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Ni(II) ions cleave and inactivate human alpha-1 antitrypsin hydrolytically, implicating nickel exposure as a contributing factor in pathologies related to antitrypsin deficiency

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Human alpha-1 antitrypsin (AAT) is an abundant serum protein, present at a concentration of 1.0–1.5 g L−1. AAT deficiency is a genetic disease, manifesting itself with emphysema and liver cirrhosis, due to accumulation of a misfolded AAT mutant in hepatocytes. Lung AAT amount is inversely correlated with chronic obstructive pulmonary disease (COPD), a serious and often deadly condition, with increasing frequency in the aging population. Exposure to cigarette smoke and products of fossil fuel combustion aggravates AAT deficiency and COPD according to mechanisms that are not fully understood. Taking into account that these fumes contain particles that can release nickel to human airways and skin, we decided to investigate interactions of AAT with Ni(II) ions within the paradigm of Ni(II)-dependent peptide bond hydrolysis. We studied AAT protein derived from human blood using HPLC, SDS-PAGE, and mass spectrometry. These studies were aided by spectroscopic experiments on model peptides. As a result, we identified three hydrolysis sites in AAT. Two of them are present in the N-terminal part of the molecule next to each other (before Thr-13 and Ser-14 residues) and effectively form one N-terminal cleavage site. The single C-terminal cleavage is located before Ser-285. The N-terminal hydrolysis was more efficient than the C-terminal one, but both abolished the ability of AAT to inhibit trypsin in an additive manner. Nickel ions bound to hydrolysis products demonstrated an ability to generate ROS. These results implicate Ni(II) exposure as a contributing factor in AAT-related pathologies.
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Cite this: Metallomics, 2015,
7, 596
Ni(II) ions cleave and inactivate human alpha-1
antitrypsin hydrolytically, implicating nickel
exposure as a contributing factor in pathologies
related to antitrypsin deficiency
Nina Ewa Wezynfeld, Arkadiusz Bonna, Wojciech Bal* and Tomasz Fra˛czyk*
Human alpha-1 antitrypsin (AAT) is an abundant serum protein present at a concentration of 1.0–1.5 g L
1
.
AAT deficiency is a genetic disease that manifests with emphysema and liver cirrhosis due to the accumu-
lation of a misfolded AAT mutant in hepatocytes. Lung AAT amount is inversely correlated with chronic
obstructive pulmonary disease (COPD), a serious and often deadly condition, with increasing frequency in
the aging population. Exposure to cigarette smoke and products of fossil fuel combustion aggravates AAT
deficiency and COPD according to mechanisms that are not fully understood. Taking into account that
these fumes contain particles that can release nickel to human airways and skin, we decided to investigate
interactions of AAT with Ni(II) ions within the paradigm of Ni(II)-dependent peptide bond hydrolysis. We
studied AAT protein derived from human blood using HPLC, SDS-PAGE, and mass spectrometry. These
studies were aided by spectroscopic experiments on model peptides. As a result, we identified three
hydrolysis sites in AAT. Two of them are present in the N-terminal part of the molecule next to each other
(before Thr-13 and Ser-14 residues) and effectively form one N-terminal cleavage site. The single
C-terminal cleavage site is located before Ser-285. The N-terminal hydrolysis was more efficient than the
C-terminal one, but both abolished the ability of AAT to inhibit trypsin in an additive manner. Nickel ions
bound to hydrolysis products demonstrated an ability to generate ROS. These results implicate Ni(II)
exposure as a contributing factor in AAT-related pathologies.
Introduction
Nickel toxicity is a well-recognized health problem in the
industrialised world. The human body has two major routes of
exposure to nickel. Inhalation of particles containing Ni(II)sulphides,
oxides and salts is a major risk factor for the occurrence of nickel-
related cancers of the upper and lower respiratory tract. In contrast,
skin contact with nickel-containing objects made of stainless steel
and other alloys, such as coins, jewellery, mobile phones and
accessories, results in nickel allergy symptoms in sensitised persons.
Implantable surgical materials made of nickel alloys may also give
rise to cancers in neighbouring tissues. The release of Ni
2+
ions from
these objects and materials is a common feature of all these
phenomena.
1–8
Several types of molecular mechanisms of nickel toxicity
have been proposed in the literature, all considering exchange-
able Ni(II) as the ultimate toxic species. None of these concepts
have gained unanimous support. Epigenetic alterations of gene
expression, inhibition of DNA repair and direct oxidative assault
on cellular biomolecules are the most common concepts in nickel
carcinogenesis supported at least partially by experimental
evidence.
9–11
Despite many efforts, the molecular mechanisms
of nickel allergies are even less clear. Scattered observations
suggest the involvement of square-planar Ni(II) complexes,
Ni(II)-catalysed redox reactions and perhaps direct interactions
of Ni(II) ions with components of T-cell-mediated immunity,
but a comprehensive picture has not yet emerged from these
studies (for review see, e.g., ref. 12–14).
In a series of studies, we developed a molecular mechanism
that can be a unifying factor for at least some of these concepts.
Nickel-dependent peptide bond hydrolysis occurs in the amino
acid sequences Aaa–Ser/Thr–Xaa–His–Zaa, where a cleavage
occurs before the Ser or Thr residue following the formation
of a square-planar Ni(II) complex involving the Ser/Thr–Xaa and
His residues. This reaction occurs in three major stages. It is
initiated by the formation of a square-planar complex in which
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin
´skiego
5a, 02-106 Warsaw, Poland. E-mail: wbal@ibb.waw.pl, tfraczyk@ibb.waw.pl
Electronic supplementary information (ESI) available: MALDI-MS spectra and
SDS-PAGE gels of AAT protein and cleavage fragments; UV-vis spectra of Ni(II)
complexes with peptide; UV-vis spectra of nickel complexes of hydrolysis product
incubated with H
2
O
2
; ESI-MS spectrum of oxidised peptide; the list of PDB codes
of non-mutated AAT structures. See DOI: 10.1039/c4mt00316k
Received 29th November 2014,
Accepted 24th December 2014
DOI: 10.1039/c4mt00316k
www.rsc.org/metallomics
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the Ni(II) ion is coordinated to the N1 imidazole nitrogen and
three preceding main chain nitrogens (those of His, Xaa, and
Ser/Thr residues). The specific geometry of this complex
enables an acyl shift reaction, transferring the carbonyl of the
amide bond linking the Aaa and Ser/Thr residues towards
the hydroxyl group of the Ser/Thr side chain. An ester formed
in this fashion is subsequently hydrolysed, completing the
cleavage.
15,16
Such a reaction can lead to the damage and loss
of function of intra- and extracellular proteins, and to the
formation of new epitopes recognised as non-native by the
immune system. In an early study, we demonstrated that
histone H2A undergoes such hydrolysis in cells exposed to
Ni(II) salts.
17
More recently, we indicated that ca. 25% of human
C2H2 zinc fingers and human annexins A1, A2, and A8 harbour
such sequences and are potential targets for Ni(II) ions.
18,19
However, sequences potentially susceptible to such hydrolysis
appear on the surfaces of many other human proteins.
Ni(II)-dependent peptide bond hydrolysis is a pH-dependent
reaction as it relies on the formation of square-planar 4N nickel
complexes. The pK
a
values characterizing such complexes in
Aaa–Ser/Thr–Xaa–His–Zaa peptides are usually above 8.0.
19
This means that at pH 7.4, which is recognized as close to
the physiological pH (e.g., in blood), the complexation levels
and hydrolysis rates are low. However, there are at least several
conditions where local pH in the organism is higher, thus
potentially facilitating hydrolysis. Values of pH as high as 8.9
were observed in chronic wounds,
20,21
and the pH in calcified
areas of human atherosclerotic plaques has a mean value of
7.55 and may reach 8.2 in some cases.
22
Alpha-1 antitrypsin (AAT) is a highly abundant serum
glycoprotein. It circulates at a concentration of 1.0–1.5 g L
1
(B25 mM).
23
AAT is serine protease inhibitor that inhibits a
range of human enzymes including neutrophil elastase and
pancreatic trypsin. It also modulates the immune system, e.g.,
by inhibiting the production of pro-inflammatory cytokines and
suppressing leukocyte migration.
