1,721,011 research outputs found
Strukturelle und funktionelle Charakterisierung von Profilin aus Schistosoma japonicum
No full textSchistosomiasis, also known as bilharzia, is considered the second most socio-economically devastating disease after malaria in the (sub)tropical areas. Schistosomiasis is rather effectively treated by praziquantel. However, drug and repeated infections urge the scientific community to search for potent vaccine targets from the Schistosoma proteome. A key to drug and vaccine development is understanding how parasite-‐host recognition works at the molecular level. Neodermatan flatworms contain a unique cellular organ, the syncytial tegument, which plays an important role in host infection. Cytoskeletal proteins form a major fraction of Schistosoma tegumental proteins and are, thus, attractive drug targets. An important cytosolic regulator of actin dynamics in eukaryotes is profilin. In addition to actin, profilins bind to polyproline stretches and acidic phospholipids, which makes them important keys in linking signal transduction to the actin cytoskeleton. This work focused on the biochemical and structural characterization of profilin of Schistosoma japonicum (SjPfn). Profilins
control a complex network of molecular interactions and bind different ligands through poly-‐L-‐proline repeats. Here, the ability of SjPfn to bind octaproline repeats was shown by fluorescence spectroscopy. On the contrary, no binding could be observed for proline-‐rich peptides derived from S. japonicum formin. The crystal structure of SjPfn shows a highly conserved overall fold but also several crucial differences in the peptide binding site compared to canonical profilins. Profilins sequester monomeric actin. This main characteristic of the profilin family was confirmed for SjPfn using polymerization kinetics and cosedimentation assays. Increasing concentrations of SjPfn decrease the rate of actin polymerization by keeping actin in its monomeric, soluble form. The crystal structure of the SjPfn-‐actin complex showed that SjPfn binds actin to the canonical actin-‐binding face, but the binding site itself is remarkably unconserved. The structural and functional information obtained here, provide insight into the profilin-‐ ediated actin dynamics in S. japonicum and clues on the immunogenicity of SjPfn.Schistosomiasis, auch als Billharziose bekannt, ist nach Malaria die sozioökonomisch verheerendste Infektionskrankheit in den (sub)tropischen Regionen. Schistosomiasis kann mit Praziquantel behandelt werden, aber Medikamentenresistenz und wiederkehrende Infektionen erfordern Entwicklung von wirksamen Impfungen gegen die Krankheit. Ein Schlüssel zur Medikamenten-‐ und Impfstoffentwicklung ist das Verständnis, wie die Parasit-‐Wirterkennung auf molekularer Ebene funktioniert. Neodermatan Plattwürmer besitzen ein einzigartiges Zellorgan, das syncytial Tegument, das eine wichtige Rolle bei der Infektion spielt. Zytoskelettproteine formen eine große Fraktion von Schistosoma tegument und sind somit interessante Ziele für Medikamentenentwicklung. Ein wichtiger zytosolischer Regulator der Aktindynamik in Eukaryoten ist Profilin. Außer an Aktin, binden Profiline auch an Polyprolinketten und sauren Phospholipiden, was sie zu wichtigen Schlüsseln zwischen Signaltransduktion und dem Aktinzytoskelett macht. Diese Dissertation befasst sich mit der biochemischen und strukturellen Charakterisierung von Profilin des Schistosoma japonicum (SjPfn). Profiline kontrollieren ein komplexes Netzwerk von molekularen Interaktionen durch Bindung an Liganden mit Poly-‐L-‐Prolinketten. Hier wurde die Fähigkeit zu binden von SjPfn Octaprolinketten gezeigt. Im Gegensatz dazu, konnte keine Bindung von Peptiden von S. japonicum Formin nachgewiesen werden. Die Kristallstruktur von SjPfn