RESUMO
Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.
Assuntos
Lactoferrina , Mannheimia haemolytica , Metaloproteases , Proteólise , Lactoferrina/metabolismo , Lactoferrina/farmacologia , Metaloproteases/metabolismo , Metaloproteases/antagonistas & inibidores , Animais , Apoproteínas/metabolismo , Apoproteínas/química , Simulação de Acoplamento Molecular , Ovinos , Bovinos , Colagenases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Zinco/metabolismoRESUMO
BACKGROUND: Giardiasis and zinc deficiency have been identified as serious health problems worldwide. Although Zn depletion is known to occur in giardiasis, no work has investigated whether changes occur in brain structures. METHODS: Three groups of gerbils were used: control (1), orogastrically inoculated on day 3 after birth with trophozoites of two isolates of Giardia intestinalis (HGINV/WB) group (2 and 3). Estimates were made at five ages covering: establishment of infection, Giardia population growth, natural parasite clearance and a post-infection age. QuantiChrome zinc assay kit, cresyl violet staining and TUNEL technique were used. RESULTS: A significant decrease (p<0.01) in tissue zinc was observed and persisted after infection. Cytoarchitectural changes were observed in 75% of gerbils in the HGINV or WB groups. Ectopic pyramidal neurons were found in the cornus ammonis (CA1-CA3). At 60 and 90 days of age loss of lamination was clearly visible in CA1. In the dentate gyrus (DG), thinning of the dorsal lamina and abnormal thickening of the ventral lamina were observed from 30 days of age. In the cerebellum, we found an increase (p<0.01) in the thickness of the external granular layer (EGL) at 14 days of age that persisted until day 21 (C 3 ± 0.3 µm; HGINV 37 ± 5 µm; WB 28 ± 3 µm); Purkinje cell population estimation showed a significant decrease; a large number of apoptotic somas were observed scattered in the molecular layer; in 60 and 90 days old gerbils we found granular cell heterotopia and Purkinje cell ectopia. The pattern of apoptosis was different in the cerebellum and hippocampus of parasitized gerbils. CONCLUSION: The morphological changes found suggest that neuronal migration is affected by zinc depletion caused by giardiasis in early postnatal life; for the first time, the link between giardiasis-zinc depletion and damaged brain structures is shown. This damage may explain the psychomotor/cognitive delay associated with giardiasis. These findings are alarming. Alterations in zinc metabolism and signalling are known to be involved in many brain disorders, including autism.
Assuntos
Cerebelo , Gerbillinae , Giardia lamblia , Giardíase , Hipocampo , Zinco , Animais , Gerbillinae/parasitologia , Zinco/deficiência , Zinco/metabolismo , Giardíase/parasitologia , Giardíase/patologia , Cerebelo/patologia , Cerebelo/parasitologia , Hipocampo/patologia , Hipocampo/parasitologia , Giardia lamblia/crescimento & desenvolvimento , Masculino , Modelos Animais de DoençasRESUMO
Dysregulation of zinc and zinc transporters families has been associated with the genesis and progression of prostate cancer. The prostate epithelium utilizes two types of zinc transporters, the ZIP (Zrt-, Irt-related Protein) and the ZnTs (Zinc Transporter), to transport zinc from the blood plasma to the gland lumen. ZIP transporters uptake zinc from extracellular space and organelle lumen, while ZnT transporters release zinc outside the cells or to organelle lumen. In prostate cancer, a commonly observed low zinc concentration in prostate tissue has been correlated with downregulations of certain ZIPs (e.g., ZIP1, ZIP2, ZIP3, ZIP14) and upregulations of specific ZnTs (e.g., ZnT1, ZnT9, ZnT10). These alterations may enable cancer cells to adapt to toxic high zinc levels. While zinc supplementation has been suggested as a potential therapy for this type of cancer, studies have yielded inconsistent results because some trials have indicated that zinc supplementation could exacerbate cancer risk. The reason for this discrepancy remains unclear, but given the high molecular and genetic variability present in prostate tumors, it is plausible that some zinc transporters-comprising 14 ZIP and 10 ZnT members-could be dysregulated in others patterns that promote cancer. From this perspective, this review highlights novel dysregulation, such as ZIP-Up/ZnT-Down, observed in prostate cancer cell lines for ZIP4, ZIP8, ZnT2, ZnT4, ZnT5, etc. Additionally, an in silico analysis of an available microarray from mouse models of prostate cancer (Nkx3.1;Pten) predicts similar dysregulation pattern for ZIP4, ZIP8, and ZnT2, which appear in early stages of prostate cancer progression. Furthermore, similar dysregulation patterns are supported by an in silico analysis of RNA-seq data from human cancer tumors available in cBioPortal. We discuss how these dysregulations of zinc transporters could impact zinc supplementation trials, particularly focusing on how the ZIP-Up/ZnT-Down dysregulation through various mechanisms might promote prostate cancer progression.
