Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a ß-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the ß-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual ß-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

Molybdenum-Copper Antagonism In Metalloenzymes And Anti-Copper Therapy.

The connection between 3d (Cu) and 4d (Mo) via the "Mo-S-Cu" unit is called Mo-Cu antagonism. Biology offers case studies of such interactions in metalloproteins such as Mo/Cu-CO Dehydrogenases (Mo/Cu-CODH), and Mo/Cu Orange Protein (Mo/Cu-ORP). The CODH significantly maintains the CO level in the atmosphere below the toxic level by converting it to non-toxic CO2 for respiring organisms. Several models were synthesized to understand the structure-function relationship of these native enzymes. However, this interaction was first observed in ruminants, and they convert molybdate (MoO4 2- ) into tetrathiomolybdate (MoS4 2- ; TTM), reacting with cellular Cu to yield biological unavailable Mo/S/Cu cluster, then developing Cu-deficiency diseases. These findings inspire the use of TTM as a Cu-sequester drug, especially for treating Cu-dependent human diseases such as Wilson diseases (WD) and cancer. It is well known that a balanced Cu homeostasis is essential for a wide range of biological processes, but negative consequence leads to cell toxicity. Therefore, this review aims to connect the Mo-Cu antagonism in metalloproteins and anti-copper therapy.

How to Build a Metalloenzyme: Lessons from a Protein-Based Model of Acetyl Coenzyme A Synthase.

"What I cannot create, I do not understand"─Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes catalyze reactions with efficiencies, rates, and selectivity that generally cannot be achieved in synthetic systems. Many of these processes represent vital building blocks for a sustainable society, including CO2 conversion, nitrogen fixation, water oxidation, and liquid fuel synthesis. Our inability as chemists to fully reproduce the functionality of naturally occurring enzymes implicates yet-unknown contributors to reactivity. To identify these properties, it is necessary to consider all of the components of naturally occurring metalloenzymes, from the active site metal(s) to large-scale dynamics. In this Account, we describe the holistic development of a metalloprotein-based model that functionally reproduces the acetyl coenzyme A synthase (ACS) enzyme.ACS catalyzes the synthesis of a thioester, acetyl coenzyme A, from gaseous carbon monoxide, a methyl group donated by a cobalt corrinoid protein, and coenzyme A. The active site of ACS contains a bimetallic nickel site coupled to a [4Fe-4S] cluster. This reaction mimics Monsanto's acetic acid synthesis and represents an ancient process for incorporating inorganic carbon into cellular biomass through the primordial Wood-Ljungdahl metabolic pathway. From a sustainability standpoint, the reversible conversion of C1 substrates into an acetyl group and selective downstream transfer to a thiolate nucleophile offer opportunities to expand this reactivity to the anthropogenic synthesis of liquid fuels. However, substantial gaps in our understanding of the ACS catalytic mechanism coupled with the enzyme's oxygen sensitivity and general instability have limited these applications. It is our hope that development of an artificial metalloenzyme that carries out ACS-like reactions will advance our mechanistic understanding and enable synthesis of robust compounds with the capacity for similar reactivity.To construct this model, we first focused on the catalytic proximal nickel (NiP) site, which has a single metal center bound by three bridging cysteine residues in a "Y"-shaped arrangement. With an initial emphasis on reproducing the general structure of a low-coordinate metal binding site, the type I cupredoxin, azurin, was selected as the protein scaffold, and a nickel center was incorporated into the mononuclear site. Using numerous spectroscopic and computational techniques, including electron paramagnetic resonance (EPR) spectroscopy, nickel-substituted azurin was shown to have similar electronic and geometric structures to the NiP center in ACS. A substrate access channel was installed, and both carbon monoxide and a methyl group were shown to bind individually to the reduced NiI center. The elusive EPR-active S = 1/2 Ni-CH3 species, which has never been detected in native ACS, was observed in the azurin-based model, establishing the capacity of a biological NiI species to support two-electron organometallic reactions. Pulsed EPR studies on the S = 1/2 Ni-CH3 species in azurin suggested a noncanonical electronic structure with an inverted ligand field, which was proposed to prevent irreversible site degradation. This model azurin protein was ultimately shown to perform carbon-carbon and carbon-sulfur bond formation using sequential, ordered substrate addition for selective, stoichiometric thioester synthesis. X-ray spectroscopic methods were used to provide characterization of the remaining catalytic intermediates, resolving some debate over key mechanistic details.The overall approach and strategies that we employed for the successful construction of a functional protein-based model of ACS are described in this Account. We anticipate that these principles can be adapted across diverse metalloenzyme classes, providing essential mechanistic details and guiding the development of next-generation, functional artificial metalloenzymes.

