Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity

Abstract

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

Figure 1. Coq8a^−/− Mice Develop Cerebellar Ataxia and Mild Exercise Intolerance

(A) Immunoblot analysis of COQ8A abundance in 30-week-old mice. (B) Accelerating rotarod retention times of 10-week-old mice (mean ± SEM, n = 8–12). ANOVA test P<0.05. (C) Left, representative footprints of 10-week-old mice. Right, “linearity coefficient”, which is proportional to non-linear movement (mean ± SEM, n = 8–12), *P<0.05, **P<0.01. (D) Number of hind limb coordination errors (mistakes) by mice on beam test (mean ± SEM, n = 8–12), *P<0.05, **P<0.01, ***P<0.01. (E) Hematoxylin and eosin (H&E) (upper) and Calbindin (lower) staining of cerebellar sections of 15- and 40-week-old mice, respectively. *Shrunken Purkinje neurons; scale bar, 20 μm. (F) Electron microscopy of 30-week-old cerebella. G, Golgi apparatus; *dilated Golgi; scale bars, 5 μm upper, 1 μm lower. (G) Electrophysiological recordings of mouse Purkinje cells. ISI and CV2 (mean ± SEM) (n = 3, > 40 cells/mouse); ** Mann-Whitney U test P<0.01. (H) Maximum speed (upper) and duration (lower) on treadmill test by 10-month-old mice (mean ± SEM, n = 8), *P<0.05. (I) H&E stained skeletal muscle sections of 7-month-old mice. Scale bar, 50 μm. (J) Electron microscopy of quadriceps showing broken mitochondria with collapsing cristae (arrows). Upper: 7-month-old mice, scale bar, 2 μm. Lower: 15-month-old mice, scale bar, 1 μm. See also Figure S1.

Figure 2. Loss of COQ8A or Coq8p Causes Deficiency of Complex Q and CoQ

(A) CoQ₉ content in 7-month-old mice (mean ± SD, n = 6). *P<0.05, **P<0.01, ***P<0.001. (B) Fold changes in mouse lipid abundances (log₂(Coq8a^−/−/WT), n = 4) versus statistical significance (−log₁₀(p-value)) as quantified by LC-MS for quadriceps, cerebellum, and serum. (C) Fold changes in mouse protein abundances (log₂(Coq8a^−/−/WT), n = 4) versus statistical significance as quantified by LC-MS/MS for cerebellum, quadriceps, heart, and kidney. (D) Expression of COQ7 in muscles of 10-week-old mice (E) Expression of COQ7 in myoblasts from 7–9-day-old pups (F) Fold changes in yeast protein abundances (log₂(Δcoq8/WT), n = 3) across the diauxic shift (Figure S2E) versus statistical significance as quantified by LC-MS/MS See also Figure S2

Figure 3. COQ8A Interacts with Complex Q but Lacks Protein Kinase Activity in trans

(A) Immunoblot (IB) analysis of interactions between COQ8A-FLAG and COQ5-HA, COQ3-HA, or COQ9-HA transfected into COS cells and immunoprecipitated (IP’d) using anti-FLAG beads. (B) Heatmap showing the top 25 endogenous proteins most enriched by COQ8A-FLAG IP’d from HEK293 cells compared to various control IPs (mean, n = 4). (C) Relative abundances of endogenous proteins co-purifying with COQ8A-FLAG compared to MLS-GFP-FLAG (mean, n = 4) IP’d from HEK293 cells as assessed by LC-MS/MS. (D) Cartoon of COQ8 active site residues (based on PDB 4PED). (E) Relative abundances of endogenous COQ proteins co-purifying with COQ8A-FLAG (log₂(mutant/WT)) IP’d from HEK293 cells as assessed by LC-MS/MS. (F) SDS-PAGE analysis of in vitro Mg[γ-³²P]ATP autophosphorylation reactions with Coq8^NΔ41 variants or PKA. BSA, bovine serum albumin (reaction buffer component). (G) Divalent cation dependence of Coq8^NΔ41 autophosphorylation. (H) Time course of Coq8^NΔ41 autophosphorylation. (I) Coq8^NΔ41 autophosphorylation sites identified by LC-MS/MS mapped onto a homology model of Coq8p (based on COQ8A structure, PDB 4PED). (J) SDS-PAGE analysis of in vitro Mg[γ-³²P]ATP autophosphorylation reactions with combinations of Coq8^NΔ41 variants. MBP, maltose binding protein tag. (K) SDS-PAGE analysis of in vitro Mg[γ-³²P]ATP kinase reactions with PKA or Coq8^NΔ41 and potential substrate proteins. See also Figure S3.

Figure 4. COQ8A and Coq8p Exhibit Unorthodox PKL Activities

(A) k_cat and k_cat/K_m values for the ATPase activity of Coq8^NΔ41 variants measured by observing production of phosphate (mean ± SD, n = 3), *P<0.05. (B) Relative protein abundances in Δcoq8 yeast transformed with the indicated coq8 (Coq8p) variants as quantified by label free quantitation LC-MS/MS analysis (mean ± SD, n = 4). (C) Immunoblot analysis of COQ8A localization in submitochondrial fractions. Mitochondrial markers: COQ7 (inner membrane, peripheral), CYC1 (intermembrane space), NFU1 (matrix), Prohibitin (inner membrane, integral), and TOM20 (outer membrane, integral). Supe., supernatant. (D) Heatmap showing enrichment of lipids co-purifying with MBP-Coq8^NΔ41 variants compared to the bacterial lysate from which the proteins were purified as assessed by LC-MS (mean, n = 6). (E) Relative abundances (mutant/WT) of CoQ biosynthesis intermediates (R, polyisoprenyl tail) co-purifying with MBP-Coq8^NΔ41 variants as assessed by LC-MS (mean ± SD, n = 6). See also Figure S4.

Figure 5. Structure and Dynamics of Nucleotide-Bound COQ8A

(A) Surface representations of COQ8A^NΔ254 R611K bound to AMPPNP (PDB 5I35), colored by domains (see Figure 3I). Two hydrophobic pockets (KxGQ pockets 1 and 2) are highlighted. (B) Surface representation of apo COQ8A^NΔ254 (PDB 4PED). (C) Surface representation of PKA (PDB 1ATP) (Knighton et al., 1991). (D) Representative snapshots of MD analyses of COQ8A showing interactions of D488 with either R611 or the KxGQ motif. Left, apo; Middle and right, MgATP-bound. See also Figure S5.

Figure 6. A Model for the Molecular Basis of COQ8 Biology

(A) Models for the molecular activity of COQ8 (COQ8A/B and Coq8p). (B) Models for COQ8A biology and its disruption in ARCA2.