Autophagy is an intracellular degradation pathway essential for cellular and energy homoeostasis. It functions in the clearance of misfolded proteins and damaged organelles, as well as recycling of ...cytosolic components during starvation to compensate for nutrient deprivation. This process is regulated by mTOR (mammalian target of rapamycin)-dependent and mTOR-independent pathways that are amenable to chemical perturbations. Several small molecules modulating autophagy have been identified that have potential therapeutic application in diverse human diseases, including neurodegeneration. Neurodegeneration-associated aggregation-prone proteins are predominantly degraded by autophagy and therefore stimulating this process with chemical inducers is beneficial in a wide range of transgenic disease models. Emerging evidence indicates that compromised autophagy contributes to the aetiology of various neurodegenerative diseases related to protein conformational disorders by causing the accumulation of mutant proteins and cellular toxicity. Combining the knowledge of autophagy dysfunction and the mechanism of drug action may thus be rational for designing targeted therapy. The present review describes the cellular signalling pathways regulating mammalian autophagy and highlights the potential therapeutic application of autophagy inducers in neurodegenerative disorders.
Autophagy, a cellular homeostatic process, which ensures cellular survival under various stress conditions, has catapulted to the forefront of innate defense mechanisms during intracellular ...infections. The ability of autophagy to tag and target intracellular pathogens toward lysosomal degradation is central to this key defense function. However, studies involving the role and regulation of autophagy during intracellular infections largely tend to ignore the housekeeping function of autophagy. A growing number of evidences now suggest that the housekeeping function of autophagy, rather than the direct pathogen degradation function, may play a decisive role to determine the outcome of infection and immunological balance. We discuss herein the studies that establish the homeostatic and anti-inflammatory function of autophagy, as well as role of bacterial effectors in modulating and coopting these functions. Given that the core autophagy machinery remains largely the same across diverse cargos, how selectivity plays out during intracellular infection remains intriguing. We explore here, the contrasting role of autophagy adaptors being both selective as well as pleotropic in functions and discuss whether E3 ligases could bring in the specificity to cargo selectivity.
Autophagy is a cellular degradation process involved in the clearance of aggregate-prone proteins associated with neurodegenerative diseases. While the mTOR pathway has been known to be the major ...regulator of autophagy, recent advancements into the regulation of autophagy have identified mTOR-independent autophagy pathways that are amenable to chemical perturbations. Several chemical and genetic screens have been undertaken to identify small molecule and genetic regulators of autophagy, respectively. The small molecule autophagy enhancers offer great potential as therapeutic candidates not only for neurodegenerative diseases, but also for diverse human diseases where autophagy acts as a protective pathway. This review highlights the various chemical screening platforms for autophagy drug discovery pertinent for the treatment of neurodegenerative diseases.
Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine ...cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.
The mammalian target of rapamycin complex 1 (mTORC1) is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. As a prerequisite for ...activation of mTORC1 by hormones and mitogens, there first has to be an available pool of intracellular amino acids. Arginine, an amino acid essential during mammalian embryogenesis and early development is one of the key activators of mTORC1. Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine cooperates with growth factor signaling which further promotes dissociation of TSC2 from lysosomes and activation of mTORC1. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling. Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling.
Maintenance of amino acid homeostasis is important for healthy cellular function, metabolism and growth. Intracellular amino acid concentrations are dynamic; the high demand for protein synthesis ...must be met with constant dietary intake, followed by cellular influx, utilization and recycling of nutrients. Autophagy is a catabolic process via which superfluous or damaged proteins and organelles are delivered to the lysosome and degraded to release free amino acids into the cytoplasm. Furthermore, autophagy is specifically activated in response to amino acid starvation via two key signaling cascades: the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and the general control nonderepressible 2 (GCN2) pathways. These pathways are key regulators of the integration between anabolic (amino acid depleting) and catabolic (such as autophagy which is amino acid replenishing) processes to ensure intracellular amino acid homeostasis. Here, we discuss the key roles that amino acids, along with energy (ATP, glucose) and oxygen, are playing in cellular growth and proliferation. We further explore how sophisticated methods are employed by cells to sense intracellular amino acid concentrations, how amino acids can act as a switch to dictate the temporal and spatial activation of anabolic and catabolic processes and how autophagy contributes to the replenishment of free amino acids, all to ensure cell survival. Relevance of these molecular processes to cellular and organismal physiology and pathology is also discussed.
Autophagy is a nonspecific bulk degradation pathway for long-lived cytoplasmic proteins, protein complexes, or damaged organelles. This process is also a major degradation pathway for many ...aggregate-prone, disease-causing proteins associated with neurodegenerative disorders, such as mutant huntingtin in Huntington's disease. In this review, we discuss factors regulating the degradation of mutant huntingtin by autophagy. We also report the growing list of new drugs/pathways that upregulate autophagy to enhance the clearance of this mutant protein, as autophagy upregulation may be a tractable strategy for the treatment of Huntington's disease.
mTOR (mammalian target of rapamycin) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes, including cellular responses to altered nutrient ...levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates. Here we report that lysosomal positioning coordinates anabolic and catabolic responses with changes in nutrient availability by orchestrating early plasma-membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, whereas starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH. Lysosomal positioning regulates mTORC1 signalling, which in turn influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting at both the initiation and termination stages of the process. Our findings provide a physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.
Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which ...H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.
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•WEE1 inhibition selectively kills H3K36me3-deficient cancer cells•These cells are killed through dNTP starvation because of RRM2 depletion•RRM2 is regulated by H3K36me3 through transcription and WEE1 via degradation•WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumors in vivo
Pfister et al. show that WEE1 inhibition selectively kills H3K36me3-deficient cancer cells through dNTP starvation resulting from RRM2 depletion. Pfister et al. further show that H3K36me3 facilitates RRM2 transcription whereas WEE1 inhibition promotes RRM2 degradation via CDK activation.
(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of ...cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.
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