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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...
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Cite this: Metallomics, 2020,
Peptide bond cleavage in the presence of
Ni-containing particles
Nina Ewa Wezynfeld, *
Tomasz Fra˛czyk,
Arkadiusz Bonna
Wojciech Bal *
NiO nanoparticles and non-stoichiometric black NiO were shown
to be effective sources of Ni
ions causing sequence-selective
peptide bond hydrolysis. NiO nanoparticles were as effective in
this reaction as their molar equivalent of soluble Ni(II) salt. These
findings highlight the efficacy of delivery of toxic Ni
by these
environmentally available particles.
The unique properties of nickel, including high melting point
and resistance to corrosion and oxidation are exploited in the
production of everyday objects such as coins, mobile phones,
kitchen utensils and cutlery,
e-cigarette lighters
as well as in
wind turbines
or in electric vehicles.
Behind the growing
demand for nickel-containing products, the numbers of people
allergic to nickel and developing cancers of the respiratory tract
are on the rise.
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
A number of human proteins, including histone
phospholipid scramblase 1,
and transcrition factors containing zinc finger domains
susceptible to the Ni(II)-dependent hydrolysis, resulting in their
Other transition metal ions, Cu
and Pd
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. Then, the N-terminal
fragment preceding the Ser residue is transferred to the hydroxyl
group of Ser forming an intermediate ester product (IP). Finally, the
ester is easily hydrolyzed to final products: the free N-terminal
fragment of the peptide/protein and the Ni(II)/Cu(II)/Pd(II) complex
with the C-terminal fragment.
are non-native (foreign) for the immune system and could initialize
the allergic response. On the other hand, the products of MdH were
shown to catalyze the production of Reactive Oxygen Species
involved in the cancer development.
Considering potential severe consequences of MdH for
human health, we decided to study this reaction in the
presence of Ni-containing particles, the poorly soluble forms
of nickel significantly present in human environment.
Their inhalation could result even in accumulating tens to
hundreds of micrograms of nickel in lung tissue as shown in
studies on nickel refinery workers
and experimental mice.
Ni nanoparticles could also easily penetrate the skin,
and be
deposited during tattooing, triggering allergic response.
Moreover, subcellular accumulation of Ni-containing particles,
for example in lysosomes or in mitochondria could signifi-
cantly enhance their dissolution and catalysis of ROS produc-
tion, respectively.
In our work we used four types of Ni-containing particles:
metallic (Ni metal) and three nickel oxides: stoichiometric
green nickel oxide (NiO green), non-stoichiometric black nickel
oxide (NiO black), and nickel oxide nanoparticles (NiO nano).
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
´skiego 5a, 02-106 Warsaw, Poland. E-mail:
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3,
00-664, Warsaw, Poland. E-mail:
Department of Immunology, Transplantology and Internal Medicine,
Medical University of Warsaw, Nowogrodzka 59, 02-006 Warsaw, Poland
Department of Biochemistry, University of Cambridge, Downing Site,
Tennis Court Road, Cambridge CB2 1QW, UK
Electronic supplementary information (ESI) available: Experimental section,
EDX results, determination of Ni
concentration. See DOI: 10.1039/d0mt00070a
Received 18th March 2020,
Accepted 29th April 2020
DOI: 10.1039/d0mt00070a
Significance to metallomics
Humankind are facing a growing incidence of nickel contact allergy and
respiratory tract cancers, possibly related to environmental exposure to
particulate nickel. In this work, we showed that poorly soluble Ni-
containing particles, the nickel forms we are mostly exposed to, could
effectively drive the cleavage of the peptide bond preceding the Ser/Thr-
Xaa-His motif present in many essential human proteins. Strikingly, NiO
nanoparticles were as effective hydrolytic agents as a soluble Ni(II) salt.
The products of this process could initiate the immune response and
increase the oxidative stress, thereby participating in initial steps of
allergy and cancer development.
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650 |Metallomics, 2020, 12, 649--653 This journal is ©The Royal Society of Chemistry 20 20
Their images obtained by scanning electron microscopy (SEM)
are presented in Fig. 2.
