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Magnetic Resonance (Gottingen, Germany) 2022The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance...
The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii) reaction of the cyano groups with -cysteine, penicillamine or -cysteine to complete the lanthanoid chelating moiety; and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with and ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron-electron resonance experiments after titration with ions.
PubMed: 37904871
DOI: 10.5194/mr-3-169-2022 -
Biology Mar 2022Assessments of sperm performance are valuable tools for the analysis of sperm fertilizing potential and to understand determinants of male fertility. Hamster species...
Assessments of sperm performance are valuable tools for the analysis of sperm fertilizing potential and to understand determinants of male fertility. Hamster species constitute important animal models because they produce sperm cells in high quantities and of high quality. Sexual selection over evolutionary time in these species seems to have resulted in the largest mammalian spermatozoa, and high swimming and bioenergetic performances. Earlier studies showed that golden hamster sperm requires motility factors such as D-penicillamine, hypotaurine and epinephrine (PHE) to sustain survival over time, but it is unknown how they affect swimming kinetics or ATP levels and if other hamster species also require them. The objective of the present study was to examine the effect of PHE on spermatozoa of five hamster species (, , , , ). In sperm incubated for up to 4 h without or with PHE, we assessed motility, viability, acrosome integrity, sperm velocity and trajectory, and ATP content. The results showed differences in the effect of PHE among species. They had a significant positive effect on the maintenance of sperm quality in and , whereas there was no consistent effect on spermatozoa of the species. Differences between species may be the result of varying underlying regulatory mechanisms of sperm performance and may be important to understand how they relate to successful fertilization.
PubMed: 35453725
DOI: 10.3390/biology11040526 -
Cellular and Molecular Life Sciences :... Aug 2020Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of...
Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.
Topics: Adenine; DNA; Extracellular Traps; Granulomatous Disease, Chronic; Humans; Leukocyte Elastase; Neutrophils; Nitric Oxide; Peroxynitrous Acid; Phosphatidylinositol 3-Kinases; Reactive Nitrogen Species; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; p38 Mitogen-Activated Protein Kinases
PubMed: 31650185
DOI: 10.1007/s00018-019-03331-x -
Scientific Reports May 2023Hydrogen sulfide (HS) is a gaseous signaling molecule that participates in various signaling functions in health and diseases. The tetrameric cystathionine γ-lyase...
Hydrogen sulfide (HS) is a gaseous signaling molecule that participates in various signaling functions in health and diseases. The tetrameric cystathionine γ-lyase (CSE) contributes to HS biogenesis and several investigations provide evidence on the pharmacological modulation of CSE as a potential target for the treatment of a multitude of conditions. D-penicillamine (D-pen) has recently been reported to selectively impede CSE-catalyzed HS production but the molecular bases for such inhibitory effect have not been investigated. In this study, we report that D-pen follows a mixed-inhibition mechanism to inhibit both cystathionine (CST) cleavage and HS biogenesis by human CSE. To decipher the molecular mechanisms underlying such a mixed inhibition, we performed docking and molecular dynamics (MD) simulations. Interestingly, MD analysis of CST binding reveals a likely active site configuration prior to gem-diamine intermediate formation, particularly H-bond formation between the amino group of the substrate and the O3' of PLP. Similar analyses realized with both CST and D-pen identified three potent interfacial ligand-binding sites for D-pen and offered a rational for D-pen effect. Thus, inhibitor binding not only induces the creation of an entirely new interacting network at the vicinity of the interface between enzyme subunits, but it also exerts long range effects by propagating to the active site. Overall, our study paves the way for the design of new allosteric interfacial inhibitory compounds that will specifically modulate HS biogenesis by cystathionine γ-lyase.
Topics: Humans; Hydrogen Sulfide; Cystathionine gamma-Lyase; Signal Transduction; Cystathionine; Computer Simulation
PubMed: 37142727
DOI: 10.1038/s41598-023-34405-3 -
The Journal of Biological Chemistry Jun 2020Sirtuins ( human Sirt1-7) catalyze the removal of acyl groups from lysine residues in proteins in an NAD-dependent manner, and loss of sirtuin deacylase activity...