Assuntos
Proteínas de Transporte de Cátions , Neoplasias da Próstata , Zinco , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Zinco/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica , Suplementos Nutricionais , Próstata/metabolismoRESUMO
The role of the carapace in the uptake and storage of newly accumulated metals was investigated in the green crab exposed to environmentally relevant concentrations of calcium ([Ca] = 389 mg L-1 or 9.7 mmol L-1), zinc ([Zn] = 82 µg L-1 or 1.25 µmol L-1), and nickel ([Ni] = 8.2 µg L-1 or 0.14 µmol L-1) in 12 °C seawater, using radio-tracers (45Ca, 65Zn, 63Ni). After 24-h exposure, carapace exhibited the highest concentration of newly accumulated Ca, whereas carapace and gills exhibited the highest concentrations of both newly accumulated Zn and Ni relative to other tissues. For all three metals, the carapace accounted for >85 % of the total body burden. Acute temperature changes (to 2 °C and 22 °C) revealed the highest overall temperature coefficient Q10 (2.15) for Ca uptake into the carapace, intermediate Q10 for Ni (1.87) and lowest Q10 (1.45) for Zn. New Ca uptake into the carapace continued linearly with time for 24 h, new Zn uptake gradually deviated from linearity, whereas Ni uptake reached a plateau by 6 h. Attachment of a rubber membrane to the dorsal carapace, thereby shielding about 20 % of the total crab surface area from the external water, eliminated both new Zn and Ni incorporation into the shielded carapace, whereas 36 % of new Ca incorporation persisted. When recently euthanized crabs were exposed, new Zn uptake into the carapace remained unchanged, whereas Ca and Ni uptake were reduced by 89 % and 71 %, respectively. We conclude that the carapace is a very important uptake and storage site for all three metals. All of the uptake of new Zn and new Ni, and most of the uptake of new Ca into this tissue comes directly from the external water. For Zn, the mechanism involves only physicochemical processes, whereas for Ca and Ni, life-dependent processes make the major contribution.
Assuntos
Braquiúros , Cálcio , Níquel , Água do Mar , Poluentes Químicos da Água , Zinco , Animais , Braquiúros/metabolismo , Níquel/metabolismo , Zinco/metabolismo , Poluentes Químicos da Água/metabolismo , Água do Mar/química , Cálcio/metabolismo , Exoesqueleto/química , Exoesqueleto/metabolismo , Monitoramento AmbientalRESUMO
The influence of metal ions on the structure of amyloid- ß (Aß) protofibril models was studied through molecular dynamics to explore the molecular mechanisms underlying metal-induced Aß aggregation relevant in Alzheimer's disease (AD). The models included 36-, 48-, and 188-mers of the Aß42 sequence and two disease-modifying variants. Primary structural effects were observed at the N-terminal domain, as it became susceptible to the presence of cations. Specially when ß-sheets predominate, this motif orients N-terminal acidic residues toward one single face of the ß-sheet, resulting in the formation of an acidic region that attracts cations from the media and promotes the folding of the N-terminal region, with implications in amyloid aggregation. The molecular phenotype of the protofibril models based on Aß variants shows that the AD-causative D7N mutation promotes the formation of N-terminal ß-sheets and accumulates more Zn2+, in contrast to the non-amyloidogenic rodent sequence that hinders the ß-sheets and is more selective for Na+ over Zn2+ cations. It is proposed that forming an acidic ß-sheet domain and accumulating cations is a plausible molecular mechanism connecting the elevated affinity and concentration of metals in Aß fibrils to their high content of ß-sheet structure at the N-terminal sequence.
