Chelation Of Zn2 Research Articles

Intracellular Zn2+ promotes extracellular matrix remodeling in dexamethasone-treated trabecular meshwork.

Given the widespread application of glucocorticoids in ophthalmology, the associated elevation of intraocular pressure (IOP) has long been a vexing concern for clinicians, yet the underlying mechanisms remain inconclusive. Much of the discussion focuses on the extracellular matrix (ECM) of trabecular meshwork (TM). It is widely agreed that glucocorticoids impact the expression of matrix metalloproteinases (MMPs), leading to ECM deposition. Since Zn2+ is vital for MMPs, we explored its role in ECM alterations induced by dexamethasone (DEX). Our study revealed that in human TM cells treated with DEX, the level of intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. This correlated with changes in several Zrt-, Irt-related proteins (ZIPs) and metallothionein. ZIP8 knockdown impaired extracellular Zn2+ uptake, but Zn2+ chelation did not affect ZIP8 expression. Resembling DEX's effects, chelation of Zn2+ decreased MMP2 expression, increased the deposition of ECM proteins, and induced structural disarray of ECM. Conversely, supplementation of exogenous Zn2+ in DEX-treated cells ameliorated these outcomes. Notably, dietary zinc supplementation in mice significantly reduced DEX-induced IOP elevation and collagen content in TM, thereby rescuing the visual function of the mice. These findings underscore zinc's pivotal role in ECM regulation, providing a novel perspective on the pathogenesis of glaucoma.NEW & NOTEWORTHY Our study explores zinc's pivotal role in mitigating extracellular matrix dysregulation in the trabecular meshwork and glucocorticoid-induced ocular hypertension. We found that in human trabecular meshwork cells treated with dexamethasone, intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. Zinc supplementation rescues visual function by modulating extracellular matrix proteins and lowering intraocular pressure, offering a direction for further exploration in glaucoma management.

Novel strategy to the characterization and enhance the glycemic control properties of walnut-derived peptides via zinc chelation

This study aimed to utilize zinc coordination to promote the hypoglycemic and antioxidant properties of walnut-derived peptides, such as walnut protein hydrolysate (WPH) and Leu-Pro-Leu-Leu-Arg (LPLLR, LP5), of which LP5 was previously identified from WPH. The optimal conditions for the chelation were a peptide-to-zinc ratio of 6:1, pH of 9, duration of 50 min, and temperature of 50 °C. The WPH-Zn and LP5-Zn complexes increased the α-glucosidase inhibition, α-amylase inhibition, and antioxidant activity more than WPH and LP5 (p < 0.05). In particular, the antioxidant activity of WPH-Zn was superior to LP5-Zn. This is attributable to the WPH containing more aromatic amino acids, carboxylate groups and the imidazole groups, which implies its capacity to potentially coordinate with Zn2+ to form the WPH-Zn complex. Moreover, particle size, zeta potential, and scanning electron microscope indicated that the chelation of Zn2+ by peptides led to intramolecular and intermolecular folding and aggregation.

Novel Hydroxamic Acids Containing Aryl-Substituted 1,2,4- or 1,3,4-Oxadiazole Backbones and an Investigation of Their Antibiotic Potentiation Activity.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc amidase that catalyzes the second step of the biosynthesis of lipid A, which is an outer membrane essential structural component of Gram-negative bacteria. Inhibitors of this enzyme can be attributed to two main categories, non-hydroxamate and hydroxamate inhibitors, with the latter being the most effective given the chelation of Zn2+ in the active site. Compounds containing diacetylene or acetylene tails and the sulfonic head, as well as oxazoline derivatives of hydroxamic acids, are among the LpxC inhibitors with the most profound antibacterial activity. The present article describes the synthesis of novel functional derivatives of hydroxamic acids-bioisosteric to oxazoline inhibitors-containing 1,2,4- and 1,3,4-oxadiazole cores and studies of their cytotoxicity, antibacterial activity, and antibiotic potentiation. Some of the hydroxamic acids we obtained (9c, 9d, 23a, 23c, 30b, 36) showed significant potentiation in nalidixic acid, rifampicin, and kanamycin against the growth of laboratory-strain Escherichia coli MG1655. Two lead compounds (9c, 9d) significantly reduced Pseudomonas aeruginosa ATCC 27853 growth in the presence of nalidixic acid and rifampicin.

Molecular Characterization and the Essential Biological Function of the Metal Chaperone Protein MtmA in Aspergillus fumigatus.

The detoxification system of reactive oxygen species (ROS) plays critical roles in the survival and virulence of fungal pathogens in infected hosts, while superoxide dismutase (SOD) is the primary ROS scavenger. In the model yeast Saccharomyces cerevisiae, the metal chaperone protein Mtm1 is required for mitochondrial Sod2 activation and responses to oxidative stress. However, the function of the S. cerevisiae Mtm1 homolog in the human fungal pathogen Aspergillus fumigatus has not yet been clarified. In this study, we found that mitochondria-localized MtmA in A. fumigatus, a putative homolog of yeast Mtm1, not only has a similar function to Mtm1 in responding to oxidative stress resistance by affecting SodB (MnSOD) activity but is also essential for hyphal growth such that repressed expression of MtmA results in severe growth defects in A. fumigatus. In addition, the chelation of Zn2+ can obviously rescue growth defects caused by repression of MtmA, suggesting that MtmA may be involved in hyphal growth by affecting cellular Zn2+ detoxification. Moreover, MtmA contains four Mito-carr domains, whereas only the first Mito-carr domain is required for the function of MtmA. Therefore, the findings in this study suggest that MtmA in A. fumigatus has an important and unique function that is different from that in yeast. IMPORTANCE Knowledge of the key factors required for the viability of pathogenic fungi can help to explore new antifungal drugs. Here, we demonstrate that MtmA is involved in responding to oxidative stress by activating mitochondrial SodB activity. MtmA, especially for the first Mito-carr domain, is essential for colony growth by regulating cellular Zn2+ equilibrium and responses to oxidative stress in A. fumigatus. This is the first report of the vital and unique role of the MtmA protein in pathogenic fungi, indicating that it might be a potential antifungal drug target.

Upregulation of p67phox in response to ischemia/reperfusion is cardioprotective by increasing ZIP2 expression via STAT3

While the zinc transporter ZIP2 (Slc39a2) is upregulated via STAT3 as an adaptive response to protect the heart from ischemia/reperfusion (I/R) injury, the precise mechanism underlying its upregulation remains unclear. The purpose of this study was to investigate the role of NADPH oxidase (NOX) isoform NOX2-derived ROS in the regulation of ZIP2 expression, focusing on the role of the NOX2 cytosolic factor p67phox. Mouse hearts or H9c2 cells were subjected to I/R. Protein expression was detected with Western blotting. Infarct size was measured with TTC staining. The cardiac-specific p67phox conditional knockout mice (p67phox cKO) were generated by adopting the CRISPR/Cas9 system. I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression was reversed by the ROS scavenger N-acetylcysteine (NAC) and the NOX inhibitor diphenyleneiodonium (DPI). p67phox but not NOX2 expression was increased 30 min after the onset of reperfusion, and downregulation of p67phox by siRNA or cKO invalidated I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression. Both NAC and DPI prevented upregulation of STAT3 phosphorylation and ZIP2 expression induced by overexpression of p67phox, whereas the STAT3 inhibitor stattic abrogated upregulation ZIP2 expression, indicating that the increase of p67phox at reperfusion is an upstream signaling event responsible for ZIP2 upregulation via STAT3. Experiments also showed that chelation of Zn2+ markedly enhanced p67phox and ZIP2 expression as well as STAT3 phosphorylation, whereas supplementation of Zn2+ had the opposite effects, indicating that cardiac Zn2+ loss upon reperfusion triggers p67phox upregulation. Furthermore, ischemic preconditioning (IPC) upregulated ZIP2 via p67phox, and cKO of p67phox aggravated cardiac injury after I/R, indicating that p67phox upregulation is cardioprotective against I/R injury. In conclusion, an increase of p67phox expression in response to Zn2+ is an intrinsic adaptive response to I/R and leads to cardioprotection against I/R by upregulating ZIP2 via STAT3.

