We are investigating the role of amino acid side chain oxidation in amyloid assemblies by comparing the kinetics of fibril formation of native and oxidized proteins.. The effects of amino
Trang 1Simin D Maleknia1, Nata`lia Reixach2and Joel N Buxbaum2
1 School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, NSW, Australia
2 Division of Rheumatology Research, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA, USA
Protein oxidation has been implicated in a wide range
of diseases, and ageing [1–4] Reactive oxygen species
(ROS) contribute to processes that induce irreversible
structural damage and alter protein activity
Oxygen-containing radicals, in particular the hydroxy radical,
react with proteins through hydrogen abstraction,
addition and elimination reactions at both the amino
acid side chains and backbone amide bonds to produce
oxidized, degraded, and cross-linked proteins [2,5,6]
The oxidized cross-linked products and protein
aggre-gates have been identified as insoluble proteins in
many diseased tissues including amyloid fibrils [7,8]
We are investigating the role of amino acid side chain
oxidation in amyloid assemblies by comparing the kinetics of fibril formation of native and oxidized proteins
Interactions between amino acid side chains help to stabilize protein structures and control folding and the assembly of complexes [9,10] The nature of amino acid side chain bonds and their thermodynamic stabil-ity direct the formation of secondary structure in pro-teins [11,12], and these types of information are useful
in predicting misfolding or aggregation events in rela-tion to disease [13,14] Oxidarela-tion of amino acids may alter their tertiary structure contacts, and oxidation can be used as a facile method of investigating the
Keywords
amyloid fibril; footprinting; radical probe
mass spectometry; reactive oxygen species;
transthyretin
Correspondence
S D Maleknia, School of Biological, Earth
and Environmental Sciences, University of
New South Wales, Sydney, NSW 2052,
Australia
E-mail: s.maleknia@unsw.edu.au
(Received 11 July 2006, revised 28
Septem-ber 2006, accepted 9 OctoSeptem-ber 2006)
doi:10.1111/j.1742-4658.2006.05532.x
The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on pro-tein structures Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing pro-tein structures Transthyretin (TTR) is a soluble human plasma propro-tein The wild-type (WT) and several of its variants are prone to fibril forma-tion, which leads to amyloidosis associated with many clinical syndromes The effects of amino acid side chain oxidations were investigated by com-paring the kinetics of fibril formation of oxidized and unoxidized proteins The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments Side chain modification of methionine residues
at position 13 and 30 (the latter for V30M TTR only) were dominant oxi-dative products Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach Oxidation inhibited the initial rates and extent of fibril forma-tion for both the WT and V30M TTR proteins In the case of WT TTR, oxidation inhibited fibril growth by 76%, and for the V30M TTR by nearly 90% These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures
Abbreviations
ROS, reactive oxygen species; TTR, transthyretin.
Trang 2residues and interactions that are critical in stabilizing
protein structures and folding
The amyloidoses are a group of protein-misfolding
diseases that result from deposition of proteins
nor-mally soluble under physiological conditions [15–18]
These include Alzheimer’s disease, Creutzfeldt–Jakob
disease, familial amyloidotic polyneuropathy, familial
amyloidotic cardiomyopathy and senile systemic
amy-loidosis Transthyretin (TTR) is a homotetrameric
plasma protein associated with the transport of
thyrox-ine and vitamin A [19] Deposition of the wild-type
(WT) protein has been associated with senile systemic
amyloidosis [20], and more than 80 TTR variants have
been linked to familial amyloidotic polyneuropathy
and familial amyloidotic cardiomyopathy when
depos-ition occurs in peripheral nerve and heart, respectively
[21] The kinetics of fibril formation of TTR and its
variants have been the subject of many studies [22–24],
and TTR makes an ideal model system for
investi-gating the effects of protein oxidation
Although the onset of amyloidogenesis is not well
understood, in vitro studies suggest that the molecular
mechanism of amyloid fibril formation is based on
dis-sociation of the tetrameric protein into its monomeric
subunits, which, upon misfolding, self-assemble to
form insoluble fibrils [25,26] Further studies have
shown that mutant proteins with modified disulfide
bonds are more susceptible to fibril formation,
suggest-ing that tetramer dissociation may not be the
rate-limiting step in fibril kinetics [27] Moreover, mutations
of single amino acids alter the kinetics of fibril
forma-tion For example, familial mutations in which valine
at position 30 has been substituted with methionine
(V30M) or leucine at position 55 has been replaced
with proline (L55P) increase fibril formation kinetics
[28,29] Accordingly in this study, we investigated the
