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(Cornish-Bowden, A. and Ca! rdenas, M. L., eds),
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and Hood, L. (2001) Science 292, 929–934
Received 20 November 2001
Triosephosphate isomerase deficiency: a neurodegenerative misfolding disease
Judit Ola! h*, Ferenc Orosz*, Gyo$ rgy M. Keseru% †, Zolta! n Kova! ri†, Ja! nos Kova! cs‡, Susan Holla! n§
and Judit Ova! di*1
*Institute of Enzymology, Hungarian Academy of Sciences, Budapest, H-1518, P.O. Box 7, Hungary, †Chemical
Works of Gedeon Richter Ltd., Budapest, Hungary, ‡Department of General Zoology, University of Eo$ tvo$ s Lora! nd,
Budapest, Hungary, and §National Institute of Blood Transfusion, Budapest, Hungary
ultrastructural (immunoelectron microscopy) data
for characterization of mutant isomerase structures and for the TPI-related metabolic processes
in normal and deficient cells. The relationships
between mutation-induced TPI misfolding and
formation of aberrant protein aggregates are
discussed.
Abstract
A number of neurodegenerative diseases are mediated by mutation-induced protein misfolding. The
resulting genetic defects, however, are expressed
in varying phenotypes. Of the several wellestablished glycolytic enzyme deficiencies, triosephosphate isomerase (TPI) deficiency is the only
one in which haemolytic anaemia is coupled with
progressive, severe neurological disorder. In a
Hungarian family with severe decrease in TPI
activity, two germ line-identical but phenotypically differing compound heterozygote brothers
inherited two independent (Phe#%! Leu and
Glu"%& stop codon) mutations. We have demonstrated recently [Orosz, Ola! h, Alvarez, Keseru% ,
Szabo! , Wa! gner, Kova! ri, Hora! nyi, Baro! ti, Martial,
Holla! n and Ova! di (2001) Blood 98, 3106–3112]
that the mutations of TPI explain in themselves
neither the severe decrease in the enzyme activity
characteristic of TPI deficiency nor the enhanced
ability of the mutant enzyme from haemolysate
of the propositus to associate with subcellular
particles. Here we present kinetic (flux analysis),
thermodynamic (microcalorimetry and fluorescence spectroscopy), structural (in silico) and
Relationship between protein
misfolding and neurodegeneration
Proteins exhibit a variety of motions due to their
conformational flexibility. These macromolecules
undergo fast dynamic interconversion between
different conformational substates. These interconversions coupled with subunit exchange between oligomers may be rather slow on the
biological time scale, resulting in a long-lived
heterogeneous population, which is an important
issue for their biological function [1]. The conformational changes of non-toxic forms of specific
proteins or fragments can produce pathological
conditions such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and other
neurodegenerative disorders. In the development
of these diseases mutations of proteins with misfolded structures play an important role.
With the aging of society, neurodegenerative
diseases are becoming more widespread. These
serious chronic diseases are the consequences of
the destruction of groups of neurons in various
areas of the brain. Research in the last 10 years has
produced significant results in the understanding
of the mechanism of the evolution of these diseases. The studies have revealed that the development of neurodegeneration is a multistep
Key words : kinetics, metabolism, microtubules, mutant protein.
Abbreviations used : DHAP, dihydroxyacetone phosphate ;
DSC, differential scanning calorimetry ; Glu-6-PDH, glucose-6phosphate dehydrogenase ; MD, molecular dynamics ; MT, microtubule ; TPI, triosephosphate isomerase ; mTPI ; human recombinant mutant (Phe240 Leu) TPI ; PFK, phosphofructokinase ;
PPP, pentose phosphate pathway ; wTPI, human recombinant
wild-type TPI ; βAPP, β-amyloid precursor protein.
1
To whom correspondence should be addressed (e-mail
ovadi!enzim.hu).
