Abstract

Mature frataxin is essential for the assembly of iron–sulfur cluster proteins including a number of mitochondrial enzymes. Reduced levels of mature frataxin (81-20) in human subjects caused by the genetic disease Friedreich’s ataxia results in decreased mitochondrial function, neurodegeneration, and cardiomyopathy. Numerous studies of mitochondrial dysfunction have been conducted using mouse models of frataxin deficiency. However, mouse frataxin that is reduced in these models, is assumed to be mature frataxin (78-207) by analogy with human mature frataxin (81-210). Using immunoaffinity purification coupled with liquid chromatography-high resolution tandem mass spectrometry, we have discovered that mature frataxin in mouse heart (77%), brain (86%), and liver (47%) is predominantly a 129-amino acid truncated mature frataxin (79-207) in which the N-terminal lysine residue has been lost. Mature mouse frataxin (78-207) only contributes 7–15% to the total frataxin protein present in mouse tissues. We have also found that truncated mature frataxin (79-207) is present primarily in the cytosol of mouse liver; whereas, frataxin (78-207) is primarily present in the mitochondria. These findings, which provide support for the role of extra-mitochondrial frataxin in the etiology of Friedreich’s ataxia, also have important implications for studies of mitochondrial dysfunction conducted in mouse models of frataxin deficiency.

Highlights

  • Mature frataxin is essential for the assembly of iron–sulfur cluster proteins including a number of mitochondrial enzymes

  • Mitochondrial dysfunction resulting from frataxin deficiency is considered to be an underlying cause of the devastating genetic disease of Friedreich’s ataxia (FRDA)10,11, which is characterized by progressive neurodegeneration and hypertrophic c­ ardiomyopathy12,13

  • We have developed methodology based on immunoaffinity purification coupled with 2-dimensional ultra-high performance liquid chromatographyparallel reaction monitoring/mass spectrometry (IP 2D-nano-UHPLC-PRM/MS) for characterizing the different circulating proteoforms of mature frataxin in human ­subjects26,27

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Summary

Introduction

Mature frataxin is essential for the assembly of iron–sulfur cluster proteins including a number of mitochondrial enzymes. We have found that truncated mature frataxin [79-207] is present primarily in the cytosol of mouse liver; whereas, frataxin [78-207] is primarily present in the mitochondria These findings, which provide support for the role of extra-mitochondrial frataxin in the etiology of Friedreich’s ataxia, have important implications for studies of mitochondrial dysfunction conducted in mouse models of frataxin deficiency. We have developed methodology based on immunoaffinity purification coupled with 2-dimensional ultra-high performance liquid chromatographyparallel reaction monitoring/mass spectrometry (IP 2D-nano-UHPLC-PRM/MS) for characterizing the different circulating proteoforms of mature frataxin in human ­subjects. We have developed methodology based on immunoaffinity purification coupled with 2-dimensional ultra-high performance liquid chromatographyparallel reaction monitoring/mass spectrometry (IP 2D-nano-UHPLC-PRM/MS) for characterizing the different circulating proteoforms of mature frataxin in human ­subjects26,27 This methodology has been applied to characterizing the major mature frataxin proteoforms that are present in mouse heart, brain, and liver

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