Shun Kageyama

p62/SQSTM1/A170: physiology and pathology.

p62/SQSTM1/A170 (hereafter referred to as p62) is a stress-inducible intracellular protein known to regulate various signal transduction pathways involved in cell survival and cell death. Comprehensive analysis of LC3 (an autophagosome localizing protein)-binding proteins resulted in the recognition of autophagy and p62. While autophagy modulates the level of p62 protein, p62 can suppress autophagy via activation of mTORC1. Moreover, growing lines of evidence point to the important role of p62 in directing ubiquitinated cargos toward autophagy as well as compaction of those cargos. Furthermore, this protein functions as a signaling hub for various signal transduction pathways, such as NF-κB signaling, apoptosis, and Nrf2 activation, whose dysregulation is associated with Paget disease of bone and tumorigenesis. In this review, we discuss the pathophysiological significance of p62 and its role in autophagy.

Differential Involvement of Atg16L1 in Crohn Disease and Canonical Autophagy ANALYSIS OF THE ORGANIZATION OF THE Atg16L1 COMPLEX IN FIBROBLASTS

A single nucleotide polymorphism in Atg16L1, an autophagy-related gene (ATG), is a risk factor for Crohn disease, a major form of chronic inflammatory bowel disease. However, it is still unknown how the Atg16L1 variant contributes to disease development. The Atg16L1 protein possesses a C-terminal WD repeat domain whose function is entirely unknown, and the Crohn disease-associated mutation (T300A) is within this domain. To elucidate the function of the WD repeat domain, we established an experimental system in which a WD repeat domain mutant of Atg16L1 is stably expressed in Atg16L1-deficient mouse embryonic fibroblasts. Using the system, we show that the Atg16L1 complex forms a dimeric complex and that the total Atg16L1 protein level is strictly maintained, possibly by the ubiquitin proteasome system. Furthermore, we show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium. Therefore, we propose that Atg16L1 T300A is differentially involved in Crohn disease and canonical autophagy.

Proteasome Dysfunction Activates Autophagy and the Keap1-Nrf2 Pathway

Background: Malfunctions in the ubiquitin-proteasome system cause accumulation of non-functional, potentially toxic protein aggregates. Results: The protein aggregates activate Nrf2 and are then excluded by autophagy in vivo. Conclusion: Both Nrf2 and autophagy serve as in vivo cellular adaptations to impaired proteasome. Significance: Cells contain networks of cellular defense mechanisms against defective proteostasis. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. These pathways are interdependent, and dysfunction in either pathway causes accumulation of ubiquitin-positive aggregates, a hallmark of human pathological conditions. To elucidate in vivo compensatory action(s) against proteasomal dysfunction, we developed mice with reduced proteasome activity in their livers. The mutant mice exhibited severe liver damage, accompanied by formation of aggregates positive for ubiquitin and p62/Sqstm1, an adaptor protein for both selective autophagy and the anti-oxidative Keap1-Nrf2 pathway. These aggregates were selectively entrapped by autophagosomes, and pathological features of livers with impaired proteasome activity were exacerbated by simultaneous suppression of autophagy. In contrast, concomitant loss of p62/Sqstm1 had no apparent effect on the liver pathology though p62/Sqstm1 was indispensable for the aggregates formation. Furthermore, defective proteasome function led to transcriptional activation of the Nrf2, which served as a physiological adaptation. Our in vivo data suggest that cells contain networks of cellular defense mechanisms against defective proteostasis.

Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures

Several autophagy proteins contain an LC3‐interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B‐positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP‐ALFY‐LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR‐binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.

The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates

Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.

LC3B is indispensable for selective autophagy of p62 but not basal autophagy

Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.

Megalin-Mediated Tubuloglomerular Alterations in High-Fat Diet–Induced Kidney Disease

Obesity, an important risk factor for metabolic syndrome (MetS) and cardiovascular disease, is often complicated by CKD, which further increases cardiovascular risk and causes ESRD. To elucidate the mechanism underlying this relationship, we investigated the role of the endocytic receptor megalin in proximal tubule epithelial cells (PTECs). We studied a high-fat diet (HFD)-induced obesity/MetS model using kidney-specific mosaic megalin knockout (KO) mice. Compared with control littermates fed a normal-fat diet, control littermates fed an HFD for 12 weeks showed autolysosomal dysfunction with autophagy impairment and increased expression of hypertrophy, lipid peroxidation, and senescence markers in PTECs of the S2 segment, peritubular capillary rarefaction with localized interstitial fibrosis, and glomerular hypertrophy with mesangial expansion. These were ameliorated in HFD-fed megalin KO mice, even though these mice had the same levels of obesity, dyslipidemia, and hyperglycemia as HFD-fed control mice. Intravital renal imaging of HFD-fed wild-type mice also demonstrated the accumulation of autofluorescent lipofuscin-like substances in PTECs of the S2 segment, accompanied by focal narrowing of tubular lumens and peritubular capillaries. In cultured PTECs, fatty acid-rich albumin induced the increased expression of genes encoding PDGF-B and monocyte chemoattractant protein-1 via megalin, with large (auto)lysosome formation, compared with fatty acid-depleted albumin. Collectively, the megalin-mediated endocytic handling of glomerular-filtered (lipo)toxic substances appears to be involved primarily in hypertrophic and senescent PTEC injury with autophagy impairment, causing peritubular capillary damage and retrograde glomerular alterations in HFD-induced kidney disease. Megalin could be a therapeutic target for obesity/MetS-related CKD, independently of weight, dyslipidemia, and hyperglycemia modification.