MTM1 displays a new function in the regulation of nickel resistance in Saccharomyces cerevisiae.

Nickel (Ni) is an essential yet toxic trace element. Although a cofactor for many metalloenzymes, nickel function and metabolism is not fully explored in eukaryotes. Molecular biology and metallomic methods were utilized to explore the new physiological functions of nickel in Saccharomyces cerevisiae. Here we showed that MTM1 knockout cells displayed much stronger nickel tolerance than wild-type cells and mitochondrial accumulations of Ni and Fe of mtm1Δ cells dramatically decreased compared to wild-type cells when exposed to excess nickel. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely attenuated and restored through Ni supplementation in media or total protein. SOD2 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain but was decreased by Ni supplementation. MTM1 knockout afforded resistance to excess nickel mediated through reactive oxygen species levels. Meanwhile, additional Ni showed no significant effect on the localization of Mtm1p. Our study reveals the MTM1 gene plays an important role in nickel homeostasis and identifies a novel function of nickel in promoting Sod2p activity in yeast cells.

Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.

Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues.

Heme proteins have proven to be a convenient platform for the development of designer proteins with novel functionalities. This is achieved by substituting the native iron porphyrin cofactor with a heme analogue that possesses the desired properties. Replacing the iron center of the porphyrin with another metal provides one inroad to novel protein function. A less explored approach is substitution of the porphyrin cofactor with an alternative tetrapyrrole macrocycle or a related ligand. In general, these ligands exhibit chemical properties and reactivity that are distinct from those of porphyrins. While these techniques have most prominently been utilized to develop artificial metalloenzymes, there are many other applications of this methodology to problems in biochemistry, health, and medicine. Incorporation of synthetic cofactors into protein environments represents a facile way to impart water solubility and biocompatibility. It circumvents the laborious synthesis of water-soluble cofactors, which often introduces substantial charge that leads to undesired bioaccumulation. To this end, the incorporation of unnatural cofactors in heme proteins has enabled the development of designer proteins as optical oxygen sensors, MRI contrast agents, spectroscopic probes, tools to interrogate protein function, antibiotics, and fluorescent proteins.Incorporation of an artificial cofactor is frequently accomplished by denaturing the holoprotein with removal of the heme; the refolded apoprotein is then reconstituted with the artificial cofactor. This process often results in substantial protein loss and does not necessarily guarantee that the refolded protein adopts the native structure. To circumvent these issues, our laboratory has pioneered the use of the RP523 strain of E. coli to incorporate artificial cofactors into heme proteins using expression-based methods. This strain lacks the ability to biosynthesize heme, and the bacterial cell wall is permeable to heme and related molecules. In this way, heme analogues supplemented in the growth medium are incorporated into heme proteins. This approach can also be leveraged for the direct expression of the apoprotein for subsequent reconstitution.These methodologies have been exploited to incorporate non-native cofactors into heme proteins that are resistant to harsh environmental conditions: the heme nitric oxide/oxygen binding protein (H-NOX) from Caldanaerobacter subterraneus (Cs) and the heme acquisition system protein A (HasA) from Pseudomonas aeruginosa (Pa). The exceptional stability of these proteins makes them ideal scaffolds for biomedical applications. Optical oxygen sensing has been accomplished using a phosphorescent ruthenium porphyrin as the artificial heme cofactor. Paramagnetic manganese and gadolinium porphyrins yield high-relaxivity, protein-based MRI contrast agents. A fluorescent phosphorus corrole serves as a heme analogue to produce fluorescent proteins. Iron complexes of nonporphyrin cofactors bound to HasA inhibit the growth of pathogenic bacteria. Moreover, HasA can deliver a gallium phthalocyanine into the bacterial cytosol to serve as a sensitizer for photochemical sterilization. Together, these examples illustrate the potential for designer heme proteins to address burgeoning problems in the areas of health and medicine. The concepts and methodologies presented in this Account can be extended to the development of next-generation biomedical sensing and imaging agents to identify and quantify clinically relevant metabolites and other key disease biomarkers.