The studied Ni-containing particles vary greatly. Ni metal
and NiO black particles are approximately spherical in shape,
but their surface is not perfectly smooth. There are a lot
of insets and dimples in their outer coat. On the other hand,
NiO green and NiO nano aggregated very easily in water
solution due to the relatively smooth surfaces. The linear
particle sizes did not usually exceed 10 mm, even for the largest
Ni metal and NiO black. Much smaller dimensions were noted
for NiO green, whose particles were limited to 1 mm. Despite the
NiO nano susceptibility to aggregation, the size of individual
particles was consistent with the manufacturer’s specification
(below 50 nm) and agreed with previous reports on the same
Results of energy-dispersive X-ray (EDX) spectro-
scopy confirmed the high molar nickel fraction expected for
Ni metal (98%) and close to equimolar Ni : O ratio for
NiO particles of both types (detailed EDX results available in
Table S1, ESI). The amount of Ni
released from the studied
particles in HEPES buffer at pH 7.4 was monitored over one-
week long incubation. During this time, small aliquots of
particle suspension were collected periodically, followed by
centrifugation to separate the supernatant from the particles,
and quantification of dissolved Ni
ions by a spectroscopic
method based on the intensity of d–d band at 460 nm of stable
Ni(DTT) complexes.
The precise description of this method is
provided in ESIincluding Fig. S1 and S2. Its results were
positively verified by ICP-OES. According to data presented in
Fig. 3A, the highest concentration of Ni
was observed for NiO
nano, followed by NiO black, ca. 6% and 2% of total Ni after a
week, respectively. It agrees with other studies on Ni
from nano and micro Ni-containing particles.
The release rate was higher during the first eight hours of
the incubation. Much lower amounts of released Ni
ions were
observed for NiO green and Ni metal, not exceeding 0.2% after
a week. The presence of the peptide did not affect the Ni
release (see Fig. 3B and C). The promotion of Ni
release by
proteins was noticed in several previous studies on stainless
Small size of the peptide and/or ten-fold molar excess
Fig. 2 SEM images of nickel particles: Ni metal (A), NiO green (B), NiO
black (C), NiO nanoparticles (D).
Fig. 3 (A) Concentration of Ni
released from Ni-containing particles
containing 5.87 g l
Ni (equivalent to 100 mM Ni concentration after
the complete dissolution) in 100 mM HEPES, pH 7.4 and 37 1C. Kinetics of
release from NiO nanoparticles (B) or non-stoichiometric black NiO
particles (C) containing 5.87 g l
Ni without (full symbols) and with (open
symbols) 1 mM Ac-GGASRHWKF-am (peptide), in 100 mM HEPES pH 7.4,
37 1C. Insets represent the kinetics from the first day of the incubation.
Fig. 1 The mechanism of Ni(II)-dependent hydrolysis of the peptide bond
preceding Ser in the R
sequence, based on ref. 12, 14
and 23.
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of particulate nickel over the peptide could make this effect
unnoticeable here.
We also monitored the hydrolysis progression for samples
containing Ni-containing particles and Ac-GGASRHWKF-am by
HPLC-UV and ESI-MS. Results of this analysis are presented
in Fig. 4.
The fastest degradation of the peptidic substrate was
noticed in the presence of NiO nano, followed by NiO black.
Much smaller amounts of the substrate were degraded in the
suspensions of NiO green and Ni metal. A similar dependence
was demonstrated for the formation of final products and
intermediate product (IP). The IP fraction was negligible for
NiO green and Ni metal as the IP hydrolysis was faster than its
formation in the presence of these particles. Surprisingly,
despite the preceding relatively slow kinetics of Ni
from the particles, all steps of MdH followed the pseudo-first-
order kinetics, used before for the description of MdH by
salts. The values of substrate decay rate constants k
the presence of the particles were in the range of 0.069–4.0
, whereas k
values for IP hydrolysis were 1.9–3.4
. The differences in k
likely originated from differ-
ences in Ni
concentration released by given particles. The
similarity of k
values was expected because this reaction step
did not depend on the Ni
source, but only on the peptide
sequence and the pH (see Fig. 1). The range of k
values was
satisfactorily similar to that observed previously for a similar
The overall MdH rates in the presence of NiO nano
and 10 mM Ni(NO
were very similar to each other, due to a
very effective Ni
release from NiO nano, yielding the 1 mM
concentration equal to that of the peptide in less than one hour
and 4 mM Ni
after 8 h (see Fig. 3). However, as the released
content differed for different particles and varied during
the incubation even for a given particle type, we performed a
more detailed analysis on the k
dependence on Ni
tration. We first calculated the amount of MdH-active 4N
complexes (step 1 in Fig. 1) for the released Ni
at selected incubation times. The basis for these calculations
was provided by the species distribution of the analogous Ni(II)-
Ac-GASRHWKFL-am system.
