Small-Molecule Allosteric Inhibitors of Human Aspartate Transcarbamoylase Suppress Proliferation of Bone Osteosarcoma Epithelial Cells.

Aspartate transcarbamoylase (ATC) is the first committed step in de novo pyrimidine biosynthesis in eukaryotes and plants. A potent transition state analog of human ATCase (PALA) has previously been assessed in clinical trials for the treatment of cancer, but was ultimately unsuccessful. Additionally, inhibition of this pathway has been proposed to be a target to suppress cell proliferation in E. coli, the malarial parasite and tuberculosis. In this manuscript we screened a 70-member library of ATC inhibitors developed against the malarial and tubercular ATCases for inhibitors of the human ATC. Four compounds showed low nanomolar inhibition (IC50 30-120 nM) in an in vitro activity assay. These compounds significantly outperform PALA, which has a triphasic inhibition response under identical conditions, in which significant activity remains at PALA concentrations above 10 µM. Evidence for a druggable allosteric pocket in human ATC is provided by both in vitro enzyme kinetic, homology modeling and in silico docking. These compounds also suppress the proliferation of U2OS osteoblastoma cells by promoting cell cycle arrest in G0/G1 phase. This report provides the first evidence for an allosteric pocket in human ATC, which greatly enhances its druggability and demonstrates the potential of this series in cancer therapy.

Beyond the C-terminal Glycine of ATG8 Proteins - The Story of Some Neglected Amino Acids.

ATG8 proteins form a family of small ubiquitin-like modifiers, well-known for their importance in both macroautophagy and autophagy-independent processes. A unique feature of this protein family is their conjugation to membrane lipids through the covalent attachment of a glycine residue at the C-terminus of ATG8 proteins. Notably, most ATG8 proteins are expressed with additional amino acids at their C-terminus, shielding the key glycine residue. Consequently, lipidation requires the activation of the ATG8 precursors through proteolytic cleavage, known as priming. ATG4 proteases catalyze this priming process, and under physiological conditions, unprimed forms of ATG8 are not detected. This raises the question about the purpose of the C-terminal extension of ATG8 proteins. While the roles of lipidated and free, primed ATG8 proteins have been extensively studied, the potential function of their precursor form or the priming process itself remains largely unexplored. Here, we summarize information from existing literature and our own experiments to contribute to the understanding of these neglected amino acids.

The GTPase activating protein Gyp7 regulates Rab7/Ypt7 activity on late endosomes.

Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function.

Molecular Insights into Aggrephagy: Their Cellular Functions in the Context of Neurodegenerative Diseases.

Protein homeostasis or proteostasis is an equilibrium of biosynthetic production, folding and transport of proteins, and their timely and efficient degradation. Proteostasis is guaranteed by a network of protein quality control systems aimed at maintaining the proteome function and avoiding accumulation of potentially cytotoxic proteins. Terminal unfolded and dysfunctional proteins can be directly turned over by the ubiquitin-proteasome system (UPS) or first amassed into aggregates prior to degradation. Aggregates can also be disposed into lysosomes by a selective type of autophagy known as aggrephagy, which relies on a set of so-called selective autophagy receptors (SARs) and adaptor proteins. Failure in eliminating aggregates, also due to defects in aggrephagy, can have devastating effects as underscored by several neurodegenerative diseases or proteinopathies, which are characterized by the accumulation of aggregates mostly formed by a specific disease-associated, aggregate-prone protein depending on the clinical pathology. Despite its medical relevance, however, the process of aggrephagy is far from being understood. Here we review the findings that have helped in assigning a possible function to specific SARs and adaptor proteins in aggrephagy in the context of proteinopathies, and also highlight the interplay between aggrephagy and the pathogenesis of proteinopathies.

Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

Phagophore closure, autophagosome maturation and autophagosome fusion during macroautophagy in the yeast Saccharomyces cerevisiae.

Macroautophagy, hereafter referred to as autophagy, is a complex process in which multiple membrane-remodeling events lead to the formation of a cisterna known as the phagophore, which then expands and closes into a double-membrane vesicle termed the autophagosome. During the past decade, enormous progress has been made in understanding the molecular function of the autophagy-related proteins and their role in generating these phagophores. In this Review, we discuss the current understanding of three membrane remodeling steps in autophagy that remain to be largely characterized; namely, the closure of phagophores, the maturation of the resulting autophagosomes into fusion-competent vesicles, and their fusion with vacuoles/lysosomes. Our review will mainly focus on the yeast Saccharomyces cerevisiae, which has been the leading model system for the study of molecular events in autophagy and has led to the discovery of the major mechanistic concepts, which have been found to be mostly conserved in higher eukaryotes.

Autophagy as an innate immunity response against pathogens: a Tango dance.

