Abstract
Suppressor of Fused (SUFU) is a highly conserved protein that acts as a negative regulator of the Hedgehog (HH) signalling pathway, a major determinant of cell differentiation and proliferation. Therefore, SUFU deletion in mammals has devastating effects on embryo development. SUFU is part of a multi-protein cytoplasmic signal-transducing complex. Its partners include the Gli family of transcription factors that function either as repressors, or as transcription activators according to the HH activation state. The crystal structure of SUFU revealed a two-domain arrangement, which undergoes a closing movement upon binding a peptide from Gli1. There remains however, much to be discovered about SUFU’s behaviour. To this end, we expressed recombinant, full-length SUFU from Drosophila, Zebrafish and Human. Guided by a sequence analysis that revealed a conserved potential metal binding site, we discovered that SUFU binds zinc. This binding was found to occur with a nanomolar affinity to SUFU from all three species. Mutation of one histidine from the conserved motif induces a moderate decrease in affinity for zinc, while circular dichroism indicates that the mutant remains structured. Our results reveal new metal binding affinity characteristics about SUFU that could be of importance for its regulatory function in HH.
Highlights
The evolutionarily conserved Hedgehog (HH) pathway is crucial during multiple steps of animal embryo development as it controls a number of cell functions including division, survival, migration and differentiation
Circular Dichroism (SRCD) spectra were collected at the DISCO beamline at the SOLEIL synchrotron
We discovered that Suppressor of Fused (SUFU) binds zinc in a manner not modifying its secondary structure and that one of the binding sites may involve two conserved histidines from the N-terminal domain
Summary
The evolutionarily conserved Hedgehog (HH) pathway is crucial during multiple steps of animal embryo development as it controls a number of cell functions including division, survival, migration and differentiation. There is a high-level of sequence conservation of SUFU: amino-acid sequence identity is 37.6% between Drosophila melanogaster and human, 36.3% between Drosophila melanogaster and Zebrafish (Danio rerio), and 81.2% between human and Zebrafish Despite this high sequence conservation, SUFU removal in mammals and Drosophila has very contrasting effects: in flies, loss-of-function SUFU mutants do not exhibit a notable developmental or reproduction phenotype[15], whereas SUFU deficient mice die before birth due to severe defects linked to HH upregulation[21, 22]. The dre loss-of-function mutant affects SUFU and results in a point mutation replacing Thr 111 by a lysine It triggers HH overactivation resulting in eye, ear and fin defects in the embryo and misformed gill reminiscent of lung defects in mice[23]. Drosophila SUFU can restore proper HH signalling in SUFU deficient mammalian cells[24], and murine SUFU can interact with FUSED and CI from Drosophila, and can be regulated by HH in flies[25, 26]
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