PMC6347510_01
Clinical findings for subject with CASKL209P mutation.. A) Subject's clinical presentation. B) T2-weighted MRI brain scans from subject displaying small cerebellum (white arrow) and C) optic nerve (white arrow) at age 5 years. D) Wide field images of the retina of the right (left panel) and left (right panel) eyes show optic atrophy and arteriolar attenuation but no significant pigment migration at age 7 years. E) Results from full-field electroretinography examination. The scotopic maximal response, representing both cones and rods, is electronegative and is moderately reduced in right (top) and left (bottom) eyes at 5 years. The dashed lines indicate the normal amplitude of trough and crest.
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F1
Here we describe a 9-year-old girl with microcephaly and mild cerebellar hypoplasia with severe intellectual and motor disability (Fig. 1A,B). However, the optic nerve looked normally developed at this stage (Fig. 1C). In view of the oculodigital reflex and arteriolar attenuation, an ERG was performed that showed severe loss of rod greater than cone photoreceptors, indicating a retinal dystrophy (Fig. 1D-E).
Impact of L209P mutation on CASK structure.. A) Schematic of the domains of CASK, including locations of known interactions with other proteins, and the site of the L209P mutation. B) Conservation of the mutated site in various animal species. C) Box and whisker plots of the radius of gyration and root mean square deviation (RMSD) of the backbone of the CAMK domain of CASK or CASKL209P over the course of three molecular dynamics (MD) trajectories, respectively. D) Structure of CASK's CAMK domain, modeled from a crystal structure (3C0H.pdb), with a proline substituted for leucine at position 209 (red) in the structurally important alphaF helix (blue). Other structurally critical regions of the core protein kinase catalytic domain are also shown: alphaH helix (pink), R-spine (red space-filling residues), and C-spine (yellow space-filling residues). The activation segment is shown in green, with regions of increased mobility due to the L209P mutation as predicted by MD simulations shown in orange. E) Average B-factors (+-SEM) for the two regions (residues 166-168 and 174-176; shown in orange in panel D) within the activation segment (green, panel D) of CASK's CAMK domain that demonstrated notable differences between CASK and CASKL209P.
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F2
The substituted leucine in CASK's CaMK domain (Fig. 2A) at position 209 is highly conserved in all species surveyed (Fig. 2B), indicating that it may be a critical part of CASK's primary structure. The leucine is situated in the alphaF helix of the C-terminus lobe of the CaM-kinase domain (Fig. 2D). MD simulations were done on the isolated CaM-kinase structure (a previously solved crystal structure, an analysis of the simulations indicates that the addition of a proline at this position does not destabilize the overall structure of the isolated domain, as indicated by the nearly identical average radii of gyration (19.37398A+- 0.20033 and 19.5173A +- 0.254 for wildtype and CASKL209P, respectively) and root mean square deviation of the backbone relative to the starting structure (1.57455A +-0.31 and 1.52034A +- 0.29 for wildtype and CASKL209P, respectively) over the course of the 100 ns MD trajectories for CASKL209P structure as compared to the wildtype structure (Fig. 2C). MD simulations, by their very nature, yield millions of individual protein conformations, but most of these conformations differ from each other very little. B-factors provide a measure of mobility at a given site, and a comparison between B-factor values by sequence position revealed very few dramatic differences throughout the domain due to the L209P mutation, with the exception of increased mobility in two specific regions (Fig. 2D,E), both located in the activation loop of the CAMK domain: residues 166-168 and 174-176. Both the alphaF helix, where the L209P mutation is located, and the activation loop, which is predicted by the MD simulations to be impacted by the L209P mutation, are considered critical structural components of the core kinase domain, but these changes do not point, in isolation, to a global unfolding of CASK's CAMK domain due to the L209P residue.
Properties of GFP-CASKL209P expressed in cell culture.. A,B) Representative images of wildtype CASK and CASKL209P GFP fusion protein expressed in HEK293 cells. Note the cotton-wool aggregates of CASKL209P evident in some cells. Scale bar= 5microm. C) Quantitation of number of cells displaying large aggregations. Results represent mean and SEM from 6 different experiments. A total of ~1000 cells were examined in each sample. D) Blot for CASK in insoluble and soluble fractions of cell lysate. CASK antibody detects both expressed GFP-CASK and endogenous CASK present in HEK cells. Note that CASKL209P is predominantly present in the insoluble fraction.
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F3
Typically GFP-CASK can be visualized using a confocal microscope and is evenly distributed throughout the cytoplasm but excluded from the nucleus (Figure 3A). Upon overexpression of CASKL209P, aggregates are readily observable, indicating that the protein has a tendency to misfold and aggregate (Figure 3B). In cells where large aggregates are not observed (67%) (Figure 3C), we observe a small punctate distribution not previously seen with the other mutations that we have tested. CASK and GFP-CASK exhibited a high degree of solubility with little protein left in pellet (Figure 3D). CASKL209P, however, was less soluble, with large amounts remaining in pellet.
Neurexin-mediated recruitment assay in HEK293 cells.. Confocal images of HEK 293 cells transfected with A) GFP-CASK alone, B) GFP-CASK with FLAG-tagged neurexin1-beta, C) GFP-CASKL209P alone, and D) GFP-CASKL209P with FLAG-tagged neurexin1-beta. Scale bar= 5microm
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F4
The recruitment can be easily observed using a confocal microscope in a large, flat cell (with a high cytosolic-to-nuclear ratio) such as HEK293 cells (Figure 4B). Although CASKL209P aggregates in many cells, neurexin is still able to recruit some CASKL209P, indicating that soluble CASKL209P retains its capacity to interact with neurexin (Figure 4D).
Pull-down assays to evaluate interactions of CASKL209P with other proteins.. A) Schematic of GST pull-down from transfected HEK293 cells. B) Schematic of immunoprecipitation assay using GFP antibody. C) Immunoblot of CASK from HEK 293 cells. Endogenous CASK can be seen in GFP sample. D) Blots of indicated antigen from immunoprecipitation assay.
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F5
This finding was confirmed using a GST pull-down experiment (Figure 5A). GFP-CASK was expressed in HEK293 cells, and the lysate from these cells was combined with rat brain lysate to allow the exogenous CASK to form complexes with brain-derived proteins (Figure 5B). GFP-CASK was then immunoprecipitated with GFP antibodies, and immunoprecipitates were blotted for Veli and Mint1 (Figure 5D), revealing that CASKL209P can still bind to Veli, but its interaction with Mint1 is disrupted.