1,721,076 research outputs found
The characterisation of trypanosomal type 1 DnaJ-like proteins
Get PDFTrypanosomes are protozoans, of which many are parasitic, and possess complex lifecycles
which alternate between mammalian and arthropod hosts. As is the case with most
organisms, molecular chaperones and heat shock proteins are encoded within the
genomes of these protozoans. These proteins are an integral part of maintaining the
structural integrity of proteins during normal and stress conditions. Heat shock protein 40
(Hsp40) is a co-chaperone of heat shock protein 70 (Hsp70) and in some cases can act as
a chaperone. These proteins work together to bind non-native polypeptide structures to
prevent unfolded protein aggregrate formation in times of stress, translocate proteins
across organelle membranes, and transport unsalvageable proteins to proteolytic
degradation by the cellular proteasome. Hsp40s are divided into four types based on their
domain structure.
Analysis of the nuclear genomes of eight trypanosomatid species revealed that less than
10 of the approximate 70 Hsp40 sequences per genome were Type 1 Hsp40s, many of
which contained putative orthologues in the other seven trypanosomatid genomes. One of
these Type 1 Hsp40s from T b. brucei, Trypanosoma brucei DnaJ 2 (Tbj2), was
functionally characterised in T brucei brucei. RNA interference knockdown of
expression in T brucei brucei showed that cells deficient in Tbj2 displayed a severe
inhibition of the growth of the cell population. The levels of the Tbj2 protein population
in T brucei brucei cells increases after exposure to 42°c and the protein was found to
have a generalized cytoplasmic subcellular localization at 37°c. These findings provide
evidence that Tbj2 is an orthologue of Yeast DnaJ 1 (Y dj l), an essential S. cerevisiae protein.
Hsp40s interact with their partner Hsp70s through their J-domain. The amino acids of the
J-domain important for a functional interaction with Hsp70 were examined in
Trypanosoma cruzi DnaJ 2 (Tcj2) (the orthologue of Tbj2) and T cruzi DnaJ protein 3
(Tcj3) by testing their ability to substitute for Y dj l in Saccharomyces cerevisae and for
DnaJ in Escherichia coli. In both systems, the positively charged amino acids of Helix II and III of the J-domain disrupted the functional interaction of these Hsp40s with their
partner Hsp70s. Substitutions in Helix I and IV of the J-domains of Tcj2 and Tcj3
produced varied results in the two different systems, possibly suggesting that these
helices serve to define with which Hsp70s a given Hsp40 can interact.
The inability of an Hsp40 and an Hsp70 to interact functionally does not necessarily
mean a total absence of physical interaction between these proteins. The amino acid
substitution of the histidine in the HPD motif (H34Q) of the J-domain of Tcj2 and Tcj3
removed the ability of these proteins to interact functionally with S. cerevisiae Hsp70
(Ssal) in vivo. However, preliminary binding studies using the quartz crystal
microbalance with dissipation monitoring (QCM-D) show that Tcj2 and Tcj2(H34Q)
both physically interact with M sativa Hsp70 in vitro.
