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Identification and mechanism of wheat protein disulfide isomerase-promoted gluten network formation.

ABSTRACT: Formation of the gluten network depends on glutenin crosslinking via disulfide bonds, and wheat protein disulfide isomerase (wPDI) plays an important role in this process. Here, we identify a substrate gluten protein of wPDI and the mechanism underlying wPDI-promoted glutenin crosslinking. Farinographic, rheologic, and alveographic analysis unambiguously proves that wPDI improves gluten network formation, which is directly observed by 3D reconstruction of the gluten network. Protein analysis and LC-MS/MS reveal that glutenin subunit 1Dx5 is primarily recruited by wPDI to participate in gluten network formation, and its cysteine-containing N-terminal domain (1Dx5-NTD), which harbors three cysteine residues for crosslinking, is purified. 1Dx5-NTD interacts with wPDI in both redox states, possibly folded by reduced wPDI and then catalyzed by oxidized wPDI, as further evidenced by wPDI-promoted self-crosslinking. Consistent with macroscopic observations, our results suggest that wPDI folds 1Dx5-NTD into β-strand structure that favors disulfide bond formation.

SUBMITTER: Gao J

PROVIDER: S-EPMC11376372 | biostudies-literature | 2024 Sep

REPOSITORIES: biostudies-literature

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Identification and mechanism of wheat protein disulfide isomerase-promoted gluten network formation.

Gao Jihui J Ma Jiayin J Yu Peixuan P Yang Dong D

PNAS nexus 20240821 9

Formation of the gluten network depends on glutenin crosslinking via disulfide bonds, and wheat protein disulfide isomerase (wPDI) plays an important role in this process. Here, we identify a substrate gluten protein of wPDI and the mechanism underlying wPDI-promoted glutenin crosslinking. Farinographic, rheologic, and alveographic analysis unambiguously proves that wPDI improves gluten network formation, which is directly observed by 3D reconstruction of the gluten network. Protein analysis and ...[more]

PMID: 39238603

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Project description:BackgroundThe major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds. In eukaryotes, the formation of most intramolecular disulfide bonds in the ER is thought to be catalyzed by protein disulfide isomerase (PDI) family proteins. The cDNAs that encode eight groups of bread wheat (Triticum aestivum L.) PDI family proteins have been cloned, and their expression levels in developing wheat grains have been determined. The purpose of the present study was to characterize the enzymatic properties of the wheat PDI family proteins and clarify their expression patterns in wheat caryopses.ResultsPDI family cDNAs, which are categorized into group I (TaPDIL1Aα, TaPDIL1Aβ, TaPDIL1Aγ, TaPDIL1Aδ, and TaPDIL1B), group II (TaPDIL2), group III (TaPDIL3A), group IV (TaPDIL4D), and group V (TaPDIL5A), were cloned. The expression levels of recombinant TaPDIL1Aα, TaPDIL1B, TaPDIL2, TaPDIL3A, TaPDIL4D, and TaPDIL5A in Escherichia coli were established from the cloned cDNAs. All recombinant proteins were expressed in soluble forms and purified. Aside from TaPDIL3A, the recombinant proteins exhibited oxidative refolding activity on reduced and denatured ribonuclease A. Five groups of PDI family proteins were distributed throughout wheat caryopses, and expression levels of these proteins were higher during grain filling than in the late stage of maturing. Localization of these proteins in the ER was confirmed by fluorescent immunostaining of the immature caryopses. In mature grains, the five groups of PDI family proteins remained in the aleurone cells and the protein matrix of the starchy endosperm.ConclusionsHigh expression of PDI family proteins during grain filling in the starchy endosperm suggest that these proteins play an important role in forming intramolecular disulfide bonds in seed storage proteins. In addition, these PDI family proteins that remain in the aleurone layers of mature grains likely assist in folding newly synthesized hydrolytic enzymes during germination.

Project description:BACKGROUND: The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species. RESULTS: Eight new non-homologous wheat genes were cloned and characterized. The nine PDI and PDI-like sequences of wheat were located in chromosome regions syntenic to those in rice and assigned to eight plant phylogenetic groups. The nine wheat genes differed in their sequences, genomic organization as well as in the domain composition and architecture of their deduced proteins; conversely each of them showed high structural conservation with genes from other plant species in the same phylogenetic group. The extensive quantitative RT-PCR analysis of the nine genes in a set of 23 wheat samples, including tissues and developmental stages, showed their constitutive, even though highly variable expression. CONCLUSIONS: The nine wheat genes showed high diversity, while the members of each phylogenetic group were highly conserved even between taxonomically distant plant species like the moss Physcomitrella patens. Although constitutively expressed the nine wheat genes were characterized by different expression profiles reflecting their different genomic organization, protein domain architecture and probably promoter sequences; the high conservation among species indicated the ancient origin and diversification of the still evolving gene family. The comprehensive structural and expression characterization of the complete set of PDI and PDI-like wheat genes represents a basis for the functional characterization of this gene family in the hexaploid context of bread wheat.

Project description:The correct formation of native disulfide bonds is critical for the proper structure and function of many proteins. Cellular disulfide bond formation pathways commonly consist of two parts: sulfhydryl oxidase-mediated oxidation and disulfide isomerase-mediated isomerization. Some large DNA viruses, such as baculoviruses, encode sulfhydryl oxidases, but viral disulfide isomerases have not yet been identified, although G4L in poxvirus has been suggested to serve such a function. Here, we report that the baculovirus core gene ac81 encodes a putative disulfide isomerase. ac81 is conserved in baculoviruses, nudiviruses, and hytrosaviruses. We found that AC81 homologs contain a typical thioredoxin fold conserved in disulfide isomerases. To determine the role of AC81, a series of Autographa californica nucleopolyhedrovirus (AcMNPV) bacmids containing ac81 knockout or point mutations was generated, and the results showed that AC81 is essential for budded virus production, multinucleocapsid occlusion-derived virus (ODV) formation, and ODV embedding in occlusion bodies. Nonreducing Western blot analysis indicated that disulfide bond formation in per os infectivity factor 5 (PIF5), a substrate of the baculoviral sulfhydryl oxidase P33, was abnormal when ac81 was knocked out or mutated. Pulldown assays showed that AC81 interacted with PIF5 and P33 in infected cells. In addition, two critical regions that harbor key amino acids for function were identified in AC81. Taken together, our results suggest that AC81 is a key component involved in the baculovirus disulfide bond formation pathway and likely functions as a disulfide isomerase. IMPORTANCE Many large DNA viruses, such as poxvirus, asfarvirus, and baculovirus, encode their own sulfhydryl oxidase to facilitate the disulfide bond formation of viral proteins. Here, we show that AC81 functions as a putative disulfide isomerase and is involved in multiple functions of the baculovirus life cycle. Interestingly, AC81 and P33 (sulfhydryl oxidase) are conserved in baculoviruses, nudiviruses, and hytrosaviruses, which are all insect-specific large DNA viruses replicating in the nucleus, suggesting that viral disulfide bond formation is an ancient mechanism shared by these viruses.

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Identification and mechanism of wheat protein disulfide isomerase-promoted gluten network formation.

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Identification and mechanism of wheat protein disulfide isomerase-promoted gluten network formation.

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