The correctness of this approach
was corroborated by comparing the pH-dependence of Ni(II)
complexation by Ac-GGASRHWKF-am (from UV-vis spectro-
scopy) and by Ac-GASRHWKFL-am (from potentiometry). The
full consistency of these profiles shown in Fig. 5 confirmed that
differences in terminal residues did not affect the coordination
sphere of respective complexes.
The resulting concentrations of 4N complexes for MdH were
very low, between 0.2 and 15 mM depending on the particle type.
Then, considering that for 10 mM Ni
1.67% of the peptide
was bound in 4N complexes, the determined k
= 4.17
led to the estimation of the theoretically maximal k
(where the whole peptide would be initially bound as the
hydrolytically active 4N complex) under given experimental
= 2.5 10
The obtained amounts of 4N complexes at different time of
incubation, together with the
value, allowed us to assess
the theoretical kinetics of peptide degradation in response to
time dependent Ni
release. The results of these calculations
were compared with the experimentally determined amounts of
the degraded peptide for NiO nano and NiO black, as presented
in Fig. 6. The hydrolysis in the presence of NiO green and Ni
metal was not investigated in such fashion due to the very low
yield of those reactions.
The analysis demonstrated a very good correlation of the
experimental peptide fractions (rainbow circles) with those
expected from the calculations (rainbow lines). One could notice
that the fit of experimental points is closer to the theoretical
curves for the Ni
concentration after longer (424 h) incubation
times, while signals measured for NiO black are more consistent
with the dependence expected after incubation for about 8 h.
Fig. 4 Kinetics of substrate decay (left), formation of intermediate pro-
duct (middle), and formation of final products (right) for metal-dependent
hydrolysis of 1 mM Ac-GGASRHWKF-am in the presence of particles
containing 5.87 g l
Ni or in the presence of 10 mM Ni(NO
100 mM HEPES, pH 7.4, 37 1C. The experimental data were fit to first
order kinetic equations.
Fig. 5 (A) UV-vis titration of 0.9 mM Ni(NO
and 0.95 mM Ac-GGASRHWKF-
am with concentrated NaOH. The spectra were codded with rainbow colors
from red (pH 4.5) to violet (pH 11.5), where the thicker orange line corresponds
to pH 7.4 and dotted grey line represents the spectrum of the peptide
alone. (B) The comparison of species distribution of previously studied
Ac-GASRHWKFL-am and Ni
with the pH dependence of the formation of
the square-planar complex of Ni(II)-Ac-GGASRHWKF-am monitored by change
in the absorbance at 455 nm from the experiments, presented in (A).
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However, considering experimental accuracy of determination
of Ni
concentration, we did not find the differences between
the theoretical and experimental kinetics to be significant.
Therefore, the Ni
release rate is the limiting factor for MdH
in the presence of Ni-containing particles.
Summarizing, our results indicate that Ni-containing parti-
cles, especially NiO nanoparticles and non-stoichiometric NiO
black, could be effective sources of Ni
ions, leading to protein
degradation via MdH. The rate of this process is limited mostly
by the amount of released Ni
ions. Due to the high number
of likely MdH targets,
the increasing human exposition to
Ni-containing objects and materials,
and the ability of NiO
nanoparticles to penetrate tissues
the protein hydrolysis in
the presence of poorly soluble Ni forms could be an important
contributor to nickel toxicity, including contact allergy and
respiratory cancers. Our test peptide was a relatively small
molecule. Therefore, we plan to continue these studies to
explore how the size and adsorption of proteinaceous MdH
targets and coincident biological molecules on the particles
could affect the studied reaction.
Conflicts of interest
There are no conflicts to declare.
