The Different Energetic State of the Intra A-Chain/Domain Disulfide of Insulin and Insulin-like Growth Factor 1 Is Mainly Controlled by Their B-Chain/Domain

Guo, Zhan‐Yun; Shen, Lu; Yang, Feng

doi:10.1021/bi020165f

Cited by 17 publications

(17 citation statements)

References 43 publications

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“…Thus, such molecules may exhibit a sufficiently folded structure to escape ER-based quality control, resulting in secretion (37). Consistent with the idea that the B-chain provides a template to guide folding of the A-chain (27), during in vitro refolding an insulin B-chain/IGF-1 A-chain chimera forms stable native disulfides and a single thermodynamically stable tertiary structure, whereas an IGF-1 B-chain/insulin A-chain chimera cannot form/maintain a single set of native disulfide bonds but instead folds into two distinct disulfide isomers (28,62). From these data, it seems reasonable to be concerned about the possibility that certain mutations in the insulin B-chain sequence might impair the fidelity of native disulfide bond formation within single-chain insulins and in proinsulin as well.…”

Section: Figsupporting

confidence: 60%

“…Because substitution of proximal B-chain residues can provoke increased conformational changes of the insulin moiety including increased B-chain flexibility (25,26), and the B-subdomain may provide a template to guide folding of the A-chain (27,28), it seemed possible that such B-chain point mutants might be recognized as misfolded by Vps10p (29 -31). We therefore employed a pulse-chase protocol to examine the behavior of ICFP constructs bearing B9D, B10D, B12E, or B28K,B29P point mutations, driven by the strong TDH3 promoter, in strains that were (or were not) deleted for VPS10.…”

Section: In Vivo Folding Of Insulins Bearing B-chain Mutationsmentioning

confidence: 99%

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Behavior in the Eukaryotic Secretory Pathway of Insulin-containing Fusion Proteins and Single-chain Insulins Bearing Various B-chain Mutations

Zhang¹,

Liu²,

Arvan³

2003

Journal of Biological Chemistry

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In the secretory pathway, endoproteolytic cleavage of the insulin precursor protein promotes a change in the biophysical properties of the processed insulin product, and this may be relevant for its intracellular trafficking. We have now studied several independent point mutants contained within the insulin B-chain, S9D, H10D, V12E (called B9D, B10D, and B12E), as well as the double point mutant P28K,K29P (B28K,B29P), that have been reported to inhibit insulin oligomerization. In yeast cells, the unprocessed precursor of each of these mutants is secreted, whereas >90% of the endoproteolytically released single-chain insulin moiety is retained intracellularly; a large portion of the B9D, B10D, and B12E single-chain insulins exhibit abnormally slow mobility upon nonreducing SDS-PAGE, despite normal mobility upon reducing SDS-PAGE. Although no free thiols can be detected, each of these mutants exhibits increased disulfide accessibility to dithiothreitol. After dithiothreitol treatment, a portion of the molecules can reoxidize to a form more compact than the original single-chain insulin mutants formed in vivo (indicating initial disulfide mispairing). Disulfide mispairing of a fraction of B9D, B10D, and B12E mutants also occurs in the context of single-chain insulin and even in authentic proinsulin expressed within the secretory pathway of mammalian cells. We conclude that analyses of the intracellular trafficking of certain oligomerization-defective insulin mutants is complicated by the formation of disulfide isomers in the secretory pathway.

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Section: Figsupporting

confidence: 60%

Section: In Vivo Folding Of Insulins Bearing B-chain Mutationsmentioning

confidence: 99%

Behavior in the Eukaryotic Secretory Pathway of Insulin-containing Fusion Proteins and Single-chain Insulins Bearing Various B-chain Mutations

Zhang¹,

Liu²,

Arvan³

2003

Journal of Biological Chemistry

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“…The different folding behavior of insulin/PIP and IGF-1 is mainly controlled by their B-chain/domain that controls the different energetic state of the intra-A-chain/domain disulfide (37,38).…”

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confidence: 99%

Refolding of Amphioxus Insulin-like Peptide: Implications of a Bifurcating Evolution of the Different Folding Behavior of Insulin and Insulin-like Growth Factor 1

et al. 2003

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Insulin and insulin-like growth factor 1 (IGF-1) share high sequence homology, but their folding behaviors are significantly different: insulin folds into one unique thermodynamically controlled structure, while IGF-1 folds into two thermodynamically controlled disulfide isomers. However, the origin of their different folding behaviors is still elusive. The amphioxus insulin-like peptide (ILP) is thought to be the common ancestor of insulin and IGF-1. A recombinant single-chain ILP has been expressed previously, and now its folding behavior is investigated. The folding behavior of ILP shows the characteristics of both insulin and IGF-1. On one hand, two thermodynamically controlled disulfide isomers of ILP have been identified; on the other hand, the content of isomer 1 (its disulfides are deduced identical to those of swap IGF-1) is much less than that of isomer 2 (its disulfides are deduced identical to those of native IGF-1); that is, more than 96% of ILP folds into the native structure. The present results suggest that the different folding behaviors of insulin and IGF-1 are acquired through a bifurcating evolution: the tendency of forming the thermodynamically controlled non-native disulfide isomer is diminished during evolution from ILP to insulin, while this tendency is amplified during evolution from ILP to IGF-1. Moreover, the N-terminal Gln residue of ILP can spontaneously form a pyroglutamate residue, and its cyclization has a significant effect on the folding behavior of ILP: the percentage of isomer 1 is approximately 2-fold that of isomer 1 of the noncyclized ILP; that is, isomer 1 becomes more favored when the N-terminal residue of ILP is cyclized. So, we deduce that the N-terminal residues have a significant effect on the folding properties of insulin, IGF-1, and ILP.In the 1960s Anfinsen and co-workers first demonstrated the three-dimensional structure of a globular protein is uniquely determined by its amino acid sequence (1). Since then, significant advances have been made in the understanding of protein folding through experimental and theoretical approaches. For small proteins with two-state folding, topology is a major determinant of the folding rate and greatly influences the structure of the transition-state ensemble (2-4). Studies on the disulfide-coupled folding of some small globular proteins, such as BPTI, 1 RNase A, and EGF, have revealed a sequence of preferred kinetic intermediates which define a folding pathway (5-12). In vivo the protein folding is assisted by the molecular chaperones, especially for large proteins (13-15); some chaperones even can provide the missing steric information for protein folding (16).Insulin is an extensively studied small globular protein with A-and B-chains linked by three disulfides (one intrachain bond, A6-A11; two interchain bonds, A7-B7 and A20-B19). Its three-dimensional structure has been well solved by X-ray crystallography (17,(18)(19)(20)(21) since the 1970s. Although the separate A-and B-chains of insulin can be recombined success...

