gene are causes of hyperphenylalaninemia (HPA), with resultant phenotypes ranging from classical phenylketonuria (PKU) through variant PKU to benign.
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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.
