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1.
Br J Pharmacol ; 2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32496597

RESUMO

BACKGROUND AND PURPOSE: Ethaninidothioic acid (R5421) has been used as a scramblase inhibitor to determine the role of phospholipid scrambling across a range of systems including platelet procoagulant activity. The selectivity of R5421 has not been thoroughly studied. Here, we characterised the effects of R5421 on platelet function and its suitability for use as a scramblase inhibitor. EXPERIMENTAL APPROACH: Human platelet activation was measured following pretreatment with R5421 and stimulation with a range of agonists. Phosphatidylserine exposure was measured using annexin V binding. Integrin αIIb ß3 activation and α-granule release were measured by flow cytometry. Cytosolic Ca2+ signals were measured using Cal520 fluorescence. An in silico ligand-based screen identified 16 compounds which were tested in these assays. KEY RESULTS: R5421 inhibited A23187-induced phosphatidylserine exposure in a time- and temperature-dependent manner. R5421 inhibited Ca2+ signalling from the PAR1, PAR4 and glycoprotein VI receptors as well as platelet αIIb ß3 integrin activation and α-granule release. R5421 is therefore not a selective inhibitor of platelet scramblase activity. An in silico screen identified the pesticide thiodicarb as similar to R5421. It also inhibited platelet phosphatidylserine exposure, Ca2+ signalling from the PAR1 and glycoprotein VI, αIIb ß3 activation and α-granule release. Thiodicarb additionally disrupted Ca2+ homeostasis in unstimulated platelets. CONCLUSION AND IMPLICATIONS: R5421 is not a selective inhibitor of platelet scramblase activity. We have identified the pesticide thiodicarb, which had similar effects on platelet function to R5421 as well as additional disruption of Ca2+ signalling which may underlie some of thiodicarb's toxicity.

2.
Metallomics ; 12(5): 649-653, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32393924

RESUMO

NiO nanoparticles and non-stoichiometric black NiO were shown to be effective sources of Ni2+ 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 Ni2+ by these environmentally available particles.

3.
Chem Biodivers ; 17(2): e1900652, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31869504

RESUMO

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.


Assuntos
Níquel/química , Peptídeos/química , Sequência de Aminoácidos , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Íons/química , Cinética , Níquel/metabolismo , Níquel/toxicidade , Peptídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Alinhamento de Sequência , Espectrofotometria
4.
Metallomics ; 11(11): 1800-1804, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31657408

RESUMO

Model peptides relevant to hCtr1 transchelate CuI from the anti-tumour [CuI(PTA)4]+ complex before metal internalization into tumor cells. ESI(+)MS experiments corroborated by DFT calculations indicate that tetracoordinated-CuII and linear-CuI arrangements of in situ generated copper-peptide products play a crucial role in promoting the transfer of copper from the terminal MDH portion into adjacent HSH peptide sequence.

5.
Sci Rep ; 9(1): 13397, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527604

RESUMO

Tissue factor (TF) plays a central role in haemostasis and thrombosis. Following vascular damage, vessel wall TF initiates the extrinsic coagulation cascade. TF can also be exposed by monocytes. Inflammatory or infectious stimuli trigger synthesis of new TF protein by monocytes over the course of hours. It has also been suggested that monocytes can expose TF within minutes when stimulated by activated platelets. Here, we have confirmed that monocytes rapidly expose TF in whole blood and further demonstrate that platelet P-selectin exposure is necessary and sufficient. Monocyte TF exposure increased within five minutes in response to platelet activation by PAR1-AP, PAR4-AP or CRP-XL. PAR1-AP did not trigger TF exposure on isolated monocytes unless platelets were also present. In whole blood, PAR1-AP-triggered TF exposure required P-selectin and PGSL-1. In isolated monocytes, although soluble recombinant P-selectin had no effect, P-selectin coupled to 2 µm beads triggered TF exposure. Cycloheximide did not affect rapid TF exposure, indicating that de novo protein synthesis was not required. These data show that P-selectin on activated platelets rapidly triggers TF exposure on monocytes. This may represent a mechanism by which platelets and monocytes rapidly contribute to intravascular coagulation.

