Alzheimer’s disease (AD) is an incurable neurodegenerative disease and cases are predicted to rise significantly in the next 30 years. The race is on to find methods to diagnose, treat, and prevent the disease, however, the root causes of AD pathogenesis remain unclear [1]. Mitochondria act as cellular batteries responsible for the generation of ATP via the fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) pathways, which are particularly important for energy-demanding neurons. Interestingly, in the brain of AD patients, amyloid-β (Aβ) peptides progressively accumulate within mitochondria and perturb Complex I (CI), the first and largest protein complex in OXPHOS. CI assembly is a complicated process, involving a range of ‘assembly factors’ responsible for integrating its 45 individual subunits and cofactors to form the functional holoenzyme. A key player is the mitochondrial CI assembly (MCIA) complex, composed of three core proteins – ACAD9, ECSIT, and NDUFAF1. How MCIA contributes to CI assembly is still unclear due to the versatile nature of the individual proteins: ECSIT participates in several signaling pathways whereas ACAD9 is a Flavin cofactor-containing redox enzyme.
We have determined the structures of ACAD9 alone and in complex with the C-terminal domain of ECSIT by an integrative biology approach [2]. Data collected at the IBS EM facility and the CM01 microscope at the ESRF have revealed the interaction site between ACAD9 and ECSITCTER, highlighting a site of functional interest (Figure 1A-B). Upon comparing the bound and unbound ACAD9 structures, a striking conformational change is observed at a site bridging the ACAD9 cofactor pocket and the ACAD9-ECSIT interaction site. A stretch of 20 residues adopts a downward-facing conformation in ACAD9 alone, acting as a barrier between external solvent and the FAD cofactor (Fig. 1A, bottom), however, upon ACAD9-ECSITCTER complex formation, an ECSIT helix induces the opening of this gatekeeper loop. This loop-flipping mechanism results in deflavination and reassigns ACAD9 from an FAO to an OXPHOS enzyme (Figure 1B, bottom) [2, 3]. In addition, we have conducted cell biology analyses that have revealed that ECSIT undergoes phosphorylation. Remarkably, we have identified an ECSIT threonine residue located at the ACAD9-ECSIT binding interface as a phosphorylation site and our biophysical analyses show that the dephosphorylation of ECSIT seems to be a prerequisite for successful MCIA formation (Figure 1C, left). Furthermore, the exposure of ECSIT to neuronal cells containing soluble Aβ oligomers (before they turn into fibrils) decreases the level of ECSIT phosphorylation (Figure 1C, middle), whereas CI activity is increased (Figure 1C, right). Therefore, our studies suggest that under early amyloidogenic conditions, there is an increased stability of a dephosphorylated MCIA complex, deactivating the fatty acid oxidising function of ACAD9 and assisting to the correct assembly of the CI holoenzyme (Figure 2). However, a sustained overactivity of CI may lead to oxidative stress, boosting the accumulation of Aβ peptides and resulting in a detrimental cycle over time, causing mitochondrial dysfunction and compromising neuronal integrity.
Overall, combining molecular and cell biology, biophysics and structural analysis, we have revealed the interactions within the MCIA subcomplex and how its assembly and activity may be regulated in the presence of amyloid toxicity. These findings will be instrumental in determining whether the MCIA proteins can be used as biomarkers for the early stages of AD.
L. McGregor (ESRF), I. Gutsche (IBS) and M. Soler Lopez (ESRF)
[1] L. McGregor and M. Soler-López (2023) Curr. Opin. Struct. Biol., 80, 102573.
[2] L. McGregor, S. Acajjaoui, A. Desfosses, M. Saïdi et al. (2023) Nat. Commun., 14, 8248.
[3] G. Giachin, M. Jessop, R. Bouverot, S. Acajjaoui et al. (2021) Angew. Chem. Int. Ed. Engl., 60, 4689-4697.
Figure 1: Cryo-EM reconstructions and atomic details of (A) ACAD9 unbound with its FAD cofactor essential for the FAO activity and (B) the ACAD9-ECSITCTER complex and the conformational arrangement of the gatekeeper loop. (C) Biophysical analyses of the MCIA complex formation upon ECSIT phosphorylation (left) and its effect on amyloid toxicity exposure in neuronal cells (right).
Figure 2: Proposed mechanism of ACAD9-ECSIT assembly and its functional implications in FAO and OXPHOS pathways.
