Functionally specified protein signatures distinctive for each of the different blue copper proteins

BACKGROUND: Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. RESULTS: Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific for each kind of blue copper protein and the false picks were minimized. The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section. CONCLUSIONS: These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences

Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea

Background Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. Results We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. Conclusion The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic differences, coupled with unknown differences in gene expression, distinguish these organisms enough to allow for co-existence. Overall this study reveals shared features of organisms from the Thermoplasmatales lineage and provides new insights into the functioning of AMD communities.United States. Dept. of Energy. Genomics:GTL (Grant DE-FG02-05ER64134)National Science Foundation (U.S.). Graduate Research Fellowshi

The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon \u3ci\u3eMetallosphaera sedula\u3c/i\u3e Provides Insights into Bioleaching-Associated Metabolism

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, _2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized

functional analyses of variants of human SCO1, a mitochondrial metallochaperone

Cytochrome c oxidase (COX) is a multimeric protein complex whose enzymatic activity contributes to the generation of an electrochemical potential required to synthesize adenosine triphosphate (ATP). Synthesis of Cytochrome c Oxidase 1 (SCO1) and SCO2 are two of the many accessory factors that are required to assemble individual structural subunits of COX into a functional holoenzyme complex. Mutations in either SCO gene cause severe, early onset forms of human disease. SCO1 and SCO2 are closely related paralogues localized to the inner mitochondrial membrane. Both proteins bind copper and exhibit a thiol disulphide oxidoreductase activity. Copper is bound by a highly conserved Cysteine x x x Cysteine motif and a histidine found within a thioredoxin fold, which is contained in the C-terminal half of the protein and projects into the mitochondrial intermembrane space. Mutations in either SCO1 or SCO2 affect their ability to deliver copper to COX II and metallate its CuA site, and also result in an increased rate of copper efflux from the cell. However, the relative importance of the ability to bind and transfer copper to SCO protein function remains poorly understood. Therefore, to investigate the significance of several cysteine residues and the conserved histidine to the copper-binding properties of SCO1, I functionally characterized a series of N- and C-terminal SCO1 mutant proteins by transducing them into control and patient fibroblasts, and quantifying their phenotypic effect on COX activity. I found that the two cysteines within the soluble, N-terminal matrix domain of SCO1 are not required for protein function. Overexpression of C-terminal SCO1 mutants only affected COX activity in SCO1-2 patient fibroblasts. To further characterize the copper-binding properties of these C-terminal mutants, soluble forms of each SCO1 variant were expressed and purified from bacteria, and the amount of total bound copper and the relative abundance of Cu(I) and Cu(II) were quantified. Although these analyses suggested that one mutant, SCO1 C169H, binds significantly more Cu(I) than the wild-type protein, none of the SCO1 variants exhibited properties that furthered our understanding of the precise role of SCO1 in the biogenesis of the CuA site of COX II

The role of SPARK-I receptor-like kinases in the arbuscular mycorrhizal symbiosis

The nutritional mutualism formed between land plants and arbuscular mycorrhizal (AM) fungi involves intracellular accommodation of the fungal symbiont and relies on concerted cellular reprograming. Maximal physical interaction is achieved during the emergence and development of the arbuscules, creating a hub for nutrient exchange and potentially for plant-fungal communication. Receptor-like kinases (RLKs) allow plant cells to sense and respond to their environment and are crucial for signalling during biotic interactions. A rice RLK, ARBUSCULAR RECEPTOR-LIKE KINASE 1 (OsARK1), had earlier been found to localize specifically to the symbiotic interface of arbusculated cells and its orthologues are present only among AM-competent plant species. In this dissertation, the symbiotic role of OsARK1 was studied using genetics, phylogenetics and cell biology approaches. Mutation in OsARK1 resulted in normal arbuscule development but reduced colonization levels that could be rescued in the presence of a wild-type AM fungal mycelium indicating OsARK1 regulates AM symbiosis after arbuscule formation by maintaining fungal fitness. Phylogenetic analyses of the OsARK1 RLK subfamily showed ARK and SIMILAR PROTEIN TO ARK 1 (SPARK1) as the two members present in non-vascular plants while the paralogues ARK1 and ARK2 arose early in the evolution of tracheophytes. The pattern of occurrence of the newly discovered SPARK domain in the extracellular region of members of this RLK subfamily, named SPARK-I, argues for ARK1 and ARK2 to have coevolved. A rice ark2 mutant displayed a reduced colonization phenotype which was quantitatively different compared to ark1. Global transcriptional signatures of the mutants showed several putative components of common as well as distinctive signalling pathways being regulated by OsARK1 and OsARK2. A global downregulation of hydrolytic enzymes in the mutant backgrounds and alterations in lipid distribution in arbusculated cells suggest these RLKs to be part of an ancient signalling pathway directing post-arbuscule development processes to sustain AM symbiosis

