Hongguang Xia

Control of basal autophagy by calpain1 mediated cleavage of ATG5

Autophagy functions as an important catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes. Although the induction of autophagy by starvation has been extensively studied, we still understand very little about how autophagy is regulated under normal nutritional conditions. Here we describe a study using a small molecule autophagy inducer, fluspirilene, as a tool to explore the mechanism of autophagy induction in normal living cells. We confirm the activity of fluspirilene in inhibiting Ca2+ flux. Furthermore, we show that reducing intracellular Ca2+ prevents the cleavage of ATG5, which in turn increases the levels of full-length ATG5 and ATG12-ATG5 conjugate. Using siRNA mediated gene silencing, we demonstrate that inhibiting calpain1 is sufficient to induce autophagy in living cells. We conclude that calpain1 plays an important role in controlling the levels of autophagy in normal living cells by regulating the levels of a key signaling molecule, ATG12-ATG5 conjugate.

Pharmacologic agents targeting autophagy.

Autophagy is an important intracellular catabolic mechanism critically involved in regulating tissue homeostasis. The implication of autophagy in human diseases and the need to understand its regulatory mechanisms in mammalian cells have stimulated research efforts that led to the development of high-throughput screening protocols and small-molecule modulators that can activate or inhibit autophagy. Herein we review the current landscape in the development of screening technology as well as the molecules and pharmacologic agents targeting the regulatory mechanisms of autophagy. We also evaluate the potential therapeutic application of these compounds in different human pathologies.

Chaperone-mediated autophagy degrades mutant p53

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells.

Recent advances in photoinduced trifluoromethylation and difluoroalkylation

Recent advances in photoinduced trifluoromethylation and difluoroalkylation under photocatalysis are summarized. Most of the photoredox reactions proceed efficiently under mild conditions with simple operation. Various fluorinated reagents are developed and applied in different transformations. Usually, the photocatalyst in a photocatalytic cycle is initially stimulated to the excited state in the presence of visible light, which then provides an electron to the fluorinated reagent to release a CF3 radical or CF2 radical. The subsequent radical addition to an unsaturated bond with further transformations would produce the corresponding fluorinated products. Alkenes, alkynes, isocyanides, and arenes are frequently employed as substrates for access to fluorochemicals. The advantages of trifluoromethylation and difluoroalkylation under photocatalysis including mild reaction conditions, good functional group tolerance, and experimental ease will make the approaches attractive for further applications.

Generation of (2-oxoindolin-3-yl)methanesulfonohydrazides via a photo-induced reaction of N-(2-iodoaryl)acrylamide, DABSO, and hydrazine

A photo-induced catalyst-free three-component reaction of N-(2-iodoaryl)acrylamide, sulfur dioxide, and hydrazine is reported. Under ultraviolet irradiation, diverse (2-oxoindolin-3-yl)methanesulfonohydrazides are generated in moderate to good yields. During the process, the sulfonyl group can be easily incorporated via insertion of sulfur dioxide in the absence of any metals or photo-redox catalysts under mild conditions. A plausible mechanism is proposed, which involves 5-exo radical cyclization and subsequent insertion of sulfur dioxide.

Copper(I)-catalyzed sulfonylation of (2-alkynylaryl)boronic acids with DABSO

The scaffold of benzo[b]thiophene 1,1-dioxides can be easily constructed through a copper(I)-catalyzed insertion of sulfur dioxide into (2-alkynylaryl)boronic acids. The reaction proceeds in the presence of 10 mol% copper(I) acetate in DMF at 100 °C with high efficiency, leading to benzo[b]thiophene 1,1-dioxides in good to excellent yields. The sulfonyl group can be easily introduced via insertion of sulfur dioxide and the subsequent intramolecular 5-endo cyclization affords the core of benzo[b]thiophene 1,1-dioxide.

Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

Metabolic stress caused by perturbation of receptor tyrosine kinase FLT3 sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy, which triggers metabolic catastrophe in cancer cells through the degradation of HK2.

Mitochondrial Electron Transport Chain Complex III Is Required for Antimycin A to Inhibit Autophagy

Autophagy is a cellular lysosome-dependent catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. We show that antimycin A, a known inhibitor of mETC complex III, can inhibit autophagy. A structural and functional study shows that four close analogs of antimycin A that have no effect on mitochondria inhibition also do not inhibit autophagy, whereas myxothiazol, another mETC complex III inhibitor with unrelated structure to antimycin A, inhibits autophagy. Additionally, antimycin A and myxothiazol cannot inhibit autophagy in mtDNA-depleted H4 and mtDNA-depleted HeLa cells. These data suggest that antimycin A inhibits autophagy through its inhibitory activity on mETC complex III. Our data suggest that mETC complex III may have a role in mediating autophagy induction.

Generation of benzosultams via a radical process with the insertion of sulfur dioxide

An efficient route to diverse 3-sulfonated-2,3-dihydrobenzo[d]isothiazole 1,1-dioxides is achieved through a three-component reaction of 2-ethynylbenzenesulfonamides, DABCO-bis(sulfur dioxide), and aryldiazonium tetrafluoroborates. The corresponding sulfonated benzosultams are produced in moderate to good yields. During the reaction process, the in situ generated arylsulfonyl radical via addition of an aryl radical to sulfur dioxide and the subsequent single electron transfer would be the key steps for the final outcome. DABCO acts as the carrier for single electron transfer, as well as a base to promote the C–N bond formation.

G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L

Autophagy is an important intracellular catabolic mechanism involved in the removal of misfolded proteins. Atg14L, the mammalian ortholog of Atg14 in yeast and a critical regulator of autophagy, mediates the production PtdIns3P to initiate the formation of autophagosomes. However, it is not clear how Atg14L is regulated. In this study, we demonstrate that ubiquitination and degradation of Atg14L is controlled by ZBTB16-Cullin3-Roc1 E3 ubiquitin ligase complex. Furthermore, we show that a wide range of G-protein-coupled receptor (GPCR) ligands and agonists regulate the levels of Atg14L through ZBTB16. In addition, we show that the activation of autophagy by pharmacological inhibition of GPCR reduces the accumulation of misfolded proteins and protects against behavior dysfunction in a mouse model of Huntingtons disease. Our study demonstrates a common molecular mechanism by which the activation of GPCRs leads to the suppression of autophagy and a pharmacological strategy to activate autophagy in the CNS for the treatment of neurodegenerative diseases. DOI: http://dx.doi.org/10.7554/eLife.06734.001