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Cytosolic Glutathione Reducing Potential is Important for Membrane Penetration of HPV16 at the Trans-Golgi NetworkLi, Shuaizhi January 2016 (has links)
High-risk human papillomaviruses (HPVs) cause 5% of all human cancers worldwide. The HPV capsid consists of 72 disulfide-linked pentamers of major capsid protein L1 and up to 72 molecules of minor capsid protein L2. The viral genome (vDNA) is 8KB circular dsDNA, condensed with histones and complexed with L2. HPV infection requires the virion particle to get access to basal layer keratinocytes, binding and entry of the cells, uncoating, and transport of the viral genomes to the host cell nucleus. During infection, L2 is important for transport of the viral genome from membrane bound vesicular compartments, through the cytosol and into the host cell nucleus. Previous work has identified a conserved disulfide bond between Cys22 and Cys28, which is necessary for HPV16 infection. We hypothesize that endosomal reduction of this disulfide might be important for L2 conformational changes that allow a hydrophobic transmembrane-like region in L2 to span across endosomal membranes, exposing sorting adaptor binding motifs within L2 to the cytosol. Prior research suggests that cytosolic glutathione (GSH) redox potential is important for reduction of disulfide-linked proteins within the lumen of endosomes. This is achieved by endosomal influx of cytosolic reduced cysteine, where it can reduce disulfide bonds in lumenal proteins. Cytosolic GSH regenerates the pool of reduced cysteine needed to maintain endosomal redox potential. Here we studied the relationship between cytosolic GSH and HPV16 infection. siRNA knockdown of critical enzymes of the GSH biosynthesis pathway or the endosomal cystine efflux pump cystinosin caused partial abrogation of HPV16 infection. Likewise, inhibition of the GSH biosynthesis pathway with L-buthionine sulfoximine (L-BSO) blocked HPV16 infection in multiple cell types, suggesting that cytosolic GSH redox may be important for HPV16 infection. Further studies have revealed that the decrease of HPV16 infection is not because of defects in binding, entry, L2 cleavage or capsid uncoating, but rather is due to inefficient cytosolic translocation of L2/viral genome from the trans-Golgi network (TGN). Contrary to our initial hypothesis, we show that L2 is able to span the endosomal membrane and direct TGN localization in the presence of BSO. Lack of cytosolic GSH causes L2/viral genome to become trapped in the TGN lumen. This suggests that there are redox-sensitive viral or cellular factors necessary for L2/viral genome translocation at the TGN. Future research will focus on the redox state of the Cys22-Cys28 disulfide bond during infection of normal and GSH-depleted cells.
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Functional characterization of two banana NPR1 genes for pathogen defense response in ArabidopsisYocgo, Rosita Endah 19 October 2011 (has links)
The Non-expressor of pathogenesis-related1 gene (NPR1) mediates the induction of pathogenesis-related (PR) gene products, vital for resistance in plants. In this study, the role of two previously isolated Cavendish banana NPR1-like genes (MNPR1A and MNPR1B) has been characterized in protection against Xanthomonas campestris, Hylaperonospora arabidopsidis, Botrytis cinerea and Pseudomonas syringae pathogens. The specific aim was to investigate if sequence differences in both genes are responsible for differential activity against pathogens because in a previous expression study, MNPR1A and not MNPR1B had been more responsive to the banana necrotrophic pathogen Fusarium oxysporum. By challenging Fusarium-tolerant GCTCV-218 and susceptible Grand Naine Cavendish banana plants (which had been used in a previous characterization study) with the hemi-biotrophic Xanthomonas pathogen (a very important economical pathogen of banana), the two MNPR1, PR-1 and PR-3 genes were found to be sequentially expressed. Expression of these genes was more pronounced in the tolerant GCTCV-218 banana cultivar than in the sensitive Grand Naine cultivar. Comparative sequence analysis further showed that these two banana NPR1-like coding sequences had dissimilarities even within conserved functional domains; they grouped closely with other defense-related NPR1-like sequences and harboured