Discovery and functional analysis of a novel ALPK1 variant in ROSAH syndrome

Abstract

Retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and migraine headache (ROSAH) syndrome is an autosomal dominant disorder and to date is known to be caused by either the Thr237Met or Tyr254Cys variant in the protein kinase ALPK1. Here, we identify a family in which ROSAH syndrome is caused by a novel variant in which Ser277 is changed to Phe. All six patients examined display ocular inflammation and optic nerve elevation, four have retinal degeneration and four are registered blind. In contrast to wild-type ALPK1, which is activated specifically by bacterial ADP-heptose, ALPK1[Ser277Phe] is also activated by the human metabolites UDP-mannose and ADP-ribose and more strongly than the most frequent ROSAH-causing variant (ALPK1[Thr237Met]) but, unlike ALPK1[Thr237Met], ALPK1[Ser277Phe] is also activated by GDP-mannose. These observations can explain why ALPK1 variants causing ROSAH syndrome display constitutive activity in human cells. The side chains of Ser277 and Tyr254 interact in the crystal structure of ALPK1, but mutational analysis established that it is not the loss of this hydrogen bond between Ser277 and Tyr254 that alters the specificity of the ADP-heptose-binding pocket in the Ser277Phe and Tyr254Cys variants. The characterization of ALPK1 variants that cause ROSAH syndrome suggests ways in which drugs that selectively inhibit these disease-causing variants may be developed.

Keywords: ADP-heptose; ALPK1; ROSAH; TIFA; UDP-mannose; nucleotide sugar.

Figure 1.

Family expressing the ALPK1[Ser277Phe] variant. Individual II1 and individual II2 have the same mother but different fathers. The daughters of individual II1 are monozygous twins. The patients that have been analysed clinically are marked by an asterisk, and the ages at which they were last evaluated are given in parentheses.

Figure 2.

The ALPK1[Ser277Phe] mutant stimulates NF-κB/AP-1-dependent gene transcription in the absence of ADP-heptose. (a) ALPK1 KO cells were transfected with plasmids encoding WT ALPK1 or the indicated ALPK1 mutants. Twenty-four hours later, cells were incubated with (blue bars) or without (grey bars) 5 μM ADP-heptose (ADP-H) and NF-κB/AP-1-dependent gene transcription was measured after another 24 h (see §2). (b) ALPK1 KO or ALPK1/TIFA double KO (DKO) cells were transfected with the plasmids indicated and analysed as in (a). (c) As in (a) but using plasmids in which each ROSAH-causing mutant was combined with Arg150Ala, which disrupts the ADP-heptose binding. (a–c) Results are shown as the mean of an experiment performed in triplicate, where individual values are denoted by filled circles and the error bars indicate plus and minus one standard error of the mean. Similar results were obtained in two additional, independent experiments.

Figure 3.

ALPK1[Ser277Phe] is activated by mammalian nucleotide sugars in cell-free phosphorylation assays. (a) FLAG-tagged WT ALPK1 (WT, grey bars), ALPK1[Thr237Met] (T237M, blue bars) or ALPK1[Ser277Phe] (S277F, pink bars) were immunoprecipitated from cell extracts and assayed for 30 min in the absence or presence of 5 µM ADP-heptose (ADPH) or 100 µM UDP-α-d-mannose (UDPM), ADP-d-ribose (ADPR) or GDP-α-d-mannose (GDPM), and the amount of phosphate (in pmol) incorporated into GST-TIFA plotted (see §2). (b) As in (a), except comparing ALPK1[Ser277Phe] (S277F, pink bars) and ALPK1[Ser277Phe/Arg150Ala] (S277F/R150A, unfilled bars). (a,b) Two independent experiments were performed, each in duplicate. The results from each individual experiment were averaged and are represented by the filled circles. The bar heights reflect the mean of these two values.

Figure 4.

Interactions between Ser277 and Tyr254, and between Thr237 and ADP-heptose, within the ADP-heptose binding domain of ALPK1. (a) Location of Ser277, Tyr254, Thr237 and Arg150 in the ADP-heptose (ADP-H) binding domain of ALPK1 (PDB: 5z2c). Thr237, Tyr254, Ser277 and Arg150 are shown in stick representation and coloured by element (carbon: bronze; nitrogen: blue; oxygen: red). ADP-H is also shown in stick representation and coloured similarly (except carbon: green; phosphate: orange). The interactions between Thr237 and ADP-H, between Arg150 and ADP-H, and between Tyr254 and Ser277 are shown by black broken lines. (b,c) ALPK1 KO cells were transfected with plasmids encoding WT ALPK1 or the indicated ALPK1 mutants and 24 h later incubated with (blue bars) or without (grey bars) 5 μM ADP-H. The activation of NF-κB/AP-1-dependent gene transcription was then measured after a further 24 h as in figure 2 (see §2). Results are shown as the mean of an experiment performed in triplicate, where individual values are denoted by filled circles and the error bars indicate plus and minus one standard error of the mean. Similar results were obtained in two additional, independent experiments. (d) FLAG-tagged WT ALPK1 (WT, grey bars), ALPK1[Thr237Ala] (T237A, blue bars), ALPK1[Tyr254Phe] (Y254F, yellow bars) and ALPK1[Ser277Ala] (S277A, pink bars) were immunoprecipitated from cell extracts and assayed and plotted as in figure 3. Two independent experiments were performed, each in duplicate. The results from each experiment were averaged and represented by filled circles. The bar heights reflect the mean of these two values.