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Molecular Genetics and Metabolism 104 (2011) S40S44

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Molecular Genetics and Metabolism

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m g m e

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The G46S-hPAH mutant protein: A model to study the rescue of aggregation-prone

PKU mutations by chaperones
Joo Leandro a, b,, Jaakko Saraste a, Paula Leandro b, c, Torgeir Flatmark a
a
Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway
b
Metabolism and Genetics Group, iMed.UL-Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
c
Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: Phenylketonuria (PKU), the most common inborn error of metabolism, is caused by dysfunction of the liver
Received 26 July 2011 enzyme phenylalanine hydroxylase (PAH), with more than 550 PAH gene mutations identied to date. A large
Accepted 26 July 2011 number of these mutations result in mutant forms of the enzyme displaying reduced stability, increased
Available online 31 July 2011
propensity to aggregate, and accelerated in cellulo degradation. Loss or reduction of human PAH activity
results in hyperphenylalaninemia (HPA) which, if untreated, results in severe mental retardation and
Keywords:
Phenylketonuria
impaired cognitive development. Until now, strict low phenylalanine diet has been the most effective therapy,
Human phenylalanine hydroxylase but as a protein misfolding disease PKU is a good candidate for treatment by natural/chemical/
Conformational disease, Aggregation-prone pharmacological chaperones. The natural cofactor of human PAH, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin
Chemical/pharmacological chaperones (BH4), has already been approved for oral treatment of HPA, giving a positive response in mild forms of the
disease showing considerable residual enzymatic activity. In the case of the most severe forms of PKU,
ongoing studies with chemical and pharmacological chaperones to rescue misfolded mutant proteins from
aggregation and degradation are providing promising results. The PKU mutation G46S is associated with a
severe form of the disease, resulting in an aggregation-prone protein. The human PAH mutant G46S is rapidly
degraded in the cellular environment and, in vitro (upon removal of its stabilizing fusion partner maltose
binding protein (MBP)) self-associates to form higher-order oligomers/brils. Here, we present an in vitro
experimental model system to study the modulation of G46S aggregation by chemical/pharmacological
chaperones, which may represent a useful approach to study the rescue of other severe PKU mutations by
chemical/pharmacological chaperones.
2011 Elsevier Inc. All rights reserved.

Contents

1. Phenylketonuria as a conformational disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S40

2. Rescuing the function of mutant forms of human PAH by chemical and pharmacological chaperones . . . . . . . . . . . . . . . . . . . . S41
3. The G46S PAH mutation: An aggregation-prone PKU mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S41
4. The G46S PAH as a model protein to study the ability of chaperones to rescue aggregation-prone PKU mutations . . . . . . . . . . . . . . S42
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S42
Conict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S43
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S43
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S43

1. Phenylketonuria as a conformational disease

The majority of human genetic diseases are caused by missense

Corresponding author at: Metabolism and Genetics Group, iMed.UL, Faculty of
Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.
Fax: + 351 217946491.
E-mail address: jptleandro@ff.ul.pt (J. Leandro).
mutations resulting in single amino acid substitutions in the expressed
proteins and their abnormal folding [1]. The misfolded proteins can
be recognized by the cellular protein quality control machinery and
targeted for degradation, thus lowering the concentration of the protein

