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Dev Growth Differ
2023 May 01; doi: 10.1111/dgd.12852.
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Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.
Okano K
,
Otsuka H
,
Nakagawa M
,
Okano T
.
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Cryptochromes (CRYs) are multifunctional molecules that act as a circadian clock oscillating factor, a blue light-sensor, and a light-driven magnetoreceptor. Cry genes are classified into several groups based on the evolutionary relationships. Cryptochrome 6 gene (Cry6) is present in invertebrates and lower vertebrates such as amphibians and fishes. Here we identified a Cry6 orthologue in Xenopus tropicalis (XtCry6). XtCRY6 retains a conserved long N-terminal extension (termed CRY N-terminal extension; CNE) that is not found in any CRY in the other groups. A structural prediction suggested that CNE contained unique structures; a tetrahelical fold structure topologically related to KaiA/RbsU domain, overlapping nuclear- and nucleolar-localizing signals (NLS/NoLS), and a novel motif (termed DI-UIM) overlapping a double-sided ubiquitin-interacting motif (DUIM) and an inverted ubiquitin-interacting motif (IUIM). Potential activities of the NLS/NoLS and DI-UIM were examined to infer the molecular function of XtCRY6. GFP-NLS/NoLS fusion protein exogeneously expressed in HEK293 cells was mostly observed in the nucleolus, while GFP-XtCRY6 was observed in the cytoplasm. A glutathione S-transferase (GST) pull-down assay suggested that the DI-UIM physically interacts with polyubiquitin. Consistently, protein docking simulations implied that XtCRY6 DI-UIM binds two ubiquitin molecules in a relationship of a two-fold rotational symmetry with the symmetry axis parallel or perpendicular to the DI-UIM helix. These results strongly suggested that XtCRY6 does not function as a circadian transcriptional repressor and that it might have another function such as photoreceptive molecule regulating light-dependent protein degradation or gene expression through a CNE-mediated interaction with ubiquitinated proteins in the cytoplasm and/or nucleolus. This article is protected by copyright. All rights reserved.
FIGURE 1
Phylogenetic analysis and molecular architectures of CRYs in X. tropicalis. (a) A molecular phylogenetic tree (NJ tree) of CRYs and photolyases. Values at the branches indicate bootstrap probabilities. Each protein is shown with combining the abbreviated species name (m, mouse; c, chicken; z, zebrafish; d, Drosophila [fruit fly]) or scientific name (Ag, Anopheles gambiae; At, Arabidopsis thaliana; Bm, Bombyx mori; Cg, Crassostrea gigas [oyster]; Cr, Chlamydomonas reinhardtii; Dp, Danaus plexippus plexippus; Ec, Escherichia coli; Tr, Takifugu rubripes [pufferfish]; Xt, X. tropicalis; Xl, X. laevis) and protein name followed by its accession ID. Alignment of N- and C-terminally deleted sequences (corresponding to Asp244-Tyr691 of XtCRY6) are shown in Figure S2. (b) Domain structures of X. tropicalis CRYs. Amino acid positions shown are start of domains/motifs and start/end of the protein. DI-UIM, double-sided/inverted ubiquitin-interacting motif; NLS, nuclear localization signal; NoLS, nucleolar localization signal
FIGURE 2
Quantitative RT-PCR analysis of tissue and temporal expression of Cry6 in Xenopus tropicalis. Each tissue was collected at ZT0-ZT1 and ZT11-ZT12. XtCry6 mRNA level was calculated as a value relative to that of the XtHprt1 and XtGusb gene. Error bars represent ± SD. XtCry6 primer pair set A (above); XtCry6 primer pair set B (below). Primer sequences are presented in Table 1
FIGURE 3
Cellular localization of GFP-fused XtCRY6 and XtCRY6NLS/NoLS expressed in HEK 293 cells. Expression vector (pcDNA-GFP-XtCRY6 or pcDNA-GFP-XtCRY6NLS/NoLS or pcDNA-GFP-XtCRY1 or pcDNA-GFP-SV40NLS) was transfected into HEK 293 cells. After 24 h, the cells were treated Hoechst 33342 (Dojindo), and observed using a fluorescence microscope for detection of GFP (left, GFP) and nuclei (right, Hoechst) or a differential interference microscope (middle, DIC, GFP signals are overlaid). Scale bars, 20 μm
FIGURE 4
Comparison of DUIM and IUIM in various ubiquitin-interacting proteins and CRY6 proteins. Amino acid sequences of putative DI-UIM of CRY6s were compared with DUIM (upper and middle alignments, UIM1 and UIM2, respectively) and IUIM (lower alignments). Consensus motifs (Penengo et al., 2006) are shown in the top of alignments, and conserved residues are colored in yellow
FIGURE 5
GST pull-down assay. GST-fusion protein (GST-XtCRY6DI-UIM_WT, GST-XtCRY6DI-UIM_SFL, GST-Rabex5_IUIM) was subjected to in vitro pull-down assay using K48Ub2–8 (GST pull-down; lanes 5–9), and the precipitant corresponding to 1.8 μg of input GST-fusion protein was loaded in each lane. Loaded as an input was 1.8 μg of each GST-fusion protein (Input; lanes 2–4, and 10–12). Antibodies used are anti-ubiquitin antibody (upper panel) and anti-GST antibody (lower panel)
FIGURE 6
Predicted molecular structures of XtCRY6. (a) Plots of pLDDT (upper) and pAE (lower) scores from structural prediction of full-length XtCRY6. Red bars above the pLDDT plot indicate two regions subjected to the molecular modeling shown in panels b and c. (b) A predicted structure of N-terminal region of XtCRY6 (a part of the CNE; Met1-Ala105). (c) A predicted structure of PHR domain and a part of CCE region of XtCRY6 (Pro182-Gle829). (d) 3D structure of KaiA from PCC7120 based on a crystal structure analysis (PDB accession code 1R5Q)
FIGURE 7
Predicted structures of XtCRY6 DI-UIM binding two ubiquitin molecules. (a) A predicted structure (ClusPro-A model) of XtCRY6 DI-UIM binding two ubiquitin molecules being related by twofold rotational symmetry with the symmetry axis in parallel to the DI-UIM helix. (b) A structure of Bos taurus Hrs DUIM binding two ubiquitin molecules (Hirano et al., 2006) being related by twofold rotational symmetry with the symmetry axis in parallel to the DUIM helix (PDB accession code 2D3G). (c) A predicted structure (ClusPro-B model) of XtCRY6 DI-UIM with two ubiquitin molecules that are related by twofold rotational symmetry with the symmetry axis being perpendicular to the DI-UIM helix. (d) A ColabFold-predicted structure of XtCRY6 DI-UIM with two ubiquitin molecules interacting in inverse directions along the helix. (e) 3D structure of human Rabex-5 based on a crystal structure analysis (PDB accession code 2C7M)
