A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions.

Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment. IMPORTANCE Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface.

A New Paradigm of Multiheme Cytochrome Evolution by Grafting and Pruning Protein Modules.

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochrome c involved in metabolic pathways that contribute to the nitrogen, sulfur, and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant octaheme nitrite reductase and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analyzed here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

Investigation of the Molecular Mechanisms of the Eukaryotic Cytochrome-c Maturation System.

Cytochromes-c are ubiquitous heme proteins with enormous impact at the cellular level, being key players in metabolic processes such as electron transfer chains and apoptosis. The assembly of these proteins requires maturation systems that catalyse the formation of the covalent thioether bond between two cysteine residues and the vinyl groups of the heme. System III is the maturation system present in Eukaryotes, designated CcHL or HCCS. This System requires a specific amino acid sequence in the apocytochrome to be recognized as a substrate and for heme insertion. To explore the recognition mechanisms of CcHL, the bacterial tetraheme cytochrome STC from Shewanella oneidensis MR-1, which is not a native substrate for System III, was mutated to be identified as a substrate. The results obtained show that it is possible to convert a bacterial cytochrome as a substrate by CcHL, but the presence of the recognition sequence is not the only factor that induces the maturation of a holocytochrome by System III. The location of this sequence in the polypeptide also plays a role in the maturation of the c-type cytochrome. Furthermore, CcHL appears to be able to catalyse the binding of only one heme per polypeptide chain, being unable to assemble multiheme cytochromes c, in contrast with bacterial maturation systems.

Preparation of end-capped pH-sensitive mesoporous silica nanocarriers for on-demand drug delivery.

Nanocarriers with a pH responsive behavior are receiving an ever growing attention due to their potential for promoting on-demand drug release and thus increase the therapeutic effectiveness of anti-tumoral pharmaceutics. However, the majority of these systems require costly, time-consuming and complex chemical modifications of materials or drugs to synthesize nanoparticles with pH triggered release. Herein, the development of dual drug loaded pH-responsive mesoporous silica nanoparticles (MSNs) with a calcium carbonate-based coating is presented as an effective alternative. This innovative approach allowed the loading of a non-steroidal anti-inflammatory drug (Ibuprofen) and Doxorubicin, with high efficiency. The resulting dual drug loaded MSNs have spherical morphology and a mean size of 171nm. Our results indicate that under acidic conditions the coating disassembles and the drugs are rapidly released, whereas at physiologic pH the release is slower and gradually increases with time. Furthermore, an improved cytotoxic effect was obtained for Doxorubicin-Ibuprofen MSNs coated with CaCO3 in comparison with non-coated particles. The cytotoxic effect of dual loaded carbonate coated particles, was similar to that of Doxorubicin+Ibuprofen free drug administration at 72h, even with the delivery of a significantly lower amount of drug by MSNs-CaCO3. These results suggest that the carbonate coating of MSNs is a promising approach to create a pH-sensitive template for a delivery system with application in cancer therapy.

Synthesis and characterization of micelles as carriers of non-steroidal anti-inflammatory drugs (NSAID) for application in breast cancer therapy.

Non-steroidal anti-inflammatory drugs (NSAIDS) are emerging as a particularly valuable class of drugs due to their recently reported anti-tumoral activity in colorectal cancer. However, despite this tremendous potential, their bioavailability at the tumor microenvironment remains rather limited. To overcome this issue, in this work we synthesized biocompatible micellar nanocarriers composed of amphiphilic chitosan to deliver ibuprofen into breast cancer cells and evaluate its anti-tumor activity, while avoiding side-effects. Our results reveal that the formulations produced herein self-assembly into spherical micelles with suitable sizes for tumor accumulation (108-252 nm). Furthermore, by using a vortex-sonication method, ibuprofen was successfully encapsulated with high efficiency. Cell uptake studies show that ibuprofen-loaded micelles are readily internalized by tumor cells and deliver their cargo in the intracellular compartment as demonstrated by confocal microscopy images. This fact led to a remarkable reduction in cancer cell viability (<13%), at a relatively low drug dosage, illustrating the anti-tumoral activity of ibuprofen when delivered to breast cancer cells. These findings demonstrate the promising potential of chitosan micelles as carriers of cost-effective NSAIDS for application in breast cancer therapy.

Nanoparticle mediated delivery of pure P53 supercoiled plasmid DNA for gene therapy.

The translation of non-viral gene replacement therapies for cancer into clinical application is currently hindered due to known issues associated with the effectiveness of plasmid DNA (pDNA) expression vectors and the production of gene delivery vehicles. Herein we report an integrative approach established on the synthesis of nanoparticulated carriers, in association with the supercoiled (sc) isoform purification of a p53 tumor suppressor encoding plasmid, to improve both delivery and transfection. An arginine-based chromatographic matrix with specific recognition for the different topoisoforms was used to completely isolate the biologically active sc pDNA. Our findings showed that the sc topoisoform is recovered under mild conditions with high purity and structural stability. In addition, to further enhance protection and transfection efficiency, the naked sc pDNA was encapsulated within chitosan nanoparticles by ionotropic gelation. The mild conditions for particle synthesis used in the former technique allowed the attainment of a high encapsulation efficiency for sc pDNA (>75%). Moreover, in vitro transfection experiments confirmed the reinstatement of the p53 protein expression and most importantly, the sc pDNA transfected cells exhibited the highest p53 expression levels when compared to other formulations. Overall, given the fact that sc pDNA topoisoform indeed enhances transgene expression rates this approach might have a profound impact on the development of a sustained nucleic acid-based therapy for cancer.

Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1.

In the model microbe Shewanella oneidensis, multi-heme proteins are utilized for respiratory metabolism where metals serve as the terminal electron acceptor. Among those is the periplasm-localized small tetraheme cytochrome (STC). STC has been extensively characterized structurally and electrochemically to which electron flow in and out of the protein has been modeled. However, until the present work, no kinetic studies have been performed to probe the route of electron flow or to determine the iron-binding site on STC. Using iron chelated by EDTA, NTA, or citrate, we have used chemical modification, site-directed mutagenesis along with isothermal titration calorimetry (ITC), and stopped-flow measurements to identify the iron binding site of STC. Chemical modifications of STC revealed that carboxyl groups on STC are involved in binding of EDTA-Fe(3+). Scanning mutagenesis was performed on Asp and Glu to probe the putative iron-binding site on STC. Two STC mutants (D21N; D80N) showed ∼70% decrease in observed electron transfer rate constant with EDTA-Fe(3+) from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe(3+) was further confirmed by a significant decrease (>10-fold) in iron binding affinity.