Crystal Structure and Functional Analysis of the Protein Disulfide Isomerase-Related Protein ERp29

Barak, Naomi; Neumann, Piotr; Sevvana, Madhumati; Schutkowski, Mike; Naumann, Kai; Malešević, Miroslav; Reichardt, Heike; Fischer, Gunter; Stubbs, Milton T.; Ferrari, David M.

doi:10.1016/j.jmb.2008.11.052

Cited by 61 publications

(61 citation statements)

References 44 publications

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“…These data provide strong functional evidence that ERp29 is, in fact, a bona fide molecular chaperone or co-chap- erone, extending recent reports by us and others (31,40). The presumed function of ERp29 as a chaperone was supported previously by cellular studies of thyroglobulin, a soluble secretory protein (27,28,30).…”

Section: Discussionsupporting

confidence: 84%

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ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

Suaud

Miller

Alvey

et al. 2011

Journal of Biological Chemistry

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Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ⌬F508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ⌬F508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ⌬F508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (ϳ1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ⌬F508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o؊ WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ⌬F508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ⌬F508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ⌬F508-CFTR trafficking in CF epithelial cells.

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Section: Discussionsupporting

confidence: 84%

“…folding substrates) remain lacking. Notably, ERp29 lacks classical chaperone and oxidoreductase activities (26) but exhibits chaperone-like properties at both the biophysical and cellular levels (27)(28)(29)(30)(31). It has also been inferred that ERp29 prefers hydrophobic substrates such as membrane proteins (29,32).…”

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confidence: 99%

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

Suaud

Miller

Alvey

et al. 2011

Journal of Biological Chemistry

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“…A tagged form of ERp29 was produced by inserting an EGFP tag between the signal sequence and the N terminus of the mature ERp29 peptide (EGFP-ERp29, Supplemental Figure S1), to preserve the Cterminal KEEL ER retention/retrieval sequence (Demmer et al, 1997). EGFP-ERp29 was further mutated by deleting the C-terminal ligand binding domain (Magnuson et al, 2005;Barak et al, 2009;Rainey-Barger et al, 2009) while retaining the N-terminal dimerization domain and KEEL ER retention motif (EGFP-ERp29-N). As shown in Figure 5, cells expressing EGFP-ERp29 continued to form gap junctions containing Cx43.…”

Section: Effect Of Normal and Mutant Erp29 On Cx43mentioning

confidence: 99%

“…The resulting PCR product was cut with HindIII and BamHI and ligated into doubly cut plasmids containing ERp29 signal-EGFP to produce EGFP-ERp29 ( Supplemental Figure S1). A putative dominant-negative EGFP-ERp29 construct (Magnuson et al, 2005;Barak et al, 2009;Rainey-Barger et al, 2009) containing the Nterminal domain of EGFP-ERp29 and the KEEL ER retention sequence (EGFPERp29-N) was produced by amplifying EGFP-ERp29 with 5Ј-GGTACCATGG CTGCCGCTGT GCCCCG-3Ј and 5Ј-TCTAGATTAC AGCTCCTCTT TCAT-ACCTAG GTAGACCCCT T-3Ј as sense and antisense primers. The resulting PCR product was cut with KpnI and XbaI and ligated into doubly cut pcDNA3.1 (Clontech).…”