Sirtuins ( human Sirt1-7) catalyze the removal of acyl groups from lysine residues in proteins in an NAD-dependent manner, and loss of sirtuin deacylase activity correlates with the development of aging-related diseases. Although multiple reports suggest that sirtuin activity is regulated by oxidative post-translational modifications of cysteines during inflammation and aging, no systematic comparative study of potential direct sirtuin cysteine oxidative modifications has been performed. Here, using IC and / analyses, we quantified the ability of nitrosothiols (-nitrosoglutathione and -nitroso--acetyl-d,l-penicillamine), nitric oxide, oxidized GSH, and hydrogen peroxide to post-translationally modify and inhibit the deacylase activity of Sirt1, Sirt2, Sirt3, Sirt5, and Sirt6. The inhibition was correlated with cysteine modification and assessed with chemical-probe and blot-based assays for cysteine -nitrosation, sulfenylation, and glutathionylation. We show that the primarily nuclear sirtuins Sirt1 and Sirt6, as well as the primarily cytosolic sirtuin Sirt2, are modified and inhibited by cysteine -nitrosation in response to exposure to both free nitric oxide and nitrosothiols (/ ≥ 5 m s), which is the first report of Sirt2 and Sirt6 inhibition by -nitrosation. Surprisingly, the mitochondrial sirtuins Sirt3 and Sirt5 were resistant to inhibition by cysteine oxidants. Collectively, these results suggest that nitric oxide-derived oxidants may causatively link nuclear and cytosolic sirtuin inhibition to aging-related inflammatory disease development.
Topics: Cysteine; Glutathione; Humans; Kinetics; Mitochondria; Nitric Oxide; Oxidants; Protein Processing, Post-Translational; Recombinant Proteins; S-Nitrosoglutathione; Sirtuin 1; Sirtuin 2; Sirtuins
PubMed: 32371394
DOI: 10.1074/jbc.RA119.011988 -
Frontiers in Plant Science 2020Autophagy plays a role in regulating important cellular functions in response to stress conditions. The role of nitric oxide (NO) in the regulation of autophagy in has...
Autophagy plays a role in regulating important cellular functions in response to stress conditions. The role of nitric oxide (NO) in the regulation of autophagy in has been not studied. Illumination of cells under a high light (HL, 1,600 μmol m s) condition induced a NO burst through NO synthase- and nitrate reductase-independent routes, and cell death. The abundance of CrATG8 protein, an autophagy marker of , increased after HL illumination along with a linear increase in the transcript abundance of autophagy-associated genes (CrVPS34, CrATG1, CrATG3, CrATG4, CrATG6, CrATG7, CrATG8, and CrATG12), which were suppressed in the presence of an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). The cells were treated with NO donors, -nitroso--acetyl-penicillamine, and -nitrosoglutathione, under a normal light (50 μmol m s) condition to elucidate the role of NO in autophagy activation and cell death. Treatment with 0.05 mM or 0.1 mM NO donors increased the abundance of ATG8 protein and CrATG transcripts, which were suppressed in the presence of cPTIO. Moreover, treatment with 0.05 mM NO donors did not affect cell viability, while 0.1 mM NO donors elicited a transient decrease in cell growth and death that recovered after 12 h. The transient effect could be prevented by the presence of cPTIO. However, treatment with 1 mM HO and 0.1 mM NO donors enhanced autophagy induction and resulted in cell death after 24 h. The interaction of HO and NO can be prevented by cPTIO treatment. This implies that NO is critical for the interaction of HO and NO that induces cell death and autophagy. Furthermore, exposure to 0.1 mM NO donors under a non-lethal HL condition (750 μmol m s) evoked autophagy and cell death. In conclusion, the present findings demonstrated that the NO-mediated autophagy pathway is activated in under lethal high intensity illumination and may interact with HO for HL-induced cell death. The relationships between autophagy and cell death are discussed.
