ER stress-induced clearance of misfolded GPI-anchored proteins via the secretory pathway.

Proteins destined for the cell surface are first assessed in the endoplasmic reticulum (ER) for proper folding before release into the secretory pathway. This ensures that defective proteins are normally prevented from entering the extracellular environment, where they could be disruptive. Here, we report that, when ER folding capacity is saturated during stress, misfolded glycosylphosphatidylinositol-anchored proteins dissociate from resident ER chaperones, engage export receptors, and quantitatively leave the ER via vesicular transport to the Golgi. Clearance from the ER commences within minutes of acute ER stress, before the transcriptional component of the unfolded protein response is activated. These aberrant proteins then access the cell surface transiently before destruction in lysosomes. Inhibiting this stress-induced pathway by depleting the ER-export receptors leads to aggregation of the ER-retained misfolded protein. Thus, this rapid response alleviates the elevated burden of misfolded proteins in the ER at the onset of ER stress, promoting protein homeostasis in the ER.

Fluctuation of lysosomal protein degradation in neural stem cells of the postnatal mouse brain.

Lysosomes are intracellular organelles responsible for degrading diverse macromolecules delivered from several pathways, including the endo-lysosomal and autophagic pathways. Recent reports have suggested that lysosomes are essential for regulating neural stem cells in developing, adult and aged brains. However, the activity of these lysosomes has yet to be monitored in these brain tissues. Here, we report the development of a new probe to measure lysosomal protein degradation in brain tissue by immunostaining. Our results indicate that lysosomal protein degradation fluctuates in neural stem cells of the hippocampal dentate gyrus, depending on age and brain disorders. Neural stem cells increase their lysosomal activity during hippocampal development in the dentate gyrus, but aging and aging-related disease reduce lysosomal activity. In addition, physical exercise increases lysosomal activity in neural stem cells and astrocytes in the dentate gyrus. We therefore propose that three different stages of lysosomal activity exist: the state of increase during development, the stable state during adulthood and the state of reduction due to damage caused by either age or disease.

The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes.

The lysosome is a degradative organelle, and its fusion with other organelles is strictly regulated. In contrast to fusion with the late endosome, the mechanisms underlying autophagosome-lysosome fusion remain unknown. Here, we identify syntaxin 17 (Stx17) as the autophagosomal SNARE required for fusion with the endosome/lysosome. Stx17 localizes to the outer membrane of completed autophagosomes but not to the isolation membrane (unclosed intermediate structures); for this reason, the lysosome does not fuse with the isolation membrane. Stx17 interacts with SNAP-29 and the endosomal/lysosomal SNARE VAMP8. Depletion of Stx17 causes accumulation of autophagosomes without degradation. Stx17 has a unique C-terminal hairpin structure mediated by two tandem transmembrane domains containing glycine zipper-like motifs, which is essential for its association with the autophagosomal membrane. These findings reveal a mechanism by which the SNARE protein is available to the completed autophagosome.

Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation.

We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions.

IL-17 is a neuromodulator of Caenorhabditis elegans sensory responses.

Interleukin-17 (IL-17) is a major pro-inflammatory cytokine: it mediates responses to pathogens or tissue damage, and drives autoimmune diseases. Little is known about its role in the nervous system. Here we show that IL-17 has neuromodulator-like properties in Caenorhabditis elegans. IL-17 can act directly on neurons to alter their response properties and contribution to behaviour. Using unbiased genetic screens, we delineate an IL-17 signalling pathway and show that it acts in the RMG hub interneurons. Disrupting IL-17 signalling reduces RMG responsiveness to input from oxygen sensors, and renders sustained escape from 21% oxygen transient and contingent on additional stimuli. Over-activating IL-17 receptors abnormally heightens responses to 21% oxygen in RMG neurons and whole animals. IL-17 deficiency can be bypassed by optogenetic stimulation of RMG. Inducing IL-17 expression in adults can rescue mutant defects within 6 h. These findings reveal a non-immunological role of IL-17 modulating circuit function and behaviour.