Fluorescence Multiplexing with Combination Probes for Biological and Diagnostic Applications

Cancer refers to a group of diseases containing more than 200 different subtypes. Cancer is a heterogeneous disease by nature, meaning that there are differences among tumors of the same type in different patients, and there are differences among cancer cells within a single tumor of one patient. Since cancer is not a single disease, nor does it have a single cause, it proves to be incredibly hard to diagnose and treat. The ability to study cellular markers, cell and tissue spatial arrangement, and gene function are all integral parts of cancer diagnostic and treatment efforts. Here, I first present a review of current techniques for quantitative tissue imaging at cellular resolution. I broadly divide current imaging techniques into three categories: fluorescence-based, mass spectrometry-based, and sequencing-based. In this work, I primarily concentrate on fluorescence-based methods, with the focus being on our recently developed theory Multiplexing using Spectral Imaging and Combinatorics (MuSIC). The basis for MuSIC is to create combinations of fluorescent molecules (whether it be small molecule fluorophores or fluorescent proteins) to create unique spectral signatures. I then present a protocol for labeling antibodies with combinations of small molecule fluorophores, which I refer to as MuSIC probes. I use fluorescent oligonucleotides (oligos) to arrange the fluorophores at specified distances and orientations from one another in order to produce complex fluorescence spectra when the probe is excited. This labeling protocol is demonstrated using a 3-probe experimental setup, bound to Protein A beads, and analyzed via spectral flow cytometry. When translating this method to staining human cells, our staining intensity was not comparable to that of a conventional antibody labeling kit. Therefore, next I present an improved method to label antibodies with MuSIC probes with increased signal intensity. I re-arrange the oligo-fluorophore arrangement of the MuSIC probe to emit an increased fluorescent signal. Then I validate this approach by comparing the staining intensity of MuSIC probe-labeled antibodies to a conventional antibody labeling kit using human peripheral blood mononuclear cells. Lastly, I present simulation theories for the multiplexing capabilities of MuSIC probes for various biological and diagnostic applications. First, I present a theory for high-throughput genetic interaction screening using MuSIC probes generated from 18 currently available fluorescent proteins. Simulation studies based on constraints of current spectral flow cytometry equipment suggest our ability to perform genetic interaction screens at the human genome-scale. Finally, I adapt this simulation protocol to generate MuSIC probes from 30 currently available small-molecule fluorophores. Using the same constraints as before, I predict that I can perform cell-type profiling of 200+ analytes. I hope that the work presented here provides a foundation for the use of combination probes for various biological and disease applications and ultimately help to better diagnose and treat different types of cancer

The compositional and evolutionary logic of metabolism

Metabolism displays striking and robust regularities in the forms of modularity and hierarchy, whose composition may be compactly described. This renders metabolic architecture comprehensible as a system, and suggests the order in which layers of that system emerged. Metabolism also serves as the foundation in other hierarchies, at least up to cellular integration including bioenergetics and molecular replication, and trophic ecology. The recapitulation of patterns first seen in metabolism, in these higher levels, suggests metabolism as a source of causation or constraint on many forms of organization in the biosphere. We identify as modules widely reused subsets of chemicals, reactions, or functions, each with a conserved internal structure. At the small molecule substrate level, module boundaries are generally associated with the most complex reaction mechanisms and the most conserved enzymes. Cofactors form a structurally and functionally distinctive control layer over the small-molecule substrate. Complex cofactors are often used at module boundaries of the substrate level, while simpler ones participate in widely used reactions. Cofactor functions thus act as "keys" that incorporate classes of organic reactions within biochemistry. The same modules that organize the compositional diversity of metabolism are argued to have governed long-term evolution. Early evolution of core metabolism, especially carbon-fixation, appears to have required few innovations among a small number of conserved modules, to produce adaptations to simple biogeochemical changes of environment. We demonstrate these features of metabolism at several levels of hierarchy, beginning with the small-molecule substrate and network architecture, continuing with cofactors and key conserved reactions, and culminating in the aggregation of multiple diverse physical and biochemical processes in cells.Comment: 56 pages, 28 figure

