ERp18 is an endoplasmic reticulum (ER)-resident thioredoxin (Trx) family protein, similar to cytosolic Trx1. The Trx-like domain occupies a major portion of the whole ERp18 structure, which is postulated to be an ER paralog of cytosolic Trx1. Here, we elucidate that zinc ion (Zn) binds ERp18 through its catalytic motif, triggering oligomerization of ERp18 from a monomer to a trimer.
View Article and Find Full Text PDFJ-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.
View Article and Find Full Text PDFThe endoplasmic reticulum (ER) maintains an oxidative redox environment that is advantageous for the oxidative folding of nascent polypeptides entering the ER. Reductive reactions within the ER are also crucial for maintaining ER homeostasis. However, the mechanism by which electrons are supplied for the reductase activity within the ER remains unknown.
View Article and Find Full Text PDFInositol 1,4,5-trisphosphate receptors (IP3Rs) are one of the two types of tetrameric ion channels that release calcium ion (Ca) from the endoplasmic reticulum (ER) into the cytosol. Ca released via IP3Rs is a fundamental second messenger for numerous cell functions. Disturbances in the intracellular redox environment resulting from various diseases and aging interfere with proper calcium signaling, however, the details are unclear.
View Article and Find Full Text PDFChinese hamster ovary (CHO) cells are widely used for manufacturing antibody drugs. We attempted to clone a novel high-expression promoter for producing monoclonal antibodies (mAbs) based on transcriptome analysis to enhance the transcriptional abundance of mAb genes. The efficacy of conventional promoters such as CMV and hEF1α decrease in the latter phase of fed-batch cell culture.
View Article and Find Full Text PDFThe endoplasmic reticulum (ER) is the organelle responsible for the folding of secretory/membrane proteins and acts as a dynamic calcium ion (Ca) store involved in various cellular signalling pathways. Previously, we reported that the ER-resident disulfide reductase ERdj5 is involved in the ER-associated degradation (ERAD) of misfolded proteins in the ER and the activation of SERCA2b, a Ca pump on the ER membrane. These results highlighted the importance of the regulation of redox activity in both Ca and protein homeostasis in the ER.
View Article and Find Full Text PDFSarco/endoplasmic reticulum (ER) Ca-ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca uptake from the cytosol to the ER. SERCA2b includes a characteristic 11 transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca-adenylyl methylenediphosphonate (AMPPCP) state.
View Article and Find Full Text PDFHsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones.
View Article and Find Full Text PDFCold Spring Harb Perspect Biol
May 2019
The endoplasmic reticulum (ER) is a dynamic organelle responsible for many cellular functions in eukaryotic cells. Proper redox conditions in the ER are necessary for the functions of many luminal pathways and the maintenance of homeostasis. The redox environment in the ER is oxidative compared with that of the cytosol, and a network of oxidoreductases centering on the protein disulfide isomerase (PDI)-Ero1α hub complex is constructed for efficient electron transfer.
View Article and Find Full Text PDFDespite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys and Lys of human PDI . Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner.
View Article and Find Full Text PDFERdj5, composed of an N-terminal J domain followed by six thioredoxin-like domains, is the largest protein disulfide isomerase family member and functions as an ER-localized disulfide reductase that enhances ER-associated degradation (ERAD). Our previous studies indicated that ERdj5 comprises two regions, the N- and C-terminal clusters, separated by a linker loop and with distinct functional roles in ERAD. We here present a new crystal structure of ERdj5 with a largely different cluster arrangement relative to that in the original crystal structure.
View Article and Find Full Text PDFThiol-based redox control is among the most important mechanisms for maintaining cellular redox homeostasis, with essential participation of cysteine thiols of oxidoreductases. To explore cellular redox regulatory networks, direct interactions among active cysteine thiols of oxidoreductases and their targets must be clarified. We applied a recently described thiol-ene crosslinking-based strategy, named divinyl sulfone (DVSF) method, enabling identification of new potential redox relay partners of the cytosolic oxidoreductases thioredoxin (TXN) and thioredoxin domain containing 17 (TXNDC17).
View Article and Find Full Text PDFCalcium ion (Ca) is an important second messenger that regulates numerous cellular functions. Intracellular Ca concentration ([Ca]i) is strictly controlled by Ca channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca from the cytosol into the ER in an ATPase activity-dependent manner.
View Article and Find Full Text PDFBackground: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity.
View Article and Find Full Text PDFChronic liver injury, often caused by alcoholism and viral hepatitis, causes liver fibrosis via the induction of collagen production. In liver fibrosis, hepatic stellate cells (HSCs) are activated and transform into myofibroblasts, which actively produce and secrete collagen into the extracellular matrix. Hsp47 (heat shock protein 47) is a collagen-specific molecular chaperone that is essential for the maturation and secretion of collagen.
View Article and Find Full Text PDFDuring endoplasmic reticulum (ER)-associated degradation (ERAD), terminally misfolded proteins are retrotranslocated from the ER to the cytosol and degraded by the ubiquitin-proteasome system. Misfolded glycoproteins are recognized by calnexin and transferred to EDEM1, followed by the ER disulfide reductase ERdj5 and the BiP complex. The mechanisms involved in ERAD of nonglycoproteins, however, are poorly understood.
View Article and Find Full Text PDFER-associated degradation (ERAD) is an ER quality-control process that eliminates terminally misfolded proteins. ERdj5 was recently discovered to be a key ER-resident PDI family member protein that accelerates ERAD by reducing incorrect disulfide bonds in misfolded glycoproteins recognized by EDEM1. We here solved the crystal structure of full-length ERdj5, thereby revealing that ERdj5 contains the N-terminal J domain and six tandem thioredoxin domains that can be divided into the N- and C-terminal clusters.
View Article and Find Full Text PDFMethods Enzymol
April 2011
The endoplasmic reticulum (ER) is an organelle where secretory or membrane proteins are correctly folded with the aid of various molecular chaperones and oxidoreductases. Only correctly folded and assembled proteins are enabled to reach their final destinations, which are called as ER quality control (ERQC) mechanisms. ER-associated degradation (ERAD) is one of the ERQC mechanisms for maintaining the ER homeostasis and facilitates the elimination of misfolded or malfolded proteins accumulated in the ER.
View Article and Find Full Text PDFThe folding of secretory and membrane proteins takes place in the endoplasmic reticulum (ER). The quality of the proteins folded in the ER is carefully monitored by an ER quality control mechanism that allows only correctly folded proteins to be transported to their final destination, and misfolded or unassembled proteins to be retained in the ER and subsequently degraded in a process termed 'ER-associated degradation' (ERAD). The ERAD pathway is conserved from yeast to mammals, and plays an essential role in the maintenance of ER homeostasis, as well as in the prevention of various diseases that arise from the accumulation of aberrant proteins in the ER.
View Article and Find Full Text PDFMembrane and secretory proteins cotranslationally enter and are folded in the endoplasmic reticulum (ER). Misfolded or unassembled proteins are discarded by a process known as ER-associated degradation (ERAD), which involves their retrotranslocation into the cytosol. ERAD substrates frequently contain disulfide bonds that must be cleaved before their retrotranslocation.
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