Journal of Biological Inorganic Chemistry最新文献

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM–NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-l-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM–heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM–NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.

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Organometallic η⁶-arene ruthenium(II) complexes with 3-chloro-6-(1H-pyrazol-1-yl)pyridazine (Ru1, Ru2, and Ru5) and 3-chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine (Ru3-4) N,N’ heterocyclic and η⁶-arene (cymene (Ru1-4) or toluene (Ru 5)) have been synthesized. The ruthenium(II) complexes have common “three-legged piano-stool” pseudo-octahedral structures known for half-sandwich complexes. Evolution of their UV–Visible absorption spectra in PBS buffer or DMSO over 24 h confirmed their good solvolysis stability. Titrations of the complexes with the calf thymus DNA (CT-DNA) were monitored using UV–Visible absorption and fluorescence spectroscopies. The complexes interact moderately with CT-DNA and their binding constants are in the order of 10⁴ M⁻¹. Competitive binding of the complexes to a DNA-Hoechst 33,258 depicted competitive displacement of Hoechst from DNA’s minor grooves. These complexes bind to glutathione forming GSH-adducts through S coordination by replacement of a halide, with the iodo-analogues having higher binding constants than the chloro-complexes. Cyclic voltammograms of the complexes exhibited one electron-transfer quasi-reversible process. Trends in the molecular docking data of Ru1-5/DNA were similar to those for DNA binding constants. Of the five, only Ru1, Ru3 and Ru5 showed some activity (moderate) against the MCF-7 breast cancer cells with IC₅₀ values in the range of 59.2–39.9 for which Ru5 was the most active. However, the more difficult-to-treat cell line, MDA-MB 231 cell was recalcitrant to the treatment by these complexes.

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Molecular docking simulations visualized the interactions of arene Ru(II) complexes with CT-DNA via minor grooving. The trends were corroborated by electrochemical and cytotoxicity data.

Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation.

We previously reported that the bismuth(III) dithiocarbamate derivative, bismuth diethyldithiocarbamate (1) exhibited greater cytotoxicity while inducing apoptosis via the intrinsic pathway in MCF-7 cells. We further evaluated the other bismuth(III) dithiocarbamate derivatives, Bi[S₂CNR]₃, with R = (CH₂CH₂OH)(ⁱPr), (CH₂)₄, and (CH₂CH₂OH)(CH₃), denoted as 2, 3, and 4, respectively, in the same MCF-7 cell line. 2–4 were found to exhibit IC₅₀ values of 10.33 ± 0.06 µM, 1.07 ± 0.01 µM and 25.37 ± 0.12 µM, respectively, compared to that of cisplatin at 30.53 ± 0.23 µM. Apoptotic promotion via the mitochondrial-dependent pathway was due to the elevation of intracellular reactive oxygen species (ROS), promotion of caspases, release of cytochrome c, fragmentation of DNA, and results of staining assay observed in all compound-treated cells. 2–4 are also capable of suppressing MCF-7 cell invasion and modulate Lys-48 also Lys-63 linked polyubiquitination, leading to proteasomal degradation. Analysis of gene expression via qRT-PCR revealed their modulation, which supported all activities conducted upon treatment with 2–4. Altogether, bismuth dithiocarbamate derivatives, with bismuth(III) as the metal center bound to ligands, isopropyl ethanol, pyrrolidine, and methyl ethanol dithiocarbamate, are potential anti-breast cancer agents that induce apoptosis and suppress metastasis. Further studies using other breast cancer cell lines and in vivo studies are recommended to clarify the anticancer effects of these compounds.