6.
Sci Rep ; 8(1): 16677, 2018 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-30420683

RESUMO

Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro. We have previously shown that this action is independent of citalopram's ability to block serotonin uptake by the serotonin transporter and must therefore be mediated via distinct pharmacological mechanisms. We now report evidence for two novel and putative mechanisms of citalopram-induced platelet inhibition. Firstly, in platelets, citalopram blocked U46619-induced Rap1 activation and subsequent platelet aggregation, but failed to inhibit U46619-induced increases in cytosolic Ca2+. Similarly, in neutrophils, citalopram inhibited Rap1 activation and downstream functions but failed to block PAF-induced Ca2+ mobilisation. In a cell-free system, citalopram also reduced CalDAG-GEFI-mediated nucleotide exchange on Rap1B. Secondly, the binding of anti-GPVI antibodies to resting platelets was inhibited by citalopram. Furthermore, citalopram-induced inhibition of GPVI-mediated platelet aggregation was instantaneous, reversible and displayed competitive characteristics, suggesting that these effects were not caused by a reduction in GPVI surface expression, but by simple competitive binding. In conclusion, we propose two novel, putative and distinct inhibitory mechanisms of action for citalopram: (1) inhibition of CalDAG-GEFI/Rap1 signalling, and (2) competitive antagonism of GPVI in platelets. These findings may aid in the development of novel inhibitors of CalDAG-GEFI/Rap1-dependent nucleotide exchange and novel GPVI antagonists.


Assuntos
Citalopram/farmacologia , Neutrófilos/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Cálcio/metabolismo , Citosol/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Modelos Biológicos , Neutrófilos/citologia , Glicoproteínas da Membrana de Plaquetas/metabolismo
7.
Sci Signal ; 11(532)2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29844053

RESUMO

Fibrillar collagens of the extracellular matrix are critical for tissue structure and physiology; however, excessive or abnormal deposition of collagens is a defining feature of fibrosis. Regulatory mechanisms that act on collagen fibril assembly potentially offer new targets for antifibrotic treatments. Tissue weakening, altered collagen fibril morphologies, or both, are shared phenotypes of mice lacking matricellular thrombospondins. Thrombospondin-1 (TSP1) plays an indirect role in collagen homeostasis through interactions with matrix metalloproteinases and transforming growth factor-ß1 (TGF-ß1). We found that TSP1 also affects collagen fibril formation directly. Compared to skin from wild-type mice, skin from Thbs1-/- mice had reduced collagen cross-linking and reduced prolysyl oxidase (proLOX) abundance with increased conversion to catalytically active LOX. In vitro, TSP1 bound to both the C-propeptide domain of collagen I and the highly conserved KGHR sequences of the collagen triple-helical domain that participate in cross-linking. TSP1 also bound to proLOX and inhibited proLOX processing by bone morphogenetic protein-1. In human dermal fibroblasts (HDFs), TSP1 and collagen I colocalized in intracellular vesicles and on extracellular collagen fibrils, whereas TSP1 and proLOX colocalized only in intracellular vesicles. Inhibition of LOX-mediated collagen cross-linking did not prevent the extracellular association between collagen and TSP1; however, treatment of HDFs with KGHR-containing, TSP1-binding, triple-helical peptides disrupted the collagen-TSP1 association, perturbed the collagen extracellular matrix, and increased myofibroblastic differentiation in a manner that depended on TGF-ß receptor 1. Thus, the extracellular KGHR-dependent interaction of TSP1 with fibrillar collagens contributes to fibroblast homeostasis.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Trombospondina 1/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Homeostase , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Homologia de Sequência , Pele/citologia , Pele/metabolismo
8.
J Inorg Biochem ; 182: 230-237, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29402466

