Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin-Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios.

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin-ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

Cellular binding analysis of recombinant hybrid heteropolymer of camel hepcidin and human ferritin H chain. The unexpected human H-ferritin binding to J774 murine macrophage cells.

Ferritin is a molecule with enormous potentiality in biotechnology that have been already used to encapsulate molecules, as contrast in magnetic resonance imaging and to carry epitopes. We proposed to use it to carry another key protein of iron metabolism, hepcidin that is a small hormone peptide that control systemic iron homeostasis. In this work, we purified the previously produced camel hepcidin and human H-ferritin heteropolymer (HepcH-FTH) and to monitor its binding capability toward J744 cell line in presence or absence of ferric ammonium citrate. Fused camel hepcidin and human H-ferritin monomer (HepcH) as well as the assembled HepcH-FTH heteropolymer (ratio 1:5) was easily purified by a one-step purification using size exclusion chromatography. SDS-PAGE electrophoresis of HepcH, purified from soluble and insoluble fractions, showed a single band of 24 kDa with an estimated purity of at least 90%. The purification yields of HepcH from the soluble and insoluble fractions was, respectively, of about 6.80 and 2 mg/L of bacterial culture. Time curse cellular binding assays of HepcH-FTH revealed its great potential to bind the J774 cells after 15 min of incubation. Furthermore, HepcH-FTH was able to degrade ferroportin, the unique hepcidin receptor, even after 30 min of incubation with J774 cells treated with 100 µM ferric ammonium citrate. In conclusion, we proposed ferritin as a peptide carrier to promote the association of the hybrid HepcH-FTH nanoparticle with a particular type of cell for therapeutic or diagnostic.

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology.

The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H- and L-type that coassemble in an almost spherical nanocage 12nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. In order to study this, we used the Fluorescence Resonance Energy Transfer (FRET) tool by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. The FRET efficiency increase from <0.3 of the disassembled to >0.7 in the assembled allowed to study the assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Moreover, we found that the H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize.

Expression and characterization of the ferritin binding domain of Nuclear Receptor Coactivator-4 (NCOA4).

Ferritinophagy is the process of autophagic degradation of ferritin that participates in the regulation of cellular iron homeostasis. This process was shown to be mediated by the selective cargo-receptor Nuclear Receptor Coactivator-4 (NCOA4) that binds ferritin and targets it to emerging autophagosome. To characterize some of the biochemical properties of the interaction between the two proteins we cloned and expressed in E. coli the ferritin-binding domain of human NCOA4, fragment 383-522. It was purified and subjected to biochemical analysis. The NCOA4(383-522) fragment was expressed in soluble and dimeric form, and CD spectra indicated low level of secondary structure. The Ferritin binding activity of the fragment was investigated by developing an electrophoretic mobility shift and an ELISA assays. They showed that the NCOA4 fragment binds the H-ferritin with an affinity in the nM range, but not the R23A H-ferritin mutant and the L-ferritin chain, confirming the high specificity for the H-chain. The H-ferritin could bind up to 24 NCOA4(383-522) fragments forming highly stable and insoluble complexes. The binding was partially inhibited only by Fe(II) among the various divalent metal ions analyzed. The iron-dependent, highly-specific formation of the remarkably stable H-ferritin-NCOA4 complex shown in this work may be important for the characterization of the mechanism of ferritinophagy.

Plasma clusterin as a candidate pre-diagnosis marker of colorectal cancer risk in the Florence cohort of the European Prospective Investigation into Cancer and Nutrition: a pilot study.

BACKGROUND: Colorectal cancer is one of the major causes of cancer mortality world-wide. Prevention would improve if at-risk subjects could be identified. The aim of this study was to characterise plasma protein biomarkers associated with the risk of colorectal cancer in samples collected prospectively, before the disease diagnosis. METHODS: After an exploratory study on the comprehensive plasma proteome analysis by liquid chromatography-tandem mass spectrometry from ten colorectal cancer cases enrolled at diagnosis, and ten matched controls (Phase 1), a similar preliminary study was performed on prospective plasma samples from ten colorectal cancer cases, enrolled years before disease development, and ten matched controls identified in a nested case-control study within the Florence cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) study (Phase 2); in Phase 3 the validation of the candidate biomarkers by targeted proteomics on 48 colorectal cancer cases and 48 matched controls from the Florence-EPIC cohort, and the evaluation of the disease risk were performed. RESULTS: Systems biology tools indicated that both in the Phase 1 and Phase 2 studies circulating protein levels differing in cases more than 1.5 times from controls, were involved in inflammation and/or immune response. Eight proteins including apolipoprotein C-II, complement C4-B, complement component C9, clusterin, alpha-2-HS-glycoprotein, mannan-binding lectin serine-protease, mannose-binding protein C, and N-acetylmuramoyl-L-alanine amidase were selected as promising candidate biomarkers. Targeted proteomics of the selected proteins in the EPIC samples showed significantly higher clusterin levels in cases than controls, but only in men (mean ± SD, 1.98 ± 0.46 and 1.61 ± 0.43 nmol/mL respectively, Mann-Whitney U, two-tailed P = 0.0173). The remaining proteins were unchanged. Using multivariate logistic models a significant positive association emerged for clusterin, with an 80% increase in the colorectal cancer risk with protein's unit increase, but only in men. CONCLUSIONS: The results show that plasma proteins can be altered years before colorectal cancer detection. The high circulating clusterin in pre-diagnostic samples suggests this biomarker can improve the identification of people at risk of colorectal cancer and might help in designing preventive interventions.