This work was financed by Preludium Grant No. 2013/11/N/
NZ1/02400 (Polish National Science Centre) and in part
by TEAM No. 2009-4/1 grant (Foundation for Polish Science)
co-financed by the European Regional Development Fund
resources within the framework of Operational Program Inno-
vative Economy (Action 1.2). N. W. was supported by ETIUDA
scholarship No. 2015/16/T/NZ1/00377 (National Science Centre
of Poland). The equipment used was sponsored in part by the
Centre for Preclinical Research and Technology (CePT), a project
co-sponsored by European Regional Development Fund and
Innovative Economy, The National Cohesion Strategy of Poland.
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Metallomics Communication
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... We chose physiological temperature (37 • C) and two pH values, 7.4 and 8.2, close to those existing in cytoplasm and mitochondria, respectively. Such pH values were also used in our previous publications [17,[19][20][21]39]. Thus, it is also possible to compare the measured hydrolysis rates with other peptides. ...
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Purpose: Since nanoparticles (NPs) are beginning to be introduced in medicine and industry, it is mendatory to evaluate their biological side-effects, among other things. The present study aimed to investigate the pathways by which nickel nanoparticles (NiNPs) enter nephrons and to evaluate their localization and effects on cellular ultrastructure. Methods: Rats were injected intraperitoneally with 20 nm NiNPs (20 mg/Kg/b.w./day) for 28 consecutive days. Transmission electron microscope technique was used to detect localization of NiNPs and their effects on cellular ultrastructure in rat kidneys. Additionally, measurements of certain biochemical parameters such as creatinine, urea, uric acid and phosphorus for investigating renal function following NiNPs treatment were taken. Results: The presence of NiNPs in the nephrons in treated rats was confirmed by transmission electron microscopy. NiNPs entered the renal tubules cells via various pathways. The results indicated that NiNPs administration induced ultrastructural changes in the proximal cells of renal tubules and certain glomerular cells (podocytes and mesangial cells). Additionally, NiNPs were found to be localized in the mitochondria, which led to a significant decrease in their density and morphology. Furthermore, cell death was induced in the glomerular cells as found with a Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay and through detection of p35 using immunohistochemical staining. Conclusion: Herein, NiNPs were found to induce various cellular ultrastructural changes in the kidneys of rats. NiNPs used diverse pathways to internalize into the cytoplasm of the proximal convoluted tubules (PT) cells across the basement membrane, and also through the plasma membrane of two adjacent PT cells. NiNPs internalization, accumulation and their alterations of the cellular ultrastructure affected rat renal function.
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Many biomedical materials used today for applications such as orthopedic, dental, and cardiovascular implants and devices are made of corrosion-resistant, ‘inert’, metallic materials of the cobalt–chromium, titanium, and stainless steel alloy groups. This perspective focuses on the role of proteins in the degradation of these materials in a human body environment. After adsorption, the proteins interact relatively slowly with the metal and metal surface oxide. A number of factors, including the individual body chemistry (especially the presence of inflammatory cells producing oxidative species), determine whether the proteins can bind to metals in the surface oxide and whether the metal–protein conjugates can detach from the surface. Metals in the forms of protein-bound metal ions or nanosized particles can also increase protein–protein interactions and aggregation, which can cause some health effects and change the material degradation mechanism. While proteins in some short-term studies (<6 h) even decrease material degradation due to shielding effects and better lubrication, they may increase degradation after longer time periods due to relatively slow binding, detachment, and combined corrosion processes. In-vitro material degradation studies of relatively corrosion-resistant alloys for biomedical applications should therefore include long-term studies, complexing agents or proteins, and realistic oxidative environments simulating inflammatory conditions.
