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Neuroblastoma tumorigenesis is regulated through the Nm23-H1/h-Prune C-terminal interaction.

Carotenuto M, Pedone E, Diana D, de Antonellis P, Džeroski S, Marino N, Navas L, Di Dato V, Scoppettuolo MN, Cimmino F, Correale S, Pirone L, Monti SM, Bruder E, Zenko B, Slavkov I, Pastorino F, Ponzoni M, Schulte JH, Schramm A, Eggert A, Westermann F, Arrigoni G, Accordi B, Basso G, Saviano M, Fattorusso R, Zollo M - Sci Rep (2013)

Bottom Line: H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers.We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation.We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP.

View Article: PubMed Central - PubMed

Affiliation: Centro di Ingegneria Genetica e Biotecnologie Avanzate-CEINGE, Naples, Italy.

ABSTRACT
Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment.

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Migration assays to assert migration properties of differential protein domains of the h-Prune protein.(a) Alignment of the h-Prune C-terminal. Amino acids involved in Nm23-H1 binding are highlighted in yellow. (b) Two-dimensional invasion assay. HEK293 cells transfected with h-Prune mutant proteins (D388A and D422A) showed decreased migration ability. Data are represented as relative (fold) increases in the number of cells migrating compared to full-length h-Prune wild-type transfected cells. (c) Affinity chromatography. The His-tagged 354-453 h-Prune D422A mutant (upper) and D388A mutant (lower) were immobilized on the resin. HEK293 total extract was loaded as control. An anti-His antibody was used as control for the protein immobilized. An anti-Nm23-H1 antibody shows that the mutated 354–453 h-Prune interacts weakly with Nm23-H1. (d) Affinity chromatography. SH-SY5Y pre-infected cells (Ad-CPP) were loaded onto the chromatography column and the eluates were loaded onto acrylamide gels. Nm23-H1 was detected using an anti-Nm23-H1 antibody. Protein 373–353 h-Prune was revealed using an antibody against the His tag. (e) Following Ad-CPP and Ad-Mock infections in SH-SY5Y and SK-N-BE cells, the protein levels of phospho-Nm23-H1, Nm23-H1 and anti-h-Prune were analyzed by Western blotting. β-Actin was used as the loading control. (f) Two-dimensional migration assay of NBL cells showing that CPP overexpression reduces the migratory properties of both SH-SY5Y and SK-N-BE cells.
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f4: Migration assays to assert migration properties of differential protein domains of the h-Prune protein.(a) Alignment of the h-Prune C-terminal. Amino acids involved in Nm23-H1 binding are highlighted in yellow. (b) Two-dimensional invasion assay. HEK293 cells transfected with h-Prune mutant proteins (D388A and D422A) showed decreased migration ability. Data are represented as relative (fold) increases in the number of cells migrating compared to full-length h-Prune wild-type transfected cells. (c) Affinity chromatography. The His-tagged 354-453 h-Prune D422A mutant (upper) and D388A mutant (lower) were immobilized on the resin. HEK293 total extract was loaded as control. An anti-His antibody was used as control for the protein immobilized. An anti-Nm23-H1 antibody shows that the mutated 354–453 h-Prune interacts weakly with Nm23-H1. (d) Affinity chromatography. SH-SY5Y pre-infected cells (Ad-CPP) were loaded onto the chromatography column and the eluates were loaded onto acrylamide gels. Nm23-H1 was detected using an anti-Nm23-H1 antibody. Protein 373–353 h-Prune was revealed using an antibody against the His tag. (e) Following Ad-CPP and Ad-Mock infections in SH-SY5Y and SK-N-BE cells, the protein levels of phospho-Nm23-H1, Nm23-H1 and anti-h-Prune were analyzed by Western blotting. β-Actin was used as the loading control. (f) Two-dimensional migration assay of NBL cells showing that CPP overexpression reduces the migratory properties of both SH-SY5Y and SK-N-BE cells.

Mentions: These NMR methodologies have identified the most significant amino acids of the h-Prune C-terminal region involved in binding to Nm23-H1. We then undertook a functional analysis of three of the most conserved amino acids (D388, D422 and C419) (Fig. 4a). The amino acids were mutated to alanine, alanine and serine, respectively. Then, we evaluated their functions on HEK293 cells in two-dimensional cell-migration assays (Fig. 4b, Supplementary Fig. 1e). Overexpression of the h-Prune-D388A and h-Prune-D422A mutated proteins did not induce cell migration, as shown by empty-vector-transfected cells, compared to the full-length h-Prune wild-type transfected cells. The affinity chromatography in Fig. 4c shows that the D388A and D422A mutant h-Prune proteins interact weakly with Nm23-H1. Moreover, the h-Prune-C419S mutant did not affect cell motility to the same extent (Supplementary Fig. 1f,g), thus indicating direct correlation of protein complex formation and enhancement of cell motility.


