Spermidine induces autophagy by inhibiting the acetyltransferase EP300

Abstract

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-microtubuleassociated 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. Macroautophagy (herein referred to as 'autophagy') consist in the sequestration of cytoplasmic material in autophagosomes, followed by their fusion with lysosomes for the bulk degradation of autophagic cargo by lysosomal hydrolases.1 This phenomenon can be measured by following the redistribution of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) fusion proteins from a diffuse location to autophagosomes (that results in the formation of the so-called GFP-LC3 'puncta'), the diminution of the overall abundance of autophagic substrates (such as sequestosome-1, p62/SQSTM1), and the stereotyped activation of proautophagic signals (such as the inhibition of the mammalian target of rapamycin complex 1, mTORC1). 2There is growing consensus that the induction of autophagy by nutritional, pharmacological or genetic interventions can reduce age-related pathologies (such as neurodegenerative diseases or type 2 diabetes) and/or extend longevity. [3][4][5][6] This applies to caloric restriction or intermediate fasting, 7 continuous or intermittent medication of rapamycin, [8][9][10] administration of the sirtuin 1-activator resveratrol, 11,12 external supply of the polyamine spermidine, 13 or genetic ablation of p53.14 In all these cases, inhibition of autophagy by deleting or silencing relevant genes abolishes the extension of health span and/or lifespan. [13][14][15][16][17] Moreover, direct induction of autophagy by transgenic expression of autophag...

Metabolic effects of fasting on human and mouse blood in vivo

“…Experiments were performed in compliance with the EU Directive 63/2010, and protocols (reference number 03981.02) were approved by the local ethics committee (C2EA n. 5, registered at the French Ministry of Research). Nine healthy human volunteers (age 24-54, 5 males, 4 females, all without any known past or present pathology and a normal body mass index of [20][21][22][23][24][25] agreed to engage in a zero-calorie diet (with water, tea and coffee ad libitum) for 4 consecutive d. Heparin blood was drawn every morning between 9 and 9:30 a.m. during the starvation period, as well as on 3 consecutive d (without prior breakfast) 1 wk before and 1 wk after the starvation period. Freshly isolated human leukocytes were incubated for 1 h at 37 C in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum in the presence or absence of 100 mM leupeptin.…”

“…19 Starvation leads to a global decrease in protein acetylation in cells due to the combined increase in the enzymatic activity of protein deacetylases (in particular sirtuins) and the decrease in the activity of acetyltransferases (in particular EP300 [E1A binding protein p300]). [20][21][22][23] Based on these premises, we decided to measure protein acetylation and autophagy in leukocytes from mice or healthy persons in conditions of obligatory or voluntary starvation, respectively. Here, we report that we were able to measure protein deacetylation in leukocytes from starved mice and healthy volunteers that agreed to start a period of up to 5 d of fasting.…”

Spermidine: a physiological autophagy inducer acting as an anti-aging vitamin in humans?

“…Spermidine induces autophagy through the inhibition of several acetyltransferases [7], including EP300 [21], one of the main negative regulators of autophagy [22]. Its potency has been recently quantified to be equivalent to that of rapamycin [23], an FDA-approved immunosuppressant with protective and autophagy-stimulatory properties [24,25].…”

Autophagy in stress and disease

“…Finally, two papers deal with the mode of action of two autophagy-inducing longevity-extending comopounds. Pietrocola et al 13 show that spermidine, which has universal …”

“…Finally, two papers deal with the mode of action of two autophagy-inducing longevity-extending comopounds. Pietrocola et al 13 show that spermidine, which has universal anti-aging effects across multiple model organisms including yeast, nematodes, flies and mice, induces autophagy owing to its capacity to inhibit the enzymatic activity of the EP300 acetyltransferase. Rockenfeller et al…”