23
Deficiency of this protein
is a genetic disease that is mostly due to a D342K mutation
(Z-AAT) that causes protein misfolding. It results in a protein
deficiency in the lungs, which leads to lung tissue destruction
due to insufficient inhibition of elastase.
24
AAT may come into contact with Ni(II) ions in blood as
relatively high concentrations of this metal were detected in the
blood of nickel-exposed humans including smokers.
25,26
Furthermore, AAT was found in the bronchoalveolar lavage
fluid.
27
This means that AAT may have contact with highly
abundant free or loosely-bound Ni
2+
ions, released locally on
the surface of lung epithelium from airborne dust and cigarette
smoke particles. AAT was also detected in sweat;
28
thus, along
with other components like urocanic acid,
29
it may interact
with Ni(II) ions released from objects made of stainless steel
and other nickel alloys.
Our study was devoted to exploring interactions of Ni(II)
with AAT within the paradigm of Ni(II)-dependent peptide
bond hydrolysis. Its purpose was to see whether this reaction
may yield molecular mechanisms pertaining to AAT-related
pathologies.
Experimental
Material
Trypsin (sequencing grade modified) was obtained from Promega.
HEPES was purchased from Carl-Roth. Alpha-1 antitrypsin isolated
from human blood plasma was purchased from Sigma, cat. no.
A9024. Nickel(II) nitrate hexahydrate (99.999% pure on the trace
metal basis) and other reagents were obtained from Sigma-Aldrich.
Peptide synthesis
Peptides were synthesised in the solid phase according to the Fmoc
protocol
30
using an automatic peptide synthesiser (Prelude, Protein
Technology). Crude peptides were purified by HPLC, and their
identities were checked by ESI-MS, as described before.
31
HPLC analysis of AAT hydrolysis
Samples containing 50 mM AAT and 2.5 mM Ni(NO
3
)
2
were
prepared in 20 mM HEPES (pH 7.4 or 8.2) and incubated at
37 1C. Forty microlitre aliquots were periodically collected from
the reaction mixtures, frozen in liquid nitrogen and stored at
20 1C until analysis with HPLC (Breeze, Waters) on a C18
column with detection at 220 and 280 nm. A linear gradient
from 100% solution A (0.1% TFA in water) to 70% solution B
(95% acetonitrile, 0.1% TFA) in 70 minutes was used. Collected
fractions were lyophilised and analysed by SDS-PAGE, ESI-MS
and MALDI-MS.
SDS-PAGE analysis of AAT hydrolysis
Samples containing 10 mM AAT and 0.5 mM Ni(NO
3
)
2
were
prepared in 20 mM HEPES, (pH 7.4 or 8.2) and incubated at
37 1C. Thirty microlitre aliquots were periodically collected from
the reaction mixtures, frozen in liquid nitrogen and stored at
20 1C until separation using SDS-PAGE in Tris-glycine buffer.
The gels were stained with SYPRO Ruby Protein Gel Stain.
HPLC analysis of peptide hydrolysis
Samples containing 0.8 mM peptide, 1 mM Ni(NO
3
)
2
and 20 mM
HEPES at pH 7.4 or 8.2 were incubated at 37 1C. Twenty
microlitre aliquots were periodically collected from the samples
and acidified by the addition of 20 mL of 2% TFA to stop the
hydrolysis reaction.
16
The products of hydrolysis were then
separated by HPLC, and their identities were checked by ESI-MS.
Kinetic analysis
From the kinetic point of view, the Ni(II)-dependent peptide
bond hydrolysis is practically a two-step reaction because the
initial Ni(II) coordination step is much faster than the other two
steps. The first slow step is the formation of an intermediate
ester (with the same mass as a substrate), and the second is the
formation of the final hydrolytic products. However, in our
protein experiments, the intermediate ester could not be separated
fromthenativeproteinbyHPLCandSDS-PAGE.Thus,weanalysed
the hydrolysis reaction as effectively a one-step process, taking the
sum of the unreacted protein and the intermediate ester as
substrate. The rate constants were determined using equations
for first order reactions:
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S(t)=S
0
e
kt
(1)
P(t)=P
0
(1 e
kt
) (2)
where S(t)andP(t) are concentrations of substrate and products,
respectively, at a given time t,S
0
and P
0
are the initial concentra-
tions of substrate and products, respectively, and kis the rate
constant. To compare results from protein and peptide studies,
we employed the same calculation method for the analysis of the
hydrolysis of peptides.
Effect of Ni(II) on AAT inhibitory activity
The samples containing 50 mM AAT, 2.5 mM Ni(NO
3
)
2
and 20 mM
HEPES (pH 8.2) were incubated at 37 1C. One hundred microlitre
aliquots were collected after 0, 6, 12, and 24 hours of incubation
andstoredat41C until the start of the enzymatic experiment.
Before the enzymatic experiment, the aliquots were diluted to
obtain an AAT concentration of 0.55 mM.
The reaction mixture for the enzymatic experiment con-
tained AAT, trypsin, and the DAEFRHDSGYEVHHQK peptide
serving as a substrate for trypsin. Trypsin preferentially cleaves
peptide bonds’ C-terminal to Arg or Lys. Therefore, the expected
products of the proteolytic activity of trypsin were peptides
DAEFR and HDSGYEVHHQK. In the inhibition assay, 5.13 mL
of diluted aliquots containing 0.55 mM AAT and collected at
given time points of incubation was added to 50 mLof0.75mM
peptide. Then, 1.2 mLof20ngmL
1
of trypsin was added, and the
reaction mixture was incubated for 15 min at 37 1C. The trypsin
activity was stopped by the addition of 2% TFA and decreasing
the temperature to 4 1C. Reagents were separated by HPLC, and
the peptides in the HPLC peaks were analysed by ESI-MS.
These experiments were repeated three times. The control
experiment without AAT (maximum activity of trypsin) was also
performed. The trypsin to peptide ratio was optimised so that
the consumption of peptide (substrate of trypsin) did not
exceed 20% after 15 min. The amount of AAT was optimised
to not exceed 65% inhibition of trypsin. The final molar ratio of
AAT to trypsin was ca. 2:1.
Oxidative properties of products of Ni(II)-dependent peptide
bond hydrolysis
Samples containing 1.2 mM peptide and 1.0 mM Ni(NO
3
)
2
were
incubated for 24 h at pH 10 and 60 1C to hydrolyse the peptide.
After the hydrolysis was completed, phosphate buffer was
added to the sample to the final concentration of 20 mM, and
pH was adjusted to 7.4. Then, a solution of hydrogen peroxide
was added to a final Ni(II):H
2
O
2
ratio of 1: 10. The control
reaction contained no Ni(II) ions. The progress of reaction was
monitored by UV-vis spectroscopy every 10 min within 16 h over
the range of 240–850 nm at 37 1C on a LAMBDA 950 UV/vis/NIR
spectrophotometer (PerkinElmer). The products of reaction were
also analysed by ESI-MS/MS.
UV-vis titrations
Samples containing 0.95 mM peptide and 0.9 mM Ni(NO
3
)
2
were titrated with small portions of concentrated NaOH in the
pH range of 3.0–11.5. The spectra were recorded on a LAMBDA
950 UV/vis/NIR spectrophotometer (PerkinElmer) over the spec-
tral range of 330–850 nm at 25 1C.
Results
Location of hydrolytic cleavage sites in AAT
AAT is biosynthesised initially as a single-chain 418 amino acid
protein. This protein is further post-translationally processed
by the cleavage of the N-terminal 1–24 signal peptide and
glycosylation of three asparagine residues (Asn46, Asn83, and
Asn247; the amino acid residue numbering throughout this
paper is for the protein without a signal peptide). Thus, the
active form of AAT in serum is a glycoprotein with 394 amino
acid residues.