effects of protein oxidation by comparing the kinetics
of fibril formation of WT and V30M TTR mutant
with their oxidized counterparts
Results and Discussion
Reactions of proteins with ROS induce predominantly
covalent modification of amino acid side chains [2,5,6]
The amino acids methionine, cysteine, phenylalanine,
tyrosine, tryptophan, proline, histidine, leucine and
lysine are most susceptible to reactions with ROS
[5,6,30,31] When reactions are restricted to millisecond
timescales, limited oxidation of amino acid side chains
occurs without structural damage This limited
oxida-tion method, termed radical probe mass spectometry
[6,30], has been utilized for probing protein structure
[32], folding [33] and interactions [34,35] As the
reac-tion timescale increases, backbone cleavage and aggre-gation reactions occur [6], resulting in the possibility of structural damage [36] The dose-dependent oxidation method has been applied to the study of protein stabil-ity and the onset of oxidative damage [36] The present study expands the utility of radical probe mass specto-metry in investigating side chain interactions that are critical in stabilizing protein assemblies
Oxidized proteins for this study were prepared by reaction with hydrogen peroxide [37] in a timescale range of 10 min to 12 h Oxidation of WT and V30M TTR proteins in these timescales increased their molecular masses by 80 Da, indicating that
up to five oxygen atoms were incorporated into the protein structure Electrospray mass spectometry (ESI-MS) analysis also revealed that, after reaction with hydrogen peroxide, these proteins were nearly all oxidized (i.e oxidized samples did not contain unre-acted proteins) To verify that this level of oxidation did not disturb the tetrameric structure of TTR, glu-taraldehyde cross-linking reactions were performed for WT and V30M TTR and their oxidized forms Products of cross-linking reactions were analyzed by gel electrophoresis (data not shown) The unoxidized and oxidized proteins contained similar cross-linking products, and a dominant band of 55 kDa signified that tetrameric structures of WT and V30M TTR were preserved after oxidation These results suggest that oxidation in these timescales did not alter the structure of TTR significantly, and the oxidized pro-teins maintained tetrameric structures
In vitro fibril formation of TTR was performed to compare the effects of amino acid side chain oxidation Structural transitions of proteins to amyloid fibrils can
be followed under laboratory conditions by exposing the folded protein to mildly denaturing conditions such
as low pH or elevated temperatures [28] TTR can be converted into amyloid fibrils through a pH-mediated tetramer-dissociation step The in vitro mechanism of fibril formation is believed to involve tertiary structural changes at low pH resulting in the formation of mono-meric amyloidogenic intermediates that can self-assem-ble into fibrils [21,26] Oxidation of amino acid side chains is used in this study to facilitate generation of new TTR variants, and the kinetics of fibril formation
of these oxidized proteins reveal the amino acid inter-actions that are critical in the onset of amyloido-genesis
The rates of amyloid fibril formation for WT and V30M TTR and their oxidized forms were monitored
by turbidity measurement at 330 nm and 400 nm These absorbance measurements detect both fibrils and aggregates [24] The results of measurements at 330
Trang 3and 400 nm in this study were similar, and therefore
only the 330-nm data are discussed here The kinetics
of fibril formation for the unoxidized proteins and
oxidized proteins resulting from the 12-h reaction with
hydrogen peroxide are shown in Fig 1 Fibril growth
was followed as a function of time for up to 14 days
These results show that both the unoxidized and
oxid-ized proteins could form fibrils The absorbance
meas-urements (Fig 1) show the normal pattern of an initial
exponential fibril growth over the 5-day period
fol-lowed by a slower growth period as a function of time
As the concentration and buffers for all samples were
similar and the oxidized samples did not contain
signi-ficant amounts of unreacted protein, differences in
tur-bidity measurements reflect the effects of amino acid
side chain oxidation on fibril growth kinetics
Oxida-tion had a dramatic affect on initial rates (slopes of
tangent lines to experimental curves up to t¼ 24 h) of
fibril growth for both WT and V30M TTR Larger
effects on the kinetics of fibril formation were seen for
oxidized V30M TTR compared to the unoxidized
V30M TTR than for oxidized WT TTR compared to
unoxidized WT TTR, consistent with the fact that in
V30M TTR there is one more methionine available for
oxidation than in WT TTR
While fibril growth progressed over the 14 days,
oxi-dation inhibited the extent of fibril formation overall
for both the WT and V30M TTR proteins The extent
of fibril formation can be calculated as the percentage
of the turbidity (absorbance at 330 nm) of the oxidized
proteins divided by the turbidity of the unoxidized
proteins Oxidation reduced fibril growth of the WT
