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1.

Background

An iron-overloaded state has been reported to be associated with insulin resistance. On the other hand, conditions such as classical hemochromatosis (where iron overload occurs primarily in the liver) have been reported to be associated with increased insulin sensitivity. The reasons for these contradictory findings are unclear. In this context, the effects of increased intracellular iron per se on insulin signaling in hepatocytes are not known.

Methods

Mouse primary hepatocytes were loaded with iron in vitro by incubation with ferric ammonium citrate (FAC). Intracellular events related to insulin signaling, as well as changes in gene expression and hepatocyte glucose production (HGP), were studied in the presence and absence of insulin and/or forskolin (a glucagon mimetic).

Results

In vitro iron-loading of hepatocytes resulted in phosphorylation-mediated activation of Akt and AMP-activated protein kinase. This was associated with decreased basal and forskolin-stimulated HGP. Iron attenuated forskolin-mediated induction of the key gluconeogenic enzyme, glucose-6-phosphatase. It also attenuated activation of the Akt pathway in response to insulin, which was associated with decreased protein levels of insulin receptor substrates 1 and 2, constituting insulin resistance.

Conclusions

Increased intracellular iron has dual effects on insulin sensitivity in hepatocytes. It increased basal activation of the Akt pathway, but decreased activation of this pathway in response to insulin.

General significance

These findings may help explain why both insulin resistance and increased sensitivity have been observed in iron-overloaded states. They are of relevance to a variety of disease conditions characterized by hepatic iron overload and increased risk of diabetes.  相似文献   

2.

Background

Tight regulation of heme homeostasis is a critical mechanism in pathogenic bacteria since heme functions as iron source and prosthetic group, but is also toxic at elevated concentrations. Hemolysin-activating lysine-acyltransferase (HlyC) from Escherichia coli is crucial for maturation of hemolysin A, which lyses several mammalian cells including erythrocytes liberating large amounts of heme for bacterial uptake. A possible impact and functional consequences of the released heme on events employing bacterial HlyC have remained unexplored.

Methods

Heme binding to HlyC was investigated using UV/vis and SPR spectroscopy. Functional impact of heme association was examined using an in vitro hemolysis assay. The interaction was further studied by homology modeling, molecular docking and dynamics simulations.

Results

We identified HlyC as potential heme-binding protein possessing heme-regulatory motifs. Using wild-type protein and a double alanine mutant we demonstrated that heme binds to HlyC via histidine 151 (H151). We could show further that heme inhibits the enzymatic activity of wild-type HlyC. Computational studies illustrated potential interaction sites in addition to H151 confirming the results from spectroscopy indicating more than one heme-binding site.

Conclusions

Taken together, our results reveal novel insights into heme-protein interactions and regulation of a component of the heme uptake system in one of the major causative agents of urinary tract infections in humans.

General significance

This study points to a possible novel mechanism of regulation as present in many uropathogenic E. coli strains at an early stage of heme iron acquisition from erythrocytes for subsequent internalization by the bacterial heme-uptake machinery.  相似文献   

3.

Background

Elevated endogenous phosphoinositide-3-kinase (PI3K) activity is critical for cell proliferation in gliomas. Iron availability is one of the essential factors for cell growth and proliferation. However, any relation between PI3K and cellular iron homeostasis has not been understood so far.

Methods

Glioma cells and human primary astrocytes were treated with class I PI3K inhibitors to examine regulation of iron homeostasis components. Regulation of ferritin was detected at mRNA and translational level. Labile iron pool (LIP) and cell proliferation were examined in glioma cells and human primary astrocytes.

Results

Blocking of PI3K activity elevated ferritin level by 6–10 folds in glioma cells by augmenting mRNA expression of ferritin subunits and also by influencing ferritin translation. IRE-IRP interaction was affected due to conversion of IRP1 to cytosolic aconitase that was influenced by increased iron-sulfur scaffold protein iron-sulfur cluster assembly enzyme (ISCU) level. Elevated ferritin sequestered LIP to affect cell proliferation that was reversed in silencing ferritin by siRNAs of ferritin-H and ISCU. Human primary astrocyte with little PI3K activity did not show any change in ferritin level, LIP and cell proliferation by PI3K inhibitors.