RESUMO

Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 1013 M-1 at pH 7.4, ~13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 1014 M-1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its reduction to Cu(I), both steps necessary for the CTR1 function.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Cobre/química , Cobre/metabolismo , Animais , Sítios de Ligação , Transportador de Cobre 1 , Humanos , Camundongos , Ligação Proteica
9.
Matrix Biol ; 63: 106-116, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28215822

RESUMO

The small leucine-rich proteoglycans (SLRPs) are important regulators of extracellular matrix assembly and cell signalling. We have determined crystal structures at ~2.2Å resolution of human fibromodulin and chondroadherin, two collagen-binding SLRPs. Their overall fold is similar to that of the prototypical SLRP, decorin, but unlike decorin neither fibromodulin nor chondroadherin forms a stable dimer. A previously identified binding site for integrin α2ß1 maps to an α-helix in the C-terminal cap region of chondroadherin. Interrogation of the Collagen Toolkits revealed a unique binding site for chondroadherin in collagen II, and no binding to collagen III. A triple-helical peptide containing the sequence GAOGPSGFQGLOGPOGPO (O is hydroxyproline) forms a stable complex with chondroadherin in solution. In fibrillar collagen I and II, this sequence is aligned with the collagen cross-linking site KGHR, suggesting a role for chondroadherin in cross-linking.


Assuntos
Proteínas da Matriz Extracelular/química , Fibromodulina/química , Sequência de Aminoácidos , Cristalografia por Raios X , Cistina/química , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Soluções
10.
Blood Adv ; 1(19): 1495-1504, 2017 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-29296791

RESUMO

Fibrin has recently been shown to activate platelets through the immunoglobulin receptor glycoprotein VI (GPVI). In the present study, we show that spreading of human platelets on fibrin is abolished in patients deficient in GPVI, confirming that fibrin activates human platelets through the immunoglobulin receptor. Using a series of proteolytic fragments, we show that D-dimer, but not the E fragment of fibrin, binds to GPVI and that immobilized D-dimer induces platelet spreading through activation of Src and Syk tyrosine kinases. In contrast, when platelets are activated in suspension, soluble D-dimer inhibits platelet aggregation induced by fibrin and collagen, but not by a collagen-related peptide composed of a repeat GPO sequence or by thrombin. Using surface plasmon resonance, we demonstrate that fibrin binds selectively to monomeric GPVI with a KD of 302 nM, in contrast to collagen, which binds primarily to dimeric GPVI. These results establish GPVI as the major signaling receptor for fibrin in human platelets and provide evidence that fibrin binds to a distinct configuration of GPVI. This indicates that it may be possible to develop agents that selectively block the interaction of fibrin but not collagen with the immunoglobulin receptor. Such agents are required to establish whether selective targeting of either interaction has the potential to lead to development of an antithrombotic agent with a reduced effect on bleeding relative to current antiplatelet drugs.

11.
Protein Sci ; 26(2): 343-354, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27870250

RESUMO

The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Queratina-8/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Medição da Troca de Deutério , Humanos , Queratina-8/genética , Queratina-8/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Domínios Proteicos
12.
Inorg Chem ; 55(16): 7829-31, 2016 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-27476515

RESUMO

α-Factor-1 (WHWLQLKPGQPMY), a peptidic pheromone of Saccharomyces cerevisiae yeast, contains a XHX type copper(II) binding N-terminal site. Using a soluble analogue, WHWSKNR-amide, we demonstrated that the W(1)H(2)W(3) site alone binds copper(II) with a Kd value of 0.18 pM at pH 7.4 and also binds imidazole (Im) in a ternary complex (Kd of 1 mM at pH 7.4). This interaction boosts the ability of the peptide to sequester copper(II) depending on the Im concentration up to a subfemtomolar range, not available for any oligopeptidic system studied before. Therefore, α-factor-1 and other XHX-type peptides are likely copper(II) carriers in biological systems.


