Significance It is known that autophagy plays a major role in cellular homeostasis and impaired autophagy has been implicated in various forms of human disease. Here we investigate one way in which ...autophagy is critically connected to cellular integrity by showing that autophagy loss diminishes DNA repair by the error-free process, homologous recombination. As a result, this causes reliance on the error-prone process of nonhomologous end joining for repair of DNA double-strand breaks, providing one explanation of why autophagy-deficient cells accumulate genomic damage. These findings therefore have important implications for diseases in which autophagy is impaired and for therapeutic strategies designed to inhibit autophagy, particularly if autophagy inhibition is undertaken in combination with agents that cause genomic damage.
(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.
Abstract
Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player of the control of numerous cellular functions, and whose defects have been ...associated with several human pathologies. To date, this cellular function is presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in nontetrapod species. However, the recent identification of expressed sequences displaying high homology with mammalian LAMP2A in several fish species challenges that view and suggests that CMA likely appeared earlier during evolution than initially thought. In the present study, we provide a comprehensive picture of the evolutionary history of the LAMP2 gene in vertebrates and demonstrate that LAMP2 indeed appeared at the root of the vertebrate lineage. Using a fibroblast cell line from medaka fish (Oryzias latipes), we further show that the splice variant lamp2a controls, upon long-term starvation, the lysosomal accumulation of a fluorescent reporter commonly used to track CMA in mammalian cells. Finally, to address the physiological role of Lamp2a in fish, we generated knockout medaka for that specific splice variant, and found that these deficient fish exhibit severe alterations in carbohydrate and fat metabolisms, in consistency with existing data in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective.
Bcl-2 family proteins control programmed cell death through a complex network of interactions within and outside of this family, that are modulated by post-translational modifications (PTM). Bcl-xL, ...an anti-apoptotic member of this family, is overexpressed in a number of cancers, plays an important role in tumorigenesis and is correlated with drug resistance. Bcl-xL is susceptible to a number of different PTMs. Here, we focus on deamidation. We will first provide an overview of protein deamidation. We will then review how the apoptotic and autophagic functions of Bcl-xL are modified by this PTM, and how this impacts on its oncogenic properties. Possible therapeutic outcomes will also be discussed. Finally, we will highlight how the specific case of Bcl-xL deamidation provides groundings to revisit some concepts related to protein deamidation in general.
•Deamidated proteins accumulate with time•Deamidation can modify protein structure and functions•Bcl-xL deamidation alters its oncogenic properties•Bcl-xL deamidation can be disrupted in cancer cells•Restoration of Bcl-xL deamidation pathways can be a therapeutic target
Bcl-x
is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-x
shows unique eligibility to deamidation, a ...time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn→Asp/IsoAsp conversions or Glu→Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-x
generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-x
remains eligible for regulation by phosphorylation.
Nowadays, aquaculture provides more than 50% of fish consumed worldwide but faces new issues that challenge its sustainability. One of them relies on the replacement of fish meal (FM) in aquaculture ...feeds by other protein sources without deeply affecting the whole organism's homeostasis. Multiple strategies have already been tested using in vivo approaches, but they hardly managed to cope with the multifactorial problems related to the complexities of fish biology together with new feed formulations. In this context, rainbow trout (RT) is particularly concerned by these problems, since, as a carnivorous fish, dietary proteins provide the amino acids required to supply most of its energetic metabolism. Surprisingly, we noticed that in vitro approaches considering RT cell lines as models to study RT amino acid metabolism were never previously used. Therefore, we decided to investigate if, and how, three major pathways described, in other species, to be regulated by amino acid and to control cellular homeostasis were functional in a RT cell line called RTH-149-namely, the mechanistic Target Of Rapamycin (mTOR), autophagy and the general control nonderepressible 2 (GCN2) pathways. Our results not only demonstrated that these three pathways were functional in RTH-149 cells, but they also highlighted some RT specificities with respect to the time response, amino acid dependencies and the activation levels of their downstream targets. Altogether, this article demonstrated, for the first time, that RT cell lines could represent an interesting alternative of in vivo experimentations for the study of fish nutrition-related questions.
The replacement of fishmeal by plant proteins in aquafeeds imposes the use of synthetic methionine (MET) sources to balance the amino acid composition of alternative diets and so to meet the ...metabolic needs of fish of agronomic interest such as rainbow trout (RT-
). Nonetheless, debates still exist to determine if one MET source is more efficiently used than another by fish. To address this question, the use of fish cell lines appeared a convenient strategy, since it allowed to perfectly control cell growing conditions notably by fully depleting MET from the media and studying which MET source is capable to restore cell growth/proliferation and metabolism when supplemented back. Thus, results of cell proliferation assays, Western blots, RT-qPCR and liquid chromatography analyses from two RT liver-derived cell lines revealed a better absorption and metabolization of DL-MET than DL-Methionine Hydroxy Analog (MHA) with the activation of the mechanistic Target Of Rapamycin (mTOR) pathway for DL-MET and the activation of integrated stress response (ISR) pathway for MHA. Altogether, the results clearly allow to conclude that both synthetic MET sources are not biologically equivalent, suggesting similar in vivo effects in RT liver and, therefore, questioning the MHA efficiencies in other RT tissues.
