Autophagy

Identifying novel genes associated with autophagy (ATG) in man remains an important task for gaining complete understanding on this fundamental physiological process. A machine-learning guided approach can highlight potentially \"missing pieces\" linking core autophagy genes with understudied, \"dark\" genes that can help us gain deeper insight into these processes. In this study, we used a set of 103 (out of 288 genes from the Autophagy Database, ATGdb), based on the presence of ATG-associated terms annotated from 3 secondary sources: GO (gene ontology), KEGG pathway and UniProt keywords, respectively. We regarded these as additional confirmation for their importance in ATG. As negative labels, we used the OMIM list of genes associated with monogenic diseases (after excluding the 288 ATG-associated genes). Data associated with these genes from 17 different public sources were compiled and used to derive a Meta Path/XGBoost (MPxgb) machine learning model trained to distinguish ATG and non-ATG genes (10-fold cross-validated, 100-times randomized models, median AUC = 0.994 +/- 0.0084). Sixteen ATG-relevant variables explain 64% of the total model gain, and 23% of the top 251 predicted genes are annotated in ATGdb. Another 15 genes have potential ATG associations, whereas 193 do not. We suggest that some of these 193 genes may represent \"autophagy dark genes\", and argue that machine learning can be used to guide autophagy research in order to gain a more complete functional and pathway annotation of this complex process.

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.

Macroautophagy (hereafter autophagy) is a cellular recycling pathway that requires different ATG (autophagy-related) proteins to generate double-membraned autophagosomes. ATG9A, a multi-spanning membrane protein, plays a crucial role in this process as the only transmembrane component of the core autophagy machinery. ATG9A functions as a lipid scramblase, redistributing lipids between membrane leaflets for the expanding autophagosome membrane. Structural studies have revealed that ATG9A forms a homotrimer with an interlocked domain-swapped architecture and a network of internal hydrophilic cavities. This configuration underlies its role in lipid transfer and membrane remodeling together with the lipid transporter ATG2A. ATG9A dysfunction has also been linked to human disease, as specific ATG9A mutations cause neurodevelopmental or neurodegenerative phenotypes. Additionally, ATG9A is altered in cancer, promoting pro-tumorigenic traits. However, most missense variants in ATG9A remain uncharacterized, posing a significant challenge for interpreting genomic data. In this study, we employed in silico saturation mutagenesis approach using the MAVISp (Multi-layered Assessment of VarIants by Structure) framework to predict the impact of every missense mutation in ATG9A. By analyzing multiple structural assemblies of ATG9A (monomer, trimer, and the ATG9A-ATG2A complex), we evaluated diverse mechanistic indicators of variant impact, including protein stability, long-range conformational changes, effects on multimerization interfaces, and alterations in post-translational modifications. We integrated the structure-based predictions with Variant Effect Predictors from recent deep-learning or evolutionary-based models and cross-referenced known variants catalogued in ClinVar, COSMIC, and cBioPortal. Finally, we predicted mechanistic indicators for all possible variants with structural coverage not yet reported in the disease-related databases supported by MAVISp. Our analyses identified a group of potentially damaging variants in ATG9A and the possible molecular mechanisms underlying their effects. Together, this work provides a roadmap for interpreting missense variants in autophagy regulators and highlights specific ATG9A mutations that deserve further investigation in the context of human disease.

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,500 genes that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology with direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. Here, we provide a curated list of 959 unique genes that comprise the protein synthesis machinery, chaperones, folding enzymes, systems for trafficking proteins into and out of organelles, and organelle-specific degradation systems. In subsequent manuscripts, we will delineate the human autophagy-lysosome pathway, the ubiquitin-proteasome system, and the proteostasis networks of model organisms.

During autophagy, the ULK complex nucleates autophagic precursors which give rise to autophagosomes. We analysed by live imaging and mathematical modelling translocation of ATG13 (part of ULK complex) to autophagic puncta in starvation-induced autophagy and ivermectin-induced mitophagy. In non-selective autophagy, the intensity and duration of ATG13 translocation approximated a normal distribution whereas wortmannin reduced this and shifted to a log-normal distribution. During mitophagy, multiple translocations of ATG13, with increasing time between peaks were observed. We hypothesised that these multiple translocations arise because engulfment of mitochondrial fragments requires successive nucleations of multiple phagophores on the same target, and a mathematical model based on this idea reproduced the oscillatory behaviour. Significantly, model and experimental data were also in agreement that the number of ATG13 translocations is directly proportional to the diameter of the targeted mitochondrial fragments. Our data provide novel insights into the early dynamics of selective and non-selective autophagy.

