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

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.

gH625 is a viral derived peptide for effective delivery of intrinsically disordered proteins

“…27,28 The conformational analysis of the C-terminal domain of h-prune performed by nuclear magnetic resonance showed that it assumes a random coil conformation with the exception of some protein regions with helical structural propensity and a disulfide bridge as unique rigid linkage. 20 In particular, the recombinant C-terminal domain of h-prune (residues 354-453, c-prune) was sufficient to bind the characterized interacting Nm23-H1 endogenous protein as verified by Western blotting analysis and nuclear magnetic resonance spectroscopy. In addition, further recent studies using a mimetic peptide, derived from the minimal interaction region of Nm23-H1 with h-prune, showed the impairment of neuroblastoma in vivo and elected it as possible therapeutic strategy.…”

“…In addition, further recent studies using a mimetic peptide, derived from the minimal interaction region of Nm23-H1 with h-prune, showed the impairment of neuroblastoma in vivo and elected it as possible therapeutic strategy. 20 Here, we report the use of gH625 for delivery of c-prune as representative of a class of intrinsically disordered proteins (IDPs). The peculiar properties of gH625 render it an optimal candidate to act as a carrier for net negatively charged molecules by comparison with the positively charged TAT.…”

“…This study has indeed been focused on the different delivery of an intrinsically disordered C-terminal domain of h-prune 20 fused to TAT and gH625. H-prune is a member of the DHH protein super-family, overexpressed in breast, colorectal, and gastric cancers.…”

“…The digested fragment was ligated to the gene encoding the C-terminal domain of h-prune (amino acid sequence 353-454, pETM11-c-prune) previously digested with the restriction enzymes NcoI and XhoI. 20 The fused segment was finally ligated to the expression vector pET28b+, which provided a cleavable N-terminal His 6 tag. The coding sequence of TAT-cprune was obtained through two consecutive PCR reactions.…”

“…20 The gH625-c-prune and TAT-c-prune were purified from inclusion bodies. Briefly, the cells were resuspended, incubated for 16 hours at room temperature in 30 mL of 50 mM Tris-HCl (pH 7.5), 8 M urea, 300 mM NaCl, and 5% glycerol (buffer A), and then sonicated to promote cell lysis.…”