1096-7192/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2011.07.024
 Author's personal copy
J. Leandro et al. / Molecular Genetics and Metabolism 104 (2011) S40S44
at its destination (loss-of-function). Alternatively, they can form
aggregates which present a cytotoxic function (gain-of-function).
Since in both cases the misfolded protein species trigger a pathological
condition, they are now recognized as new therapeutic targets.
The autosomal recessive disorder phenylketonuria (PKU; OMIM
261600) is caused by mutations in the PAH gene encoding for
phenylalanine hydroxylase (PAH; EC 1.14.16.1). Mammalian PAH is
an iron dependent homotetrameric enzyme catalyzing the hydroxyl-
ation of L-phenylalanine (L-Phe) into L-tyrosine (L-Tyr) in the presence
of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and
dioxygen. It assembles as a dimer of dimers and each monomer
comprises a N-terminal regulatory domain, a catalytic domain, and a
C-terminal region which includes the dimerization motif and the
tetramerization domain [24]. Mutations in human PAH lead to a wide
spectrum of clinical and biochemical phenotypes, ranging from
classical or severe PKU to mild PKU and mild hyperphenylalaninemia
(HPA) [5,6]. About 2/3 of the more than 550 mutations identied to
date are missense mutations [6]. Analysis of the mutant proteins
expressed in vitro in different eukaryotic and prokaryotic cells or in
cell free systems have demonstrated that only a small fraction of
mutant proteins present changes in their afnity for the substrate or
the cofactor [7,8]. When transiently expressed in eukaryotic cells, the
majority shows reduced half-lives, and when overexpressed in
Escherichia coli they demonstrate a high tendency to form soluble
and insoluble aggregates [913]. The tetrameric form of recombinant
human wild-type (WT) PAH shows only marginal stability [14],
explaining the high frequency of misfolding human PAH mutations
associated with PKU/HPA. In addition, co-expression studies of several
PKU mutant proteins in E. coli with bacterial chaperonins (GroES/
GroEL) led to a considerable increase in the amount of functional
protein and residual activity unveiling also the deleterious mutational
effects on stability and folding properties of human PAH [10,13]. In
vitro studies with molecular chaperones (Hsp70/40 and Hsp90) also
points to a likely role of the chaperone machinery in vivo [15]. In silico
predictions of the energetic impact of mutations on the native-state
stability of human PAH have also provided evidence in support of
decreased protein stability as the molecular basis of PKU [16],
connecting it with the growing number of genetic diseases classied
as conformational disorders with a loss-of-function.
2. Rescuing the function of mutant forms of human PAH by
chemical and pharmacological chaperones
In conformational disorders the misfolded protein represents the
therapeutic target. Thus, small molecules able to affect the protein
folding reaction or to stabilize folding intermediates and native-like
states, are potential candidates as therapeutic compounds. Such
molecules include enzyme cofactors and inhibitors, as well as
pharmacological and chemical chaperones. Phenylketonuria, which
is a paradigm of inherited metabolic diseases, provides an example in
this new area of therapeutic intervention.
In 1999 Kure and co-workers [17] rst described the natural
cofactor BH4 as a potential therapeutic agent, and after many years of
research a stable form of the human PAH natural cofactor is now
available for the treatment of PKU [18]. Mild forms, with a con-
siderable residual enzyme activity, correlate with the best response to
BH4 intake, whereas patients with a severe form of PKU, presenting
two inactive alleles, generally respond poorly [19,20]. When mild PKU
mutations are expressed in a coupled in vitro transcription-translation
system in the presence of BH4, the proteins display an increased
catalytic activity and stability [8]. Thus, the underlying molecular
mechanism of BH4-responsiveness is intrinsically related to the
inhibitory regulation of human PAH by its cofactor. Upon binding
BH4, in the absence of the substrate, the PAH protein assumes a more
closed conformation, which protects it from proteolytic degradation
