Cu homeostasis in bacteria: The ins and outs

Copper (Cu) is an essential trace element for all living organisms and used as cofactor in key enzymes of important biological processes, such as aerobic respiration or superoxide dismutation. However, due to its toxicity, cells have developed elaborate mechanisms for Cu homeostasis, which balance Cu supply for cuproprotein biogenesis with the need to remove excess Cu. This review summarizes our current knowledge on bacterial Cu homeostasis with a focus on Gram-negative bacteria and describes the multiple strategies that bacteria use for uptake, storage and export of Cu. We furthermore describe general mechanistic principles that aid the bacterial response to toxic Cu concentrations and illustrate dedicated Cu relay systems that facilitate Cu delivery for cuproenzyme biogenesis. Progress in understanding how bacteria avoid Cu poisoning while maintaining a certain Cu quota for cell proliferation is of particular importance for microbial pathogens because Cu is utilized by the host immune system for attenuating pathogen survival in host cells

Bioinorganic Chemistry

This book covers material that could be included in a one-quarter or one-semester course in bioinorganic chemistry for graduate students and advanced undergraduate students in chemistry or biochemistry. We believe that such a course should provide students with the background required to follow the research literature in the field. The topics were chosen to represent those areas of bioinorganic chemistry that are mature enough for textbook presentation. Although each chapter presents material at a more advanced level than that of bioinorganic textbooks published previously, the chapters are not specialized review articles. What we have attempted to do in each chapter is to teach the underlying principles of bioinorganic chemistry as well as outlining the state of knowledge in selected areas. We have chosen not to include abbreviated summaries of the inorganic chemistry, biochemistry, and spectroscopy that students may need as background in order to master the material presented. We instead assume that the instructor using this book will assign reading from relevant sources that is appropriate to the background of the students taking the course. For the convenience of the instructors, students, and other readers of this book, we have included an appendix that lists references to reviews of the research literature that we have found to be particularly useful in our courses on bioinorganic chemistry

Biophysical and functional characterization of wheat metallothionein at molecular level

Metallothioneins (MTs) are small, cysteine-rich proteins with high binding capacity for metals including Zn, Cu and Cd. MTs exist in a wide range of organisms and are classified in one super-family according to the distribution of cysteine motifs in their sequences. Plant Type 1 MTs are low molecular weight (7-8 kDa) aromatic residue lacking metal-binding proteins. They have two metal-binding domains separated by a conserved specifically long spacer region of about 30-45 amino acids. Recent studies indicate that all members of this family do not have a single unifying function; while some MTs participate in metal homeostasis others play a role in detoxification of heavy metals. Despite the large literature on MT sequences and functional roles, lack of direct biochemical and biophysical data on purified proteins hinders a comprehensive understanding of sequence, structure, metal-binding and function relationships in MTs from different sources. Due to their aggregation propensity, sensitivity to oxidation, proteolytic cleavage especially in the spacer region and difficulties in quantification, standard methods could not be directly used for purification of plant MTs. A Type 1 MT from Triticum durum, dMT, was expressed in E. coli cells as a fusion protein with GST (Bilecen et al., 2005). In the present study structure-function relationship of dMT is investigated using the fusion protein GSTdMT as a model system. The procedure developed for GSTdMT purification required strict anaerobic conditions and critical parameters including concentration of Cd and specific reducing agents, as well as choice of buffers was optimized. The purified GSTdMT was characterized by size exclusion chromatography, SDS- and native-PAGE, UV-vis absorption spectrophotometry, inductively coupled plasma optical emission spectroscopy (ICP-OES), dynamic light scattering (DLS), circular dichroism (CD), Extended X-ray Absorption Fine Structure (EXAFS) and small-angle solution X-ray scattering (SAXS). Size exclusion chromatography revealed a stable dimeric form of GSTdMT in solution. Purified GSTdMT solutions were monodisperse and homogeneous and thus suitable for structural and functional studies. The Cd binding ability of GSTdMT was initially characterized by UV-vis absorption spectroscopy and through the ICP-OES measurements the Cd2+ binding ratio was found to 3.5 Cd2+/protein. This finding was further confirmed by EXAFS measurement which strongly indicated Cd-S coordination with four sulphurs. SAXS measurements revealed that GSTdMT has an elongated shape with a radius of gyration of 3.57 nm. ab initio models resulted in a structure in which two GST molecules form an electron dense region at one end of the dimer and the two dMT molecules extend from this region. dMT structure appears to be independent of GST in the GSTdMT fusion. The combination of SAXS results with biochemical data lead to the proposal of a hairpin like model for dMT structure. Results show that the metal content and structure of dMT in the fusion protein are preserved, thus biologically relevant structural parameters can be determined using the GSTdMT construct. In a previous study (Bilecen et al., 2005) the predicted structure for the spacer region had shown similarity with that of a family that includes DNA binding proteins. In this thesis DNA binding possibility of dMT protein was examined through the whole genome PCR-based screening method. It was found that application of this method resulted in several artifacts and in our hands the method could not be used for investigation of DNA binding possibility of any protein. In part of this work response of Triticum durum cv. Balcalı-85 to environmental Cd was investigated. Balcalı-85 was subjected to increasing Cd concentrations (e.g., 0, 2, 5, 10, and 20 μM Cd). As a result, reduction in dry weight matter was observed both in roots and shoot. Also, it was found that Balcalı-85 has high capacity to retain Cd in roots. These studies were carried out as a part of an investigation which will focus on the correlation between mt-d gene expression and Cd response. In addition, southern blot analysis revealed that the mt-d gene, having 2 exons and a non-coding intron region, has a single copy in T. durum genome