zeigt eine hohe Konservierung von der Gesamtstruktur, aber auch wesentliche Unterschiede in der Peptidbindungsstelle im Vergleich zu kanonischen Profilinen. Profilin sequestriert monomeres Aktin. Diese Hauptcharakteristik der Profilinfamilie wurde für SjPfn bestätigt. SjPfn reduziert die Geschwindigkeit der Aktinpolymerisation dadurch, dass es Aktin in seiner monomeren, löslichen Form hält. Die Kristallstruktur des Actin-‐ jPfn-‐Komplex zeigt, dass das SjPfn-‐Aktin an die bekannte Bindungsstelle bindet, aber die Interaktionen bemerkenswert unkonserviert sind. Die strukturellen und funktionellen Informationen geben Einblick in die Profilinvermittelte Aktindynamik in S. japonicum und in Hinweisen auf die Immunogenität von SjPfn
Apicomplexan Aktin depolymerisierende Faktoren und Capping-Proteine in der Regulation der Aktin-Filament-Dynamik
No full textThe phylum Apicomplexa consists mainly of intracellular parasites. The parasite motility and invasion involves a complex and uncharacterized process called gliding motility, which is thought to be driven by the actin-myosin motor of the parasite. Though most of the actins are highly conserved, apicomplexan actins are divergent by 20% from conventional actins. Distinctively Plasmodium, the most harmful member of this phylum, expresses only a small sub-set of actin regulatory proteins. The current work focusses on the effect of actin depolymerizing factors (ADFs) and capping proteins (CP), the two most important regulators of actin filament dynamics.
Plasmodium expresses two actins and ADFs with stage specific expression profiles; actin2 and ADF2 are expressed in the sexual stages, while actin1 and ADF1 are expressed all through the life cycle. Conventional ADFs sever actin filaments, decrease the nucleotide exchange rate on G-actin and sequester monomers. Current results show that both Plasmodium ADFs bind G-actin with comparable affinities and accelerate nucleotide exchange, indicating they function analogous to conventional profilins. Analysis of SAXS data indicate that ADF2 acts as monomer sequestering protein, and ADF1 forms only a transient complex in vitro. Additionally, ADF1 binds to and severs filaments.
ADFs are regulated by phosphoinositols. In case of Plasmodium ADFs, current results confirm that ADF1 binds PIP2 specifically. Very weak or negligible binding between ADF2 and PIP2 was observed. Binding sites of PIP2 and actin on ADF1 are mutually exclusive and might also involve an additional step of recognition, mediated by a loop to helix transition in the loop preceding ADF1 α-helix1.
Conventional CPs form heterodimers of α and β subunits and bind to the fast growing end of filaments, thus inhibiting addition or of loss of monomers. Here, it is snown that the α subunit of Plasmodium CP, in contrast to conventional CPs, forms stable homodimers in vitro. The homodimers were found to inhibit actin polymer elongation and had no impact on actin nucleation, indicating independent function of the two subunits in certain stages of the parasite.Der Stamm der Apicomplexa besteht hauptsächlich aus intrazellulären Parasiten. Motilität und Invasion der Parasiten beinhalten einen komplexen und bislang unvollständig verstanden Prozess, der als “gleitende Motilität” bezeichnet wird und von dem man ausgeht, dass er durch den Aktin-Myosin Motor der Parasiten angetrieben wird. Obwohl die Sequenzen der meisten Aktine hochkonserviert sind, zeigen die Aktine der Apicomplexa eine Divergenz gegenüber den konventionellen Aktinen von 20%. Insbesondere Plasmodium, der gefährlichste Vertreter dieses Phylums, exprimiert lediglich einen kleinen Teil der aktinregulierenden Proteine. Die vorliegende Arbeit untersucht den Einfluss von actin depolymerizing factors “ADFs” und capping proteins “CP” auf die Dynamik der Aktinfilamente.
