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Role of low native state kinetic stability and interaction of partially unfolded states with molecular chaperones in the mitochondrial protein mistargeting associated with primary hyperoxaluria

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Abstract

The G170R variant of the alanine:glyoxylate aminotransferase (AGT) is the most common pathogenic allele associated to primary hyperoxaluria type I (PH1), leading to mitochondrial mistargeting when combined with the P11L and I340M polymorphisms (minor allele; AGTLM). In this work, we have performed a comparative analysis on the conformation, unfolding energetics and interaction with molecular chaperones between AGTwt, AGTLM and AGTLRM (G170R in the minor allele) proteins. Our results show that these three variants share similar conformational and functional properties as folded dimers. However, kinetic stability analyses showed a ≈1,000-fold increased unfolding rate for apo-AGTLRM compared to apo-AGTwt, as well as a reduced folding efficiency upon expression in Escherichia coli. Pyridoxal 5′-phosphate (PLP)-binding provided a 4–5 orders of magnitude enhancement of the kinetic stability for all variants, suggesting a role for kinetic stabilization in pyridoxine-responsive PH1. Conformational studies at mild acidic pH and moderate guanidium concentrations showed the formation of a molten-globule-like unfolding intermediate in all three variants, which do not reactivate to the native state and strongly interact with Hsc70 and Hsp90 chaperones. Additional expression analyses in a mammalian cell-free system at neutral pH showed enhanced interaction of AGTLRM with Hsc70 and Hsp90 proteins compared to AGTwt, suggesting kinetic trapping of the mutant by chaperones along the folding process. Overall, our results suggest that mitochondrial mistargeting of AGTLRM may involve the presentation of AGT partially folded states to the mitochondrial import machinery by molecular chaperones, which would be facilitated by the low native state kinetic stability (partially corrected by PLP binding) and kinetic trapping during folding of the AGTLRM variant with molecular chaperones.

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Abbreviations

AGT:

Alanine:glyoxylate aminotranferase

ANS:

8-Anilinonaphthalene-1-sulfonic acid

CD:

Circular dichroism

DSC:

Differential scanning calorimetry

PH1:

Primary hyperoxaluria type 1

PLP:

Pyridoxal 5′-phosphate

SEC:

Size-exclusion chromatography

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Acknowledgments

We want to thank Prof. Aurora Martinez at the University of Bergen for support at the initial stages of this work. This work was supported by grants from the Spanish Ministry of Education (SAF2007-62343 to E.S. and BIO2009–09562 and CSD2009-00088 to J.M.S-R). Angel L. Pey is supported by a Ramon y Cajal research contract from the Spanish Ministry of Science and Innovation (MICINN) and also acknowledges a short-term fellowship from the Federation of European Biochemical Societies (FEBS).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Departamento de Química-Física, Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain

    Angel L. Pey & Jose M. Sanchez-Ruiz

  2. Hospital Universitario Canarias, Center Biomedical Research on Rare Diseases (CIBERER) and Institute of Biomedical Technologies (ITB), Tenerife, Spain

    Eduardo Salido

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  1. Angel L. Pey
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  2. Eduardo Salido
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Correspondence to Angel L. Pey.

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Pey, A.L., Salido, E. & Sanchez-Ruiz, J.M. Role of low native state kinetic stability and interaction of partially unfolded states with molecular chaperones in the mitochondrial protein mistargeting associated with primary hyperoxaluria. Amino Acids 41, 1233–1245 (2011). https://doi.org/10.1007/s00726-010-0801-2

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