Insulin‐like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions<sup>1</sup>

Hober, Sophia; Ljung, Johanna; Uhlén, Mathias; Nilsson, Björn

doi:10.1016/s0014-5793(98)01737-2

Cited by 28 publications

(47 citation statements)

References 42 publications

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“…As analyzed by C4 reverse-phase HPLC, the local hybrid incubated in the redox buffer (the ratio of GSH to GSSG is 1:10 and 5:5, respectively) appeared as a single peak on the HPLC profile with the retention time identical to that of the control. This result is identical to that of the wild-type PIP but different from that of IGF-1 significantly since IGF-1 cannot maintain its intact disulfides in the above redox buffer (34). The present result suggested that the local swap had little effect on the disulfide stability of PIP.…”

Section: Construction Expression and Purification Of The Localsupporting

confidence: 54%

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

2002

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Insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar threedimensional structure, and weakly overlapping biological activity, but different folding information is stored in their homologous sequences: the sequence of insulin encodes one unique thermodynamically stable three-dimensional structure while that of IGF-1 encodes two disulfide isomers with different threedimensional structure but similar thermodynamic stability. Their different folding behavior probably resulted from the different energetic state of the intra A-chain/domain disulfide: the intra A-chain disulfide of insulin is a stable bond while that of IGF-1 is a strained bond with high energy. To find out the sequence determinant of the different energetic state of their intra A-chain/domain disulfide, the following experiments were carried out. First, a local chimeric single-chain insulin (PIP) with the A8-A10 residues replaced by the corresponding residues of IGF-1 was prepared. Second, the disulfide stability of two global hybrids of insulin and IGF-1, Ins(A)/IGF-1(B) and Ins(B)/IGF-1(A), was investigated. The local segment swap had no effect on the fidelity of disulfide pairing and the disulfide stability of PIP molecule although the swapped segment is close to the intra A-chain/domain disulfide. In redox buffer which favors the disulfide formation for most proteins, Ins(A)/IGF-1(B) cannot form and maintain its native disulfides just like that of IGF-1, while the disulfides of Ins(B)/IGF-1(A) are stable in the same condition. One major equilibrium intermediate with two disulfides of Ins(A)/IGF-1(B) was purified and characterized. V8 endoproteinase cleavage and circular dichroism analysis suggested that the intra A-chain/domain disulfide was reduced in the intermediate. Our present results suggested that the energetic state of the intra A-chain/domain disulfide of insulin and IGF-1 was not controlled by the A-chain/domain sequence close to this disulfide but was mainly controlled by the sequence of the B-chain/domain.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-defined by X-ray crystallography (1, 2) and NMR (3-5) since the 1970s. IGF-1 1 is an insulin-like 70-residue singlechain protein composed of B-, C-, A-, and D-domains (6). The B-and A-domains of IGF-1 are homologous to the Band A-chains of insulin, respectively; the C-domain is analogous to the C-peptide of proinsulin, but they share no sequence homology; the D-domain has no counterparts in insulins. The three-dimensional structure of IGF-1 is very similar with that of insulin (7). The architecture of insulin and IGF-1 mainly consists 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-chain/domains ( Figure 1A,B). The three R-helical segments are stabilized by the three disulfides (A6-A11, A7-B7, A20-B19 in insul...

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Section: Construction Expression and Purification Of The Localsupporting

confidence: 54%

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

2002

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“…In a pioneering study, Nillson and co-workers (27) demonstrated that under the redox conditions of the Golgi apparatus, secretory granules, and bloodstream, isolated IGF-I undergoes in vitro disulfide rearrangement to form an equilibrium distribution of native and swapped species. Because, to our knowledge, the biological implications of these findings have not been explored, it would be of future interest to extend this study to molecular analysis of the state of IGF-I during cellular biosynthesis.…”

Section: B5mentioning

confidence: 99%

“…Co-expression or RNA interference knockdown of IGFBPs might be found to modulate the fidelity of disulfide pairing. Nillson and co-workers (27) further speculated that isomerization of free IGF-I in the bloodstream and tissues might provide a mechanism to down-regulate its growth-promoting activity (27). The proposed mechanism thus exploits the instability of free IGF-I and the low activity of IGFswap as a biological defense against excessive mitogenic signaling.…”

Section: B5mentioning

confidence: 99%

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Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

Sohma

Hua

Liu

et al. 2010

Journal of Biological Chemistry

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Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6 -A11, A7-B7, and A20 -B19) maintained by IGFbinding proteins; IGF-swap has alternative pairing (A7-A11, A6 -B7, and A20 -B19) and impaired activity. Studies of minidomain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His The vertebrate insulin-related superfamily consists of insulin and insulin-related growth factors (IGF-I 5 and IGF-II) (1, 2), relaxin (3-5), and relaxin-related factors (6 -9). Insulin and IGFs function as ligands for receptor tyrosine kinases (the insulin receptor and type 1 IGF receptor; IR and IGF-1R) (10), whereas relaxin and related factors bind to G-protein-coupled receptors (11). Interest in the evolution and folding properties of insulin-related polypeptides has recently been invigorated by the discovery of mutations in the human insulin gene associated with permanent neonatal-onset diabetes mellitus (12). These dominant mutations impair the foldability of variant and (in trans) wild-type proinsulin, leading to ␤-cell dysfunction, endoplasmic reticular (ER) stress, and impaired ␤-cell viability (13). One such mutation occurs at position B5 (12, 14). Insulin contains a conserved His at B5, whereas IGF-I contains a conserved Thr (Table 1). Here, we investigate reciprocal substitutions in these proteins, Thr B5 in insulin and His B5 in IGF-I, as probes of competing evolutionary constraints among otherwise homologous sequences. 6IGFs are single-chain polypeptides containing A-and B-domains, an intervening connecting (C)-domain, and a C-terminal D-domain (Fig. 1A) (1, 2); insulin (like relaxin and related factors) contains two chains (designated A and B; Fig. 1B) as a consequence of proteolytic processing in the trans-Golgi network (3-5, 15). Crystal structures of IGF-I and insulin exhibit similar ␣-helical domains (Fig. 1, A and B) (1-9). Protein folding is linked to specific disulfide pairing. The canonical cystines in insulin are A6 -A11, A7-B7, and A20 -B19, and the corresponding cystines in IGF-I are at polypeptide positions 47-52,

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“…Native IGF-1 has three R-helical segments (6,7), while swap IGF-1 has only two R-helical segments (15). The bifurcating folding behavior of IGF-1 is also present under in vivo-like conditions (16). Different from IGF-1, the swap form of insulin (with the disulfide pairings A7-B6, A6-A11, and A20-B19) was only obtained as a kinetic trap with the existence of guanidine hydrochloride and DTT (17).…”

mentioning

confidence: 98%

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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Insulin‐like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions¹

Cited by 28 publications

References 42 publications

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

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

Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

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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Insulin‐like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions1

Cited by 28 publications

References 42 publications

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

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

Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

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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Insulin‐like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions¹