PubMed: 32587598
DOI: 10.3389/fpls.2020.00772 -
Vascular Pharmacology 2020Stem cell-derived cardiac myocytes are potential sources for testing cardiocytoprotective molecules against ischemia/reperfusion injury in vitro. (Comparative Study)
Comparative Study
INTRODUCTION
Stem cell-derived cardiac myocytes are potential sources for testing cardiocytoprotective molecules against ischemia/reperfusion injury in vitro.
MATERIALS AND METHODS
Here we performed a systematic analysis of two different induced pluripotent stem cell lines (iPSC 3.4 and 4.1) and an embryonic stem cell (ESC) line-derived cardiac myocytes at two different developmental stages. Cell viability in simulated ischemia/reperfusion (SI/R)-induced injury and a known cardiocytoprotective NO-donor, S-nitroso-n-acetylpenicillamine (SNAP) was tested.
RESULTS
After analysis of full embryoid bodies (EBs) and cardiac marker (VCAM and cardiac troponin I) positive cells of three lines at 6 conditions (32 different conditions altogether), we found significant SI/R injury-induced cell death in both full EBs and VCAM+ cardiac cells at later stage of their differentiation. Moreover, full EBs of the iPS 4.1 cell line after oxidative stress induction by SNAP was protected at day-8 samples.
CONCLUSION
We have shown that 4.1 iPS-derived cardiomyocyte line could serve as a testing platform for cardiocytoprotection.
Topics: Cell Differentiation; Cell Line; Cell Survival; Humans; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitric Oxide Donors; Oxidative Stress; Phenotype; Pluripotent Stem Cells; S-Nitroso-N-Acetylpenicillamine; Troponin I; Vascular Cell Adhesion Molecule-1
PubMed: 32827678
DOI: 10.1016/j.vph.2020.106781 -
Frontiers in Microbiology 2022Low-molecular-mass (LMM) thiol compounds are known to be important for many biological processes in various organisms but LMM thiols are understudied in anaerobic...
Low-molecular-mass (LMM) thiol compounds are known to be important for many biological processes in various organisms but LMM thiols are understudied in anaerobic bacteria. In this work, we examined the production and turnover of nanomolar concentrations of LMM thiols with a chemical structure related to cysteine by the model iron-reducing bacterium . Our results show that tightly controls the production, excretion and intracellular concentration of thiols depending on cellular growth state and external conditions. The production and cellular export of endogenous cysteine was coupled to the extracellular supply of Fe(II), suggesting that cysteine excretion may play a role in cellular trafficking to iron proteins. Addition of excess exogenous cysteine resulted in a rapid and extensive conversion of cysteine to penicillamine by the cells. Experiments with added isotopically labeled cysteine confirmed that penicillamine was formed by a dimethylation of the C-3 atom of cysteine and not indirect metabolic responses to cysteine exposure. This is the first report of metabolic synthesis of this compound. Penicillamine formation increased with external exposure to cysteine but the compound did not accumulate intracellularly, which may suggest that it is part of ' metabolic strategy to maintain cysteine homeostasis. Our findings highlight and expand on processes mediating homeostasis of cysteine-like LMM thiols in strict anaerobic bacteria. The formation of penicillamine is particularly noteworthy and this compound warrants more attention in microbial metabolism studies.
PubMed: 36713222
DOI: 10.3389/fmicb.2022.1085214 -
ACS Applied Materials & Interfaces Jul 2022Physical incorporation of nitric oxide (NO) releasing materials in biomedical grade polymer matrices to fabricate antimicrobial coatings and devices is an economically...
