HIV-1 Nef disrupts antigen presentation early in the secretory pathway

Human immunodeficiency virus, type 1 Nef disrupts viral antigen presentation and promotes viral immune evasion from cytotoxic T lymphocytes. There is evidence that Nef acts early in the secretory pathway to redirect major histocompatibility complex class I (MHC-I) from the trans-Golgi network to the endolysosomal pathway. However, a competing model suggests that Nef acts much later by accelerating MHC-I turnover at the cell surface. Here we demonstrate that Nef targets early forms of MHC-I molecules in the endoplasmic reticulum by preferentially binding hypophosphorylated cytoplasmic tails. The Nef-MHC-I complex migrates normally into the Golgi apparatus but subsequently fails to arrive at the cell surface and become phosphorylated. Cell type-specific differences in the rate of MHC-I transport through the secretory pathway correlate with responsiveness to Nef and co-precipitation of adaptor protein 1 with the Nef.MHC-I complex. We propose that the assembly of a Nef.MHC-I.adaptor protein 1 complex early in the secretory pathway is important for Nef activity.     

Differential regulation by ATP versus ADP further links CaMKII aggregation to ischemic conditions

CaMKII, a major mediator of synaptic plasticity, forms extra-synaptic clusters under ischemic conditions. This study further supports self-aggregation of CaMKII holoenzymes as the underlying mechanism. Aggregation in vitro was promoted by mimicking ischemic conditions: low pH (6.8 or less), Ca²⁺ (and calmodulin), and low ATP and/or high ADP concentration. Mutational analysis showed that high ATP prevented aggregation by a mechanism involving T286 auto-phosphorylation, and indicated requirement for nucleotide binding but not auto-phosphorylation also for extra-synaptic clustering within neurons. These results clarify a previously apparent paradox in the nucleotide and phosphorylation requirement of aggregation, and support a mechanism that involves inter-holoenzyme T286-region/T-site interaction.     

Inhomogeneous Encoding of the Visual Field in the Mouse Retina

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RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins

We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin-binding site of thrombin, anion-binding exosite-2 (exosite-2). We localized the Toggle-25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of exosite-2. We conclude that a RNA aptamer to thrombin exosite-2 might provide an effective clinical reagent to control heparin's anticoagulant action.     

Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source

The increasing prevalence of insecticide resistance and the ongoing global burden of vector-borne diseases have encouraged new efforts in mosquito control. For Aedes aegypti, the most important arboviral vector, integration rates achieved in Cas9-based knock-ins so far have been rather low, highlighting the need to understand gene conversion patterns and other factors that influence homology-directed repair (HDR) events in this species. In this study, we report the effects of sequence mismatches or donor template forms on integration rates. We found that modest sequence differences between construct homology arms [DNA sequence in the donor template which resembles the region flanking the target cut] and genomic target comprising 1.2% nucleotide dissimilarity (heterology) significantly reduced integration rates. While most integrations (59–88%) from plasmid templates were the result of canonical [on target, perfect repair] HDR events, no canonical events were identified from other donor types (i.e. ssDNA, biotinylated ds/ssDNA). Sequencing of the transgene flanking region in 69 individuals with canonical integrations revealed 60% of conversion tracts to be unidirectional and extend up to 220 bp proximal to the break, though in three individuals bidirectional conversion of up to 725 bp was observed.     

Murine Efficacy and Pharmacokinetic Evaluation of the Flaviviral NS5 Capping Enzyme 2-Thioxothiazolidin-4-One Inhibitor BG-323

Arthropod-borne flavivirus infection continues to cause significant morbidity and mortality worldwide. Identification of drug targets and novel antiflaviviral compounds to treat these diseases has become a global health imperative. A previous screen of 235,456 commercially available small molecules identified the 2-thioxothiazolidin-4-one family of compounds as inhibitors of the flaviviral NS5 capping enzyme, a promising target for antiviral drug development. Rational drug design methodologies enabled identification of lead compound BG-323 from this series. We have shown previously that BG-323 potently inhibits NS5 capping enzyme activity, displays antiviral effects in dengue virus replicon assays and inhibits growth of West Nile and yellow fever viruses with low cytotoxicity in vitro. In this study we further characterized BG-323’s antiviral activity in vitro and in vivo. We found that BG-323 was able to reduce replication of WNV (NY99) and Powassan viruses in culture, and we were unable to force resistance into WNV (Kunjin) in long-term culture experiments. We then evaluated the antiviral activity of BG-323 in a murine model. Mice were challenged with WNV NY99 and administered BG-323 or mock by IP inoculation immediately post challenge and twice daily thereafter. Mice were bled and viremia was quantified on day three. No significant differences in viremia were observed between BG-323-treated and control groups and clinical scores indicated both BG-323-treated and control mice developed signs of illness on approximately the same day post challenge. To determine whether differences in in vitro and in vivo efficacy were due to unfavorable pharmacokinetic properties of BG-323, we conducted a pharmacokinetic evaluation of this small molecule. Insights from pharmacokinetic studies indicate that BG-323 is cell permeable, has a low efflux ratio and does not significantly inhibit two common cytochrome P450 (CYP P450) isoforms thus suggesting this molecule may be less likely to cause adverse drug interactions. However, the T_1/2 of BG-323 was suboptimal and the percent of drug bound to plasma binding proteins was high. Future studies with BG-323 will be aimed at increasing the T_1/2 and determining strategies for mitigating the effects of high plasma protein binding, which likely contribute to low in vivo efficacy.     