Disruption of macroautophagy/autophagy has emerged as a common feature in many neurodegenerative diseases. Autophagy is a membrane-dependent pathway that requires many key regulators to quickly localize on and off membranes during induction promoting membrane fusion. Previously, our bioinformatic approaches have shown that autophagy and Huntington disease (HD) are enriched in S-acylated proteins. S-acylation involves the reversible addition of long chain fatty acids to promote membrane binding. Herein, we show that inhibition of S-acylation regulates the abundance of several key regulators of autophagy and leads to a partial block of autophagic flux. We show that the autophagy receptor SQSTM1/p62 (sequestosome 1) is S-acylated and directed to the lysosome. Importantly, we see that SQSTM1 S-acylation is significantly reduced in HD patient and mouse model brains, thus providing a novel mechanism for the generation of empty autophagosomes previously seen in HD models and patient cells.

Autophagy is a highly conserved homeostatic process essential for the bulk degradation of cytoplasmic components and aggregated proteins. Multiple evidence indicates that impairment of (macro)autophagy leads to neurodegeneration, such as Parkinson disease (PD). Our previous work showed that p21 activated kinase 6 (PAK6) interacts with the PD-associated leucine-rich repeat kinase (LRRK2) to promote neurite outgrowth in the mouse striatum; still the function of PAK6 in the brain is largely unknown. Here, we found that downregulation of neuronal but not glial mbt, the D. melanogaster homolog of PAK6, impairs autophagy-lysosomal function. PAK6 overexpression in cells and in C. elegans increases transcription factor EB (TFEB) nuclear translocation in a kinase activity-dependent manner. Mechanistically, PAK6 forms a complex with TFEB to regulate its nuclear localization in a manner dependent on phosphorylation of and binding to 14-3-3 proteins and phosphorylation of TFEB at S467. In line with its ability to promote neuronal autophagy, mbt downregulation exacerbates alpha-synuclein toxicity in Drosophila dopaminergic neurons. Moreover, PAK6 overexpression in the substantia nigra of mutant LRRK2 mice reduces the burden of phosphorylated alpha-synuclein in dopaminergic neurons. Altogether, our study uncovers a novel role of PAK6 as a positive regulator of autophagy via TFEB and suggests that modulating its activity may represent a way to selectively turn on autophagy in neurons, with implications for the treatment of neurodegenerative disorders.

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

Autophagy is a lysosome-dependent degradation process that involves autophagosome formation, typically mediated by mammalian ATG8 proteins (mATG8s). Autophagosomes can still form in their absence, suggesting alternative mechanisms. NBR1, a selective autophagy receptor, has been shown to compensate for autophagy defects. Here, we examined NBR1 regulation under proteotoxic stress in mATG8s-deficient HeLa cells. NBR1 levels were elevated in mATG8s knockout (KO) cells under basal conditions but decreased significantly after treatment with the proteasome inhibitor MG132. This reduction was not prevented by lysosomal inhibition with BafA1, indicating a non-lysosomal mechanism. Silencing of RAB27A reduced basal NBR1 levels, and the effects of MG132 were no longer observed, likely due to already diminished NBR1. Remaining NBR1 localized to puncta positive for ubiquitin and the ESCRT-0 component HRS, suggesting involvement of a ubiquitin-dependent endosomal pathway. Overall, our results suggest that under proteotoxic stress and impaired autophagy, cells activae alternative routes, potentially involving unconventional secretion, to regulate NBR1 levels. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/664987v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@10568bcorg.highwire.dtl.DTLVardef@1f3d53org.highwire.dtl.DTLVardef@f33508org.highwire.dtl.DTLVardef@11ec28e_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender https://BioRender.com/geoh3sz In the absence of mATG8s, NBR1 may be recruited to multivesicular bodies (MVBs) via HRS, for later degradation in lysosomes (left panel). Under proteasomal inhibition (right panel), NBR1 associated with MVBs (with ubiquitinated cargos) is downregulated, independent of lysosomes. Possibly, this downregulation is due to a secretion process, through a RAB27A-independent mechanism.