# 2002 Biochemical Society
30
Metabolite Channelling and Metabolic Complexity
In this paper we present data on the structural
and associative consequences and metabolic processes related to TPI mutation obtained with
human recombinant enzymes and with haemolysates. We focus on the mutation-related structural and functional alterations of TPI occurring
at the atomic, molecular, ultrastructural and metabolic levels.
process during which one or a few specific mutant
protein species of altered conformation initiate
non-physiological protein–protein interactions,
resulting in enhanced aggregation. The aggregates
form plaques, which are typical morphological
hallmarks of the neurodegenerative process.
The hetero-associations of the mutant proteins with subcellular structures, such as cytoskeleton, cell membranes or glycolytic enzymes,
may be crucial in the initiation of neurodegeneration. Aberrant associations of mutant proteins
with glycolytic enzymes are well documented; for
example, glyceraldehyde-3-phosphate dehydrogenase associates with mutant huntingtin protein
[2], and phosphofructokinase (PFK) binds to βamyloid fragments [3] or to ataxin proteins [4].
The potential involvement of a truncated fragment
of the mutant huntingtin protein in the formation of Huntington’s disease was suggested recently [5]. Nevertheless, details of these processes,
the mechanisms causing differences in the phenotypes of identical mutations and the different
sensitivity of the neuronal structures towards
neurotoxic effects are still unclear.
Catalytic and associative properties of
the mutant enzymes
We have recently reported that the specific activity
of human recombinant mutant (Phe#%! Leu)
TPI (mTPI) relative to human recombinant wildtype TPI (wTPI) is much higher (30 %) than
expected from the activity (3 %) measured in
haemolysates of normal control and deficient red
cells (Table 1). Concerning the associative properties of the normal and mutant enzymes in purified
form compared with in haemolysates, similar
discrepancies were observed with both enzymes.
Associations of TPIs with red cell membranes and
microtubules (MTs) were investigated. The latter,
which are the major constituent of the axon in
neuronal cells, are likely to be a potent target of
TPIs, as suggested by data obtained with a
reconstituted bovine brain system [9]. We found
that wTPI and mTPI showed similar capabilities
of associating with MTs. The binding of the
mutant enzyme from the haemolysate of deficient
cells was more extensive compared with the
normal control (Table 1). Qualitatively similar
binding results were obtained with inside-outvesicles prepared from red cell membranes instead
of MTs (results not shown). At high physiological
protein concentrations one would expect a much
higher binding ratio. This assumption was supported by similar experiments carried out in the
presence of poly(ethylene glycol), a crowding
agent that mimics the high physiological protein
concentration favouring protein–protein interactions [10]. The hetero-association of TPIs with
subcellular membranes decreases the catalytic
activity of both wild-type and mutant enzymes [9].
This decrease of the catalytic activity may not be
important in the case of normal cells, where the
wild-type enzyme exhibits high excess catalytic
activity compared with other glycolytic enzymes.
However, in the case of deficient cells the additional decrease may result in metabolic blockage
at the interconversion of triosephosphates.
The hetero-association of TPI with MTs
forms a superstructure, which can be visualized by
electron microscopy. MTs assembled in the pres-
Triosephosphate isomerase (TPI)
deficiency
TPI is a glycolytic enzyme, which catalyses the
interconversion of -glyceraldehyde 3-phosphate
to dihydroxyacetone phosphate (DHAP). The rate
of the catalysis is diffusion-limited [6], and the
equilibrium favours the formation of DHAP by
1 : 20. Large numbers of glycolytic enzymopathies
were found in red blood cells, resulting in haemolysis and other aberrations [7]. However, TPI
deficiency is unique among the glycolytic enzyme
defects since the chronic haemolytic anaemia in
this case is associated with progressive neurological dysfunction and death in early childhood
[8]. Patients with various inherited mutations have
been identified and all but one of the homozygotes
and several compound heterozygotes carry the
Glu"!% Asp mutation. Compound heterozygote
Hungarian brothers with their missense (Phe#%!
Leu) and nonsense (Glu"%& stop) mutations are
unique because (i) the sites and their combination
of the mutations differ from all others identified
so far, (ii) the elder brother of the two germline-identical compound heterozygotes is free of
neurological manifestations and (iii) both, even
the propositus with neurological symptoms, are
over 20 years old. Mother, father and a simple
heterozygote brother are symptom-free heterozygotes.