There are two amino acid sequences potentially susceptible
to Ni(II)-dependent peptide bond hydrolysis in AAT. The hydrolysis
within the first of them,
12
DTSHHD
17
, can occur before Thr13 or
Ser14; in both cases, an active Aaa–Ser/Thr–Xaa–His–Zaa complex
may be formed. Thus, the cleavage in this region can release either
EDPQGDAAQKTD or EDPQGDAAQKTDT as N-terminal products,
with masses 1274 Da or 1375 Da, respectively. The other hydrolysis
site present in the sequence is
284
ASLHL
288
,anditscleavagewould
release a C-terminal 285–394 fragment of the protein with a mass of
11 841 Da. The middle part of the protein contains all three
glycosylated asparagine residues. Thus, the theoretical mass of
the middle part is a sum of 31 245 Da or 31 144 Da for the amino
acid part and the additional mass resulting from glycosylation.
Protein hydrolysis
We used commercial samples of AAT purified from human
blood plasma as substrates for Ni(II)-dependent peptide bond
hydrolysis. The protein was subjected to this reaction in the
presence of Ni(II) ions at 37 1C and pH 7.4 or 8.2. HPLC analyses
in conjunction with ESI-MS and MALDI-MS demonstrated that
both N- and C-terminal fragments of AAT were cleaved off, as
expected from the sequence analysis above. We used absorbance
at 220 nm, characteristic of amide bonds, to detect reaction
products using HPLC. Non-hydrolysed AAT gave a peak at 67 min
(Fig. 1A). The mass of the protein measured by MALDI-MS was
50.2 kDa (Fig. S1, ESI). We also observed a small MALDI-MS
signal from a dimeric form of AAT at 100 kDa. New peaks
appeared gradually at 23 and 62 min (Fig. 1B) in samples
collected at increasing times of incubation with Ni(II)ions.ESI-MS
measurements of the peak at 23 min showed the presence of
two molecules with masses of 1274 and 1375 Da. Both match the
theoretical masses of cleavable N-terminal fragments of AAT;
the lighter corresponds to the 1–12 peptide, and the heavier to the
1–13 peptide. ESI-MS analysis of the peak at 62 min showed the
presence of a molecule with a mass of 11 838 Da, corresponding to
the theoretical mass of the cleaved-off C-terminal 285–394 frag-
ment of the AAT sequence. The peak at 67 min from Ni(II)-treated
samples contained two protein fragments with masses of 37.0 and
48.8kDa,asmeasuredbyMALDI-MS.Wealsodetectedaweak
signal from the dimeric form of the 37.0 kDa fragment (74 kDa)
and probably the mixed dimer of the 37.0 kDa fragment
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with 48.8 kDa and/or 50.2 kDa molecules (86–87 kDa). Example
spectra are shown in Fig. S1 (ESI). The mass of 37.0 kDa
matches the central glycosylated fragment of AAT with the
N- and C-termini cleaved off. The 48.8 kDa molecule corre-
sponds to AAT with only the N-terminus cleaved off. We also
detected a very small constant peak at 57 min in all chromato-
grams. MALDI-MS measurements showed that it contains a
molecule with a mass of 67.5 kDa, which we consider as an
unreactive impurity (Fig. S1, ESI).
The absorbance at 220 nm is proportional to the number of
amide bonds in peptides (those of main chain and of asparagine
and glutamine side chains). Using this relationship, we calculated
molar fractions of N- and C-terminal fragments accumulating during
the hydrolysis reaction. The time-wise increase of the amount of
N- and C-terminal fragments cleaved from AAT is presented in Fig. 2.
The hydrolysis is faster at the N-terminus than at the C-terminus.
The cleavage at the N-terminus is a biphasic process (Fig. 2),
and the first phase is much faster than the second one.
However, the first phase consumes only half of the protein.
Taking into account the fact that we detected dimeric forms of
AAT by MALDI-MS, this result suggests that only one monomer
in the dimer can undergo fast N-terminal hydrolysis. The
second phase is much slower, but results in the complete
cleavage of the N-terminal part of all AAT molecules. At pH
7.4, we observed only the first phase because of the slower rate
of reaction, which required impractically long incubation times
in the order of several months. After 24 hours, 47% and 4% of
cleaved N-terminal fragments appeared at pH 8.2 and 7.4,
respectively.
The hydrolysis at the C-terminal part of AAT proceeded more
slowly than that at the N-terminus (Fig. 2). We observed one
phase of this process, reaching half of the AAT monomers.
These results suggest that the hydrolysis of the C-terminus
occurs only in half of AAT molecules; however, the time course
of our experiments did not allow us to exclude the biphasic
pattern of hydrolysis at the C-terminus, analogous to that
confirmed for the N-terminus. After 24 hours, 11% and 1% of
the cleaved C-terminal fragment appeared at pH 8.2 and 7.4,
respectively.
We employed SDS-PAGE to monitor the Ni(II)-dependent hydro-
lysis of AAT complementarily to the HPLC data. The C-terminal
hydrolysis was easily observed on the gels as the difference between
native AAT and that cleaved at Ser-285 is high enough (12 kDa) to
discriminate between the two forms (Fig. 3). Furthermore, the
cleaved-off C-terminal part was also detected in the gels. As pre-
sented above, the N-terminal hydrolysis leads to two products of a
very similar low molecular weight of ca. 1 kDa, and the difference
between the non-hydrolysed and hydrolysed proteins was only
1 kDa. Neither could be detected in our SDS-PAGE gels. Thus, the
electrophoretic analysis was limited to the C-terminal cleavage of
AAT. Its reaction rate constant at pH 8.2 calculated from SDS-PAGE
experiments (29 210
7
s
1
)isconsistentwiththevalue
calculated from HPLC experiments (27 210
7
s
1
). The
analogous calculation from SDS-PAGE data for pH 7.4 gave a rate
constant of 2.9 0.4 10
7
s
1
, identical to that calculated from
the HPLC data.
We performed an additional SDS-PAGE analysis of samples
collected from peaks separated by HPLC (Fig. S2, ESI). It allowed us
to confirm the identity of protein bands. The control incubation of
AAT without Ni(II)ionsundertheconditionsofhydrolysisexperi-
ments showed that the bands of hydrolysis products did not appear
in the absence of Ni(II) (Fig. S3, ESI). Thus, the observed cleavage is
theresultofinteractionsofAATwithNi(
II)ions.
Influence of Ni(II)-dependent hydrolysis on the function of AAT
Inhibition of proteases is the physiological function of AAT.
Thus, we checked whether the Ni(II)-dependent peptide bond
hydrolysis of AAT could compromise this activity. We chose
trypsin as the protease and applied the DAEFRHDSGYEVHHQK
peptide as its substrate. Using HPLC, we found that the incuba-
tion of AAT with Ni(II) ions decreases the inhibition of trypsin by
AAT (Fig. 4). The extent of impairment of AAT inhibitory activity
correlates very well with the extent of cleavage of AAT by Ni(II).
Peptide hydrolysis
We synthetised two peptides, Ac-KTDTSHHDQ-am and
Ac-RSASLHLPK-am; they represent the 10–18 and 282–290
Fig. 1 HPLC chromatograms of the products of Ni(II)-dependent hydrolysis
of AAT for the initial reaction time (A) and after 28 days of incubation at pH
8.2 and 37 1C (B). The sample initially contained 50 mMAAT,2.5mM
Ni(NO
3
)
2
, and 20 mM HEPES. Absorbance at 220 nm was used for detection.
Fig. 2 Kinetics of Ni(II)-dependent peptide bond hydrolysis in AAT at pH 8.2
(A) and 7.4 (B), both at 37 1C, followed by HPLC. The samples contained 50 mM
AAT, 2.5 mM Ni(NO
3
)
2
and 20 mM HEPES. The cleavage of the N-terminal
(black squares) and C-terminal (red dots) fragments of the protein is shown.
The inset in panel A shows the first two days of incubation at pH 8.2.
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fragments of the AAT sequence, respectively, covering sites of
Ni(II)-dependent hydrolysis. The N- and C-termini of these peptides
were blocked by acetylation (Ac-) and amidation (-am), respectively,
to make them more reliable models of protein sequence with respect
to Ni(II) binding and hydrolysis. The Ac-KTDkTkSHHDQ-am peptide
has two cleavage sites located next to each other: between Asp and
ThrandbetweenThrandSer(markedwitharrows).Wedidnot
observe a significant preference of cleavage between these sites. For
Ac-RSAkSLHLPK-am, we observed the expected hydrolysis of the
bond between Ala and Ser (arrow).