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Previous studies reported that Pb exposure causes a negative association with delta-aminolevulinic acid dehydratase activity (δ-ALAD), but the impact of Pb exposure (dose and time), B vitamin deficiencies, and lifestyle factors needs to be explored. In this study, the impact of Pb exposure, B vitamin deficiencies, and lifestyle factors on δ-ALAD activity among workers exposed to Pb from the Pb-recycling process was evaluated. Blood lead levels (BLLs), B vitamins (B6, B9, and B12), hematological factors (Hb% and HCT), lifestyle factors, and δ-ALAD activity was assessed in 170 male Pb-exposed workers engaged in the Pb recycling process. BLLs are estimated using the ICP-OES method. B vitamins in serum samples from workers were determined using the ELISA method. The δ-ALAD activity in whole blood samples was determined using the spectrophotometer method. The lifestyle factors were collected using a standard questionnaire. The δ-ALAD activity was significantly decreased in workers with the habits of alcohol use, tobacco consumption, hematocrit < 41%, mild and moderate categories of anemia, vitamin B6 and B12 deficiency, and BLL categories of 10–30, 30–50, and > 50 µg/dL. Multiple regression analysis revealed that the independent variables of alcohol consumption (β = − 0.170; P = 0.025), BLLs (β = − 0.589; P = 0.001) and Hb% (β = 0.183; P = 0.001) significantly influenced the δ-ALAD activity with 44.2% (R² = 0.442). Among the workers exposed to Pb from the Pb recycling plant, δ-ALAD activity was considerably reduced by Pb exposure, B vitamin deficiency, hematological parameters, and lifestyle factors.

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Human calprotectin (CP) is an innate immune protein that participates in the metal-withholding response to infection by sequestering essential metal nutrients from invading microbial pathogens. CP is comprised of S100A8 (α subunit, 10.8 kDa) and S100A9 (β subunit, 13.2 kDa). Two transition-metal binding sites of CP form at the S100A8/S100A9 dimer interface. Site 1 is a His₃Asp motif comprised of His83 and His87 from the S100A8 subunit and His20 and Asp30 from the S100A9 subunit. Site 2 is an unusual hexahistidine motif composed of S100A8 residues His17 and His27 and S100A9 residues His91, His95, His103, and His105. In the present study, the His₃Asp and His₆ sites of CP were further characterized by utilizing Co²⁺ as a spectroscopic probe. Magnetic circular dichroism spectroscopy was employed in conjunction with electron paramagnetic resonance spectroscopy and density functional theory computations to characterize the Co²⁺-bound S100A8(C42S)/S100A9(C3S) CP-Ser variant and six site variants that allowed the His₃Asp and His₆ sites to be further probed. Our results provide new insight into the metal-binding sites of CP-Ser and the effect of amino acid substitutions on the structure of site 2.

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Hyperthermophilic (‘superheat-loving’) archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (k_cat/K_m) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

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Transition metal complexes with characteristics of unique packaging in nanoparticles and remarkable cancer cell cytotoxicity have emerged as potential alternatives to platinum-based antitumor drugs. Here we report the synthesis, characterization, and antitumor activities of three new Ruthenium complexes that introduce 5-fluorouracil-derived ligands. Notably, encapsulation of one such metal complex, Ru3, within pluronic^® F-127 micelles (Ru3-M) significantly enhanced Ru3 cytotoxicity toward A549 cells by a factor of four. To determine the mechanisms underlying Ru3-M cytotoxicity, additional in vitro experiments were conducted that revealed A549 cell treatment with lysosome-targeting Ru3-M triggered oxidative stress, induced mitochondrial membrane potential depolarization, and drastically reduced intracellular ATP levels. Taken together, these results demonstrated that Ru3-M killed cells mainly via a non-apoptotic pathway known as oncosis, as evidenced by observed Ru3-M-induced cellular morphological changes including cytosolic flushing, cell swelling, and cytoplasmic vacuolation. In turn, these changes together caused cytoskeletal collapse and activation of porimin and calpain1 proteins with known oncotic functions that distinguished this oncotic process from other cell death processes. In summary, Ru3-M is a potential anticancer agent that kills A549 cells via a novel mechanism involving Ru(II) complex triggering of cell death via oncosis.

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