Protein N-Glycosylation, Protein Folding, and Protein Quality Control

Jürgen Roth,Christian Zuber,박수진,Insook Jang,Yangsin Lee,Katarina Gaplovska Kysela,Valérie Le Fourn,Roger Santimaria,Bruno Guhl,조진원 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.6

Quality control of protein folding represents a funda-mental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.

Glycobiology leads to the discovery of a novel vesicular ER exit pathway

Jürgen Roth 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1

The cloning of glucosidase II (1) permitted the identification of a glyco-code promoting protein folding (2) and another one for dislocation of misfolded glycoproteins to degradation (3). Based on these works, EDEM1 (yeast ortholog Htm1p) was discovered and shown to be a lectin-like glycoprotein directing terminally misfolded glycoproteins for degradation (4,5). Endogenous EDEM1 existed mainly as a soluble glycoprotein and was sequestered in buds forming along rough ER cisternae outside of the transitional ER (6). This resulted in the formation of ~150 nm vesicles in the cytoplasm lacking a recognizable COP I or II coat. Surprisingly, EDEM1 in the cytosol became de-glycosylated and formed detergent-insoluble aggregates that were degraded by autophagy and not be proteasomes (7). EDEM1 vesicles also containedmisfolded Hong Kong variant of -1-antitrypsin (6). Together, these findings demonstrated the existence of a novel vesicular pathway out of the rough ER to remove misfolded luminal glycoproteins for subsequent proteasomal degradation. When the fate of incompletely assembled fibrinogen was studied (8), we made the following observations. Naturally occurring Aa-g assembly intermediates of fibrinogen were substrate for EDEM1 and exited the ER in EDEM1 vesicles. In contrast to the proteasomal degradation of free single chains, surplus Aa-g assembly intermediates of fibrinogen formed detergent-insoluble cytosolic aggregates that were degraded by autophagy. In summary, EDEM1 dislocates not only misfolded glycoproteins but also an assembly intermediate of an oligomeric glycoprotein. This indicates that ER-to-cytosol dislocation of such EDEM1 substrates occurs by a vesicular mechanism rather than by a channel of the ER membrane. Depending on the propensity of the glycoprotein to form aggregates in the cytosol. proteasomal degradation or autophagic elimination ensues.

Selective autophagy receptors are involved in degradation of EDEM1

Sujin Park,Insook Jang,Jin Won Cho,Jürgen Roth 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Misfolded and mis-assembled glycoproteins are retained in the endoplasmic reticulum (ER) where they are exposed to the protein folding machinery and protein quality control. The UPR (Unfoled Protein Response) is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum. Eventually, misfolded and mis-assembled glycoproteins are targeted for degradation by a process called ER-associated protein degradation (ERAD). EDEM1 is an ERAD component that recognizes misfolded luminal glycoproteins and is routing them for dislocation to the cytosol. This is classically followed by their degradation. Although EDEM1 was initially proposed to be lectin–like and to react with Man8GlcNAc2 oligosaccharides of glycoproteins, its complex mode of interaction with substrates has become clear only recently. However, still very little is known about the turnover and degradation mechanism of EDEM1 and how this relates to the fate of its substrates. We already reported that EDEM1 becomes rapidly degraded and that this occurs by basal autophagy. Here, we provide detailed insight into the mechanism by which EDEM1 becomes degraded. After its dislocation to the cytosol, EDEM1 is apparently making complexes with the selective autophagy receptors p62, NBR1 and Alfy. We observed co-distribution of EDEM1 and selective autophagy receptors by double or triple confocal laser scanning immunofluorescence. By quantifying the relationship of EDEM1 and the selective autophagy receptors as visualized by confocal laser scanning immunofluorescence, dramatical changes were observed in HepG2 cells following inhibition of autophagy by wortmannin treatment. These changes were fully reversible upon wortmannin wash-out. In addition, we observed its ubiquitination after dislocation to the cytosol. This demonstrates that the ERAD component EDEM1 itself undergoes ERAD involving selective autophagy

ERADication of EDEM1 occurs by selective autophagy

Sujin Park,Insook Jang,Jin Won Cho,Jürgen Roth 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1