N-Terminally Truncated and Pyroglutamate-Modified Aß Forms Are Measurable in Human Cerebrospinal Fluid and Are Potential Markers of Disease Progression in Alzheimer's Disease.

Alzheimer's disease (AD) is a pathology characterized by the accumulation in the brain of intracellular and extracellular amyloid-ß (Aß) aggregates, especially of Aß1-40 and Aß1-42 peptides. It is known that N-terminally truncated or modified Aß forms also exist in AD brains and cerebrospinal fluid (CSF), and they play a key role in the pathogenesis of the disease. Herein, we developed an antibody-free method based on Solid-Phase Extraction and Electrospray Ionization Liquid Chromatography Mass Spectrometry for the identification and quantitation in human CSF of Aß isoforms. In human CSF, we could detect and quantify a panel of 19 Aß isoforms, including N-terminally truncated and pyroglutamate-modified forms, never quantified before in CSF. Among these, we identified novel N-terminally truncated Aß species: four bound to copper and two phosphorylated forms, which were found to be the most common proteoforms in human CSF along with Aß1-40, Aß3-40, and AßpE11-42. We tested the newly developed and validated method in a pilot study on CSF from elderly individuals with subjective memory complaints (SMCs, n = 9), mild cognitive impairment (MCI, n = 18), and AD (n = 15); along with Aß1-42, five N-terminally truncated forms (Aß11-40, Aß3-42, AßpE11-42, AßpE3-40, and Aß4-40 Cu2+) are altered in AD/MCI. Thus, we demonstrated that N-terminally truncated and pyroglutamate-modified Aß can be quantified in human CSF, and five of them, along with Aß1-42, are potential markers of AD progression. The described method could represent a useful tool for patients' stratification and monitoring. Moreover, the newly identified Aß CSF species might represent new potential therapeutic targets.

Enlightening Gliotoxin Biological System in Agriculturally Relevant Trichoderma spp.

Gliotoxin (GT) is a dual fungal secondary metabolite (SM). It displays pleiotropic activities and possesses medicinal properties and biocontrol abilities but, unfortunately, has toxic properties in humans. Various Trichoderma species are used as fungal biological control agents (BCAs), as a sustainable alternative for crop protection worldwide. Among them is Trichoderma virens, a GT-producing fungus. Since no information was available on the genetically coded prerequisites for the production of GT in other Trichoderma spp., genome analyses were carried out in 10 Trichoderma spp. genomes. Moreover, a real-time PCR assay setup ad hoc and high-performance liquid chromatography (HPLC) analyses were employed to understand the GT-producing biological systems in T. virens GV29-8 (TvGv29-8) and Trichoderma afroharzianum T6776 (TaT6776), two relevant biocontrol fungi. The structure of the GT biosynthesis genes (GT-BG) is polymorphic, with two distinct types associated with the ability to produce GT. GliH, a key protein for GT synthesis, is absent in most of the Trichoderma GT biosynthetic pathways, which may be the reason for their inability to produce GT. The GT-BG are expressed in TvGv29-8 as expected, while they are silent in TaT6776. Interestingly, in the GT-non-producing TaT6776, only gliA (putative GT transporter) and gtmA (putative GT S-methyltransferase) were induced by exogenous GT, underlining the ability of this strain to reduce the deleterious effect of the toxin. This ability is confirmed by growth assays and by the detection of the bis-thiomethylated form of GT catalyzed by GtmA in the culture medium supplemented with GT. To the best of our knowledge, this is the first general description of the GT biological system in different Trichoderma spp. as far as the GT-BG content and organization is concerned and a preliminary insight into their functionality.

Adrenocortical Carcinoma Xenograft in Zebrafish Embryos as a Model To Study the In Vivo Cytotoxicity of Abiraterone Acetate.