Plasmodium exprimiert zwei Aktine und ADFs mit stadienspezifischen Expressionsprofilen; Aktin2 und ADF2 werden in den geschlechtlichen Stadien exprimiert, während Aktin1 und ADF1 den gesamten Lebenszyklus hindurch exprimiert werden. Konventionelle ADFs zertrennen Aktinfilamente, verringern den Nukleotidaustausch am G-Aktin und sequestrieren Monomere. Die vorliegenden Ergebnisse zeigen, dass ADFs aus Plasmodium sowohl G-Aktine mit vergleichbarer Affinität binden als auch den Nukleotidaustausch beschleunigen, was auf eine Funktion analog der konventionellen Profiline hinweist. Die Analyse der SAXS-Daten zeigt, dass ADF2 als monomersequestrierendes Protein fungiert und dass ADF1 in vitro einen transienten Komplex bildet. Zusätzlich bindet ADF1 an Filamente und abbricht diese.
ADFs werden durch Phosphoinositole reguliert werden. Die vorliegenden Ergebnisse bestätigen, dass ADF1 aus Plasmodium spezifisch an PIP2 bindet. Für ADF2 konnte nur eine sehr schwache Bindung an PIP2 beobachtet werden. Die Bindung von PIP2 und Aktin an ADF1 schliesst sich gegenseitig aus und könnten einen zusätzlichen Erkennungsschritt benötigen, der durch einen Übergang der Sekundärstruktur von Loop zu Helix im Bereich der α-Helix1 vorangehenden Loops vermittelt wird.
Konventionelle CPs bilden ein Heterodimer, bestehend aus α- und β-Untereinheiten, und binden an das schnell wachsenden Ende der Filamente, wodurch Anlagerung oder Verlust von Monomeren inhibiert wird. Anhand der vorliegenden Arbeit konnte gezeigt werden, dass die Plasmodium CP α-Untereinheit, im Gegensatz zu herkömmlichen CPs, stabile Homodimere in vitro bilden. Diese Homodimere inhibieren die Elongation der Aktinpolymere und haben keinen Einfluss auf die Aktinnukleation, was auf eine unabhängige Funktion der beiden Untereinheiten in bestimmten Stadien des Parasiten hinweist
Crystallographic studies on the structure-function relationships in triosephosphate isomerase
Full text linkAbstractThe triosephosphate isomerase (TIM) barrel superfamily is a broad family of proteins, most of which are enzymes. At the amino-acid-sequence level, many of the members of this family share little, if any, homology. Yet, they adopt the same three-dimensional (βα)8 fold. The TIM barrel fold seems to be a good framework for many different kinds of enzymes, providing unique possibilities for both natural and human-designed evolution, as the catalytic center and the stabilizing features are separated to different ends of the barrel. Indeed, in the light of most recent studies, it seems likely that at least most of the different TIM barrel enzymes, catalyzing a huge variety of reactions, have evolved from a common ancestor.TIM can be considered a real text-book enzyme — its catalytic properties and stucture-function relationships have been studied for decades. Still, at present, we are quite far from understanding the structural features that make TIM and other enzymes such superior catalysts in both efficiency and precision. TIM is a dimeric enzyme that consists of two identical subunits of 250 residues. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in glycolysis. The basics of this reaction are well known, but there is ongoing discussion about the details of the proton transfer steps, and three alternative pathways have been suggested. In addition, it is a fascinating question how the enzyme succeeds in abstracting a highly stable proton from a carbon atom of the substrate.This study was undertaken to shed light on some of the questions concerning the structure-function relationships in TIM. The most important findings are the elucidation of the role of Asn11 as a catalytic residue and the meaning of the flexibility of both the catalytic Glu167 side chain as well as the substrate during catalysis, and the presence of a low-barrier hydrogen bond between Glu167 and a transition-state analogue, 2-phosphoglycolate. Furthermore, significant results were obtained on the importance of a conserved salt bridge, 20 Å away from the active site and the dimer interface, for the stability and folding of TIM as well as on the factors influencing the opening of the flexible loop 6 upon product release.Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Raahensali (Auditorium L10), Linnanmaa, on May 16th, 2003, at 12 noon.Abstract
The triosephosphate isomerase (TIM) barrel superfamily is a broad family of proteins, most of which are enzymes. At the amino-acid-sequence level, many of the members of this family share little, if any, homology. Yet, they adopt the same three-dimensional (βα)8 fold. The TIM barrel fold seems to be a good framework for many different kinds of enzymes, providing unique possibilities for both natural and human-designed evolution, as the catalytic center and the stabilizing features are separated to different ends of the barrel. Indeed, in the light of most recent studies, it seems likely that at least most of the different TIM barrel enzymes, catalyzing a huge variety of reactions, have evolved from a common ancestor.