This study is the first report to provide evidence that certain trypanosoma! Type 1 Hsp40s
are essential proteins. Futhermore, the interaction of these Hsp40s with Hsp70 identified
important features of the functional interface of this chaperone machinery
The screening and characterisation of compounds for modulators of heat shock protein (Hsp90) in a breast cancer cell model
No full textBreast cancer is a leading cause of cancer death in Africa. Hsp90 has been identified as a target for anti-cancer treatments as its inhibition results in the disruption and ubiquitin–proteasome degradation of activated oncoproteins. Currently, there are no US Food and Drug Administration approved Hsp90 inhibitor drugs and existing Hsp90 inhibitors such as geldanamycin and novobiocin are hepatotoxic and display a low affinity for Hsp90, respectively. Therefore, there is a need for the development of Hsp90 inhibitors with improved inhibitory properties. In this study twelve natural compounds bearing a quinone nucleus were screened and characterised for the modulation of Hsp90. The compounds analysed formed three series; the sargaquinoic acid (SQA), naphthoquinone, and pyrroloiminoquinone alkaloid series. Certain compounds exhibited half maximal inhibitory concentrations of between 3.32 μM and 12.4 μM, while others showed no antiproliferative activity
at concentrations of up to 500 μM in the MDA-MB-231 breast adenocarcinoma cell line. Immunofluorescence and Western analyses indicated that the modulation of Hsp90 and partner proteins by SQA was more similar to that of novobiocin. Isothermal titration calorimetry analyses suggested that SQA interacted with Hsp90β with a low affinity, and saturation-transfer difference nuclear magnetic resonance confirmed that this interaction with Hsp90β occurred through the methyl moiety bound to 1, 4 benzoquinone of SQA. Pulldown assays indicated SQA disrupted the
association between Hsp90 and Hop dose-dependently, more similarly to novobiocin. Immunofluorescence and Western analyses performed on naphthoquinone and
pyrroloiminoquinone alkaloid compounds indicated modulation of Hsp90 and Hsp90 partner proteins by the compounds. Naphthoquinone compounds were prioritised for analysis for binding to Hsp90β over the pyrroloiminoquinone alkaloid compounds. Lapachol interacted with Hsp90β with a low affinity however; this interaction was thought to be too weak to disrupt the association of Hsp90 and Hop. The remaining naphthoquinone compounds showed no interaction with Hsp90β, thus allowing the determination of a preliminary structure-activity relationship for these compounds. To the best of our knowledge, this is the first study to describe a systematic subcellular analysis of the effects of geldanamycin and novobiocin in comparison to sargaquinoic acid and compounds of the naphthoquinone and pyrroloquinoline scaffold on Hsp90 and its partner proteins
Characterization of the Hsp40 partner proteins of Plasmodium falciparum Hsp70
Get PDFHuman malaria is an economically important disease caused by single-celled parasites of the
Plasmodium genus whose biology displays great evolutionary adaptation to both its
mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein
(Hsp70) and J protein chaperone complements in malaria parasites affecting humans,
primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and
structurally related proteins that play a crucial role in maintaining the structural integrity of
proteins during normal and stress conditions. They are considered future therapeutic targets
in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically
partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins
for degradation. However, in P. falciparum, these classes of proteins have also been
implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol
following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in
tremendous modifications of the infected erythrocyte, imparting properties that allow it to
adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum
encodes six Hsp70 homologues and a large number of J proteins that localize to the various
intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding
the Hsp70-J protein interactions and/or partnerships is an essential step for drug target
validation and illumination of parasite biology. A review of these chaperone complements
across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J
protein complement compared to the rodent and primate infecting species. It further
highlights how unique the P. falciparum chaperone complement is compared to the other
Plasmodium species included in the analysis. In silico analysis showed that the genome of P.
falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has
been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL
containing J proteins are unique with no homologues in the human system and are considered
as attractive drug targets.
Very few of the predicted J proteins in P. falciparum have been experimentally characterized.
To this end, cell biological and biochemical approaches were employed to characterize
PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in
literature regarding its localization formed the basis for the experimental work. This is the
first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70
partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are
described. It is also the first study that details the successful expression and purification of
PfHsp70-3. Further, research findings have described for the first time the expression and
localization of PFB0595w in the intraerythrocytic stages of P. falciparum development.
Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone –
cochaperone partnership was proposed that formed the basis for the in vitro experiments.
PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1
pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has
revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need
for further related experiments to support the findings. Gel filtration analysis showed that
PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these
observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1
in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion.
Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships
in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding
of parasite biology
Functions of the Hsp90-binding FKBP Immunophilins.
No full textHsp90 functionally interacts with a broad array of client proteins, but in every case examined Hsp90 is accompanied by one or more co-chaperones. One class of co-chaperone contains a tetratricopeptide repeat domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is now clear that the client protein influences, and is influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members
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
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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