Autophagy is required during high MUC2 mucin biosynthesis in colonic goblet cells to contend metabolic stress

Goblet cells are specialized for the production and secretion of MUC2 glycoproteins that forms a thick layer covering the mucosal epithelium as a protective barrier against noxious substances and invading microbes. High MUC2 mucin biosynthesis induces endoplasmic reticulum (ER) stress and apoptosis in goblet cells during inflammatory and infectious diseases. Autophagy is an intracellular degradation process required for maintenance of intestinal homeostasis. In this study, we hypothesized that autophagy was triggered during high MUC2 mucin biosynthesis from colonic goblet cells to cope with metabolic stress. To interrogate this, we analyzed the autophagy process in high MUC2-producing human HT29-H and a clone HT29-L silenced for MUC2 expression by lentivirus-mediated shRNA, and WT and CRISPR/Cas9 MUC2 KO LS174T cells. Autophagy was constitutively increased in high MUC2 producing cells characterized by elevated pULK1S555 expression and increased numbers of autophagosomes as compared to MUC2 silenced or gene edited cells. Similarly, colonoids from Muc2+/+ but not Muc2-/- littermates differentiated into goblet cells showed increased autophagy. IL-22 treatment corrected misfolded MUC2 protein and alleviated the autophagy process in LS174T cells. This study highlights that autophagy plays an essential role in goblet cells to survive during high mucin biosynthesis by regulating cellular homeostasis.

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p62 targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

The Journal of Cell Biology ◽

10.1083/jcb.201009067 ◽

2011 ◽

Vol 192 (1) ◽

pp. 17-27 ◽

Cited By ~ 252

Author(s):

Eisuke Itakura ◽

Noboru Mizushima

Keyword(s):

Endoplasmic Reticulum ◽

Degradation Process ◽

Related Protein ◽

Autophagosome Formation ◽

Intracellular Degradation ◽

Cargo Receptor ◽

Specific Proteins

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)–associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.

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Autophagy: Dance of the yeast ATGs and worm EPGs

The Biochemist ◽

10.1042/bio03305020 ◽

2011 ◽

Vol 33 (5) ◽

pp. 20-22

Author(s):

Fan Wu ◽

Hong Zhang

Keyword(s):

Dynamic Process ◽

Metabolic Stress ◽

Degradation Process ◽

Stress Conditions ◽

Inner Membrane ◽

Intracellular Degradation ◽

Physiological Processes ◽

Higher Eukaryotes ◽

Autophagy Activity

Macroautophagy (hereinafter referred to simply as autophagy) is a lysosome-mediated intracellular degradation process that involves the bulk segregation and digestion of portions of the cytoplasm in membrane-enclosed vacuoles1. The dynamic process of autophagy starts with the formation of a cup-shaped membrane sac (known as the isolation membrane or phagophore) which engulfs a portion of cytoplasm, and then elongates and seals off to form a double-membraned autophagosome. The autophagosome fuses with lysosomes to form the autolysosome, where the cargo together with the inner membrane of the autophagosome is degraded. Finally, the digested material is released back into the cytosol through lysosomal permeases for recycling1. Autophagy plays an important role in a wide variety of physiological processes in higher eukaryotes, including adaptation to various metabolic stress conditions, removal of aggregate-prone proteins and damaged organelles, and elimination of invading pathogens. Dysregulated autophagy activity has been linked to various pathological diseases such as neurodegeneration, cardiomyopathy and tumorigenesis2.

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Maternal high-fat diet induces metabolic stress response disorders in offspring hypothalamus

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0056 ◽

2017 ◽

Vol 59 (1) ◽

pp. 81-92 ◽

Cited By ~ 7

Author(s):

Long The Nguyen ◽

Sonia Saad ◽

Yi Tan ◽

Carol Pollock ◽

Hui Chen

Keyword(s):

