1,720,987 research outputs found
Biochemical analysis of collagen binding by invertebrate SPARC and engineered monomeric decorin
Get PDFCollagens are a family of extracellular matrix proteins that are critically important for providing structural support to tissues and for regulating cell behaviour. The thesis describes the biochemical analysis of collagen binding by two globular proteins that profoundly influence collagen fibril formation in vivo, decorin and SPARC.
Decorin is the archetypal small leucine-rich repeat proteoglycan and an important regulator of collagen fibrillogenesis. The crystal structure of decorin, published in 2004, revealed a dimeric structure in which the presumed collagen binding site was not accessible. Whether the dimer is functional has been controversial, however, and it has been claimed that biologically active decorin is monomeric. In order to resolve this controversy, I designed a number of decorin mutants to disrupt the crystallographic dimer, including two mutants which introduced glycosylation sites into the dimer interface. Size exclusion chromatography with multi-angle laser light scattering and analytical ultracentrifugation were used to determine the oligomeric states of decorin and the designed mutants. I found that wild-type decorin dimerises in solution with a dissociation constant of ~1 μM. The mutants with engineered glycosylation sites were pure monomers while other mutants remained dimeric. Thermal unfolding experiments showed that the engineered decorin monomers were as stable as wild-type decorin. Mutations on the concave face of decorin abolished collagen binding, regardless of whether the mutant proteins retained the ability to dimerise or not. Thus the concave face of decorin is involved in collagen binding and the dimer must dissociate in order to bind collagen.
The crystal structure of human SPARC bound to a collagen-like triple-helical peptide was determined in the Hohenester lab in 2008. The key collagen binding residues are conserved between human and invertebrate (Drosophila and C. elegans) SPARCs. There is a key difference between the orthologues, however: high-affinity collagen binding to human SPARC requires proteolytic cleavage of an inhibitory loop that is absent from invertebrate SPARCs. To investigate the functional consequences of this structural difference for collagen binding, I compared the interactions of the different SPARCs with collagen I and IV, using solid-phase assays and surface plasmon resonance. I found that invertebrate SPARCs did not bind collagen more tightly than human SPARC, suggesting that the absence of the inhibitory loop does not confer a higher affinity for collagen in invertebrate SPARCs. I made several unsuccessful attempts to crystallise an invertebrate SPARC, including experiments in which the glycan was trimmed by endoglycosidase digestion or removed by mutagenesis of acceptor sites.Open Acces
An investigation into the role of TbZC3H20: a zinc finger protein that acts as a post-transcriptional regulator of gene expression in Trypanosoma brucei
No full textTrypanosomes rely on post-transcriptional mechanisms to regulate gene
expression, thus RNA-binding proteins are of particular significance in these
parasites. This project focuses on a CCCH zinc finger protein TbZC3H20, which
is hypothesised to regulate gene expression via mRNA stability, and
consequently control normal cell functions. Using reverse genetics such as RNAi
down-regulation and ectopic over-expression, the role of TbZC3H20 in insect
procyclic form and mammalian bloodstream form Trypanosoma brucei has been
investigated. Phenotypes resulting from perturbing the expression of TbZC3H20
in both life forms were examined and characterised, and these provided clues
about the mRNA species that might be regulated by this protein. In combination
with microarray analysis, specific mRNA targets were selected for further
investigation: to verify that TbZC3H20 regulates transcripts via mRNA stability
and turnover, to demonstrate their direct interaction with TbZC3H20, and to show
that the 3’ UTR is responsible for regulation by TbZC3H20. This analysis also
addressed the possibility of creating TbZC3H20 null mutants in bloodstream
forms and procyclic forms, which indicated the essential nature of TbZC3H20
itself in either life form, as well as providing a useful tool in characterising its role
within the cell. In conclusion, this project reveals a novel RNA-binding protein
that is able to regulate mRNAs involved in the highly coordinated process of
differentiation from one life form to another, as well as regulating life cycle stage
enriched transcripts essential for trypanosome cell survival
Laminin G-like domains: dystroglycan-specific lectins
Get PDFA unique O-mannose-linked glycan on the transmembrane protein dystroglycan binds a number of extracellular matrix proteins containing laminin G-like (LG) domains. The dystroglycan-matrix interaction is essential for muscle function: disrupted biosynthesis of the matrix-binding modification causes several forms of muscular dystrophy. The complete chemical structure of this modification has been deciphered in the past few years. We now know that LG domains bind to a glycosaminoglycan-like polysaccharide of [-3GlcAβ1,3Xylα1-] units, termed matriglycan, that is attached to a highly unusual heptasaccharide linker. X-ray crystallography has revealed the principles of Ca2+-dependent matriglycan binding by LG domains. In this review, the new structural insights are applied to the growing number of LG domain-containing proteins that bind dystroglycan. It is proposed that LG domains be recognised as ‘D-type’ lectins to indicate their conserved function in dystroglycan binding
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Structural biology of laminins
Get PDFLaminins are large cell-adhesive glycoproteins that are required for the formation and function of basement membranes in all animals. Structural studies by electron microscopy in the early 1980s revealed a cross-shaped molecule, which subsequently was shown to consist of three distinct polypeptide chains. Crystallographic studies since the mid-1990s have added atomic detail to all parts of the laminin heterotrimer. The three short arms of the cross are made up of continuous arrays of disulphide-rich domains. The globular domains at the tips of the short arms mediate laminin polymerization; the surface regions involved in this process have been identified by structure-based mutagenesis. The long arm of the cross is an α-helical coiled coil of all three chains, terminating in a cell-adhesive globular region. The molecular basis of cell adhesion to laminins has been revealed by recent structures of heterotrimeric integrin-binding fragments and of a laminin fragment bound to the carbohydrate modification of dystroglycan. The structural characterization of the laminin molecule is essentially complete, but we still have to find ways of imaging native laminin polymers at molecular resolution
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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