E3 Ligase RNF126 Directly Ubiquitinates Frataxin, Promoting Its Degradation: Identification of a Potential Therapeutic Target for Friedreich Ataxia
Friedreich ataxia (FRDA) is a severe genetic neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. To date, there is no therapy to treat this condition. The amount of residual frataxin critically affects the severity of the disease; thus, attempts to restore physiological frataxin levels are considered therapeutically relevant. Frataxin levels are controlled by the ubiquitin-proteasome system; therefore, inhibition of the frataxin E3 ligase may represent a strategy to achieve an increase in frataxin levels. Here, we report the identification of the RING E3 ligase RNF126 as the enzyme that specifically mediates frataxin ubiquitination and targets it for degradation. RNF126 interacts with frataxin and promotes its ubiquitination in a catalytic activity-dependent manner, both in vivo and in vitro. Most importantly, RNF126 depletion results in frataxin accumulation in cells derived from FRDA patients, highlighting the relevance of RNF126 as a new therapeutic target for Friedreich ataxia.
Friedreich ataxia·frataxin·E3 ligase·ubiquitin·RNF126·protein degradation·therapeutic target
Friedreich ataxia (FRDA, OMIM: 229300) is a debilitating, life-shortening, neurodegenerative disorder affecting mainly the nervous system and the heart. It is classified as a rare disease; however, it is the most common form of inherited ataxia, with an estimated prevalence of 1 in 50,000 individuals in the Caucasian population (Alper and Narayanan, 2003, Delatycki et al., 2000). Symptoms are progressive and usually begin around puberty, although late-onset cases have been described. Early signs include sensory deficit with consequent loss of movement coordination and gait ataxia. Patients are usually wheelchair bound within 15 years after diagnosis and require assistance to accomplish daily activities (Collins, 2013). Other hallmarks of disease progression include visual impairment because of optic nerve atrophy, dysarthria, and dysphagia. Skeletal abnormalities such as scoliosis and pes cavus are also present. Patients develop a hypertrophic cardiomyopathy that often leads to premature death (Koeppen et al., 2015, Lane et al., 2013, Weidemann et al., 2013). Moreover, about 25% of patients develop diabetes mellitus (Cnop et al., 2013, Igoillo-Esteve et al., 2015). Neurological symptoms are caused by degeneration of sensory neurons in the dorsal root ganglia and in the dentate nucleus of the cerebellum (Koeppen and Mazurkiewicz, 2013). So far, there is no approved therapy for Friedreich ataxia, and only palliative treatments are available for patients. Identification of a treatment for Friedreich ataxia represents a major unmet medical need (Evans-Galea et al., 2014, Strawser et al., 2014, Wilson, 2012).
The disease is caused by reduced expression of the essential mitochondrial protein frataxin (Campuzano et al., 1997). The underlying mutation primarily consists of a homozygous trinucleotide guanine-adenine-adenine (GAA) repeat expansion within the first intron of the corresponding gene (Campuzano et al., 1996). The presence of the GAA tract severely impairs transcription initiation (Chutake et al., 2014a) and elongation (Kim et al., 2011, Li et al., 2015) of the frataxin gene, mainly due to formation of an atypical triplex non-B DNA structure (sticky DNA) (Vetcher et al., 2002) or DNA-RNA hybrids (R loops) (Grabczyk et al., 2007, Groh et al., 2014) and epigenetic modifications that induce a non-permissive chromatin conformation of this DNA region (Al-Mahdawi et al., 2008, Herman et al., 2006), resulting in reduced levels of frataxin protein. Patients live with 5%–30% residual frataxin, the severity of the disease being correlated to the extent of frataxin reduction, which in turn correlates to the length of the GAA expansion (Chutake et al., 2014b). Thus, any increase in frataxin levels is considered therapeutically relevant. Strategies to cure Friedreich ataxia are therefore mostly aimed at increasing the amount of frataxin in patients’ cells (Soragni et al., 2014, Wilson, 2012). Frataxin is a nuclear-encoded mitochondrial protein that plays a crucial role in the biosynthesis of iron-sulfur clusters (Bulteau et al., 2004, Tsai and Barondeau, 2010, Vaubel and Isaya, 2013) and in iron metabolism (Anzovino et al., 2014, Richardson et al., 2010). Iron-sulfur clusters are important cofactors required for proper functioning of enzymes such as aconitase and complexes I, II, and III of the mitochondrial electron transport chain (Rouault and Tong, 2008). Frataxin deficiency therefore results in defective aconitase activity (Condò et al., 2010), impaired mitochondrial respiration, reduced ATP production, imbalance of iron metabolism, mitochondrial iron overload, and increased sensitivity to oxidative stress (Martelli and Puccio, 2014, Pastore and Puccio, 2013). These events are eventually responsible for neuronal degeneration, particularly in the dorsal root ganglia (Koeppen and Mazurkiewicz, 2013, Lynch et al., 2012).
