Screen for Chemical Modulators of Autophagy Reveals Novel Therapeutic Inhibitors of mTORC1 Signaling

Background

Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties.

Methodology/Principal Findings

Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation.

Conclusion/Significance

The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.     

Comparative Analysis of RNA Families Reveals Distinct Repertoires for Each Domain of Life

The RNA world hypothesis, that RNA genomes and catalysts preceded DNA genomes and genetically-encoded protein catalysts, has been central to models for the early evolution of life on Earth. A key part of such models is continuity between the earliest stages in the evolution of life and the RNA repertoires of extant lineages. Some assessments seem consistent with a diverse RNA world, yet direct continuity between modern RNAs and an RNA world has not been demonstrated for the majority of RNA families, and, anecdotally, many RNA functions appear restricted in their distribution. Despite much discussion of the possible antiquity of RNA families, no systematic analyses of RNA family distribution have been performed. To chart the broad evolutionary history of known RNA families, we performed comparative genomic analysis of over 3 million RNA annotations spanning 1446 families from the Rfam 10 database. We report that 99% of known RNA families are restricted to a single domain of life, revealing discrete repertoires for each domain. For the 1% of RNA families/clans present in more than one domain, over half show evidence of horizontal gene transfer (HGT), and the rest show a vertical trace, indicating the presence of a complex protein synthesis machinery in the Last Universal Common Ancestor (LUCA) and consistent with the evolutionary history of the most ancient protein-coding genes. However, with limited interdomain transfer and few RNA families exhibiting demonstrable antiquity as predicted under RNA world continuity, our results indicate that the majority of modern cellular RNA repertoires have primarily evolved in a domain-specific manner.     

Global Analysis of ATM Polymorphism Reveals Significant Functional Constraint

ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%-100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure.     

Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.     

Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity

1. Download high-res image (186KB)
2. Download full-size image

     

Meta-Analysis of Arabidopsis thaliana Phospho-Proteomics Data Reveals Compartmentalization of Phosphorylation Motifs

Protein (de)phosphorylation plays an important role in plants. To provide a robust foundation for subcellular phosphorylation signaling network analysis and kinase-substrate relationships, we performed a meta-analysis of 27 published and unpublished in-house mass spectrometry–based phospho-proteome data sets for Arabidopsis thaliana covering a range of processes, (non)photosynthetic tissue types, and cell cultures. This resulted in an assembly of 60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data for quality and consistency generated a set of medium and a set of high confidence phospho-proteins and their assigned phospho-sites. The relation between single and multiphosphorylated peptides is discussed. The distribution of p-proteins across cellular functions and subcellular compartments was determined and showed overrepresentation of protein kinases. Extensive differences in frequency of pY were found between individual studies due to proteomics and mass spectrometry workflows. Interestingly, pY was underrepresented in peroxisomes but overrepresented in mitochondria. Using motif-finding algorithms motif-x and MMFPh at high stringency, we identified compartmentalization of phosphorylation motifs likely reflecting localized kinase activity. The filtering of the data assembly improved signal/noise ratio for such motifs. Identified motifs were linked to kinases through (bioinformatic) enrichment analysis. This study also provides insight into the challenges/pitfalls of using large-scale phospho-proteomic data sets to nonexperts.     

Network-Based Elucidation of Human Disease Similarities Reveals Common Functional Modules Enriched for Pluripotent Drug Targets

Current work in elucidating relationships between diseases has largely been based on pre-existing knowledge of disease genes. Consequently, these studies are limited in their discovery of new and unknown disease relationships. We present the first quantitative framework to compare and contrast diseases by an integrated analysis of disease-related mRNA expression data and the human protein interaction network. We identified 4,620 functional modules in the human protein network and provided a quantitative metric to record their responses in 54 diseases leading to 138 significant similarities between diseases. Fourteen of the significant disease correlations also shared common drugs, supporting the hypothesis that similar diseases can be treated by the same drugs, allowing us to make predictions for new uses of existing drugs. Finally, we also identified 59 modules that were dysregulated in at least half of the diseases, representing a common disease-state “signature”. These modules were significantly enriched for genes that are known to be drug targets. Interestingly, drugs known to target these genes/proteins are already known to treat significantly more diseases than drugs targeting other genes/proteins, highlighting the importance of these core modules as prime therapeutic opportunities.     

Structural and Inhibition Analysis Reveals the Mechanism of Selectivity of a Series of Aggrecanase Inhibitors

