Abstract
Nitrilase1 was classified as a tumour suppressor in association with the fragile histidine-triad protein Fhit. However, knowledge about nitrilase1 and its tumour suppressor function is still limited. Whereas nitrilase1 and Fhit are discrete proteins in mammals, they are merged in Drosophila melanogaster and Caenorhabditis elegans. According to the Rosetta-Stone hypothesis, proteins encoded as fusion proteins in one organism and as separate proteins in another organism may act in the same signalling pathway. Although a direct interaction of human nitrilase1 and Fhit has not been shown, our previous finding that Fhit interacts with β-catenin and represses its transcriptional activity in the canonical Wnt pathway suggested that human nitrilase1 also modulates Wnt signalling. In fact, human nitrilase1 forms a complex with β-catenin and LEF-1/TCF-4, represses β-catenin-mediated transcription and shows an additive effect together with Fhit. Knockdown of human nitrilase1 enhances Wnt target gene expression. Moreover, our experiments show that β-catenin competes away human nitrilase1 from LEF-1/TCF and thereby contributes to the activation of Wnt-target gene transcription. Inhibitory activity of human nitrilase1 on vertebrate Wnt signalling was confirmed by repression of Wnt-induced double axis formation in Xenopus embryogenesis. In line with this finding, the Drosophila fusion protein Drosophila NitFhit directly binds to Armadillo and represses the Wingless pathway in reporter gene assays. Genetic experiments confirmed the repressive activity of Drosophila NitFhit on Wingless signalling in the Drosophila wing imaginal disc. In addition, colorectal tumour microarray analysis revealed a significantly reduced expression of human nitrilase1 in poorly differentiated tumours. Taken together, repression of the canonical Wnt pathway represents a new mechanism for the human nitrilase1 tumour suppressor function.
Highlights
Human nitrilase1 was recognized during the characterization of the human fragile histidine triad (Fhit) tumour suppressor protein, when it was realizedNit1 represses Wnt/β-catenin signalling dimeric structure was previously reported for the human Fhit protein [3]
To test whether Human nitrilase1 (hNit1) can form a complex with β-catenin, co-immunoprecipitation experiments were performed in HEK-293 cells transiently transfected with β-catenin-FLAG and hNit1-myc6
A strong correlation exists for loss of hFhit expression and overall survival, both in adenocarcinoma and squamous cell cancers
Summary
Human nitrilase (hNit1) was recognized during the characterization of the human fragile histidine triad (Fhit) tumour suppressor protein, when it was realizedNit represses Wnt/β-catenin signalling dimeric structure was previously reported for the human Fhit protein [3]. Fhit and Nit show similar expression patterns [4]. Based on these observations, Fhit and Nit were defined as Rosetta-Stone proteins [5] with a postulated common tumour suppressive function, a direct interaction of both proteins has not been shown till now. In contrast to Fhit, little is known about the function of Nit and interaction partners have not been studied. Branch 1 enzymes are known to have specific nitrilase activity performing nitrile hydrolysis, whereas most other branches have apparent or postulated amidase or amide condensation activities [6]. Mammalian Nit was reported to cleave several synthetic dipeptide substrates, but has no amidase activity as reported for mammalian nitrilase2 [7, 8]. No physiological substrate of Nit is known
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