Endoplasmic Reticulum ◽

Body Weight ◽

Er Stress ◽

High Fat Diet ◽

Maternal Obesity ◽

Mrna Level ◽

Metabolic Stress ◽

High Fat ◽

Chemical Chaperone ◽

Protein Levels

Maternal obesity has been shown to increase the risk of obesity and related disorders in the offspring, which has been partially attributed to changes of appetite regulators in the offspring hypothalamus. On the other hand, endoplasmic reticulum (ER) stress and autophagy have been implicated in hypothalamic neuropeptide dysregulation, thus may also play important roles in such transgenerational effect. In this study, we show that offspring born to high-fat diet-fed dams showed significantly increased body weight and glucose intolerance, adiposity and plasma triglyceride level at weaning. Hypothalamic mRNA level of the orexigenic neuropeptide Y (NPY) was increased, while the levels of the anorexigenic pro-opiomelanocortin (POMC), NPY1 receptor (NPY1R) and melanocortin-4 receptor (MC4R) were significantly downregulated. In association, the expression of unfolded protein response (UPR) markers including glucose-regulated protein (GRP)94 and endoplasmic reticulum DNA J domain-containing protein (Erdj)4 was reduced. By contrast, protein levels of autophagy-related genes Atg5 and Atg7, as well as mitophagy marker Parkin, were slightly increased. The administration of 4-phenyl butyrate (PBA), a chemical chaperone of protein folding and UPR activator, in the offspring from postnatal day 4 significantly reduced their body weight, fat deposition, which were in association with increased activating transcription factor (ATF)4, immunoglobulin-binding protein (BiP) and Erdj4 mRNA as well as reduced Parkin, PTEN-induced putative kinase (PINK)1 and dynamin-related protein (Drp)1 protein expression levels. These results suggest that hypothalamic ER stress and mitophagy are among the regulatory factors of offspring metabolic changes due to maternal obesity.

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Emc3 maintains intestinal homeostasis by preserving secretory lineages

Mucosal Immunology ◽

10.1038/s41385-021-00399-2 ◽

2021 ◽

Author(s):

Meina Huang ◽

Li Yang ◽

Ning Jiang ◽

Quanhui Dai ◽

Runsheng Li ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Dominant Role ◽

Paneth Cell ◽

Intestinal Barrier ◽

Goblet Cells ◽

Paneth Cells ◽

Intestinal Barrier Function ◽

Endoplasmic Reticulum Membrane ◽

Mucus Production

AbstractIntestinal exocrine secretory lineages, including goblet cells and Paneth cells, provide vital innate host defense to pathogens. However, how these cells are specified and maintained to ensure intestinal barrier function remains poorly defined. Here we show that endoplasmic reticulum membrane protein complex subunit 3 (Emc3) is essential for differentiation and function of exocrine secretory lineages. Deletion of Emc3 in intestinal epithelium decreases mucus production by goblet cells and Paneth cell population, along with gut microbial dysbiosis, which result in spontaneous inflammation and increased susceptibility to DSS-induced colitis. Moreover, Emc3 deletion impairs stem cell niche function of Paneth cells, thus resulting in intestinal organoid culture failure. Mechanistically, Emc3 deficiency leads to increased endoplasmic reticulum (ER) stress. Mitigating ER stress with tauroursodeoxycholate acid alleviates secretory dysfunction and restores organoid formation. Our study identifies a dominant role of Emc3 in maintaining intestinal mucosal homeostasis.

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Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

The Journal of Cell Biology ◽

10.1083/jcb.115.1.31 ◽

1991 ◽

Vol 115 (1) ◽

pp. 31-43 ◽

Cited By ~ 235

Author(s):

H Plutner ◽

A D Cox ◽

S Pind ◽

R Khosravi-Far ◽

J R Bourne ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Essential Role ◽

Secretory Pathway ◽

Binding Protein ◽

Vesicular Transport ◽

Initial Step ◽

Gtp Binding Protein ◽

Gtp Binding ◽

Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

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Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2

Biochemical Journal ◽

10.1042/bj2940735 ◽

1993 ◽

Vol 294 (3) ◽

pp. 735-743 ◽

Cited By ~ 129

Author(s):

S Benjannet ◽

N Rondeau ◽

L Paquet ◽

A Boudreault ◽

C Lazure ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Brefeldin A ◽

Pulse Labelling ◽

Prohormone Convertases ◽

Post Translational Modifications ◽

Intracellular Degradation ◽

Golgi Compartment ◽

Carbonyl Cyanide ◽

Insulinoma Cells

We present herein the pulse-chase analysis of the biosynthesis of the prohormone convertases PC1 and PC2 in the endocrine GH4C1 cells infected with vaccinia virus recombinants expressing these convertases. Characterization of the pulse-labelled enzymes demonstrated that pro-PC1 (88 kDa) is cleaved into PC1 (83 kDa) and pro-PC2 (75 kDa) into PC2 (68 kDa). Secretion of glycosylated and sulphated PC1 (84 kDa) occurs about 30 min after the onset of biosynthesis, whereas glycosylated and sulphated PC2 (68 kDa) is detected in the medium after between 1 and 2 h. Furthermore, in the case of pro-PC2 only, we observed that a fraction of this precursor escapes glycosylation. A small proportion (about 5%) of the intracellular glycosylated pro-PC2 (75 kDa) is sulphated, and it is this glycosylated and sulphated precursor that is cleaved into the secretable 68 kDa form of PC2. Major differences in the carbohydrate structures of PC1 and PC2 are demonstrated by the resistance of the secreted PC1 to endoglycosidase H digestion and sensitivity of the secreted PC2 to this enzyme. Inhibition of N-glycosylation with tunicamycin caused a dramatic intracellular degradation of these convertases within the endoplasmic reticulum, with the net effect of a reduction in the available activity of PC1 and PC2. These results emphasize the importance of N-glycosylation in the folding and stability of PC1 and PC2. Pulse-labelling experiments in uninfected mouse beta TC3 and rat Rin m5F insulinoma cells, which endogenously synthesize PC2, showed that, as in infected GH4C1 cells, pro-PC2 predominates intracellularly. In order to define the site of prosegment cleavage, pulse-chase analysis was performed at low temperature (15 degrees C) or after treatment of GH4C1 cells with either brefeldin A or carbonyl cyanide m-chlorophenylhydrazone. These results demonstrated that the onset of the conversions of pro-PC1 into PC1 and non-glycosylated pro-PC2 into PC2 (65 kDa) occur in a pre-Golgi compartment, presumably within the endoplasmic reticulum. In contrast, pulse labelling in the presence of Na(2)35SO4 demonstrated that the processing of glycosylated and sulphated pro-PC2 occurs within the Golgi apparatus. In order to test the possibility that zymogen processing is performed by furin, we co-expressed this convertase with either pro-PC1 or pro-PC2. The data demonstrated the inability of furin to cleave either proenzyme.

