Synonymous Codon Variant Analysis for Autophagic Genes Dysregulated in Neurodegeneration.

Neurodegenerative disorders are often a culmination of the accumulation of abnormally folded proteins and defective organelles. Autophagy is a process of removing these defective proteins, organelles, and harmful substances from the body, and it works to maintain homeostasis. If autophagic removal of defective proteins has interfered, it affects neuronal health. Some of the autophagic genes are specifically found to be associated with neurodegenerative phenotypes. Non-functional, mutated, or gene copies having silent mutations, often termed synonymous variants, might explain this. However, these synonymous variant which codes for exactly similar proteins have different translation rates, stability, and gene expression profiling. Hence, it would be interesting to study the pattern of synonymous variant usage. In the study, synonymous variant usage in various transcripts of autophagic genes ATG5, ATG7, ATG8A, ATG16, and ATG17/FIP200 reported to cause neurodegeneration (if dysregulated) is studied. These genes were analyzed for their synonymous variant usage; nucleotide composition; any possible nucleotide skew in a gene; physical properties of autophagic protein including GRAVY and AROMA; hydropathicity; instability index; and frequency of acidic, basic, neutral amino acids; and gene expression level. The study will help understand various evolutionary forces acting on these genes and the possible augmentation of a gene if showing unusual behavior.

Using GBP Nanotrap to Restore Autophagy in the Rab5/Vps21 Mutant by Forcing Snf7 and Atg17 Interaction.

Protein-protein interactions are important for physiology performance. Green fluorescent protein (GFP) is a widely used protein tag to show protein localization in vivo. GFP binding protein (GBP) is a specific domain with high affinity to GFP. A novel technique with GBP fused protein X tagged with red fluorescence protein binding to GFP of GFP fused protein Y to establish a close association for proteins X and Y independently from other proteins has recently been developed. It is found that the interaction and colocalization between Snf7 and Atg17 is impaired in Saccharomyces cerevisiae vps21Δ cells, which are defective in autophagy. In order to determine whether the interaction between Snf7 and Atg17 is important for autophagy, we forced the interaction between Snf7 and Atg17 through GBP-GFP binding. Snf7-GBP-mCherry and/or GFP-Atg17 tagged wild-type and vps21Δ cells were compared for autophagy process under starvation by determining the maturation of proprotein of Ape1 (prApe1). Our results showed that the defective autophagy in vps21Δ cells was significantly suppressed when both Snf7-GBP-mCherry and GFP-Atg17 were installed. Our results indicate that the GBP-GFP nanotrap technique is a powerful tool to restore colocalization/interaction in vivo and the Snf7-Atg17 interaction is important for yeast autophagy.

Snx4-assisted vacuolar targeting of transcription factors defines a new autophagy pathway for controlling ATG expression.

Autophagy, in part, is controlled by the repression and activation of autophagy-related (ATG) genes. Here, we describe a new selective autophagy pathway that targets functional transcriptional regulators to control their activity. This pathway is activated in response to nitrogen starvation and recycles transcriptional activators (Msn2 and Rim15) and a repressor (Ssn2/Med13) of ATG expression. Further analysis of Ssn2/Med13 vacuolar proteolysis revealed that this pathway utilizes the core autophagic machinery. However, it is independent of known nucleophagy mechanisms, receptor proteins, and the scaffold protein Atg11. Instead, Ssn2/Med13 exits the nucleus through the nuclear pore complex (NPC) and associates with the cytoplasmic nucleoporin Gle1, a member of the RNA remodeling complex. Dbp5 and Nup159, that act in concert with Gle1, are also required for Ssn2/Med13 clearance. Ssn2/Med13 is retrieved from the nuclear periphery and degraded by Atg17-initiated phagophores anchored to the vacuole. Efficient transfer to phagophores depends on the sorting nexin heterodimer Snx4/Atg24-Atg20, which binds to Atg17, and relocates to the perinucleus following nitrogen starvation. To conclude, this pathway defines a previously undescribed autophagy mechanism that targets select transcriptional regulators for rapid vacuolar proteolysis, utilizing the RNA remodeling complex, the sorting nexin heterodimer Snx4-Atg20, Atg17, and the core autophagic machinery. It is physiologically relevant as this Snx4-assisted vacuolar targeting pathway permits cells to fine-tune the autophagic response by controlling the turnover of both positive and negative regulators of ATG transcription.Abbreviations: AIM: Atg8 interacting motif; ATG: autophagy-related; CKM: CDK8 kinase module; IDR: intrinsically disordered region; IP6: phosphoinositide inositol hexaphosphate; NPC: nuclear pore complex; PAS: phagophore assembly site; UPS: ubiquitin-proteasomal system.

Scaffold proteins in bulk and selective autophagy.

Autophagy is a crucial cellular degradation and recycling pathway. During autophagy double-membrane vesicles, called autophagosomes, encapsulate cellular components and deliver their cargo to the lytic compartment for degradation. Formation of autophagosomes is regulated by the Atg1 kinase complex in yeast and the homologous ULK1 kinase complex in mammals. While research on Atg1 and ULK1 has advanced our understanding of how these protein kinases function in autophagy, the other Atg1/ULK1 kinase complex members have received much less attention. Here, we focus on the functions of the Atg1 kinase complex members Atg11 and Atg17 as well as the ULK1 kinase complex member FIP200 in autophagy. These three proteins act as scaffolds in their respective complexes. Recent studies have made it evident that they have similar but also distinct functions. In this article, we review our current understanding of how these scaffold proteins function from autophagosome formation to fusion and also discuss their possible roles in diseases.