Implications of Metal Coordination in Damage and Recognition of Nucleic Acids and Lipid Bilayers

Metal ions have a myriad of biological functions from structural stability to enzymatic (de)activation and metabolic electron transfer. Redox-active metals also mediate the formation of reactive oxygen species which may either cause oxidative damage or protect cellular components. Computational modeling is used here to investigate the role of (1) metal-ion binding to antimicrobial peptides, (2) metal-ion removal and disulfide formation on zinc finger (ZF) proteins, and (3) coordination of thiones/selones for the prevention of metal-mediated redox damage. Piscidins, natural-occurring antimicrobial peptides, efficiently kill bacteria by targeting their membranes. Their efficacy is enhanced in vitro by metal-binding and the presence of membrane-destabilizing oxidized phospholipids (oxPLs). Molecular dynamics (MD) simulations are used to model insertion of Ni2+-bound piscidins 1 (P1:Ni2+) and 3 (P3:Ni2+) into lipid bilayers in the presence and absence of oxPLs. Metallation promotes deeper peptide insertion in the membrane, and P1:Ni2+ is suggested to interact more with anionic lipid headgroups in the presence of oxPLs. The release of Zn2+ from ZF proteins through oxidation of the cysteine thiolates is associated with inhibition of viral replication, disruption of cancer gene expression, and DNA repair preventing tumor growth. Multi-microsecond MD simulations were performed to examine the effect of cysteine oxidation on the ZF456 fragment of transcription factor IIIA and its complex with 5S RNA. Upon oxidation in the absence of RNA, the individual ZF domains unfold yielding a globular ZF456 peptide. Oxidation of the RNA-bound ZF456 peptide disrupts key hydrogen bonding interactions between ZF5/ZF6 and 5S RNA. The antioxidant properties of sulfur and selenium compounds prevent metal-mediated (i.e., Fenton chemistry) oxidative damage. The effect of the coordination of sulfur/selenium derivatives of imidazolidinone (thiones/selones) on the electronic structure and reduction potential of Fe2+ ions solvated or coordinated to guanine are examined using density functional theory. The highest occupied molecular orbital (HOMO) for the Fe(II)-aqua complex is metal-centered but localized on the nucleobase in the Fe2+-guanine complex. Complexation of the thione/selone shifts the HOMO to the sulfur/selenium center suggesting a mechanism for protection of DNA by sacrificial oxidation of the sulfur/selenium ligand

