DOI: 10.5061/dryad.rbnzs7hqh
title: Interaction between the TBC1D24 TLDc domain and the KIBRA C2 domain is disrupted by two epilepsy-associated TBC1D24 missense variants

description: Mutations of human TBC1D24 are associated with deafness,
 epilepsy, or DOORS syndrome (deafness, onychodystrophy, osteodystrophy,
 cognitive disability, and seizures). The causal relationships
 between TBC1D24 variants and the different clinical
 phenotypes are not understood. Our hypothesis is that phenotypic
 heterogeneity of missense mutations of TBC1D24 results,
 in part, from perturbed binding of different protein partners. To discover
 novel protein partners of TBC1D24, we conducted yeast two-hybrid (Y2H)
 screen using mouse full-length TBC1D24 as bait. Kidney and brain protein
 (KIBRA), a scaffold protein encoded by Wwc1, was identified as a
 partner of TBC1D24. KIBRA functions in the Hippo signaling pathway and is
 important for human cognition and memory. The TBC1D24 TLDc domain binds to
 KIBRA full-length and to its C2 domain, confirmed by Y2H assays. No
 interaction was detected with Y2H assays between the KIBRA C2 domain and
 TLDc domains of NCOA7, MEAK7, and OXR1. Two epilepsy-associated recessive
 variants (Gly511Arg and Ala515Val) in the TLDc domain of human TBC1D24
 disrupt the interaction with the human KIBRA C2 domain. In the case of
 mouse constructs, Y2H assays show only mouse TBC1D24 Ala517Val
 corresponding to Ala515Val of human TBC1D24 abolished the interaction
 between mouse TBC1D24 and mouse KIBRA. This study reveals a pathogenic
 mechanism of TBC1D24-associated epilepsy, linking the TBC1D24 and KIBRA
 pathways. The interaction of TBC1D24-KIBRA is physiologically meaningful
 and necessary to reduce the risk of epilepsy.
description: This folder contains the data files for the article: Tona, R., Inagaki,
 S., Ishibashi, Y., Faridi, R., Yousaf, R., Roux, I., Wilson, E.,
 Fenollar-Ferrer, C., Chien, W.W., Belyantseva, I.A. and Friedman, T.B.
 (2024). Interaction between the TBC1D24 TLDc domain and the KIBRA C2
 domain is disrupted by two epilepsy-associated TBC1D24 missense variants.
 *Journal of Biological Chemistry*, 300, 107725.\ DOI:
 10.1016/j.jbc.2024.107725 This dataset contains previously unpublished
 data collected by the Laboratory of Molecular Genetics, National Institute
 on Deafness and Communication Disorders, National Institutes of Health,
 and associated with published data in Journal of Biological Chemistry
 (DOI: 10.1016/j.jbc.2024.107725). If you use any of the data in this
 dataset, please cite the associated publication **Description of the
 data** Yeast two-hybrid (Y2H) assays presented here show our published
 results are reproducible and verified by a separate source, Hybrigenics
 Service in France [Link](https://www.hybrigenics-services.com). The
 experimental protocol for the Y2H assays, yeast strains and sequence
 information of bait and prey constructs were described in “yeast two
 hybrid screening” in Experimental procedures and Supplementary Table S6 of
 the original paper (DOI: 10.1016/j.jbc.2024.107725). **Data** The **data**
 folder contains four files. Original data are provided by Hybrigenics
 Service. **Figure_1-2-9.pdf**: Y2H assays showing that mouse TBC1D24 TLDc
 domain interacts with mouse KIBRA C2 domain, and human TBC1D24 TLDc domain
 interacts with human KIBRA C2 domain as well. **Figure_4-S3.pdf**: Y2H
 assays using the TLDc domains of the five mouse TLDc domain-containing
 proteins and mouse KIBRA C2 domain as bait and prey, respectively, to
 indicate only the mouse TBC1D24 TLDc domain interacts with the mouse KIBRA
 C2 domain. **Figure_10A.pdf**: Y2H assay showing human TBC1D24 Arg515Val
 abolished the interaction between human TBC1D24 and human KIBRA, and human
 TBC1D24 Gly511Arg reduces the interaction between human TBC1D24 and human
 KIBRA. **Figure_10B.pdf**: Y2H assay showing only mouse TBC1D24 A517V
 abolished the interaction between mouse TBC1D24 and mouse KIBRA.