Several inhibitors of a series of cis-1(S)2(R)-amino-2-indanol-based compounds were reported to be selective for the aggrecanases, ADAMTS-4 and -5 over other metalloproteases. To understand the nature of this selectivity for aggrecanases, the inhibitors, along with the broad spectrum metalloprotease inhibitor marimastat, were independently bound to the catalytic domain of ADAMTS-5, and the corresponding crystal structures were determined. By comparing the structures, it was determined that the specificity of the relative inhibitors for ADAMTS-5 was not driven by a specific interaction, such as zinc chelation, hydrogen bonding, or charge interactions, but rather by subtle and indirect factors, such as water bridging, ring rigidity, pocket size, and shape, as well as protein conformation flexibility.Osteoarthritis (OA)³ pathology includes degradation of articular cartilage, along with subchondral bone sclerosis and osteophyte formation, all contributing to impaired joint function. Pain, restricted movement, and joint instability accompany these structural changes and often result in the need for total joint replacement. Current therapies alleviate the mild to moderate pain and inflammation associated with OA, but do not protect the cartilage from further damage and have not demonstrated an effect on disease progression (1). Therefore, therapeutics that prevent or slow the alteration of joint structure and function will address a major unmet medical need.Loss of aggrecan, a macromolecular proteoglycan providing cartilage with its properties of compressibility and resilience, is a major phenotype associated with OA and is believed to be a critical event in driving disease progression (2, 3). Both ex vivo and in vivo proof of concept studies support ADAMTS-4 and ADAMTS-5, commonly referred to as aggrecanase-1 and -2, respectively, as the two major enzymes responsible for the proteolytic breakdown of cartilage aggrecan (reviewed in Ref. 4). Blocking their activity may be an attractive strategy to stop or slow down the progression of the disease, as suggested by studies in knock-out mice (5). Given the chronic nature of the disease, long term treatment will be likely, demanding very safe therapeutic interventions only achievable with ADAMTS-4- and ADAMTS-5-specific inhibitors lacking off-target side effects. Designing selectivity has been very challenging and a major source of difficulty is that at least 57 metalloproteases (MP) divided in three major families, 1) matrix metalloproteases (MMP); 2) a disintegrin and metalloproteinase (ADAM); and 3) a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), are present in humans. ADAMTS-4 and -5 belong to the ADAMTS family and share common catalytic and structural features with the other MP members. These features include the highly conserved amino acid sequence, HEXXHXXGXXH, harboring a catalytic zinc cation, required for activation of the peptide bond toward hydrolysis. In addition, many MPs share significant structural topology in the active site, such as a flexible S1′ loop. To complicate matters further, only a handful of MP structures, mostly in the MMP family, have been determined, and the functions of most MPs still remain unknown, a fact that has earned them an orphan status denomination. Lack of structural information has hindered the design for inhibitor specificity and without known substrates for many MPs, assays for screening inhibitors are often not available, making determinations of selectivity difficult (6, 7).Yao et al. (8, 9) reported the discovery of a series of (2R)-N4-hydroxy-2-(3-hydroxybenzyl)-N1-[(1S,2R)-2-hydroxy-,3-dihydro-1H-inden-1-yl]butanediamide derivatives as potent and selective inhibitors of aggrecanase activity. Using a homology model of aggrecanase based on the active site of atrolysin C and adamalysin II and docking compound 8, shown in Fig. 1, the authors concluded that the 3-hydroxyl group of inhibitor 8 achieved selectivity through a specific hydrogen-bonding interaction with Thr⁴⁴⁰ (numerical numbering is based on the human sequence of ADAMTS-5) in the S1′ pocket of aggrecanase.Open in a separate windowFIGURE 1.Aggrecanase inhibitors. Structures of the inhibitors evaluated in these studies. The P1 moiety of each inhibitor is marked in red.Whereas both ADAMTS-4 and -5 have a threonine at this position, MMP-1, -2, -3, -7, -8, -9, -10, -13, -14, -16, and ADAM-17 have a valine, lending credence to the proposed hypothesis around selectivity. Recently, our group has established a protocol for crystallizing the catalytic domain of ADAMTS-5 and determined its three-dimensional structure (10). Thus, experimental validation or invalidation of the hypothesis that inhibitor 8 and related molecules form a specific hydrogen bond with the hydroxyl group of threonine in the S1′ pocket of aggrecanases could now be determined by protease-inhibitor crystallographic analysis.In the current study, we wanted to confirm and extend the selectivity profile of compound 8 and 11 against a wide array of MPs. Moreover, we wanted to elucidate the key molecular interactions responsible for the enhanced selectivity profile of this series of compounds. For this purpose we generated co-crystals and solved the structures of marimastat, compound 8, and compound 11 in complex with the catalytic domain of recombinant human ADAMTS-5.     

Global Analysis of Apicomplexan Protein S‐Acyl Transferases Reveals an Enzyme Essential for Invasion

The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S‐acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp‐His‐His‐Cys cysteine‐rich domain (DHHC‐CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan‐specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite's ability to egress.     

Defining A Global Map of Functional Group-based 3D Ligand-binding Motifs

Uncovering conserved 3D protein–ligand binding patterns on the basis of functional groups (FGs) shared by a variety of small molecules can greatly expand our knowledge of protein–ligand interactions. Despite that conserved binding patterns for a few commonly used FGs have been reported in the literature, large-scale identification and evaluation of FG-based 3D binding motifs are still lacking. Here, we propose a computational method, Automatic FG-based Three-dimensional Motif Extractor (AFTME), for automatic mapping of 3D motifs to different FGs of a specific ligand. Applying our method to 233 naturally-occurring ligands, we define 481 FG-binding motifs that are highly conserved across different ligand-binding pockets. Systematic analysis further reveals four main classes of binding motifs corresponding to distinct sets of FGs. Combinations of FG-binding motifs facilitate the binding of proteins to a wide spectrum of ligands with various binding affinities. Finally, we show that our FG–motif map can be used to nominate FGs that potentially bind to specific drug targets, thus providing useful insights and guidance for rational design of small-molecule drugs.