Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin.

Joseph F Nabhan,Eugene L Piatnitski Chekler,Christine E Bulawa,Renea L Gooch,Betsy Pierce

doi:10.1038/srep18251

Abstract

Friedreich’s Ataxia is a genetic disease caused by expansion of an intronic trinucleotide repeat in the frataxin (FXN) gene yielding diminished FXN expression and consequently disease. Since increasing FXN protein levels is desirable to ameliorate pathology, we explored the role of major cellular proteostasis pathways and mitochondrial proteases in FXN processing and turnover. We targeted p97/VCP, the ubiquitin proteasome pathway (UPP), and autophagy with chemical inhibitors in cell lines and patient-derived cells. p97 inhibition by DBeQ increased precursor FXN levels, while UPP and autophagic flux modulators had variable effects predominantly on intermediate FXN. Our data suggest that these pathways cannot be modulated to influence mature functional FXN levels. We also targeted known mitochondrial proteases by RNA interference and discovered a novel protease PITRM1 that regulates intermediate FXN levels. Treatment with the aforementioned chemical and genetic modulators did not have a differential effect in patient cells containing lower amounts of FXN. Interestingly, a number of treatments caused a change in total amount of FXN protein, without an effect on mature FXN. Our results imply that regulation of FXN protein levels is complex and that total amounts can be modulated chemically and genetically without altering the absolute amount of mature FXN protein.

Highlights

In the genetic disease Friedreich’s Ataxia (FRDA), a homozygous GAA (Guanine-Adenine-Adenine) trinucleotide expansion in intron 1 of the FXN nuclear locus partly silences de novo transcription leading to a reduction in intracellular levels of FXN protein[1,2]
FXN is expressed in the cytoplasm as a 210 amino acid (AA) precursor protein that is translocated into mitochondria where it is processed by two consecutive steps into iFXN (FXN 42–210; 19 KDa) and mFXN (81–210; 14.2 KDa), which is functional[12,13]
We first eliminated the possibility that the FXN maturation machinery may limit steady state levels of mFXN. 293T cells were transfected with increasing amounts of a construct expressing full length human FXN

Summary

Introduction

In the genetic disease Friedreich’s Ataxia (FRDA), a homozygous GAA (Guanine-Adenine-Adenine) trinucleotide expansion in intron 1 of the FXN nuclear locus partly silences de novo transcription leading to a reduction in intracellular levels of FXN protein[1,2]. The FXN transcript is translated into a cytosolic precursor protein (pFXN) that is rapidly imported into mitochondria, where it is further processed from an intermediate form (iFXN) to a mature protein (mFXN), resident in the mitochondrial matrix. While UPP inhibition did not increase levels of FXN, some treatments augmented total FXN levels www.nature.com/scientificreports/. Through upregulation of pFXN and/or iFXN, suggesting complex modulation of FXN import and processing in mitochondria. Uncoupling of mitochondrial membrane potential and suspected alteration of mitochondrial pH, both of which are known to impact mitochondrial import[9,10] and processing[11], reproduced some of the phenotypes elicited by proteostasis modulators. We further carried out an siRNA screen targeting known mitochondrial proteases and discovered that knockdown of PITRM1 augmented total FXN, again by increasing iFXN. Measurement of total FXN does not predict mFXN level, underscoring the need to characterize potential FXN enrichment therapies using methods that monitor FXN processing

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