Physical incorporation of nitric oxide (NO) releasing materials in biomedical grade polymer matrices to fabricate antimicrobial coatings and devices is an economically viable process. However, achieving long-term NO release with a minimum or no leaching of the NO donor from the polymer matrix is still a challenging task. Herein, (-acetyl--nitrosopenicillaminyl)--nitrosopenicillamine (SNAP-SNAP), a penicillamine dipeptide NO-releasing molecule, is incorporated into a commercially available biomedical grade silicone rubber (SR) to fabricate a NO-releasing coating (SNAP-SNAP/SR). The storage stabilities of the SNAP-SNAP powder and SNAP-SNAP/SR coating were analyzed at different temperatures. The SNAP-SNAP/SR coatings with varying wt % of SNAP-SNAP showed a tunable and sustained NO release for up to 6 weeks. Further, -nitroso--acetylpenicillamine (SNAP), a well-explored NO-releasing molecule, was incorporated into a biomedical grade silicone polymer to fabricate a NO-releasing coating (SNAP/SR) and a comparative analysis of the NO release and -nitrosothiol (RSNO) leaching behavior of 10 wt % SNAP-SNAP/SR and 10 wt % SNAP/SR was studied. Interestingly, the 10 wt % SNAP-SNAP/SR coatings exhibited ∼36% higher NO release and 4 times less leaching of NO donors than the 10 wt % SNAP/SR coatings. Further, the 10 wt % SNAP-SNAP/SR coatings exhibited promising antibacterial properties against and due to the persistent release of NO. The 10 wt % SNAP-SNAP/SR coatings were also found to be biocompatible against NIH 3T3 mouse fibroblast cells. These results corroborate the sustained stability and NO-releasing properties of the SNAP-SNAP in a silicone polymer matrix and demonstrate the potential for the SNAP-SNAP/SR polymer in the fabrication of long-term indwelling biomedical devices and implants to enhance biocompatibility and resist device-related infections.
Topics: Animals; Anti-Bacterial Agents; Escherichia coli; Mice; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Polymers; S-Nitroso-N-Acetylpenicillamine; Silicone Elastomers
PubMed: 35759508
DOI: 10.1021/acsami.2c06712 -
Pharmaceutics Oct 2021Actinium-225 (Ac) is a promising radionuclide used in targeted alpha therapy (TAT). Although Ac labeling of bifunctional chelating ligands is effective, previous in vivo...
Evaluation of Aminopolycarboxylate Chelators for Whole-Body Clearance of Free Ac: A Feasibility Study to Reduce Unexpected Radiation Exposure during Targeted Alpha Therapy.
Actinium-225 (Ac) is a promising radionuclide used in targeted alpha therapy (TAT). Although Ac labeling of bifunctional chelating ligands is effective, previous in vivo studies reported that free Ac can be released from the drugs and that such free Ac is predominantly accumulated in the liver and could cause unexpected toxicity. To accelerate the clinical development of Ac TAT with a variety of drugs, preparing methods to deal with any unexpected toxicity would be valuable. The aim of this study was to evaluate the feasibility of various chelators for reducing and excreting free Ac and compare their chemical structures. Nine candidate chelators (D-penicillamine, dimercaprol, Ca-DTPA, Ca-EDTA, CyDTA, GEDTA TTHA, Ca-TTHA, and DO3A) were evaluated in vitro and in vivo. The biodistribution and dosimetry of free Ac were examined in mice before an in vivo chelating study. The liver exhibited pronounced Ac uptake, with an estimated human absorbed dose of 4.76 Sv/MBq. Aminopolycarboxylate chelators with five and six carboxylic groups, Ca-DTPA and Ca-TTHA, significantly reduced Ac retention in the liver (22% and 30%, respectively). Significant Ac reductions were observed in the heart and remainder of the body with both Ca-DTPA and Ca-TTHA, and in the lung, kidney, and spleen with Ca-TTHA. In vitro interaction analysis supported the in vivo reduction ability of Ca-DTPA and Ca-TTHA. In conclusion, aminopolycarboxylate chelators with five and six carboxylic groups, Ca-DTPA and Ca-TTHA, were effective for whole-body clearance of free Ac. This feasibility study provides useful information for reducing undesirable radiation exposure from free Ac.
PubMed: 34683999
DOI: 10.3390/pharmaceutics13101706