Frataxin is produced in the cytosol as a precursor form with an N-terminal mitochondrial localization signal, which allows the precursor to be directed to mitochondria. During mitochondrial import, the precursor undergoes a two-step catalytic processing that, through the generation of an intermediate form, yields the mature functional form of frataxin (Condò et al., 2007), which is localized in the mitochondrial matrix. We have previously shown that during the normal maturation process, a significant amount of frataxin precursor is degraded through the ubiquitin (Ub)-proteasome pathway, before its mitochondrial import (Rufini et al., 2011). Most current therapeutic approaches aim at promoting frataxin gene transcription (Strawser et al., 2014); however, molecular characterization of the frataxin degradation pathway suggested the possibility to increase frataxin protein by preventing its degradation. We have identified small molecules named ubiquitin-competing molecules that, by docking on the frataxin ubiquitination site, prevent frataxin precursor degradation and eventually promote accumulation of functional mature frataxin (Rufini et al., 2011, Rufini et al., 2015). These molecules provide the rational basis for the development of therapeutic approaches that aim at preventing frataxin degradation. Another strategy to prevent frataxin degradation could be the inhibition of the enzyme responsible for frataxin ubiquitination. Protein ubiquitination is a finely regulated process that ensures tight control of intracellular proteins levels, in particular through the ability of E3 ligase enzymes to selectively recognize their substrates. E3 ligases are therefore considered attractive targets for the development of specific therapies. However, the E3 ligase that specifically recognizes frataxin and targets it for degradation was unknown.
Here we report on the identification of really interesting new gene (RING) finger protein 126 (RNF126) as the E3 ligase that ubiquitinates frataxin. We show that RNF126 interacts with frataxin and directly promotes its ubiquitination, both in vitro and in vivo. We show that RNF126 knockdown results in frataxin accumulation in cells derived from FRDA patients, suggesting the therapeutic potential of strategies aimed at inhibiting RNF126. This enzyme therefore represents a novel important therapeutic target for Friedreich ataxia.
IDENTIFICATION OF THE FRATAXIN-SPECIFIC E3 LIGASE
To identify the E3 ligase responsible for frataxin ubiquitination, we performed a functional screening of a small interfering RNA (siRNA) library targeting more than 600 cellular E3 ligases. By preventing the ubiquitin-dependent degradation of frataxin, the silencing of the critical E3 ligase is expected to yield an increase in frataxin abundance. To measure variation in frataxin levels upon siRNA transfection, we generated a fusion construct between frataxin and ProLabel that was used as a reporter in a cell-based assay. The system is based on β-galactosidase enzyme fragment complementation (Eglen, 2002). The 6 kDa ProLabel tag encodes the inactive α fragment of the β-galactosidase enzyme. When the Ω subunit of the enzyme is added, together with the substrate, the two subunits form an active enzyme that generates a chemiluminescent signal whose intensity correlates to the amount of frataxin-ProLabel fusion present in the cells (Figure 1A). Regular processing of the frataxin precursor into intermediate and mature forms in the ProLabel fusion construct was verified by western blot analysis of transfected cells (Figure S1A). To validate this system, cells transfected with frataxin-ProLabel were treated with the proteasome inhibitor MG132, as a positive control. As previously reported (Rufini et al., 2011), accumulation of the frataxin precursor can be observed 24 hr after MG132 treatment. In this case, up to an 8-fold increase (5.37 ± 2.52) in the intensity of the luminescence signal could be detected by luminometer reading, confirming the sensitivity and the wide dynamic range of the system. The Z′ value for the assay was 0.4, indicating an acceptable to good assay (Birmingham et al., 2009). The library consisted of pools of four siRNAs per gene prearrayed into nine 96-wells plates (Figure S1B). HEK293 cells transiently transfected with frataxin-ProLabel were reverse transfected with the siRNA library. Then, 48 hr after siRNA transfection, the luminescence signal was assessed by luminometer reading, upon addition of the complementary β-galactosidase subunit and substrate (Figure 1B). The increase in the luminescence signal by siRNA transfection was most likely due to suppression of the expression of the critical E3 ligase. Six replicates of each plate were screened independently. From the screening, we isolated six candidate genes potentially involved in the regulation of frataxin stability that were selected for further validation (Figures S1C and S1D).
Full article at http://www.cell.com/cell-reports/fulltext/S2211-1247(17)30149-3