Labeling and measuring stressed mitochondria using a PINK1-based ratiometric fluorescent sensor.

Mitochondria are essential organelles that carry out a number of pivotal metabolic processes and maintain cellular homeostasis. Mitochondrial dysfunction caused by various stresses is associated with many diseases such as type 2 diabetes, obesity, cancer, heart failure, neurodegenerative disorders, and aging. Therefore, it is important to understand the stimuli that induce mitochondrial stress. However, broad analysis of mitochondrial stress has not been carried out to date. Here, we present a set of fluorescent tools, called mito-Pain (mitochondrial PINK1 accumulation index), which enable the labeling of stressed mitochondria. Mito-Pain uses PTEN-induced putative kinase 1 (PINK1) stabilization on mitochondria and quantifies mitochondrial stress levels by comparison with PINK1-GFP, which is stabilized under mitochondrial stress, and RFP-Omp25, which is constitutively localized on mitochondria. To identify compounds that induce mitochondrial stress, we screened a library of 3374 compounds using mito-Pain and identified 57 compounds as mitochondrial stress inducers. Furthermore, we classified each compound into several categories based on mitochondrial response: depolarization, mitochondrial morphology, or Parkin recruitment. Parkin recruitment to mitochondria was often associated with mitochondrial depolarization and aggregation, suggesting that Parkin is recruited to heavily damaged mitochondria. In addition, many of the compounds led to various mitochondrial morphological changes, including fragmentation, aggregation, elongation, and swelling, with or without Parkin recruitment or mitochondrial depolarization. We also found that several compounds induced an ectopic response of Parkin, leading to the formation of cytosolic puncta dependent on PINK1. Thus, mito-Pain enables the detection of stressed mitochondria under a wide variety of conditions and provides insights into mitochondrial quality control systems.

Forced lipophagy reveals that lipid droplets are required for early embryonic development in mouse.

Although autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.

Reversible DNA damage checkpoint activation at the presenescent stage in telomerase-deficient cells of Saccharomyces cerevisiae.

The telomere protects the ends of eukaryotic linear chromosomes, and its shortening or erosion is recognized as DNA damage, leading to loss of proliferation activity and, thus, cellular senescence at the population level. Here, using a GFP-based DNA damage checkpoint marker suited for single-cell observation of Saccharomyces cerevisiae cells, we correlated the checkpoint status of telomere-shortened cells with their behavior. We show that some cells possessing short telomeres retain proliferation capacity even after the DNA damage checkpoint is activated. At the presenescent stage, the activation of the checkpoint causes cell cycle delay, but does not induce permanent cell cycle arrest, eventually leading to the expansion of cell size that is characteristic of cellular senescence. Moreover, the proliferation capacity of checkpoint-activated cells is not dependent on homologous recombination or the checkpoint adaptation pathway. The retention of proliferation capacity is specific to the telomere-derived DNA damage response, suggesting that damaged telomeres differ functionally from other types of DNA damage. Our data establish the role of the presenescent stage in telomere shortening-induced senescence, which proceeds gradually and is associated with a variety of changes, including altered cell morphology and metabolism.

DA-Raf, a dominant-negative regulator of the Ras-ERK pathway, is essential for skeletal myocyte differentiation including myoblast fusion and apoptosis.

