Download Degradation rates differ between mutant and wild

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Biochemical Society Transactions (1998) 26
Degradation rates differ between mutant
and wild-type forms of phenylalanine
hydroxylase expressed in vitro.
Table 1. Rates of deeradation of wild-twe and mutant
PAH exvressed in vitro
Paula J. Waters*, Charles R. Scriver* and Michael A.
Parniakt.
*de Belle Laboratory for Biochemical Genetics, McGill
University / Montreal Children’s Hospital Research
Institute, 2300 Tupper St., H3H 1P3, and §Lady Davis
Institute for Medical Research, 3755, CGte St. Catherine,
H3T 1E2, Montreal, Quebec, Canada.
Phenylalanine hydroxylase (PAH) catalyses the
conversion of phenylalanine (phe) to tyrosine and is the
major determinant of flux through the pathway leading to
complete oxidation of phe. Mutations in the human PAH
gene are causes of hyperphenylalaninemia (HPA), with
resultant
phenotypes ranging
from
classical
phenylketonuria (PKU) through variant PKU to benign
forms of mild HPA. Most mutations analysed lead to
reduced levels of PAH protein and enzyme activity in
transient expression systems (reviewed in [l]). In
previous studies these parameters have been assayed in
lysates of transfected cells harvested at a single arbitrary
time point. The abundance of a protein at any moment is
however a function of the rates of both synthesis and
degradation. We have therefore examined both synthesis
and degradation of PAH, and the effects of PAH
mutations upon these enzymic properties.
We studied wild-type PAH and two PKU-associated
mutant forms of the enzyme, A104D and R157Nt. The
A104D mutation produces relatively mild effects, both in
vivo and in multiple in vitro systems ( human embryonal
kidney cells, rabbit reticulocyte lysate and E. coli ),
whereas the R157N mutation has a more severe impact
on PAH function and structure [l-31. In this study the
three proteins were expressed by coupled in vitro
transcription-translation using rabbit reticulocyte lysate
(TNT-T7 system, Promega). After a one-hour “pulse”
with [35S]-cysteine,“chase” was initiated by addition of
unlabelled cysteine. PAH present at various time points
was resolved by SDS-PAGE and quantitated by
phosphorimaging.
Abbreviations used: PAH (phenylalanine hydroxylase),
PKU (phenylketonuria), PAH (gene encoding PAH), HPA
(hyperphenylalaninemia).
tA104D and R157N are the trivial ( amino acid ) names
for these mutations in PAH.
The corresponding
systematic ( nucleotide ) names are c.311CjA and
[c.470G+A;c.471A+C].
Wild-type
0.08
8.9
A104D
0.16
4.5
R157N
0.23
3.0
Table 1 shows the rates of degradation of the 52 kDa
PAH subunit obtained in a representative experiment.
PAH degradation was fastest for the “severe” mutant
form, R157N, slowest for the wild-type enzyme, and
intermediate for the “mild” mutant protein, A104D.
Rates of synthesis appeared similar for all three proteins
( data not shown ).
Summary: The PKU-associated PAH mutations A104D
and R157N cause increased degradation of PAH in vitro.
We have thus identified a basic mechanism for effects of
these PAH mutations.
Degradation rates correlate with the severity of these
mutations’ effects in vitro and in vivo. Pulse-chase
approaches should therefore be useful to correlate other
PAH genotypes with HPA phenotypes.
The extent to which increased PAH degradation may be
a common phenomenon underlying the effects of different
PAH mutations remains to be established. Further details
of the mechanism also need to be elucidated. It has been
suggested that PAH turnover might involve ubiquitindependent mediation by proteasomes, and that mutant
PAH enzymes with abnormal structure could be prone to
increased destruction by this pathway [4,5]. Studies on
factors involved in degradation of wild-type and mutant
forms of PAH, both in vitro and in cultured cells, are in
progress.
Acknowledaements: We thank Per Knappskog (Bergen) for the kind gift
of wild-type expression plasmid pcDNA3-PAH. This work and PJW
(postdoctoral Fellowship) were supported by the Medical Research
Council (Canada).
1. Waters, P.J., Parniak, M.A., Nowacki, P. and Scriver, C.R. (1998)
Hum. Mutat. 11,4-17.
2. Waters, P.J., Hewson, A.S., Scriver, C.R., Treacy, E.P., Martinez, A,,
Knappskog, P.M. and Pamiak, M.A. (1997) Biochem. Soc. Trans. 25,
3628.
3. Waters. P.J., Hewson,AS., Pamiak, MA, Carter, K., Kayaalp, E.,
L a h b o i s e , R., Treacy, E.P. and Scriver, C.R. (1997) Am. J. Hum.
Genet. 61 Suppl. A263.
4. Eiken, H.G., Knappskog, P.M., Apold, J. and Flatmark, T. (1996)
Hw. Mutat. 7,228-238.
5 . Doskeland, A.P. and Flatmark, T. (1996) Biochem. J. 319,941-945.