Fig. 4: In silico analyses regarding the missense p.Asp798Asn and western blot analyses. | Genetics in Medicine

Fig. 4: In silico analyses regarding the missense p.Asp798Asn and western blot analyses.

From: Haploinsufficiency of ARFGEF1 is associated with developmental delay, intellectual disability, and epilepsy with variable expressivity

Fig. 4

(a) The resolved fragments available in the RSCB Protein Data Bank are shown in the cartoon together with the position of the discussed mutation from leucine 695. (b) Alignment of the sequence region surrounding the variation in S. cerevisiae, S. pombe, C. elegans, M. musculus, D. melanogaster and H. sapiens. Sequences were fetched from NCBI RefSeq database. Fasta file combining the extracted protein sequences were then subjected to EMBOSS Clustal tool with default settings33. For mutation at position 798, the alignment shows a highly conserved region spanning on 18 amino acids in all 6 aligned sequences. The aspartic acid in position 798 represent the wild-type situation in all considered sequences including the yeast S. cerevisiae. (c) Modeling of mutation calculated in PyMol. The resolved fragment 3LTL was loaded with PyMol. The sequence spanning from proline 730 to lysine 885 was analyzed and modelled. The identied F, H and J loops of ARFGEF1 are rendered in red, orange and blue respectively. Asp798 was highlighted and is shown to probably interact with two tyrosines (Tyr825 and Tyr829) belonging to loop H (left panel). (d) Protein contact potential calculated in PyMol exhibits the charges modification when the mutation is simulated in the structure (from proline 730 to lysine 885). The top two insets show an enlargement of the aspartic acid 798 region. In the wild-type situation (WT), the area appears largely colored in red, while after simulating the Asp798Asn replacement, the same region charges are exhibiting a large channel colored in blue (dotted ovals). Similarly, 90° rotation in both horizontal and vertical directions of the structure give view on the rendered simulation of the Met884Val replacement as shown in the two bottom insets. The charge modification observed exhibits a clear shift (dotted circles). Black stars indicate the position of the amino acids subject to mutation Asp798 (top star), Met884 (bottom star). (e) western blot from individual 1 (p.(Asp798Asn)) and individuals 8 and 9’s father (p.(Gln648*)). Note the reduced levels of protein as compared to control samples (CTRL). Vinculin is used as a loading control.

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