Fig. 2

Missense variants do not alter KICS2 levels or influence KICSTOR components, but the p.Lys260Asnfs?18 variant is ubiquitinated and rapidly degraded by the UPS system (A) Western blot analysis of HEK293T cells overexpressing wild-type (WT) HA-tagged KICS2 or its variants, along with KICSTOR components FLAG-tagged SZT2 and MYC-tagged KPTN, shows a strong reduction in levels of the C-terminally truncated variant p.Lys260Asnfs?18, while missense variants p.Tyr393Cys and p.Asp296Glu do not affect KICS2 amounts. High-intensity display reveals multiple high-molecular-weight, potentially post-translationally modified forms of KICS2 p.Lys260Asnfs?18 (KICS2-fs [frameshift], marked with a dashed gray line). Overexpressed proteins were detected using tag-specific antibodies. GAPDH served as the loading control. LI, low intensity; HI, high intensity. (B) Relative quantification of HA-KICS2, FLAG-SZT2, and MYC-KPTN from n = 3 biological replicates (???p < 0.001 and ?p < 0.05, one-sample t test and two-way ANOVA followed by Tukey?s multiple comparison test). (C?E) Analysis of putatively ubiquitinated (Ub) forms of KICS2 using Ub-Trap immunoprecipitation (IP) of lysates obtained from HEK293T cells transfected with HA-tagged WT KICS2 or KICS2 p.Lys260Asnfs?18 (KICS2-fs), or an empty control vector (mock), along with MYC-tagged KPTN and FLAG-tagged SZT2. Control beads (ctrl) were used for confirming specificity. Western blot of input samples and eluates, followed by SYPRO Ruby total protein staining, shows successful purification (C). Immunodetection with a Ub-specific antibody shows precipitation of polyubiquitinated (pUb) proteins (marked with a solid black line) (D), while HA-tag-specific immunodetection (E) demonstrates the presence of Ub forms of KICS2-WT and -fs (indicated by solid and dashed gray lines, respectively). GAPDH served as the loading control. The asterisk marks an unspecific protein band present in the input. Unmodified/non-Ub HA-KICS2-WT and -fs were detectable in both the IP:ctrl and IP:Ub lanes, likely due to non-specific or indirect binding of the protein, regardless of its modification status. (F) Investigation of the proteolytic mechanism responsible for the degradation of WT KICS2 and, in particular, the KICS2 p.Lys260Asnfs?18 (KICS2-fs) variant. HEK293T cells expressing KICS2-WT and KICS2-fs, along with MYC-tagged KPTN and FLAG-tagged SZT2, were treated with the proteasome inhibitor epoxomicin (Epoxo) or autophagy inhibitor bafilomycin A1 (BafA1). DMSO served as the vehicle control. Western blot analysis confirms the accumulation of K48-linked pUb (K48-pUb) chains and LC3B-II as markers of successful proteasomal or autophagosomal inhibition, respectively. HA-tag-specific detection shows a strong accumulation of KICS2-WT and KICS2-fs, as well as their Ub forms (Ub-WT and Ub-fs), upon proteasomal, but not autophagosomal, inhibition. GAPDH served as the loading control. The asterisk marks an unspecific protein band. (G) Relative quantification of KICS2 and Ub KICS2 amounts, relative to vehicle-treated control from n = 3 biological replicates, shows significantly increased levels upon proteasomal inhibition. Calculating the ratios between Ub-KICS2 and KICS2 shows that baseline ubiquitination of KICS-fs is significantly higher compared to KICS2-WT (????p < 0.0001, ??p < 0.01, and ?p < 0.05, one-sample t test and two-way ANOVA followed by ?ídák?s multiple comparison test).