TIM can be considered a real text-book enzyme — its catalytic properties and stucture-function relationships have been studied for decades. Still, at present, we are quite far from understanding the structural features that make TIM and other enzymes such superior catalysts in both efficiency and precision. TIM is a dimeric enzyme that consists of two identical subunits of 250 residues. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in glycolysis. The basics of this reaction are well known, but there is ongoing discussion about the details of the proton transfer steps, and three alternative pathways have been suggested. In addition, it is a fascinating question how the enzyme succeeds in abstracting a highly stable proton from a carbon atom of the substrate.
This study was undertaken to shed light on some of the questions concerning the structure-function relationships in TIM. The most important findings are the elucidation of the role of Asn11 as a catalytic residue and the meaning of the flexibility of both the catalytic Glu167 side chain as well as the substrate during catalysis, and the presence of a low-barrier hydrogen bond between Glu167 and a transition-state analogue, 2-phosphoglycolate. Furthermore, significant results were obtained on the importance of a conserved salt bridge, 20 Å away from the active site and the dimer interface, for the stability and folding of TIM as well as on the factors influencing the opening of the flexible loop 6 upon product release
Myeliinientsyymi 2′,3′-syklisten nukleotidien 3′-fosfodiesteraasin rakenne ja toiminta
No full textAbstractThe myelin sheath is a crucial component of vertebrate nervous systems. Myelin is formed as the plasma membrane of a glial cell is wrapped around a neuronal axon. The presence of myelin enables the fast transmission of neuronal impulses, and degradation or dysfunction of myelin results in severe neurological symptoms. Molecular composition of myelin is unique, and many myelin proteins are not present elsewhere in the body. A myelin enzyme, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), is found in specific regions within the myelin sheath and is one of the most abundant proteins in the brain. Substrates for CNPase catalytic activity are formed during brain damage. CNPase also interacts with the cytoskeleton and cell membranes, and it is thought to play a role during myelin formation. Mice that lack CNPase suffer from axonal degeneration and die early.The aim of this study was to characterise CNPase structure and function. To this end, a system was first developed to produce the protein for subsequent analyses. The aim was to characterise the catalytic mechanism of CNPase by determining its three-dimensional molecular structure at different stages of the catalytic reaction. The interactions between CNPase and other molecules related to its function would also be characterised. Finally, the structure of the full-length protein would be used to understand of the function of the uncharacterised N-terminal domain. Using X-ray crystallography, the structure of the CNPase catalytic domain was determined in the presence of substrate and product molecules. These data, complemented with analyses of mutationally inactivated enzyme variants, were used to examine the catalytic reaction at the molecular level. The catalytic domain structure was compared to homologous enzymes from diverse organisms. The interaction between CNPase and the calcium-sensing protein calmodulin was characterised. The solution structure of full-length CNPase was determined using small-angle X-ray scattering, and protein sequence databases were utilised to determine CNPase conservation during animal evolution. The results provide novel information on the catalytic activity and overall function of CNPase. Further studies will be necessary to determine its specific role, but it is increasingly clear that CNPase can perform multiple important tasks within the