protein by 76% after 1 day to 60% after 14 days
In the case of V30M TTR protein, oxidation reduced
fibril growth by 90% after 1 day and 74% after
14 days After 1 day of incubation, 60% of the
unoxi-dized V30M TTR was in the supernatant, whereas
80% of the oxidized protein was in the supernatant
After 3 days of incubation, the values were 27% for the unoxidized V30M TTR and 44% for the oxidized protein These data show that the decrease in turbidity
is not due to different properties of the fibril formed
by oxidized relative to unoxidized protein, rather the differences observed reflect true inhibition of fibril formation
A similar effect was observed for both the WT and V30M TTR when they were reacted with ROS on shorter timescales The percentages of fibril formation over time for V30M TTR are compared in Fig 2 for unoxidized and oxidized proteins from reactions with hydrogen peroxide for 10 min and 1 h These results show that shorter reaction times of 10 min are suffi-cient to inhibit the growth of fibrils, although the extent is somewhat smaller; for example, after 1 day, inhibition of fibril formation decreased from 90% for the 1 h oxidation treatment to 84% for the 10 min oxi-dation preparation
Oxidation of amino acid side chains follows their order of solvent accessibility when oxidative reactions are performed in millisecond timescales [6,30–36] The reaction time influences the level of oxidation at each reactive residue The site of oxidation of amino acid side chains was investigated after proteolysis by mass spectometry sequencing Methionine residues are highly reactive and oxidize readily in the presence of ROS [5,6,37] The WT contains methionine at posi-tions )1 (methionine resulting from the recombinant preparation) and 13 V30M TTR contains an additional methionine at position 30 [38] These methionine residues were highly oxidized to their mono-oxidized and di-oxidized forms The oxidation
of Met13 can be explained by an accessible surface area of 22.8 A˚2 [solvent accessible surface area calcu-lated for V30M TTR monomer (Protein Data Bank entry1TTC) and based on the percentage of the maximum possible exposure of the C-terminal Glu127
350
300
250
200
150
100
50
0
0.0
0.1
0.2
0.3
0.4
0.5
incubation time (h)
V30M TTR V30M TTR Oxidized
WT TTR
WT TTR Oxidized
Fig 1 Kinetics of fibril formation monitored at 330 nm for WT TTR,
V30M TTR and their oxidation products after reaction with
hydro-gen peroxide for 1 h.
0 25 50 75 100
336 120
72 24
6
incubation time (h)
60 min oxidation
10 min oxidation unoxidized
Fig 2 Percentage of TTR fibril formation over time for V30M TTR and its oxidized forms from reaction with hydrogen peroxide for
10 min and 1 h Absorbance measurements (A330) for each dataset normalized to absorbance of unoxidized V30M TTR on day 14.
%Fibrils ¼ [A 330nm (oxidized) ⁄ A 330nm (unoxidized)] x 100.
Trang 4residue] However, Met30 is not solvent accessible
and was completely oxidized [39]
Oxidation of the methionine residues to their
mono-oxidized and di-mono-oxidized forms was confirmed by mass
spectometry sequencing Figure 3 shows post-source
decay sequencing mass spectra for the di-oxidized
(after reaction with ROS) and unoxidized tryptic
pep-tides covering residues 23–35 for V30M TTR The
protonated di-oxidized tryptic peptide is observed at
m/z 1430.5 Oxidation of the methionine residue is
verified, as C-terminus fragment ions from y5
(MHVFR) to y8 (NVAMHVFR) are shifted by 32u,
indicating the addition of two oxygen atoms on this
methionine residue The y1 to y4 remain unchanged,
signifying that the C-terminal HVFR portion of this
peptide was not oxidized The N-terminal fragment
ions b3 to b8 remain unchanged, indicating that the
GSPAINVA portion is not oxidized, and (b10 +32)
and (b11 +32) ions signify that oxidation is exclusive
to the methionine residue These results confirm that
the methionine residues of WT TTR and V30M TTR are highly reactive toward oxidative modification The inhibition effects of fibril formation for these oxidized proteins are intriguing and show that side chain oxidation can be used as a method of inducing mutations in protein sequences to investigate amino acids that are critical in preserving a protein’s structure and stability [36] Interestingly, in vitro studies of a 17-residue peptide showed that replacement of methi-onine residues with their oxidized forms eliminated fibril formation [40] In the case of TTR, dissociation
of the tetramers into monomers is believed to be a pre-liminary and limiting step of the fibril formation pro-cess [26] This inhibition of fibril formation seen in the oxidized proteins suggests that they are more stable than the unoxidized forms Whereas changing the valine residue at position 30 to methionine increases the amyloidogenesis of TTR [28,29], oxidation of the methionine is shown here to partially inhibit fibril growth The amino acid side chain oxidation may have
Fig 3 Post-source decay sequencing mass
spectra for (top) di-oxidized and (bottom)
unoxidized tryptic peptides showing the
oxidation of methionine after reaction of
V30M TTR with ROS.