Assuntos
Cobre/metabolismo , Fator de Acasalamento/química , Fator de Acasalamento/metabolismo , Amidas/química , Sítios de Ligação , Dicroísmo Circular , Imidazóis/química , Imidazóis/metabolismo , Ligantes , Conformação Proteica , Espectrofotometria Ultravioleta
13.
PLoS One ; 11(8): e0160256, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27517864

RESUMO

The influence of cation-π interactions on the electrochemical properties of copper(II) complexes with synthesized pentapeptide C-terminal fragment of Atrial Natriuretic Factor (ANF) hormone was studied in this work. Molecular modeling performed for Cu(II)-NSFRY-NH2 complex indicated that the cation-π interactions between Tyr and Cu(II), and also between Phe-Arg led to specific conformation defined as peptide box, in which the metal cation is isolated from the solvent by peptide ligand. Voltammetry experiments enabled to compare the redox properties and stability of copper(II) complexes with NSFRY-NH2 and its analogues (namely: NSFRA-NH2, NSFRF-NH2, NSAAY-NH2, NSAAA-NH2, AAAAA-NH2) as well as to evaluate the contribution of individual amino acid residues to these properties. The obtained results led to the conclusion, that cation-π interactions play a crucial role in the effective stabilization of copper(II) complexes with the fragments of ANF peptide hormone and therefore could control the redox processes in other metalloproteins.


Assuntos
Fator Natriurético Atrial/química , Cobre/química , Fragmentos de Peptídeos/química , Fator Natriurético Atrial/metabolismo , Sítios de Ligação , Cobre/metabolismo , Estabilidade de Medicamentos , Humanos , Modelos Moleculares , Oxirredução , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
14.
J Biol Chem ; 291(15): 7951-60, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-26893379

RESUMO

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.


Assuntos
Colágeno/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Proteoglicanas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Colágeno/análise , Ativação Enzimática , Proteínas da Matriz Extracelular/genética , Fibromodulina , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mapas de Interação de Proteínas , Proteína-Lisina 6-Oxidase/análise , Proteoglicanas/genética
15.
Angew Chem Int Ed Engl ; 54(36): 10460-4, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26178596

RESUMO

Accumulation of the ß-amyloid (Aß) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aß1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aß4-42 peptide is a major Aß isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aß1-x peptides. By using Aß4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aß4-16 and Aß4-42 possess negligible redox activity. Combined with the predominance of Aß4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.


Assuntos
Peptídeos beta-Amiloides/fisiologia , Cobre/metabolismo , Homeostase , Peptídeos beta-Amiloides/metabolismo
16.
Chem Res Toxicol ; 28(2): 191-201, 2015 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-25549802

RESUMO

Poly(ADP-ribose) polymerase 1 (PARP-1) is a key eukaryotic enzyme,catalyzing the NAD+ dependent poly(ADP-ribosyl)ation of protein substrates, crucial for major DNA repair pathways, and involved in other fundamental cellular processes, such as transcription, cell cycle control, and apoptosis. Its ability to bind DNA depends on two CCHC zinc finger domains, in short, PARPzf1 and PARPzf2. Using spectroscopic methods and competitive titrations with Zn(II), Co(II), and Ni(II) ions, we determined conditional dissociation constants for Zn(II) complexes of PARPzf1 and PARPzf2 at pH 7.4 (HEPESbuffer) as 26 ± 4 nM and 4 ± 1 pM, respectively. The former value indicates an extremely low affinity of PARPzf1 toward metal ions, meaning that under cellular conditions PARP1zf might be largely present in a "metal-free" state. This finding provides a clue to the high susceptibility of PARP-1 to oxidative stress but also raises questions regarding the activation of PARPzf1 under cellular conditions. We also determined conditional dissociation constants for Ni(II) complexes of PARPzf1 and PARPzf2 under the same conditions as 0.78 ± 0.04 µM and 0.26 ± 0.05 nM, respectively.


