Biophysical characterisation of the recombinant human frataxin precursor

Abstract

Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin ( FXN ). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN 81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor ( FXN 1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐ TAT ‐ FXN 1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐ TAT ‐ FXN 1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro : the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐ TAT ‐ FXN 1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS 1/ ISD 11/ ISCU was activated in vitro by His6‐ TAT ‐ FXN 1‐210. Moreover, the results showed that His6‐ TAT ‐ FXN 1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF 126, in a similar way as the FXN 1‐210, suggesting that the His6‐ TAT extension does not interfere with the ubiquitination machinery.

E3 ubiquitin ligase RNF126 affects bladder cancer progression through regulation of PTEN stability

“…RNF126 is a RING domain-containing protein that plays an important role in different physiological processes dependent or independent of the E3 ligase activity. A bunch of substrates of E3 ligase RNF126 have been identified, including G0/G1 switch gene 2 (G0S2) 6 , frataxin (FXN) 7 , 8 , epidermal growth factor receptor (EGFR) 9 , pyruvate dehydrogenase kinases (PDKs) 10 , insulin-like growth factor II receptor (IGF-IIR) 11 , mechanistic target of rapamycin (mTOR) 12 , CDK inhibitor CDKN1A/p21 13 and Bag6 complex 14 . As a transcription factor, RNF126 promotes DNA homologous recombination (HR) through interacting with E2F1 and promoting the transcription of BRCA1 15 .…”

Regulation of Fibroblast Activation Protein-α Expression: Focus on Intracellular Protein Interactions

“…RNF126 is recruited to the N -terminal UBL domain of BAG6 and preferentially ubiquitinates juxtahydrophobic lysine residues of BAG6 protein clients. Substrates of RNF126 include p21, epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), and frataxin (responsible of Friedreich ataxia), promoting their degradation. , Knockdown of RNF126 using siRNA technology increases the level of these proteins in diseased cells. RNF126 specifically regulates PTEN stability through ubiquitination, functioning as an oncogene.…”

Drug Repositioning in Friedreich Ataxia

“…Strategies to elevate endogenous frataxin levels focus on either promoting its gene expression at the transcriptional ( Herman et al, 2006 ; Li et al, 2016 ; Erwin et al, 2017 ; Shen et al, 2020 ) or translational ( Bon et al, 2019 ) level, or preventing its degradation ( Rufini et al, 2011 , 2015 ; Benini et al, 2017 ). Alternatively, gene therapy ( Perdomini et al, 2014 ; Piguet et al, 2018 ) or protein replacement approaches ( Vyas et al, 2012 ; Britti et al, 2018 ; Castro et al, 2018 ) are being considered by various groups, reporting encouraging results ( Delatycki and Bidichandani, 2019 ). Pharmacological treatments under development also focus on reducing the downstream consequences of frataxin deficiency, such as mitochondrial dysfunction and oxidative stress ( Zesiewicz et al, 2018a , b ; Lynch and Johnson, 2021 ).…”