Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction.

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 µM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1ß (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy.Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Acute appendicitis in children: Reexamining indications for conservative treatment - A large prospective analysis.

INTRODUCTION: Conservative antibiotic treatment (CAT) for uncomplicated acute appendicitis (AUA) in children has been proven safe and efficacious. However, as data accumulate, high rates of recurrent appendicitis and subsequent appendectomy have been reported. This prospective longitudinal study evaluated risk-factors for recurrent AUA after CAT in a large cohort, with long-term follow-up. MATERIALS AND METHODS: Children ages 5 to 16 years admitted to the Department of Pediatric Surgery from 2014 through 2018, diagnosed with AUA were eligible for CAT. We recorded their age, appendix outer diameter, white blood cell counts, C-reactive protein and other related signs and symptoms associated with AUA. Clinical and ultrasonographic follow-up was carried out until follow-up data collection ceased according to the study design (2014-2019). RESULTS: The cohort included 646 children who were initially treated successfully with CAT. Among them, 180 (28%) were readmitted for recurrent acute appendicitis during the follow-up period and 138 (21%) eventually had appendectomy. Overall success of 79% for CAT was recorded in this cohort. A multivariable model including; age, sex, appendiceal diameter, WBC and CRP, found the factors of older age, larger outer appendiceal diameter and high WBC counts significantly related to appendectomy during the follow-up period. We offer a decision tree model to predict appendectomy probabilities for patients based on their prognostic measurements. CONCLUSION: CAT in AUA in children should consider older age, larger outer appendiceal diameter and high WBC counts as risk-factors for recurrent AUA and subsequent appendectomy. The proposed decision tree model may help both clinicians and parents before CAT is chosen. LEVEL OF EVIDENCE: Level 2.

ETS Proteins Bind with Glucocorticoid Receptors: Relevance for Treatment of Ewing Sarcoma.

The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR's transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.

A cancer associated somatic mutation in LC3B attenuates its binding to E1-like ATG7 protein and subsequent lipidation.

Macroautophagy/autophagy is a conserved catabolic process that maintains cellular homeostasis under basal growth and stress conditions. In cancer, autophagy can either prevent or promote tumor growth, at early or advanced stages, respectively. We screened public databases to identify autophagy-related somatic mutations in cancer, using a computational approach to identify cancer mutational target sites, employing exact statistics. The top significant hit was a missense mutation (Y113C) in the MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) protein, which occurred at a significant frequency in cancer, and was detected in early stages in primary tumors of patients with known tumor lineage. The mutation reduced the formation of GFP-LC3B puncta and attenuated LC3B lipidation during Torin1-induced autophagy. Its effect on the direct physical interaction of LC3B with each of the 4 proteins that control its maturation or lipidation was tested by applying a protein-fragment complementation assay and co-immunoprecipitation experiments. Interactions with ATG4A and ATG4B proteases were reduced, yet without perturbing the cleavage of mutant LC3B. Most importantly, the mutation significantly reduced the interaction with the E1-like enzyme ATG7, but not the direct interaction with the E2-like enzyme ATG3, suggesting a selective perturbation in the binding of LC3B to some of its partner proteins. Structure analysis and molecular dynamics simulations of LC3B protein and its mutant suggest that the mutation changes the conformation of a loop that has several contact sites with ATG4B and the ATG7 homodimer. We suggest that this loss-of-function mutation, which attenuates autophagy, may promote early stages of cancer development.

Non-canonical activation of DAPK2 by AMPK constitutes a new pathway linking metabolic stress to autophagy.

Autophagy is an intracellular degradation process essential for adaptation to metabolic stress. DAPK2 is a calmodulin-regulated protein kinase, which has been implicated in autophagy regulation, though the mechanism is unclear. Here, we show that the central metabolic sensor, AMPK, phosphorylates DAPK2 at a critical site in the protein structure, between the catalytic and the calmodulin-binding domains. This phosphorylation activates DAPK2 by functionally mimicking calmodulin binding and mitigating an inhibitory autophosphorylation, providing a novel, alternative mechanism for DAPK2 activation during metabolic stress. In addition, we show that DAPK2 phosphorylates the core autophagic machinery protein, Beclin-1, leading to dissociation of its inhibitor, Bcl-XL. Importantly, phosphorylation of DAPK2 by AMPK enhances DAPK2's ability to phosphorylate Beclin-1, and depletion of DAPK2 reduces autophagy in response to AMPK activation. Our study reveals a unique calmodulin-independent mechanism for DAPK2 activation, critical to its function as a novel downstream effector of AMPK in autophagy.