[2123]. It is now well accepted that BH4 acts as an in cellulo stabilizer
S41
of both wild-type and several human mutant PAH proteins [7,24,25].
This mechanism has been conrmed using Pah enu1 mice harboring the
V106A mutation associated with a mild HPA phenotype [26]. By
contrast, administration of BH4 to Pah enu2 mice with the F263S
mutation associated with a severe PKU phenotype did not restore L-
Phe levels [26].
Using a uorescence-based high-throughput thermal stability
assay Pey et al. [27] screened a library of N1000 organic compounds.
Two of them, Compound III (3-amino-2-benzyl-7-nitro-4-(2-quino-
lyl)-1,2-dihydroisoquinolin-1-one) and Compound IV (5,6-dimethyl-
3-(4-methyl-2-pyri-dinyl)-2-thioxo-2,3-dihydrothienol[2,3-d]pyri-
midin-4(1H)-one), enhanced the thermal stability of the WT PAH
(T0.5 of 13.6 and 7.2 C, respectively) [27]. Further in cellulo studies
with mild forms of PKU (I65T, R68S and R261Q mutations)
demonstrated that these compounds signicantly increased the
enzyme activity and steady-state protein levels in transiently
transfected cells, as well as the activity of WT PAH in mouse liver
[27]. The effect of these compounds has also been evaluated on the
structurally and functionally related tyrosine and tryptophan hydrox-
ylase, where Compound III demonstrated a large stabilizing effect on
mutant R202H of tyrosine hydroxylase, a protein variant with reduced
stability and residual activity associated with L-DOPA-responsive
dystonia [28].
The natural osmolytes glycerol and trimethylamine N-oxide
(TMAO) have been shown to rescue the catalytic activity of the mild
forms of PKU associated with the mutations I65T, R261Q and V388M
as well as the severe form R270K mutation [29,30]. Expression of the
mutant proteins in E. coli in the presence of 1% glycerol resulted in
decreased formation of aggregated forms and increased recovery of
tetramers (R261Q, R270K and V388M) or dimers (I65T) [30].
Moreover, when synthesized in the presence of glycerol both the
V388M and I65T dimers exhibited more structured forms resembling
a native-like state as measured by far-UV circular dichroism. These
results are compatible with rescued folding of intracellular human
PAH, resulting from stabilization of folding intermediates or native-
like states by glycerol [30,31].
Although considerable progress has been made in the search for
PAH chaperones and the understanding of the underlying stabilizing
mechanisms, the advances in the case of the most severe forms of the
disease have been modest. In general, the evaluation of in vitro
stabilizing effects of chaperones has been hampered by the high levels
of aggregated forms.
3. The G46S PAH mutation: An aggregation-prone PKU mutation
The G46S PAH mutation belongs to the group of PKU mutations
which result in a propensity of the protein to self-associate and form
soluble/insoluble aggregates when over-expressed in prokaryotic
systems, and its rapid degradation when expressed in eukaryotic cells
[32]. It is a relatively common mutation in Scandinavia (i.e. ~10%
relative frequency for PKU alleles in Southeast Norway [32,33]) and is
associated with a severe form of the disease [32].
The G46S mutation is localized in the regulatory domain of PAH
that contains an ACT module (domain which name derives from
aspartokinase, chorismate mutase, and TyrA (prephenate dehydro-
genase)) [34]. The mutation is located at the start of -helix 1 (A47
E57), in a ve residue (L41G46) loop stabilized by a network of
hydrogen bonds and a salt-bridge (K42E44). When the mutant
protein was transiently expressed in human embryonic kidney
(HEK293) cells, the level of immunoreactive protein was only ~ 3%
of the WT protein, at identical PAH mRNA levels [32]. However, its
expression as a fusion protein in E. coli, coupled to the maltose binding
protein (MBP-pep(Xa)-G46S-PAH), gave rise to a metastable tetra-
meric form characterized by a near normal catalytic efciency [15,32].
Cleavage of the fusion partner by the restriction protease factor Xa
lead to destabilization and self-association of the G46S tetramer (and
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S42 J. Leandro et al. / Molecular Genetics and Metabolism 104 (2011) S40S44