nervous system. TiivistelmäMyeliinituppi on tärkeä osa selkärankaisten hermostoa. Myeliiniä muodostuu, kun gliasolun solukalvo kiertyy hermosolun aksonin ympärille. Myeliini mahdollistaa hermoimpulssien nopean välityksen, ja sen tuhoutuminen ja vajaatoiminta aiheuttavat vakavia neurologisia oireita. Myeliinin molekyylikoostumus on ainutlaatuinen, ja monet myeliiniproteiineista eivät esiinny muualla elimistössä. Myeliinissä esiintyvää entsyymiä, 2′,3′-syklisten nukleotidien 3′-fosfodiesteraasia (CNPaasi), esiintyy runsaasti tietyillä myeliinialueilla, ja se on yksi aivojen runsaslukuisimmista proteiineista. Substraatteja CNPaasin katalyyttiselle aktiivisuudelle muodostuu aivovaurion aikana. CNPaasi on myös vuorovaikutuksessa solun tukirangan ja solukalvon kanssa, ja sen uskotaan vaikuttavan myeliinin muodostumiseen. Hiiret, joilta puuttuu CNPaasi, kärsivät aksonien rappeumista ja kuolevat ennenaikaisesti. Tämän tutkimuksen tavoite oli karakterisoida CNPaasin rakennetta ja toimintaa. Tätä tarkoitusta varten ensin kehitettiin menetelmä analysoitavan proteiinin tuottamiseksi. Tavoitteena oli karakterisoida CNPaasin katalyyttinen mekanismi määrittämällä sen kolmiulotteinen molekyylirakenne katalyysireaktion eri vaiheissa. Myös CNPaasin vuorovaikutuksia sen toimintaan liittyvien molekyylien kanssa tutkittiin. Lopuksi kokopitkän proteiinin rakenteen avulla selvitettiin karakterisoimattoman aminoterminaalisen alayksikön toimintaa. CNPaasin katalyyttisen alayksikön rakenne määritettiin käyttäen röntgenkristallografiaa substraatti- ja tuotemolekyylien läsnäollessa. Rakennetta, täydennettynä mutaatioilla inaktivoitujen entsyymimuunnosten analyysillä, käytettiin katalyyttisen reaktion molekyylitason karakterisointiin. Katalyyttisen alayksikön rakennetta verrattiin eri organismeissa esiintyviin homologisiin entsyymeihin. CNPaasin ja kalsiumia sitovan kalmoduliinin vuorovaikutusta karakterisoitiin. Kokopitkän CNPaasin liuosrakenne selvitettiin pienkulmaröntgensironnan avulla, ja CNPaasin sekvenssin säilymistä eläinten evoluution aikana tarkasteltiin proteiinisekvenssitietokantoja käyttämällä. Tulokset antavat uutta tietoa CNPaasin katalyyttisestä aktiivisuudesta ja tämän arvoituksellisen entsyymin toiminnasta. Jatkotutkimukset ovat tarpeen sen täsmällisen roolin selvittämiseksi, mutta on kasvavassa määrin selvää, että CNPaasi pystyy suorittamaan useita tärkeitä tehtäviä hermostossa. Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in OP-sali (Auditorium L10), Linnanmaa, on 6 June 2013, at 12 noonAbstract
The myelin sheath is a crucial component of vertebrate nervous systems. Myelin is formed as the plasma membrane of a glial cell is wrapped around a neuronal axon. The presence of myelin enables the fast transmission of neuronal impulses, and degradation or dysfunction of myelin results in severe neurological symptoms. Molecular composition of myelin is unique, and many myelin proteins are not present elsewhere in the body. A myelin enzyme, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), is found in specific regions within the myelin sheath and is one of the most abundant proteins in the brain. Substrates for CNPase catalytic activity are formed during brain damage. CNPase also interacts with the cytoskeleton and cell membranes, and it is thought to play a role during myelin formation. Mice that lack CNPase suffer from axonal degeneration and die early.
The aim of this study was to characterise CNPase structure and function. To this end, a system was first developed to produce the protein for subsequent analyses. The aim was to characterise the catalytic mechanism of CNPase by determining its three-dimensional molecular structure at different stages of the catalytic reaction. The interactions between CNPase and other molecules related to its function would also be characterised. Finally, the structure of the full-length protein would be used to understand of the function of the uncharacterised N-terminal domain.
