Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2

Zhang, Suode; Hughes, Richard A.; Bathgate, Ross A. D.; Shabanpoor, Fazel; Hossain, Mohammed Akhter; Lin, Feng; Lierop, Bianca van; Robinson, Andrea J.; Wade, John D.

doi:10.1016/j.peptides.2010.05.021

Cited by 35 publications

(31 citation statements)

References 21 publications

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“…Relaxin/insulin family peptides contain three α-helices that are functionally required 22 , 23 . The secondary structure of recombinant pINSL3 was analyzed by circular dichroism (CD) spectroscopy.…”

Section: Resultsmentioning

confidence: 99%

“…The A- and B-domains are joined by three disulfide bonds that are required for INSL3 biological activity. For example, the conversion of three disulfide bonds into isopeptide bonds decreased binding affinity for the receptor 22 , and analogs lacking the intra-chain disulfide bond showed no activity 23 . To date, the challenge of disulfide bond formation in prokaryotic cells has precluded the preparation of biologically active INSL3 using a recombinant expression system without post-expression treatment.…”

Section: Introductionmentioning

confidence: 99%

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Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity

Miyazaki

Ishizaki

Dohra

et al. 2017

Sci Rep

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Insulin-like peptide 3 (INSL3) is a member of the relaxin/insulin superfamily and is expressed in testicular Leydig cells. Essential for fetal testis descent, INSL3 has been implicated in testicular and sperm function in adult males via interaction with relaxin/insulin-like family peptide receptor 2 (RXFP2). The INSL3 is typically prepared using chemical synthesis or overexpression in Escherichia coli followed by oxidative refolding and proteolysis. Here, we expressed and purified full-length porcine INSL3 (pINSL3) using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system. Biophysical measurements and proteomic analysis revealed that this recombinant pINSL3 exhibited the correct conformation, with the three critical disulfide bonds observed in native pINSL3, although partial cleavage occurred. In cAMP stimulation assays using RXFP2-expressing HEK293 cells, the recombinant pINSL3 possessed full biological activity. This is the first report concerning the production of fully active pINSL3 without post-expression treatments and provides an efficient production platform for expressing relaxin/insulin superfamily peptides.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity

Miyazaki

Ishizaki

Dohra

et al. 2017

Sci Rep

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“…The amino acid residues essential for the function of relaxin are marked in bold relaxin should contain both A and B chains. We believe uPTI-1 preserves the possibility to interact with A-chain of relaxin since it contains all cysteins responsible for such interaction [39] (Fig. 3, C).…”

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confidence: 85%

PTI-1: novel way to oncogenicity

et al. 2012

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Aim. The prostate tumor-inducing oncogene (PTI-1), presumably encoding a truncated form of eukaryotic translation elongation factor 1A1 (eEF1A1), was discovered as a gene overexpressed in prostate tumor samples and absent in normal tissues. The mechanism of PTI-1 oncogenicity remains obscure. Methods. Several bioinformatics methods were applied to analyze the PTI-1 mRNA structure, translation efficiency and coding potential. Results. In silico analysis of 5'UTR of its mRNA suggest that PTI-1 mRNA most probably belongs to the class of templates with low translation efficiency. Additionally, novel open reading frame (ORF) starting with alternati- ve initiation site situated upstream of the main ORF start codon was found. Finally, the peptide that does not resemble eEF1A1 but is partially homologous to relaxin can be synthesized. Conclusions. We suggest that the alternative upstream start codon may initiate synthesis of a peptide (uPTI-1) homologous to relaxin, the hormone shown to promote the prostate cancer progression. uPTI-1 protein may interact with the respective relaxin-specific receptors, suggesting that the tumorigenic outcome of PTI-1 is possibly realized via the relaxin-dependent pathway

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“…Numerous dicarba analogues of cystine-containing peptides have also been prepared [ 12 – 15 ] and shown to possess near-native structure and extended in vivo stability. Monosubstituted dicarba bond analogues of the heterodimeric peptides relaxin-3 [ 16 ] and insulin-like peptide 3 (INSL3) [ 17 ] have also been prepared and evaluated.…”

Section: Introductionmentioning

confidence: 99%

Total Chemical Synthesis of a Heterodimeric Interchain Bis-Lactam-Linked Peptide: Application to an Analogue of Human Insulin-Like Peptide 3

Karas

Shabanpoor

Hossain

et al. 2013

International Journal of Peptides

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Nonreducible cystine isosteres represent important peptide design elements in that they can maintain a near-native tertiary conformation of the peptide while simultaneously extending the in vitro and in vivo half-life of the biomolecule. Examples of these cystine mimics include dicarba, diselenide, thioether, triazole, and lactam bridges. Each has unique physicochemical properties that impact upon the resulting peptide conformation. Each also requires specific conditions for its formation via chemical peptide synthesis protocols. While the preparation of peptides containing two lactam bonds within a peptide is technically possible and reported by others, to date there has been no report of the chemical synthesis of a heterodimeric peptide linked by two lactam bonds. To examine the feasibility of such an assembly, judicious use of a complementary combination of amine and acid protecting groups together with nonfragment-based, total stepwise solid phase peptide synthesis led to the successful preparation of an analogue of the model peptide, insulin-like peptide 3 (INSL3), in which both of the interchain disulfide bonds were replaced with a lactam bond. An analogue containing a single disulfide-substituted interchain lactam bond was also prepared. Both INSL3 analogues retained significant cognate RXFP2 receptor binding affinity.

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Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2

Cited by 35 publications

References 21 publications

Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity

Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity

PTI-1: novel way to oncogenicity

Total Chemical Synthesis of a Heterodimeric Interchain Bis-Lactam-Linked Peptide: Application to an Analogue of Human Insulin-Like Peptide 3

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