Clathrin plays a critical role in clathrin-mediated endocytosis (CME) in plants, and it is required for autophagy in mammals. However, the functional interconnection of clathrin with autophagy has not been firmly established in plants. Here, we demonstrate that loss of function of clathrin light chain (CLC) subunit 2 and 3 results in salicylic acid (SA)- and H2O2-dependent accelerated senescence and activated defense responses in Arabidopsis, which are hallmarks of the autophagy-related gene (ATG) mutants. Similar to atg mutants, the clc2-1clc3-1 double mutant has enhanced sensitivity to both carbon and nitrogen starvation and enhanced resistance to biotrophic bacterial and fungal pathogens. In addition, the autophagy flux was significantly reduced in the roots of clc2-1clc3-1 mutant plants relative to Col-0 plants under carbon starvation conditions. Furthermore, our Yeast-2-hybrid (Y2H) and Luciferase complementation assays showed that CLC2 directly interacted with ATG8h and ATG8i. Mutations within the unique ATG8-interacting motif (AIM) of CLC2 as well as at the LIR/AIM-docking site (LDS) of ATG8h abolished the interaction between CLC2 and ATG8h. As anticipated, both GFP-ATG8h/GFP-ATG8i and CLC2 were subjected to autophagic degradation in the vacuoles. Together, our data revealed that the accelerated senescence and activated immune responses observed in Arabidopsis clc2-1clc3-1 mutant plants result from impaired autophagy, and CLC2 participates in autophagy through direct interactions with ATG8h and ATG8i in an AIM1- and LDS-dependent manner. Our results unveil a previously unidentified link between the function of CLCs and autophagy.

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

The mitochondrion is an essential organelle in eukaryotic cells, playing crucial roles in cellular respiration and intracellular signaling pathways. To maintain a healthy population of mitochondria, dysfunctional and excess mitochondria are selectively removed through an autophagic process known as mitophagy. Over the past few decades, various autophagy-related (ATG) proteins involved in mitophagy have been well characterized in yeast and mammalian cells since it has significance to the survival of eukaryotes. While the core autophagy machinery responsible for autophagosome formation is conserved among eukaryotes, the homologs of key regulators of mammalian system is absent in plant. In this study, we identified a unique mitophagy mechanism in plant, that three voltage-dependent anion channel (VDAC) family proteins in the mitochondria outer membrane -- specifically VDAC1, VDAC2, and VDAC3 -- as mitophagy receptors in Arabidopsis. These proteins were required for translocation of ATG8 from the cytosol to the mitochondria surface, when Arabidopsis cells were treated with an uncoupler, 2,4-dinitrophenol (DNP). The VDACs interacted directly with ATG8 through an ATG8-interacting motif (AIM) located in their amino (N) termini. Furthermore, vdac mutants exhibited impaired uncoupler-induced mitophagy and accumulated damaged mitochondria. These mitophagy-related phenotypes were more pronounced in vdac double and triple mutant lines. Altogether, our results indicated that VDAC1, 2, and 3 recruit ATG8 to depolarized mitochondria, facilitating the formation of mitophagosomes, presenting a distinguishing mitophagy pathway with mammalian system.

Autophagy is an evolutionarily conserved, highly regulated catabolic process critical to neuronal homeostasis, function and survival throughout organismal lifespan. However, the external factors and signals that control autophagy in neurons are still poorly understood. Here we report that the G protein-coupled metabotropic glutamate receptor 1 (mGlu1) contributes to control basal autophagy in the brain. Autophagy is upregulated in the brain of adult mGlu1 knockout mice and genetic deletion or pharmacological inhibition of native mGlu1 receptors enhances autophagy flux in neurons. The evolutionarily conserved adaptor protein FEZ1, identified by a genome-wide screen as mGlu1 receptor interacting partner, was found to participate in the regulation of neuronal autophagy and to be required for repression of autophagy flux by the mGlu1 receptor. Furthermore, FEZ1 appears to enable association of mGlu1 with Ulk1, a core component of the autophagy pathway. Thus, we propose that the mGlu1 receptor contributes to restrain constitutive autophagy in neurons.

The selective autophagy receptors SQSTM1/p62 and NBR1 are evolutionary related and involved in autophagy of different substrates including proteins, protein aggregates and organelles. The two proteins interact via their PB1 domains, and essential for their roles in autophagy is the formation of a specific type of condensates named p62 bodies. The scaffold of these structures is formed by the interaction of polymeric p62 with polyubiquitin, but NBR1 is recruited and essential for their formation. Previous studies have shown that p62 contains nuclear export signal (NES) and nuclear localization signal (NLS) motifs and shuttles between the cytoplasm and the nucleus. Its nuclear roles are not fully understood, but there is evidence that p62 is involved in protein quality control in the nucleus. No previous studies have tested if NBR1 is transported into the nucleus. We show here that NBR1 contains two NES motifs and one NLS motif, and like p62, the protein shuttles between the cytoplasm and the nucleus. NBR1 also accumulates in nuclear p62 bodies and the formation of nuclear p62 bodies depends on NBR1.

