. We will explain how AMPK-mediated signaling is mechanistically involved in autophagy regulation both through specific phosphorylation of autophagy-relevant proteins or by indirectly impacting in the activity of additional autophagy regulators.
Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic ...activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABA
receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.
Renal ischemia-reperfusion injury (IRI) leads to endoplasmic reticulum (ER) stress, thereby initiating the unfolded protein response (UPR). When sustained, this response may trigger the inflammation ...and tubular cell death that acts to aggravate the damage. Here, we show that knockdown of the BET epigenetic reader BRD4 reduces the expression of ATF4 and XBP1 transcription factors under ER stress activation. BRD4 is recruited to the promoter of these highly acetylated genes, initiating gene transcription. Administration of the BET protein inhibitor, JQ1, one hour after renal damage induced by bilateral IRI, reveals reduced expression of
and
genes, low KIM-1 and NGAL levels and recovery of the serum creatinine and blood urea nitrogen levels. To determine the molecular pathways regulated by ATF4 and XBP1, we performed stable knockout of both transcription factors using CRISPR-Cas9 and RNA sequencing. The pathways triggered under ER stress were mainly XBP1-dependent, associated with an adaptive UPR, and partially regulated by JQ1. Meanwhile, treatment with JQ1 downmodulated most of the pathways regulated by ATF4 and related to the pathological processes during exacerbated UPR activation. Thus, BRD4 inhibition could be useful for curbing the maladaptive UPR activation mechanisms, thereby ameliorating the progression of renal disease.
In recent years, the study of single nucleotide polymorphisms (SNPs) has gained increasing importance in biomedical research, as they can either be at the molecular origin of a determined disorder or ...directly affect the efficiency of a given treatment. In this regard, sequence variations in genes involved in pro-survival cellular pathways are commonly associated with pathologies, as the alteration of these routes compromises cellular homeostasis. This is the case of autophagy, an evolutionarily conserved pathway that counteracts extracellular and intracellular stressors by mediating the turnover of cytosolic components through lysosomal degradation. Accordingly, autophagy dysregulation has been extensively described in a wide range of human pathologies, including cancer, neurodegeneration, or inflammatory alterations. Thus, it is not surprising that pathogenic gene variants in genes encoding crucial effectors of the autophagosome/lysosome axis are increasingly being identified. In this review, we present a comprehensive list of clinically relevant SNPs in autophagy-related genes, highlighting the scope and relevance of autophagy alterations in human disease.
Lysosomes are the main organelles responsible for the degradation of macromolecules in eukaryotic cells. Beyond their fundamental role in degradation, lysosomes are involved in different ...physiological processes such as autophagy, nutrient sensing, and intracellular signaling. In some circumstances, lysosomal abnormalities underlie several human pathologies with different etiologies known as known as lysosomal storage disorders (LSDs). These disorders can result from deficiencies in primary lysosomal enzymes, dysfunction of lysosomal enzyme activators, alterations in modifiers that impact lysosomal function, or changes in membrane-associated proteins, among other factors. The clinical phenotype observed in affected patients hinges on the type and location of the accumulating substrate, influenced by genetic mutations and residual enzyme activity. In this context, the scientific community is dedicated to exploring potential therapeutic approaches, striving not only to extend lifespan but also to enhance the overall quality of life for individuals afflicted with LSDs. This review provides insights into lysosomal dysfunction from a molecular perspective, particularly in the context of human diseases, and highlights recent advancements and breakthroughs in this field.
In the last years, autophagy has been revealed as an essential pathway for multiple biological processes and physiological functions. As a catabolic route, autophagy regulation by nutrient ...availability has been evolutionarily conserved from yeast to mammals. On one hand, autophagy induction by starvation is associated with a significant loss in body weight in mice. Here, we demonstrate that both genetic and pharmacological inhibition of the autophagy process compromise weight loss induced by starvation. Moreover, autophagic potential also impacts on weight gain induced by distinct hypercaloric regimens. Atg4b-deficient mice, which show limited autophagic competence, exhibit a major increase in body weight in response to distinct obesity-associated metabolic challenges. This response is characterized by the presence of larger adipocytes in visceral fat tissue, increased hepatic steatosis, as well as reduced glucose tolerance and attenuated insulin responses. Similarly, autophagy-deficient mice are more vulnerable to experimentally induced type-I diabetes, showing an increased susceptibility to acute streptozotocin administration. Notably, pharmacological stimulation of autophagy in wild-type mice by spermidine reduced both weight gain and obesity-associated alterations upon hypercaloric regimens. Altogether, these results indicate that systemic autophagic activity influences the resilience of the organism to weight gain induced by high-calorie diets, as well as to the obesity-associated features of both type-1 and type-2 diabetes.
Analysis of ATG4C function in vivo Tamargo-Gómez, Isaac; Martínez-García, Gemma G.; Suárez, María F. ...
Autophagy,
11/2023, Volume:
19, Issue:
11
Journal Article
Peer reviewed
Open access
Despite the great advances in macroautophagy/autophagy research in the last years, the in vivo role of the different members of the four mammalian orthologs of yeast Atg4 protease (ATG4A-D) remain ...unclear. To gain further insights into the functional relevance of Atg4 orthologs, we have generated mutant mice deficient in Atg4c. These mice are viable and fertile, and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, they show tissue-specific autophagy alterations, including reduced autophagic flux in diaphragm and show decreased breathing and locomotor activity after fasting. In addition, atg4c
-/-
mice show reduced number of circulating T and B lymphocytes, which is associated with accumulation of apoptotic cells in the spleen and an increased susceptibility to develop chemically-induced fibrosarcomas. Moreover, through the analysis of cells and mice simultaneously deficient for ATG4C and ATG4D proteases we also reveal a role for ATG4C in mATG8 proteins delipidation.
ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1).
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