31
# 2002 Biochemical Society
Kinetic and binding properties of recombinant TPIs and TPIs from blood cells
DHAP levels in whole blood [27], enzyme activities [28], TPI binding to MTs [9] and heat stability [18] were measured. The pentose phosphate pathway (PPP) was measured with 5 mM ribose 5-phosphate
as a substrate, in the presence of 0.1 mM thiamine pyrophosphate, 1 mM NADP+ and 5 mM MgCl2 [29]. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PEG, poly(ethylene glycol).
Binding to MTs (%)
Heat stability (1/min*)
PPP
PEGk
PEG+
MTk
MT+
–
–
1.6
1.7
–
11.6
0.014
0.099
0.007
0.074
Catalytic properties (units/mg)
32
wTPI
mTPI
DHAP metabolic level (µM)
TPI
Aldolase
GAPDH
–
–
8000
2400
–
–
–
–
Glu-6-PDH
–
–
Catalytic properties (units/g of Hb)
Control
Propositus
Binding to MTs (%)
Heat stability (1/min*)
DHAP metabolic level (µM)
TPI
Aldolase
GAPDH
Glu-6-PDH
PPP
PEGk
PEG+
MTk
MT+
11.4
701
1400
33
3.2
5.2
156
208
10
15.7
9.5
14.8
1.5
11
3.1
11
0.011
0.111
–
–
*First-order rate constant of inactivation.
Biochemical Society Transactions (2002) Volume 30, part 2
# 2002 Biochemical Society
Table 1
Metabolite Channelling and Metabolic Complexity
Figure 1
Transmission (A, B) and immuno- (C, D) electron microscopic images of MTs assembled in the absence (A) or
presence of wild-type (C) or mutant TPI (B, D)
The structure of MTs is similar on sections prepared from glutaraldehyde/osmium tetroxide-fixed samples (A, B). Immunogold decoration
suggests that both types of the enzyme can bind to the surface of MTs (C, D). Scale bar, 150 nm. Transmission electron microscopy and
immunoelectron microscopy were carried out as described in [11] and [12], respectively.
ence of either mutant or wild-type TPI, as described previously [11,12], were apparently intact
and similar to those polymerized without TPI
(Figures 1A and 1B). As TPI is a relatively small
protein with a molecular mass of 53 kDa, its direct
detection by conventional transmission electron
microscopy is rather difficult. To see whether
wTPI or mTPI was present on the surface of
MTs, immunoelectron microscopic images were
taken by the use of an anti-TPI antibody, which
recognizes both TPIs. As shown in Figure 1, the
immunogold conjugates are clearly seen on
the surface of MTs prepared with addition of
wTPI (Figure 1C) and mTPI (Figure 1D). Nonspecific labelling by gold-linked secondary antibody was minimal on control sections incubated
in the absence of primary antibody and no immunostaining was seen on MTs polymerized
without addition of TPI (results not shown).
from persistent conformational heterogeneity in
the ensemble of native oligomers [17]. A prerequisite for such heterogeneity is that the dynamics of subunit exchange\unfolding should be
slow relative to the experimental time scale [1].
Our recent heat-inactivation experiments
showed that the stability of mTPI is significantly reduced compared with wTPI (the t .