Misfolded and misassembled glycoproteins are retained in the endoplasmic reticulum (ER) where they are exposed to the protein folding machinery and protein quality control. The Unfolded Protein Response is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the ER. Eventually, misfolded and mis-assembled glycoproteins are targeted for degradation by a process called ER-associated protein degradation (ERAD). EDEM1 is an ERAD component that interacts with misfolded luminal glycoproteins and routes them for dislocation. This is followed by their ubiquitination and proteasomal degradation. Although EDEM1 was proposed to be a lectin–like protein and to react with Man8GlcNAc2 oligosaccharides of glycoproteins, still very little is known about the turnover and degradation mechanism of EDEM1 and how this relates to the fate of its substrates. Previously, we reported that EDEM1 exits the ER by a vesicular non-COPII-mediated mechanism and becomes rapidly degraded by basal autophagy. Here, we provide detailed insight into the mechanism by which EDEM1 becomes degraded. After its dislocation to the cytosol, EDEM1 is apparently making complexes with the selective autophagy receptors p62, NBR1 and Alfy. We observed co-distribution of EDEM1 and selective autophagy receptors by double and triple confocal laser scanning immunofluorescence. By quantifying the relationship of EDEM1 and the selective autophagy receptors as visualized by confocal laser scanning immunofluorescence and double immunogold electron microscopy, dramatical changes were observed in HepG2 cells. Following inhibition of autophagy by wortmannin, the number and size of cytoplasmic clusters composed of EDEM1 and the selective autophagy cargo receptors dramatically increased and this aggregate formation was independent of the activity of HDAC6. We observed that deglycosylation of EDEM1 occurred by the action of the cytosolic peptide N-glycanase since treatment with inhibitors resulted in a strong increase in the amount of glycosylated EDEM1. Inhibition of cytosolic peptide N-glycanase also inhibited wortmannin-induced aggregation of EDEM1 and its complex formation with p62. This indicates that deglycosylation of EDEM1 is a prerequisite for subsequent ubiquitination and interaction with selective autophagy receptors. This demonstrates that the ERAD component EDEM1 itself undergoes ERAD involving selective autophagy

Protein O-GlcNAcylation regulates developmental growth of Drosophilathrough the insulin signaling pathway

Sujin Park,Si-Hyoung Park,Ju Yuel Baek,Kwan Soo Kim,Jürgen Roth,Jin Won Cho,Kwang-Min Choe 한국당과학회 2010 한국당과학회 학술대회 Vol.2010 No.1

Modification of nuclear and cytosolic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is ubiquitous in cells. The in vivo function of the protein O-GlcNAcylation, however, is not well understood. Here, we genetically manipulated the cellular O-GlcNAcylation level in Drosophila and found that it promotes developmental growth by enhancing insulin signaling. This alteration of growth is due to cell growth and apoptosis and not to cell proliferation, and is mediated, at least in part, through O-GlcNAcylation of Akt. These results indicate that O-GlcNAcylation is one of the crucial mechanisms involved in control of insulin signaling in normal Drosophila development.

Protein O-GlcNAcylation regulates developmental growth of Drosophila through the insulin-IGF signaling pathway

Sujin Park,Yeon Jung Kim,Si-Hyoung Park,JuYuel Baek,KwanSoo Kim,Jürgen Roth,Jin Won Cho,Kwang-Min Choe 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

Modification of nuclear and cytosolic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is ubiquitous in cells. The in vivo function of the protein O-GlcNAcylation, however, is not well understood. Here, we genetically manipulated the cellular O-GlcNAcylation level in Drosophila and found that it promotes developmental growth by enhancing insulin signaling. This alteration of growth is due to cell growth and apoptosis and not to cell proliferation, and is mediated, at least in part, through O-GlcNAcylation of Akt. These results indicate that O-GlcNAcylation is one of the crucial mechanisms involved in control of insulin signaling in normal Drosophila development.

