SUMMARY Autophagy is an important intracellular catabolic mechanism that mediates the degradation of cytoplasmic proteins and organelles. We report a potent small molecule inhibitor of autophagy named “spautin-1” for specific and potent autophagy inhibitor-1. Spautin-1 promotes the degradation of Vps34 PI3 kinase complexes by inhibiting two ubiquitin-specific peptidases, USP10 and USP13, that target the Beclin1 subunit of Vps34 complexes. Beclin1 is a tumor suppressor and frequently monoallelically lost in human cancers. Interestingly, Beclin1 also controls the protein stabilities of USP10 and USP13 by regulating their deubiquitinating activities. Since USP10 mediates the deubiquitination of p53, regulating deubiquitination activity of USP10 and USP13 by Beclin1 provides a mechanism for Beclin1 to control the levels of p53. Our study provides a molecular mechanism involving protein deubiquitination that connects two important tumor suppressors, p53 and Beclin1, and a potent small molecule inhibitor of autophagy as a possible lead compound for developing anticancer drugs.

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.A utophagy is a cellular catabolic mechanism mediating the turnover of intracellular organelles and proteins through a lysosome-dependent but proteasome-independent degradative pathway (1, 2). An autophagosome sequesters cytoplasmic constituents, such as mitochondria, endoplasmic reticulum, and ribosomes, by forming a double-membrane vesicle. The outer membrane of the autophagosome then fuses with the lysosome in mammalian cells delivering the sequestered content to the lumen of lysosome for degradation. Autophagy is critical for the survival of yeast and mammalian cells under starvation conditions because it functions to recycle intracellular material for macromolecular synthesis and energy production (3).Autophagy occurs in all cells at low basal levels under normal conditions to perform homeostatic functions, but it can be rapidly up-regulated under starvation or stress conditions (3). Elegant genetic analysis has identified 17 genes that are essential for autophagy in yeast (referred to as the ATG genes) (4, 5). In mammalian cells, mTOR kinase, the target of rapamycin, mediates the major inhibitory signal that shuts off autophagy under nutrient-rich conditions (3). On the other hand, mammalian type III PI3-kinase, the homolog of yeast VPS34 and inhibitable by 3-methyladenine (3-MA) (a nonspecific inhibitor of PI3-kinase), is required for the onset of autophagy. In this regard, rapamycin and 3-MA, the most commonly used chemicals to induce and inhibit autophagy, respectively, provide convenient tools to study autophagy.To explore the mechanism of autophagy and identify additional small molecules that can activate it, we developed a high-throughput image-based screen. This system takes advantage of the local...

Balloon cells (BCs) in focal cortical dysplasia (FCD) and giant cells (GCs) in tubers of the tuberous sclerosis complex (TSC) share phenotypic similarities. TSC1 or TSC2 gene mutations in TSC lead to mTOR pathway activation and p70S6kinase (phospho-S6K) and ribosomal S6 (phospho-S6) protein phosphorylation. Phospho-S6K, phospho-S6, and phospho-S6K-activated proteins phospho-STAT3 and phospho-4EBP1 were detected immunohistochemically in GCs, whereas only phospho-S6 was observed in BCs. Expression of four candidate gene families (cell signaling, cell adhesion, growth factor/receptor, and transcription factor mRNAs) was assayed in single, microdissected phospho-S6-immunolabeled BCs and GCs as a strategy to define whether BCs and GCs exhibit differential transcriptional profiles. Among 60 genes, differential expression of 24 mRNAs distinguished BCs from GCs and only 4 genes showed similar expression profiles between BCs and GCs. Tuberin mRNA levels were reduced in GCs from TSC patients with TSC2 gene mutations but were unchanged in BCs. Phospho-S6K, -S6, -STAT3, and -4EBP1 expression in GCs reflects loss of hamartin-tuberin-mediated mTOR pathway inhibition. Phospho-S6 expression alone in BCs does not support mTOR cascade activation in FCD. Differential gene expression profiles in BCs and GCs supports the hypothesis that these cell types derive by distinct pathogenic mechanisms.