Ras-activated ERK pathway (Raf-MEK-ERK phosphorylation cascade) regulates a variety of cellular responses including cell proliferation, differentiation, survival, and apoptosis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative manner. Here we show that DA-Raf plays essential roles in skeletal myocyte differentiation including myoblast fusion and in apoptosis, which are suppressed by the Ras-ERK pathway. Expression of DA-Raf was highly induced in C2C12 skeletal myocytes in a low serum concentration of differentiation condition and in NIH3T3 fibroblasts under a serum starvation apoptosis-inducing condition. Stable knockdown of DA-Raf resulted in suppression of muscle-specific gene expression, myoblast fusion, and apoptosis. In contrast, exogenous overexpression of DA-Raf prominently caused apoptosis. DA-Raf induces apoptosis by preventing ERK-RSK-mediated inhibitory phosphorylation of Bad. Although it has been reported that apoptosis triggers myoblast fusion, DA-Raf-induced apoptosis was not involved in myoblast fusion in C2C12 cells. These results imply that suppression of the Ras-ERK pathway by DA-Raf is essential for both myocyte differentiation including myoblast fusion and apoptosis but that apoptosis is not a prerequisite for myoblast fusion.

An ER complex of ODR-4 and ODR-8/Ufm1 specific protease 2 promotes GPCR maturation by a Ufm1-independent mechanism.

Despite the importance of G-protein coupled receptors (GPCRs) their biogenesis is poorly understood. Like vertebrates, C. elegans uses a large family of GPCRs as chemoreceptors. A subset of these receptors, such as ODR-10, requires the odr-4 and odr-8 genes to be appropriately localized to sensory cilia. The odr-4 gene encodes a conserved tail-anchored transmembrane protein; the molecular identity of odr-8 is unknown. Here, we show that odr-8 encodes the C. elegans ortholog of Ufm1-specific protease 2 (UfSP2). UfSPs are cysteine proteases identified biochemically by their ability to liberate the ubiquitin-like modifier Ufm1 from its pro-form and protein conjugates. ODR-8/UfSP2 and ODR-4 are expressed in the same set of twelve chemosensory neurons, and physically interact at the ER membrane. ODR-4 also binds ODR-10, suggesting that an ODR-4/ODR-8 complex promotes GPCR folding, maturation, or export from the ER. The physical interaction between human ODR4 and UfSP2 suggests that this complex's role in GPCR biogenesis may be evolutionarily conserved. Unexpectedly, mutant versions of ODR-8/UfSP2 lacking catalytic residues required for protease activity can rescue all odr-8 mutant phenotypes tested. Moreover, deleting C. elegans ufm-1 does not alter chemoreceptor traffic to cilia, either in wild type or in odr-8 mutants. Thus, UfSP2 proteins have protease- and Ufm1-independent functions in GPCR biogenesis.

Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells.

Autophagy is mediated by a unique organelle, the autophagosome. Autophagosome formation involves a number of autophagy-related (ATG) proteins and complicated membrane dynamics. Although the hierarchical relationships of ATG proteins have been investigated, how individual ATG proteins or their complexes contribute to the organization of the autophagic membrane remains largely unknown. Here, systematic ultrastructural analysis of mouse embryonic fibroblasts (MEFs) and HeLa cells deficient in various ATG proteins reveals that the emergence of the isolation membrane (phagophore) requires FIP200 (also known as RB1CC1), ATG9A and phosphatidylinositol (PtdIns) 3-kinase activity. By contrast, small premature isolation-membrane-like and autophagosome-like structures were generated in cells lacking VMP1 and both ATG2A and ATG2B, respectively. The isolation membranes could elongate in cells lacking ATG5, but did not mature into autophagosomes. We also found that ferritin clusters accumulated at the autophagosome formation site together with p62 (also known as SQSTM1) in autophagy-deficient cells. These results reveal the specific functions of these representative ATG proteins in autophagic membrane organization and ATG-independent recruitment of ferritin to the site of autophagosome formation.

Stearoyl-CoA desaturase 1 activity is required for autophagosome formation.

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.

Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy.

