Homomeric and heteromeric interactions between wild-type and mutant phenylalanine hydroxylase subunits: Evaluation of two-hybrid approaches for functional analysis of mutations causing hyperphenylalaninemia
Pj. Waters et al., Homomeric and heteromeric interactions between wild-type and mutant phenylalanine hydroxylase subunits: Evaluation of two-hybrid approaches for functional analysis of mutations causing hyperphenylalaninemia, MOL GEN MET, 73(3), 2001, pp. 230-238
Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxyla
se gene (PAH), while mutations in genes encoding the two enzymes (dihydropt
eridine reductase, DHPR, and pterin-4-alpha -carbinolamine dehydratase, PCD
) required for recycling of its cofactor, tetrahydrobiopterin (BH4), cause
other rarer disease forms of hyperphenylalaninemia. We have applied a yeast
two-hybrid method, in which protein-protein interactions are measured by f
our reporter gene constructs, to the analysis of six PKU-associated PAH mis
sense mutations (F39L, K42I, L48S, I65T, A104D, and R157N). By studying hom
omeric interactions between mutant PAH subunits, we show that this system i
s capable of detecting quite subtle aberrations in PAH oligomerization caus
ed 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 inter
act with wild-type PAH subunits, pointing to an explanation for apparent do
minant negative effects previously observed in obligate heterozygotes for P
KU mutations. Based on our findings, the applications and limitations of tw
o-hybrid approaches in understanding mechanisms by which PAH missense mutat
ions exert their pathogenic effects are discussed. We have also used this t
echnique to demonstrate homomeric interactions between wild-type DHPR subun
its and between wild-type PCD subunits. These data provide a basis for func
tional studies on HPA-associated mutations affecting these enzymes. (C) 200
1 Academic Press.