The Angiotensin Metabolite His-Leu Is a Strong Copper Chelator Forming Highly Redox Active Species.

His-Leu is a hydrolytic byproduct of angiotensin metabolism, whose concentration in the bloodstream could be at least micromolar. This encouraged us to investigate its Cu(II) binding properties and the concomitant redox reactivity. The Cu(II) binding constants were derived from isothermal titration calorimetry and potentiometry, while identities and structures of complexes were obtained from ultraviolet-visible, circular dichroism, and room-temperature electronic paramagnetic resonance spectroscopies. Four types of Cu(II)/His-Leu complexes were detected. The histamine-like complexes prevail at low pH. At neutral and mildly alkaline pH and low Cu(II):His-Leu ratios, they are superseded by diglycine-like complexes involving the deprotonated peptide nitrogen. At His-Leu:Cu(II) ratios of ≥2, bis-complexes are formed instead. Above pH 10.5, a diglycine-like complex containing the equatorially coordinated hydroxyl group predominates at all ratios tested. Cu(II)/His-Leu complexes are also strongly redox active, as demonstrated by voltammetric studies and the ascorbate oxidation assay. Finally, numeric competition simulations with human serum albumin, glycyl-histydyl-lysine, and histidine revealed that His-Leu might be a part of the low-molecular weight Cu(II) pool in blood if its abundance is >10 µM. These results yield further questions, such as the biological relevance of ternary complexes containing His-Leu.

Concentrations of Transition Metal Ions in Rat Lungs after Tobacco Smoke Exposure and Treatment with His-Leu Dipeptide.

Tobacco smoking is deleterious to the lungs because it exposes them to many toxic substances. These include transition metal ions, such as cadmium. However, there is a lack of information about the influence of endogenous metal-binding peptides, such as His-Leu (HL), on the lung distribution of transition metals in smokers. To address this, we administered HL subcutaneously to rats exposed to tobacco smoke for six weeks, then we measured the concentrations of transition metal ions in the lungs. We found that exposure to tobacco smoke elevates the concentrations of Cd(II) and Cu(II). Administration of the HL peptide, whose elevation is a consequence of angiotensin receptor blocker anti-hypertension therapy, increases the concentration of Fe in the lungs of rats exposed to smoke. These findings suggest that smoking is a risk factor for patients receiving angiotensin receptor blockers to treat hypertension.

Corrigendum: Ni2+-assisted hydrolysis may affect the human proteome; filaggrin degradation ex vivo as an example of possible consequences.

[This corrects the article DOI: 10.3389/fmolb.2022.828674.].

Ni(II) Ions May Target the Entire Melatonin Biosynthesis Pathway-A Plausible Mechanism of Nickel Toxicity.

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.

Neglected N-Truncated Amyloid-ß Peptide and Its Mixed Cu-Zn Complexes.

Amyloid-ß (Aß) peptides are involved in Alzheimer's disease (AD) development. The interactions of these peptides with copper and zinc ions also seem to be crucial for this pathology. Although Cu(II) and Zn(II) ions binding by Aß peptides has been scrupulously investigated, surprisingly, this phenomenon has not been so thoroughly elucidated for N-truncated Aß4-x-probably the most common version of this biomolecule. This negligence also applies to mixed Cu-Zn complexes. From the structural in silico analysis presented in this work, it appears that there are two possible mixed Cu-Zn(Aß4-x) complexes with different stoichiometries and, consequently, distinct properties. The Cu-Zn(Aß4-x) complex with 1:1:1 stoichiometry may have a neuroprotective superoxide dismutase-like activity. On the other hand, another mixed 2:1:2 Cu-Zn(Aß4-x) complex is perhaps a seed for toxic oligomers. Hence, this work proposes a novel research direction for our better understanding of AD development.

Ni2+-Assisted Hydrolysis May Affect the Human Proteome; Filaggrin Degradation Ex Vivo as an Example of Possible Consequences.