dimer) [15], and the formation of higher-order oligomers and large

twisted non-amyloid brils (Fig. 1). The self-association process has
been well characterized in terms of the intrinsic physico-chemical
properties of the mutant protein, as well as extrinsic factors (e.g. ionic
strength, temperature, pH, protein concentration, phosphorylation
state and deamidation), thus offering a well controlled in vitro system
to evaluate the effect of chaperones [15].

4. The G46S PAH as a model protein to study the ability of

chaperones to rescue aggregation-prone PKU mutations

The mutation G46S is considered a non-responsive BH4 mutation

[35]. This conclusion was conrmed by our in vitro light scattering
analyses showing that the cofactor did not inhibit the self-association
of the mutant protein upon factor Xa-mediated cleavage of the
metastable MBP fusion protein [15]. Moreover, the chemical chaper-
ones glycerol, TMAO and ()-epigallocatechin gallate (EGCG), the
major polyphenol in green tea, gave variable effects (Fig. 2). Glycerol
and TMAO have been shown to rescue the enzyme activity of mild and
severe (R270K) PKU mutations, when added to the culture medium
during protein expression in E. coli [30]. In case of the R270K
mutation, a 6.5-fold increase in enzymatic activity with 5% glycerol,
and a 4.6-fold increase with 5 mM TMAO were observed. Concomi-
tantly, the presence of the natural osmolytes reduced the amount of
aggregates and increased the recovery of enzyme tetramer. For
example, 5% glycerol decreased the amount of R270K mutant
aggregates (from 65 to 4%) resulting in corresponding increase in
the tetrameric fraction (from 30 to 95%) [30]. By contrast, in the study
of the rescue of the G46S protein, only glycerol showed a
concentration-dependent inhibition of self-association, whereas
TMAO promoted the process (Fig. 2). Higher concentrations of
TMAO (N10 mM) have been found to have a negative effect on the
enzyme activity of some PKU mutations [30]. Additionally, the green
tea polyphenol EGCG gave a similar effect as the osmolyte TMAO, and
did not inhibit bril formation (Fig. 2), in contrast to what has been
reported for certain amyloidogenic proteins [36,37]. The proposed
pharmacological chaperone of aromatic amino acid hydroxylases,
Compound III [27,28], partly inhibits the self-association process of
the G46S mutant and disrupts the formation of brils (Fig. 3). Thus,
this compound (or a derivative thereof) seems to be a promising
candidate to rescue severe and aggregation-prone PKU mutations.

Fig. 2. The effect of chemical chaperones on the self-association of G46S PAH was
followed by the change in light scattering following cleavage of the metastable,
tetrameric MBP-pep(Xa)-G46S-PAH fusion protein by the restriction protease factor Xa.
(A) Effect of glycerol on the self-association of G46S PAH: control (), 1% glycerol (),
2.5% glycerol () and 5% glycerol (). (B) Effect of trimethylamine N-oxide (TMAO) on
the self-association of G46S PAH: control (), 5 mM TMAO (), 150 mM TMAO () and
250 mM TMAO (). (C) Effect of ()-epigallocatechin gallate (EGCG) on the self-
association of G46S PAH (expressed as molar ratios of G46S PAH fusion protein:EGCG):
1:0 control (), 1:0.1 () and 1:1 (). The chemical structure of the compounds is
represented inside each panel, respectively.
Data taken from [15].

5. Conclusions

Although promising, a therapy of PKU based on chaperoning of

Fig. 1. Electron micrograph showing negatively stained brils of tetrameric G46S PAH.
misfolded mutant proteins may be characterized by its mutant
The protein was negatively stained with aqueous uranyl acetate and analyzed in JEOL specicity and compound-dependency. Consequently, a panoply of
1230 electron microscope operated at 80 kV. Scale bar: 500 nm. experimental approaches including prokaryotic and eukaryotic
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J. Leandro et al. / Molecular Genetics and Metabolism 104 (2011) S40S44 S43

Fig. 3. The effect of pharmacological chaperones on the self-association of G46S PAH was followed by the change in light scattering after cleavage of the metastable, tetrameric MBP-
pep(Xa)-G46S-PAH fusion protein by the restriction protease factor Xa in the absence of any added compound (), in the presence of 0.83% dimethyl sulfoxide (DMSO) () or 100 M
3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one (in 0.83% DMSO) (). Error bars represent mean SD, (n = 3). The electron micrographs on the right
correspond to negatively stained G46S PAH in the absence of any added compound after 3 h of cleavage of its MBP fusion partner by factor Xa (upper panel), and in the presence of
the pharmacological chaperone Compound III, 3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one in 0.83% DMSO at t = 3 h of cleavage (lower panel). The
proteins were negatively stained with aqueous uranyl acetate and examined in JEOL 1230 electron microscope operated at 80 kV. Scale bar: 500 nm.
Data taken and gure adapted from [15].

expression systems, mouse models and in vitro assays will be
necessary to screen and identify the most promising chaperones and
elucidate their underlying mechanisms. In this context, our studies on
the aggregation-prone G46S mutant could be a useful model in
systematic in vitro studies of misfolded, aggregation-prone PKU/HPA-
associated mutant proteins.
Conict of interest
The authors declare that there are no conicts of interest.
Acknowledgments
This work was supported by Fundao para a Cincia e a
Tecnologia, Portugal, grant SFRH/BD/19024/2004 and the University
of Bergen, Norway.
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