Waters PJ, Scriver CR, Parniak MA

Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase gene (PAH), while mutations in genes encoding the two enzymes (dihydropteridine reductase, DHPR, and pterin-4-alpha-carbinolamine dehydratase, PCD) required for recycling of its cofactor, tetrahydrobiopterin (BH(4)), cause other rarer disease forms of hyperphenylalaninemia. We have applied a yeast two-hybrid method, in ...

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which protein--protein interactions are measured by four reporter gene constructs, to the analysis of six PKU-associated PAH missense mutations (F39L, K42I, L48S, I65T, A104D, and R157N). By studying homomeric interactions between mutant PAH subunits, we show that this system is capable of detecting quite subtle aberrations in PAH oligomerization caused by missense mutations and that the observed results generally correlate with the severity of the mutation as determined by other expression systems. The mutant PAH subunits are also shown in this system to be able to interact with wild-type PAH subunits, pointing to an explanation for apparent dominant negative effects previously observed in obligate heterozygotes for PKU mutations. Based on our findings, the applications and limitations of two-hybrid approaches in understanding mechanisms by which PAH missense mutations exert their pathogenic effects are discussed. We have also used this technique to demonstrate homomeric interactions between wild-type DHPR subunits and between wild-type PCD subunits. These data provide a basis for functional studies on HPA-associated mutations affecting these enzymes.

Date: Jul. 01, 2001

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