1. Fig. 5. Annexin A11 mutations disrupt binding to calcyclin.
Annexin A11 mutations disrupt binding to calcyclin. (A) Western blot demonstrating lack of binding of FLAG-tagged calcyclin to GFP-tagged mutant annexin A11 (ANXA11D40G, ANXA11G189E, and ANXA11R235Q) compared to ANXA11WT after IP (n = 3). Top: GFP intensities of input and immunoprecipitated fractions, with immunoglobulin G (IgG) heavy [50 kDA (*)] and light [25 kDa (**)] chains indicated. Bottom: Input and IP of FLAG-tagged calcyclin alone. Ctrl, control. (B) Cross section of postmortem spinal cord tissue showing calcyclin expression in (i) controls and (ii) a SALS case with the p.D40G mutation. High calcyclin expression can be seen in the lateral corticospinal tracts (black arrows). (iii) SALS case with no known ALS causing mutation but displaying strong expression of calcyclin in the lateral corticospinal tracts in postmortem spinal cord (black arrows). Scale bars, 40 μm (i), 30 μm (ii), and 25 μm (iii). (C) To determine the effect of calcyclin overexpression on aggregation, we conducted an NP-40 solubility assay by coexpressing 500 ng of GFP-tagged ANXA11WT or ANXA11R235Q and 500 ng of FLAG-tagged calcyclin in HEK cells. Clearance of insoluble p.R235Q mutant annexin A11 aggregates by calcyclin was observed. Addition of the proteasomal inhibitor MG132 restored the aggregation event by blocking degradation of the aggregated proteins by the ubiquitin proteosomal pathway. An equivalent amount of empty GFP vector (500 ng) was added to HEK cells expressing WT or p.R235Q mutant annexin A11 as a loading control. (D) Quantification of the proportion of insoluble fractions from (C) relative to untreated ANXA11GFP (WT) including calcyclin overexpression (+C) and calcyclin overexpression plus MG132 treatment (+C + M). Bands were quantified with ImageJ, and intensity was calculated with respect to untreated GFP-tagged ANXA11WT. A one-way ANOVA test demonstrated significance for increased aggregation due to the p.R235Q mutation and rescue of aggregation in p.R235Q mutant cells by calcyclin overexpression and MG132 treatment (R235Q + C + M). *P < 0.05 and ***P < 0.001, respectively, one-way ANOVA and Dunnett’s post hoc test (n = 3).
Bradley N. Smith et al., Sci Transl Med 2017;9:eaad9157
Copyright © 2017, American Association for the Advancement of Science