DOPA Melanin Research Articles

Amorosia littoralis Mantle & D. Hawksw. survives as root endophyte of mangroves and produces water soluble melanin pigment

Melanins are heterogenous biopolymers produced by many macro and microorganisms. They enhance the ecological fitness of the producer organisms by improving their virulence and protecting them from radiation, osmotic and heat stresses. Fungi synthesise either DOPA or DHN melanin and deposit them on their cell walls. Some fungal isolates produce water soluble melanin (pyomelanin) which is excerted out of the hyphae into the surrounding environment. Pyomelanin, a polymer of homogentisate, exhibits antimicrobial, UV screening, antioxidative, and anti-inflammatory activities and recent studies also show that pyomelanin could find use in space travel as radiation shield. However, scant literature is available on fungi with ability to produce pyomelanin. We report for the first time that Amorosia littoralis occurs as a root endophyte in mangrove species including Avicennia marina, Bruguiera cylindrica and Bruguiera gymnorhiza and produces pyomelanin. Considering the various technological uses of pyomelanin, this study underscores the need to explore fungi of different habitats to identify hyperproducers and to obtain chemically diverse pyomelanin.

Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans.

The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of the important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. While the production of DOPA melanin is catalyzed by laccase that is predominantly encoded by LAC1 gene. Therefore, regulating the genetic expression of C. neoformans is conducive to exploring the impact of interested molecules on the host. In this work, we established two systems that were constructed quickly and easily for the knock-down/knock-out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats CRISPR-Cas9. The RNAi system was constructed by pSilencer 4.1-CMV neo plasmid and short hairpin RNA to achieve effective transcriptional suppression. The CRISPR-Cas9 system was used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, quantitative real-time polymerase chain reaction, transmission electron microscope, and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA was more powerful and lasted longer. An albino strain produced by CRISPR-Cas9 was completely unable to synthesize melanin. In conclusion, strains with different capacities of melanin production were obtained by RNAi and CRISPR-Cas9 systems, which might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, the two systems in this article might be convenient to quickly screen the possible trait-regulating genes of other serotypes of C. neoformans.

Fungal melanin-induced metabolic reprogramming in macrophages is crucial for inflammation.

The overuse of antifungal and immunosuppressant drugs and the higher frequency of organ transplantation has resulted in mycosis being increasingly intractable, and there is a great need for the development of new therapies. Melanin is an important virulence factor that can inhibit the inflammatory response in the host and facilitate fungal survival by several methods. However, a recent study showed that the Akt/mTOR/HIF1α axis in macrophages was activated after melanin-binding proteins recognised the DHN melanin of Aspergillus fumigatus, with a resulting metabolic shift towards glycolysis (i.e., metabolic reprogramming). As a result, antimicrobial compounds (e.g., inflammatory mediators and reactive oxygen species) were increased to fight the fungal invasion. Actually, DHN melanin from other fungi and DOPA melanin can induce inflammation and stimulate the production of melanin-binding antibodies. In addition, DOPA melanin contains conserved repeating units that are similar to those of DHN melanin. Therefore, we evaluated the associated evidence to propose an interesting and reasonable hypothesis that melanin promotes inflammation by metabolic reprogramming, which could provide a research direction for antifungal therapy. It suggests that regulating the metabolism of immune cells can guide the inflammatory response against fungi, despite the presence of immunosuppressant melanin. Since the biochemical molecules of glycolysis are clearly described, regulating glycolysis in macrophages may be easier than inventing new antifungal drugs. Further clarification of our hypothesis may strengthen the candidacy of melanin for future antifungal vaccines.

Photodynamics of Melanin Radicals: Contribution to Photoprotection by Melanin†.

The mechanism of very efficient relaxation of the melanin-photoexcited states, responsible for the photoprotective action of the pigment, remains a subject for intense investigation. The most recent study by C. Grieco, F. Kohl, and B. Kohler, entitled "Ultrafast radical photogeneration pathways in eumelanin," addresses key issues of melanin photophysics and photochemistry. By using femtosecond broad-band pump probe-transient absorption measurements, the researchers were able to identify the absorption spectrum of DOPA melanin radicals for the first time and proposed two distinct mechanisms of radical formation-photoionization and photoinduced charge separation. The observed photodynamic of melanin radicals suggests a new paradigm in which the ultrafast excited state deactivation is due to the efficient recombination of melanin radicals created promptly by photoexcitation.