The Life of Prion: an investigation into the physiological role of a prion-like protein in the nematode Caenorhabditis elegans

For centuries, the threat of prion disease has plagued populations – whether it be in the form of scrapie ravaging through the sheep populations of Spain in the eighteenth century, fatal familial insomnia afflicting families in Italy, or an outbreak of Creutzfeldt-Jakob disease in the UK triggered by the consumption of contaminated beef. The ability of prions to spread with such pathogenic intent whilst remaining incurable fuels the fire of public concern - yet delving beyond that classical view of prions has led to an enlightened appreciation of the role of such proteins throughout the biota of life. Prion-like proteins subvert the dogma of the one-to-one structure-function relationship traditionally held onto when considering the physiology of proteins. Instead, these proteins display structural polymorphism, capable of adopting different conformations under different conditions; this expands the capabilities of protein function and represents a mechanism for epigenetic coding, with protein conformation possessing the ability convey and propagate specific states for biological advantage without the need for DNA modification. The properties observed in many of these proteins, including those of multivalency and low complexity, predispose them towards phase separation, whereby localised concentration of biomolecules compartmentalise cellular activity. This ability confers upon an organism the ability to respond to stimuli in dynamic and transient fashions without needing to use cumbersome membrane-bound organelles. Using the Prion-Like Amino Acid Composition program, we have identified the stress-responsive protein ABU-13 as a potential prion in the nematode species, Caenorhabditis elegans. Phenotypic analysis of ABU-13 knockout animals has identified a non-redundant role for this protein in ER stress and innate immune responses – in keeping with previous evidence that this family of proteins is involved with a non-canonical unfolded protein response pathway of the endoplasmic reticulum. These knockout animals do not display defects in the activation of the stress-response gene hsp-4 upon tunicamycin treatment, suggesting that these effects occur independently of the canonical UPR pathway. In vivo and in vitro characterisation of ABU-13 has demonstrated a propensity for coalescence, reminiscent of a phase-separated organelle. This is further supported by the ability of this protein to bind RNA – a common feature of prion-like proteins involved with such transitions. Co-immunoprecipitation of ABU-13 confirmed an enrichment of ER and RNA-related protein interactions, supporting a role for this protein in ER stress responses. In addition to this, a number of glycosylation related terms were identified, pointing towards a role for ABU-13 in the folding quality control of such proteins. These punctated structures do, however, appear less mobile than traditional liquid-like condensates, as demonstrated by a slow FRAP recovery, indicative of a more hydrogel-like structure. To further investigate this, we extracted hydrogel-forming proteins from N2 animals using a biotinylated isoxazole precipitation, identifying a number of novel proteins that may be involved with in the formation of such solid-like structures. ABU-13, however, was not amongst this dataset, suggesting that if it were indeed involved in a physiological hydrogel, this transition is driven by another protein component. Overall, we propose that ABU-13 represents a phase-separating prion-like protein capable of modulating ER stress and immune responses, potentially via the regulation of RNA processing