Autophagy is a pleiotropic and evolutionarily conserved process in eukaryotes that encompasses different types of mechanisms by which cells deliver cytoplasmic constituents to the lysosome for ...degradation. Interestingly, in mammals, two different and specialized autophagic pathways, (i) the chaperone-mediated autophagy (CMA) and (ii) the endosomal microautophagy (eMI), both rely on the use of the same cytosolic chaperone HSPA8 (also known as HSC70) for targeting specific substrates to the lysosome. However, this is not true for all organisms, and differences exist between species with respect to the coexistence of these two autophagic routes. In this paper, we present an in-depth analysis of the evolutionary history of the main components of CMA and eMI and discuss how the observed discrepancies between species may contribute to improving our knowledge of these two functions and their interplays.
There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein ...turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contributes to the pathogenesis of spinocerebellar ataxia type 7 (SCA7), caused by expansion of a polyglutamine tract in the ataxin-7 protein, we looked for biochemical, histological and transcriptomic abnormalities in components of the autophagy/lysosome pathway in a knock-in mouse model of the disease, postmortem brain and peripheral blood mononuclear cells (PBMC) from patients. In the mouse model, mutant ataxin-7 accumulated in inclusions immunoreactive for the autophagy-associated proteins mTOR, beclin-1, p62 and ubiquitin. Atypical accumulations of the autophagosome/lysosome markers LC3, LAMP-1, LAMP2 and cathepsin-
D
were also found in the cerebellum of the SCA7 knock-in mice. In patients, abnormal accumulations of autophagy markers were detected in the cerebellum and cerebral cortex of patients, but not in the striatum that is spared in SCA7, suggesting that autophagy might be impaired by the selective accumulation of mutant ataxin-7. In vitro studies demonstrated that the autophagic flux was impaired in cells overexpressing full-length mutant ataxin-7. Interestingly, the expression of the early autophagy-associated gene
ATG12
was increased in PBMC from SCA7 patients in correlation with disease severity. These results provide evidence that the autophagy/lysosome pathway is impaired in neurons undergoing degeneration in SCA7. Autophagy/lysosome-associated molecules might, therefore, be useful markers for monitoring the effects of potential therapeutic approaches using modulators of autophagy in SCA7 and other autophagy/lysosome-associated neurodegenerative disorders.
Amino acid (AA) transporters (AAT) control AA cellular fluxes across membranes, contributing to maintain cellular homeostasis. In this study, we took advantage of rainbow trout metabolic feature, ...which highly relies on dietary AA, to explore the cellular and physiological consequences of unbalanced diets on AAT dysregulations with a particular focus on cationic AAs (CAA), frequently underrepresented in plant-based diets. Results evidenced that 24 different CAAT are expressed in various trout tissues, part of which being subjected to AA- and CAA-dependent regulations, with y+LAT2 exchanger being prone to the strongest dysregulations. Moreover, CAA were shown to control two major AA-dependent activation pathways (namely mTOR and GCN2) but at different strength according to the CAA considered. A new feed formulation strategy has been put forward to improve specifically the CAA supplemented absorption in fish together with their growth performance. Such “precision formulation” strategy reveals high potential for nutrition practices, especially in aquaculture.
Display omitted
•Dietary glycine supplementation decreases fish feed intake but preserves fish growth•Cationic amino acids (CAA) availabilities tightly control mTOR and GCN2 activities•GCN2 pathway regulates the expression of CAA transporters, including y+LAT2 exchanger•Glycine supplementation improves CAA bioavailability likely through exchange mechanism
It is now well established that tumours undergo changes in cellular metabolism
. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake
, we have been ...interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy. We then show that these effects also occur in vivo in mice following the oral administration of mannose, without significantly affecting the weight and health of the animals. Mechanistically, mannose is taken up by the same transporter(s) as glucose
but accumulates as mannose-6-phosphate in cells, and this impairs the further metabolism of glucose in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and glycan synthesis. As a result, the administration of mannose in combination with conventional chemotherapy affects levels of anti-apoptotic proteins of the Bcl-2 family, leading to sensitization to cell death. Finally we show that susceptibility to mannose is dependent on the levels of phosphomannose isomerase (PMI). Cells with low levels of PMI are sensitive to mannose, whereas cells with high levels are resistant, but can be made sensitive by RNA-interference-mediated depletion of the enzyme. In addition, we use tissue microarrays to show that PMI levels also vary greatly between different patients and different tumour types, indicating that PMI levels could be used as a biomarker to direct the successful administration of mannose. We consider that the administration of mannose could be a simple, safe and selective therapy in the treatment of cancer, and could be applicable to multiple tumour types.
PDF