Iron Protein Succinylate in the Management of Iron Deficiency Anemia: A Comparative Study with Ferrous Sulphate at Low and High Therapeutic Doses.

Oral iron supplementation constitutes the first line treatment for iron deficiency anemia (IDA), with daily doses between 80 mg and 200 mg of elemental iron. Ferrous salts, such as ferrous sulphate (FeSO4), while efficacious, frequently give rise to gastrointestinal side effects. In the present paper we attempted to directly compare the efficacy of an alternative to the FeSO4 formulation, which presents a better tolerability profile, iron protein succinylate (Ferplex®). In a diet-induced anemia model, rats were treated by oral gavage with vehicle, FeSO4, or Ferplex® at a human-dose equivalent of 80 mg and 200 mg of elemental iron. We evaluated the change in anemia-related hematological and biochemical parameters, conducting a histological examination of the intestine at sacrifice. Results indicate that both types of iron supplementation are equally effective in the treatment of IDA, restoring hemoglobin, hematocrit, erythrocytes, free iron and transferrin levels in 15 days, with no statistical differences between treated groups and control. The impact of anemia on body weight was also attenuated following treatment with both iron supplements. Thrombocyte and reticulocyte levels, altered by the anemic condition, returned to homeostasis after 15 days of either FeSO4 or Ferplex® treatment. Importantly, the lower and higher doses of iron were equally effective, thus supporting the current school of thought which states that lower therapeutic doses are sufficient for management of IDA. In addition, the study shows for the first time that oral treatment with Ferplex® does not increase serum hepcidin. Finally, Ferplex® induced minimal iron depositions in the intestinal tissue compared to FeSO4.

Learning from the worm: the effectiveness of protein-bound Moco to treat Moco deficiency.

Molybdenum cofactor (Moco) is synthesized endogenously in humans and is essential for human development. Supplementation of Moco or its precursors has been explored as a therapy to treat Moco-deficient patients but with significant limitations. By using the nematode C. elegans as a model, Warnhoff and colleagues (pp. 212-217) describe the beneficial impact of protein-bound Moco supplementation to treat Moco deficiency. If such an effect is conserved, this advance from basic research in worms may have significant clinical implications as a novel therapy for molybdenum cofactor deficiency.

Biogeographic and Evolutionary Patterns of Trace Element Utilization in Marine Microbial World.

Trace elements are required by all organisms, which are key components of many enzymes catalyzing important biological reactions. Many trace element-dependent proteins have been characterized; however, little is known about their occurrence in microbial communities in diverse environments, especially the global marine ecosystem. Moreover, the relationships between trace element utilization and different types of environmental stressors are unclear. In this study, we used metagenomic data from the Global Ocean Sampling expedition project to identify the biogeographic distribution of genes encoding trace element-dependent proteins (for copper, molybdenum, cobalt, nickel, and selenium) in a variety of marine and non-marine aquatic samples. More than 56,000 metalloprotein and selenoprotein genes corresponding to nearly 100 families were predicted, becoming the largest dataset of marine metalloprotein and selenoprotein genes reported to date. In addition, samples with enriched or depleted metalloprotein/selenoprotein genes were identified, suggesting an active or inactive usage of these micronutrients in various sites. Further analysis of interactions among the elements showed significant correlations between some of them, especially those between nickel and selenium/copper. Finally, investigation of the relationships between environmental conditions and metalloprotein/selenoprotein families revealed that many environmental factors might contribute to the evolution of different metalloprotein and/or selenoprotein genes in the marine microbial world. Our data provide new insights into the utilization and biological roles of these trace elements in extant marine microbes, and might also be helpful for the understanding of how these organisms have adapted to their local environments.

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a Celegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Adverse effects of iron deficiency anemia on pregnancy outcome and offspring development and intervention of three iron supplements.