Copper maturation of nitrous oxide reductase in Paracoccus denitrificans

Nitrous oxide (N2O) is an important greenhouse gas that is also responsible for stratospheric ozone depletion [1, 2]. Human activity is the main source of N2O due to the use of fertilisers in agriculture. Nitrous oxide reductase (N2OR) is the only enzyme to destroy N2O as part of a biological process termed denitrification. This enzyme has a unique catalytic CuZ centre, an electron transfer CuA centre and a high demand for Cu with 12 atoms required per functional dimer. A previous transcriptomic study revealed that two putative Cu chaperones, ScoB and PCuC were upregulated under Cu limiting conditions [3]. Here we demonstrate that ScoB/PCuC is a high-affinity Cu system essential for N2O respiration. Deletion of scoB causes N2O accumulation under anoxic and Cu-limited growth. N2O respiration could be restored complementation in trans with recombinant full-length, or soluble, periplasmic ScoB proteins (ScoBFL and ScoBsol, respectively). ScoBsol was biochemically characterised and found to be a monomeric protein of _25 kDa that can bind Cu1+ or Cu2+ with an apparent KD value within the subfemtomolar range. In contrast, PCuC is a multidomain protein with a Ycn-like N-terminal domain [4], and a PCuAC-like C-terminal domain [5]. Recombinant periplasmic proteins for each individual domain and full-length protein were generated (i.e., PCuCNt, PCuCCt and PCuCFL). The pcuC deletion strain has an N2O-genic phenotype. Only complementation in trans with PCuCFL restored N2O reduction under anaerobic and Cu-limited conditions. In addition, the crystallographic structure of Cu-bound PCuCNt was solved to a resolution of 1.5 Å revealing a trimeric protein of _56 kDa with a novel histidine brace metal binding site. PCuCNt can bind 1+ or 2+ and competition assays with 1+ chelators revealed that metallation occurs with femtomolar affinity. Analysis of YcnI-type proteins revealed the presence of two defined families. Family A contains a HX22HX101W consensus Cu-binding motif and was principally found among alphaproteobacteria, while Family B contain a HX22DX90WX13H motif and are distributed in actinobacteria and firmicutes. The Cu-bound structure of PCuCCt was also solved to a resolution of 1.6 Å and reveals a _18 kDa monomer that contains a defined H(M)X10MX21HXM Cu-binding site that can bind Cu1+ with subfemtomolar affinity. Further biochemical studies of native PCuC confirmed that the full-length protein forms a _100 kDa homotrimer in solution regardless of metallation state, with the N-terminal domain driving oligomerization exposing individual C-terminal domains to bulk solution through a flexible linker region. Each trimer can bind up to 6 Cu atoms with binding affinities within the subfemtomolar range. Finally, the maturation of the Cu centres of N2OR was studied in P. denitrificans WT, scoB and pcuC deletion strains. A periplasmic and readily isolatable affinity-tagged N2OR protein was expressed in cis under two different Cu regimes in P. denitrificans. N2OR purified from WT cells grown under anaerobic and Cu-limited conditions only contained a recognisable CuA centre. However, N2OR from scoB and pcuC mutants lacked both Cu-centres, had significantly lower Cu content and impaired enzymatic activity. A model for the metallation process of the CuA centre of N2OR by the high affinity Cu-maturation system ScoB/PCuC has been proposed

A histidine-rich Pseudomonas metallothionein with a disordered tail displays higher binding capacity for cadmium than zinc

Metallothioneins (MTs) are crucial players in metal-related physiological processes. They are characterized by a high cysteine content and unique metal binding properties resulting in specific metal–thiolate clusters formation. Here we present the first NMR solution structure of a Pseudomonas MT, PflQ2 MT, using the strain P. fluorescens Q2-87. It consists of a metal binding domain and an intrinsically disordered C-terminal tail, that was not observed in other MTs so far. While not influencing the structure or function of the metal binding domain, the tail contains a potential binding motif that might be important in so far undiscovered biological interactions. Unusual is the different metal binding capacity for three ZnIIversus four CdII ions that results in two novel metal-cluster topologies. Nevertheless, the affinity for the fourth CdII ion is reduced due to transient coordination. PflQ2 MT contains an unusually large number of four histidine residues, of which only one is involved in metal ion binding. The three non-coordinating histidine residues influence neither the protein fold nor the stability in vitro. We demonstrate that reinstatement of a second coordinating histidine residue, observed for cyanobacterial MTs, in place of a non-coordinating residue in Pseudomonas MTs, decreases the kinetic lability of the cluster, while preserving the overall metal ion binding stability and the protein fold. Since high thermodynamic stability combined with high kinetic lability of metal binding are mechanistic features critical for the function of MTs, the observed replacement might be advantageous for Pseudomonas MTs with respect to metal ion handling in vivo

