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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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Three-dimensional model of h-Prune protein based on protein similarities at the N-H2 terminal domain region combined with NMR h-Prune C-terminal structural studies.(a) The h-Prune C-terminal sequence. The amino acids given in bold represent those that are more exposed according to limited proteolysis experiments. (b) Affinity chromatography. His-tagged amino acids 354-453 of h-Prune was immobilized on the resin. SH-SY5Y and SH-SY5Y-Nm23-H1 total extracts were loaded as control. Elution E3 (from 200 mM imidazole) and E4 (from elution of the complex) were loaded. An anti-His antibody was used as control of immobilized protein. The anti-Nm23-H1 antibody shows that h-Prune C-terminal interacts with Nm23-H1. (c) The secondary chemical shift index, corrected for sequence-dependent contributions, based on1Hα,13Cβ,13Cα and13CO chemical shifts of the h-Prune C-terminal. Protein regions with a propensity to a helical structure (α1, α2 and α3) are highlighted. The1H-15N steady-state heteronuclear NOE values are also reported, according to the h-Prune C-terminal amino acids.
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f2: Three-dimensional model of h-Prune protein based on protein similarities at the N-H2 terminal domain region combined with NMR h-Prune C-terminal structural studies.(a) The h-Prune C-terminal sequence. The amino acids given in bold represent those that are more exposed according to limited proteolysis experiments. (b) Affinity chromatography. His-tagged amino acids 354-453 of h-Prune was immobilized on the resin. SH-SY5Y and SH-SY5Y-Nm23-H1 total extracts were loaded as control. Elution E3 (from 200 mM imidazole) and E4 (from elution of the complex) were loaded. An anti-His antibody was used as control of immobilized protein. The anti-Nm23-H1 antibody shows that h-Prune C-terminal interacts with Nm23-H1. (c) The secondary chemical shift index, corrected for sequence-dependent contributions, based on1Hα,13Cβ,13Cα and13CO chemical shifts of the h-Prune C-terminal. Protein regions with a propensity to a helical structure (α1, α2 and α3) are highlighted. The1H-15N steady-state heteronuclear NOE values are also reported, according to the h-Prune C-terminal amino acids.

Mentions: The recombinant C-terminal domain of h-Prune (amino acids 354–453) is stable and soluble, and it contains a disulfide bridge that links cysteines 419 and 437, as shown by liquid chromatography–mass spectrometry analysis (Fig. 2a). This domain has a low overall hydrophobicity and a high net negative charge, and analyses according to several algorithms have suggested that it is mostly unfolded in the native protein. Far-UV circular dichroism spectrometry has confirmed this lack of secondary structure (see Supplementary Experimental Procedures). Western blotting revealed that the h-Prune C-terminal was sufficient for binding to the endogenous Nm23-H1 protein (Fig. 2b).


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)

Three-dimensional model of h-Prune protein based on protein similarities at the N-H2 terminal domain region combined with NMR h-Prune C-terminal structural studies.(a) The h-Prune C-terminal sequence. The amino acids given in bold represent those that are more exposed according to limited proteolysis experiments. (b) Affinity chromatography. His-tagged amino acids 354-453 of h-Prune was immobilized on the resin. SH-SY5Y and SH-SY5Y-Nm23-H1 total extracts were loaded as control. Elution E3 (from 200 mM imidazole) and E4 (from elution of the complex) were loaded. An anti-His antibody was used as control of immobilized protein. The anti-Nm23-H1 antibody shows that h-Prune C-terminal interacts with Nm23-H1. (c) The secondary chemical shift index, corrected for sequence-dependent contributions, based on1Hα,13Cβ,13Cα and13CO chemical shifts of the h-Prune C-terminal. Protein regions with a propensity to a helical structure (α1, α2 and α3) are highlighted. The1H-15N steady-state heteronuclear NOE values are also reported, according to the h-Prune C-terminal amino acids.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Three-dimensional model of h-Prune protein based on protein similarities at the N-H2 terminal domain region combined with NMR h-Prune C-terminal structural studies.(a) The h-Prune C-terminal sequence. The amino acids given in bold represent those that are more exposed according to limited proteolysis experiments. (b) Affinity chromatography. His-tagged amino acids 354-453 of h-Prune was immobilized on the resin. SH-SY5Y and SH-SY5Y-Nm23-H1 total extracts were loaded as control. Elution E3 (from 200 mM imidazole) and E4 (from elution of the complex) were loaded. An anti-His antibody was used as control of immobilized protein. The anti-Nm23-H1 antibody shows that h-Prune C-terminal interacts with Nm23-H1. (c) The secondary chemical shift index, corrected for sequence-dependent contributions, based on1Hα,13Cβ,13Cα and13CO chemical shifts of the h-Prune C-terminal. Protein regions with a propensity to a helical structure (α1, α2 and α3) are highlighted. The1H-15N steady-state heteronuclear NOE values are also reported, according to the h-Prune C-terminal amino acids.
Mentions: The recombinant C-terminal domain of h-Prune (amino acids 354–453) is stable and soluble, and it contains a disulfide bridge that links cysteines 419 and 437, as shown by liquid chromatography–mass spectrometry analysis (Fig. 2a). This domain has a low overall hydrophobicity and a high net negative charge, and analyses according to several algorithms have suggested that it is mostly unfolded in the native protein. Far-UV circular dichroism spectrometry has confirmed this lack of secondary structure (see Supplementary Experimental Procedures). Western blotting revealed that the h-Prune C-terminal was sufficient for binding to the endogenous Nm23-H1 protein (Fig. 2b).

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