Neurodevelopmental disorder mutations in the exchange factor DENN/MADD disrupt activation of Rab GTPases

Abstract

DENN/MADD (mitogen-activated protein kinase-activating death domain), a differentially expressed in normal and neoplastic cells (DENN) domain-containing protein functions in membrane trafficking. DENN domain-bearing proteins have guanine nucleotide exchange factor activity toward Rab GTPases. Here, we identify Rab GTPase substrates for DENN/MADD using a cell-based assay involving DENN domain-mediated recruitment of Rab substrates to mitochondria. We confirmed known interactions of DENN/MADD with Rab3A, Rab3B, Rab3C, Rab3D, and Rab27B and identified four new potential substrates, Rab8B, Rab15, Rab26, and Rab37, results confirmed with biochemical experiments. Mutations in the DENN domain of DENN/MADD result in diverse pathophysiological manifestations, ranging from predominant neurological dysfunction to a multisystem disorder. Structural analysis using AlphaFold suggested that these mutations affect DENN/MADD's interaction with Rab GTPases. Introducing such mutations into DENN/MADD's DENN domain influenced the mitochondrial recruitment of Rabs. This study identifies new DENN/MADD protein interactions and cellular pathways, the disruption of which results in human disorders.

Keywords: DENN/MADD; Rab GTPases; cell biology; genetic disease; guanine nucleotide exchange factor; imaging; membrane trafficking; neurodevelopmental disorder; protein–protein interaction.

Figure 1

Cell-based mitochondrial recruitment assay.A, schematic model of the cell-based assay. This model is adapted from Rahul et al. and generated via BioRender. B, tabular representation of all the screened and identified Rab GTPases via mitochondrial recruitment assay.

Figure 2

Mitochondrial-targeted DENN/MADD recruits corresponding Rab GTPases. HeLa cells cotransfected with GFP-Rabs and mito-mScarlet (A) or mito-mScarlet-DENN/MADD (B). Scale bars represent 10 μm. C, quantification of Rab GTPase colocalization with mito-mScarlet and mito-mScarlet-DENN/MADD using Pearson's correlation coefficient measuring cells from three independent experiments (n = 25) per condition; means ± SEM; unpaired t test (for Rab3A, Rab3C, Rab26, Rab27B, and Rab37), Welch t test (for Rab8B) and Mann–Whitney U test (Rab3B, Rab3D and Rab15); ∗∗∗∗p < 0.001. DENN, differentially expressed in normal and neoplastic cells domain; MADD, mitogen-activated protein kinase–activating death domain.

Figure 3

DENN/MADD preferentially interacts with the inactive (TN) Rab mutants. Interaction of DENN/MADD with QL and TN mutants of the identified Rabs via coimmunoprecipitation. HEK-293T cells were cotransfected with FLAG-DENN/MADD and GFP alone (control/ctrl) or GFP-Rab QL/TN. At 24 h post-transfection, cells were lysed and incubated with FLAG antibody. Bound proteins were identified through immunoblotting using either an anti-GFP antibody to identify active/inactive Rabs or an anti-FLAG antibody that recognizes DENN/MADD. Five percent of the lysate used for coimmunoprecipitation was loaded as starting material (SM). DENN, differentially expressed in normal and neoplastic cells domain; HEK-293, human embryonic kidney 293 cell line; MADD, mitogen-activated protein kinase–activating death domain.

Figure 4

Cell-based assay reveals disruption of Rab GTPase recruitment because of the mutation in the conserved catalytic residue of DENN/MADD’s DENN domain. HeLa cells were cotransfected with mito-DENN/MADD (A) or mito-scarlet DENN/MADD P372L (B) and GFP Rabs. Scale bars represent 10 μm. C, quantification of Rab GTPase colocalization with mito-mScarlet-DENN/MADD and mito-scarlet DENN/MADD P372L using Pearson's correlation coefficient measuring cells from three independent experiments (n = 25) per condition; means ± SEM; unpaired t test (for Rab3A, Rab8B, and Rab15), Welch's t test (for Rab3C, Rab3D, Rab27B, and Rab37) and Mann–Whitney U test (for Rab3B and Rab26); ∗∗∗∗p < 0.001. DENN, differentially expressed in normal and neoplastic cells domain; MADD, mitogen-activated protein kinase–activating death domain.

Figure 5

Cell-based assay reveals disruption of Rab GTPase recruitment because of the mutation in the unconserved residue of DENN/MADD’s DENN domain. HeLa cells were cotransfected with mito-mScarlet DENN/MADD (A) or mito-mScarlet DENN/MADD L346P (B) and GFP Rabs. Scale bars represent 10 μm. C, quantification of Rab GTPase colocalization with mito-mScarlet-DENN/MADD and mito-mScarlet DENN/MADD L346P using PCC measuring cells from three independent experiments (n = 25) per condition; means ± SEM; unpaired t test (for Rab3D, Rab8B, and Rab27B), Welch's t test (for Rab3A, Rab15, and Rab37) and Mann–Whitney U test (for Rab3B, Rab3C, and Rab26); ∗∗∗∗p < 0.001. DENN, differentially expressed in normal and neoplastic cells domain; MADD, mitogen-activated protein kinase–activating death domain; PCC, Pearson's correlation coefficient.

Figure 6

Structural analysis of DENN/MADD in complex with Rab GTPases.A, structural model of DENN/MADD in complex with Rab3C bound to GDP–Mg²⁺ generated by AlphaFold 3. The side chains of Leu346 and Pro372 are shown as spheres. B, modeling of the P372L mutation. C, modeling of the L346P mutation. D, superposition of Rab3A, Rab3B, Rab3C, and Rab3D bound to GDP-Mg²⁺ in complex with DENN/MADD. E, superposition of Rab8B, Rab15, Rab26, Rab27, and Rab37 bound to GDP–Mg²⁺ in complex with DENN/MADD. DENN, differentially expressed in normal and neoplastic cells domain; MADD, mitogen-activated protein kinase–activating death domain.