!&
value of heat inactivation was approximately one
order of magnitude lower) [18]. In the present
study the unfolding process of the recombinant
enzymes were investigated by differential scanning
calorimetry (DSC). The thermograms recorded
in the temperature range of 10–75 mC (see Figure 2, upper panel) indicate distinct thermal
unfolding processes. Both enzymes undergo a temperature-induced transition from the native state
to the unfolded one. Whereas in the case of wTPI
the heat-absorption curve shows a single sharp
transition with its melting point at 66.2 mC,
the unfolding of the mutant enzyme is induced
at a lower temperature (65.4 mC) ; furthermore, it
shows an additional transition at 54.8 mC. These
findings suggest that the mutation of Phe#%! results
in a protein with lowered stability, probably due
to an altered conformational state. Different
mechanisms could be responsible for the complex
behaviour of the mutant enzyme. A plausible explanation is that the heat-assisted conformational
changes of the mutant enzyme are coupled with
dissociation of the dimeric species, which is more
extensive in the case of the mutant enzyme. Such
Folding and stability of mutant TPI
TPI, which conserved its structural and functional
properties across species [13], is a stable homodimer [14]. This is supported by its marked
resistance against oxidation and proteolysis, during which it maintains the native dimeric structure
[15]. Unfolding\refolding experiments revealed
that the refolding of TPI proceeds from unfolded
monomer to folded inactive monomer, and then
to native, active dimers [16]. The dissociation\
unfolding processes are independent of the protein
concentration [15]. This behaviour can be derived
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# 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 2
a complex thermal-stability profile has not been
documented for TPI previously, although similar
types of study were carried out with other mutant
TPIs [19,20].
The conformational stability of mTPI compared with the wild-type enzyme was also investigated by fluorescence spectroscopy. The effect
of the mutation on TPI stability was assessed by
monitoring changes in the fluorescence emission
peak wavelength as a function of temperature
elevation. The heat-induced denaturation resulted
in a red-shifted emission peak wavelength from
325 to 348 nm for both recombinant enzymes
(Figure 2, lower panel); however, the inflection
points of the unfolding curves were different, but
similar to those obtained with DSC. Our recent
CD measurements have indicated that the α-helix
content of mTPI, which is similar to that of wTPI
at 30 mC, is almost abolished at 60 mC, whereas
only a small decrease in α-helix content of wTPI
could be detected at this temperature [18]. All
these data reveal the crucial role of Phe at
Figure 2
Thermal stability of recombinant mTPI and wTPI monitored by DSC (upper
panel) and intrinsic fluorescence (lower panel)
Upper panel : DSC thermograms of wild-type (solid line) and mutant (dotted line) TPIs in
20 mM Tris buffer, pH 8.0. Enzyme concentration, 10 µg/ml ; heating rate, 1 mC/min. Lower
panel : dependence of fluorescence emission peak wavelength (λmax) on temperature. The
concentration of TPIs was 50 µg/ml. Excitation wavelength was 280 nm. Curves are drawn to
visualize the difference between mTPI (#, dashed line) and wTPI ($, solid line).
(°C)
# 2002 Biochemical Society
34
Metabolite Channelling and Metabolic Complexity
duced by the nonsense mutation (Glu"%& stop)
can bind to wild-type monomer, forming a heterodimer, has been supported by our molecular
dynamics (MD) simulation [18]. The calculations,
based upon the X-ray crystal structure of wTPI,
suggested that the wild-type monomer–truncatedfragment heterodimer is a stable species (Figure 3). However, in the deficient cells two kinds of
polypeptide chain could be synthesized : a whole
chain with a missense mutation and a truncated
fragment. Figure 3 also shows that a heterodimer
can be formed from these polypeptide chains. Our
MD model reveals a distinct conformational state
position of 240 in maintaining the conformational
stability of TPI.
Searching for misfolding promoting
factors
Two experimental observations need explanation
concerning the mutation-induced effects : (i) the
activity of TPI in deficient cells is significantly
lower, as compared with that caused by mutation
and (ii) the association of the mutant TPI with
subcellular particles is enhanced in haemolysate
compared with the relevant recombinant mutant
enzyme. The idea that the truncated peptide pro-
Figure 3
MD structures of the truncated heterodimeric TPIs
MD simulations were based on the crystal structure of human recombinant TPI dimer [30]. The
crystal structure was mutated to have the mutant starting models (one dimer from a wild-type
and a truncated monomer, another dimer from a Phe240 Leu mutant and a truncated
monomer). The backbone atoms of the dimer structures were fitted with a root mean square
distance of 6.54 AH , using the Sybyl 6.7 software package (Tripos, St Louis, MO, U.S.A.). The
dimers consist of a wild-type (red) plus a truncated monomer (orange) and a mutant
(Phe240 Leu ; blue) plus a truncated (cyan) monomer. MD calculations were carried out as
described previously [18].