To compare results for the hydrolysis of model peptides and the
protein, we carried out the reaction at 37 1CandpH7.4or8.2and
calculated the rate constants of hydrolysis, treating the sum of
native peptide and intermediate ester as a formal reaction sub-
strate. The rate constants for the hydrolysis of peptides and AAT are
shown in Table 1. At pH 7.4, the rate constant for hydrolysis at the
N-terminal sequence calculated from the data recorded over the
initial 24 h for the model peptide is similar to that of AAT. At pH
8.2, this constant for AAT is even higher than that for the peptide.
However, the hydrolysis at the N-terminal part of AAT monitored
from the 2nd to the 28th day at pH 8.2 was much slower compared
to the hydrolysis of Ac-KTDTSHHDQ-am. This second reaction
phase was not seen at pH 7.4, despite prolonging the incubation
to over 30 days. The hydrolysis at the C-terminal part of AAT was
monophasic and uniformly much slower than that for its model
peptide at pH 7.4 and 8.2.
Ni(II) complexes with peptides
The formation of a 4N Ni(II) complex is a prerequisite for the
hydrolysistooccur.Thehydrolysisrateisdirectlyproportionalto
its concentration, which is strongly dependent on pH. The 4N
complexes are low-spin species with a characteristic absorption band
at ca. 450 nm. For some peptides, 3N complexes are also low-spin,
and their absorbance adds up to the mentioned band. To decipher
the pH-dependence of complexation, we performed UV-vis titrations.
The results for Ac-RSASLHLPK-am are shown in Fig. 5, and those for
Ac-KTDTSHHDQ-am are shown in Fig. S4 (ESI). The pKvalues
for the formation of 4N low-spin Ni(II) complexes are 8.6 for
Ac-KTDTSHHDQ-am and 8.4 for Ac-RSASLHLPK-am. The analysis
of low-spin d–d bands obtained in these titrations indicated that
they belonged to a 4N geometry.
19
It has to be noted that the low-
spin complex of Ac-KTDTSHHDQ-am peptide is in fact a sum of two
complexes spanning the TSH and SHH residues. The shapes of the
low-spin spectra obtained for this peptide did not change through-
out the titration, indicating that the pKvalues for these two complex
species were very similar to each other.
Oxidative properties of 4N Ni(II) complexes with hydrolysed
peptides
Low-spin 4N Ni(II) complexes often exhibit pro-oxidative proper-
ties. Using model peptides, we aimed to find out whether this
Fig. 3 SDS-PAGE gels of products of the Ni(II)-dependent hydrolysis of
AAT at 37 1C and pH 8.2 (A) or 7.4 (B) performed for 10 mM AAT and 0.5 mM
Ni(NO
3
)
2
in 20 mM HEPES for time points indicated above the lanes. The
first lane contains the molecular weight markers with weights indicated on
the left side of the gels. Four species were detected: impurity; x–394
AAT – native 1–394 AAT and 13/14–394 AAT; x–284 AAT – 1–284 and 13/14–
284–394 AAT and 285–394 AAT (numbers indicate the amino acid fragments).
Proteins were visualised by SYPRO Ruby Protein Gel Stain.
Fig. 4 The correlation of the N- and C-terminal hydrolysis of AAT by Ni(II)
with the AAT inhibitory activity towards trypsin. The left side axis shows
molar fractions of AAT cleaved at the N-terminus (black squares) and
C-terminus (red dots) determined for samples containing 50 mMAAT,2.5mM
Ni(NO
3
)
2
and 20 mM HEPES at pH 8.2 and incubated at 37 1C. The dashed
line shows the arithmetic sum of these two fractions. The right side axis
shows the residual activity of trypsin in the presence of Ni(II)-treated AAT
tested for 15 min at 37 1C with a trypsin to AAT ratio of B1 : 2 (green
triangles). Error bars of triplicate repetitions of the assay are contained within
the symbols. The activity of trypsin in the absence of AAT is represented
by a dotted green line at the top of the graph.
Table 1 First-order rate constants determined for the Ni(II)-dependent
hydrolysis of model peptides and AAT protein at 37 1C
a
Site in AAT or peptide sequence
k(s
1
10
7
)
pH 8.2 pH 7.4
AAT
12
DTSHHD
17
273(3)
b
/2.8(4)
c
7.2(8)
Peptide Ac-KTDTSHHDQ-am 180(2) 8.3(4)
AAT
284
ASLHL
288
27(2) 2.9(4)
Peptide Ac-RSASLHLPK-am 390(2) 19(3)
a
Standard deviations on the least significant digits are given in
parentheses.
b
Calculated for initial 24 h.
c
Calculated for days 2–28.
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may be the case for Ni(II) complexes formed upon interaction with
AAT. We focused on the complexes of hydrolysis products rather
than substrates because their abundance at pH 7.4 approaches
100%, which is much higher than that of pre-hydrolytic 4N
complexes. This is due to the fact that in the C-terminal reaction
product, the crucial His residue is at the 3
rd
position in the peptide
chain, enabling highly cooperative chelate ring formation.
32,33
Thus, we checked the oxidative properties of 4N Ni(II) complexes
with C-terminal products of hydrolysis of both model peptides at
pH 7.4. For this purpose, they were incubated with a ten-fold excess
of H
2
O
2
for 16 hours. This led to changes in their UV-vis spectra,
shownforAc-RSASLHLPK-aminFig. 6 and for Ac-KTDTSHHDQ-am
in Fig. S5 (ESI). For both peptides, we observed a decrease in
the initial d–d band at ca. 420 nm accompanied by a transient
appearance of a band at 375 nm, which was particularly pronounced
for the hydrolysis products of Ac-RSASLHLPK-am. However, the
biggest changes were observed at 310 nm, where a new, intense
and permanent band appeared gradually. The majority of the
changes in the spectra were accomplished within 6 hours.
The ESI-MS/MS analysis of reaction products after 30 min for
Ac-RSASLHLPK-am demonstrated the appearance of a modified
peptide containing a mono-oxidized Leu residue (Fig. S6, ESI).
Discussion
Hydrolysis of AAT and model peptides
The results presented in Fig. 1–3 and Table 1 demonstrate that
Ni(II) ions cleave AAT hydrolytically at all three peptide bonds
predicted by our sequence analysis: D12–T13, T13–S14, and
A284–S285. The Ni(II) reactive domains for the first two sites,
DkTSH and TkSHH, overlap, and cleavage at one of them
precludes another. This is because the cleavage of the T13–
S14 bond leaves the D12–T13 bond in the N-terminal hydrolysis
product, decomposing its reactive domain, while the cleavage
of the D12–T13 bond yields a 4N Ni(II) complex with the now
N-terminal TSH sequence, which locks the T13 and S14 residues
in an unreactive conformation.
15,31,34
We detected the expected
products of cleavage by ESI-MS for both the model peptide and
the protein, but the products of both reactions were too similar
to be easily separated and quantified. Therefore, we considered
the reactions at these two sites jointly as one cleavage process.
The rate constants for model peptides measured at pH 7.4 and
8.2 differ by a factor of ca. 20 for both model peptides; this is in
satisfactory agreement with the relative abundances of the active
complexes derived from the pKvalues for the formation of 4N
complexes.
The comparison of rate constants between model peptides
and protein sites reveals a high similarity in reaction rates
for the N-terminal cleavage site. The concentration of active
species for individual cleavage sites in AAT could not be
determined for the relatively low concentration of protein used,
but they should be similar to those of the model peptide and
the protein sequence at a given pH. The protein was studied at
a lower concentration than the peptide, but with a higher Ni(II)
concentration, which compensates for the protein dilution.
15,31
Indeed, the rate constants for the N-terminal protein cleavage
and the Ac-KTDTSHHDQ-am peptide (Table 1) are very similar
at both tested pH values. Therefore, this region of the protein
must be able to freely wrap around Ni(II) ion to form the active
species. This finding is consistent with the available data on the
conformation of AAT. The first 22 amino acid residues are not
resolved in the crystallographic structures of AAT available in
the RCSB PDB database (relevant PDB codes are provided in
Table S1, ESI), indicating that the N-terminal part of AAT is
flexible/unstructured.