Deficiency in a principal epidermal barrier protein, filaggrin (FLG), is associated with multiple allergic manifestations, including atopic dermatitis and contact allergy to nickel. Toxicity caused by dermal and respiratory exposures of the general population to nickel-containing objects and particles is a deleterious side effect of modern technologies. Its molecular mechanism may include the peptide bond hydrolysis in X1-S/T-c/p-H-c-X2 motifs by released Ni2+ ions. The goal of the study was to analyse the distribution of such cleavable motifs in the human proteome and examine FLG vulnerability of nickel hydrolysis. We performed a general bioinformatic study followed by biochemical and biological analysis of a single case, the FLG protein. FLG model peptides, the recombinant monomer domain human keratinocytes in vitro and human epidermis ex vivo were used. We also investigated if the products of filaggrin Ni2+-hydrolysis affect the activation profile of Langerhans cells. We found X1-S/T-c/p-H-c-X2 motifs in 40% of human proteins, with the highest abundance in those involved in the epidermal barrier function, including FLG. We confirmed the hydrolytic vulnerability and pH-dependent Ni2+-assisted cleavage of FLG-derived peptides and FLG monomer, using in vitro cell culture and ex-vivo epidermal sheets; the hydrolysis contributed to the pronounced reduction in FLG in all of the models studied. We also postulated that Ni-hydrolysis might dysregulate important immune responses. Ni2+-assisted cleavage of barrier proteins, including FLG, may contribute to clinical disease associated with nickel exposure.

Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction.

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV-vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.

Ternary Cu2+ Complexes of Human Serum Albumin and Glycyl-l-histidyl-l-lysine.

Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu2+ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu2+ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)NImHSA complexes are formed between GHK and the imino nitrogen (NIm) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant cKCu(GHK)NImHis3Cu(GHK) = 2900 M-1 at pH 7.4), with the second site (cKCu(GHK)NImHisXCu(GHK) = 1700 M-1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu2+ pool and the functional form of GHK.

Phosphorylation Impacts Cu(II) Binding by ATCUN Motifs.

ATCUN (amino terminal Cu(II) and Ni(II) binding) motifs chelate Cu(II) ions strongly. However, the impact of the phosphorylation of neighboring residues on such complexation has not been elucidated. The copper(II) dissociation constants of original and phosphorylated peptides from human histatin-1 and human serum albumin were compared using spectroscopic methods. Phosphorylation markedly weakened Cu(II) binding. Thus, these results indicate that phosphorylation may be a vital mechanism governing metal ion binding.

Cu(II)-Binding N-Terminal Sequences of Human Proteins.

Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1 , His2 , and His3 ) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2 , thus, non-saturating the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) reduction to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those containing His1 , His2 , or His3 . Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the analysis. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the respective sequences with the probability of interaction with divalent copper.

Design and in Vitro Characterization of Tricyclic Benzodiazepine Derivatives as Potent and Selective Antileukemic Agents.

Currently available chemotherapeutic treatments for blood cancers (leukemia) usually have strong side effects. More selective, efficient, and less toxic anticancer agents are needed. We synthesized seven, new, optically pure (12aS)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione derivatives and examined their cytotoxicity towards eight cancer cell lines, including urinary bladder (TCC-SUP, UM-UC-3, KU-19-9), colon (LoVo), and breast (MCF-7, MDA-MB-231) cancer representatives, as well as two leukemic cell lines (MV-4-11, CCRF-CEM) and normal murine fibroblasts (Balb/3T3) as reference cell line. Three of the seven newly-obtained compounds ((12aS)-8-bromo-2-(3-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, (12aS)-8,9-dimethoxy-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione and (12aS)-8-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, showed enhanced activity and selectivity toward the leukemic MV-4-11 cell lines when compared to our previously reported compounds, with IC50 values in the range of 2.9-5.6 µM. Additionally, (12aS)-9-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione exhibited a strong cytotoxic effect against the leukemic CCRF-CEM (IC50 =6.1 µM) and MV-4-11 (IC50 =11.0 µM) cell lines, a moderate cytotoxic effect toward other tumor lines (IC50 =31.8-55.0 µM) and very weak cytotoxic effect toward the Balb/3T3 reference cell lines. Selected compounds were further evaluated for their potential to induce apoptotic cell death in MV-4-11 cells by measuring caspase-3 activity. We also established the crystal structure of three products and investigated the effect of 22 derivatives of 1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione on the activity of the cancer-associated enzyme autotaxin. All compounds proved to be weak inhibitors of autotaxin, although some (R) and (S) enantiomers had Ki values of 10-19 µM. The obtained results showed that the tested compounds exhibited a selective antileukemic effect, which appeared not to be related directly to autotaxin. Molecular targets responsible for this effect remain to be identified. The newly obtained compounds can be used in the search for new, selective anticancer therapies.