35
# 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 2
the disease. Generation of βAPP starts a vicious
circle because it binds in nanomolar concentrations to the glycolytic key enzyme PFK ([3]
and references therein). The inhibition of PFK
decreases the glucose turnover and subsequently
the oxidative phosphorylation. Once oxidative
phosphorylation has dropped below a critical level,
βAPP can only partly be inserted into the synaptic
membranes and β-amyloid is generated. In this
way the decrease of PFK activity in senile demented brain cortex can be caused by the PFK–
β-amyloid interaction.
In brain the major energy source is the glucose
that is metabolised by glycolysis. Similarly, mature red cells depend almost solely on anaerobic
glycolysis to produce the energy required for their
functions. Therefore, it is obvious that deficiency
of glycolysis may induce pathological processes.
Consequently, the glycolytic pathway has been the
objective of many theoretical studies, and computer models that simulate this network in normal
and deficient red blood cells have been evaluated
in the active-site region due to the mutation
(results not shown) ; however, it may not extend to
the subunit contact surface since the two heterodimeric structures display similar global conformational states. Therefore, our MD simulation
provided in silico evidence for the formation of the
mutation-containing heterodimeric TPI. This
artificial species, if present in vivo, may display
very different catalytic and aggregation properties.
Metabolism in the TPI-deficient
diseased cell
In many diseases the structural changes of proteins
result in functional alteration, from which metabolic disorders originate. However, the causeconsequence relations in these processes remain
obscure in many cases. A typical example is
Alzheimer’s disease, in which alteration of the
critical levels of glucose and ATP turnovers
ensuring the proper insertion of β-amyloid precursor protein (βAPP) into the cellular or synaptic
membranes seems to be crucial in the evolution of
Scheme 1
The PPP cross-talking with glycolysis
Glu-6-PDH, glucose-6-phosphate dehydrogenase ; GAP, D-glyceraldehyde 3-phosphate.
# 2002 Biochemical Society
36
Metabolite Channelling and Metabolic Complexity
[21–23]. Nevertheless, no clear data are available
on the energy metabolism of patients with severe
TPI deficiency.
It is an extensively discussed issue that the
low TPI activity in deficient cells may lead to a
metabolic block in the glycolytic pathway that
results in an increased (20–60-fold) concentration
of DHAP (for reviews see [24,25]), especially if the
hetero-association of the mutant TPI further
reduces the catalytic activity, as we have suggested
previously [9]. This phenomenon can explain why
the DHAP level is so high in red cells. However, in
platelets from TPI-deficient individuals only
modest elevation of DHAP level was detected
[18], probably because in platelets, just as in many
other cells including brain cells, DHAP is not a
dead-end product but can be converted in the
direction of lipid synthesis. This fact, plus an early
finding [26] that another enzymopathy coupled
with high DHAP levels does not cause neurological disorder, suggest that the high level of
DHAP may not be responsible for the clinical
symptoms. On the other hand, it has been reported
that the concentrations of 2,3-diphosphoglycerate,
and more importantly the ATP level, in TPIdeficient red blood cells do not differ significantly
from those of normal controls [27]. To assess this
apparent inconsistency, the activities of other
glycolytic enzymes as well as the flux of the pentose
phosphate pathway (PPP) cross-talking with glycolysis via common intermediates (Scheme 1) were
measured in normal and in deficient cell-free
haemolysates. As shown in Table 1, we have found
that while the activity of TPI was much lower
in the deficient cell haemolysate as compared with
the normal control, the activity of aldolase and
glyceraldehyde-3-phosphate dehydrogenase was
higher. These two enzymes catalyse the conversion of hexose (fructose 6-phosphate) and
triose (-glyceraldehyde 3-phosphate) phosphates, which are involved in both pathways. The
key regulatory enzyme of the PPP is glucose6-phosphate dehydrogenase (Glu-6-PDH), catalysing the oxidative conversion of glucose
6-phosphate in the direction of ribose 5-phosphate
formation. The further conversion of this metabolite results in the formation of the common
intermediates of the two pathways. We have found
that the activity of Glu-6-PDH in the haemolysate
of the deficient cells is significantly higher than
that of the control, which may result in a more
active PPP in the deficient cells. This idea is
supported by direct flux measurement when the
reduction of NADPH to NADP+ catalysed by
Glu-6-PDH was monitored, initiating the pathway by adding ribose 5-phosphate as substrate. As
shown in Table 1 the PPP flux is significantly
higher in deficient cells as compared with the
normal controls. Therefore, the interconnection
of the two pathways can compensate for the
reduced TPI activity of the deficient cells, and it
could be responsible for the normal ATP level
found in the TPI-deficient erythrocytes.
