Valentina Sica

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.

Unsaturated fatty acids induce non‐canonical autophagy

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy‐inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate‐induced, but not oleate‐induced, autophagy in human cancer cells. Moreover, Becn1+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non‐canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.

Phosphatidylethanolamine positively regulates autophagy and longevity

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy.

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-XL is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-XL may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.

The oncolytic peptide LTX-315 kills cancer cells through Bax/Bak-regulated mitochondrial membrane permeabilization

LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes.

Chapter Five – High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy

Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3+ dots by automated fluorescence microscopy.

Evaluation of autophagy inducers in epithelial cells carrying the ΔF508 mutation of the cystic fibrosis transmembrane conductance regulator CFTR

Cystic Fibrosis (CF) due to the ΔF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) can be treated with a combination of cysteamine and Epigallocatechin gallate (EGCG). Since ECGC is not a clinically approved drug, we attempted to identify other compounds that might favourably interact with cysteamine to induce autophagy and thus rescuing the function of ΔF508 CFTR as a chloride channel in the plasma membrane. For this, we screened a compound library composed by chemically diverse autophagy inducers for their ability to enhance autophagic flux in the presence of cysteamine. We identified the antiarrhythmic Ca2+ channel blocker amiodarone, as an FDA-approved drug having the property to cooperate with cysteamine to stimulate autophagy in an additive manner. Amiodarone promoted the re-expression of ΔF508 CFTR protein in the plasma membrane of respiratory epithelial cells. Hence, amiodarone might be yet another compound for the etiological therapy of CF in patients bearing the ΔF508 CFTR mutation.

Chapter Nine - Assessment of Glycolytic Flux and Mitochondrial Respiration in the Course of Autophagic Responses

Autophagy is an evolutionarily conserved process that mediates prominent homeostatic functions, both at the cellular and organismal level. Indeed, baseline autophagy not only ensures the disposal of cytoplasmic entities that may become cytotoxic upon accumulation, but also contributes to the maintenance of metabolic fitness in physiological conditions. Likewise, autophagy plays a fundamental role in the cellular and organismal adaptation to homeostatic perturbations of metabolic, physical, or chemical nature. Thus, the molecular machinery for autophagy is functionally regulated by a broad panel of sensors that detect indicators of metabolic homeostasis. Moreover, increases in autophagic flux have a direct impact on core metabolic circuitries including (but not limited to) glycolysis and mitochondrial respiration. Here, we detail a simple methodological approach to monitor these two processes in cultured cancer cells that mount a proficient autophagic response to stress.

Rapid methods to create a positive control and identify the PAX8/PPARγ rearrangement in FNA thyroid samples by molecular biology

Thyroid cancer is the most common malignancy of the endocrine system and includes well-differentiated forms, namely papillary and follicular carcinomas, and the poorly differentiated and undifferentiated forms that result from the transformation of thyroid follicular cells (anaplastic carcinomas). Notably, 5–10% of all thyroid cancers are medullary thyroid cancers that arise from parafollicular cells also known as C cells. The most common genetic mutations in papillary and follicular thyroid cancers are point mutations of the BRAF or RAS genes, while the most common chromosomal alterations are RET/PTC and PAX8/PPARγ rearrangements. The most frequent initial manifestation of thyroid cancer is the appearance of a nodule most of which are benign; indeed, less than 5% are malignant. However, some cases are misdiagnosed, and many patients undergo unnecessary surgery. Therefore, an accurate pre-surgery evaluation is crucial. The most reliable diagnostic test for thyroid nodules is fine needle aspiration (FNA) cytology, which accurately distinguishes between a benign and malignant lesion in most cases. However, cytological discrimination between malignant and benign follicular cancer is often difficult because of poor quality samples. Here we describe rapid methods to create a positive control and identify the PAX8/PPARγ rearrangement in FNA thyroid samples by molecular biology.