Selective Autophagy Receptors Interact with EDEM1 During itsDegradation

Insook Jang,Sujin Park,Bruno Guhl,Jin Won Cho,Jürgen Roth 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

EDEM1 is an endoplasmic reticulum-associated protein degradation (ERAD) component. ERAD is a cellular pathway that targets misfolded and misassembled glycoproteins for degradation. EDEM1 is involved in the recognition of misfolded luminal glycoproteins and in routing them for dislocation to the cytosol, followed by their degradation. EDEM1 interacts with substrate glycoproteins after their exit from the calnexin/calreticulin cycle and after processing by ER-mannosidase I. Although EDEM1 was proposed to be lectin–like and to react with Man8GlcNAc2 oligosaccharides, itsmechanism of action and its fate are still largely unknown. In a previous report, we found that EDEM1 becomes rapidly degraded and that this occurs by basal autophagy. Here, we show that EDEM1 forms complexes with the selective autophagy receptor p62, NBR1 and Alfy. This was demonstrated in HepG2 cells by double immunogold electron microscopy. Furthermore, we show the interaction between p62 and EDEM1 by immunoprecipitation- Western blot experiments. By serial section analysis, the origin of the phagophore for selective autophagy of EDEM1 could be identified as modified parts of rough ER cisternae. Hence, we provide new insight into the details of EDEM1 degradation process.

Glucose deprivation increases O-GlcNAc protein modification in cancer cells through glycogen breakdown

Jeong Gu Kang,Sang Yoon Park,Suena Ji,Insook Jang,Sujin Park,Hyun Sil Kim,Sung-Min Kim,Jong In Yook,Yong-Il Park,Jürgen Roth,Jin Won Cho 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

In general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases when cellular glucose concentrations fall below normal levels. However, recent reports demonstrated that O-GlcNAcylation was increased by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was not changed, it contained less O-GlcNAc and the activity was increased. Also, the activity of O-GlcNAcase was reduced. The studied glycogen containing cells, and we show that its degradation by glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and it may be useful for cancer cells to survive.

O-GlcNAc Biology

Jeong Gu Kang,Sang Yoon Park,Suena Ji,In Sook Jang,Su Jin Park,Hyeon Gyu Seo,Hanbyeol Kim,Eun Ah Kim,Ho Jung Seo,Yang Shin Lee,Jürgen Roth,Jin Won Cho 한국당과학회 2010 한국당과학회 학술대회 Vol.2010 No.1

The O-GlcNAc modification is a quite different fro m conventional glycosylation in two aspects. First, it occurs in cytoplasm and nucleus and does not in endoplasmic reticulum or Golgi apparatus. Second, this is a single sugar modification and is not a long chain oligosaccharide modification. O-GlcNAc is covalently modified on hydroxyl group of serine and threonine and usually this modification affects or competes with phosphorylation. Thus this modification might modulate many cellular events due to inhibiting or sometimes accelerating phosphorylation. O-GlcNAc transferase and O-GlcNAcase are two important enzymes for modifying proteins with O-GlcNAc. More than 800 proteins have been identified as O-GlcNAcylated proteins. Today I am going to summarize results obtained last 10 years and discuss about future aspects in O-GlcNAc biology.

Snail1 is Stabilized by O-GlcNAc Modification in Hyperglycemic Condition and Causes Epithelial-Mesenchymal Transition

Sang Yoon Park,Hyun Sil Kim,Nam Hee Kim,Suena Ji,So Young Cha,Jeong Gu Kang,Ichiro Ota,Keiji Shimada,Noboru Konishi,Hyung Wook Nam,Won Ho Yang,Jürgen Roth,Jong In Yook,Jin Won Cho 한국당과학회 2010 한국당과학회 학술대회 Vol.2010 No.1

Protein O-phosphorylation can occur reciprocal with O-GlcNAc modification and represents a regulatory principle for proteins. O-phosphorylation of serine by GSK-3β on Snail1, a transcriptional repressor of E-cadherin and a key regulator of the epithelial-mesenchymal transition (EMT) program, results in its proteasomal degradation. We show that Snail1 carries O-GlcNAc at serine112 that stabilizes it by suppressing O-phosphorylation-mediated degradation. Stabilization by O-GlcNAc of Snail1 results in attenuation of E-cadherin mRNA expression. Enhanced O-GlcNAc modification occurred under hyperglycemic conditions and initiated EMT by transcriptional suppression of E-cadherin through Snail1. Thus, a molecular link exists between cellular glucose metabolism and the control of EMT by dynamic reciprocal O-phosphorylation and O-GlcNAc modification of Snail1.