Identification of the frataxin-specific E3 ligase as a potential therapeutic target for Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is a rare debilitating, life-shortening, autosomal recessive inherited disease that leads to progressive damage to the nervous system. Onset is usually around the puberty and patients develop a progressive loss of motor coordination, inability to walk, slurred speech, and a cardiac hypertrophy that often leads to premature death. The particular genetic mutation – expansion of an intronic GAA triplet repeat in the FXN gene – leads to reduced expression of the mitochondrial protein frataxin involved in iron-sulfur cluster biogenesis. The subsequent frataxin insufficiency causes mitochondrial dysfunction and oxidative damage with ultimately cell death, particularly in peripheral sensory ganglia. No therapy to prevent or slow down the progression of the disease has been found yet. Since there is an inverse correlation between the amount of residual frataxin and the severity of disease progression, therapeutic approaches aiming at increasing frataxin levels are expected to improve patients’ conditions. We have recently proven the therapeutic relevance of increasing frataxin levels by preventing its degradation. Indeed, we have recently shown that a significant amount of frataxin precursor is degraded by the ubiquitin-proteasome system before its functional mitochondrial maturation and we have described the therapeutic potential of small molecules that promote frataxin accumulation by docking on the frataxin ubiquitination site, thus interfering with its ubiquitination and degradation. In light of these data, inhibition of frataxin E3 ubiquitin ligase, the enzyme responsible for frataxin ubiquitination, could represent another attractive strategy to prevent frataxin degradation. We therefore pursued the identification of such enzyme by performing a functional screening of an E3 ubiquitin ligase small interfering RNA library. HIT2 was identified and validated as a candidate from this screening. Consistently, knockdown of HIT2 promotes frataxin accumulation in cells. Most importantly, silencing of this candidate gene results in frataxin accumulation also in cells derived from FRDA patients, suggesting the therapeutic potential of strategies aimed at inhibiting this E3 enzyme. Additionally, we demonstrated that HIT2 directly interacts with frataxin and its overexpression increased frataxin protein ubiquitination in a catalytic activity-dependent manner, both in cells and in in vitro assay, indicating that this enzyme may actually represent the frataxin E3 ubiquitin ligase. These findings suggest that HIT2 could be a novel important therapeutic target for Friedreich’s ataxia.