Intracellular infections as well as changes in the cell nutritional environment are main events that trigger cellular stress responses. One crucial cell response to stress conditions is autophagy. During the last 30 years, several scenarios involving autophagy induction or inhibition over the course of an intracellular invasion by pathogens have been uncovered. In this review, we will present how this knowledge was gained by studying different microorganisms. We intend to discuss how the cell, via autophagy, tries to repel these attacks with the objective of destroying the intruder, but also how some pathogens have developed strategies to subvert this. These two fates can be compared with a Tango, a dance originated in Buenos Aires, Argentina, in which the partner dancers are in close connection. One of them is the leader, embracing and involving the partner, but the follower may respond escaping from the leader. This joint dance is indeed highly synchronized and controlled, perfectly reflecting the interaction between autophagy and microorganism.

The yeast dynamin-like GTPase Vps1 mediates Atg9 transport to the phagophore assembly site in Saccharomyces cerevisiae.

Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal compartments called phagophores are generated to sequester intracellular components; these structures mature into autophagosomes, which deliver the cargo into lysosomes/vacuoles for degradation. Numerous autophagy-related (Atg) proteins are part of the core machinery that mediates autophagosome biogenesis. Atg9, a lipid scramblase and the only multispanning transmembrane protein among the core Atg machinery, traffics between cytoplasmic reservoirs and the phagophore assembly site (PAS) to provide membranes, recruit other Atg proteins and rearrange lipids on the phagophore membrane. However, the factors mediating Atg9 trafficking remain to be fully understood. In our recent study, we found that the yeast dynamin-like GTPase Vps1 (vacuolar protein sorting 1) is involved in autophagy and is important for Atg9 transport to the PAS. Moreover, we showed that Vps1 function in autophagy requires its GTPase and oligomerization activities. Interestingly, specific mutations in DNM2 (dynamin 2), one of the human homologs of Vps1 that have been linked with specific human diseases such as microcytic anemia and Charcot-Marie-Tooth, also impairs Atg9 transport to the PAS, suggesting that a defect in autophagy may underlay the pathophysiology of these severe human pathologies.

Mitochondrial protein BNIP3 regulates Chikungunya virus replication in the early stages of infection.

Chikungunya virus (CHIKV) is a human pathogen causing outbreaks of febrile illness for which vaccines and specific treatments remain unavailable. Autophagy-related (ATG) proteins and autophagy receptors are a set of host factors that participate in autophagy, but have also shown to function in other unrelated cellular pathways. Although autophagy is reported to both inhibit and enhance CHIKV replication, the specific role of individual ATG proteins remains largely unknown. Here, a siRNA screen was performed to evaluate the importance of the ATG proteome and autophagy receptors in controlling CHIKV infection. We observed that 7 out of 50 ATG proteins impact the replication of CHIKV. Among those, depletion of the mitochondrial protein and autophagy receptor BCL2 Interacting Protein 3 (BNIP3) increased CHIKV infection. Interestingly, BNIP3 controls CHIKV independently of autophagy and cell death. Detailed analysis of the CHIKV viral cycle revealed that BNIP3 interferes with the early stages of infection. Moreover, the antiviral role of BNIP3 was found conserved across two distinct CHIKV genotypes and the closely related Semliki Forest virus. Altogether, this study describes a novel and previously unknown function of the mitochondrial protein BNIP3 in the control of the early stages of the alphavirus viral cycle.

The Organization and Function of the Phagophore-ER Membrane Contact Sites.

Macroautophagy is characterized by the de novo formation of double-membrane vesicles termed autophagosomes. The precursor structure of autophagosomes is a membrane cistern called phagophore, which elongates through a massive acquisition of lipids until closure. The phagophore establishes membrane-contact sites (MCSs) with the endoplasmic reticulum (ER), where conserved ATG proteins belonging to the ATG9 lipid scramblase, ATG2 lipid transfer and Atg18/WIPI4 ß-propeller families concentrate. Several recent in vivo and in vitro studies have uncovered the relevance of these proteins and MCSs in the lipid supply required for autophagosome formation. Although important conceptual advances have been reached, the functional interrelationship between ATG9, ATG2 and Atg18/WIPI4 proteins at the phagophore-ER MCSs and their role in the phagophore expansion are not completely understood. In this review, we describe the current knowledge about the structure, interactions, localizations, and molecular functions of these proteins, with a particular emphasis on the yeast Saccharomyces cerevisiae and mammalian systems.

Mammalian ATG8 proteins maintain autophagosomal membrane integrity through ESCRTs.

The canonical autophagy pathway in mammalian cells sequesters diverse cytoplasmic cargo within the double membrane autophagosomes that eventually convert into degradative compartments via fusion with endolysosomal intermediates. Here, we report that autophagosomal membranes show permeability in cells lacking principal ATG8 proteins (mATG8s) and are unable to mature into autolysosomes. Using a combination of methods including a novel in vitro assay to measure membrane sealing, we uncovered a previously unappreciated function of mATG8s to maintain autophagosomal membranes in a sealed state. The mATG8 proteins GABARAP and LC3A bind to key ESCRT-I components contributing, along with other ESCRTs, to the integrity and imperviousness of autophagic membranes. Autophagic organelles in cells lacking mATG8s are permeant, are arrested as amphisomes, and do not progress to functional autolysosomes. Thus, autophagosomal organelles need to be maintained in a sealed state in order to become lytic autolysosomes.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.

Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.

TBK1 phosphorylation activates LIR-dependent degradation of the inflammation repressor TNIP1.

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0-4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.

Digest it all: the lysosomal turnover of cytoplasmic aggregates.

Aggrephagy describes the selective lysosomal transport and turnover of cytoplasmic protein aggregates by macro-autophagy. In this process, protein aggregates and conglomerates are polyubiquitinated and then sequestered by autophagosomes. Soluble selective autophagy receptors (SARs) are central to aggrephagy and physically bind to both ubiquitin and the autophagy machinery, thus linking the cargo to the forming autophagosomal membrane. Because the accumulation of protein aggregates is associated with cytotoxicity in several diseases, a better molecular understanding of aggrephagy might provide a conceptual framework to develop therapeutic strategies aimed at delaying the onset of these pathologies by preventing the buildup of potentially toxic aggregates. We review recent advances in our knowledge about the mechanism of aggrephagy.

--Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy.

During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes in vitro, it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer in vitro, although this is not the only requirement for its function in vivo. In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9F627A self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9F627A has identical scramblase activity in vitro like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9's role as a lipid scramblase.Abbreviations: BafA1: bafilomycin A1; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type.

Getting on the right track: Interactions between viruses and the cytoskeletal motor proteins.

The cytoskeleton is an essential component of the cell and it is involved in multiple physiological functions, including intracellular organization and transport. It is composed of three main families of proteinaceous filaments; microtubules, actin filaments and intermediate filaments and their accessory proteins. Motor proteins, which comprise the dynein, kinesin and myosin superfamilies, are a remarkable group of accessory proteins that mainly mediate the intracellular transport of cargoes along with the cytoskeleton. Like other cellular structures and pathways, viruses can exploit the cytoskeleton to promote different steps of their life cycle through associations with motor proteins. The complexity of the cytoskeleton and the differences among viruses, however, has led to a wide diversity of interactions, which in most cases remain poorly understood. Unveiling the details of these interactions is necessary not only for a better comprehension of specific infections, but may also reveal new potential drug targets to fight dreadful diseases such as rabies disease and acquired immunodeficiency syndrome (AIDS). In this review, we describe a few examples of the mechanisms that some human viruses, that is, rabies virus, adenovirus, herpes simplex virus, human immunodeficiency virus, influenza A virus and papillomavirus, have developed to hijack dyneins, kinesins and myosins.

Membrane Contact Sites in Autophagy.

Eukaryotes utilize different communication strategies to coordinate processes between different cellular compartments either indirectly, through vesicular transport, or directly, via membrane contact sites (MCSs). MCSs have been implicated in lipid metabolism, calcium signaling and the regulation of organelle biogenesis in various cell types. Several studies have shown that MCSs play a crucial role in the regulation of macroautophagy, an intracellular catabolic transport route that is characterized by the delivery of cargoes (proteins, protein complexes or aggregates, organelles and pathogens) to yeast and plant vacuoles or mammalian lysosomes, for their degradation and recycling into basic metabolites. Macroautophagy is characterized by the de novo formation of double-membrane vesicles called autophagosomes, and their biogenesis requires an enormous amount of lipids. MCSs appear to have a central role in this supply, as well as in the organization of the autophagy-related (ATG) machinery. In this review, we will summarize the evidence for the participation of specific MCSs in autophagosome formation, with a focus on the budding yeast and mammalian systems.

Phospholipid imbalance impairs autophagosome completion.

Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.

Transcriptomic changes in autophagy-related genes are inversely correlated with inflammation and are associated with multiple sclerosis lesion pathology.

Autophagy is a lysosomal degradative pathway essential for maintaining cellular homeostasis and is also implicated in multiple aspects of both innate and adaptive immunity. Neuroinflammation, along with demyelination and axonal loss, is an important component of multiple sclerosis (MS). Induction of autophagy ameliorated disease progression in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, underlying a possible link between autophagy and MS pathology. However, it is still unclear how autophagy is affected during different stages of MS. Here, we show a decreased expression of the autophagy-related (ATG) genes during the acute phase of EAE development in mice as well as in mixed active/inactive lesions of post-mortem human MS brain tissues. Using spatial transcriptomics, we observed that this decreased ATG gene expression is most prominent in the core of mixed active/inactive lesions. Furthermore, we observed a hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1) in lesions, which could inhibit both the initiation of autophagy and the transcription factors that regulate the expression of the ATG genes. Thus, based on our data, we propose a negative regulation of autophagy in MS, possibly through persistent mTORC1 activation, which depends on the lesion stage. Our results contribute to the understanding of the role of autophagy in different stages of MS pathology and point to the mTORC1 pathway as a potential modulator that likely regulates central nervous system (CNS) homeostasis and neuroinflammation in MS.