Ultrafast Radical Photogeneration Pathways in Eumelanin.

Eumelanin is a ubiquitous biological pigment that rapidly and efficiently deactivates excited states created by UV or visible radiation. Paradoxically, photoirradiation of eumelanin also generates radicals and harmful reactive oxygen species, but the relationship between these pathways and excited-state deactivation is uncertain. Here, greatly expanding the excitation tuning range (225-620 nm) and probing window (400-1500 nm) in femtosecond transient absorption spectroscopy of the synthetic eumelanin, DOPA melanin, enables the detection of photogenerated radials with ultrafast time resolution for the first time. Despite its heterogeneous nature, the transient absorption signals can be modeled by two spectral components assigned to solvated electrons and photogenerated radicals. Radical absorbance measured several nanoseconds after excitation increases exponentially with increasing photon energy, matching the trend in radical yields measured in electron paramagnetic resonance spectroscopy experiments. Spectral modeling of the transient signals reveals two radical generation mechanisms: (1) photoionization by UV light; and (2) photoinduced charge transfer among eumelanin chromophores by UVA and visible wavelengths capable of reaching the pigment in skin. Concurrent ultrafast relaxation and radical generation underlie the ability of eumelanin to be both photoprotective and photodamaging, and the branching between these pathways likely depends on the wavelength of the absorbed light.

Raman Characterization of Fungal DHN and DOPA Melanin Biosynthesis Pathways.

Fungal melanins represent a resource for important breakthroughs in industry and medicine, but the characterization of their composition, synthesis, and structure is not well understood. Raman spectroscopy is a powerful tool for the elucidation of molecular composition and structure. In this work, we characterize the Raman spectra of wild-type Aspergillus fumigatus and Cryptococcus neoformans and their melanin biosynthetic mutants and provide a rough “map” of the DHN (A. fumigatus) and DOPA (C. neoformans) melanin biosynthetic pathways. We compare this map to the Raman spectral data of Aspergillus nidulans wild-type and melanin biosynthetic mutants obtained from a previous study. We find that the fully polymerized A. nidulans melanin cannot be classified according to the DOPA pathway; nor can it be solely classified according to the DHN pathway, consistent with mutational analysis and chemical inhibition studies. Our approach points the way forward for an increased understanding of, and methodology for, investigating fungal melanins.

Metabolomics reveals dopa melanin involved in the enzymatic browning of the yellow cultivars of East Asian golden needle mushroom (Flammulina filiformis)

Browning seriously causes postharvest deterioration of the yellow cultivars of Flammulina filiformis, yet the browning process and its mechanism have not been described. Changes of L*, a*, b* values, the browning and whiteness index during air contacted storage were evaluated, uncovering the great loss of brightness and meanwhile the accumulation of yellowness and redness. Browning tissue showed an increase of malondialdehyde, total phenolics, and browning-related enzyme activities of polyphenol oxidase and peroxidase, in contrast to the decrease of bioprotective catalase, superoxide, and dismutase. Non-targeted metabolomics revealed an upregulation of melanin synthesis under oxidation stress, and targeted LC-MS/MS verified the upregulation of l-dopa (3,4-dihydroxy-l-phenylalanine) during browning. Pyrrole-2,3,5-tricarboxylic acid was identified in the degradation products of browning pigments after alkaline hydrogen peroxide by LC-MS/MS, suggesting the existence of 5,6-dihydroxyindole-2-carboxylic acid derived units of eumelanin. Therefore, the biosynthesis of eumelanin via l-dopa pathway could participate in the enzymatic browning of postharvest F. filiformis.

Regulatory effects of nitric oxide on reproduction and melanin biosynthesis in onion pathogenic fungus Stemphylium eturmiunum

