p62 bodies: cytosolic zoning by phase separation.

Cellular zoning or partitioning is critical in preventing macromolecules from random diffusion and orchestrating the spatiotemporal dynamics of biochemical reactions. Along with membranous organelles, membraneless organelles contribute to the precise regulation of biochemical reactions inside cells. In response to environmental cues, membraneless organelles rapidly form through liquid-liquid phase separation, sequester certain proteins and RNAs, mediate specific reactions and dissociate. Among membraneless organelles, ubiquitin-positive condensates, namely, p62 bodies, maintain cellular homeostasis through selective autophagy of themselves to contribute to intracellular quality control. p62 bodies also activate the anti-oxidative stress response regulated by the KEAP1-NRF2 system. In this review, we present an overview of recent advancements in cellular and molecular biology related to p62 bodies, highlighting their dynamic nature and functions.

Vault-phagy: a phase-separation-mediated selective autophagy of vault, a non-membranous organelle.

SQSTM1/p62 bodies are phase-separated condensates that play a fundamental role in intracellular quality control and stress responses. Despite extensive studies investigating the mechanism of formation and degradation of SQSTM1/p62 bodies, the constituents of SQSTM1/p62 bodies remain elusive. We recently developed a purification method for intracellular SQSTM1/p62 bodies using a cell sorter and identified their constituents by mass spectrometry. Combined with mass spectrometry of tissues from selective autophagy-deficient mice, we identified vault, a ubiquitous non-membranous organelle composed of proteins and non-coding RNA, as a novel substrate for selective autophagy. Vault directly binds to NBR1, an SQSTM1/p62 binding partner recruited to SQSTM1/p62 bodies, and is subsequently degraded by selective autophagy dependent on the phase separation of SQSTM1/p62. We named this process "vault-phagy" and found that defects in vault-phagy are related to nonalcoholic steatohepatitis (NASH)-derived hepatocellular carcinoma. Our method for purifying SQSTM1/p62 bodies will contribute to elucidating the mechanisms of several stress responses and diseases mediated by SQSTM1/p62 bodies.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex.

In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis.

AUTOPHAGY IN THE EYE: FROM PHYSIOLOGY TO PATHOPHYSOLOGY.

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.

Regulation of ER-derived membrane dynamics by the DedA domain-containing proteins VMP1 and TMEM41B.

The endoplasmic reticulum (ER) is a central hub for the biogenesis of various organelles and lipid-containing structures. Recent studies suggest that vacuole membrane protein 1 (VMP1) and transmembrane protein 41B (TMEM41B), multispanning ER membrane proteins, regulate the formation of many of these ER-derived structures, including autophagosomes, lipid droplets, lipoproteins, and double-membrane structures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. VMP1 and TMEM41B possess a DedA domain that is widely distributed not only in eukaryotes but also in prokaryotes and predicted to adopt a characteristic structure containing two reentrant loops. Furthermore, recent studies show that both proteins have lipid scrambling activity. Based on these findings, the potential roles of VMP1 and TMEM41B in the dynamic remodeling of ER membranes and the biogenesis of ER-derived structures are discussed.

A new insight into the lens: cytosolic PLAAT phospholipases degrade organelles to make the lens transparent.

All membrane-bound organelles are degraded during the terminal differentiation of lens fiber cells. How these organelles are degraded has been a long-standing question in biology. We recently revealed that PLAAT (phospholipase A and acyltransferase)-family phospholipases degrade organelles in the lens independently of macroautophagy. Here, we discuss the mechanism and physiological relevance of this new mode of intracellular degradation.

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.

Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.

Organelle degradation in the lens by PLAAT phospholipases.

The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.

Evolution and insights into the structure and function of the DedA superfamily containing TMEM41B and VMP1.

TMEM41B and VMP1 are endoplasmic reticulum (ER)-localizing multi-spanning membrane proteins required for ER-related cellular processes such as autophagosome formation, lipid droplet homeostasis and lipoprotein secretion in eukaryotes. Both proteins have a VTT domain, which is similar to the DedA domain found in bacterial DedA family proteins. However, the molecular function and structure of the DedA and VTT domains (collectively referred to as DedA domains) and the evolutionary relationships among the DedA domain-containing proteins are largely unknown. Here, we conduct a remote homology search and identify a new clade consisting mainly of bacterial proteins of unknown function that are members of the Pfam family PF06695. Phylogenetic analysis reveals that the TMEM41, VMP1, DedA and PF06695 families form a superfamily with a common origin, which we term the DedA superfamily. Coevolution-based structural prediction suggests that the DedA domain contains two reentrant loops facing each other in the membrane. This topology is biochemically verified by the substituted cysteine accessibility method. The predicted structure is topologically similar to that of the substrate-binding region of Na+-coupled glutamate transporter solute carrier 1 (SLC1) proteins. A potential ion-coupled transport function of the DedA superfamily proteins is discussed. This article has an associated First Person interview with the joint first authors of the paper.