Outlook
In recent years many observations have revealed
that the protein aggregation induced by protein
misfolding into fibrils and deposition in senile
plaques is primarily related to neurotoxicity in
the cases of several neurological diseases. TPI
enzymopathy is a unique glycolytic enzyme
deficiency coupled with neurodegeneration. We
present data on the mutation-induced misfolding
process, which probably plays an important role
in the enhanced associations of mTPI. The mutant
enzyme may interact with the truncated fragment
via subunit exchange, or with MTs in the brain.
No dependence on ionic strength was found in
these protein–protein associations. Aberrant
hetero-associations primarily involved in the
process may lead to fibrillar aggregation, a potent
constituent of the senile plaque formation similar
to that detected in the case of Alzheimer’s disease
and other neurodegenerative disorders.
However, on the basis of recent clinical and
experimental results obtained with the compound
heterozygote Hungarian brother (results not
shown), it became obvious that the mutations
alone are not sufficient to explain the extreme
variability of neurodegenerative diseases. That
phenotype is not determined necessarily by genotype is demonstrated by our recent data. Additional investigations of protein conformational
dynamics and their relation to folding and protein–
protein interaction by means of basic physicochemical methods are necessary to understand the
human syndrome. Our studies presented here may
be relevant to the understanding of the molecular
basis of human ‘ conformational ’ or ‘ misfolding ’
diseases.
This work was supported by grants from the Hungarian National
Science Foundation OTKA (T-025291 and T-031892 to J. Ova! di,
T-029910 to J. K., T-029924 and T-035019 to F. O., T-30044 to
G. M. K. and T-033138 to S. H.) and from the Hungarian Ministry of
Education (NKFP 1/47 to J. Ova! di). We thank Professor Joseph
A. Martial for kindly providing recombinant TPIs.
37
# 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 2
17
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Received 19 November 2001
Multiple glucose 6-phosphate pools or channelling of flux in diverse pathways ?
Loranne Agius*1, Josep Centelles† and Marta Cascante†
*Department of Diabetes and Metabolism, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH,
U.K., and †Departament de Bioquı! mica i Biologia Molecular, Facultat de Quı! mica, Universitat de Barcelona,
Martı! i Franque' s, 1, 08028 Barcelona, Spain
have suggested that there are multiple subcellular
pools of glucose 6-phosphate. It is proposed that
this data can be interpreted in terms of channelling
of metabolic intermediates through multiple pathways of glucose metabolism with leakage of glucose 6-phosphate from the channels into a single
free pool. It is also proposed that measurement
of total tissue content of glucose 6-phosphate
approximates the free pool.
Abstract
Glucose 6-phosphate is an intermediate of pathways of glucose utilization and production as well
as a regulator of enzyme activity and gene expression. Studies on the latter functions are in part
based on measurement of the glucose 6-phosphate
content in a whole-cell extract. Several studies
Key words : compartmentation, glycolysis, intermediates.
Abbreviations used : glucose 6-P, glucose 6-phosphate ; glucose
1-P, glucose 1-phosphate ; fructose 1-P, fructose 1-phosphate ;
fructose 6-P, fructose 6-phosphate.
1
To whom correspondence should be addressed (e-mail
Loranne.Agius!ncl.ac.uk).
# 2002 Biochemical Society
Introduction
Glucose 6-phosphate (glucose 6-P) is the first
intermediate in the metabolism of glucose by
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