The formation of propagules is the critical stage for transmission of the pathogenic fungus Stemphylium eturmiunum. However, how the development of these propagules is regulated remains to be fully understood. Here, we show that nitric oxide (NO) is necessary for reproduction in S. eturmiunum.Application of NO scavenger carboxy-CPTIO (cPTIO) or soluble guanylate cyclase (sGC) inhibitor NS-2028 abolishes propagules formation, which was increased by a supplement of sodium nitroprusside (SNP). SNP supplement also triggered increased biosynthesis of melanin, which can be inhibited upon the addition of arbutin or tricyclazole, the specific inhibitors for DOPA and DHN synthetic pathway, respectively. Intriguingly, enhanced melanin biosynthesis corelates with an increased propagules formation; The SNP-induced increment propagules formation can be also compromised upon the supplement of cPTIO or NS-2028. RT-PCR analysis showed that SNP promoted transcription of brlA, abA and wetA at 0.2 mmol/L, but inhibited at 2 mmol/L. In contrast, SNP increased transcription of mat1, and mat2, and the synthetic genes for DHN and DOPA melanins at 2 mmol/L. However, the increased transcription of these genes is down-regulated upon the supplement of cPTIO or NS-2028. Thus, NO regulates reproduction and melanin synthesis in S. eturmiunum possibly through the NO-sGC-GMP signaling pathway.

Paramagnetism and Relaxation Dynamics in Melanin Biomaterials.

Spectroscopical characterization of melanins is a prior requirement for the efficient tailoring of their radical scavenging, ultraviolet-visible radiation absorption, metal chelation, and natural pigment properties. Electron paramagnetic resonance (EPR), exploiting the common persistent paramagnetism of melanins, represents the elective standard for the structural and dynamical characterization of their constituting radical species. Although melanins are mainly investigated using X-band (9.5 GHz) continuous wave (CW)-EPR, an integration with the application of Q-band (34 GHz) in CW and pulse EPR for the discrimination of melanin pigments of different compositions is presented here. The longitudinal relaxation times measured highlight faster relaxation rates for cysteinyldopa melanin, compared to those of the most common dopa melanin pigment, suggesting pulse EPR spin-lattice relaxation time measurements as a complementary tool for characterization of pigments of interest for biomimetic materials engineering.

Biomimetic Oxygen-Evolving Photobacteria Based on Amino Acid and Porphyrin Hierarchical Self-Organization.

Biomimetic organization provides a promising strategy to develop functional materials and understand biological processes. However, how to mimic complex biological systems using simple biomolecular units remains a great challenge. Herein, we design and fabricate a biomimetic cyanobacteria model based on self-integration of small bioinspired molecules, including amphiphilic amino acid, 3,4-dihydroxyphenylalanine (DOPA), and metalloporphyrin and cobalt oxide nanoparticles (Co3O4 NPs), with the assistance of chemical conjugation and molecular self-assembly. The assembled amino acid fiber can be modified by DOPA to form covalently bound DOPA melanin containing hydroxyl and quinone species via Schiff base reaction. The adhering template can further tune the self-assembly of metalloporphyrin and Co3O4 NPs into J-aggregation and dispersive distribution, respectively, mainly via coordination binding. Metalloporphyrin molecules in the resulting hybrid fibers capture light; quinone species accept the excited electrons, and Co3O4 NPs catalyze water oxidation. Thus, the essential components of the photosystem-II protein complex in cyanobacteria are simplified and engineered into a simple framework, still retaining a similar photosynthetic mechanism. In addition, this architecture leads to efficient coupling of antenna, quinone-type reaction center, and photocatalyst, which increases the flux of light energy from antenna to reaction center for charge separation, resulting in enhanced oxygen evolution rate with excellent sustainability.

Evidence for glycosylation as a regulator of the pigmentary system: key roles of sialyl(α2-6)gal/GalNAc-terminated glycans in melanin synthesis and transfer.

The major regulators of melanogenesis are glycoproteins, however no role for glycosylation in the pathway has yet been described. We stained skin biopsies and melanocyte-keratinocyte co-cultures with a panel of 20 lectins as oligosaccharide markers. Notably, the Elderberry Bark Lectin (EBL/SNA) stained melanocytes in both systems. EBL binds the sequence Neu5Ac(α(2-6)Gal/GalNAc)- at the termini of some oligosaccharide antennae. We used inhibitors of synthesis and/or binding of this sequence to assess effects on pigmentation. METHODS. Cell culture, lectin histochemistry, siRNA transfection, and assays for dopa oxidase and melanin were carried out by standard techniques. RESULTS. 6′-sialyllactose, a short homolog of the sequence in question, anti-sialyltransferase 6 (ST6) siRNA, and cytidine, a sialyltransferase (ST) inhibitor, each inhibited EBL binding, melanogenesis and melanosome transfer. Unexpectedly, 3′-sialyllactose and siRNA for ST3, chosen as a negative controls, also inhibited these processes. Though strong inhibitors of melanization, none of the agents affected tyrosinase/dopa oxidase activity, indicating previously unrecognized post-tyrosinase regulation of melanization. CONCLUSIONS. We report for the first time that Neu5Ac (α(2-6)Gal/GalNAc)- and possibly Neu5Ac(α(2–3)Gal/GalNAc)-terminated oligosaccharides play multiple roles in melanin synthesis and transfer.