IMMP2L: a mitochondrial protease suppressing cellular senescence

Cellular senescence contributes to organismal aging and tumor suppression. A recent paper by Yuan et al. published in Cell Research now unveils that switching off a mitochondrial protease can simultaneously suppress phospholipid biosynthesis and lethal signaling for senescence induction. Organismal aging is coupled to the accumulation of senescent cells that, by definition, are permanently arrested in their cell cycle and often manifest the senescence-associated secretory phenotype (SASP), hence favoring chronic tissue inflammation. One prominent biomarker of cellular senescence is the enhanced expression of cyclin-dependent kinase (CDK) inhibitor 2A (CDKN2A, best known as p16). Genetic ablation of senescent cells by expressing inducible suicide genes under the control of the CDKN2A promoter can retard and reverse aging phenotypes in mice. That said, the phenomenon of cellular senescence also has important physiological functions, for instance in the suppression of oncogenesis (by irreversible arrest of the cell cycle in potentially malignant cells) and in wound healing (presumably because the SASP can favor local tissue repair). Much of the research on senescence has focused on the tumor suppressor p53 protein (TP53)-dependent or -independent mechanisms leading to the activation of CDKN2A or other CDKs. In a recent paper in Cell Research, Yuan et al. reveal the surprising finding that one major mechanism leading to senescence may consist in the shut down of inner mitochondrial membrane peptidase subunit 2 (IMMP2L), through downregulation of protein expression or inactivation by an endogenous clade B serine protease inhibitor named SERPINB4. Indeed, in normal bronchial epithelial cells, induction of senescence by blockade of epidermal growth factor receptor was linked to the transcriptional upregulation of SERPINB4, and SERPINB4 knockdown could prevent senescence in this model, while its overexpression was sufficient to trigger senescence. Similarly, knockdown of IMMP2L, which turned out to be the principal target of SERPINB4, stimulated the senescent program in various human cell types in vitro echoing the prior observation that mice bearing a mutation in the IMMP2L locus display a progeroid (early aging) phenotype. In the muscle from aged normal mice, as well as in peripheral blood from fragile old humans, IMMP2L was diminished (while CDKN2A protein was increased) as compared to young controls. More importantly, deletions of intron 3 of IMMP2L were less frequent in centenarians than in unrelated control individuals, indicating that longevity is associated with genomic integrity of the IMMP2L locus All these findings argue in favor of the conjecture that organismal aging and cellular senescence are coupled to the downregulation or inhibition of IMMP2L (Fig. 1). It remains to be seen, however, whether transgene-enforced upregulation of IMMP2L or depletion/deletion of its inhibitor SERPIN4B may reduce the manifestations of aging in whole organisms. Moreover, it might be interesting to develop pharmacological inhibitors of the IMMP2L/SERPIN4B interaction to allow newly synthesized IMMP2L to escape from its irreversible blockade by SERPIN4B, thus reactivating its function. By which mechanism can IMMP2L suppress senescence? Through a series of elegantly conducted experiments, Yuan et al. demonstrate that, in non-senescent cells, IMMP2L assures the proteolytic maturation of two major substrates, namely glycerol-3-phosphate dehydrogenase 2 (GPD2), a metabolic enzyme, and apoptosis-inducing factor (AIF), a redox-active flavoprotein that contributes to the biogenesis of the respiratory chain complex I, but also has a potentially lethal function. After its processing by IMMP2L, GPD2 locates at the inner mitochondrial membrane where it catalyzes the conversion of glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP). Together with GPD1, GPD2 constitutes the glycerol phosphate shuttle, which reoxidizes NADH formed during glycolysis, coupling this reaction to the reduction of coenzyme Q (ubiquinone to ubiquinol), which enters into oxidative phosphorylation. Apparently, knockdown of GPD2 is sufficient to recapitulate many features of cellular senescence, phenocopying the effect of IMMP2L depletion. GPD2 may have two effects on cellular metabolism that have anti-senescent effects. On one hand, it may increase NAD levels, knowing that NAD has prominent pro-longevity effects. On the other hand, GPD2 may favor mitochondrial phospholipid synthesis by providing the precursor G3P. Indeed, the abundance of mitochondrial phospholipids and phospholipid-binding proteins such as protein kinase C-δ (PKC-δ) diminished upon depletion of either IMMP2L or GPD2. This effect was coupled to reduced phosphorylation of the PKC-δ substrate pyruvate dehydrogenase kinase (PDK), resulting in subsequent dephosphorylation and enhanced activity of the PDK substrate pyruvate dehydrogenase (PDH). PDH favors the catabolism of pyruvate via the tricarboxylic acid cycle and has previously been implicated as a key effector of oncogene-induced senescence. Altogether, this cascade illustrates how failed processing of one metabolic enzyme, GPD2, by IMMP2L can precipitate a cascade of