Using X-ray crystallography, the structure of the CNPase catalytic domain was determined in the presence of substrate and product molecules. These data, complemented with analyses of mutationally inactivated enzyme variants, were used to examine the catalytic reaction at the molecular level. The catalytic domain structure was compared to homologous enzymes from diverse organisms. The interaction between CNPase and the calcium-sensing protein calmodulin was characterised. The solution structure of full-length CNPase was determined using small-angle X-ray scattering, and protein sequence databases were utilised to determine CNPase conservation during animal evolution.
The results provide novel information on the catalytic activity and overall function of CNPase. Further studies will be necessary to determine its specific role, but it is increasingly clear that CNPase can perform multiple important tasks within the nervous system.Tiivistelmä
Myeliinituppi on tärkeä osa selkärankaisten hermostoa. Myeliiniä muodostuu, kun gliasolun solukalvo kiertyy hermosolun aksonin ympärille. Myeliini mahdollistaa hermoimpulssien nopean välityksen, ja sen tuhoutuminen ja vajaatoiminta aiheuttavat vakavia neurologisia oireita. Myeliinin molekyylikoostumus on ainutlaatuinen, ja monet myeliiniproteiineista eivät esiinny muualla elimistössä. Myeliinissä esiintyvää entsyymiä, 2′,3′-syklisten nukleotidien 3′-fosfodiesteraasia (CNPaasi), esiintyy runsaasti tietyillä myeliinialueilla, ja se on yksi aivojen runsaslukuisimmista proteiineista. Substraatteja CNPaasin katalyyttiselle aktiivisuudelle muodostuu aivovaurion aikana. CNPaasi on myös vuorovaikutuksessa solun tukirangan ja solukalvon kanssa, ja sen uskotaan vaikuttavan myeliinin muodostumiseen. Hiiret, joilta puuttuu CNPaasi, kärsivät aksonien rappeumista ja kuolevat ennenaikaisesti.
Tämän tutkimuksen tavoite oli karakterisoida CNPaasin rakennetta ja toimintaa. Tätä tarkoitusta varten ensin kehitettiin menetelmä analysoitavan proteiinin tuottamiseksi. Tavoitteena oli karakterisoida CNPaasin katalyyttinen mekanismi määrittämällä sen kolmiulotteinen molekyylirakenne katalyysireaktion eri vaiheissa. Myös CNPaasin vuorovaikutuksia sen toimintaan liittyvien molekyylien kanssa tutkittiin. Lopuksi kokopitkän proteiinin rakenteen avulla selvitettiin karakterisoimattoman aminoterminaalisen alayksikön toimintaa.
CNPaasin katalyyttisen alayksikön rakenne määritettiin käyttäen röntgenkristallografiaa substraatti- ja tuotemolekyylien läsnäollessa. Rakennetta, täydennettynä mutaatioilla inaktivoitujen entsyymimuunnosten analyysillä, käytettiin katalyyttisen reaktion molekyylitason karakterisointiin. Katalyyttisen alayksikön rakennetta verrattiin eri organismeissa esiintyviin homologisiin entsyymeihin. CNPaasin ja kalsiumia sitovan kalmoduliinin vuorovaikutusta karakterisoitiin. Kokopitkän CNPaasin liuosrakenne selvitettiin pienkulmaröntgensironnan avulla, ja CNPaasin sekvenssin säilymistä eläinten evoluution aikana tarkasteltiin proteiinisekvenssitietokantoja käyttämällä.
