| 1. |
- Haugaard-Kedström, Linda M., et al.
(författare)
-
Solution Structure, Aggregation Behavior, and Flexibility of Human Relaxin-2.
- 2015
-
Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 10:3, s. 891-900
-
Tidskriftsartikel (refereegranskat)abstract
- Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.
|
|
| 2. |
- Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M., et al.
(författare)
-
Structural Properties of Relaxin Chimeras: NMR Characterization of the R3/I5 Relaxin Peptide
- 2009
-
Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 1160, s. 27-30
-
Tidskriftsartikel (refereegranskat)abstract
- Relaxin-3 interacts with high potency with three relaxin family peptide receptors (RXFP1, RXFP3, and RXFP4). Therefore, the development of selective agonist and antagonist analogs is important for in vivo studies characterizing the biological significance of the different receptor-ligand systems and for future pharmaceutical applications. Recent reports demonstrated that a peptide selective for RXFP3 and RXFP4 over RXFP1 can be generated by the combination of the relaxin-3 B chain with the A chain from insulin-like peptide 5 (INSL5), creating an R3/I5 chimera. We have used NMR spectroscopy to determine the three-dimensional structure of this peptide to gain structural insights into the consequences of combining chains from two different relaxins. The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B chain compared to native relaxin-3, and the INSL5 A chain displays a relaxin/insulin-like fold with two parallel helices. The findings indicate that binding and activation of RXFP3 and RXFP4 mainly require the B chain and that the A chain functions as structural support. RXFP1, however, demonstrates a more complex binding mechanism, involving both the A chain and the B chain. The creation of chimeras is a promising strategy for generating new structure-activity data on relaxins.
|
|
| 3. |
- Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M., et al.
(författare)
-
Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist
- 2008
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:35, s. 23811-23818
-
Tidskriftsartikel (refereegranskat)abstract
- The human relaxin family comprises seven peptide hormones with various biological functions mediated through interactions with G-protein-coupled receptors. Interestingly, among the hitherto characterized receptors there is no absolute selectivity toward their primary ligand. The most striking example of this is the relaxin family ancestor, relaxin-3, which is an agonist for three of the four currently known relaxin receptors: GPCR135, GPCR142, and LGR7. Relaxin-3 and its endogenous receptor GPCR135 are both expressed predominantly in the brain and have been linked to regulation of stress and feeding. However, to fully understand the role of relaxin-3 in neurological signaling, the development of selective GPCR135 agonists and antagonists for in vivo studies is crucial. Recent reports have demonstrated that such selective ligands can be achieved by making chimeric peptides comprising the relaxin-3 B-chain combined with the INSL5 A-chain. To obtain structural insights into the consequences of combining A-and B-chains from different relaxins we have determined the NMR solution structure of a human relaxin-3/INSL5 chimeric peptide. The structure reveals that the INSL5 A-chain adopts a conformation similar to the relaxin-3 A-chain, and thus has the ability to structurally support a native-like conformation of the relaxin-3 B-chain. These findings suggest that the decrease in activity at the LGR7 receptor seen for this peptide is a result of the removal of a secondary LGR7 binding site present in the relaxin-3 A-chain, rather than conformational changes in the primary B-chain receptor binding site.
|
|
| 4. |
- Hossain, M. Akhter, et al.
