Assisted Protein Folding

Ruddon, Raymond W.; Bedows, Elliott

doi:10.1074/jbc.272.6.3125

Cited by 140 publications

(89 citation statements)

References 58 publications

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“…These observations have raised questions about the validity of earlier conclusion from the unfolding and refolding experiments of ribonuclease. Formation of misfolded and non-native species during the refolding process of various proteins has been reported in the literature [5,6]. The conformation of the refolded protein depends not only on the refolding conditions, but also on the overall size and domain structure of the protein.…”

Section: Introductionmentioning

confidence: 99%

Unfolding Studies of Escherichia coli Maltodextrin Glucosidase Monitored by Fluorescence Spectroscopy

et al. 2008

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Equilibrium unfolding of a 69-kDa monomeric Escherichia coli maltodextrin glucosidase (MalZ) was studied using intrinsic and extrinsic fluorescence spectroscopy. The unfolding transition of MalZ followed a three-state process, involving the formation of a stable intermediate state having more exposed hydrophobic surface. It was found that the protein structure can be easily perturbed by low concentration of guanidium hydrochloride (GdnHCl) and, at a GdnHCl concentration of 2 M, MalZ was denatured completely. The active site of the protein also has been proved to be sensitive to a low concentration of GdnHCl since MalZ deactivated at 0.5 M GdnHCl completely. The surface hydrophobicity and ANS-binding site of the protein have been determined to be 150.7 and 0.24, respectively. Perhaps the formation of the stable unfolding intermediate, having higher surface hydrophobicity, may be one of the reasons for aggregation of MalZ and its recognition by chaperonin GroEL during the assisted folding pathway.

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Section: Introductionmentioning

confidence: 99%

Unfolding Studies of Escherichia coli Maltodextrin Glucosidase Monitored by Fluorescence Spectroscopy

et al. 2008

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“…Secretory proteins are completely transferred into the ER lumen, whereas membrane proteins integrate into the lipid bilayer by lateral exit of hydrophobic sequences from the translocon (High, 1995). During translocation, ␣-helical packing (Lemmon et al, 1997), interaction with molecular chaperones and cotranslational modifications (Ruddon and Bedows, 1997) and, in the case of oligomeric proteins, assembly with partner subunits (Geering, 1997) favor the correct folding into a tertiary protein structure. For many proteins, it is well documented that this maturation process in the ER is necessary for intracellular trafficking and function.…”

Section: Introductionmentioning

confidence: 99%

Endoplasmic Reticulum Quality Control of Oligomeric Membrane Proteins: Topogenic Determinants Involved in the Degradation of the Unassembled Na,K-ATPase α Subunit and in Its Stabilization by β Subunit Assembly

Béguin

Hasler

Staub

et al. 2000

MBoC

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The molecular nature of determinants that mediate degradation of unassembled, polytopic subunits of oligomeric membrane proteins and their stabilization after partner subunit assembly is largely unknown. Expressing truncated Na,K-ATPase ␣ subunits alone or together with ␤ subunits, we find that in unassembled ␣ subunits neither the four N-terminal transmembrane segments acting as efficient alternating signal anchor-stop transfer sequences nor the large, central cytoplasmic loop exposes any degradation signal, whereas poor membrane insertion efficiency of C-terminal membrane domains M5, M7, and M9 coincides with the transient exposure of degradation signals to the cytoplasmic side. ␤ assembly with an ␣ domain comprising at least D902 up to Y910 in the extracytoplasmic M7/M8 loop is necessary to stabilize Na,K-ATPase ␣ subunits by favoring M7/M8 membrane pair formation and by protecting a degradation signal recognized from the endoplasmic reticulum (ER) lumenal side. Thus our results suggest that ER degradation of Na,K-ATPase ␣ subunits is 1) mainly mediated by folding defects caused by inefficient membrane insertion of certain membrane domains, 2) a multistep process, which involves proteolytic and/or chaperone components acting from the ER lumenal side in addition to cytosolic, proteasome-related factors, and 3) prevented by partner subunit assembly because of direct protection and retrieval of degradation signals from the cytoplasm to the ER lumenal side. These results likely represent a paradigm for the ER quality control of unassembled, polytopic subunits of oligomeric membrane proteins.

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“…Recent findings have also indicated that oligomers formed in LS174T cells by both MUC2 [16] and MUC5AC [17] are probably dimers. In general, proteins in the ER fold and undergo subunit assembly with the assistance of a number of chaperones such as calreticulin (CRT), calnexin (CLN), the immunoglobulin-binding protein (BiP) and protein disulphide-isomerase (PDI) [18,19]. Chaperones prevent the aggregation of unfolded and misfolded proteins and play a role in quality control of the ER synthetic pathway [20].…”

Section: Introductionmentioning

confidence: 99%

Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells

McCool

Okada

Forstner

et al. 1999

Biochemical Journal

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Molecular chaperones are presumed to associate with large secretory mucin glycoproteins during their synthesis in the endoplasmic reticulum (ER), but have not been identified to date. We decided to look for possible involvement of the chaperones calreticulin (CRT) and calnexin (CLN) during synthesis of two similar gastrointestinal mucins, MUC2 and MUC5AC. Pulse-chase labelling of MUC2 and MUC5AC with [$&S]methionine\cysteine ([$&S]Promix) was performed using LS180 and HT29\A1 colonic carcinoma cell lines and was followed by immunoprecipitation with anti-mucin and antichaperone antibodies. The precipitated labelled mucin precursors were analysed by SDS\PAGE and autoradiography. Using antibodies specific for each mucin, newly synthesized monomeric precursors of both MUC2 and MUC5AC were detected after a 15 min pulse and then disappeared as oligomers were formed during a 2 h chase period. Only homo-oligomers of MUC2 and MUC5AC were present in the cells. Using anti-CRT, the MUC2 monomeric precursor and oligomer were co-precipitated from both cell lines after a 15 min pulse and the oligomer less strongly after a 0.5 h chase, but there was little co-precipitation after a 2 h chase. At this time, MUC2 immunoprecipitated by anti-

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Assisted Protein Folding

Cited by 140 publications

References 58 publications

Unfolding Studies of Escherichia coli Maltodextrin Glucosidase Monitored by Fluorescence Spectroscopy

Unfolding Studies of Escherichia coli Maltodextrin Glucosidase Monitored by Fluorescence Spectroscopy

Endoplasmic Reticulum Quality Control of Oligomeric Membrane Proteins: Topogenic Determinants Involved in the Degradation of the Unassembled Na,K-ATPase α Subunit and in Its Stabilization by β Subunit Assembly

Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells

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