Tulokset antavat uutta tietoa CNPaasin katalyyttisestä aktiivisuudesta ja tämän arvoituksellisen entsyymin toiminnasta. Jatkotutkimukset ovat tarpeen sen täsmällisen roolin selvittämiseksi, mutta on kasvavassa määrin selvää, että CNPaasi pystyy suorittamaan useita tärkeitä tehtäviä hermostossa
Recombinant Production, Purification and Crystallization of the Toxoplasma Gondii Coronin WD40 Domain
No full textToxoplasma gondii is one of the most widely spread parasitic organisms in the world. Together with other apicomplexan parasites, it utilizes a special actin-myosin motor for its cellular movement, called gliding motility. This actin-based process is regulated by a small set of actin-binding proteins, which in Apicomplexa comprises only 10-15 proteins, compared with >150 in higher eukaryotes. Coronin is a highly conserved regulator of the actin cytoskeleton, but its functions, especially in parasites, have remained enigmatic. Coronins consist of an N-terminal actin-binding -propeller WD40 domain, followed by a conserved region, and a C-terminal coiled-coil domain implicated in oligomerization. Here, the WD40 domain and the conserved region of coronin from . were produced recombinantly and crystallized. A single-wavelength diffraction data set was collected to a resolution of 1.65 Å. The crystal belonged to the orthorhombic space group , with unit-cell parameters = 55.13, = 82.51, = 156.98 Å
Functional insights into Plasmodium actin depolymerizing factor interactions with phosphoinositides
Full text linkMalaria is caused by protozoan parasites, Plasmodium spp., that belong to the phylum Apicomplexa. The life cycle of these parasites depends on two different hosts; the definitive host, or vector, is a mosquito, and the intermediate host is a vertebrate, such as human. Malaria parasites use a unique form of substrate-dependent motility for host cell invasion and egress, which is dependent on an actomyosin motor complex called the glideosome. Apicomplexa have a small set of actin regulators, which are poorly conserved compared to their equivalents in higher eukaryotes. Actin-depolymerizing factors (ADFs) are key regulators responsible for accelerating actin turnover in eukaryotic cells. The activity of ADFs is regulated by membrane phosphoinositides. Malaria parasites express two ADF isoforms at different life stages. ADF1 differs substantially from canonical ADF/cofilins and from Plasmodium ADF2 in terms of both structure and function. Here, we studied the interaction of both Plasmodium ADFs with phosphoinositides using biochemical and biophysical methods and mapped their binding sites on ADF1. Both Plasmodium ADFs bind to different phosphoinositides, and binding in vitro requires the formation of vesicles or micelles. Interaction with phosphoinositides increases the α-helical content of the parasite ADFs, and the affinities are in the micromolar range. The binding site for phosphatidylinositol 4,5-bisphosphate in PfADF1 involves a small, positively charged surface patch.Malaria is caused by protozoan parasites, Plasmodium spp., that belong to the phylum Apicomplexa. The life cycle of these parasites depends on two different hosts; the definitive host, or vector, is a mosquito, and the intermediate host is a vertebrate, such as human. Malaria parasites use a unique form of substrate-dependent motility for host cell invasion and egress, which is dependent on an actomyosin motor complex called the glideosome. Apicomplexa have a small set of actin regulators, which are poorly conserved compared to their equivalents in higher eukaryotes. Actin-depolymerizing factors (ADFs) are key regulators responsible for accelerating actin turnover in eukaryotic cells. The activity of ADFs is regulated by membrane phosphoinositides. Malaria parasites express two ADF isoforms at different life stages. ADF1 differs substantially from canonical ADF/cofilins and from Plasmodium ADF2 in terms of both structure and function. Here, we studied the interaction of both Plasmodium ADFs with phosphoinositides using biochemical and biophysical methods and mapped their binding sites on ADF1. Both Plasmodium ADFs bind to different phosphoinositides, and binding in vitro requires the formation of vesicles or micelles. Interaction with phosphoinositides increases the α-helical content of the parasite ADFs, and the affinities are in the micromolar range. The binding site for phosphatidylinositol 4,5-bisphosphate in PfADF1 involves a small, positively charged surface patch
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Variations on the Author
Full text link“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
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