The reaction at the C-terminal cleavage site for AAT was
much slower than that at the N-terminal site, although its
model peptide Ac-RSASLHLPK-am was hydrolysed more than
two-fold faster than Ac-KTDTSHHDQ-am. This observation can
be easily explained by the involvement of the RSASLHLPK
sequence in the b-sheet structure (cf. structures with PDB codes
given in Table S1, ESI), a conformation inconsistent with the
steric requirements of the active Ni(II) complex.
Strikingly, the above picture is only valid for about half
(exactly 47%) of the N-terminal cleavage sites available in the
Fig. 5 (A) UV-vis spectra of Ni(II) complexes of the Ac-RSASLHLPK-am
peptide at different pH values coded with rainbow colours from red (the
lowest pH, 4.0) to dark blue (the highest pH, 11.5); and (B) pH-dependence
of the absorbance at 457 nm derived from the spectra shown in panel A.
The spectrum of the peptide in the absence of Ni(II) ions is shown as a
dotted grey line. The spectra were recorded at 25 1C for samples containing
0.95 mM peptide and 0.9 mM Ni(NO
3
)
2
.
Fig. 6 (A) UV-vis spectra of nickel complexes of the C-terminal product
of the hydrolysis of Ac-RSASLHLPK-am (1.0 mM) incubated with 10 mM
H
2
O
2
for 16 hours at 37 1C. The spectra were recorded every 10 min. The
spectrum recorded prior to H
2
O
2
addition is marked with a thick black line.
Arrows indicate the direction of changes at 310 nm, 375 nm, and 480 nm.
(B) Time course of spectral changes at 310 nm (black), 375 (red), and
480 nm (green).
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protein. Afterwards, the reaction at this site slows down by ca.
two orders of magnitude (judged from the pH 8.2 data). A very
similar behaviour was seen for the C-terminal site, but a much
slower reaction precluded the observation of the second reac-
tion phase (Fig. 2). This behaviour can be explained with help
from the MALDI-MS results, which showed a propensity of AAT
and its forms truncated by hydrolytic cleavage to dimerise. In
particular, we detected mixed dimers of the native AAT mono-
mer with molecules truncated at the either N- or C-terminus.
Apparently, the cleavage of one monomer changes the confor-
mation of a dimer so that the corresponding cleavage site in the
other monomer becomes less accessible to Ni(II) ions. Judging
from the masses of dimers, the dimerisation domain is located
at the glycosylated central part of AAT. The issue of AAT
dimerisation and its relation to the biological activity of this
protein requires further research, especially as we are not aware
of any prior research indicating the existence of such species.
Effects of hydrolysis on AAT activity
We tested the effect of N- and C-terminal truncation of AAT on
its ability to inhibit trypsin, one of its natural targets. As shown
in Fig. 4, the loss of AAT activity is initially directly proportional
to the sum of N- and C-terminal truncation; however, at longer
hydrolysis times, the effect is somewhat stronger than additive.
The loss of AAT activity due to either of the hydrolysis phenomena
can be readily understood on the basis of the known mechanism of
action of this inhibitor. In brief, native AAT has a metastable
conformation. The attack of a protease on the reactive centre loop
of AAT triggers a dramatic conformational rearrangement of AAT
and traps the protease in a distorted and catalytically inactive
form.
35,36
While the metastability of the protein’s tertiary structure
is a fundamental aspect of the inhibitory activity of AAT, it also
renders the protein highly susceptible to loss of function when the
conformational ensemble is altered. Such an effect is inevitable
upon a significant lesion such as the deletion of the N-terminal
12- or 13-peptide. Furthermore, the C-terminal part contains the
reactive centre loop; thus, its separation from the rest of the protein
molecule can impair the fundamental mechanism of protease
inhibition.
A stronger than additive decrease of AAT activity against trypsin
at longer hydrolysis times can be provisionally explained by the
formation of heterodimers between native AAT and products of its
Ni(II)-dependent hydrolysis, which would accumulate to a concen-
tration enabling them to bind to native AAT molecules and,
perhaps, turning them inactive. Z-AAT mutant polymerisation is
considered to be one of causes of pathology in AAT deficiency.
36
Our finding that native AAT and its truncated forms can form
dimers with altered activities suggests an interesting direction of
further research into the possible pathological effects of Ni(II)–AAT
interaction.
Oxidative reactivity of hydrolysis products
Some square-planar Ni(II) complexes are known to induce oxidative
damage in macromolecules in the presence of naturally occurring
oxidants such as H
2
O
2
.
37–42
These reactions proceed via strongly
oxidizing Ni(III) species.
43–45
In order to find out whether products
of AAT hydrolysis might undergo such reactivity, we incubated
Ni(II)-hydrolysed model peptides with a ten-fold molar excess of
H
2
O
2
and followed the course of reactions using UV-vis spectra and
ESI-MS. The spectra of samples recorded prior to the H
2
O
2
addition
confirmed the presence of 4N Ni(II) complexes with C-terminal
hydrolysis products. For Ac-RSASLHLPK-am, the d–d band
parameters are e= 177 M
1
cm
1
at l
max
= 418 nm and e=
97 M
1
cm
1
at l
sh
= 480 nm (e= 159 M
1
cm
1
at l
max
=423nm
and e=81M
1
cm
1
at l
sh
= 480 nm for Ac-KTDTSHHDQ-am). For
Ac-RSASLHLPK-am peptide, the H
2
O
2
addition resulted in the
appearance of a new transient spectroscopic species at 375 nm
(eZ260 M
1
cm
1
) formed at the expense of the original complex,
as evidenced by a decrease in its d–d band. The transient species
peaked at about 30 min of incubation and then decomposed slowly,
giving rise to a new intense band at ca. 310 nm (eZ1200 M
1
cm
1
)
and a residual absorption at 418 nm, about one-third of the initial
absorption of the Ni(II) complex. An isosbestic point at 351.5 nm
confirmed the strictly stepwise character of the second reaction,
which proceeded roughly according to first-order kinetics (k=1.4
0.1 10
4
s
1
). The spectral changes were qualitatively similar for
the Ac-KTDTSHHDQ-am peptide with a very similar final product
(Fig. S5, ESI).
Based on the literature,
43–45
we can propose that H
2
O
2
oxidised the initial Ni(II) complexes to a corresponding Ni(III)
species with a similar geometry, which then rearranged into a
tetragonal, likely square-pyramidal geometry by the addition of
an oxygen-type axial donor. These reactions were accompanied
by the generation of reactive oxygen species (ROS), as evidenced
by oxygen insertion in the side-chain of the Leu residue (Fig. S6,
ESI). This experiment proved the potential of the products of
Ni(II)-dependent AAT hydrolysis to exert oxidative damage by
ROS generation.
Biological relevance and research perspective
Alpha-1 antitrypsin deficiency is a genetic disorder manifesting
itself with emphysema and, less frequently, liver cirrhosis.
Cigarette smoke is a very strong factor for emphysema.
24
Among the many mutations in the AAT gene, the Z-mutation,
D342K, causes the disease because the mutated protein tends
to polymerise and remain confined in liver cells instead of
being released to the bloodstream and lungs.
46
In turn, the
accumulation of Z-AAT in liver cells causes liver disease including
liver cirrhosis and carcinogenesis.
47
Metal ions were implicated in AAT loss in some early
studies, with only cadmium implicated as an AAT-destroying
agent.
48
To our best knowledge, the mechanism for this process
was never provided, and little research followed this initial
observation. Nickel was indicated to bind to AAT,
49
but no
effect on the AAT inhibitory activity was detected.
48
Our results
clearly show that this negative finding was due to an insufficient
incubation time, which allowed our predecessors to only detect
the reversible coordination step of the Ni(II)–AAT interaction.
The deleterious effects of contact between Ni(II) and AAT
depend on the initial amount of 4N complex and thus are
strongly dependent on the pH, concentrations of AAT and Ni(II)
and incubation time. The hydrolysis reactions are very slow at
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pH 7.4; only a few percent of AAT is hydrolysed per day at a high
Ni(II) concentration. The reactions are 10–40 times faster at pH
8.2. Such high pH values can be found locally in the human
organism as a result of several pathologies. For example, the
local pH of calcified atherosclerotic plaques can reach 8.2, and
the pH of chronic wounds can be as high as 9.