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Background Studies on sensitization to metals in the general population are scarce. Objectives To determine the prevalence of sensitization to metals in the general population, and factors associated with nickel sensitization. Methods In 5 European countries (The Netherlands, Germany, Italy, Portugal and Sweden), a random sample (N = 3119) from the general population (aged 18‐74 years) was patch tested and interviewed by use of a questionnaire on exposure to metals, piercing, and jewellery. Results Overall, the age‐standardized prevalences of sensitization to nickel, cobalt and chromium were, respectively, 14.5%, 2.1%, and 0.8%. The highest prevalence of nickel sensitization was seen in Portugal (18.5%) and the lowest (8.3%) in Sweden. The prevalence of cobalt sensitization varied between 3.8% (The Netherlands) and 0.9% (Italy), and the prevalence of chromium sensitization varied between 1.3% (Portugal) and 0.2% (Sweden). Significant associations were observed between nickel allergy and female sex (odds ratio [OR] 5.19; 95% confidence interval [95%CI]: 3.99‐6.74), past piercing use (OR 3.86; 95%CI: 2.85‐5.24), and currently having ≥3 piercings (OR 5.58; 95%CI: 4.02‐7.76). Conclusions The prevalence of sensitization to metals in the European general population was high, mostly because of nickel. The lowest prevalence of contact allergy to nickel and chromium observed in Sweden supports the effectiveness of long‐standing regulation.
Introduction: E-cigarettes deliver an aerosol of nicotine by heating a liquid and are promoted as an alternative to combustible tobacco. This study determines the longitudinal associations between e-cigarette use and respiratory disease controlling for combustible tobacco use. Methods: This was a longitudinal analysis of the adult Population Assessment of Tobacco and Health Waves 1, 2, and 3. Multivariable logistic regression was performed to determine the associations between e-cigarette use and respiratory disease, controlling for combustible tobacco smoking, demographic, and clinical variables. Data were collected in 2013-2016 and analyzed in 2018-2019. Results: Among people who did not report respiratory disease (chronic obstructive pulmonary disease, chronic bronchitis, emphysema, or asthma) at Wave 1, the longitudinal analysis revealed statistically significant associations between former e-cigarette use (AOR=1.31, 95% CI=1.07, 1.60) and current e-cigarette use (AOR=1.29, 95% CI=1.03, 1.61) at Wave 1 and having incident respiratory disease at Waves 2 or 3, controlling for combustible tobacco smoking, demographic, and clinical variables. Current combustible tobacco smoking (AOR=2.56, 95% CI=1.92, 3.41) was also significantly associated with having respiratory disease at Waves 2 or 3. Odds of developing respiratory disease for a current dual user (e-cigarette and all combustible tobacco) were 3.30 compared with a never smoker who never used e-cigarettes. Analysis controlling for cigarette smoking alone yielded similar results. Conclusions: Use of e-cigarettes is an independent risk factor for respiratory disease in addition to combustible tobacco smoking. Dual use, the most common use pattern, is riskier than using either product alone.
Nickel is the most frequent cause of contact allergy worldwide and has been studied extensively. This clinical review provides an updated overview of the epidemiology, exposure sources, methods for exposure quantification, skin deposition and penetration, immunology, diagnosis, thresholds for sensitization and elicitation, clinical expression, prevention and treatment. The implementation of a nickel regulation in Europe led to a decrease in the prevalence of nickel allergy, as well as a change in the clinical expression and disease severity. Still, the prevalence of nickel allergy in the European general population is approximately 8‐19% in adults and 8‐10% in children and adolescents, with a strong female predominance. Well‐known consumer items such as jewellery and metal in clothing are still the main causes of nickel allergy and dermatitis, although a wide range of items for both private and occupational use may cause dermatitis. Allergic nickel dermatitis may be localized to the nickel exposure site, be more widespread or present as hand eczema. Today, efficient methods for exposure quantification exist, and new insights regarding the associated risk factors and the immunological mechanisms underlying the disease have been accomplished. Nevertheless, questions remain in relation to the pathogenesis, the persistent high prevalence and treatment of severe cases. This article is protected by copyright. All rights reserved.
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 incidence and mortality from lung cancer is decreasing in the US due to decades of public education and tobacco control policies, but are increasing elsewhere in the world related to the commencement of the tobacco epidemic in various countries and populations in the developing world. Individual cigarette smoking is by far the most common risk factor for lung carcinoma; other risks include passive smoke inhalation, residential radon, occupational exposures, infection and genetic susceptibility. The predominant disease burden currently falls on minority populations and socioeconomically disadvantaged people. In the US, the recent legalization of marijuana for recreational use in many states and the rapid growth of commercially available electronic nicotine delivery systems (ENDS) present challenges to public health for which little short term and no long term safety data is available.