Trang 5altered tertiary contacts in a manner that stabilized the
oxidized tetramers We speculate that oxidation may
have introduced new tertiary contacts that stabilized
the folded monomeric structure of the oxidized
pro-teins and inhibited the formation of the unfolded
monomer, which has been proposed [25,26] to be a
prerequisite for fibril growth Together these effects
caused a delay in the onset of amyloid fibril formation
Alternatively, the inhibition of fibril formation may
purely be the result of an increase in solubility of
oxid-ized proteins [6,41] Limited oxidation increases the
hydrophilicity of proteins as determined by their
elu-tion times from hydrophobic columns [6,31,32] On the
basis of liquid chromatography⁄ ESI-MS analysis under
similar conditions, oxidized TTR proteins were eluted
40 s faster than their unoxidized forms, indicating
an increase in their hydrophilicity
These results show that amino acid side chain
oxida-tion can be used as a method of investigating regions of
proteins that are critical in the onset of amyloid
forma-tion This study reveals that domains neighboring
methionine residues are critical in the formation of fibril
assemblies These oxidation reactions are being followed
in shorter timescales to possibly distinguish between the
oxidation of Met13 and Met30 in order to more
accu-rately define the key residues of amyloid fibril inhibition
The timescales of reactions with hydrogen peroxide are
limiting, yet ROS can be generated by an electrospray
discharge source [30] that has been shown to generate a
high flux of ROS on millisecond timescales for studies of
protein structures [6,30–36] Alternatively, other mutant
proteins could be designed to further investigate the
effect of fibril formation by substituting amino acids
neighboring methionine residues
Studies revealing the onset and growth of amyloid
fibrils are necessary to understand the pathological
con-ditions that lead to many diseases Valuable information
can be gained on why certain mutants have a greater
propensity to form fibrils or to inhibit fibrils in
compar-ison with their respective native proteins Identifying
protein sequences or domains that are critical in
preser-ving protein stability and function should provide
opportunities for prevention and treatment of diseases
Experimental procedures
Two variants of WT TTR and V30M TTR were selected
for this study These proteins were expressed in an
Escherichia coli system as described elsewhere [29] The
proteins were purified by gel-filtration chromatography on
a Superdex 75 column (Amersham Biosciences, Uppsala,
Sweden) in 10 mm sodium phosphate buffer (pH 7.6)⁄
100 mm KCl⁄ 1 mm EDTA Oxidized proteins were
pre-pared by allowing the proteins (35 lm) to react with hydrogen peroxide (reagent-grade; 30 mgÆmL)1; Sigma Chemicals, St Louis, MO, USA) at a concentration of 2.7% peroxide The oxidation reactions were performed at
pH 7.6 in a timescale range of 10 min to 12 h The oxid-ized proteins were then purified from the hydrogen perox-ide reagent through extensive buffer exchange [10 mm phosphate buffer (pH 7.6)⁄ 100 mm KCl ⁄ 1 mm EDTA] with centriprep devices with 10-kDa filters (Millipore, Bill-erica, MA, USA) The concentrations of all protein solu-tions were adjusted to 10 lm with the sodium phosphate buffer at pH 7.6 based on A280 The proteins were ana-lyzed by liquid chromatography⁄ ESI-MS to verify their molecular masses and extent of oxidation Proteins were also digested with trypsin, and post-source decay sequen-cing experiments identified the site of amino acid side chain modification
Kinetics of amyloid fibril formation
Chemical cross-linking was performed to check that the tetrameric structure of proteins was preserved after the oxi-dation reactions Glutaraldehyde (25%) was added to pro-tein solutions (10% v⁄ v), and incubated for 4 min The reaction was quenched by the addition of NaBH4 (7%
in 0.1 m NaOH) The samples were analyzed by 1D SDS⁄ PAGE, and protein bands were visualized with Coomassie blue stain
The in vitro amyloid fibril formation procedure is well established [42] and was initiated by diluting the protein solutions with an equal volume of 200 mm acetate buffer (pH 4.2)⁄ 100 mm KCl ⁄ 1 mm EDTA The protein solutions were then distributed into a series of cluster tubes and incu-bated at 37C The rates of fibril formation were monit-ored over the course of 14 days by measuring absorbance
at 330 and 400 nm in UV 96-well plates; triplicate experi-ments were used for each time point The results are expressed as mean ± SD from triplicate determinations
Acknowledgements The MALDI-TOF MS instrument (Axima-CFR; Shimadzu Biotech, Manchester, UK) utilized for post-source decay experiments was purchased through a Griffith University Infrastructure grant provided to Simin D Maleknia
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