Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

Reithinger, Johannes H.,Kim, Ji Eun Hani,Kim, Hyun American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.25

<P>Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N<SUB>in</SUB>-C<SUB>out</SUB> (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.</P>

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.

A Misfolded Thyroglobulin is Retained by Molecular Chaperones in the Endoplasmic Reticulum

Kwon, O-Yu,Seong, Yeon-Mun,Shong, Min-Bo Korean Society of Life Science 1999 Journal of Life Science Vol.9 No.1

Deficient thyroglobulin is one of the important causes of congenital hypothyroid goiter with a prevalence of -1/40,000 humans. We now demonstrate that in cog/cog mice showing hypothyroidism, four endoplasmic reticulum-molecular chaperones stably bind to thyroglobulin, providing insight into physiologic regulation of endoplasmic reticulum storage diseases.

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

Roth, Jurgen,Zuber, Christian,Park, Su-Jin,Jang, In-Sook,Lee, Yang-Sin,Kysela, Katarina Gaplovska,Le Fourn, Valerie,Santimaria, Roger,Guhl, Bruno,Cho, Jin-Won Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.30 No.6

Quality control of protein folding represents a fundamental 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.

The enfichment of endoplasmic reticulum-mitochondria contact contrubuts to inflammasome-associated severe neutrophilic asthma

( So Ri Kim ),( Hae Jin Park ),( Yong Chul Lee ),( Kyeong Hwa Park ),( Yeong Hun Choe ),( Seung Yong Park ),( Jae Seok Jeong ) 대한결핵 및 호흡기학회 2018 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.126 No.-

Each asthma phenotype may have distinct observable molecular, cellular, morphological, functional, and clinical features, all of which can be possibly integrated into specific biological mechanisms, called as endotypes. Recently, one of the new molecular phenotypes of severe asthma has been reported as inflammasome-associated severe asthma. In this study, we investigated which mechanisms contribute to NLRP3 inflammasome activation in severe neutrophilic asthma focusing on the mechanical and functional link between the endoplasmic reticulum (ER) and mitochondria. The mice sensitized with OVA and LPS and then challenged with OVA (OVA_LPS-OVA mice) mice showed the typical features of neutrophilic asthma. Interestingly, confocal analysis and electron-microscopic findings revealed that in lung cells from OVALPS-OVA mice, the ER and mitochondria get closed each other even seemed to be united one compared to the finding of cells from control mice. An ER stress inhibitor, mitochondrial ROS inhibitor, or MCC950 significantly reduced the increases in inflammatory cytokines, mitochondrial ROS generation, NLRP3 inflammasome activation, airway inflammation, and bronchial hyperresponsiveness. Interestingly, the treatment restored the physical changes and distances of ER and mitochondria near normally. These findings indicate that the development of ER-mitochondria complex in airway inflammatory cells may be implicated in the pathogenesis of neutrophilic bronchial asthma through NLRP3 inflammasome activation, providing the novel therapeutic target for bronchial asthm.

Exposure to aflatoxin B1 attenuates cell viability and induces endoplasmic reticulum-mediated cell death in a bovine mammary epithelial cell line (MAC-T)

Park, Wonhyoung,Park, Min Young,Song, Gwonhwa,Lim, Whasun Elsevier 2019 Toxicology in vitro Vol.61 No.-

<P><B>Abstract</B></P> <P>Mammary gland epithelial cells play a key role in milk production in dairy cattle, which are prone to feed contamination from microorganisms, especially pathogenic fungi and their mycotoxin products. When mycotoxins enter the body, they cause a reduction in feed intake and milk production, leading to the retardation of growth and productivity in dairy cattle. Among them, aflatoxin B1 is a ubiquitous food contaminant frequently found in stored crops, which are used as animal feed, and causes many adverse health effects in domestic animals. Unfortunately, there is a lack of knowledge on the mechanisms underlying the detrimental effects of aflatoxin B1. Therefore, in this study, we found that aflatoxin B1 reduced cellular proliferation, induced apoptosis by DNA fragmentation, and disrupted intracellular homeostasis, including mitochondrial membrane potential and calcium concentration, in an immortalized bovine mammary epithelial cell line (MAC-T). Additionally, we identified aflatoxin B1-mediated cell signaling pathways associated with cell survival and the endoplasmic reticulum stress response. These results revealed that exposure to aflatoxin B1 attenuates cell viability and induces endoplasmic reticulum-mediated cell death in MAC-T cells.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Mechanisms underlying the detrimental effects of aflatoxin B1 were studied. </LI> <LI> Aflatoxin B1 reduced cellular proliferation and induced apoptosis. </LI> <LI> It disrupted intracellular homeostasis. </LI> <LI> Aflatoxin B1 regulated cell survival related signaling pathways. </LI> <LI> It also regulated ER stress response related signaling pathways. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>Schematic illustration of the cell signaling pathways induced by aflatoxin B1 treatment in bovine mammary epithelial cells. Aflatoxin B1 exposure increases ER stress by activation of GRP78, ATF6α, IRE1α, PERK, and eIF2α proteins. Aflatoxin B1 also suppresses cell viability through inhibition of the PI3K and ERK1/2 cell signaling pathways, whereas it activates the mitochondrial-mediated apoptotic pathway. Taken together, exposure to aflatoxin B1 induces cell death in bovine mammary gland cells.</P> <P>[DISPLAY OMISSION]</P>

Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation

Lee, Jea Hwang,Park, Ki Jun,Jang, Jun Ki,Jeon, Yeong Ha,Ko, Kwan Young,Kwon, Joon Hyun,Lee, Seung-Rock,Kim, Ick Young American Society for Biochemistry and Molecular Bi 2015 The Journal of biological chemistry Vol.290 No.50

<P>Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP). However, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Therefore, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.</P>

Endoplasmic reticulum‐directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

Kallehauge, Thomas Beuchert,Kol, Stefan,Rørdam Andersen, Mikael,Kroun Damgaard, Christian,Lee, Gyun Min,Faustrup Kildegaard, Helene WILEY‐VCH Verlag 2016 Biotechnology Journal Vol.11 No.10

<P><B>Abstract</B></P><P>When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide‐dependent manner, to acquire specific post‐translational modifications and to facilitate secretion into the culture medium. One key premise for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER‐directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER‐directed mRNA, with no cytoplasmic translation of ER‐directed recombinant proteins observed. These observations indicate that the recombinant mRNA, encoding ER‐directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also recombinant protein trafficking between the ER and cytosol in CHO cells.</P>

Ufd1 phosphorylation at serine 229 negatively regulates endoplasmic reticulum-associated degradation by inhibiting the interaction of Ufd1 with VCP

Nguyen, Quynh-Anh Thi,Choi, Juyong,Yang, Jin Kuk,Lee, Sang Yoon Portland Press Ltd. 2019 Biochemical journal Vol.476 No.18

<P>Misfolded proteins in the endoplasmic reticulum (ER) are removed through multistep processes termed ER-associated degradation (ERAD). Valosin-containing protein (VCP) plays a crucial role in ERAD as the interaction of ubiquitin fusion degradation protein 1 (Ufd1) with VCP via its SHP box motif (<SUP>228</SUP>F-S-G-S-G-N-R-L<SUP>235</SUP>) is required for ERAD. However, the mechanisms by which the VCP–Ufd1 interaction is regulated are not well understood. Here, we found that the serine 229 residue located in the Ufd1 SHP box is phosphorylated <I>in vitro</I> and <I>in vivo</I> by cyclic adenosine monophosphate-dependent protein kinase A (PKA), with this process being enhanced by either forskolin (an adenylyl cyclase activator) or calyculin A (a protein phosphatase inhibitor). Moreover, a phosphomimetic mutant (S229D) of Ufd1 as well as treatment by forskolin, calyculin A, or activated PKA strongly reduced Ufd1 binding affinity for VCP. Consistent with this, the Ufd1 S229D mutant significantly inhibited ERAD leading to the accumulation of ERAD substrates such as a tyrosinase mutant (C89R) and 3-hydroxy-3-methylglutaryl coenzyme A reductase. However, a non-phosphorylatable Ufd1 mutant (S229A) retained VCP-binding ability and was less effective in blocking ERAD. Collectively, our results support that Ufd1 S229 phosphorylation status mediated by PKA serves as a key regulatory point for the VCP–Ufd1 interaction and functional ERAD.</P>

Differential Role for BiP3 in Rice Immune Receptor-Mediated Resistance

Kim, Chi-Yeol,Han, Muho,Park, Chang-Jin,Jeon, Jong-Seong The Korean Society for Applied Biological Chemistr 2014 Applied Biological Chemistry (Appl Biol Chem) Vol.57 No.4

Endoplasmic reticulum-bound chaperone luminal-binding protein 3 (BiP3) has been found to regulate the immunity mediated by the membrane-bound extracellular immune receptors Xa3/Xa26 and Xa21, that encode non-arginine-aspartate (non-RD) kinases, against the bacterial pathogen Xanthomonas oryzae pathovar oryzae (Xoo). In contrast, BiP3 appeared not to regulate the immunity mediated by the intracellular immune receptor Pi5, which encodes a nucleotide-binding domain and leucine-rich repeat (NB-LRR) protein, against the fungal pathogen Magnaporthe oryzae. To further examine this differential role for BiP3 in rice immunity, we generated transgenic rice plants overexpressing BiP3 in the background of the NB-LRR intracellular immune receptor Xa1 that confers resistance to Xoo. Our molecular genetic and phenotype analyses revealed that BiP3 overexpression does not affect Xa1-mediated rice resistance to Xoo. Our current results thus provide evidence that BiP3 regulates membrane-bound non-RD kinase-mediated, but not the intracellular NB-LRR-mediated, rice immune responses and that its function does not depend on the type of pathogen.