An electron microscopic study of developing facial and hypoglossal motor nuclei in the Brazilian opossum

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IDENTIFICATION AND EXPLORATION OF NOVEL MOLECULAR SIGNATURES IN BIOLOGICAL SYSTEMS THROUGH GENOMICS AND BIOINFORMATICS

The last two decades have witnessed rapid developments in –omics technologies which enable the study of biological and disease processes in a high throughput manner. Among the -omics approaches, genomics and the related bioinformatic methods have emerged as most popular applications able to accelerate science discoveries in basic research and drug discovery and therapeutics. Genomics is an interdisciplinary field of science focusing on the structure, function, evolution, mapping, and editing of genomes (Wikipedia, url: https://en.wikipedia.org/wiki/Genomics). Over the years, the field of genomics has undergone several revolutions. Prior to the advent of Next Generation Sequencing (NGS), genomics was limited to the characterization of single disease-associated genes (e.g. Huntington disease, cystic fibrosis, cancer) or to the study of small genomes (e.g. bacteria, viruses). As physical mapping with large-insert clones became possible, the subcloned fragments of large genomes could be sequenced as individual projects, and their finished sequences combined together to reconstruct the sequence of entire chromosomes. Using this approach and beginning from 1985, in 2003 the Human Genome Project was able to complete the sequence of the DNA in the human genome (I. H. G. S. Consortium et al., 2001; Venter et al., 2001), thus providing a basic platform for the development of new technologies. In the same period, other large genomes, including those of model organisms, were also decoded (M. G. S. Consortium et al., 2002; R. G. S. P. Consortium et al., 2004; Myers et al., 2000). Hybridization-based methods such as microarrays exploited the information gained from genome projects to develop rapid, high throughput assays to allow the measurement of genetic variation, gene expression and chromatin binding, which spread rapidly in all fields of research. Most recently, these methods were quickly replaced by NGS, which allows similar studies to be conducted with much higher sensitivity and in an unbiased whole-genome and –transcriptome fashion. As a result, sequencing has become an essential and obligatory tool and not only for biologists. In the early days of NGS, the initial focus of every genomic scientist was on the de-novo assembly of novel genomes for species that were never sequenced before. These efforts led to the completion of many novel genomic sequences which include even large genomes of mammals and plants. In the case of de-novo assembly, the genomic sequence is built from scratch without the use of an existing scaffold. Advances in sequencing technology have recently led to a dramatic increase in speed and throughput capacity, and a sharp reduction in costs. These improvements enabled the shift from de-novo to re-sequencing of entire genomes from additional individuals of species already sequenced. In the case of re-sequencing, short reads can be aligned to reference genomes as a substrate for variation discovery or gene expression analysis. Re-sequencing applications provide the scientific community with an unprecedented opportunity to address fundamental evolutionary questions, as well as to extend the use of sequencing to population genetic studies to infer ancient population history. The availability of new data types given by an always increasing number of NGS applications continues to engage and excite the computational biology community working on software development and on the analysis of new data types generated to solve complex biomedical problems. In this context, the main objective of my research was to explore different biological systems to identify new molecular signals through the development and implementation of genomic and bioinformatic methods. This objective was accomplished by participating to three different research projects where I applied genomic and bioinformatic solutions to different areas of biology: genome composition, organization and regulation, malaria biology, and cancer. The first chapter provides an introduction to the main technology and biology concepts explored in my research, while the following three chapters describe in details the research work conducted during my studies