Neuroblastoma tumorigenesis is regulated through the Nm23-H1/h-Prune C-terminal interaction.

Carotenuto M, Pedone E, Diana D, de Antonellis P, Džeroski S, Marino N, Navas L, Di Dato V, Scoppettuolo MN, Cimmino F, Correale S, Pirone L, Monti SM, Bruder E, Zenko B, Slavkov I, Pastorino F, Ponzoni M, Schulte JH, Schramm A, Eggert A, Westermann F, Arrigoni G, Accordi B, Basso G, Saviano M, Fattorusso R, Zollo M - Sci Rep (2013)

Migration assays to assert migration properties of differential protein domains of the h-Prune protein.(a) Alignment of the h-Prune C-terminal. Amino acids involved in Nm23-H1 binding are highlighted in yellow. (b) Two-dimensional invasion assay. HEK293 cells transfected with h-Prune mutant proteins (D388A and D422A) showed decreased migration ability. Data are represented as relative (fold) increases in the number of cells migrating compared to full-length h-Prune wild-type transfected cells. (c) Affinity chromatography. The His-tagged 354-453 h-Prune D422A mutant (upper) and D388A mutant (lower) were immobilized on the resin. HEK293 total extract was loaded as control. An anti-His antibody was used as control for the protein immobilized. An anti-Nm23-H1 antibody shows that the mutated 354–453 h-Prune interacts weakly with Nm23-H1. (d) Affinity chromatography. SH-SY5Y pre-infected cells (Ad-CPP) were loaded onto the chromatography column and the eluates were loaded onto acrylamide gels. Nm23-H1 was detected using an anti-Nm23-H1 antibody. Protein 373–353 h-Prune was revealed using an antibody against the His tag. (e) Following Ad-CPP and Ad-Mock infections in SH-SY5Y and SK-N-BE cells, the protein levels of phospho-Nm23-H1, Nm23-H1 and anti-h-Prune were analyzed by Western blotting. β-Actin was used as the loading control. (f) Two-dimensional migration assay of NBL cells showing that CPP overexpression reduces the migratory properties of both SH-SY5Y and SK-N-BE cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3584926&req=5

f4: Migration assays to assert migration properties of differential protein domains of the h-Prune protein.(a) Alignment of the h-Prune C-terminal. Amino acids involved in Nm23-H1 binding are highlighted in yellow. (b) Two-dimensional invasion assay. HEK293 cells transfected with h-Prune mutant proteins (D388A and D422A) showed decreased migration ability. Data are represented as relative (fold) increases in the number of cells migrating compared to full-length h-Prune wild-type transfected cells. (c) Affinity chromatography. The His-tagged 354-453 h-Prune D422A mutant (upper) and D388A mutant (lower) were immobilized on the resin. HEK293 total extract was loaded as control. An anti-His antibody was used as control for the protein immobilized. An anti-Nm23-H1 antibody shows that the mutated 354–453 h-Prune interacts weakly with Nm23-H1. (d) Affinity chromatography. SH-SY5Y pre-infected cells (Ad-CPP) were loaded onto the chromatography column and the eluates were loaded onto acrylamide gels. Nm23-H1 was detected using an anti-Nm23-H1 antibody. Protein 373–353 h-Prune was revealed using an antibody against the His tag. (e) Following Ad-CPP and Ad-Mock infections in SH-SY5Y and SK-N-BE cells, the protein levels of phospho-Nm23-H1, Nm23-H1 and anti-h-Prune were analyzed by Western blotting. β-Actin was used as the loading control. (f) Two-dimensional migration assay of NBL cells showing that CPP overexpression reduces the migratory properties of both SH-SY5Y and SK-N-BE cells.
Mentions: These NMR methodologies have identified the most significant amino acids of the h-Prune C-terminal region involved in binding to Nm23-H1. We then undertook a functional analysis of three of the most conserved amino acids (D388, D422 and C419) (Fig. 4a). The amino acids were mutated to alanine, alanine and serine, respectively. Then, we evaluated their functions on HEK293 cells in two-dimensional cell-migration assays (Fig. 4b, Supplementary Fig. 1e). Overexpression of the h-Prune-D388A and h-Prune-D422A mutated proteins did not induce cell migration, as shown by empty-vector-transfected cells, compared to the full-length h-Prune wild-type transfected cells. The affinity chromatography in Fig. 4c shows that the D388A and D422A mutant h-Prune proteins interact weakly with Nm23-H1. Moreover, the h-Prune-C419S mutant did not affect cell motility to the same extent (Supplementary Fig. 1f,g), thus indicating direct correlation of protein complex formation and enhancement of cell motility.

Bottom Line: H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers.We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation.We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP.

View Article: PubMed Central - PubMed

Affiliation: Centro di Ingegneria Genetica e Biotecnologie Avanzate-CEINGE, Naples, Italy.

ABSTRACT
Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment.

Show MeSH
Related in: MedlinePlus