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The C-terminal region Mesd peptide mimics full-length Mesd and acts as an inhibitor of Wnt/β-catenin signaling in cancer cells.

Lin C, Lu W, Zhang W, Londoño-Joshi AI, Buchsbaum DJ, Bu G, Li Y - PLoS ONE (2013)

Bottom Line: In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface.We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too.Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States of America.

ABSTRACT
While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

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Related in: MedlinePlus

Structural representation of C-terminal peptides in cartons.(A) The NMR structure of mouse Mesd C-terminal domain (155–191) based on the study of Chen et al. (24). (B) The most representative snapshot of the peptide mMesd (155–191). The segments of 160–169 and 183–191 were highlighted in orange and green colors. Residues that form the positive surface were shown in solid sticks. The two surface regions that consist of residues from 160–169 segment or from 183–191segment were marked with blue and red dashed circles. Residues that are unique in the positive surface of A) or B) were labeled with red-color. (C–E) The most populated conformations based on clustering analysis of the simulation snapshots of three C-terminal peptides mMesd (155–191) (C), mMesd (160–169) (D) and mMesd (183–191) (E).
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pone-0058102-g002: Structural representation of C-terminal peptides in cartons.(A) The NMR structure of mouse Mesd C-terminal domain (155–191) based on the study of Chen et al. (24). (B) The most representative snapshot of the peptide mMesd (155–191). The segments of 160–169 and 183–191 were highlighted in orange and green colors. Residues that form the positive surface were shown in solid sticks. The two surface regions that consist of residues from 160–169 segment or from 183–191segment were marked with blue and red dashed circles. Residues that are unique in the positive surface of A) or B) were labeled with red-color. (C–E) The most populated conformations based on clustering analysis of the simulation snapshots of three C-terminal peptides mMesd (155–191) (C), mMesd (160–169) (D) and mMesd (183–191) (E).

Mentions: Our results of binding assays are consistent with recent NMR study on the full-length Mesd [24], which shows that the C-terminal flexible helical domain (156–195) is structurally distinct from the core domain (1–155), and is responsible for the escort function of Mesd by binding to LRP5/6. Nevertheless, peptides can have significant structural changes when isolated from proteins. To further explore the structural insights, we performed molecular dynamics simulations on all three peptides [mMesd (155–191), mMesd (160–169) and mMesd (183–191)] and compared them with the NMR structure of the full-length Mesd protein. Clustering analysis of the simulation results indicated that all three peptides adopted relative stable conformation. The most populated conformations of the three peptides were illustrated in Figure 2C–2E. Instead of the extended loop-like shape of C-terminal domain in the full-length Mesd (Figure 2A), mMesd (155–191) folds into a more compact structure with both its C- and N-termini bending towards the center (Figure 2B). Despite the apparent structural differences, both structures maintained a positive surface consisted of positively charged residues with their sidechains locate at the same side and fully exposed to solvent (Figure 2A and 2B). Such positive surface is crucial for Mesd's bind capacity to mature LRP5/6. Interestingly, this positive surface can be spatially divided into two regions that mainly consist of the positively charged residues from mMesd (160–169) and mMesd (183–190), respectively (Figure 2A and 2B). Simulation results show that ∼45% and ∼58% of all simulation snapshots of mMesd (160–169) and Mesd (182–190), respectively, adopted similar conformations as their corresponding ones in the full-length Mesd structure (Figure 2D and 2E). The similar structural characteristics and positive surface explain that the two shorter peptides display a similar function on binding to LRP5/6.


The C-terminal region Mesd peptide mimics full-length Mesd and acts as an inhibitor of Wnt/β-catenin signaling in cancer cells.

Lin C, Lu W, Zhang W, Londoño-Joshi AI, Buchsbaum DJ, Bu G, Li Y - PLoS ONE (2013)

Structural representation of C-terminal peptides in cartons.(A) The NMR structure of mouse Mesd C-terminal domain (155–191) based on the study of Chen et al. (24). (B) The most representative snapshot of the peptide mMesd (155–191). The segments of 160–169 and 183–191 were highlighted in orange and green colors. Residues that form the positive surface were shown in solid sticks. The two surface regions that consist of residues from 160–169 segment or from 183–191segment were marked with blue and red dashed circles. Residues that are unique in the positive surface of A) or B) were labeled with red-color. (C–E) The most populated conformations based on clustering analysis of the simulation snapshots of three C-terminal peptides mMesd (155–191) (C), mMesd (160–169) (D) and mMesd (183–191) (E).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585277&req=5

pone-0058102-g002: Structural representation of C-terminal peptides in cartons.(A) The NMR structure of mouse Mesd C-terminal domain (155–191) based on the study of Chen et al. (24). (B) The most representative snapshot of the peptide mMesd (155–191). The segments of 160–169 and 183–191 were highlighted in orange and green colors. Residues that form the positive surface were shown in solid sticks. The two surface regions that consist of residues from 160–169 segment or from 183–191segment were marked with blue and red dashed circles. Residues that are unique in the positive surface of A) or B) were labeled with red-color. (C–E) The most populated conformations based on clustering analysis of the simulation snapshots of three C-terminal peptides mMesd (155–191) (C), mMesd (160–169) (D) and mMesd (183–191) (E).
Mentions: Our results of binding assays are consistent with recent NMR study on the full-length Mesd [24], which shows that the C-terminal flexible helical domain (156–195) is structurally distinct from the core domain (1–155), and is responsible for the escort function of Mesd by binding to LRP5/6. Nevertheless, peptides can have significant structural changes when isolated from proteins. To further explore the structural insights, we performed molecular dynamics simulations on all three peptides [mMesd (155–191), mMesd (160–169) and mMesd (183–191)] and compared them with the NMR structure of the full-length Mesd protein. Clustering analysis of the simulation results indicated that all three peptides adopted relative stable conformation. The most populated conformations of the three peptides were illustrated in Figure 2C–2E. Instead of the extended loop-like shape of C-terminal domain in the full-length Mesd (Figure 2A), mMesd (155–191) folds into a more compact structure with both its C- and N-termini bending towards the center (Figure 2B). Despite the apparent structural differences, both structures maintained a positive surface consisted of positively charged residues with their sidechains locate at the same side and fully exposed to solvent (Figure 2A and 2B). Such positive surface is crucial for Mesd's bind capacity to mature LRP5/6. Interestingly, this positive surface can be spatially divided into two regions that mainly consist of the positively charged residues from mMesd (160–169) and mMesd (183–190), respectively (Figure 2A and 2B). Simulation results show that ∼45% and ∼58% of all simulation snapshots of mMesd (160–169) and Mesd (182–190), respectively, adopted similar conformations as their corresponding ones in the full-length Mesd structure (Figure 2D and 2E). The similar structural characteristics and positive surface explain that the two shorter peptides display a similar function on binding to LRP5/6.

Bottom Line: In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface.We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too.Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States of America.

ABSTRACT
While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

Show MeSH
Related in: MedlinePlus