Molecular mechanisms of Strawberry Plant Defence against colletotrichum acutatum

This thesis is focused on strawberry molecular studies aimed by the strong economic impact and social staple that represents this crop. With an annual production of 500000 tons and an economic weigh of 650 million €, Spain is the third producing country in world (FAOSTAT Agriculture Data [http://faostat.fao.org/]). Important losses in strawberry yields occur due to diseases and pests. Although resistant cultivars are a priority of most strawberry breeding programs, completely resistant cultivars have not yet been reported, relying pathogen control on the excessive use of chemical products (mostly environmental contaminants). Despite of the immediate necessity on developing new strategies to improve resistance in this crop plant, molecular knowledge is still scarce and most components and mechanisms of the strawberry defense network remain unknown and poorly understood. The main aim of this work is to get molecular clues about how plant immunity is activated in strawberry to face pathogen attack. Understanding the molecular interplay between strawberry plant and microbes will successful contribute to identify candidate genes useful for developing biotechnological strategies and help breeding to increase resistance against specific pathogens. This work has been structured in eight chapters which contributed to mount and exploit new technical platforms to subsequently uncover the strawberry defense response at molecular level. Chapter I is an intense and comprehensive compendium of all available information on strawberry immunity, and contribute to the field by discussing, updating and compiling research focused on the molecular aspects and events of the strawberry defense mechanisms against pathogens, concluding that major progress in the physiology, genetics and molecular biology of strawberry, is still needed to fully uncover the logic of its elaborate plant innate immune system. In this context, the use of high-throughput technologies will provide large amount of molecular information related with defense response in strawberry. However, the lack on commercially-available tools focused on crop plants such as strawberry, aimed us to produce our private platforms to be exploited in strawberry-based studies, as it is described in Chapter II. Thus, the generation of a ESTs collection enriched on defense-related genes, which allow the identification of new components of strawberry immunity, and provides information on those biological...Esta tesis está enfocada en estudios moleculares de la planta de fresa animada por el importante impacto económico y estabilizador social que representa este cultivo. Con una producción anual de 500000 toneladas y una relevancia económica de 650 millones de euros, España es el tercer país productor en el mundo (FAOSTAT Agriculture Data [http://faostat.fao.org/]). La producción de fresa sufre importantes pérdidas como consecuencia de las enfermedades y las plagas. Aunque la obtención de cultivares resistentes son una prioridad para la mayoría de los programas de mejora, aún no se han obtenido cultivares completamente resistentes, por lo que el control de los patógenos ha recaído en el uso excesivo de productos químicos (mayoritariamente contaminantes ambientales). Aún con la urgente necesidad de desarrollar nuevas estrategias para mejorar la resistencia en este cultivo, el conocimiento a nivel molecular de la mayoría de los componentes y mecanismos de la respuesta de defensa de la planta de fresa permanecen desconocidos y difícilmente entendibles. El objetivo principal de este trabajo consiste en la obtención de pistas a nivel molecular a cerca de cómo la inmunidad de la planta de fresa es activada para enfrentarse al ataque de los patógenos. Conocer la interacción molecular entre la planta de fresa y los microbios contribuirá eficazmente a la identificación de genes candidatos útiles en el desarrollo de estrategias biotecnológicas y ayudará en los programas de mejora para incrementar la resistencia contra patógenos específicos. Este trabajo se ha estructurado en ocho capítulos que contribuyen en la tarea de montar y explotar nuevas plataformas técnicas para descubrir secuencialmente, a un nivel molecular, los distintos pasos en la respuesta de defensa desplegados por la planta de fresa. El Capítulo I es un amplio e intenso compendio de toda la información disponible sobre la inmunidad de la fresa, y contribuye a este campo con la discusión, actualización y compilación de toda la investigación enfocada en los aspectos moleculares de los mecanismos de la defensa de esta planta. Este capítulo concluye que aún es necesario un importante esfuerzo en los estudios de la fisiología, la genética y la biología molecular en fresa, para llegar a discernir por completo la lógica de su muy elaborado sistema de inmunidad innata. En este contexto, el uso de tecnologías de gran escala proporcionará una gran cantidad de información molecular relacionada con la respuesta de defensa en..

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease