Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

The Role of One-Carbon Metabolism and Methyl Donors in Medically Assisted Reproduction: A Narrative Review of the Literature.

One-carbon (1-C) metabolic deficiency impairs homeostasis, driving disease development, including infertility. It is of importance to summarize the current evidence regarding the clinical utility of 1-C metabolism-related biomolecules and methyl donors, namely, folate, betaine, choline, vitamin B12, homocysteine (Hcy), and zinc, as potential biomarkers, dietary supplements, and culture media supplements in the context of medically assisted reproduction (MAR). A narrative review of the literature was conducted in the PubMed/Medline database. Diet, ageing, and the endocrine milieu of individuals affect both 1-C metabolism and fertility status. In vitro fertilization (IVF) techniques, and culture conditions in particular, have a direct impact on 1-C metabolic activity in gametes and embryos. Critical analysis indicated that zinc supplementation in cryopreservation media may be a promising approach to reducing oxidative damage, while female serum homocysteine levels may be employed as a possible biomarker for predicting IVF outcomes. Nonetheless, the level of evidence is low, and future studies are needed to verify these data. One-carbon metabolism-related processes, including redox defense and epigenetic regulation, may be compromised in IVF-derived embryos. The study of 1-C metabolism may lead the way towards improving MAR efficiency and safety and ensuring the lifelong health of MAR infants.

Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO4 (0, 12.5, 25, 50, and 100 µM) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 µM ZnSO4 was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 µM ZnSO4. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 µM ZnSO4 increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO4.

Hesperidin alleviates zinc-induced nephrotoxicity via the gut-kidney axis in swine.

Introduction: Zinc (Zn) is an essential trace element in animals, but excessive intake can lead to renal toxicity damage. Thus, the exploration of effective natural antagonists to reduce the toxicity caused by Zn has become a major scientific problem. Methods: Here, we found that hesperidin could effectively alleviate the renal toxicity induced by Zn in pigs by using hematoxylin-eosin staining, transmission electron microscope, immunohistochemistry, fluorescence quantitative PCR, and microfloral DNA sequencing. Results: The results showed that hesperidin could effectively attenuate the pathological injury in kidney, and reduce autophagy and apoptosis induced by Zn, which evidenced by the downregulation of LC3, ATG5, Bak1, Bax, Caspase-3 and upregulation of p62 and Bcl2. Additionally, hesperidin could reverse colon injury and the decrease of ZO-1 protein expression. Interestingly, hesperidin restored the intestinal flora structure disturbed by Zn, and significantly reduced the abundance of Tenericutes (phylum level) and Christensenella (genus level). Discussion: Thus, altered intestinal flora and intestinal barrier function constitute the gut-kidney axis, which is involved in hesperidin alleviating Zn-induced nephrotoxicity. Our study provides theoretical basis and practical significance of hesperidin for the prevention and treatment of Zn-induced nephrotoxicity through gut-kidney axis.

Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics.

Ryanodine receptor type 1 (RyR1) is a Ca2+-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn2+ in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn2+. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn2+ in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca2+, and caffeine binding, shedding light on the intricate relationship between Zn2+ coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.

The 8-17 DNAzyme can operate in a single active structure regardless of metal ion cofactor.

DNAzymes - synthetic enzymes made of DNA - have long attracted attention as RNA-targeting therapeutic agents. Yet, as of now, no DNAzyme-based drug has been approved, partially due to our lacking understanding of their molecular mode of action. In this work we report the solution structure of 8-17 DNAzyme bound to a Zn2+ ion solved through NMR spectroscopy. Surprisingly, it turned out to be very similar to the previously solved Pb2+-bound form (catalytic domain RMSD = 1.28 Å), despite a long-standing literature consensus that Pb2+ recruits a different DNAzyme fold than other metal ion cofactors. Our follow-up NMR investigations in the presence of other ions - Mg2+, Na+, and Pb2+ - suggest that at DNAzyme concentrations used in NMR all these ions induce a similar tertiary fold. Based on these findings, we propose a model for 8-17 DNAzyme interactions with metal ions postulating the existence of only a single catalytically-active structure, yet populated to a different extent depending on the metal ion cofactor. Our results provide structural information on the 8-17 DNAzyme in presence of non-Pb2+ cofactors, including the biologically relevant Mg2+ ion.

Exogenous zinc application mitigates negative effects of salinity on barley (Hordeum vulgare) growth by improving root ionic homeostasis.

Detrimental effects of salinity could be mitigated by exogenous zinc (Zn) application; however, the mechanisms underlying this amelioration are poorly understood. This study demonstrated the interaction between Zn and salinity by measuring plant biomass, photosynthetic performance, ion concentrations, ROS accumulation, antioxidant activity and electrophysiological parameters in barley (Hordeum vulgare L.). Salinity stress (200mM NaCl for 3weeks) resulted in a massive reduction in plant biomass; however, both fresh and dry weight of shoots were increased by ~30% with adequate Zn supply. Zinc supplementation also maintained K+ and Na+ homeostasis and prevented H2 O2 toxicity under salinity stress. Furthermore, exposure to 10mM H2 O2 resulted in massive K+ efflux from root epidermal cells in both the elongation and mature root zones, and pre-treating roots with Zn reduced ROS-induced K+ efflux from the roots by 3-4-fold. Similar results were observed for Ca2+ . The observed effects may be causally related to more efficient regulation of cation-permeable non-selective channels involved in the transport and sequestration of Na+ , K+ and Ca2+ in various cellular compartments and tissues. This study provides valuable insights into Zn protective functions in plants and encourages the use of Zn fertilisers in barley crops grown on salt-affected soils.

Targeting bacterial nickel transport with aspergillomarasmine A suppresses virulence-associated Ni-dependent enzymes.

Microbial Ni2+ homeostasis underpins the virulence of several clinical pathogens. Ni2+ is an essential cofactor in urease and [NiFe]-hydrogenases involved in colonization and persistence. Many microbes produce metallophores to sequester metals necessary for their metabolism and starve competing neighboring organisms. The fungal metallophore aspergillomarasmine A (AMA) shows narrow specificity for Zn2+, Ni2+, and Co2+. Here, we show that this specificity allows AMA to block the uptake of Ni2+ and attenuate bacterial Ni-dependent enzymes, offering a potential strategy for reducing virulence. Bacterial exposure to AMA perturbs H2 metabolism, ureolysis, struvite crystallization, and biofilm formation and shows efficacy in a Galleria mellonella animal infection model. The inhibition of Ni-dependent enzymes was aided by Zn2+, which complexes with AMA and competes with the native nickelophore for the uptake of Ni2+. Biochemical analyses demonstrated high-affinity binding of AMA-metal complexes to NikA, the periplasmic substrate-binding protein of the Ni2+ uptake system. Structural examination of NikA in complex with Ni-AMA revealed that the coordination geometry of Ni-AMA mimics the native ligand, Ni-(L-His)2, providing a structural basis for binding AMA-metal complexes. Structure-activity relationship studies of AMA identified regions of the molecule that improve NikA affinity and offer potential routes for further developing this compound as an anti-virulence agent.

Microbes vary strategically in their metalation of mononuclear enzymes.

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide.

Zinc and manganese homeostasis closely interact in mammalian cells.

Understanding the homeostatic interactions among essential trace metals is important for explaining their roles in cellular systems. Recent studies in vertebrates suggest that cellular Mn metabolism is related to Zn metabolism in multifarious cellular processes. However, the underlying mechanism remains unclear. In this study, we examined the changes in the expression of proteins involved in cellular Zn and/or Mn homeostatic control and measured the Mn as well as Zn contents and Zn enzyme activities to elucidate the effects of Mn and Zn homeostasis on each other. Mn treatment decreased the expression of the Zn homeostatic proteins metallothionein (MT) and ZNT1 and reduced Zn enzyme activities, which were attributed to the decreased Zn content. Moreover, loss of Mn efflux transport protein decreased MT and ZNT1 expression and Zn enzyme activity without changing extracellular Mn content. This reduction was not observed when supplementing with the same Cu concentrations and in cells lacking Cu efflux proteins. Furthermore, cellular Zn homeostasis was oppositely regulated in cells expressing Zn and Mn importer ZIP8, depending on whether Zn or Mn concentration was elevated in the extracellular milieu. Our results provide novel insights into the intricate interactions between Mn and Zn homeostasis in mammalian cells and facilitate our understanding of the physiopathology of Mn, which may lead to the development of treatment strategies for Mn-related diseases in the future.

Multiomics Analysis of PCB126's Effect on a Mouse Chronic-Binge Alcohol Feeding Model.

BACKGROUND: Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. Our group recently demonstrated that PCB126 promoted steatosis, hepatomegaly, and modulated intermediary metabolism in a rodent model of alcohol-associated liver disease (ALD). OBJECTIVE: To better understand how PCB126 promoted ALD in our previous model, the current study adopts multiple omics approaches to elucidate potential mechanistic hypotheses. METHODS: Briefly, male C57BL/6J mice were exposed to 0.2mg/kg polychlorinated biphenyl (PCB) 126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were collected and prepared for mRNA sequencing, phosphoproteomics, and inductively coupled plasma mass spectrometry for metals quantification. RESULTS: Principal component analysis showed that PCB126 uniquely modified the transcriptome in EtOH-fed mice. EtOH feeding alone resulted in >4,000 differentially expressed genes (DEGs), and PCB126 exposure resulted in more DEGs in the EtOH-fed group (907 DEGs) in comparison with the pair-fed group (503 DEGs). Top 20 significant gene ontology (GO) biological processes included "peptidyl tyrosine modifications," whereas top 25 significantly decreasing GO molecular functions included "metal/ion/zinc binding." Quantitative, label-free phosphoproteomics and western blot analysis revealed no major significant PCB126 effects on total phosphorylated tyrosine residues in EtOH-fed mice. Quantified hepatic essential metal levels were primarily significantly lower in EtOH-fed mice. PCB126-exposed mice had significantly lower magnesium, cobalt, and zinc levels in EtOH-fed mice. DISCUSSION: Previous work has demonstrated that PCB126 is a modifying factor in metabolic dysfunction-associated steatotic liver disease (MASLD), and our current work suggests that pollutants also modify ALD. PCB126 may, in part, be contributing to the malnutrition aspect of ALD, where metal deficiency is known to contribute and worsen prognosis. https://doi.org/10.1289/EHP14132.

Systematic Analysis of the Relationship Between Elevated Zinc and Epilepsy.

Previous studies have indicated a potential relationship between zinc and epilepsy. The aim of this study is to investigate the causal relationship between zinc, zinc-dependent carbonic anhydrase, and gray matter volume in brain regions enriched with zinc and epilepsy, as well as explore the possible mechanisms by which zinc contributes to epilepsy. First, this study assessed the risk causality between zinc, carbonic anhydrase, and gray matter volume alterations in zinc-enriched brain regions and various subtypes of epilepsy based on Two-sample Mendelian randomization analysis. And then, this study conducted GO/KEGG analysis based on colocalization analysis, MAGMA analysis, lasso regression, random forest model, and XGBoost model. The results of Mendelian randomization analyses showed a causal relationship between zinc, carbonic anhydrase-4, and generalized epilepsy (p = 0.044 , p = 0.010). Additionally, carbonic anhydrase-1 and gray matter volume of the caudate nucleus were found to be associated with epilepsy and focal epilepsy (p = 0.014, p = 0.003 and p = 0.022, p = 0.009). A colocalization relationship was found between epilepsy and focal epilepsy (PP.H4.abf = 97.7e - 2). Meanwhile, the MAGMA analysis indicated that SNPs associated with epilepsy and focal epilepsy were functionally localized to zinc-finger-protein-related genes (p < 1.0e - 5). The genes associated with focal epilepsy were found to have a molecular function of zinc ion binding (FDR = 2.3e - 6). After the onset of epilepsy, the function of the gene whose expression changed in the rats with focal epilepsy was enriched in the biological process of vascular response (FDR = 4.0e - 5). These results revealed mechanism of the increased risk of epilepsy caused by elevated zinc may be related to the increase of zinc ion-dependent carbonic anhydrase or the increase of the volume of zinc-rich caudate gray matter.

Design, synthesis, insilco study and biological evaluation of new isatin-sulfonamide derivatives by using mono amide linker as possible as histone deacetylase inhibitors.

OBJECTIVE: Aim: To evaluate the cytotoxic activity of newly synthesized a series of novel HDAC inhibitors comprising sulfonamide as zinc binding group and Isatin derivatives as cap group joined by mono amide linker as required to act as HDAC inhibitors. PATIENTS AND METHODS: Materials and Methods: The utilization of sulfonamide as zinc binding group joined by N-alkylation reaction with ethyl-bromo hexanoate as linker group that joined by amide reaction with Isatin derivatives as cap groups which known to possess antitumor activity in the designed of new histone deacetylase inhibitors and using the docking and MTT assay to evaluate the compounds. RESULTS: Results: Four compounds have been synthesized and characterized successfully by ART-FTIR, NMR and ESI-Ms. the compounds were synthesized and characterized by successfully by ART-FTIR, NMR and ESI- Ms. Assessed for their cytotoxic activity against human colon adenocarcinoma MCF-7 (IC50, I=105.15, II=60.00, III=54.11, IV=56.57, vorinostat=28.41) and hepatoblastoma HepG2 (IC50, I=63.91, II=135.18, III=118.85, IV=51.46, vorinostat=37.50). Most of them exhibited potent HDAC inhibitory activity and significant cytotoxicity. CONCLUSION: Conclusions: The synthesized compounds (I, II, III and IV) showed cytotoxicity toward MCF-7 and HepG2 cancer cell lines and their docking analysis provided a preliminary indication that they are viable [HDAC6] candidates.

Structural insights into the calcium-coupled zinc export of human ZnT1.

Cellular zinc (Zn2+) homeostasis is essential to human health and is under tight regulations. Human zinc transporter 1 (hZnT1) is a plasma membrane-localized Zn2+ exporter belonging to the ZnT family, and its functional aberration is associated with multiple diseases. Here, we show that hZnT1 works as a Zn2+/Ca2+ exchanger. We determine the structure of hZnT1 using cryo-electron microscopy (cryo-EM) single particle analysis. hZnT1 adopts a homodimeric structure, and each subunit contains a transmembrane domain consisting of six transmembrane segments, a cytosolic domain, and an extracellular domain. The transmembrane region displays an outward-facing conformation. On the basis of structural and functional analysis, we propose a model for the hZnT1-mediated Zn2+/Ca2+ exchange. Together, these results facilitate our understanding of the biological functions of hZnT1 and provide a basis for further investigations of the ZnT family transporters.

Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells.

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

Intestinal inflammation marker calprotectin regulates epithelial intestinal zinc metabolism and proliferation in mouse jejunal organoids.

Calprotectin (CP), a heterodimer of S100A8 and S100A9, is expressed by neutrophils and a number of innate immune cells and is used widely as a marker of inflammation, particularly intestinal inflammation. CP is a ligand for toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE). In addition, CP can act as a microbial modulatory agent via a mechanism termed nutritional immunity, depending on metal binding, most notably Zn2+. The effects on the intestinal epithelium are largely unknown. In this study we aimed to characterize the effect of calprotectin on mouse jejunal organoids as a model epithelium, focusing on Zn2+ metabolism and cell proliferation. CP addition upregulated the expression of the Zn2+ absorptive transporter Slc39a4 and of methallothionein Mt1 in a Zn2+-sensitive manner, while downregulating the expression of the Zn2+ exporter Slc30a2 and of methallothionein 2 (Mt2). These effects were greatly attenuated with a CP variant lacking the metal binding capacity. Globally, these observations indicate adaptation to low Zn2+ levels. CP had antiproliferative effects and reduced the expression of proliferative and stemness genes in jejunal organoids, effects that were largely independent of Zn2+ chelation. In addition, CP induced apoptosis modestly and modulated antimicrobial gene expression. CP had no effect on epithelial differentiation. Overall, CP exerts modulatory effects in murine jejunal organoids that are in part related to Zn2+ sequestration and partially reproduced in vivo, supporting the validity of mouse jejunal organoids as a model for mouse epithelium.

Mercury and selenium distribution in human brain tissue using synchrotron micro-X-ray fluorescence.

Mercury is a well-recognized environmental contaminant and neurotoxin, having been associated with a number of deleterious neurological conditions including neurodegenerative diseases, such as Alzheimer's disease. To investigate how mercury and other metals behave in the brain, we used synchrotron micro-X-ray fluorescence to map the distribution pattern and quantify concentrations of metals in human brain. Brain tissue was provided by the Rush Alzheimer's Disease Center and samples originated from individuals diagnosed with Alzheimer's disease and without cognitive impairment. Data were collected at the 2-ID-E beamline at the Advanced Photon Source at Argonne National Laboratory with an incident beam energy of 13 keV. Course scans were performed at low resolution to determine gross tissue features, after which smaller regions were selected to image at higher resolution. The findings revealed (1) the existence of mercury particles in the brain samples of two subjects; (2) co-localization and linear correlation of mercury and selenium in all particles; (3) co-localization of these particles with zinc structures; and (4) association with sulfur in some of these particles. These results suggest that selenium and sulfur may play protective roles against mercury in the brain, potentially binding with the metal to reduce the induced toxicity, although at different affinities. Our findings call for further studies to investigate the relationship between mercury, selenium, and sulfur, as well as the potential implications in Alzheimer's disease and related dementias.

A dynamic regulatory switch for phase separation of FUS protein: Zinc ions and zinc finger domain.

Zinc is an important trace element in the human body, and its homeostasis is closely related to amyotrophic lateral sclerosis (ALS). Cytoplasmic FUS proteins from patients with ALS aggregate their important pathologic markers. Liquid-liquid phase separation (LLPS) of FUS can lead to its aggregation. However, whether and how zinc homeostasis affects the aggregation of disease-associated FUS proteins in the cytoplasm remains unclear. Here, we found that zinc ion enhances LLPS and promotes the aggregation in the cytoplasm for FUS protein. In the FUS, the cysteine of the zinc finger (ZnF), recognizes and binds to zinc ions, reducing droplet mobility and enhancing protein aggregation in the cytoplasm. The mutation of FUS cysteine disrupts the dynamic regulatory switch of zinc ions and ZnF, resulting in insensitivity to zinc ions. These results suggest that the dynamic regulation of LLPS by binding with zinc ions may be a widespread mechanism and provide a new understanding of neurological diseases such as ALS and other ZnF protein-related diseases.

Transcriptomic investigation and biomarker discovery for zinc response in oysters Crassostrea gasar.

In an era of unprecedented industrial and agricultural growth, metal contamination in marine environments is a pressing concern. Sentinel organisms such as the mangrove oyster Crassostrea gasar provide valuable insights into these environments' health. However, a comprehensive understanding of the molecular mechanisms underlying their response to metal exposure remains elusive. To address this gap, we reanalyzed the 454-sequencing data of C. gasar, utilizing an array of bioinformatics workflow of CDTA (Combined De Novo Transcriptome Assembly) to generate a more representative assembly. In parallel, C. gasar individuals were exposed to two concentrations of zinc (850 and 4500 µg L-1 Zn) for 48 h to understand their molecular responses. We utilized Trinotate workflow for the 11,684-CDTA unigenes annotation, with most transcripts aligning with the genus Crassostrea. Our analysis indicated that 67.3% of transcript sequences showed homology with Pfam, while 51.4% and 54.5%, respectively had GO and KO terms annotated. We identified potential metal pollution biomarkers, focusing on metal-related genes, such as those related to the GSH biosynthesis (CHAC1 and GCLC-like), to zinc transporters (ZNT2-like), and metallothionein (MT-like). The evolutionary conservation of these genes within the Crassostrea genus was assessed through phylogenetic analysis. Further, these genes were evaluated by qPCR in the laboratory exposed oysters. All target genes exhibited significant upregulation upon exposure to Zn at both 850 and 4500 µg L-1, except for GCLC-like, which showed upregulation only at the higher concentration of 4500 µg L-1. This result suggests distinct activation thresholds and complex interactions among these genes in response to varying Zn concentrations. Our study provides insights into the molecular responses of C. gasar to Zn, adding valuable tools for monitoring metal pollution in marine ecosystems using the mangrove oyster as a sentinel organism.

Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies.

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.