Mitochondria can be degraded by autophagy in a process termed mitophagy. The Parkinson-disease-associated ubiquitin ligase Parkin can trigger mitophagy of depolarized mitochondria. However, it remains to be determined how the autophagy machinery is involved in this specific type of autophagy. It has been speculated that adaptor proteins such as p62 might mediate the interaction between the autophagosomal LC3 family of proteins and ubiquitylated proteins on mitochondria. Here, we describe our systematic analysis of the recruitment of Atg proteins in Parkin-dependent mitophagy. Structures containing upstream Atg proteins, including ULK1, Atg14, DFCP1, WIPI-1 and Atg16L1, can associate with depolarized mitochondria even in the absence of membrane-bound LC3. Atg9A structures are also recruited to these damaged mitochondria as well as to the autophagosome formation site during starvation-induced canonical autophagy. In the initial steps of Parkin-mediated mitophagy, the structures containing the ULK1 complex and Atg9A are independently recruited to depolarized mitochondria and both are required for further recruitment of downstream Atg proteins except LC3. Autophagosomal LC3 is important for efficient incorporation of damaged mitochondria into the autophagosome at a later stage. These findings suggest a process whereby the isolation membrane is generated de novo on damaged mitochondria as opposed to one where a preformed isolation membrane recognizes mitochondria.

Novel autophagy inducers by accelerating lysosomal clustering against Parkinson's disease.

The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including α-synuclein (αSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble αSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.

Alpha 2-macroglobulin acts as a clearance factor in the lysosomal degradation of extracellular misfolded proteins.

Proteostasis regulates protein folding and degradation; its maintenance is essential for resistance to stress and aging. The loss of proteostasis is associated with many age-related diseases. Within the cell, molecular chaperones facilitate the refolding of misfolded proteins into their bioactive forms, thus preventing undesirable interactions and aggregation. Although the mechanisms of intracellular protein degradation pathways for intracellular misfolded proteins have been extensively studied, the protein degradation pathway for extracellular proteins remain poorly understood. In this study, we identified several misfolded proteins that are substrates for alpha 2-macroglobulin (α2M), an extracellular chaperone. We also established a lysosomal internalization assay for α2M, which revealed that α2M mediates the lysosomal degradation of extracellular misfolded proteins. Comparative analyses of α2M and clusterin, another extracellular chaperone, indicated that α2M preferentially targets aggregation-prone proteins. Thus, we present the degradation pathway of α2M, which interacts with aggregation-prone proteins for lysosomal degradation via selective internalization.

CCPG1 recognizes endoplasmic reticulum luminal proteins for selective ER-phagy.

The endoplasmic reticulum (ER) is a major cell compartment where protein synthesis, folding, and posttranslational modifications occur with assistance from a wide variety of chaperones and enzymes. Quality control systems selectively eliminate abnormal proteins that accumulate inside the ER due to cellular stresses. ER-phagy, that is, selective autophagy of the ER, is a mechanism that maintains or reestablishes cellular and ER-specific homeostasis through removal of abnormal proteins. However, how ER luminal proteins are recognized by the ER-phagy machinery remains unclear. Here, we applied the aggregation-prone protein, six-repeated islet amyloid polypeptide (6xIAPP), as a model ER-phagy substrate and found that cell cycle progression 1 (CCPG1), which is an ER-phagy receptor, efficiently mediates its degradation via ER-phagy. We also identified prolyl 3-hydroxylase family member 4 (P3H4) as an endogenous cargo of CCPG1-dependent ER-phagy. The ER luminal region of CCPG1 contains several highly conserved regions that we refer to as cargo-interacting regions (CIRs); these interact directly with specific luminal cargos for ER-phagy. Notably, 6xIAPP and P3H4 interact directly with different CIRs. These findings indicate that CCPG1 is a bispecific ER-phagy receptor for ER luminal proteins and the autophagosomal membrane that contributes to the efficient removal of aberrant ER-resident proteins through ER-phagy.

Formation of aggresomes with hydrogel-like characteristics by proteasome inhibition.