defense cis-regulatory elements. Transformation of the coding sequences of both genes under the control of the 35S CaMV promoter/terminator sequences into npr1-2 Arabidopsis mutant complimented the phenotype of this mutant following infection with distinct classes of pathogens (biotrophic Hyaloperonospora, necrotrophic Botrytis and hemi-biotrophic Pseudomonas pathogens). These Infected-MNPR1-expressing plants had higher PR-1 transcript amounts with more reduced pathogen growth compared to non-transgenic npr1-2 Arabidopsis mutant plants. However, the difference in the two banana coding sequences did not translate into a differential pattern of response against the three different classes of pathogens used in this study. Further detailed studies are suggested to investigate the role of the MNPR1 promoter-coding sequences in the differential response to pathogens using a bananapathogen system. This study also addressed the question of whether cystosolic glutathione (GSH) is necessary for NPR1 transcription during systemic acquired resistance. Using Arabidopsis mutants (clt1clt2clt3) defective in cytosolic GSH biosynthesis and following infection with either Pseudomonas or Botrytis, NPR1 and PR-1 transcription was much reduced rendering the mutants more sensitive to pathogens compared to infected-wild-type i>Arabidopsis plants. Results from this study therefore implicate cytosolic glutathione as an essential antioxidant for the establishment of an effective defense response cascade. / Thesis (PhD)--University of Pretoria, 2011. / Plant Science / unrestricted
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A mixed-charge cluster facilities glutathione transferase dimerisationWalters, John Clive 14 November 2006 (has links)
Student Number : 0213014A -
MSc dissertation -
School of Molecular and Cell Biology -
Faculty of Science / Cytosolic glutathione transferases (GSTs) are obligate stable homo- and heterodimers
comprising two GST subunits. Interactions across the subunit interface play an
important role in stabilising the subunit tertiary structure and maintain the dimeric
structure required for activity. The crystal structure of a rat Mu class GST consisting
of two type one subunits (rGST M1-1) reveals a lock-and-key motif and a mixedcharge
cluster at the subunit interface. Previous investigations revealed the lock-andkey
motif was not essential for dimerisation. It was therefore postulated that the
mixed-charge cluster at the dimer interface is primarily responsible for subunit
association. Statistical analyses of individual rGST M1-1 chains did not predict the
presence of any charge clusters. This suggests that the mixed-charge cluster forms
only upon dimerisation and reinforces the probability that quaternary structure
stabilisation is a major role of the mixed-charge cluster. Arginine 81 (Arg-81), a
structurally conserved residue in the GST family involved in the mixed-charge
cluster, was mutated to alanine. Phenylalanine 56 (Phe-56), the ‘key’ residue in the
lock-and-key motif, was mutated to serine. These changes were engineered to disrupt
the mixed-charge cluster and the lock-and-key motif situated at the dimer interface of
rGST M1-1. Sizing by gel filtration chromatography of the mutant GST identified
that these engineered amino acids resulted in a stable monomeric protein
(F56S/R81A rGST M1). The F56S/R81A rGST M1 displayed almost no catalytic
activity, suggesting perturbations of the active site or substrate binding sites.
Structural investigations of the monomer by far- and near-UV circular dichroism
revealed a similar secondary structural content to the wild-type. However, the
tryptophan fluorescence properties suggested the tryptophans were situated in more
hydrophilic environments than in the wild-type. ANS binding studies indicated a
large increase in the accessible hydrophobic surface area of the monomer. Ureainduced
equilibrium unfolding of F56S/R81A rGST M1 follows a cooperative twostate
unfolding model. The unfolding data indicates decreased conformational
stability and a large increase in the solvent exposed surface area of the monomer. In
conclusion, the mixed-charge cluster at the dimer interface of rGST M1-1 is essential for monomeric association, which subsequently contributes to catalytic activity of the
dimer and the stabilities of individual rGST M1-1 subunits.
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