Conclusions

PI3K inhibition promotes ferritin synthesis by dual mechanism resulting sequestration of iron to limit its availability for cell proliferation in glioma cells but not in primary astrocytes.General Significance: This observation establishes a relation between PI3K signalling and iron homeostasis in glioma cells. It also implies that activated PI3K controls ferritin expression to ensure availability of adequate iron required for cell proliferation.  相似文献   

4.

Background

Ferritin detoxifies excess of free Fe(II) and concentrates it in the form of ferrihydrite (Fe2O3·xH2O) mineral. When in need, ferritin iron is released for cellular metabolic activities. However, the low solubility of Fe(III) at neutral pH, its encapsulation by stable protein nanocage and presence of dissolved O2 limits in vitro ferritin iron release.

Methods

Physiological reducing agent, NADH (E1/2?=??330?mV) was inefficient in releasing the ferritin iron (E1/2?=?+183?mV), when used alone. Thus, current work investigates the role of low concentration (5–50?μM) of phenazine based electron transfer (ET) mediators such as FMN, PYO - a redox active virulence factor secreted by Pseudomonas aeruginosa and PMS towards iron mobilization from recombinant frog M ferritin.

Results

The presence of dissolved O2, resulting in initial lag phase and low iron release in FMN, had little impact in case of PMS and PYO, reflecting their better ET relay ability that facilitates iron mobilization. The molecular modeling as well as fluorescence studies provided further structural insight towards interaction of redox mediators on ferritin surface for electron relay.

Conclusions

Reductive mobilization of iron from ferritin is dependent on the relative rate of NADH oxidation, dissolved O2 consumption and mineral core reduction, which in turn depends on E1/2 of these mediators and their interaction with ferritin.

General significance

The current mechanism of in vitro iron mobilization from ferritin by using redox mediators involves different ET steps, which may help to understand the iron release pathway in vivo and to check microbial growth.  相似文献   

5.

Background

Selenoprotein synthesis requires the reinterpretation of a UGA stop codon as one that encodes selenocysteine (Sec), a process that requires a set of dedicated translation factors. Among the mammalian selenoproteins, Selenoprotein P (SELENOP) is unique as it contains a selenocysteine-rich domain that requires multiple Sec incorporation events.

Scope of review

In this review we elaborate on new data and current models that provide insight into how SELENOP is made.

Major conclusions

SELENOP synthesis requires a specific set of factors and conditions.

General significance

As the key protein required for proper selenium distribution, SELENOP stands out as a lynchpin selenoprotein that is essential for male fertility, proper neurologic function and selenium metabolism.  相似文献   

6.

Background

The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1–4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins.

Methods

We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography.

Results

Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between ?65 and ?100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment.

Conclusions

Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species.

General significance

Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.  相似文献   

7.

Background

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that is currently investigated as an important target to extend lifespan and health span. Age-related NAD+ depletion due to the accumulation of oxidative stress is associated with reduced energy production, impaired DNA repair and genomic instability.

Scope of review

NAD+ levels can be elevated therapeutically using NAD+ precursors or through lifestyle modifications including exercise and caloric restriction. However, high amounts of NAD+ may be detrimental in cancer progression and may have deleterious immunogenic roles.

Major conclusions

Standardized quantitation of NAD+ and related metabolites may therefore represent an important component of NAD+ therapy.

General significance

Quantitation of NAD+ may serve dual roles not only as an ageing biomarker, but also as a diagnostic tool for the prevention of malignant disorders.  相似文献   

8.
Iron transport kinetics through blood-brain barrier endothelial cells   总被引:1,自引:0,他引:1  

Background

Transferrin and its receptors play an important role during the uptake and transcytosis of iron through blood-brain barrier (BBB) endothelial cells (ECs) to maintain iron homeostasis in BBB endothelium and brain. Any disruptions in the cell environment may change the distribution of transferrin receptors on the cell surface, which eventually alter the homeostasis and initiate neurodegenerative disorders. In this paper, we developed a comprehensive mathematical model that considers the necessary kinetics for holo-transferrin internalization and acidification, apo-transferrin recycling, and exocytosis of free iron and transferrin-bound iron through basolateral side of BBB ECs.

