Extended Data Fig. 1: GIMAP5 mutations cause protein destabilization in patients’ T cells.
From: GIMAP5 deficiency reveals a mammalian ceramide-driven longevity assurance pathway
a, Computerized tomography scan of P7.1 showing liver nodules (left, magenta arrowheads) and esophageal varices (right, cyan arrowheads) suggesting liver failure. b, Absolute lymphocyte counts versus patient age (years, yrs), shaded area representing 95% confidence interval for normal range. c, Patient amino acid substitutions on GIMAP5 protein schematic (top). GTPase (G) domain: green; switch I (SwI) and switch II (SwII): blue; conserved box (CB): cyan; alpha helices (α) 6 and 7: orange; hydrophobic segment: maroon. Amino acid alignment in GIMAP5 orthologues and 7 human paralogues (bottom). Mutations marked in purple and boxed. d, Omit Fo-Fc density map for GDP and the Mg2+ ion bound to GIMAP51-276, contoured at 3σ (green). The refined GDP•Mg2+ molecules shown in ball-and-stick. e, Comparison of GIMAP51-276-GDP (left), GIMAP21-260-GDP (middle, PDB code 2xto), and GIMAP7 L100Q-GMPPNP (right, PDB code 3zjc). G domains: green; switch I (SwI) and switch II (SwII): purple; P-loops (PL): red; conserved box (CB): cyan; nucleotides: space-filling spheres; secondary structure elements differing from the core G domain of H-Ras, helices α3* and the C-terminal helices α6 and α7 (C-terminal extension, ext.): orange; unstructured loops; dotted lines. Root mean square deviation of aligned Cα atoms: 1.4 Å (GIMAP5 vs. GIMAP2) and 2.0 Å (GIMAP5 vs. GIMAP7). f, Thermal denaturation profiles. GST- WT GIMAP51-276 displayed two temperature transitions - GIMAP51-276 unfolding (red arrow) and GST unfolding (blue arrow). GST-GIMAP51-276 variants (left: I47T, L204P and N221S; right: L223F) showed a single transition, representing GST unfolding, with no WT GIMAP51-276 transition. Left and right experiments were conducted in separate laboratories. g, Single-cell GIMAP5 RNA expression in human peripheral blood mononuclear cells (PBMCs). h, Immunoblots of GIMAP5 in primary human CD19+ B cells, CD14+ monocytes, CD56+ NK cells, and T cells with β-actin loading control (n = 3). i, Immunoblots of GIMAP5 from patients (P), family, normal control (NC) or shipping control (TC) T cell lysates with a b-actin or an HSP90 loading control (n = 3). j, GIMAP5 RNA expression by RT-qPCR in patients (n = 6) and controls (n = 6) normalized to 18S. Samples were examined over at least 3 independent experiments (mean ± s.d.).