(författare)
-
Solid phase synthesis and structural analysis of novel A-chain dicarba analogs of human relaxin-3 (INSL7) that exhibit full biological activity
- 2009
-
Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 7:8, s. 1547-1553
-
Tidskriftsartikel (refereegranskat)abstract
- Replacement of disulfide bonds with non-reducible isosteres can be a useful means of increasing the in vivo stability of a protein. We describe the replacement of the A-chain intramolecular disulfide bond of human relaxin-3 (H3 relaxin, INSL7), an insulin-like peptide that has potential applications in the treatment of stress and obesity, with the physiologically stable dicarba bond. Solid phase peptide synthesis was used to prepare an A-chain analogue in which the two cysteine residues that form the intramolecular bond were replaced with allylglycine. On-resin microwave-mediated ring closing metathesis was then employed to generate the dicarba bridge. Subsequent cleavage of the peptide from the solid support, purification of two isomers and their combination with the B-chain via two intermolecular disulfide bonds, then furnished two isomers of dicarba-H3 relaxin. These were characterized by CD spectroscopy, which suggested a structural similarity to the native peptide. Additional analysis by solution NMR spectroscopy also identified the likely cis/trans form of the analogs. Both peptides demonstrated binding affinities that were equivalent to native H3 relaxin on RXFP1 and RXFP3 expressing cells. However, although the cAMP activity of the analogs on RXFP3 expressing cells was similar to the native peptide, the potency on RXFP1 expressing cells was slightly lower. The data confirmed the use of a dicarba bond as a useful isosteric replacement of the disulfide bond.
|
|
| 5. |
- Hossain, M. Akhter, et al.
(författare)
-
Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5)
- 2008
-
Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 9:11, s. 1816-1822
-
Tidskriftsartikel (refereegranskat)abstract
- Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5–RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as Nα-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function.
|
|
| 6. |
- Hossain, M. Akhter, et al.
(författare)
-
The A-chain of the human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors
- 2008
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:25, s. 17287-17297
-
Tidskriftsartikel (refereegranskat)abstract
- The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.
|
|
| 7. |
- Hossain, M. Akhter, et al.
(författare)
-
The structural and functional role of the B-chain C-terminal arginine in the relaxin-3 peptide antagonist, R3(B Delta 23-27)R/I5.
- 2009
-
Ingår i: Chemical Biology and Drug Design. - : Wiley. - 1747-0277 .- 1747-0285. ; 73:1, s. 46-52
-
Tidskriftsartikel (refereegranskat)abstract
- Relaxin-3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin-3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin-3/RXFP3 pair. The analog R3(B Delta 23-27)R/I5, in which a C-terminally truncated human relaxin-3 (H3) B-chain is combined with the INSL5 A-chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B-chain C-terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(B Delta 23-27)R/I5 and R3(B Delta 23-27)R containing the B-chain C-terminal Arg as well as R3(B Delta 23-27)/I5 and R3(B Delta 23-27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(B Delta 23-27)R and R3(B Delta 23-27)R/I5, the peptide R3(B Delta 23-27) is a weak agonist. This suggests that the C-terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(B Delta 23-27)R/I5 its potent antagonistic activity.
|
|
| 8. |
- Rosengren, K. Johan, et al.
(författare)
-
Structural insights into the function of relaxins
- 2009
-
Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 1160, s. 20-26
-
Tidskriftsartikel (refereegranskat)abstract
- The relaxin peptide hormones are members of the insulin superfamily and share a structural fold that is characterized by two peptide chains which are cross-braced by three disulfide bonds. On this framework, various amino acid side chains are presented, allowing specific interactions with different receptors. The relaxin receptors belong to two unrelated classes of G-protein-coupled receptors, but interestingly they are not selective for a single relaxin peptide. Relaxin-3, which is considered to be an extreme example of the relaxin family, can activate receptors from both classes and in fact interacts to some degree with all four receptors identified to date. To deduce how changes in the primary sequence can fine-tune the overall structure and thus the ability to interact with the various receptors, we have studied a range of relaxin-like peptides using solution nuclear magnetic resonance analysis. Three-dimensional structures of relaxin-3, insulin-like peptide 3 (INSL3), and INSL5 were determined and revealed a number of interesting features. All peptides showed a significant amount of line-broadening in certain regions, in particular around the intra-A-chain disulfide bond, suggesting that despite the disulfide bonds the fold is rather dynamic. Although the peptides share a common structural core there are significant differences, particularly around the termini. The structural data in combination with mutational studies provide valuable insights into the structure-activity relationships of relaxins.
|
|