Methods

Ordinary differential equations are formulated based on the first order reaction kinetics to model the iron transport considering their interactions with transferrin and transferrin receptors. Unknown kinetics rate constants are determined from experimental data by applying a non-linear optimization technique.

Results

Using the estimated kinetic rate constants, the presented model can effectively reproduce the experimental data of iron transports through BBB ECs for many in-vitro studies. Model results also suggest that the BBB ECs can regulate the extent of the two possible iron transport pathways (free and transferrin-bound iron) by controlling the receptor expression, internalization of holo-transferrin-receptor complexes and acidification of holo-transferrin inside the cell endosomes.

Conclusion

The comprehensive mathematical model described here can predict the iron transport through BBB ECs considering various possible routes from blood side to brain side. The model can also predict the transferrin and iron transport behavior in iron-enriched and iron-depleted cells, which has not been addressed in previous work.  相似文献   

9.

Background

It has been almost three decades since the removal of oxidized proteins by the free 20S catalytic unit of the proteasome (20SPT) was proposed. Since then, experimental evidence suggesting a physiological role of proteolysis mediated by the free 20SPT has being gathered.

Scope of review

Experimental data that favors the hypothesis of free 20SPT as playing a role in proteolysis are critically reviewed.

Major conclusions

Protein degradation by the proteasome may proceed through multiple proteasome complexes with different requirements though the unequivocal role of the free 20SPT in cellular proteolysis towards native or oxidized proteins remains to be demonstrated.

General significance

The biological significance of proteolysis mediated by the free 20SPT has been elusive since its discovery. The present review critically analyzes the available experimental data supporting the proteolytic role of the free or single capped 20SPT.  相似文献   

10.

Background

Flavonoids possess a rich polypharmacological profile and their biological role is linked to their oxidation state protecting DNA from oxidative stress damage. However, their bioavailability is hampered due to their poor aqueous solubility. This can be surpassed through encapsulation to supramolecular carriers as cyclodextrin (CD). A quercetin- 2HP-β-CD complex has been formerly reported by us. However, once the flavonoid is in its 2HP-β-CD encapsulated state its oxidation potential, its decomplexation mechanism, its potential to protect DNA damage from oxidative stress remained elusive. To unveil this, an array of biophysical techniques was used.

Methods

The quercetin-2HP-β-CD complex was evaluated through solubility and dissolution experiments, electrochemical and spectroelectrochemical studies (Cyclic Voltammetry), UV–Vis spectroscopy, HPLC-ESI-MS/MS and HPLC-DAD, fluorescence spectroscopy, NMR Spectroscopy, theoretical calculations (density functional theory (DFT)) and biological evaluation of the protection offered against H2O2-induced DNA damage.

Results

Encapsulation of quercetin inside the supramolecule's cavity enhanced its solubility and retained its oxidation profile. Although the protective ability of the quercetin-2HP-β-CD complex against H2O2 was diminished, iron serves as a chemical stimulus to dissociate the complex and release quercetin.

Conclusions

We found that in a quercetin-2HP-β-CD inclusion complex quercetin retains its oxidation profile similarly to its native state, while iron can operate as a chemical stimulus to release quercetin from its host cavity.

General significance

The oxidation profile of a natural product once it is encapsulated in a supramolecular carrier was unveiled as also it was discovered that decomplexation can be triggered by a chemical stimilus.  相似文献   

11.

Background

Spontaneous intracranial hypotension (SIH) is caused by cerebrospinal fluid (CSF) leakage. Definitive diagnosis can be difficult by clinical examinations and imaging studies.

Methods

SIH was diagnosed with the following criteria: (i) evidence of CSF leakage by cranial magnetic resonance imaging (MRI) findings of intracranial hypotension and/or low CSF opening pressure; (ii) no recent history of dural puncture. We quantified CSF proteins by ELISA or Western blotting.

Results

Comparing with non-SIH patients, SIH patients showed significant increase of brain-derived CSF glycoproteins such as lipocalin-type prostaglandin D synthase (L-PGDS), soluble protein fragments generated from amyloid precursor protein (sAPP) and “brain-type” transferrin (Tf). Serum-derived proteins such as albumin, immunoglobulin G, and serum Tf were also increased. A combination of L-PGDS and brain-type Tf differentiated SIH from non-SIH with sensitivity 94.7% and specificity 72.6%.