Autophagosomes deliver cytosolic material to lysosomes to provide amino acids during starvation and to degrade damaged proteins and organelles to maintain tissue homeostasis. Delivery to lysosomes requires LC3/ATG8 (LC3), the major membrane protein of the autophagosome, that uses adaptor proteins to capture cargo and recruits tethering and SNARE proteins to promote fusion with lysosomes. LC3 is also recruited to endo-lysosome compartments in response to increases in vacuolar pH to facilitate degradation of material entering cells by endocytosis. A series of ubiquitin-like reactions conjugate LC3 to amino groups exposed by phosphatidylethanolamine (PE) or phosphatidylserine (PS) in target membranes. The E1 and E2-ubiquitin-like activities of ATG7 and ATG3 use thioester bonds to transfer LC3 to the ATG5-ATG12 conjugate. At the same time binding of ATG16L1 to ATG5-ATG12 provides the E3 ubiquitin-ligase like activity necessary to conjugate LC3 to PE or PS. The ATG5-ATG12 conjugate can also bind TECPR1 (tectonin beta propeller repeat-containing protein) which shares an ATG5 interaction (AIR) region with ATG16L1 and can binds LC3 directly through a LC3 interaction region (LIR). In this study we have used cells lacking ATG16L1 to determine if TECPR1 can substitute for ATG16L1 during LC3 conjugation. The results show that ATG16L1-/-MEFS can conjugate LC3 to lysosomes damaged by chloroquine or LLOMes and conjugation is dependent on the ubiquitin-like enzymes ATG3, ATG5 and ATG7 upstream of ATG16L1. TECPR1, ATG5 and galectin 3 are recruited to LAMP positive damaged lysosomes in the absence of ATG16L1 suggesting that TECPR-1 recruits ATG5-ATG12 to conjugate LC3 to damaged lysosomes. This was confirmed when truncation of TECPR1 at the central PH domain required for lysosome binding prevented LC3 conjugation, and LC3 conjugation could be restored by full length TECPR1. Recruitment of TECPR1 to damaged lysosomes required the N-terminal LIR motif and was partially dependent on the central PH domain that binds PI4P exposed during lysosome repair. TECPR1 can therefore conjugate LC3 to damaged lysosomes independently of ATG16L1 by providing E3 ligase-like activity to ATG5-ATG12. Direct conjugation of LC3 by TECPR1 may contribute to the autophagosome tethering functions reported for TECPR1 by increasing recruitment of cargo receptors, tethering proteins and SNARE proteins required for fusion with lysosomes and protein degradation. TECPR1-dependent conjugation of LC3 may also facilitate lysosome repair pathways involving autophagic lysosomal reformation and transcriptional activation of autophagy by TFEB.

Dependence receptors (DRs) induce cell death by apoptosis when unbound by their cognate ligands. Among them, Kremen1 was first described to induce cancer cell death in the absence of its ligand, DKK1. However, the precise mechanism of Kremen1-induced cell death remains unclear. In this study, we demonstrate that Kremen1 induces cell death with autophagic features, contrasting with the apoptotic process typically associated with dependence receptors. Specifically, the pharmacological inhibition of autophagy, or genetic silencing of key autophagy effectors, efficiently suppresses this cell death process. A biotin proximity labeling for protein-protein interactions identified SEC24C, a component of the COP-II complex, as a critical effector in Kremen1-induced autophagy and cell death. Our findings further reveal that Kremen1 is in proximity with SEC24C and ATG9A after vesicular trafficking and fosters the interaction of SEC24C with ATG8, ERGIC and ATG9A. This potentially underlies the increased number of autophagosomes leading to cell death. The induction of aberrant autophagy by Kremen1 deserves particular attention, especially as the Kremen1/DKK1 pair is frequently altered in cancers. Thus, targeting this pathway may offer a potential strategy for treating cancers resistant to current therapies.

Autophagy is a vital catabolic process responsible for the degradation of cytosolic components, playing a key role in cellular homeostasis and survival. At synapses, autophagy is crucial for regulating neuronal activity and utilizes a specialized machinery. While considerable progress has been made in understanding the initiation of autophagy and autophagosome formation, the mechanisms governing the clearance of autophagosomes from synaptic sites remain poorly understood. Here, we identify a novel pathway in which astrocytes actively participate in the clearance of pre-synaptic autophagosomes. Using neurons derived from human induced pluripotent stem cell (hiPSC) lines expressing fluorescent autophagy markers and chimeric mouse models, we demonstrate that neuronal autophagosomal vesicles are physically transferred to astrocytes, a process that is enhanced when synaptic activity is suppressed. Autophagosome transfer does not require direct physical cellular contact, but it does require Dynamin and cholesterol-dependent endocytosis for the internalized neuronal autophagosomes to ultimately fuse with astrocytic lysosomes. Our findings reveal a previously unrecognized mechanism of neuronal autophagosome clearance that does not require slow axonal retrograde transport but their transfer to nearby astrocytes.

