A Computer-Based Methodology to Design Non-Standard Peptides Potentially Able to Prevent HOX-PBX1-Associated Cancer Diseases.

In the last decades, HOX proteins have been extensively studied due to their pivotal role in transcriptional events. HOX proteins execute their activity by exploiting a cooperative binding to PBX proteins and DNA. Therefore, an increase or decrease in HOX activity has been associated with both solid and haematological cancer diseases. Thus, inhibiting HOX-PBX interaction represents a potential strategy to prevent these malignancies, as demonstrated by the patented peptide HTL001 that is being studied in clinical trials. In this work, a computational study is described to identify novel potential peptides designed by employing a database of non-natural amino acids. For this purpose, residue scanning of the HOX minimal active sequence was performed to select the mutations to be further processed. According to these results, the peptides were point-mutated and used for Molecular Dynamics (MD) simulations in complex with PBX1 protein and DNA to evaluate complex binding stability. MM-GBSA calculations of the resulting MD trajectories were exploited to guide the selection of the most promising mutations that were exploited to generate twelve combinatorial peptides. Finally, the latter peptides in complex with PBX1 protein and DNA were exploited to run MD simulations and the ΔGbinding average values of the complexes were calculated. Thus, the analysis of the results highlighted eleven combinatorial peptides that will be considered for further assays.

The TALE homeodomain of PBX1 is involved in human primary testis-determination.

Human sex-determination is a poorly understood genetic process, where gonad development depends on a cell fate decision that occurs in a somatic cell to commit to Sertoli (male) or granulosa (female) cells. A lack of testis-determination in the human results in 46,XY gonadal dysgenesis. A minority of these cases is explained by mutations in genes known to be involved in sex-determination. Here, we identified a de novo missense mutation, p.Arg235Gln in the highly conserved TALE homeodomain of the transcription factor Pre-B-Cell Leukemia Transcription Factor 1 (PBX1) in a child with 46,XY gonadal dysgenesis and radiocubital synostosis. This mutation, within the nuclear localization signal of the protein, modifies the ability of the PBX1 protein to localize to the nucleus. The mutation abolishes the physical interaction of PBX1 with two proteins known to be involved in testis-determination, CBX2 and EMX2. These results provide a mechanism whereby this mutation results specifically in the absence of testis-determination.

A hydrophobic residue in the TALE homeodomain of PBX1 promotes epithelial-to-mesenchymal transition of gastric carcinoma.

Pre-B-cell leukemia homeobox 1 (PBX1) was originally identified as a proto-oncogene in human leukemia. Although this protein has been shown to contribute to cellular development and tumorigenesis, the role of PBX1 in gastric carcinoma (GC) remains unclear. In this study, we observed increased expression of PBX1 in GC tissues compared with adjacent normal tissues. This increase in PBX1 expression levels negatively correlated with HOXB9 mRNA expression and was also associated with malignancy and metastasis. PBX1 promoted proliferation and metastasis of GC cells both in vitro and in vivo.These phenomena were also accompanied by epithelial-to-mesenchymal transition (EMT). Additionally, we observed that PBX1 promotes the expression of tumor growth and angiogenic factors. A structural model of the PBX1-HOX complex revealed that hydrophobic binding between PBX1 and the hexapeptide motif might be required for EMT induction. This analysis also demonstrated that the Phe252 residue in the first helix of the TALE homeodomain is involved in the latter hydrophobic binding reaction. In vitro data from PBX1 mutants suggest that PBX1 cannot promote tumorigenesis of GC cells via EMT induction when Phe252 residues lose hydrophobicity. It is likely that the presence of this residue is essential in facilitating hydrophobic binding with the hexapeptide motif. These findings suggest that PBX1 may be a potential target for GC treatment and this study provides a platform to elucidate the molecular mechanisms that underpin the role of PBX1 in GC tumorigenesis.

New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA.

PREP1 and PBX1 are homeodomain (HD) transcription factors that play crucial roles in embryonic development. Here, we present the first biophysical characterization of a PREP1 HD, and the NMR spectroscopic study of its DNA binding pocket. The data show that residues flanking the HD participate in DNA binding. The kinetic parameters for DNA binding of individual PREP1 and PBX1 HDs, and of their combination, show that isolated PREP1 and PBX1 HDs bind to DNA in a cooperative manner. A novel PREP1 motif, flanking the HD at the C-terminus, is required for cooperativity.