Nanofibers comprising polystyrene and zinc acetate for extraction of salivary histidine prior to its determination by HPLC-DAD.

Anovel nanofiber-based solid phase extraction(NFSPE)coupled with HPLC-DAD was developed for the determination of salivary histidine, a new potential index of physiological events. Thetreatment of the samplewas based on a coupling reaction of histidinewith the diazotization products of sulfanilic acid and sodium nitritein a slightly alkaline medium,followed byNFSPE.Nanofibers comprising polystyrene and zinc acetate were used as a novelsolid-phasesorbent to extract derived histidine via the chelation of Zn2+with the imidazole group in the analyte. The eluted target compound was analyzed by HPLC-DAD. Goodlinearity was achieved with the concentrations of histidine ranging from 0.645 to 64.5µM; the detection limit was 0.193µM. Theintra-day and inter-day coefficients of variation were less than 8.0%.The method was successfully applied tothe determination of salivary histidine in the subjects exposed to a public speaking stress.

A Quick Responsive Chitosan‐Oxine Based Thin Film to Recognize and Remove Zn2+ Ions from Potable Water

AbstractThrough the functionalization of chitosan (CS) with an oxine derivative, 8‐hydroxy‐2‐methyl‐quinoline5,7‐ sulfonyl chloride (HQSC), a thin film was prepared (CS‐HQSC). The film can recognize and adsorb Zn2+ ions from the aqueous samples. The sensor performances were characterized by UV‐Visible and fluorescence spectroscopic analysis. The weakly fluorescent thin film becomes highly fluorescent in the presence of Zn2+ ions. The solution‐phase study of the functionalized material shows a 30‐fold fluorescence enhancement in the presence of Zn2+ ions. The chelation of Zn2+ ions with CS‐HQSC could be the reason for the fluorescence enhancement. The chelation restricts the excited‐state proton transfer in the quinoline ring and facilitates chelation enhanced fluorescence. The sensor showed a linear detection of Zn2+ ions from 0.2 to 2 ppm, with the lowest detection limit of 0.08 ppm. The practical applicability studies of the sensor prove that the sensor film is quick responsive (30 seconds) and recyclable in acid solution. Also, the functionalized material CS‐HQSC shows 96 % removal of Zn2+ ions from the water samples.

Preparation and structural characterization of peanut peptide–zinc chelate

ABSTRACT Peanut protein isolate was hydrolyzed by trypsin. The resulting peanut peptide fractions (PPF) were then divided into three molecular weight components by ultrafiltration (PPF1, MW >5 kDa; PPF2, 3 kDa < MW <5 kDa; PPF3, MW <3 kDa; and untreated PPF). Experiments showed that PPF3 had the best capacity for chelation with zinc. PPF3 and the PPF3–Zn chelate were characterized by Ultraviolet spectrophotometry, Fourier-transform infrared spectroscopy, X-ray diffraction, Scanning electron microscopy, Energy disperse spectroscopy, Fluorescence spectroscopy, and Thermogravimetric analysis. The results showed that the apparent characteristics and internal structures of PPF3 were changed by chelation of Zn2+. PPF3 contained sufficient metal-binding sites, with – COOH, – OH, and – NH2 potentially participating in coordination with Zn2+ to form the PPF3-Zn chelate. Furthermore, the results suggested that peanut peptides might chelate with zinc to act as an emerging carrier for zinc supplements.

Diketopyrrolopyrrole-based fluorescence probes for the imaging of lysosomal Zn2+ and identification of prostate cancer in human tissue.

A series of diketopyrrolopyrrole-based fluorescent probes (DPP-C2, LysoDPP-C2, LysoDPP-C3, and LysoDPP-C4) have been developed for the detection of low pH and Zn2+ in an AND logic fashion. The chelation of Zn2+ or the protonation of a morpholine moiety within these probes results in a partial increase in the fluorescence intensity, an effect ascribed to suppression of one possible photo-induced electron transfer (PET) pathway. In contrast, a large increase in the observed fluorescence intensity is observed at low pH and in the presence of Zn2+; this is rationalized in terms of both possible PET pathways within the probes being blocked. Job plots, fluorescence titration curves, and isothermal titration calorimetry proved consistent with a 1 : 1 Zn2+ complexation stoichiometry. Each probe demonstrated an excellent selectivity towards Zn2+ and the resulting Zn2+ complexes demonstrated pH sensitivity over the 3.5-9 pH range. Fluorescence imaging experiments confirmed that LysoDPP-C4 was capable of imaging lysosomal Zn2+ in live cells. Little evidence of cytotoxicity was seen. LysoDPP-C4 was successfully applied to the bioimaging of nude mice, wherein it was shown capable of imaging the prostate. Histological studies using a human sample revealed that LysoDPP-C4 can discriminate cancerous prostate tissue from healthy prostate tissue.

X-ray structures of human bile-salt activated lipase conjugated to nerve agents surrogates

The efficiency of human butyrylcholinesterase (BChE) as a stoichiometric bioscavenger of nerve agents is well established. However, wide use is currently limited by production and purification costs. Aiming at identifying an alternative human protein bioscavenger, we looked for an original scaffold candidate by virtual screening of the Protein Data Bank for functional similarity using the "Surfing the Molecules" software (sumo-pbil.ibcp.fr) and a search model based on the BChE active site topology. Besides the expected acetylcholinesterase and butyrylcholinesterase, we identified a set of bile salt activated lipases structures, among which the human pancreatic lipase (hBAL) that shares 34% identity with BChE. We produced the recombinant enzyme in mammalian cells, purified it, and measured the inhibition constants for paraoxon and surrogates of VX, sarin and tabun. We solved the X-ray structure of apo hBAL and conjugates with paraoxon and the surrogates at resolutions in the 2-Å range. These structures allow the assessment of hBAL for scavenging nerve agents. They revealed that hBAL has inverted stereoselectivity for the surrogates of nerve agent compared to human cholinesterases. We observed a remarkable flip of the catalytic histidine driven by the chelation of Zn2+. Dealkylation of the conjugate, aka aging, was solely observed for paraoxon.

TRPM2-mediated rise in mitochondrial Zn2+ promotes palmitate-induced mitochondrial fission and pancreatic \u03b2-cell death in rodents