Abiraterone acetate (AbiAc) inhibits tumor growth when administered to immunodeficient mice engrafted with the in vitro cell model of human adrenocortical carcinoma (ACC). Here, we developed and validated a zebrafish model engrafted with cortisol-secreting ACC cells to study the effects of AbiAc on tumor growth. The experimental conditions for AbiAc absorption in AB zebrafish embryos including embryo number, AbiAc concentration, and absorption time curve by liquid chromatography-tandem mass spectrometry were set up. The AbiAc effect on steroid production in AB zebrafish embryos was measured as well. ACC cells (the NCI-H295R cell line, the primary cell ACC29, and the negative control cell SW13) were treated with drug-induced liver injury fluorescent dye, and ∼240 cells per 4 nL was injected in the subperidermal space of the yolk sac of AB zebrafish embryos (n = 80 ± 10). The cell area was measured with Noldus DanioScopeTM software. AbiAc absorption in AB zebrafish embryos was stage dependent. Abiraterone (Abi) concentration decreased, whereas its main metabolite, Δ4A, increased. Accordingly, we demonstrated that zebrafish expressed mRNA encoding the enzyme 3ß-hydroxysteroid dehydrogenase, which converts Abi in Δ4A. Furthermore, ABiAc reduced cortisol production and increased progesterone in zebrafish embryos. Three days after cell injection, the cortisol-secreting ACC cell area in solvent-treated embryos was significantly higher than that in 1 µM AbiAC‒treated embryos, whereas no AbiAc effect was observed in SW13 cells, which lack the Abi target enzyme CYP17A1.Zebrafish embryos xenografted with ACC tumor cells could be a useful, fast, and reproducible experimental model to preclinically test the activity of new drugs in human ACC.

Production and characterization of functional recombinant hybrid heteropolymers of camel hepcidin and human ferritin H and L chains.

Hepcidin is a liver-synthesized hormone that plays a central role in the regulation of systemic iron homeostasis. To produce a new tool for its functional properties the cDNA coding for camel hepcidin-25 was cloned at the 5'end of human FTH sequence into the pASK-IBA43plus vector for expression in Escherichia coli The recombinant fusion hepcidin-ferritin-H subunit was isolated as an insoluble iron-containing protein. When alone it did not refold in a 24-mer ferritin molecule, but it did when renatured together with H- or L-ferritin chains. We obtained stable ferritin shells exposing about 4 hepcidin peptides per 24-mer shell. The molecules were then reduced and re-oxidized in a controlled manner to allow the formation of the proper hepcidin disulfide bridges. The functionality of the exposed hepcidin was confirmed by its ability to specifically bind the mouse macrophage cell line J774 that express ferroportin and to promote ferroportin degradation. This chimeric protein may be useful for studying the hepcidin-ferroportin interaction in cells and also as drug-delivery agent.

"RaMassays": Synergistic Enhancement of Plasmon-Free Raman Scattering and Mass Spectrometry for Multimodal Analysis of Small Molecules.

SiO2/TiO2 core/shell (T-rex) beads were exploited as "all-in-one" building-block materials to create analytical assays that combine plasmon-free surface enhanced Raman scattering (SERS) and surface assisted laser desorption/ionization (SALDI) mass spectrometry (RaMassays). Such a multi-modal approach relies on the unique optical properties of T-rex beads, which are able to harvest and manage light in both UV and Vis range, making ionization and Raman scattering more efficient. RaMassays were successfully applied to the detection of small (molecular weight, M.W. <400 Da) molecules with a key relevance in biochemistry and pharmaceutical analysis. Caffeine and cocaine were utilized as molecular probes to test the combined SERS/SALDI response of RaMassays, showing excellent sensitivity and reproducibility. The differentiation between amphetamine/ephedrine and theophylline/theobromine couples demonstrated the synergistic reciprocal reinforcement of SERS and SALDI. Finally, the conversion of L-tyrosine in L-DOPA was utilized to probe RaMassays as analytical tools for characterizing reaction intermediates without introducing any spurious effects. RaMassays exhibit important advantages over plasmonic nanoparticles in terms of reproducibility, absence of interference and potential integration in multiplexed devices.

An Integrated Approach for a Structural and Functional Evaluation of Biosimilars: Implications for Erythropoietin.

BACKGROUND: Authorization to market a biosimilar product by the appropriate institutions is expected based on biosimilarity with its originator product. The analogy between the originator and its biosimilar(s) is assessed through safety, purity, and potency analyses. OBJECTIVE: In this study, we proposed a useful quality control system for rapid and economic primary screening of potential biosimilar drugs. For this purpose, chemical and functional characterization of the originator rhEPO alfa and two of its biosimilars was discussed. METHODS: Qualitative and quantitative analyses of the originator rhEPO alfa and its biosimilars were performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The identification of proteins and the separation of isoforms were studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and two-dimensional gel electrophoresis (2D-PAGE), respectively. Furthermore, the biological activity of these drugs was measured both in vitro, evaluating the TF-1 cell proliferation rate, and in vivo, using the innovative experimental animal model of the zebrafish embryos. RESULTS: Chemical analyses showed that the quantitative concentrations of rhEPO alfa were in agreement with the labeled claims by the corresponding manufacturers. The qualitative analyses performed demonstrated that the three drugs were pure and that they had the same amino acid sequence. Chemical differences were found only at the level of isoforms containing N-glycosylation; however, functional in vitro and in vivo studies did not show any significant differences from a biosimilar point of view. CONCLUSION: These rapid and economic structural and functional analyses were effective in the evaluation of the biosimilarity between the originator rhEPO alfa and the biosimilars analyzed.