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“…Our previous results demonstrated that the different folding behavior of insulin and IGF-1 is mainly controlled by their B-chain/domains (24,26) (28). They proposed that the N-terminal extension imparts a steric constraint at a crucial point in folding, thus allowing native disulfide bonds to form efficiently.…”

Section: Discussionmentioning

confidence: 99%

Sequences of B-Chain/Domain 1−10/1−9 of Insulin and Insulin-like Growth Factor 1 Determine Their Different Folding Behavior

et al. 2004

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Although insulin and insulin-like growth factor-1 (IGF-1) belong to one family, insulin folds into one thermodynamically stable structure, while IGF-1-folds into two thermodynamically stable structures (native and swap forms). We have demonstrated previously that the bifurcating folding behavior of IGF-1 is mainly controlled by its B-domain. To further elucidate which parts of the sequences determine their different folding behavior, by exchanging the N-terminal sequences of mini-IGF-1 and recombinant porcine insulin precursor (PIP), we prepared four peptide models: [1-9]PIP, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1 by means of protein engineering, and their disulfide rearrangement, V8 digestion, circular dichroic spectra, disulfide stability, and in vitro refolding were investigated. Among them only [1-9]-PIP, like mini-IGF-1/IGF-1, was expressed in yeast as two isomers: isomer 1 (corresponding to swap IGF-1) and isomer 2 (corresponding to native IGF-1), which are supported by the experimental results of disulfide rearrangements, peptide mapping of V8 endoprotenase digests, circular dichroic analysis, in vitro refolding, and disulfide stability analysis. The other peptide models, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1, fold into one stable structure as PIP does, which indicates that sequence 1-4 of mini-IGF-1 is important for the folding behavior of mini-IGF-1/IGF-1 but not sufficient to lead to a bifurcating folding. The results demonstrated that the folding information, by which mini-IGF-1/IGF-1-folds into two thermodynamically structures, is encoded/written in its sequence 1-9, while sequences 1-10 of B chain in insulin/PIP play an important role in the guide of its unique disulfide pairing during the folding process.Insulin is a structurally and functionally well-characterized small globular protein with A-and B-chains linked by three disulfides. Its three-dimensional structure has been well defined by X-ray crystallography (1) and NMR (2-4) since the 1970s. Insulin-like growth factor-1 (IGF-1) 1 is a 70-residue single-chain globular protein composed of B-, C-, A-, and D-domains from the N-terminus to the C-terminus (5). Its B-and A-domains are homologous to the B-and A-chains of insulin, respectively; its 12-residue C-domain is analogous to the C-peptide of proinsulin, but they share no homology; its C-terminal 8-residue D-domain has no counterpart in insulin. The three-dimensional structure of IGF-1 has also been well-defined by X-ray crystallography (6, 7) and NMR (8, 9). Insulin and IGF-1 belong to the same superfamily. They have a common structural motif (10, 11) and share homologous sequence, similar three-dimensional structure (1,6,7), and a common ancestor (12,13). Both mainly consist of three R-helical segments (A2-A8, A13-A19, and B9-B19 in insulin; 8-18, 42-49, and 54-61 in IGF-1) in the A-and B-chains/domains; the three R-helical segments are stabilized by three identical disulfides (A6-A11, A7-B7, and A20-B19 in insulin; 47-52, 6-48, and 18-61 in IGF-1); and when IGF-1...

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The Different Energetic State of the Intra A-Chain/Domain Disulfide of Insulin and Insulin-like Growth Factor 1 Is Mainly Controlled by Their B-Chain/Domain

Cited by 17 publications

References 43 publications

Behavior in the Eukaryotic Secretory Pathway of Insulin-containing Fusion Proteins and Single-chain Insulins Bearing Various B-chain Mutations

Behavior in the Eukaryotic Secretory Pathway of Insulin-containing Fusion Proteins and Single-chain Insulins Bearing Various B-chain Mutations

Refolding of Amphioxus Insulin-like Peptide: Implications of a Bifurcating Evolution of the Different Folding Behavior of Insulin and Insulin-like Growth Factor 1

Sequences of B-Chain/Domain 1−10/1−9 of Insulin and Insulin-like Growth Factor 1 Determine Their Different Folding Behavior

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