Fluorescent FYVE Chimeras to Quantify PtdIns3P Synthesis During Autophagy.

Autophagy is the major cellular process of degradation and is modulated by several signaling pathways. Phosphatidylinositol 3-kinase (PtdIns3K) class III (Vps34) and PtdIns3K class I regulate the autophagy pathway positively and negatively, respectively. Both classes of PtdIns3K participate in the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P), which plays a crucial role in autophagosome biogenesis and membrane traffic. PtdIns3P is a membrane phospholipid that is associated with endogenous FYVE domain-containing proteins. Indeed, such interactions facilitate autophagosome fusion with lysosomes and subsequent cargo degradation. During starvation-induced autophagy, the expression of FYVE domain-containing proteins increases, and their binding to PtdIns3P is strengthened. Nonetheless, not all FYVE domain proteins are related to the induction of autophagy. This method report presents the quantification of PtdIns3P synthesis by using cells either transiently transfected with or stably expressing FYVE-dsRed.

Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells.

Macroautophagy (usually referred to as autophagy) is the most important degradation system in mammalian cells. It is responsible for the elimination of protein aggregates, organelles, and other cellular content. During autophagy, these materials (i.e., cargo) must be engulfed by a double-membrane structure called an autophagosome, which delivers the cargo to the lysosome to complete its degradation. Autophagy is a very dynamic pathway called autophagic flux. The process involves all the steps that are implicated in cargo degradation from autophagosome formation. There are several techniques to monitor autophagic flux. Among them, the method most used experimentally to assess autophagy is the detection of LC3 protein processing and p62 degradation by Western blotting. In this chapter, we provide a detailed and straightforward protocol for this purpose in cultured mammalian cells, including a brief set of notes concerning problems associated with the Western-blotting detection of LC3 and p62.

Mitochondria-Associated Membranes (MAMs): Overview and Its Role in Parkinson's Disease.

Mitochondria-associated membranes (MAMs) are structures that regulate physiological functions between endoplasmic reticulum (ER) and mitochondria in order to maintain calcium signaling and mitochondrial biogenesis. Several proteins located in MAMs, including those encoded by PARK genes and some of neurodegeneration-related proteins (huntingtin, presenilin, etc.), ensure this regulation. In this regard, MAM alteration is associated with neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and Huntington's diseases (HD) and contributes to the appearance of the pathogenesis features, i.e., autophagy dysregulation, mitochondrial dysfunction, oxidative stress, and lately, neuronal death. Moreover,, ER stress and/or damaged mitochondria can be the cause of these disruptions. Therefore, ER-mitochondria contact structure and function are crucial to multiple cellular processes. This review is focused on the molecular interaction between ER and mitochondria indispensable to MAM formation and on MAM alteration-induced etiology of neurodegenerative diseases.

Pompe Disease and Autophagy: Partners in Crime, or Cause and Consequence?

Pompe disease or glycogen storage disease type II (OMIM: 232300) is a lysosomal storage disorder resulting from a partial or total lack of acid alphaglucosidase, which may produce muscle weakness, gait abnormalities, or even death by respiratory failure. In the last decade, autophagy has been proposed as a mechanism involved in the severity of symptoms related to this disorder and as a potential therapeutic target to alleviate disease progression. This review summarizes the relationship between autophagy and Pompe disease, including what information has been recently discovered and what remains unclear.

Pharmacological inhibition of fatty-acid oxidation synergistically enhances the effect of l-asparaginase in childhood ALL cells.

l-asparaginase (ASNase), a key component in the treatment of childhood acute lymphoblastic leukemia (ALL), hydrolyzes plasma asparagine and glutamine and thereby disturbs metabolic homeostasis of leukemic cells. The efficacy of such therapeutic strategy will depend on the capacity of cancer cells to adapt to the metabolic challenge, which could relate to the activation of compensatory metabolic routes. Therefore, we studied the impact of ASNase on the main metabolic pathways in leukemic cells. Treating leukemic cells with ASNase increased fatty-acid oxidation (FAO) and cell respiration and inhibited glycolysis. FAO, together with the decrease in protein translation and pyrimidine synthesis, was positively regulated through inhibition of the RagB-mTORC1 pathway, whereas the effect on glycolysis was RagB-mTORC1 independent. As FAO has been suggested to have a pro-survival function in leukemic cells, we tested its contribution to cell survival following ASNase treatment. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, constitutive activation of the mammalian target of rapamycin pathway increased apoptosis in leukemic cells treated with ASNase, but did not increase FAO. Our study uncovers a novel therapeutic option based on the combination of ASNase and FAO inhibitors.

Spermidine induces autophagy by inhibiting the acetyltransferase EP300.

Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.