Conclusion

L-PGDS and brain-type Tf can be biomarkers for diagnosing SIH.

General significance

L-PGDS and brain-type Tf biosynthesized in the brain appears to be markers for abnormal metabolism of CSF.  相似文献   

12.

Background

High levels of blood cholesterol are conventionally linked to an increased risk of developing cardiovascular disease (Grundy, 1986). Here we examine the molecular mode of action of natural products with known cholesterol-lowering activity, such as for example the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys.

Methods

Molecular Dynamics simulations are used to gain insight into the formation process of mixed micelles and, correspondingly, how active agents epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys could possibly interfere with it.

Results

Self-assembly of physiological micelles occurs on the order of 35–50?ns; most of the structural properties of mixed micelles are unaffected by epigallocatechin gallate or Ile-Ile-Ala-Glu-Lys which integrate into the micellar surface; the diffusive motion of constituting lipids palmitoyl-oleoyl-phosphatidylcholine and cholesterol is significantly down-regulated by both epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys;

Conclusions

The molecular mode of action of natural compounds epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys is a significant down-regulation of the diffusive motion of micellar lipids.

General significance

Natural compounds like the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys, lead to a significant down-regulation of the diffusive motion of micellar lipids thereby modulating cholesterol absorption into physiological micelles.  相似文献   

13.
14.

Background

The CIA2A protein, in complex with CIAO1, has been proposed to be exclusively implicated in the maturation of cytosolic aconitase. However, how the CIA2A-CIAO1 complex generates active aconitase is still unknown and the available structural information has not provided any crucial insights into the molecular function of CIA2A.

Methods

In this work we have characterized the Fe/S cluster binding properties of CIA2A and of the CIA2A-CIAO1 complex via NMR, UV???vis absorption and EPR spectroscopies and we have investigated how the Fe/S cluster is transferred to inactive aconitase/IRP1 protein.

Results

We found that an heterotrimeric species formed by two molecules of CIA2A and one of CIAO1 can bind one [4Fe-4S] cluster and that residue Cys90 of CIA2A is one of the cluster ligand. The holo trimeric complex is able to transfer the [4Fe-4S] cluster to apo-IRP1 thus generating the active form of aconitase.

Conclusions and general significance

These findings, which highlight a functional role for CIA2A-CIAO1 complex in aconitase maturation, raises a broad interest and can have a high impact on the community studying metal trafficking and iron?sulfur protein biogenesis. The present study can provide solid bases for further investigation of the molecular mechanisms involving also other CIA machinery proteins.  相似文献   

15.

Background

The N-Myc Downstream-Regulated Gene (NDRG) family comprises four members that function in cellular processes like proliferation and differentiation. While NDRG1 and NDRG2 are extensively studied, knowledge regarding NDRG3 and NDRG4, despite its recognition as a well-established early-detection marker for colorectal cancer (Cologuard®), is sparse.

Scope of review

To summarize expression, biomarker potential and functional mechanisms of the NDRGs in the developing, mature and cancerous gut, we combine current literature and in silico analyses from the TCGA-database, GTEX Project, E14.5 mouse intestine and enteric neural crest cells, and an RNA-sequencing time-series of human embryonic colonic samples.

Major conclusions

This study reveals that all members display a differential expression pattern in the gut and that NDRG1, NDRG2 and NDRG4 (1) can serve as biomarker for colorectal cancer and (2) have tumor suppressive properties mainly affecting cell proliferation and epithelial-mesenchymal transition.

General significance

Similar effects of the NDRGs on the key-hallmarks of cancer, could implicate analogous functions in other tissue/cancer types.  相似文献   

16.
17.

Background

Malaria is a serious parasitic infection affecting millions of people worldwide each year. Cerebral malaria is the most severe complication of Plasmodium infections, predominantly affecting children. Extracellular vesicles are essential mediators of intercellular communication and include apoptotic bodies, microvesicles and exosomes. Microvesicle numbers increase during disease pathogenesis and inhibition of their release can prevent brain pathology and mortality.