No air without autophagy: autophagy is important for lung and swim bladder inflation.

Macroautophagy is a catabolic process critical for the degradation of intracellular material, but its physiological functions in vertebrates are not fully understood. Here, we discuss our recent finding that macroautophagy plays a role in lamellar body maturation. The lamellar body is a lysosome-related organelle and stores phospholipid-containing surfactant complexes that reduce the surface tension of the air-water interface in order to inflate the airspace in lungs and swim bladders. In the epithelial cells of these organs, autophagosomes fuse with immature lamellar bodies to increase their size and lipid contents. This function is essential for respiration after birth in mice and for maintaining buoyancy in zebrafish. These findings unveil a novel function of macroautophagy in the maturation of surfactant-containing lamellar bodies.

Autophagy Is Required for Maturation of Surfactant-Containing Lamellar Bodies in the Lung and Swim Bladder.

Autophagy is an intracellular degradation system, but its physiological functions in vertebrates are not yet fully understood. Here, we show that autophagy is required for inflation of air-filled organs: zebrafish swim bladder and mouse lung. In wild-type zebrafish swim bladder and mouse lung type II pulmonary epithelial cells, autophagosomes are formed and frequently fuse with lamellar bodies. The lamellar body is a lysosome-related organelle that stores a phospholipid-containing surfactant complex that lines the air-liquid interface and reduces surface tension. We find that autophagy is critical for maturation of the lamellar body. Accordingly, atg-deficient zebrafish fail to maintain their position in the water, and type-II-pneumocyte-specific Fip200-deficient mice show neonatal lethality with respiratory failure. Autophagy suppression does not affect synthesis of the surfactant phospholipid, suggesting that autophagy supplies lipids and membranes to lamellar bodies. These results demonstrate an evolutionarily conserved role of autophagy in lamellar body maturation.

A critical role of VMP1 in lipoprotein secretion.

Lipoproteins are lipid-protein complexes that are primarily generated and secreted from the intestine, liver, and visceral endoderm and delivered to peripheral tissues. Lipoproteins, which are assembled in the endoplasmic reticulum (ER) membrane, are released into the ER lumen for secretion, but its mechanism remains largely unknown. Here, we show that the release of lipoproteins from the ER membrane requires VMP1, an ER transmembrane protein essential for autophagy and certain types of secretion. Loss of vmp1, but not other autophagy-related genes, in zebrafish causes lipoprotein accumulation in the intestine and liver. Vmp1 deficiency in mice also leads to lipid accumulation in the visceral endoderm and intestine. In VMP1-depleted cells, neutral lipids accumulate within lipid bilayers of the ER membrane, thus affecting lipoprotein secretion. These results suggest that VMP1 is important for the release of lipoproteins from the ER membrane to the ER lumen in addition to its previously known functions.

Diverse Cellular Roles of Autophagy.

Macroautophagy is an intracellular degradation system that delivers diverse cytoplasmic materials to lysosomes via autophagosomes. Recent advances have enabled identification of several selective autophagy substrates and receptors, greatly expanding our understanding of the cellular functions of autophagy. In this review, we describe the diverse cellular functions of macroautophagy, including its essential contribution to metabolic adaptation and cellular homeostasis. We also discuss emerging findings on the mechanisms and functions of various types of selective autophagy.

Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation.

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.

Autophagy is essential for hearing in mice.

Hearing loss is the most frequent sensory disorder in humans. Auditory hair cells (HCs) are postmitotic at late-embryonic differentiation and postnatal stages, and their damage is the major cause of hearing loss. There is no measurable HC regeneration in the mammalian cochlea, and the maintenance of cell function is crucial for preservation of hearing. Here we generated mice deficient in autophagy-related 5 (Atg5), a gene essential for autophagy, in the HCs to investigate the effect of basal autophagy on hearing acuity. Deletion of Atg5 resulted in HC degeneration and profound congenital hearing loss. In autophagy-deficient HCs, polyubiquitinated proteins and p62/SQSTM1, an autophagy substrate, accumulated as inclusion bodies during the first postnatal week, and these aggregates increased in number. These findings revealed that basal autophagy has an important role in maintenance of HC morphology and hearing acuity.

Prognosis of infantile food protein-induced enterocolitis syndrome in Japan.