20–22
Taking into
account that AAT hydrolysis under such conditions may be very
fast, and that the half-life of AAT in human serum is approximately
4–5 days,
50
one can expect that the presence of Ni(II)ionsmay
aggravate AAT deficiency if accompanied by other conditions.
Among organs, lungs are the most sensitive to AAT deficiency.
They may be exposed to Ni(II) ions by the inhalation of nickel-
containing particles resulting from industrial processes, com-
bustion of fossil fuels, and cigarette smoke. The latter was
recently confirmed to deposit significant amounts of nickel in
the lungs.
7,8,25,26,51,52
One important feature of this exposure is
that micrometre-sized nickel containing particles settle locally
on the bronchial or lung surface and can be incorporated to
phagocytosing cells as a whole.
53–55
This leads to extremely high
local Ni(II) concentrations and may result in fast AAT destruc-
tion. Notably, smoking was shown to lead to more severe
pulmonary disease
56
and is an important factor in the develop-
ment of lung disease associated with AAT deficiency.
24
For these reasons, one can envision local AAT deficiency and
resulting lung tissue damage by neutrophil elastase upon
exposure to nickel-containing particles even if the overall AAT
level is hardly affected. The diseases that can be aggravated
according to the molecular mechanism proposed in this work
include COPD and asthma.
The physiological concentration of AAT in serum is ca. 25 mM.
23
However, there is a threshold level of 11 mM below which (by
definition, in AAT deficiency) the risk of pulmonary emphysema
rises significantly.
50
Taking these facts together, despite the rela-
tively low amounts of ATT potentially cleaved by Ni(II)incirculation,
the Ni(II)-dependent hydrolysis of AAT can become important in
AAT deficiency, where even a small decrease in the amount of AAT
can have severely adverse effects.
Conclusions
We have shown that human alpha-1 antitrypsin is cleaved by
Ni(II) ions into specific fragments. The cleavage abolished the
ability of AAT to inhibit trypsin and yielded ROS-generating
species. Our results suggest that Ni(II) exposure can contribute
to AAT-related pathologies, particularly those of the respiratory
tract. Further research is warranted to verify this hypothesis.
Acknowledgements
This study was partially financed by the project ‘‘Metal dependent
peptide hydrolysis. Tools and mechanisms for biotechnology,
toxicology and supramolecular chemistry’’, carried out as part of
the Foundation for Polish Science TEAM/2009-4/1 program and
co-financed from European Regional Development Fund resources
within the framework of Operational Program Innovative Economy.
The equipment used was sponsored in part by the Centre for
Preclinical Research and Technology (CePT), a project cosponsored
by the European Regional Development Fund and Innovative
Economy, The National Cohesion Strategy of Poland.
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Metallomics Paper
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... ligand or metalbinding regulatory sequences or an endonuclease catalytic domain) linked to ZF proteins, thereby providing an opportunity to generate artificial transcription factors or enzymes for specific DNA actions. 2,[15][16][17][18][19][20][21][22][23][24][25] Previously we reported on Ni(II)-induced peptide bond hydrolysis that occurs at the N-terminal side of Ser or Thr residues in peptides, [26][27][28][29][30][31][32][33][34][35][36] designed recombinant proteins 37,38 and flexible regions of native proteins 37,39 bearing X-(Ser/Thr)-X-His-X sequences (X being any amino acid except for Cys or Pro before or after Ser/Thr). The method found application in the affinity tag removal from fusion proteins by Ni(II) ions. ...
... To overcome this effect we used at least a 10-fold molar excess of Ni(II), as demonstrated before to be effective for protein cleavage. [37][38][39] The hydrolysis removes the His-tag, while the catalytic Ni(II) ion remains coordinated to the (Ser/Thr)-X-His motif of the protein (C-terminal product). ...
... [65][66][67][68][69][70][71] The advantage of our approach compared to previous reports is that the ATCUN site of the ZF protein is initially masked by the Ni(II) sensitive sequence, and can be activated and regulated by the addition of Ni(II) ions, temperature jump or pH jump (as the rate of the peptide bond cleavage itself is strictly dependent on these three factors). [30][31][32][37][38][39] Furthermore, the Ni(II) induced peptide-bond hydrolysis can be applied for the removal of any fusion proteins (such as affinity tags, inhibitory or activating domains) from sensitive ZF metalloproteins. Thereby it provides a chance for simple regulation and replacement of the expensive specific protease enzymes. ...
Article
In this work we demonstrate that the previously described reaction of sequence specific Ni(II)-dependent hydrolytic peptide bond cleavage can be performed in complex metalloprotein molecules, such as the Cys2His2 zinc finger proteins. The cleavage within a zinc finger unit possessing a (Ser/Thr)-X-His sequence is not hindered by the presence of the Zn(II) ions. It results in loss of the Zn(II) ion, oxidation of the SH groups and thus, in a collapse of the functional structure. We show that such natural Ni(II)-cleavage sites in zinc finger domains can be edited out without compromising the DNA binding specificity. Inserting a Ni(II)-susceptible sequence between the edited zinc finger and an affinity tag allows for easy removal of the latter sequence by Ni(II) ions after the protein purification. We have shown that this reaction can be executed even when a metal ion binding N-terminal His-tag is present. The cleavage product maintains the native zinc finger structure involving Zn(II) ions. Mass spectra revealed that a Ni(II) ion remains coordinated to the hydrolyzed protein product through the N-terminal (Ser/Thr)-X-His tripeptide segment. The fact that the Ni(II)-dependent protein hydrolysis is influenced by the Ni(II) concentration, pH and temperature of the reaction provides a platform for novel regulated DNA effector design.
... The Ser/Thr-Xaa-His sequence occurs in several human proteins. We have shown that histone H2A [18], annexins A1, A2, and A8 [19], alpha-1-antitrypsin [20], phospholipid scramblase 1, Sam68-like mammalian protein 2, and CLK3 kinase [21] contain surface-exposed fragments cleavable by Ni(II) ions. Ni(II)-prone sequences are also present in all of the melatonin biosynthesis pathway enzymes, i.e., tryptophan 5-hydroxylase 1 (TPH1), aromatic-L-amino-acid decarboxylase (AADC), serotonin N-acetyltransferase (SNAT), and acetylserotonin O-methyltransferase (ASMT). ...
... The Ser/Thr-Xaa-His sequence occurs in several human proteins. We have shown that histone H2A [18], annexins A1, A2, and A8 [19], alpha-1-antitrypsin [20], phospholipid scramblase 1, Sam68-like mammalian protein 2, and CLK3 kinase [21] contain surfaceexposed fragments cleavable by Ni(II) ions. Ni(II)-prone sequences are also present in all of the melatonin biosynthesis pathway enzymes, i.e., tryptophan 5-hydroxylase 1 (TPH1), aromatic-L-amino-acid decarboxylase (AADC), serotonin N-acetyltransferase (SNAT), and acetylserotonin O-methyltransferase (ASMT). ...
... Hence, we worked on four peptides named after the source enzyme: Ac-HALSGHAKV-am (pTPH1), Ac-TVESAHVQR-am (pAADC), Ac-ERFSFHAVG-am (pSNAT), and Ac-VRASAHGTE-am (pASMT). We have previously proven that such properly selected peptides could be good models for Ni(II)-assisted cleavage of whole proteins [20]. ...