Scope of review

We explore the current knowledge on microvesicles and exosomes in cerebral malaria pathogenesis.

Major conclusions

Microvesicles and exosomes are implicated in cerebral malaria pathogenesis, in the modulation of host immunity to Plasmodium, and in cell-cell communication. Blocking their production is protective in models of cerebral malaria, both in vivo and in vitro.

General significance

While anti-malarial treatments exist to combat Plasmodium infections, increasing drug resistance presents a major challenge. In order to improve diagnosis and treatment outcomes, further research is required to better appreciate extracellular vesicle involvement in cerebral malaria.  相似文献   

18.

Background

Earthworms are widely used in basic and applied research in medicine, food, environment and agriculture, in which for instance earthworm protease has its own biochemical features.

Scope of review

This review summarizes earthworm protease biochemical features in anti-thrombosis and anti-fibrosis, and provides new perspectives for earthworm to be used in biochemical and pharmaceutical studies.

Major conclusions

Earthworm protease functions in anti-thrombosis by its fibrinolytic activity and inhibiting platelets aggregation, and anti-fibrosis by its decreasing fibronectin, collagen and laminin, showing a broad substrate specificity. The protease regulators (U3EE) from earthworm also has multiple functions acting as an activator and an inhibitor on different target proteins. Nonetheless, the protease improves the substrate selectivity through substrate-induced changes in the protease active site conformation impact on subsequent reactions with substrates.

General significance

It is predictable that both biochemical and applied studies of earthworm proteins including protease will be wider and deeper in the future.  相似文献   

19.

Background

Although the thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative properties, it is possible to reduce the first and enhance the second one by suitable chemical modifications.

Methods

Two oligonucleotides (TBA353 and TBA535) based on the TBA sequence (GGTTGGTGTGGTTGG) and containing inversion of polarity sites have been investigated by CD, UV and electrophoretic techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay), antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against Calu-6 cells have been tested and compared with TBA.

Results

CD, UV and electrophoresis data indicate that both ODNs are able to form G-quadruplex structures. Particularly, results suggest that TBA535 adopts a G-quadruplex structure characterized by a loop arrangement different from that of TBA. Both TBA analogues drop the anticoagulant activity. However, TBA535 is endowed with a significant antiproliferative activity against lung cancer Calu-6 cells. Importantly, both TBA and TBA535 possess a remarkable anti-motility property against the same cell line.

Conclusions

Both TBA analogues TBA353 and TBA535 are able to form G-quadruplex structures with no anticoagulant activity. However only TBA535 is endowed with noteworthy antiproliferative and anti-motility properties against lung cancer Calu-6 cells.

General significance

The switching from the anticoagulant to antiproliferative property can be obtained also in TBA derivatives not adopting the “chair-like” G-quadruplex structure typical of TBA. Furthermore, results have highlighted an unprecedented anti-cell-motility property of TBA and TBA535 reinforcing the potential of these ODNs as anticancer drugs.  相似文献   

20.

Background

It remains an open question whether plant phloem sap proteins are functionally involved in plant defense mechanisms.

Methods

The antifungal effects of two profilin proteins from Arabidopsis thaliana, AtPFN1 and AtPFN2, were tested against 11 molds and 4 yeast fungal strains. Fluorescence profiling, biophysical, and biochemical analyses were employed to investigate their antifungal mechanism.

Results

Recombinant AtPFN1 and AtPFN2 proteins, expressed in Escherichia coli, inhibited the cell growth of various pathogenic fungal strains at concentrations ranging from 10 to 160?μg/mL. The proteins showed significant intracellular accumulation and cell-binding affinity for fungal cells. Interestingly, the AtPFN proteins could penetrate the fungal cell wall and membrane and act as inhibitors of fungal growth via generation of cellular reactive oxygen species and mitochondrial superoxide. This triggered the AtPFN variant-induced cell apoptosis, resulting in morphological changes in the cells.

Conclusion

PFNs may play a critical role as antifungal proteins in the Arabidopsis defense system against fungal pathogen attacks.

General significance

The present study indicates that two profilin proteins, AtPFN1 and AtPFN2, can act as natural antimicrobial agents in the plant defense system.  相似文献   

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