Iron deficiency anemia (IDA) is a common micronutrient deficiency among pregnant women with severe consequences including impaired immuno-inflammatory system, premature birth, fetal death etc. The present study aimed to investigate the effects of three iron supplements on IDA female rats and their offspring. The IDA female rat model was established with low iron diet and the rats were then mated. After pregnancy, rats were fed diets containing different iron supplements (iron polysaccharide complex, iron protein succinylate and ferrous sulfate) until their offspring were 42 days old. Pregnancy outcomes, haematological, iron metabolism, physical and neurological development indexes were determined. The results showed that all three iron supplements improved the levels of hematological parameters of both mother and offspring rats. After iron supplementation, serum iron, transferrin saturation and serum ferritin levels were increased compared with the IDA group. The level of ferritin light chain in the liver and spleen of both mother and offspring rats in iron supplemented groups was significantly higher than that of the IDA group. The average number of born alive per litter in the iron treatment groups was significantly higher than that in the IDA group. Iron supplements also improved the physical growth and neurobehavioral development of offspring rats. It was also found that iron supplementation improved the expression of ferritin light chain and the synaptic growth associated proteins in the brain and hippocampus. No significant difference was found in the efficacy of three iron supplements. These results suggest that pregnant and postpartum IDA affects pregnancy outcomes, offspring physical development and causes neural impairment. Sufficient iron supplementation can significantly improve IDA and its adverse effects on both mother and offspring.

In Vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis*.

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey.

The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT: Although they have as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.

Artificial Metalloprotein Nanoanalogues: In Situ Catalytic Production of Oxygen to Enhance Photoimmunotherapeutic Inhibition of Primary and Abscopal Tumor Growth.

Photoimmunotherapy (PIT) has shown enormous potential in not only eliminating primary tumors, but also inhibiting abscopal tumor growth. However, the efficacy of PIT is greatly limited by tumor hypoxia, which causes the attenuation of phototherapeutic efficacy and is a feature of the immunosuppressive tumor microenvironment (TME). In this study, one type of brand-new artificial metalloprotein nanoanalogues is developed via reasonable integration of a "phototherapy-enzymatic" RuO2 and a model antigen, ovalbumin (OVA) for enhanced PIT of cancers, namely, RuO2 -hybridized OVA nanoanalogues (RuO2 @OVA NAs). The RuO2 @OVA NAs exhibit remarkable photothermal/photodynamic capabilities under the near-infrared light irradiation. More importantly, the photoacoustic imaging and immunofluorescence staining confirm that RuO2 @OVA NAs can remarkably alleviate hypoxia via in situ catalysis of hydrogen peroxide overexpressed in the TME to produce oxygen (O2 ). This ushers a prospect of concurrently enhancing photodynamic therapy and reversing the immunosuppressive TME. Also, OVA, as a supplement to the immune stimulation induced by phototherapy, can activate immune responses. Finally, further combination with the cytotoxic T-lymphocyte-associated protein 4 checkpoint blockade is reported to effectively eliminate the primary tumor and inhibit distant tumor growth via the abscopal effect of antitumor immune responses, prolonging the survival.

Matrix metalloprotein-triggered, cell penetrating peptide-modified star-shaped nanoparticles for tumor targeting and cancer therapy.

BACKGROUND: Specific targeting ability and good cell penetration are two critical requirements of tumor-targeted delivery systems. In the present work, we developed a novel matrix metalloprotein-triggered, cell-penetrating, peptide-modified, star-shaped nanoparticle (NP) based on a functionalized copolymer (MePEG-Peptide-Tri-CL), with the peptide composed of GPLGIAG (matrix metalloprotein-triggered peptide for targeted delivery) and r9 (cell-penetrating peptide for penetration improvement) to enhance its biological specificity and therapeutic effect. RESULTS: Based on the in vitro release study, a sustained release profile was achieved for curcumin (Cur) release from the Cur-P-NPs at pH 7.4. Furthermore, the release rate of Cur was accelerated in the enzymatic reaction. MTT assay results indicated that the biocompatibility of polymer NPs (P-NPs) was inversely related to the NP concentration, while the efficiency toward tumor cell inhibition was positively related to the Cur-P-NP concentration. In addition, Cur-P-NPs showed higher fluorescence intensity than Cur-NPs in tumor cells, indicating improved penetration of tumor cells. An in vivo biodistribution study further demonstrated that Cur-P-NPs exhibited stronger targeting to A549 xenografts than to normal tissue. Furthermore, the strongest tumor growth inhibition (76.95%) was observed in Cur-P-NP-treated A549 tumor xenograft nude mice, with slight pulmonary toxicity. CONCLUSION: All results demonstrated that Cur-P-NP is a promising drug delivery system that possesses specific enzyme responsiveness for use in anti-tumor therapy.