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Nickel is toxic to humans. Its compounds are carcinogenic. Furthermore, nickel allergy is a severe health problem that affects approximately 10–20% of humans. The mechanism by which these conditions develop remains unclear, but it may involve the cleavage of specific proteins by nickel ions. Ni(II) ions cleave the peptide bond preceding the Ser/Thr-Xaa-His sequence. Such sequences are present in all four enzymes of the melatonin biosynthesis pathway, i.e., tryptophan 5-hydroxylase 1, aromatic-l-amino-acid decarboxylase, serotonin N-acetyltransferase, and acetylserotonin O-methyltransferase. Moreover, fragments prone to Ni(II) are exposed on surfaces of these proteins. Our results indicate that all four studied fragments undergo cleavage within tens of hours at pH 8.2 and 37 °C, corresponding with the conditions in the mitochondrial matrix. Since melatonin, a potent antioxidant and anti-inflammatory agent, is synthesized within the mitochondria of virtually all human cells, depleting its supply may be detrimental, e.g., by raising the oxidative stress level. Intriguingly, Ni(II) ions have been shown to mimic hypoxia through the stabilization of HIF-1α protein, but melatonin prevents the action of HIF-1α. Considering all this, the enzymes of the melatonin biosynthesis pathway seem to be a toxicological target for Ni(II) ions.
... [8][9][10] The molecular mechanisms behind these diseases are not fully elucidated, but the ability of nickel to hydrolyze proteins and peptides containing a specific sequence Ser/Thr-Xaa-His could be an important step in their development. [11][12][13][14] A number of human proteins, including histone H2A, 15 annexins, 16 a-antitrypsin, 17 phospholipid scramblase 1, 18 and transcrition factors containing zinc finger domains 19,20 are susceptible to the Ni(II)-dependent hydrolysis, resulting in their deactivation. 17 Other transition metal ions, Cu 2+ and Pd 2+ , 14,21 were also shown to drive the peptide bond cleavage via an analogous mechanism of metal-dependent hydrolysis (MdH), presented in Fig. 1. ...
... [11][12][13][14] A number of human proteins, including histone H2A, 15 annexins, 16 a-antitrypsin, 17 phospholipid scramblase 1, 18 and transcrition factors containing zinc finger domains 19,20 are susceptible to the Ni(II)-dependent hydrolysis, resulting in their deactivation. 17 Other transition metal ions, Cu 2+ and Pd 2+ , 14,21 were also shown to drive the peptide bond cleavage via an analogous mechanism of metal-dependent hydrolysis (MdH), presented in Fig. 1. In brief: the susceptible peptide/protein site initially binds the metal ion in a square-planar complex. ...
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NiO nanoparticles and non-stoichiometric black NiO were shown to be effective sources of Ni ²⁺ ions causing sequence-selective peptide bond hydrolysis. NiO nanoparticles were as effective in this reaction as their...
... Three hydrolysis sites were identified in AAT and cleavage at any of them abolished the activity of AAT in an additive manner. Also, Ni(II) ions bound to hydroly- sis products demonstrated an ability to generate reactive oxygen species [112]. AAT was hydrolyzed not only by soluble Ni(II) salts, but also in the presence of Ni(II) oxide particles, which are actual forms of nickel entry into the respiratory tract. ...
Article
Metal-assisted hydrolysis of peptide bond is a promising alternative for enzymatic cleavage of proteins with prospective applications in biochemistry and bioengineering. Many metal ions and complexes have been tested for such reactivity with a number of targets, from dipeptides through oligopeptides through proteins. The majority of reaction mechanisms reported so far is based on the Lewis acidity of a given metal ion. In the alternative hydrolysis reaction the metal ion, Cu(II), Ni(II) or Pd(II), plays a structural role by forming a square planar complex with Ser/Thr-His or Ser/Thr-Xaa-His sequence, which enables a N→O rearrangement of the acyl moiety in the peptide bond downstream from the Ser/Thr residue. Both Lewis acid and N→O acyl rearrangement reaction types are discussed in detail, including molecular mechanisms, the chemical character of hydrolytic agents, reaction conditions, and the origins of differences between the results obtained for peptide and protein models. Toxicological implications and practical applications of metal assisted peptide bond hydrolysis are also presented, with a focus on the Ni(II) assisted N→O acyl rearrangement in Ser/Thr-Xaa-His sequences.
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Amyloid-beta (Aβ) peptides, potentially relevant in the pathology of Alzheimer’s disease, possess distinctive coordination properties, enabling an effective binding of transition-metal ions, with a preference for Cu(II). In this work, we found that a N-truncated Aβ analogue bearing a His-2 motif, Aβ5–9, forms a stable Ni(II) high-spin octahedral complex at a physiological pH of 7.4 with labile coordination sites and facilitates ternary interactions with phosphates and nucleotides. As the pH increased above 9, a spin transition from a high-spin to a low-spin square-planar Ni(II) complex was observed. Employing electrochemical techniques, we showed that interactions between the binary Ni(II)–Aβ5–9 complex and phosphate species result in significant changes in the Ni(II) oxidation signal. Thus, the Ni(II)–Aβ5–9 complex could potentially serve as a receptor in electrochemical biosensors for phosphate species. The obtained results could also be important for nickel toxicology.
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Nickel is harmful to humans, being both carcinogenic and allergenic. However, the mechanisms of this toxicity are still unresolved. We propose that Ni(II) ions disintegrate proteins by hydrolysis of peptide bonds preceding the Ser/Thr−Xaa−His sequences. Such sequences occur in nuclear localization signals (NLSs) of human phospholipid scramblase 1, Sam68‐like mammalian protein 2, and CLK3 kinase. We performed spectroscopic experiments showing that model nonapeptides derived from these NLSs bind Ni(II) at physiological pH. We also proved that these sequences are prone to Ni(II) hydrolysis. Thus, the aforementioned NLSs may be targets for nickel toxicity. This implies that Ni(II) ions disrupt the transport of some proteins from cytoplasm to cell nucleus.
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The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3′-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.
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Our recent study (Angew. Chem. Int. Ed. 2015, 54, 7391-7394) has shown that Horse heart myoglobin (HHM) was selectively hydrolyzed by a range of Zr(IV)-substituted polyoxometalates (POMs) under mild conditions. In this study the molecular interactions between the Zr-POM catalysts and HHM were investigated by using a range of complementary techniques that include circular dichroism (CD), UV-Vis spectroscopy, tryptophan fluorescence spectroscopy, 1H NMR and 31P NMR spectroscopy. Trp fluorescence quenching study revealed that among all examined Zr-POMs, the most reactive POM, 2:2 Zr(IV)-Keggin, exhibited the strongest interaction with HHM. 31P NMR studies have shown that this POM dissociates in solution resulting in formation of monomeric 1:1Zr(IV)-Keggin structure, which is a likely catalytically active species. In the presence of Zr(IV)-POMs the HHM does not undergo complete denaturation, as evidenced by CD, UV-Vis, tryptophan fluorescence and 1H NMR spectroscopy. CD spectroscopy showed gradual decrease of the -helical content of HHM upon addition of Zr(IV)-POMs, with and the biggest effect was observed in the presence of large Zr(IV)-Wells-Dawson structure, while small Zr(IV)-Lindqvist POM had smallest influence on the decrease of the -helical content of HHM. In all cases the maintaining of the Soret band at 409 nm in the presence of all examined Zr-POMs indicated that no conformational changes in the protein occurred near the heme.
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Acute phase proteins constitute an essential component of the innate immune response during infection or inflammation. Human alpha-1-antitrypsin (A1AT) is one of the major acute-phase proteins found in the circulation that has originally been named for its ability to inhibit trypsin. Nonetheless, research on inhibitory functions of A1AT revealed that this protein is a major natural inhibitor of elastase, proteinase 3, and other serine proteases released from activated neutrophils. Nowadays, A1AT is viewed as a broad-spectrum inhibitor of proteases and also as a protein with antiinflammatory/immunomodulatory properties. In spite of a large amount of literature about the antiinflammatory properties ascribed to A1AT, defining the mechanisms of A1AT action remains an important priority. At least some of the pleiotropic activities of A1AT are linked to the specific molecular forms caused by oxidative modifications, nitrosylation, polymerization, cleavage, and/or interaction with other molecules.