Autophagosomes are formed at a distinct cellular structure.

Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which deliver bulk cytoplasmic material to the lytic compartment of the cell for degradation. Autophagosome formation is initiated by assembly and recruitment of the core autophagy machinery to distinct cellular sites, referred to as phagophore assembly sites (PAS) in yeast or autophagosome formation sites in other organisms. A large number of autophagy proteins involved in the formation of autophagosomes has been identified; however, how the individual components of the PAS are assembled and how they function to generate autophagosomes remains a fundamental question. Here, we highlight recent studies that provide molecular insights into PAS organization and the role of the endoplasmic reticulum and the vacuole in autophagosome formation.

Mechanistic Insights into the Role of Atg11 in Selective Autophagy.

Macroautophagy (referred to hereafter as autophagy) is an intracellular degradation pathway in which the formation of a double-membrane vesicle called the autophagosome is a key event in the transport of multiple cytoplasmic cargo (e.g., proteins, protein aggregates, lipid droplets or organelles) to the vacuole (lysosome in mammals) for degradation and recycling. During this process, autophagosomes are formed de novo by membrane fusion events leading to phagophore formation initiated at the phagophore assembly site. In yeast, Atg11 and Atg17 function as protein scaffolds, essential for selective and non-selective types of autophagy, respectively. While Atg17 functions in non-selective autophagy are well-defined in the literature, less attention is concentrated on recent findings regarding the roles of Atg11 in selective autophagy. Here, we summarize current knowledge about the Atg11 scaffold protein and review recent findings in the context of its role in selective autophagy initiation and autophagosome formation.

Expression of LC3B and FIP200/Atg17 in brain metastases of breast cancer.

BACKGROUND: Macroautophagy/autophagy is considered to play key roles in tumor cell evasion of therapy and establishment of metastases in breast cancer. High expression of LC3, a residual autophagy marker, in primary breast tumors has been associated with metastatic disease and poor outcome. FIP200/Atg17, a multi-functional pro-survival molecule required for autophagy, has been implicated in brain metastases in experimental models. However, expression of these proteins has not been examined in brain metastases from patients with breast cancer. METHODS: In this retrospective study, specimens from 44 patients with brain metastases of infiltrating ductal carcinoma of the breast (IDC), unpaired samples from 52 patients with primary IDC (primary-BC) and 16 matched-paired samples were analyzed for LC3 puncta, expression of FIP200/Atg17, and p62 staining. RESULTS: LC3-puncta+ tumor cells and FIP200/Atg17 expression were detected in greater than 90% of brain metastases but there were considerable intra- and inter-tumor differences in expression levels. High numbers of LC3-puncta+ tumor cells in brain metastases correlated with a significantly shorter survival time in triple-negative breast cancer. FIP200/Atg17 protein levels were significantly higher in metastases that subsequently recurred following therapy. The percentages of LC3 puncta+ tumor cells and FIP200/Atg17 protein expression levels, but not mRNA levels, were significantly higher in metastases than primary-BC. Meta-analysis of gene expression datasets revealed a significant correlation between higher FIP200(RB1CC1)/Atg17 mRNA levels in primary-BC tumors and shorter disease-free survival. CONCLUSIONS: These results support assessments of precision medicine-guided targeting of autophagy in treatment of brain metastases in breast cancer patients.

Conserved and unique features of the fission yeast core Atg1 complex.

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.

How the Atg1 complex assembles to initiate autophagy.

The Atg1 complex, comprising Atg1, Atg13, Atg17, Atg29, and Atg31, is a key initiator of autophagy. The Atg17-Atg31-Atg29 subcomplex is constitutively present at the phagophore assembly site (PAS), while Atg1 and Atg13 join the complex when autophagy is triggered by starvation or other signals. We sought to understand the energetics and dynamics of assembly using isothermal titration calorimetry (ITC), sedimentation velocity analytical ultracentrifugation, and hydrogen-deuterium exchange (HDX). We showed that the membrane and Atg13-binding domain of Atg1, Atg1EAT, is dynamic on its own, but is rigidified in its high-affinity (∼100 nM) complex with Atg13. Atg1EAT and Atg13 form a 2:2 dimeric assembly and together associate with lower affinity (∼10 µM) with the 2:2:2 Atg17-Atg31-Atg29 complex. These results lead to an overall model for the assembly pathway of the Atg1 complex. The model highlights the Atg13-Atg17 binding event as the weakest link in the assembly process and thus as a natural regulatory checkpoint.

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila.

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila.

Structural characterization of the Saccharomyces cerevisiae autophagy regulatory complex Atg17-Atg31-Atg29.

Atg17, in complex with Atg29 and Atg31, constitutes a key module of the Atg1 kinase signaling complex and functions as an important organizer of the phagophore assembly site in the yeast Saccharomyces cerevisiae. We have determined the three-dimensional reconstruction of the full S. cerevisiae Atg17-Atg31-Atg29 complex by single-particle electron microscopy. Our structure shows that Atg17-Atg31-Atg29 is dimeric and adopts a relatively rigid and extended "S-shape" architecture with an end-to-end distance of approximately 345 Å. Subunit mapping analysis indicated that Atg17 mediates dimerization and generates a central rod-like scaffold, while Atg31 and Atg29 form two globular domains that are tethered to the concave sides of the scaffold at the terminal regions. Finally, our observation that Atg17 adopts multiple conformations in the absence of Atg31 and Atg29 suggests that the two smaller components play key roles in defining and maintaining the distinct curvature of the ternary complex.