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Emerging roles of endoplasmic reticulum-resident selenoproteins in the regulation of cellular stress responses and the implications for metabolic disease

Biochemical Journal ◽

10.1042/bcj20170920 ◽

2018 ◽

Vol 475 (6) ◽

pp. 1037-1057 ◽

Cited By ~ 21

Author(s):

Alex B. Addinsall ◽

Craig R. Wright ◽

Sof Andrikopoulos ◽

Chris van der Poel ◽

Nicole Stupka

Keyword(s):

Metabolic Disease ◽

Endoplasmic Reticulum ◽

Stress Responses ◽

Cellular Stress ◽

Metabolic Stress ◽

Metabolic Dysfunction ◽

Iodothyronine Deiodinase ◽

Inflammatory Stress ◽

Cellular Stress Responses

Chronic metabolic stress leads to cellular dysfunction, characterized by excessive reactive oxygen species, endoplasmic reticulum (ER) stress and inflammation, which has been implicated in the pathogenesis of obesity, type 2 diabetes and cardiovascular disease. The ER is gaining recognition as a key organelle in integrating cellular stress responses. ER homeostasis is tightly regulated by a complex antioxidant system, which includes the seven ER-resident selenoproteins — 15 kDa selenoprotein, type 2 iodothyronine deiodinase and selenoproteins S, N, K, M and T. Here, the findings from biochemical, cell-based and mouse studies investigating the function of ER-resident selenoproteins are reviewed. Human experimental and genetic studies are drawn upon to highlight the relevance of these selenoproteins to the pathogenesis of metabolic disease. ER-resident selenoproteins have discrete roles in the regulation of oxidative, ER and inflammatory stress responses, as well as intracellular calcium homeostasis. To date, only two of these ER-resident selenoproteins, selenoproteins S and N have been implicated in human disease. Nonetheless, the potential of all seven ER-resident selenoproteins to ameliorate metabolic dysfunction warrants further investigation.

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The Role of Beclin 1-Dependent Autophagy in Cancer

Biology ◽

10.3390/biology9010004 ◽

2019 ◽

Vol 9 (1) ◽

pp. 4 ◽

Cited By ~ 10

Author(s):

Silvia Vega-Rubín-de-Celis

Keyword(s):

Dna Damage ◽

Tumor Suppression ◽

Protein Aggregates ◽

Degradation Process ◽

Beclin 1 ◽

Cancer Type ◽

Intracellular Degradation

Autophagy (self-eating) is an intracellular degradation process used by cells to keep a “clean house”; as it degrades abnormal or damaged proteins and organelles, it helps to fight infections and also provides energy in times of fasting or exercising. Autophagy also plays a role in cancer, although its precise function in each cancer type is still obscure, and whether autophagy plays a protecting (through the clearing of damaged organelles and protein aggregates and preventing DNA damage) or a promoting (by fueling the already stablished tumor) role in cancer remains to be fully characterized. Beclin 1, the mammalian ortholog of yeast Atg6/Vps30, is an essential autophagy protein and has been shown to play a role in tumor suppression. Here, an update of the tumorigenesis regulation by Beclin 1-dependent autophagy is provided.

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RAB6 and microtubules restrict protein secretion to focal adhesions

The Journal of Cell Biology ◽

10.1083/jcb.201805002 ◽

2019 ◽

Vol 218 (7) ◽

pp. 2215-2231 ◽

Cited By ~ 32

Author(s):

Lou Fourriere ◽

Amal Kasri ◽

Nelly Gareil ◽

Sabine Bardin ◽

Hugo Bousquet ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Cell Surface ◽

Protein Secretion ◽

Golgi Complex ◽

Essential Role ◽

Focal Adhesions ◽

Secreted Proteins ◽

Cell Compartments ◽

Secretion Assay

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

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