Three-Axis Model for Atg Recruitment in Autophagy against Salmonella

Salmonella enterica serovar Typhimurium enter epithelial cells and take up residence there. Within epithelial cells, a portion of the bacteria are surrounded by an autophagosome-like double-membrane structure, and they are still residing within the Salmonella-containing vacuole (SCV). In this paper, we will discuss how the autophagy machinery is recruited in proximity to Salmonella. The formation of this double membrane requires Atg9L1 and FIP200; these proteins are important for autophagy-specific recruitment of the PI3-kinase complex. In the absence of Atg9L1, FIP200, and PI3-kinase activity, LC3 is still recruited to the vicinity of Salmonella. We propose a novel model in which the mechanism of LC3 recruitment is separate from the generation of the isolation membrane. There exist at least three axes in Atg recruitment: ULK1 complex, Atg9L1, and Atg16L complex.

Impaired G1-Arrest, Autophagy, and Apoptosis in Atg7-Knockout Mice

Exit from cell division cycle, induction of autophagy, and activation of apoptosis in response to metabolic stress occur simultaneously or sequentially. However, the molecular interrelation(s) between these intracellular events remains obscure. Recent work by Finkel et al1 provides evidence that Atg7, an autophagy-related protein, regulates G1-arrest by interacting with p53 and that p53-mediated apoptosis is activated under autophagy-deficient conditions. Macroautophagy (hereafter referred to as autophagy) is a self-eating system conserved among eukaryotes. In this process, cellular components, including organelles, are entrapped into a double-membrane structure called the autophagosome and then degraded by lysosomal hydrolases (Figure 1).2 In addition to its role in supplying amino acids in response to nutrient starvation, autophagy is involved in quality control to maintain cell health. Thus, inactivation of autophagy results in the accumulation of cytoplasmic protein aggregates of misfolded proteins and damaged and degenerated organelles, which compromise cell function and often result in life-threatening diseases.2 Figure 1. Autophagy . The initial steps of autophagy include the formation and subsequent elongation of the isolation membrane. The isolation membrane then enwraps various cytoplasmic constituents, such as organelles, until its edges fuse with each other to form a double-membrane structure called the autophagosome. Finally, the outer membrane of the autophagosome fuses with the lysosome and endosome. The sequestered cytoplasmic components, together with the inner membrane of the autophagosome, are completely degraded by lysosomal hydrolases (Mizushima et al4 for details on the molecular players). The ubiquitin-like modifier, LC3, covalently conjugates with phosphatidylethanolamine (PE) through an enzymatic cascade consisting of Atg7 (E1-like enzyme), Atg3 (E2-like enzyme), and Atg12-Atg5-Atg16L complex (function as an E3-like enzyme). The PE-conjugated LC3, named LC3-II, is localized to the inner and outer membranes of the isolation membrane and is essential for membrane biogenesis and closure of the isolation membrane. The activated ULK1 complex …

Sqstm1-GFP knock-in mice reveal dynamic actions of Sqstm1 during autophagy and under stress conditions in living cells

ABSTRACT Sqstm1 serves as a signaling hub and receptor for selective autophagy. Consequently, dysregulation of Sqstm1 causes imbalances in signaling pathways and disrupts proteostasis, thereby contributing to the development of human diseases. Environmental stresses influence the level of Sqstm1 by altering its expression and/or autophagic degradation, and also changes the localization of Sqstm1, making it difficult to elucidate the actions and roles of this protein. In this study, we developed knock-in mice expressing Sqstm1 fused to GFP (Sqstm1-GFPKI/+). Using these Sqstm1-GFPKI/+ mice, we revealed for the first time the dynamics of endogenous Sqstm1 in living cells. Sqstm1–GFP was translocated to a restricted area of LC3-positive structures, which primarily correspond to the inside of autophagosomes, and then degraded. Moreover, exposure to arsenite induced expression of Sqstm1–GFP, followed by accumulation of the fusion protein in large aggregates that were degraded by autophagy. Furthermore, suppression of autophagy in Sqstm1-GFPKI/+ mouse livers caused accumulation of Sqstm1–GFP and formation of GFP-positive aggregate structures, leading to severe hepatic failure. These results indicate that Sqstm1-GFPKI/+ mice are a useful tool for analyzing Sqstm1 in living cells and intact animals. Highlighted Article: We have developed Sqstm1-GFP knock-in mice, which are useful for elucidating the molecular mechanisms of cellular events, including autophagy, and various diseases in which Sqstm1 plays a role.