BACKGROUND: Although serum C-reactive protein (CRP) and the percentage of eosinophils in peripheral blood (Eo) are increased at onset in infants with food protein-induced enterocolitis syndrome (FPIES), the relationship of these laboratory findings to prognosis is presently unknown. METHODS: Correlation of serum CRP and Eo at onset with prognosis was analyzed in 32 patients with FPIES caused by cow's milk (CM). RESULTS: The rate of tolerance acquisition was 18.8%, 56.3%, 87.5%, and 96.9% at the ages of 6, 12, 24, and 36 months, respectively. Serum CRP increased in 50% of subjects at onset (median, 0.21 mg/dL; range, <0.20-18.2 mg/dL) and Eo was elevated in 71.9% of subjects at onset (median, 7.1%; range, 1.0-50.5%). Age at tolerance acquisition was significantly positively correlated with serum CRP at onset (r = 0.45, P < 0.01), and significantly negatively correlated with Eo at onset (r = -0.36, P < 0.05). Although CM-specific immunoglobulin E antibody (sIgE) was positive in nine of 32 FPIES patients at onset (median, 0.93; range, 0.38-18.9 kU/L), it decreased thereafter. CM-sIgE at onset did not correlate significantly with prognosis (r = 0.22, P > 0.05). CONCLUSIONS: Serum CRP is not only an indicator of the activity of intestinal inflammation, it is also a useful parameter of poor prognosis in FPIES. In contrast, eosinophilia at onset could be used as a marker of good prognosis, suggesting that it has some beneficial effects in the pathophysiology of FPIES.

Cytokine profile after oral food challenge in infants with food protein-induced enterocolitis syndrome.

BACKGROUND: Although food protein-induced enterocolitis syndrome (FPIES) is supposed to be caused by inflammation, the role of cytokines has not yet been clarified. METHODS: To elucidate the role of cytokines in the development of symptoms and abnormal laboratory findings at an oral food challenge (OFC), changes in serum cytokine levels were analyzed for 6 OFCs in 4 patients with FPIES. The result of OFC was judged positive if any gastrointestinal (GI) symptoms (vomiting, diarrhea, or bloody stool) were induced. RESULTS: Among 11 cytokines profiled, serum levels of interleukin (IL)-2, IL-5, and IL-8 were clearly increased in all 4 positive OFCs in which elevations of the serum level of C-reactive protein (CRP) and peripheral blood neutrophilia were also seen. The level of serum IL-10 also rose in 2 positive OFCs. Remarkable increases in the serum level of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), IL-6, and IL-12 were observed in a positive OFC where the serum level of CRP rose markedly (6.75 mg/dL). The serum levels of IL-5 were also elevated in 2 negative OFCs. No apparent specific correlations were found between cytokines and GI symptoms. CONCLUSIONS: These results suggest that IL-2 and IL-8 are involved in the antigen-specific immune responses in most patients with FPIES. Further studies are needed to elucidate the significance of these cytokine in the pathogenesis of FPIES.

A new probe to measure autophagic flux in vitro and in vivo.

Macroautophagy is a catabolic process that delivers cytoplasmic components via the autophagosome to lysosomes for degradation. Measuring autophagic activity is critical to dissect molecular mechanisms and functions of autophagy but remains challenging due to the lack of a definitive method. We have recently developed a new fluorescent probe, GFP-LC3-RFP-LC3ΔG, to assess autophagic flux. Upon intracellular expression, the probe is cleaved by ATG4 family proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. The former is degraded by autophagy while the latter persists as an internal control in the cytosol. Autophagic flux can thus be quantified by obtaining the ratio of GFP:RFP signals. Using this method, we have identified several autophagy-modulating drugs by screening an approved drug library. We have also demonstrated that induced and basal autophagic flux can be monitored in zebrafish and mice.

Eosinophilia in infants with food protein-induced enterocolitis syndrome in Japan.

BACKGROUND: Many Japanese infants with food protein-induced enterocolitis syndrome (FPIES) show eosinophilia, which has been thought to be a characteristic of food protein-induced proctocolitis (FPIP). METHODS: To elucidate the characteristics of eosinophilia in Japanese FPIES patients, 113 infants with non-IgE-mediated gastrointestinal food allergy due to cow's milk were enrolled and classified into FPIES (n = 94) and FPIP (n = 19). RESULTS: The percentage of peripheral blood eosinophils (Eo) was increased in most FPIES patients (median, 7.5%), which was comparable with that in FPIP patients (9.0%). Among FPIES patients, Eo was the highest in patients who had vomiting, bloody stool, and diarrhea simultaneously (12.9%) and lowest in patients with diarrhea alone (3.2%). Eo showed a significant positive correlation with the incidence of vomiting (Cramer's V = 0.31, p < 0.005) and bloody stool (Cramer's V = 0.34, p < 0.0005). A significant difference was found in Eo between early- (≤10 days, n = 56) and late-onset (>10 days, n = 38) FPIES (median, 9.8% vs. 5.4%; p < 0.005). IL-5 production by peripheral blood T cells stimulated with cow's milk protein in early-onset FPIES was significantly higher than that in late-onset FPIES (67.7 pg/mL vs. 12.5 pg/mL, p < 0.01), and showed a significant positive correlation with Eo (rs = 0.60, p < 0.01). CONCLUSIONS: This study demonstrated two types of eosinophilia in Japanese FPIES infants: conspicuous and mild eosinophilia in early- and late-onset FPIES patients, respectively. Conspicuous eosinophilia in early-onset FPIES is suggested to be caused by abnormally high IL-5 production.