Progetto CRYinEYE

  • Progetti di eccellenza - bando 2011 - 2012 Fondazione CARIPARO
  • CRYinEYE: Fly cryptochrome and the visual system: a link with the mammalian eye?

Cryptochromes (CRYs) are flavoproteins involved in development and temporal organization of a variety of organisms [1,2]. Drosophila CRY (dCRY) mediates light synchronization of the master circadian clock and is an integral component of the main feedback loop of the circadian clocks located in peripheral tissues. We suggested that dCRY C-terminus is a potential hotspot for molecular interactions as it harbours several "Linear Motifs" (LMs) important for protein-protein interaction[3]. We identified a specific LM which mediates interactions with proteins containing class III PDZ domains. In silico analyses suggested a number of PDZ containing proteins as putative partners of dCRY. We performed a co-immunoprecipitation assay, followed by mass spectrometry analysis (Mazzotta et al, in preparation), which revealed that dCRY is bound to NINAC, an element of the fly's visual cascade assembled in a multiprotein signaling complex (Signalplex)[4] organized by INAD, a scaffolding protein with five structural PDZs[5]. A Yeast Two Hybrid assay allowed us to prove the CRY-INAD interaction and identify the subset of PDZ motifs involved (PDZ 2-3 domains plus a short N-terminal extension). Moreover, dCRY mutants (cry0 and cryM) showed impaired optomotor behaviour, which implies the existence of abnormalities in the visual pathway (Mazzotta et al, in preparation). The presence of dCRY associated with the visual cascade complex could underline a role for this molecule in fly vision which has, as yet, not been entertained. In mammals, “intrinsically photosensitive retinal ganglion cells“ (ipRGCs) use melanopsin as a photopigment and send their axons directly to the suprachiasmatic nucleus (SCN)[6], the site of the circadian clockwork. Because the ipGRCs and the fly phototransduction mechanisms share several signaling similarities[7,8], we wonder whether mammalian CRYs (expressed in ipRGCs) may also serve as a component of a mammalian Signalplex.

Study objectives and expected results:

  • (A) In silico identification of putative dCRY LMs which could mediate interactions with other specific proteins involved in phototransduction and circadian regulation. Extension of the analysis to mammalian CRYs.
  • (B) Validation of the predicted interactions by yeast two/three-hybrid assays and Co-immunoprecipitation from Drosophila overexpressing dCRY. In vivo functional characterization of the interactions. Extension of the molecular analysis to mammals.
  • (C) Characterization of the structural basis for the dCRY/INAD interaction.  Assignment  of  backbone  resonances  of  the  INAD  fragment  essential  for  dCRY recognition. Identification of the ligand binding epitope and the critical residues responsible for the interaction.

Principal Research Institution

Keywords

light photoreceptors, circadian clocks, visual cascade, bioinformatics, protein interactions, NMR

Partnership

  • Dipartimento di Biologia, Università di Padova: in silico determination, molecular and functional characterization of LM interactions.
  • Dipartimento di Scienze Chimiche, Università di Padova: characterization of the structural basis for the interaction between dCRY and INAD.