Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b′). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb′, we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb′, the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.

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Differential activity of rice protein disulfide isomerase family members for disulfide bond formation and reduction

FEBS Open Bio ◽

10.1016/j.fob.2014.07.007 ◽

2014 ◽

Vol 4 (1) ◽

pp. 730-734 ◽

Cited By ~ 9

Author(s):

Yayoi Onda ◽

Yohei Kobori

Keyword(s):

Disulfide Bond ◽

Protein Disulfide Isomerase ◽

Bond Formation ◽

Family Members ◽

Disulfide Bond Formation ◽

Disulfide Isomerase ◽

Rice Protein ◽

Protein Disulfide ◽

Protein Disulfide Isomerase Family

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PDI Family Members as Guides for Client Folding and Assembly

International Journal of Molecular Sciences ◽

10.3390/ijms21249351 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9351

Author(s):

Shingo Kanemura ◽

Motonori Matsusaki ◽

Kenji Inaba ◽

Masaki Okumura

Keyword(s):

Endoplasmic Reticulum ◽

Cell Viability ◽

Molecular Chaperones ◽

Protein Disulfide Isomerase ◽

Family Members ◽

Secretory Proteins ◽

Efficient Production ◽

Protein Homeostasis ◽

Disulfide Isomerase ◽

Protein Disulfide

Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.

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A di-arginine motif contributes to the ER localization of the type I transmembrane ER oxidoreductase TMX4

Biochemical Journal ◽

10.1042/bj20091064 ◽

2009 ◽

Vol 425 (1) ◽

pp. 195-208 ◽

Cited By ~ 24

Author(s):

Doris Roth ◽

Emily Lynes ◽

Jan Riemer ◽

Henning G. Hansen ◽

Nils Althaus ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Disulfide Isomerase ◽

Bond Formation ◽

Living Cells ◽

Type I ◽

Disulfide Bond Formation ◽

Reporter Protein ◽

Disulfide Isomerase ◽

Protein Disulfide ◽

Protein Disulfide Isomerase Family

The thiol-disulfide oxidoreductases of the PDI (protein disulfide isomerase) family assist in disulfide-bond formation in the ER (endoplasmic reticulum). In the present study, we have shown that the previously uncharacterized PDI family member TMX4 (thioredoxin-like transmembrane 4) is an N-glycosylated type I membrane protein that localizes to the ER. We also demonstrate that TMX4 contains a single ER-luminal thioredoxin-like domain, which, in contrast with similar domains in other PDIs, is mainly oxidized in living cells. The TMX4 transcript displays a wide tissue distribution, and is strongly expressed in melanoma cells. Unlike many type I membrane proteins, TMX4 lacks a typical C-terminal di-lysine retrieval signal. Instead, the cytoplasmic tail has a conserved di-arginine motif of the RXR type. We show that mutation of the RQR sequence in TMX4 to KQK interferes with ER localization of the protein. Moreover, whereas the cytoplasmic region of TMX4 confers ER localization to a reporter protein, the KQK mutant of the same protein redistributes to the cell surface. Overall, features not commonly found in other PDIs characterize TMX4 and suggest unique functional properties of the protein.

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Functional Relationship between Protein Disulfide Isomerase Family Members during the Oxidative Folding of Human Secretory Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e10-04-0356 ◽

2010 ◽

Vol 21 (18) ◽

pp. 3093-3105 ◽

Cited By ~ 62

Author(s):

Lori A. Rutkevich ◽

Myrna F. Cohen-Doyle ◽

Ulf Brockmeier ◽

David B. Williams

Keyword(s):

Protein Disulfide Isomerase ◽

High Efficiency ◽

Family Members ◽

Secretory Proteins ◽

Human Hepatoma Cells ◽

Oxidative Folding ◽

Disulfide Isomerase ◽

The Impact ◽

Protein Disulfide ◽

Protein Disulfide Isomerase Family

To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding.

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