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.

[primary hyperoxaluria type 1]

The G 170 R variant of the alanine :glyoxylate aminotransferase (AGT ) is the most common pathogenic allele associated to primary hyperoxaluria type I (PH 1 ), leading to mitochondrial mistargeting when combined with the P 11 L and I 340 M polymorphisms (minor allele ; AGT (LM )). In this work , we have performed a comparative analysis on the conformation , unfolding energetics and interaction with molecular chaperones between AGT (wt ), AGT (LM ) and AGT (LRM ) (G 170 R 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 â‰ˆ 1 ,000 -fold increased unfolding rate for apo-AGT (LRM ) compared to apo-AGT (wt ), 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 PH 1 . 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 Hsc 70 and Hsp 90 chaperones . Additional expression analyses in a mammalian cell-free system at neutral pH showed enhanced interaction of AGT (LRM ) with Hsc 70 and Hsp 90 proteins compared to AGT (wt ), suggesting kinetic trapping of the mutant by chaperones along the folding process . Overall , our results suggest that mitochondrial mistargeting of AGT (LRM ) 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 AGT (LRM ) variant with molecular chaperones .

Diseases
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Diseases presenting "mitochondrial import machinery" symptom

primary hyperoxaluria type 1

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