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Nickel is harmful for humans, but molecular mechanisms of its toxicity are far from being fully elucidated. One of such mechanisms may be associated with the Ni(II)-dependent peptide bond hydrolysis, which occurs before Ser/Thr in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8, proteins modulating the immune system, contain several such sequences. To test if these proteins are potential molecular targets for nickel toxicity we characterized the binding of Ni(II) ions and hydrolysis of peptides Ac-KALTGHLEE-am (A1-1), Ac-TKYSKHDMN-am (A1-2), and Ac-GVGTRHKAL-am (A1-3), from annexin A1, Ac-KMSTVHEIL-am (A2-1), and Ac-SALSGHLET-am (A2-2), from annexin A2, and Ac-VKSSSHFNP-am (A8-1), from annexin A8, using UV-vis and CD spectroscopies, potentiometry, isothermal titration calorimetry, HPLC and ESI-MS. We found that at physiological conditions (pH 7.4 and 37 °C) peptides A1-2, A1-3, A8-1, and to some extent A2-2, bind Ni(II) ions sufficiently strongly in 4N complexes and are hydrolyzed at sufficiently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in the context of specific biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II) binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel toxicity and help design appropriate cellular studies.
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The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.
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This review discusses the relevance of oxidative damage to metal-induced toxicity and carcinogenesis. Presented are important facts and mechanistic concepts on the capacity of selected transition metals, mainly Ni, but also Cu, Co, Cr, and briefly several others, to generate active oxygen species and other reactive intermediates under physiological conditions. These metals are known to be toxic and/or carcinogenic contaminants of the occupational and general environments. Their redox activity may underlay the mechanism of mediation of oxidative damage to cell constituents. The presentation is focused on selected issues relative to genetic and epigenetic toxicity and illustrated with examples of metal-mediated oxidative damage to the principal components of chromatin, i.e., DNA, histones, and protamines.
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Epidemiological evidence suggests that certain paternal exposures to metals may increase the risk of cancer in the progeny. This effect may be associated with promutagenic damage to the sperm DNA. The latter is packed with protamines which might sequester carcinogenic metals and moderate the damage. Human protamine P2 has an amino acid motif at its N-terminus that can serve as a heavy metal trap, especially for Ni(II) and Cu(II). We have synthesized a pentadecapeptide modeling this motif, Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-CysSer-Arg-amide (HP21‐15) and described its complexes with Ni(II) and Cu(II), including their capacity to mediate oxidative DNA degradation [Bal et al. (1997) Chem. Res. Toxicol., 10, 906‐914 and 915‐921]. In the present study, effects of HP21‐15 on Ni(II)- and Cu(II)-mediated DNA oxidation by H2O2 at pH 7.4 were investigated in more detail using the circular plasmid pUC19 DNA as a target, and the single/double-strand breaks and production of oxidized DNA bases, as end points. Ni(II) alone was found to promote oxidative DNA strand scission (mostly single strand breaks) and base damage, while Cu(II) alone produced the same effects, but to a much greater extent. Both metals were relatively more damaging to the pyrimidine bases than to purine bases. HP21‐15 tended to increase the Ni(II)/H2O2-induced DNA breakage. In sharp contrast, the destruction of DNA strands by Cu(II)/H2O2 was almost completely prevented by HP21‐15. The effect of HP21‐15 on the oxidative DNA base damage varied from a limited enhancement (5-hydroxyhydantoin and thymine glycol) to slight suppression (5-hydroxycytosine, 5-hydroxyuracil, 8-oxoguanine, 8-oxoadenine, 2-hydroxyadenine, fapyguanine and fapyadenine) toward Ni(II)/H2O2. HP21‐15 strongly suppressed the oxidative activity of Cu(II)/H 2O2 in regard to all bases in DNA. Consistently with the Abbreviations: 2-OH-Ade, 2-hydroxyadenine (isoguanine); 5-OH-Cyt, 5-hydroxycytosine; 5-OH-Hyd, 5-hydroxyhydantoin; 8-oxo-Ade, 7,8-dihydro8-oxoadenine; 8-oxo-dG, 7,8-dihydro-8-oxo-29-deoxyguanosine (8-oxo-29deoxyguanosine); 8-oxo-Gua, 7,8-dihydro-8-oxoguanine (8-oxoguanine); BSTFA, bis(trimethylsilyl)trifluoroacetamide; dG, 2 9-deoxyguanosine; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ESR, electron spin resonance; HP2, human protamine 2; HP21‐15, Arg-Thr-His-GlyGln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide; PBS, phosphatebuffered saline; TBE buffer, 0.1 M Tris, 0.09 M boric acid and 0.001 M EDTA, pH 8.4; ThyGlycol, thymine glycol.
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Background Nickel allergy is common worldwide. It is associated with hand dermatitis, and sensitization is often induced by nickel-releasing jewellery. The European Union (EU) introduced legislation to control nickel content and release from jewellery and other consumer items through the EU Nickel Directive 1994, which came into force in 2001 and is now part of the REACH regulation. Objectives To examine the effects of the EU nickel regulations on the prevalence of nickel allergy in four European countries. Methods Nickel patch-test data from 180 390 patients were collected from national databases in Denmark, Germany, Italy and the U.K. from between 1985 and 2002 to 2010. Patients with suspected allergic contact dermatitis who had been patch tested with nickel sulfate 5% in petrolatum were included in the analysis. The main outcomes studied were the percentage of positive results to nickel patch tests, and changes in trends with time in an age- and sex-stratified analysis. ResultsA statistically significant decrease in nickel allergy was observed in Danish, German and Italian women aged below 30 years. In female patients in the U.K. this was observed between 2004 and 2010. In young men, a statistically significant decrease in nickel allergy was observed in Germany and the U.K., whereas a nonsignificant increase was observed in Italy. Conclusions There has been a reduction in the prevalence of nickel allergy in young women, contemporaneous with the introduction of the nickel regulation. A reduction is also suggested in men in Germany and the U.K. A causative effect of the regulatory intervention is the most likely explanation.
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Nickel is widely applied in industrial settings and Ni(II) compounds have been classified as group one human carcinogens. The molecular basis of Ni(II) carcinogenicity has proved complex, for many stress response pathways are activated and yield unexpected Ni(II)-specific toxicology profile. Ni(II)-induced toxicogenomic change has been associated with altered activity of HIF, p53, c-MYC, NFκB and iron and 2-oxoglutarate-dependent dioxygenases. Advancing high-throughput technology has indicated the toxicogenome of Ni(II) involves crosstalk between HIF, p53, c-MYC, NFκB and dioxygenases. This paper is intended to review the network of Ni(II)-induced common transcription-factor-governed pathways by discussing transcriptome alteration, its governing transcription factors and the underlying mechanism. Finally, we propose a putative target network of Ni(II) as a human carcinogen.
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Abstract Alpha-1 antitrypsin (AAT) is a circulating serine protease inhibitor (serpin) that inhibits neutrophil elastase in the lung, and AAT deficiency is associated with early-onset emphysema. AAT is also a liver-derived acute phase protein that, in vitro and in vivo, reduces production of pro-inflammatory cytokines, inhibits apoptosis, blocks leukocyte degranulation and migration, and modulates local and systemic inflammatory responses. In monocytes, AAT has been shown to increase intracellular cAMP, regulate expression of CD14, and suppress NFκB nuclear translocation. These effects may be mediated by AAT's serpin activity or by other protein-binding activities. In preclinical models of autoimmunity and transplantation, AAT therapy prevents or reverses autoimmune disease and graft loss, and these effects are accompanied by tolerogenic changes in cytokine and transcriptional profiles and T cell subsets. This review highlights advances in our understanding of the immunemodulating effects of AAT and their potential therapeutic utility.
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Background The possible impact of metal release from coronary artery stents has, with their increased use, become a concern.Objectives To study in vitro metal release in biologically relevant milieu from coronary stents made of different alloys.Materials and methodCoronary stents in common use in a department of cardiology at the time of the study were tested. A previously described in vitro technique was used, whereby the stents were kept in the extraction media for a week. Two different extraction media were used to show the necessity of studying the actual biological surrounding of the implant when metal release is investigated. Metal release was determined with atomic absorption spectrometry.ResultsIn this study, we show metal release from stents after immersion in extraction media of artificial sweat and cysteine solution, as illustrative media.Conclusion Metal release from coronary stents is shown. The magnitude of release is influenced by several factors. The extent to which metal release in vitro has potential biological effects, in terms of elicitation of an allergic reaction or induction of sensitization, in vivo needs to be explored. However, as metal release from an implant in a biologically appropriate medium has been established, better risk assessments in relation to delayed hypersensitivity may be undertaken.