Rise in plasma free fatty acids (FFAs) represents a major risk factor for obesity-induced type 2 diabetes. Saturated FFAs cause a progressive decline in insulin secretion by promoting pancreatic β-cell death through increased production of reactive oxygen species (ROS). Recent studies have demonstrated that palmitate (a C16-FFA)-induced rise in ROS causes β-cell death by triggering mitochondrial fragmentation, but the underlying mechanisms are unclear. Using the INS1-832/13 β-cell line, here we demonstrate that palmitate generates the ROS required for mitochondrial fission by activating NOX (NADPH oxidase)-2. More importantly, we show that chemical inhibition, RNAi-mediated silencing and knockout of ROS-sensitive TRPM (transient receptor potential melastatin)-2 channels prevent palmitate-induced mitochondrial fission. Although TRPM2 activation affects the intracellular dynamics of Ca2+ and Zn2+, chelation of Zn2+ alone was sufficient to prevent mitochondrial fission. Consistent with the role of Zn2+, palmitate caused a rise in mitochondrial Zn2+, leading to Zn2+-dependent mitochondrial recruitment of Drp-1 (a protein that catalyses mitochondrial fission) and loss of mitochondrial membrane potential. In agreement with the previous reports, Ca2+ caused Drp-1 recruitment, but it failed to induce mitochondrial fission in the absence of Zn2+. These results indicate a novel role for Zn2+ in mitochondrial dynamics. Inhibition or knockout of TRPM2 channels in mouse islets and RNAi-mediated silencing of TRPM2 expression in human islets prevented FFA/cytokine-induced β-cell death, findings that are consistent with the role of abnormal mitochondrial fission in cell death. To conclude, our results reveal a novel, potentially druggable signalling pathway for FFA-induced β-cell death. The cascade involves NOX-2-dependent production of ROS, activation of TRPM2 channels, rise in mitochondrial Zn2+, Drp-1 recruitment and abnormal mitochondrial fission.

Zinc chelation decreases IFN-β-induced STAT1 upregulation and iNOS expression in RAW 264.7 macrophages

One consequence of lipopolysaccharide (LPS)-induced stimulation of macrophages is the release of Interferon (IFN)-β, and subsequently the activation of the JAK-STAT1 pathway, resulting in the expression of inducible nitric oxide synthase (iNOS). Free intracellular zinc ions (Zn2+) have a profound impact as a second messenger in LPS-dependent gene expression. Previous work had indicated a Zn2+-dependent upregulation of STAT1 mRNA in response to LPS and IFN-β, potentially affecting STAT1-dependent downstream signaling upon pre-incubation with these agents. The aim of the present study was to investigate the long-term influence of Zn2+ chelation on cellular STAT1 levels and their effect on protein levels and activity of iNOS. The LPS- and IFN-β-mediated increase of STAT1 mRNA and protein levels was abrogated by chelation of Zn2+ with the membrane permeable chelator N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) in RAW 264.7 macrophages. After 48h pre-incubation together with IFN-β, TPEN also led to reduced nitric monoxide formation in response to a second stimulation with LPS. Nonetheless, the latter was observed regardless of any pre-incubation with IFN-β, suggesting that the effect of treatment with TPEN negatively affects iNOS induction independently from cellular STAT1 levels. In conclusion, long term Zn2+ chelation does affect STAT1 protein expression, but interferes with NO production by a different, yet unknown pathway not involving STAT1. However, as there are many additional STAT1-dependent genes, there might still be effects on targets other than iNOS.

Inhibition of T-type Ca2+ Channels by Hydrogen Sulfide.

T-type Ca(2+) channels are a distinct family of low voltage-activated Ca(2+) channels which serve many roles in different tissues. Several studies have implicated them, for example, in the adaptive responses to chronic hypoxia in the cardiovascular and endocrine systems. Hydrogen sulfide (H(2)S) was more recently discovered as an important signalling molecule involved in many functions, including O(2) sensing. Since ion channels are emerging as an important family of target proteins for modulation by H(2)S, and both T-type Ca(2+) channels and H(2)S are involved in cellular responses to hypoxia, we have investigated whether recombinant and native T-type Ca(2+) channels are a target for modulation by H(2)S. Using patch-clamp electrophysiology, we demonstrate that the H(2)S donor, NaHS, selectively inhibits Cav3.2 T-type Ca(2+) channels heterologously expressed in HEK293 cells, whilst Cav3.1 and Cav3.3 channels were unaffected. Sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) using TPEN prevented channel inhibition by H(2)S. H2S also selectively inhibited native T-type channels (primarily Cav3.2) in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H(2)S regulation, the T-type Ca(2+) channel Cav3.2. Results have important implications for the proposed pro-nociceptive effects of this gasotransmitter. Implications for the control of cellular responses to hypoxia await further study.

Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels.

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca(2+) channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM-1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) with N,N,N',N'-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn(2+) binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca(2+) channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.

Differential Regulation of TLR-Dependent MyD88 and TRIF Signaling Pathways by Free Zinc Ions

Zinc signals are utilized by several immune cell receptors. One is TLR4, which causes an increase of free zinc ions (Zn(2+)) that is required for the MyD88-dependent expression of inflammatory cytokines. This study investigates the role of Zn(2+) on Toll/IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent signals, the other major intracellular pathway activated by TLR4. Chelation of Zn(2+) with the membrane-permeable chelator N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine augmented TLR4-mediated production of IFN-β and subsequent synthesis of inducible NO synthase and production of NO. The effect is based on Zn(2+) acting as a negative regulator of the TRIF pathway via reducing IFN regulatory factor 3 activation. This was also observed with TLR3, the only TLR that signals exclusively via TRIF, but not MyD88, and does not trigger a zinc signal. In contrast, IFN-γ-induced NO production was unaffected by N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine. Taken together, Zn(2+) is specifically involved in TLR signaling, where it differentially regulates MyD88 and TRIF signaling via a zinc signal or via basal Zn(2+) levels, respectively.

Metallomic Analysis of Macrophages Infected withHistoplasma capsulatumReveals a Fundamental Role for Zinc in Host Defenses

The fungal pathogen Histoplasma capsulatum evades the innate and adaptive immune responses and thrives within resting macrophages. Cytokines that induce antimicrobial activity, such as granulocyte macrophage colony-stimulating factor (GM-CSF), inhibit H. capsulatum growth in macrophages. Conversely, interleukin 4 inhibits the killing of intracellular pathogens. Using inductively coupled plasma mass spectrometry, we examined alterations in the metal homeostasis of murine H. capsulatum-infected macrophages that were exposed to activating cytokines. Decreases in the levels of iron (Fe(2+) and Fe(3+)) and zinc (Zn(2+)) were observed in infected, GM-CSF-treated macrophages compared with those in infected controls. Interleukin 4 reversed the antifungal activity of GM-CSF-activated macrophages and was associated with increased intracellular Zn(2+) levels. Chelation of Zn(2+) inhibited yeast replication in both the absence of macrophages and the presence of macrophages. Treatment of cells with GM-CSF altered the host Zn(2+) binding species profile. These results establish that Zn(2+) deprivation may be a host defense mechanism utilized by macrophages.

Zinc Released from Injured Cells Is Acting via the Zn2+-sensing Receptor, ZnR, to Trigger Signaling Leading to Epithelial Repair

A role for Zn(2+) in accelerating wound healing is established, yet, the signaling pathways linking Zn(2+) to tissue repair are not well known. We show that in the human HaCaT keratinocytes extracellular Zn(2+) induces a metabotropic Ca(2+) response that is abolished by silencing the expression of the G-protein-coupled receptor GPR39, suggesting that this Zn(2+)-sensing receptor, ZnR, is mediating the response. Keratinocytic-ZnR signaling is highly selective for Zn(2+) and can be triggered by nanomolar concentrations of this ion. Interestingly, Zn(2+) was also released following cellular injury, as monitored by a specific non-permeable fluorescent Zn(2+) probe, ZnAF-2. Chelation of Zn(2+) and scavenging of ATP from conditioned medium, collected from injured epithelial cultures, was sufficient to eliminate the metabotropic Ca(2+) signaling. The signaling triggered by Zn(2+), via ZnR, or by ATP further activated MAP kinase and induced up-regulation of the sodium/proton exchanger NHE1 activity. Finally, activation of ZnR/GPR39 signaling or application of ATP enhanced keratinocytes scratch closure in an in vitro model. Thus our results indicate that extracellular Zn(2+), which is either applied or released following injury, activates ZnR/GPR39 to promote signaling leading to epithelial repair.