Assuntos
Poli(ADP-Ribose) Polimerases/química , Dedos de Zinco , Zinco/química , Dicroísmo Circular , Humanos , Simulação de Dinâmica Molecular , Poli(ADP-Ribose) Polimerase-1 , Estrutura Terciária de Proteína , Prótons , Espectrometria de Fluorescência
17.
Metallomics ; 7(4): 596-604, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25579632

RESUMO

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 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 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.


Assuntos
Íons , Níquel/química , Deficiência de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/química , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Humanos , Hidrólise , Estresse Oxidativo , Espectrofotometria Ultravioleta , Tripsina/química
18.
Chem Res Toxicol ; 27(11): 1996-2009, 2014 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-25330107

RESUMO

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 circular dichroism (CD) spectroscopies, potentiometry, isothermal titration calorimetry, high-performance liquid chromatography (HPLC), and electrospray ionization mass spectrometry (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.


Assuntos
Anexina A1/química , Anexina A2/química , Níquel/química , Níquel/toxicidade , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Anexina A1/metabolismo , Anexina A2/metabolismo , Anexinas/química , Anexinas/metabolismo , Calorimetria , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Humanos , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Termodinâmica
19.
J Inorg Biochem ; 139: 1-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24950384

RESUMO

The purpose of our research was to obtain peptidomimetics possessing Cu(II) and Ni(II) binding properties, which would be useful for biomedical applications. In this context we used potentiometry, UV-VIS and CD spectroscopies to characterize the Cu(II) and Ni(II) binding properties of pentapeptide analogs of the N-terminal sequence of histatin 5. The peptides investigated had a general sequence DSXAK-am (am stands for C-terminal amide), with X including His and its three synthetic analogs, (4-thiazolyl)-L-alanine (1), (2-pyridyl)-L-alanine (2), and (pyrazol-1-yl)-L-alanine (3). The heterocyclic nitrogens present in these analogs were significantly more acidic than that of the His imidazole. We found that DSXAK-am peptides were able to bind Cu(II) and Ni(II) and form 4N complexes in a cooperative fashion, with similar affinities. These results indicate that acidic heterocyclic amino acids provide a viable alternative for histidine in peptidomimetics designed for metal ion binding.


Assuntos
Complexos de Coordenação/química , Cobre/química , Histidina/análogos & derivados , Histidina/química , Níquel/química , Oligopeptídeos/química , Albuminas/química , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Secundária de Proteína
20.
Inorg Chem ; 53(9): 4639-46, 2014 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-24735221

RESUMO

Potentiometry and UV-vis and circular dichroism spectroscopies were applied to characterize Cu(II) coordination to the Ac-GASRHWKFL-NH2 peptide. Using HPLC and ESI-MS, we demonstrated that Cu(II) ions cause selective hydrolysis of the Ala-Ser peptide bond in this peptide and characterized the pH and temperature dependence of the reaction. We found that Cu(II)-dependent hydrolysis occurs solely in 4N complexes, in which the equatorial coordination positions of the Cu(II) ion are saturated by peptide donor atoms, namely, the pyridine-like nitrogen of the His imidazole ring and three preceding peptide bond nitrogens. Analysis of the reaction products led to the conclusion that Cu(II)-dependent hydrolysis proceeds according to the mechanism demonstrated previously for Ni(II) ions (Kopera, E.; Krezel, A.; Protas, A. M.; Belczyk, A.; Bonna, A.; Wyslouch-Cieszynska, A.; Poznanski, J.; Bal, W. Inorg. Chem. 2010, 49, 6636-6645). However, the pseudo-first-order reaction rate found for Cu(II) is, on average, 100 times lower than that for Ni(II) ions. The greater ability of Cu(II) ions to form 4N complexes at lower pH partially compensates for this difference in rates, resulting in similar hydrolytic activities for the two ions around pH 7.


Assuntos
Cobre/química , Níquel/química , Peptídeos/química , Cromatografia Líquida de Alta Pressão , Hidrólise , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta
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