The Palladium(II) Complex of Aß4-16 as Suitable Model for Structural Studies of Biorelevant Copper(II) Complexes of N-Truncated Beta-Amyloids.

The Aß4-42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aß4-16 and Aß4-9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aß4-x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aß4-16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aß4-16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aß4-16, Pd(II)-Aß4-16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aß complexes.

Ternary Cu(II) Complex with GHK Peptide and Cis-Urocanic Acid as a Potential Physiologically Functional Copper Chelate.

The tripeptide NH2-Gly-His-Lys-COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.

Peptide bond cleavage in the presence of Ni-containing particles.

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.

The Sub-picomolar Cu2+ Dissociation Constant of Human Serum Albumin.

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+ -binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room-temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log cKCuCu(HSA) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.

Peptide Bond Cleavage by Ni(II) Ions within the Nuclear Localization Signal Sequence.

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.

Thymidylate synthase-catalyzed, tetrahydrofolate-dependent self-inactivation by 5-FdUMP.

In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.

Gly-His-Thr-Asp-Amide, an Insulin-Activating Peptide from the Human Pancreas Is a Strong Cu(II) but a Weak Zn(II) Chelator.

The Cu(II) and Zn(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide coreleased from the pancreas along with insulin, were studied using UV-vis and circular dichroism spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu(II) chelator, forming a three-nitrogen complex with a conditional affinity constant C K at pH 7.4 of 4.5 × 1012 M-1. The fourth coordination site can be occupied by a solvent molecule or a ternary ligand, such as imidazole, with C K on the order of several hundred reciprocal molar. The Zn(II) binding ability of GHTD-am is relatively weak, with C K values at pH 7.4 of 3.0 × 104 and 2.0 × 103 M-1 for the first and second GHTD-am molecule coordinated, respectively. These results are discussed in light of the modes of interactions of Zn(II) and Cu(II) ions with insulin. A direct effect of GHTD-am on the Zn(II) interactions with insulin is unlikely, but its Cu(II) complex may have a biological relevance because of its high affinity and ability to form ternary complexes.

Interplay between Copper, Neprilysin, and N-Truncation of ß-Amyloid.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the ß-amyloid (Aß) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aß levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aß fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 µM),  while proteolysis of Aß yielded the soluble, Aß4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aß4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 µM) further suggests a mechanism for modulating NEP activity, Aß4-9 production, and Cu2+ homeostasis.

Crystal structures of nematode (parasitic T. spiralis and free living C. elegans), compared to mammalian, thymidylate synthases (TS). Molecular docking and molecular dynamics simulations in search for nematode-specific inhibitors of TS.

Three crystal structures are presented of nematode thymidylate synthases (TS), including Caenorhabditis elegans (Ce) enzyme without ligands and its ternary complex with dUMP and Raltitrexed, and binary complex of Trichinella spiralis (Ts) enzyme with dUMP. In search of differences potentially relevant for the development of species-specific inhibitors of the nematode enzyme, a comparison was made of the present Ce and Ts enzyme structures, as well as binary complex of Ce enzyme with dUMP, with the corresponding mammalian (human, mouse and rat) enzyme crystal structures. To complement the comparison, tCONCOORD computations were performed to evaluate dynamic behaviors of mammalian and nematode TS structures. Finally, comparative molecular docking combined with molecular dynamics and free energy of binding calculations were carried out to search for ligands showing selective affinity to T. spiralis TS. Despite an overall strong similarity in structure and dynamics of nematode vs mammalian TSs, a pool of ligands demonstrating predictively a strong and selective binding to TsTS has been delimited. These compounds, the E63 family, locate in the dimerization interface of TsTS where they exert species-specific interactions with certain non-conserved residues, including hydrogen bonds with Thr174 and hydrophobic contacts with Phe192, Cys191 and Tyr152. The E63 family of ligands opens the possibility of future development of selective inhibitors of TsTS and effective agents against trichinellosis.