 **Files:** **File 1: Figure_1-2-9.pdf** 1. Content: one pdf file 2.
 Description: Y2H assays showing that mouse TBC1D24 TLDc domain interacts
 with mouse KIBRA C2 domain, and human TBC1D24 TLDc domain interacts with
 the human KIBRA C2 domain as well.  * Page 1: Title * Page 2:
 Abbreviations * Page 3: Summary interaction matrix and results * Page 4-5:
 Results of growth assays using mouse TBC1D24 and mouse KIBRA as bait and
 prey, respectively. Data is associated with Figures 1B and 2B of the
 original paper [Link](https://doi.org/10.1016/j.jbc.2024.107725). * Page
 5-6: Results of growth assays using human TBC1D24 and human KIBRA as bait
 and prey, respectively. Data is associated with Figure 9 of the original
 paper [Link](https://doi.org/10.1016/j.jbc.2024.107725). **File 2:
 Figure_4-S3.pdf** 1. Content: one pdf file 2. Description: Y2H assays
 using the TLDc domains of the five mouse TLDc domain-containing proteins
 and mouse KIBRA C2 domain as bait and prey, respectively, to indicate only
 the mouse TBC1D24 TLDc domain interacts with the mouse KIBRA C2 domain.  *
 Page 1: Title * Page 2: Abbreviations * Page 3: Summary interaction matrix
 and results * Page 4-6: Results of growth assays using TLDc domains of
 five TLDc domain-containing proteins and mouse KIBRA as bait and prey,
 respectively. Data is associated with Figures 4, S3D, and S3E of the
 original paper [Link](https://doi.org/10.1016/j.jbc.2024.107725). **File
 3: Figure_10A.pdf** 1. Content: one pdf file 2. Description: Y2H assays
 showing that human TBC1D24 Ala515Val abolishes the interaction with human
 KIBRA, while the human TBC1D24 Gly511Arg disrupts the interaction only
 with a higher concentration of an inhibitor, 3-amino-1,2,4-triazole.  *
 Page 1: Title * Page 2: Abbreviations * Page 3: Summary interaction matrix
 and results * Page 4: Results of growth assays using human TBC1D24 WT,
 Gly511Arg, and Ala515Val and mouse KIBRA as bait and prey, respectively. *
 Page 5-6: Results of semiquantitative Y2H analysis using human TBC1D24
 wild type (WT), G511R, and A515V and mouse KIBRA as bait and prey,
 respectively. Part of data was presented in Figure 10A of the original
 paper [Link](https://doi.org/10.1016/j.jbc.2024.107725). **File 4:
 Figure_10B.pdf** 1. Content: one pdf file 2. Description: Y2H assays using
 mouse TBC1D24 WT, Gly513Arg and Ala517Val and mouse KIBRA, as bait and
 prey, respectively. Mouse TBC1D24 Gly513 and Ala517 correspond to Gly511
 and Ala515 of human TBC1D24, respectively. Mouse TBC1D24 Ala517Val
 abolishes the interaction with mouse KIBRA in all conditions tested.
 However, mouse TBC1D24 Gly513Arg allows for an interaction with mouse
 KIBRA unlike human TBC1D24 Gly511Arg presented in Figure 10A of the
 original paper [Link](https://doi.org/10.1016/j.jbc.2024.107725). * Page
 1: Title * Page 2: Abbreviations * Page 3: Summary interaction matrix and
 results * Page 4: Results of growth assays using human TBC1D24 WT, G511R,
 and A515V and mouse KIBRA as bait and prey, respectively. * Page 5-6:
 Results of semiquantitative Y2H analysis using mouse TBC1D24 WT,
 Gly513Arg, and Ala515Val and mouse KIBRA as bait and prey, respectively.
 Part of data was presented in Figure 10B of the original paper
 [Link](https://doi.org/10.1016/j.jbc.2024.107725).