The spatiotemporal sequestration of misfolded proteins is a mechanism by which cells counterbalance proteome homeostasis upon exposure to various stress stimuli. Chronic inhibition of proteasomes results in a large, juxtanuclear, membrane-less inclusion, known as the aggresome. Although the molecular mechanisms driving its formation, clearance, and pathophysiological implications are continuously being uncovered, the biophysical aspects of aggresomes remain largely uncharacterized. Using fluorescence recovery after photobleaching and liquid droplet disruption assays, we found that the aggresomes are a homogeneously blended condensates with liquid-like properties similar to droplets formed via liquid-liquid phase separation. However, unlike fluidic liquid droplets, aggresomes have more viscosity and hydrogel-like characteristics. We also observed that the inhibition of aggresome formation using microtubule-disrupting agents resulted in less soluble and smaller cytoplasmic speckles, which was associated with marked cytotoxicity. Therefore, the aggresome appears to be cytoprotective and serves as a temporal reservoir for dysfunctional proteasomes and substrates that need to be degraded. Our results suggest that the aggresome assembles through distinct and potentially sequential processes of energy-dependent retrograde transportation and spontaneous condensation into a hydrogel.

Disruption of actin dynamics induces autophagy of the eukaryotic chaperonin TRiC/CCT.

Autophagy plays important role in the intracellular protein quality control system by degrading abnormal organelles and proteins, including large protein complexes such as ribosomes. The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC), also called chaperonin-containing TCP1 (CCT), is a 1-MDa hetero-oligomer complex comprising 16 subunits that facilitates the folding of ~10% of the cellular proteome that contains actin. However, the quality control mechanism of TRiC remains unclear. To monitor the autophagic degradation of TRiC, we generated TCP1α-RFP-GFP knock-in HeLa cells using a CRISPR/Cas9-knock-in system with an RFP-GFP donor vector. We analyzed the autophagic degradation of TRiC under several stress conditions and found that treatment with actin (de)polymerization inhibitors increased the lysosomal degradation of TRiC, which was localized in lysosomes and suppressed by deficiency of autophagy-related genes. Furthermore, we found that treatment with actin (de)polymerization inhibitors increased the association between TRiC and unfolded actin, suggesting that TRiC was inactivated. Moreover, unfolded actin mutants were degraded by autophagy. Taken together, our results indicate that autophagy eliminates inactivated TRiC, serving as a quality control system.

Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells.

Endocytic internalization of extracellular proteins plays roles in signaling, nutrient uptake, immunity, and extracellular protein quality control. However, there are few protocols for analyzing the lysosomal degradation of extracellular protein. Here, we purified secreted proteins fused with pH-sensitive GFP and acid- and protease-resistant RFP from mammalian cells and describe an internalization assay for mammalian cells. This protocol enables quantification of cellular uptake and lysosomal degradation of protein-of-interest (POI) via cell biological and biochemical analyses. For full details on the use and execution of this protocol, please refer to Itakura et al. (2020).

Heparan sulfate is a clearance receptor for aberrant extracellular proteins.

The accumulation of aberrant proteins leads to various neurodegenerative disorders. Mammalian cells contain several intracellular protein degradation systems, including autophagy and proteasomal systems, that selectively remove aberrant intracellular proteins. Although mammals contain not only intracellular but also extracellular proteins, the mechanism underlying the quality control of aberrant extracellular proteins is poorly understood. Here, using a novel quantitative fluorescence assay and genome-wide CRISPR screening, we identified the receptor-mediated degradation pathway by which misfolded extracellular proteins are selectively captured by the extracellular chaperone Clusterin and undergo endocytosis via the cell surface heparan sulfate (HS) receptor. Biochemical analyses revealed that positively charged residues on Clusterin electrostatically interact with negatively charged HS. Furthermore, the Clusterin-HS pathway facilitates the degradation of amyloid ß peptide and diverse leaked cytosolic proteins in extracellular space. Our results identify a novel protein quality control system for preserving extracellular proteostasis and highlight its role in preventing diseases associated with aberrant extracellular proteins.