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confidence: 99%

ERp29 Restricts Connexin43 Oligomerization in the Endoplasmic Reticulum

Das

Smith

Sarma

et al. 2009

MBoC

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Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus. INTRODUCTIONConnexins form gap junction channels that mediate intercellular communication by allowing the direct transfer of ions and small aqueous molecules between neighboring cells or by serving as hemichannels at the plasma membrane (Harris, 2001;Goodenough and Paul, 2003;Saez et al., 2003;Laird, 2006). There are Ͼ20 human connexin genes and one of the most ubiquitously expressed is connexin43 (Cx43). Several diseases have been linked to mutations in different connexin genes, including Cx43 (Kelsell et al., 2001;Laird, 2008). Many of these mutant connexins are not efficiently processed and lack the ability to form functional gap junction channels. Determining how mutant connexins are mistargeted requires a more complete understanding of how normal connexins are processed by the cell.A gap junction hemichannel consists of six connexins, which oligomerize before delivery to the plasma membrane (Martin and Evans, 2004;Segretain and Falk, 2004;Koval, 2006). Unlike most multimeric membrane channels, Cx43 is unusual in that it does not oligomerize in the endoplasmic reticulum (ER) (Musil and Goodenough, 1993), which is more commonly a prerequisite to further transport along the secretory pathway (Ellgaard and Helenius, 2003;Anelli and Sitia, 2008). Instead, Cx43 is transported out of the ER as an apparent monomer that then oligomerizes in the Golgi complex (Musil and Goodenough, 1993;Koval et al., 1997). Although other connexins, such as Cx46 (Koval et al., 1997), also oligomerize in the Golgi apparatus, this is not a universal pathway for connexin oligomerization because other gap junction proteins, such as Cx32, preferentially oligomerize in the ER (Martin and Evans, 2004;Koval, 2006). Understanding the molecular basis for this difference, as well as the ability of monomeric Cx43 to be transported from the ER to the Golgi apparatus has proven difficult, because little is known about chaperones that regulate connexin folding. In particular, it seemed likely that one or more chaperones would be requir...

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“…ERp29 consists of a b domain and a D domain (C-terminal half). 20 The b domain of ERp29 was sufficient for peptide binding. As a natural substrate, thyroglobulin, which is precursor of T 4 and T 3 , was found, and ERp29 was essential for Total cellular proteins from pcDNA-transfected GH3 cells (mock) or GH3 cells overexpressing PDI, ERp57, ERp72, and ERp29 were isolated and immunoblotting was performed using antibodies against human PDI, ERp57, ERp72, and ERp29, which can recognize rat homologues.…”

Section: ■ Discussionmentioning

confidence: 99%

Endoplasmic Reticulum Protein (ERp) 29 Binds As Strongly As Protein Disulfide Isomerase (PDI) to Bisphenol A

Miyake

Hashimoto

Sasaki

et al. 2014

Chem. Res. Toxicol.

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Bisphenol A (BPA), which is used in polycarbonate and epoxy resins, affects the development or function of the central nervous system. Previously, we isolated a BPA-binding protein from rat brain, identified it as protein disulfide isomerase (PDI), and found that BPA binds to the b' domain of PDI and inhibits its activity. There are 20 kinds of PDI family proteins in mammalian endoplasmic reticulum. The member proteins each have a different length and domain arrangement. Here we investigated the binding of BPA and T3 to ERp29, ERp57, and ERp72, which each have the b or b' domain. BPA/T3 binding of ERp57 and that of ERp72 were lower than that of PDI, and BPA did not inhibit the oxidase or reductase activity of these proteins. On the other hand, BPA and T3 bound to ERp29 as strongly as to PDI. The CD spectrum of PDI was changed in the presence of BPA in a dose-dependent manner, while that of ERp29 was not, suggesting that BPA did not affect the conformation of ERp29. We found that PDI suppresses GH expression in rat GH3 cells stimulated by thyroid hormone (T3) overexpression of PDI and that ERp57 reduced the GH level, but overexpression of ERp29 did not change GH expression. These results suggested that affinity to T3 does not involve the reduction of the T3 response. In this study, ERp29 was first identified as a BPA-binding protein but is not involved in the T3 response of GH3 cells.

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Crystal Structure and Functional Analysis of the Protein Disulfide Isomerase-Related Protein ERp29

Cited by 61 publications

References 44 publications

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

ERp29 Restricts Connexin43 Oligomerization in the Endoplasmic Reticulum

Endoplasmic Reticulum Protein (ERp) 29 Binds As Strongly As Protein Disulfide Isomerase (PDI) to Bisphenol A

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