Efficacy and tolerability of oral iron protein succinylate: a systematic review of three decades of research.

Objective: Oral supplementation with iron is a standard intervention for treating or preventing iron deficiency with or without anemia. Over the last few decades, various forms of oral iron have been developed to improve treatment tolerability and iron bioavailability. In this review, we gathered research data regarding the use of iron protein succinylate since it was first marketed in the 1980s.Methods: Electronic databases - PubMed and the Cochrane Library - were searched for studies published up to March 2019. Clinical or observational studies reporting data on the tolerability of oral iron protein succinylate were included. Results were statistically described to evaluate and compare the efficacy and safety of iron protein succinylate with the comparators under study.Results: Iron protein succinylate was investigated in 54 studies: 38 randomized clinical trials and 16 observational studies, with a total of 8454 subjects. Of them, 8142 were included in the efficacy analysis: patients were divided into three population subtypes: general (n = 1899), gynecological/obstetric (n = 5283), and pediatric (n = 960). In total, 6450 patients received iron protein succinylate, experiencing a significant change in hemoglobin and ferritin in all populations. The change in all parameters was similar or higher with iron protein succinylate compared to other iron treatments evaluated. Overall, study groups receiving iron protein succinylate reported the lowest rate of adverse events.Conclusions: Although all iron treatments analyzed are effective and safe, our results suggest that iron protein succinylate may be an excellent choice to treat iron deficiency and anemia due to its superior effectiveness and tolerability.

OsmiR528 regulates rice-pollen intine formation by targeting an uclacyanin to influence flavonoid metabolism.

The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. The mir528 knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin gene OsUCL23 (encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition. OsUCL23 overexpression phenocopied the mir528 mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targeting OsUCL23 and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.

Comparative Assessment of Seven Docking Programs on a Nonredundant Metalloprotein Subset of the PDBbind Refined.

Extensive usage of molecular docking for computer-aided drug discovery resulted in development of numerous programs with versatile scoring and posing algorithms. Selection of the docking program among these vast number of options is central to the outcome of drug discovery. To this end, comparative assessment studies of docking offer valuable insights into the selection of the optimal tool. Despite the availability of various docking assessment studies, the performance difference of docking programs has not been well addressed on metalloproteins which comprise a substantial portion of the human proteome and have been increasingly targeted for treatment of a wide variety of diseases. This study reports comparative assessment of seven docking programs on a diverse metalloprotein set which was compiled for this study. The refined set of the PDBbind (2017) was screened to gather 710 complexes with metal ion(s) closely located to the ligands (<4 Å). The redundancy was eliminated by clustering and overall 213 complexes were compiled as the nonredundant metalloprotein subset of the PDBbind refined. The scoring, ranking, and posing powers of seven noncommercial docking programs, namely, AutoDock4, AutoDock4Zn, AutoDock Vina, Quick Vina 2, LeDock, PLANTS, and UCSF DOCK6, were comprehensively evaluated on this nonredundant set. Results indicated that PLANTS (80%) followed by LeDock (77%), QVina (76%), and Vina (73%) had the most accurate posing algorithms while AutoDock4 (48%) and DOCK6 (56%) were the least successful in posing. Contrary to their moderate-to-high level of posing success, none of the programs was successful in scoring or ranking of the binding affinities (r2 ≈ 0). Screening power was further evaluated by using active-decoy ligand sets for a large compilation of metalloprotein targets. PLANTS stood out among other programs to be able to enrich the active ligand for every target, underscoring its robustness for screening of metalloprotein inhibitors. This study provides useful information for drug discovery studies targeting metalloproteins.

Eugenol inhibits non-small cell lung cancer by repressing expression of NF-κB-regulated TRIM59.

