Summary Xenophagy, a selective autophagy pathway that protects the cytosol against bacterial invasion, relies on cargo receptors that juxtapose bacteria and phagophore membranes. Whether phagophores are recruited from a constitutive pool or are generated de novo at prospective cargo remains unknown. Phagophore formation in situ would require recruitment of the upstream autophagy machinery to prospective cargo. Here, we show that, essential for anti-bacterial autophagy, the cargo receptor NDP52 forms a trimeric complex with FIP200 and SINTBAD/NAP1, which are subunits of the autophagy-initiating ULK and the TBK1 kinase complex, respectively. FIP200 and SINTBAD/NAP1 are each recruited independently to bacteria via NDP52, as revealed by selective point mutations in their respective binding sites, but only in their combined presence does xenophagy proceed. Such recruitment of the upstream autophagy machinery by NDP52 reveals how detection of cargo-associated “eat me” signals, induction of autophagy, and juxtaposition of cargo and phagophores are integrated in higher eukaryotes.
Cells and organisms must coordinate their metabolic activity with changes in their environment to ensure their growth only when conditions are favourable. In order to maintain cellular homoeostasis, a tight regulation between the synthesis and degradation of cellular components is essential. At the epicentre of the cellular nutrient sensing is the mechanistic target of rapamycin complex 1 (mTORC1) which connects environmental cues, including nutrient and growth factor availability as well as stress, to metabolic processes in order to preserve cellular homoeostasis. Under nutrient-rich conditions mTORC1 promotes cell growth by stimulating biosynthetic pathways, including synthesis of proteins, lipids and nucleotides, and by inhibiting cellular catabolism through repression of the autophagic pathway. Its close signalling interplay with the energy sensor AMP-activated protein kinase (AMPK) dictates whether the cell actively favours anabolic or catabolic processes. Underlining the role of mTORC1 in the coordination of cellular metabolism, its deregulation is linked to numerous human diseases ranging from metabolic disorders to many cancers. Although mTORC1 can be modulated by a number of different inputs, amino acids represent primordial cues that cannot be compensated for by any other stimuli. The understanding of how amino acids signal to mTORC1 has increased considerably in the last years; however this area of research remains a hot topic in biomedical sciences. The current ideas and models proposed to explain the interrelationship between amino acid sensing, mTORC1 signalling and autophagy is the subject of the present review.
Cellular senescence, a state of irreversible cell cycle arrest, is thought to help protect an organism from cancer, yet also contributes to ageing. The changes which occur in senescence are controlled by networks of multiple signalling and feedback pathways at the cellular level, and the interplay between these is difficult to predict and understand. To unravel the intrinsic challenges of understanding such a highly networked system, we have taken a systems biology approach to cellular senescence. We report a detailed analysis of senescence signalling via DNA damage, insulin-TOR, FoxO3a transcription factors, oxidative stress response, mitochondrial regulation and mitophagy. We show in silico and in vitro that inhibition of reactive oxygen species can prevent loss of mitochondrial membrane potential, whilst inhibition of mTOR shows a partial rescue of mitochondrial mass changes during establishment of senescence. Dual inhibition of ROS and mTOR in vitro confirmed computational model predictions that it was possible to further reduce senescence-induced mitochondrial dysfunction and DNA double-strand breaks. However, these interventions were unable to abrogate the senescence-induced mitochondrial dysfunction completely, and we identified decreased mitochondrial fission as the potential driving force for increased mitochondrial mass via prevention of mitophagy. Dynamic sensitivity analysis of the model showed the network stabilised at a new late state of cellular senescence. This was characterised by poor network sensitivity, high signalling noise, low cellular energy, high inflammation and permanent cell cycle arrest suggesting an unsatisfactory outcome for treatments aiming to delay or reverse cellular senescence at late time points. Combinatorial targeted interventions are therefore possible for intervening in the cellular pathway to senescence, but in the cases identified here, are only capable of delaying senescence onset.
Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders.
SQSTM1/p62 (sequestosome 1) selectively targets polyubiquitinated proteins for degradation via macroautophagy and the proteasome. Additionally, SQSTM1 shuttles between the cytoplasmic and nuclear compartments, although its role in the nucleus is relatively unknown. Here, we report that SQSTM1 dynamically associates with DNA damage foci (DDF) and regulates DNA repair. Upon induction of DNA damage SQSTM1 interacts with FLNA (filamin A), which has previously been shown to recruit DNA repair protein RAD51 (RAD51 recombinase) to double-strand breaks and facilitate homologous recombination (HR). SQSTM1 promotes proteasomal degradation of FLNA and RAD51 within the nucleus, resulting in reduced levels of nuclear RAD51 and slower DNA repair. SQSTM1 regulates the ratio between HR and nonhomologous end joining (NHEJ) by promoting the latter at the expense of the former. This SQSTM1-dependent mechanism mediates the effect of macroautophagy on DNA repair. Moreover, nuclear localization of SQSTM1 and its association with DDF increase with aging and are prevented by life-span-extending dietary restriction, suggesting that an imbalance in the mechanism identified here may contribute to aging and age-related diseases.
Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.
Age‐related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low‐risk genotype and two patients with advanced AMD and high‐risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H‐like protein 1. The iPSC RPE cells derived from high‐risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of “drüsen”‐like deposits. The low‐ and high‐risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high‐risk AMD‐RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305–2320
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