Autophagy is a conserved quality control mechanism that removes damaged proteins, organelles and invading bacteria through lysosome-mediated degradation. During autophagy several organelles including endoplasmic reticulum, mitochondria, plasma membrane and endosomes contribute membrane for autophagosome formation. However, the mechanisms and proteins involved in membrane delivery to autophagosomes are not clear. Optineurin (OPTN), a cytoplasmic adaptor protein, is involved in promoting maturation of phagophores into autophagosomes; it is also involved in regulating endocytic trafficking and recycling of transferrin receptor (TFRC). Here, we have examined the role of optineurin in the delivery of membrane from TFRC-positive endosomes to autophagosomes. Only a small fraction of autophagosomes was positive for TFRC, indicating that TFRC-positive endosomes could contribute membrane to a subset of autophagosomes. The percentage of TFRC-positive autophagosomes was reduced in Optineurin knockout mouse embryonic fibroblasts (Optn-/- MEFs) in comparison with normal MEFs. Upon over-expression of optineurin, the percentage of TFRC-positive autophagosomes was increased in Optn-/- MEFs. Unlike wild-type optineurin, a disease-associated mutant, E478G, defective in ubiquitin binding, was not able to enhance formation of TFRC-positive autophagosomes in Optn-/- MEFs. TFRC degradation mediated by autophagy was decreased in optineurin deficient cells. Our results suggest that optineurin mediates delivery of TFRC and perhaps associated membrane from TFRC-positive endosomes to autophagosomes, and this may contribute to autophagosome formation.

Autophagy is a conserved pro-survival pathway for delivering misfolded proteins and damaged organelles to lysosomes for degradation and protein homeostasis. Anomaly in autophagy leads to aberrant protein aggregation in neuronal cells, which is a common etiology of neurodegenerative disorders. Endo-lysosomal cation channel TRPML3 (Transient Receptor Potential Mucolipin-3) has been shown to induce autophagy in cell line models. However, the mechanism of TRPML3 mediated autophagy induction and the underlying gene expression changes are not clearly understood. Here, by using Ca2+-and electrical-current measurements, RNA sequencing and RT PCR studies, we explored the cellular function of TRPML3 and the global transcriptomic profile in a cell-based serum starvation model of autophagy. We report that serum starvation leads to downregulation of neuronal developmental genes during autophagy induction. TRPML3 overexpression further amplifies the effect of starvation in downregulating neuronal gene expression. But, when nutrition is not a limiting condition, TRPML3 overexpression upregulated neuronal genes including those responsible for axon guidance, synaptogenesis, and dendritic arborization. TRPML3 mediated neuronal gene expression changes were, presumably, due to transcription factors (TF) TFEB, FOXO1 and neuron-specific TFs such as SOX2, and ETV5. To further validate the role of TRPML3 in neuronal gene regulation, we performed meta-analysis of publicly available RNAseq datasets on neurodegenerative disorders which provided insight into the heterogeneity in the molecular mechanisms of autophagy and corroborated the TFEB-mediated autophagy induction and neuronal gene expression in TRPML3 overexpression condition. Based on our results, we propose that TRPML3 may act as a potential genetic marker for familial neurodegenerative disorders.

Map1Lc3b is a protein that has pivotal functions in cellular autophagy. At least three groups in the past decade have reported its presence in the nucleoli of cells, but its functions in that organelle remain unknown. We isolated a few clonal populations of cells stably expressing V5-tagged mouse Lc3b highly enriched in the nucleoli, but the frequency of occurrence of such clones was strikingly low. The phenomenon was readily reproducible, though the protein in the nucleolus puzzlingly had varying molecular masses in different clones but consistently displayed a very strong interaction with the mitochondrial protein C1qbp, which has well-documented functions in the nucleolus. We investigated further and discovered that, in at least one of the clones, Lc3b had formed a chimera with the puromycin resistance gene in the plasmid, plausibly by illegitimate recombination during or after integration of the construct into the cellular genomic DNA. The -1 shifted reading frame of puromycin N-acetyltransferase (pac) can encode a protein that is equally long as the one encoded by the complete pac ORF, but is targeted to the nucleoli due to a drastic shift in the isoelectric point (pI). Notably, this set of events again brings into focus the low threshold often reported for recombination events to occur in eukaryotic cells, the multiple factors influencing them, and calls for increased vigilance in experiments involving DNA transfection and gene targeting.