In view of the recognized anti-tumor properties of eugenol against non-small cell lung cancer (NSCLC) in cell culture, here we further set out to investigate the potential therapeutic effect of eugenol in vivo and elucidate the underlying molecular mechanism. The relative expression levels of TRIM59 and p65 in NSCLC were quantified by real-time polymerase chain reaction. Xenograft tumor model was established with TRIM59-deficient H1975 cells, and tumor progression was monitored. Kaplan-Meier's analysis was performed to measure overall survival. Protein levels of TRIM59 and p65 in xenograft tumor were determined by western blot. Direct binding of p65 on the TRIM59 promoter was analyzed by chromatin immunoprecipitation assay, and the regulatory effect was interrogated with luciferase reporter assay. Both TRIM59 and p65 were up-regulated in NSCLC. Eugenol treatment significantly inhibited xenograft tumor progression and prolonged the overall survival of tumor-bearing mice. Mechanistically, eugenol suppressed p65 expression, which subsequently decreased TRIM59 expression. TRIM59 deficiency fully recapitulated the anti-tumoral phenotype elicited by eugenol. Ectopic expression of TRIM59 completely abolished the tumor suppressive effect of eugenol, which underlined the predominant role of TRIM59 in mediating the signaling downstream of eugenol treatment. Eugenol inhibited NSCLC via repression NF-κB-TRIM59 pathway.

Effect of Iron Supplementation on the Modulation of Iron Metabolism, Muscle Damage Biomarkers and Cortisol in Professional Cyclists.

BACKGROUND: The intense efforts made during 3-week stage races may reduce iron metabolism and hematological parameters. These efforts may increase the levels of circulating muscle damage markers and some hormones. All of these physiological changes may have negative consequences not only for the performance of athletes but also for their health. The main aim of this study was to evaluate the effects of supplementation with 80 mg/day of iron on haematological parameters, serum cortisol and biochemical muscle indicators on elite male cyclists during the 3-week stage race the Vuelta a España. Our secondary aim was to examine whether the hematological profile is associated with muscular damage parameters and cortisol. METHODS: Eighteen elite male cyclists from two teams were randomly assigned to one of two groups: (1) control group (CG, n = 9; age: 26.1 ± 4.6 years; maximum oxygen uptake per kg: 78.0 ± 5.4 mL/kg/min) or (2) group treated with 80 mg/day iron (800 mg of iron protein succinylate, ITG, n = 9; age: 25.7 ± 6.4 years; maximum oxygen uptake per kg: 77.6 ± 6.5 mL/kg/min). The cyclists were subjected to blood tests one week before the start of the race (T1) and after 4 weeks of treatment, coinciding with the end of the competition (T2). Iron metabolism parameters, muscle damage indicators and serum cortisol were assessed. Repeated-measures ANOVA with group as a factor (GC and ITG) were used to examine the differences between groups throughout the study (time × group) after iron supplementation treatment. RESULTS: Significant differences were observed between groups throughout the study in the group-by-time interaction and changes in serum iron (GC: -8.93 ± 10.35% vs. ITG: 0.60 ± 8.64%; p = 0.018), ferritin (GC: -13.88 ± 23.53% vs. ITG: 91.08 ± 118.30%; p = 0.004), haemoglobin (GC: 10.00 ± 3.32% vs. ITG: 13.04 ± 5.64%; p < 0.001), haematocrit (GC: -1.17 ± 3.78% vs. ITG: 7.32 ± 3.92%; p < 0.001) and cortisol (GC: 24.74 ± 25.84% vs. ITG: ⁻13.54 ± 13.61%; p = 0.005). However, no significant group-by-time interaction was observed for the circulating muscle biomarkers. Additionally, significant negative correlations of serum iron, haemoglobin and haematocrit with muscle circulating biomarkers and cortisol (p < 0.05) were observed. CONCLUSIONS: Oral iron supplementation with 80 mg/day iron (800 mg of iron protein succinylate) effectively prevented a decline in haematological parameters (serum iron, ferritin, haemoglobin and haematocrit) and maintained optimal levels of recovery in elite cyclists during the Vuelta a España. Moreover, the hematological values were shown to have relationship with muscular recovery parameters.