Assuntos
Peptídeos beta-Amiloides , Simulação de Dinâmica Molecular , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/genética , Conformação Proteica em Folha beta , Humanos , Zinco/metabolismo , Zinco/química , Doença de Alzheimer/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/genética , Metais/metabolismo , Metais/químicaRESUMO
DapE is a Zn2+-metallohydrolase recognized as a drug target for bacterial control. It is a homodimer that requires the exchange of interface strands by an induced fit essential for catalysis. Identifying novel anti-DapE agents requires greater structural details. Most of the characterized DapEs are from the Gram-negative group. Here, two high-resolution DapE crystal structures from Enterococcus faecium are presented for the first time with novel aspects. A loosened enzyme intermediate between the open and closed conformations is observed. Substrates may bind to loose state, subsequently it closes, where hydrolysis occurs, and finally, the change to the open state leads to the release of the products. Mutation of His352 suggests a role, along with His194, in the oxyanion stabilization in the mono-metalated Zn2+ isoform, while in the di-metalated isoform, the metal center 2 complements it function. An aromatic-π box potentially involved in the interaction of DapE with other proteins, and a peptide flip could determine the specificity in the Gram-positive ArgE/DapE group. Finally, details of two extra-catalytic cavities whose geometry changes depending on the conformational state of the enzyme are presented. These cavities could be a target for developing non-competitive agents that trap the enzyme in an inactive state.
Assuntos
Proteínas de Bactérias , Enterococcus faecium , Enterococcus faecium/enzimologia , Especificidade por Substrato , Ligantes , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Conformação Proteica , Zinco/química , Zinco/metabolismo , Domínio Catalítico , Amidoidrolases/química , Amidoidrolases/metabolismo , Amidoidrolases/genética , Cristalografia por Raios X , Sequência de Aminoácidos , Ligação ProteicaRESUMO
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.
Assuntos
Crassostrea , Transcriptoma , Poluentes Químicos da Água , Zinco , Animais , Crassostrea/genética , Crassostrea/metabolismo , Zinco/metabolismo , Poluentes Químicos da Água/toxicidade , Biomarcadores/metabolismoRESUMO
Zinc (Zn) plays essential roles in numerous cellular processes. However, there is limited understanding of Zn homeostasis within the bovine reproductive system. This study investigated the influence of estradiol (E2) and progesterone (P4) on Zn transporter expression and intracellular free Zn levels in bovine oviduct epithelial cells (BOEC). For this purpose, cells were harvested from slaughtered cows and cultured in vitro. Intracellular Zn concentrations were measured using FluoZin-3AM staining, while real-time polymerase chain reaction assessed Zn transporter gene expression and quantification. Overall, our results confirmed the gene expression of all the evaluated Zn transporters (ZIP6, ZIP8, ZIP14, ZnT3, ZnT7 and ZnT9), denoted and the active role of E2 and P4 in intracellular Zn regulation. Our findings suggest an interaction between Zn, E2 and P4.
Assuntos
Proteínas de Transporte , Progesterona , Zinco , Feminino , Bovinos , Animais , Progesterona/farmacologia , Progesterona/metabolismo , Zinco/farmacologia , Zinco/metabolismo , Oviductos/metabolismo , Células Epiteliais/metabolismo , Estrogênios/farmacologiaRESUMO
Supplementing minerals beyond dietary requirements can increase the risk of toxicity and mineral excretion, making the selection of more bioavailable sources crucial. Thus, this work aimed to use metalloproteomics tools to investigate possible alterations in the hepatic proteome of broilers fed with diets containing two sources (sulfate and hydroxychloride) and two levels of copper (15 and 150 ppm) and manganese (80 and 120 ppm), totaling four treatments: low Cu/Mn SO4, high Cu/Mn SO4, low Cu/Mn (OH)Cl and high Cu/Mn (OH)Cl. The difference in abundance of protein spots and copper and manganese concentrations in liver and protein pellets were analyzed by analysis of variance with significance level of 5%. The Cu and Mn concentrations determined in liver and protein pellets suggested greater bioavailability of hydroxychloride sources. We identified 19 Cu-associated proteins spots, 10 Mn-associated protein spots, and 5 Cu and/or Mn-associated protein spots simultaneously. The analysis also indicated the induction of heat shock proteins and detoxification proteins in broilers fed with high levels of copper and manganese, suggesting the involvement of these proteins in metal tolerance and stress.
Assuntos
Cobre , Manganês , Animais , Manganês/metabolismo , Cobre/metabolismo , Galinhas/metabolismo , Suplementos Nutricionais/análise , Zinco/metabolismo , Minerais/metabolismo , Dieta , Fígado/metabolismo , Ração Animal/análiseRESUMO
The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.