TbMP42 is a structure-sensitive ribonuclease that likely follows a metal ion catalysis mechanism.

RNA editing in African trypanosomes is characterized by a uridylate-specific insertion and/or deletion reaction that generates functional mitochondrial transcripts. The process is catalyzed by a multi-enzyme complex, the editosome, which consists of approximately 20 proteins. While for some of the polypeptides a contribution to the editing reaction can be deduced from their domain structure, the involvement of other proteins remains elusive. TbMP42, is a component of the editosome that is characterized by two C2H2-type zinc-finger domains and a putative oligosaccharide/oligonucleotide-binding fold. Recombinant TbMP42 has been shown to possess endo/exoribonuclease activity in vitro; however, the protein lacks canonical nuclease motifs. Using a set of synthetic gRNA/pre-mRNA substrate RNAs, we demonstrate that TbMP42 acts as a topology-dependent ribonuclease that is sensitive to base stacking. We further show that the chelation of Zn2+ cations is inhibitory to the enzyme activity and that the chemical modification of amino acids known to coordinate Zn2+ inactivates rTbMP42. Together, the data are suggestive of a Zn2+-dependent metal ion catalysis mechanism for the ribonucleolytic activity of rTbMP42.

Glycinergic Transmission Shaped by the Corelease of GABA in a Mammalian Auditory Synapse

The firing pattern of neurons is shaped by the convergence of excitation and inhibition, each with finely tuned magnitude and duration. In an auditory brainstem nucleus, glycinergic inhibition features fast decay kinetics, the mechanism of which is unknown. By applying glycine to native or recombinant glycine receptors, we show that response decay times are accelerated by addition of GABA, a weak partial agonist of glycine receptors. Systematic variation in agonist exposure time revealed that fast synaptic time course may be achieved with submillisecond exposures to mixtures of glycine and GABA at physiological concentrations. Accordingly, presynaptic terminals generally contained both transmitters, and depleting terminals of GABA slowed glycinergic synaptic currents. Thus, coreleased GABA accelerates glycinergic transmission by acting directly on glycine receptors, narrowing the time window for effective inhibition. Packaging both weak and strong agonists in vesicles may be a general means by which presynaptic neurons regulate the duration of postsynaptic responses.

Oxidation of Macromolecules

Sirtuin-1 (SIRT1) is an NAD+-dependent histone deacetylase (HDAC) that regulates metabolism. SIRT1 contains a Zn2+ atom coordinated by four cysteines, which is essential for enzyme activity. We hypothesized that the lower pKa of these thiolate center cysteines makes SIRT1 sensitive to reactive oxygen species, possibly lowering its activity in metabolic diseases. Methods: SIRT1 deacetylase activity was measured by cleavage of a fluorogenic p53 substrate peptide. Affinity of Zn2+ intact recombinant SIRT1 protein was determined on Zn2+-NTA beads, and thiolate cysteines were labeled in bovine aortic endothelial cells with biotinylated iodoacetamide (bIAM) after blocking free thiols and chelation of Zn2+. Results: the strong oxidant, peroxynitrite (10 to 1000 μM) dose-dependently inhibited SIRT1 binding to the Zn2+ NTA beads as well as SIRT1 activity. Both binding and activity were blocked by Zn2+ chelators. Treating endothelial cells with the peroxynitrite donor, SIN1, or with TNFα, decreased bIAM labeling of thiolate cysteines. Conclusions: the methods developed can detect the integrity of the Zn2+ thiolate center of recombinant and cellular SIRT1. Our results indicate that disruption of the Zn2+ thiolate domain of SIRT1 by a strong oxidant or metal chelator is associated with a negative effect on activity. Studies in endothelial cells indicate that oxidants added directly, or those induced by TNFα can disrupt a substantial fraction of the SIRT1 thiolate centers. This suggests that oxidants associated with metabolic diseases might directly regulate SIRT1.