Discovering protein-protein interactions within the programmed cell death network using a protein-fragment complementation screen.

Apoptosis and autophagy are distinct biological processes, each driven by a different set of protein-protein interactions, with significant crosstalk via direct interactions among apoptotic and autophagic proteins. To measure the global profile of these interactions, we adapted the Gaussia luciferase protein-fragment complementation assay (GLuc PCA), which monitors binding between proteins fused to complementary fragments of a luciferase reporter. A library encompassing 63 apoptotic and autophagic proteins was constructed for the analysis of ∼3,600 protein-pair combinations. This generated a detailed landscape of the apoptotic and autophagic modules and points of interface between them, identifying 46 previously unknown interactions. One of these interactions, between DAPK2, a Ser/Thr kinase that promotes autophagy, and 14-3-3τ, was further investigated. We mapped the region responsible for 14-3-3τ binding and proved that this interaction inhibits DAPK2 dimerization and activity. This proof of concept underscores the power of the GLuc PCA platform for the discovery of biochemical pathways within the cell death network.

The programmed cell death GLuc PCA library - a powerful tool for pathway discovery and drug screening.

A programmed cell death library based on the Gaussia luciferase protein-fragment complementation assay (GLuc PCA) enables detection of protein-protein interactions (PPI) within the cell death network and quantitative assessments of these interactions. Among future applications for the GLuc PCA cell death library is its potential use as a platform for PPI-targeted drug screening.

Combining an EGFR directed tyrosine kinase inhibitor with autophagy-inducing drugs: a beneficial strategy to combat non-small cell lung cancer.

The potential therapeutic value of combinatorial regimens based on an EGF receptor tyrosine kinase inhibitor (TKI) and autophagy inducing drugs was evaluated by comparing their molecular impacts on H1299 and A549 non-small cell lung cancer (NSCLC) cells, which overexpress wild type EGF receptor, but are either deficient or have wild type p53 alleles, respectively. We show that H1299 cells display a considerably lower sensitivity to erlotinib treatment, which can be restored by combining erlotinib with rapamycin or with imatinib, though to a lesser extent. Cytotoxicity was associated with increased autophagy and hyperpolarization of the mitochondrial membrane potential. Therefore, combining an EGF receptor directed TKI with an autophagy-inducing drug, preferably, rapamycin, might be beneficial in treating poor responding NSCLC patients.

Glycoside hydrolases as components of putative carbohydrate biosensor proteins in Clostridium thermocellum.

The composition of the cellulase system in the cellulosome-producing bacterium, Clostridium thermocellum, has been reported to change in response to growth on different carbon sources. Recently, an extensive carbohydrate-sensing mechanism, purported to regulate the activation of genes coding for polysaccharide-degrading enzymes, was suggested. In this system, CBM modules, comprising extracellular components of RsgI-like anti-σ factors, were proposed to function as carbohydrate sensors, through which a set of cellulose utilization genes are activated by the associated σ(I)-like factors. An extracellular module of one of these RsgI-like proteins (Cthe_2119) was annotated as a family 10 glycoside hydrolase, RsgI6-GH10, and a second putative anti-σ factor (Cthe_1471), related in sequence to Rsi24, was found to contain a module that resembles a family 5 glycoside hydrolase (termed herein Rsi24C-GH5). The present study examines the relevance of these two glycoside hydrolases as sensors in this signal-transmission system. The RsgI6-GH10 was found to bind xylan matrices but exhibited low enzymatic activity on this substrate. In addition, this glycoside hydrolase module was shown to interact with crystalline cellulose although no hydrolytic activity was detected on cellulosic substrates. Bioinformatic analysis of the Rsi24C-GH5 showed a glutamate-to-glutamine substitution that would presumably preclude catalytic activity. Indeed, the recombinant module was shown to bind to cellulose, but showed no hydrolytic activity. These observations suggest that these two glycoside hydrolases underwent an evolutionary adaptation to function as polysaccharide binding agents rather than enzymatic components and thus serve in the capacity of extracellular carbohydrate sensors.