The acid-catalyzed interaction of melanin with nitrite ions. An EPR investigation

Abstract The interaction of synthetic dihydroxyphenylalanine (DOPA) melanin (DM) with nitrite ions, NO2 −, in the pH 3.6–7.0 range, has been investigated using electron paramagnetic resonance (EPR). We found that especially at pH <5.5 (from ca. 5.5 to 3.6) the reaction of DM with nitrite generated large quantities of new melanin radicals, which implies the involvement of nitrous acid, HNO2, in the radical formation process. Measurements carried out at constant pH of 3.6 showed that the melanin signal increased together with nitrite concentration, reaching a plateau level which was more than fourfold larger compared to the initial signal amplitude observed in a nitrite-free buffer of the same pH. The effects of nitrite and DM concentrations on the melanin-free radical content were also investigated. It is proposed that the radicals are generated by one electron oxidation of melanin ortho-hydroquinone groups to ortho-semiquinones by HNO2 or related nitrogen oxides such as NO2 • radicals. The possible involvement of nitric oxide (•NO) and peroxynitrite (ONOO−) in DM oxidation was also examined. In air-free solutions, nitric oxide per se did not generate melanin radicals; however, in the presence of oxygen a marked increase in the melanin EPR signal intensity was observed. This result is interpreted in terms of the generation of radicals via the oxidation of DM by peroxynitrite. Our findings suggest that melanin can function as a natural scavenger of nitrous acid and some nitrous acid-derived species. This property may be relevant to physiological functions of melanin pigments in vivo.

Mussel-Inspired Modification of Nanofibers for REST siRNA Delivery: Understanding the Effects of Gene-Silencing and Substrate Topography on Human Mesenchymal Stem Cell Neuronal Commitment.

In this study, we promote neuronal differentiation of human mesenchymal stem cells (MSCs) through scaffold-mediated sustained release of siRNA targeting RE-1 silencing transcription factor (REST). Poly (ϵ-caprolactone) nanofibers were surface modified with mussel inspired DOPA-melanin (DM) coating for adsorption of REST siRNA. DM modification increased siRNA-loading efficiency and reduced the initial burst release. Fiber alignment and DM modification enhanced REST knockdown efficiencies. Under non-specific differentiation condition, REST silencing and fiber topography enhanced MSC neuronal markers expressions and reduced glial cell commitment. Such scaffolds may find useful applications in enhancing MSCs neuronal differentiation under non-specific conditions such as an in vivo environment.

Intracellular growth is dependent on tyrosine catabolism in the dimorphic fungal pathogen Penicillium marneffei.

During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host’s defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells.

Pyomelanin production in Penicillium chrysogenum is stimulated by L-tyrosine.

From a tomb in Upper Egypt we isolated a strain of Penicillium chrysogenum that was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some micro-organisms in adverse environments. We tested type strains of Pe. chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. Fourier transform IR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of Pe. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription.

The genetic control of aposematic black pigmentation in hemimetabolous insects: insights from Oncopeltus fasciatus

Variations in body pigmentation, encompassing both the range of specific colors as well as the spatial arrangement of those colors, are among the most noticeable and lineage-specific insect features. However, the genetic mechanisms responsible for generating this diversity are still limited to several model species that are primarily holometabolous insects. To address this lack of knowledge, we utilize Oncopeltus fasciatus, an aposematic hemimetabolous insect, as a new model to study insect pigmentation. First, to determine the genetic regulation of black pigment production in Oncopeltus, we perform an RNAi analysis on three core genes involved in the melanin pathway, tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and laccase 2 (lac2). The black pigmentation is affected in all instances, showing that the black pigments in this species are derived from the melanin pathway. The results of the DDC RNAi are particularly informative because they reveal that it is Dopamine melanin, not DOPA melanin, which is the predominant component of black pigments in Oncopeltus. Second, we test whether pigmentation follows a two-step model where the spatial pre-mapping of enzymatic activity is followed by vein-dependent transportation of melanin substances. We confirm the existence of the first step by observing that premature wings develop black pigmentation when exposed to melanin precursors. In addition, we provide evidence for the second step by showing that wing melanin patterning is disrupted when vein transportation is halted. These findings bring novel insights from a hemimetabolous species and establish a framework for subsequent studies on the mechanisms of pigment production and patterning responsible for variations in insect coloration.

Interaction of free radicals of DOPA-melanin-streptomycin complexes with paramagnetic oxygen O2

Oxygen plays an important role in the metabolism of all tissues. The level of oxygen in the environment of melanin biopolymer in human body may change toxic effects resulted from free radicals formation in melanin–drug complexes.Using electron paramagnetic resonance (EPR) spectroscopy we measured the effect of molecular oxygen on concentration and properties of free radicals in 3,4-dihydroxyphenylalanine–melanin (DOPA–melanin) complexes with streptomycin. Studies have shown that free radical properties of melanin are heavily dependent on the presence of oxygen in the environment of the sample. Streptomycin increases the concentration of free radicals in DOPA–melanin but in the presence of oxygen this concentration is reduced.The EPR studies confirmed the existence of stable o-semiquinone free radicals (∼1018–1019spin/g) in DOPA-melanin-streptomycin complexes, both in air and in vacuum, and the formation of quasi-chemical bonds between the DOPA-melanin-streptomycin complexes and oxygen molecules, which are responsible for the decrease in the concentration of free radicals. Our study showed that oxygen exerts an influence on spin–spin and spin–lattice interactions in the investigated complexes. Such effects can also be expected in a living organism, because DOPA–melanin is a model eumelanin mainly present in human tissues.

Preparation, Characterization, Cytotoxicity and Antioxidant Activity of DOPA Melanin Modified by Amino Acids: Melanin-Like Oligomeric Aggregates

Two new synthetic melanin-like oligomers inspired by the synthesis of pheomelanin (cys-DOPA) were prepared using L-DOPA as a precursor and the amino acids serine (ser-DOPA) and threonine (thr-DOPA) for modifications. The products obtained exhibited appreciable antioxidant activity. The compounds were characterized by liquid chromatography-mass spectrometry (LC-MS), elemental analysis and infrared spectroscopy. The pKa values of the groups present in each melanin suspension were determined in aqueous solution by potentiometric and spectrophotometric titration. Cytotoxicity assays showed an IC50 > 500 mg L -1 resulting in non-toxic compounds for living cells. Finally, cys-DOPA, ser-DOPA and thr-DOPA exhibited antioxidant activity determined by the DPPH scavenging assay, with EC50 = 7.69, 1.02 and 27.60 mg L -1 , respectively.

EPR characteristics of free radicals in DOPA–melanin–moxifloxacin complexes at ambient level of UVA radiation

EPR studies pointed out that o-semiquinone free radicals with g-values 2.0038–2.0040 take part in moxifloxacin–melanin complex formation. The process contributed to increase in free radicals concentration in nonirradiated complexes. This effect was observed for the complexes with 1×10−4M, 1×10−3M and 4×10−3M drug concentrations. UV irradiation contributed to decrease in free radicals concentration in DOPA–melanin complexes with moxifloxacin, besides the complexes with the drug concentration of 1×10−4M. The strongest decrease was observed for DOPA–melanin–moxifloxacin complexes with the drug concentration of 1×10−3M. Homogeneous broadening of EPR lines, strong dipolar interactions and slow spin–lattice relaxation processes characterized all the tested melanin samples.

Influence of Microwave Power on EPR Signal of Melanin Radical and Copper(II) Ions in DOPA-Melanin Complexes

In uence of Microwave Power on EPR Signal of Melanin Radical and Copper(II) Ions in DOPA Melanin Complexes L. Najder-Kozdrowskaa,∗, B. Pilawa, A.B. Wi¦ckowski, E. Buszman and D. Wrze±niok Division of Spectroscopy of Functional Materials, Institute of Physics, Faculty of Physics and Astronomy University of Zielona Gora, Z. Szafrana 4a, 65-516 Zielona Gora, Poland Department of Biophysics, School of Pharmacy, Medical University of Silesia, Sosnowiec, Poland Department of Pharmaceutical Chemistry, School of Pharmacy, Medical University of Silesia, Sosnowiec, Poland