Divalent cations increase lipid order in erythrocytes and susceptibility to secretory phospholipase A2

Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree to which ions affected membrane properties correlated with the ionic radius and electronegativity of the ions. Lastly, erythrocytes became more susceptible to enzyme hydrolysis in the presence of elevated intracellular levels of nickel and manganese, but not magnesium. These differences appeared related to the ability of the ions to induce a transition in erythrocyte shape.     

Composition,Assembly, and Trafficking of a Wheat Xylan Synthase Complex

Xylans play an important role in plant cell wall integrity and have many industrial applications. Characterization of xylan synthase (XS) complexes responsible for the synthesis of these polymers is currently lacking. We recently purified XS activity from etiolated wheat (Triticum aestivum) seedlings. To further characterize this purified activity, we analyzed its protein composition and assembly. Proteomic analysis identified six main proteins: two glycosyltransferases (GTs) TaGT43-4 and TaGT47-13; two putative mutases (TaGT75-3 and TaGT75-4) and two non-GTs; a germin-like protein (TaGLP); and a vernalization related protein (TaVER2). Coexpression of TaGT43-4, TaGT47-13, TaGT75-3, and TaGT75-4 in Pichia pastoris confirmed that these proteins form a complex. Confocal microscopy showed that all these proteins interact in the endoplasmic reticulum (ER) but the complexes accumulate in Golgi, and TaGT43-4 acts as a scaffold protein that holds the other proteins. Furthermore, ER export of the complexes is dependent of the interaction between TaGT43-4 and TaGT47-13. Immunogold electron microscopy data support the conclusion that complex assembly occurs at specific areas of the ER before export to the Golgi. A di-Arg motif and a long sequence motif within the transmembrane domains were found conserved at the NH₂-terminal ends of TaGT43-4 and homologous proteins from diverse taxa. These conserved motifs may control the forward trafficking of the complexes and their accumulation in the Golgi. Our findings indicate that xylan synthesis in grasses may involve a new regulatory mechanism linking complex assembly with forward trafficking and provide new insights that advance our understanding of xylan biosynthesis and regulation in plants.It is believed that Golgi-localized, multiprotein complexes synthesize plant hemicellulosic polysaccharides, including xylans. Such complexes are not well characterized in plants (Zeng et al., 2010; Atmodjo et al., 2011; Chou et al., 2012), which is in sharp contrast with mammalian and yeast cells (Jungmann and Munro, 1998; McCormick et al., 2000; Giraudo et al., 2001). Xylans are the most abundant plant hemicellulosic polysaccharides on Earth and play an important role in the integrity of cell walls, which is a key factor in plant growth. Any mutations affecting xylan backbone biosynthesis seem to result in abnormal growth of plants due mostly to thinning and weakening of secondary xylem walls, described as the irregular xylem (irx) phenotype. Thus, characterizing the xylan synthase complex (XSC) would have an impact on plant improvement, as well as many industrial applications related to food, feed, and biofuel production (Yang and Wyman, 2004; Faik, 2010). Although the Arabidopsis (Arabidopsis thaliana) irx mutants have revealed the involvement of several glycosyltransferase (GT) gene families in xylan biosynthesis (Brown et al., 2007, 2009; Lee et al., 2007, 2010; Wu et al., 2009, 2010), no XSCs have been purified/isolated from Arabidopsis tissues, and we still do not know whether some of the identified Arabidopsis GTs can assemble into functional XSCs. Furthermore, if GTs do assemble into XSCs, we don’t know the mechanisms by which plant cells control their assembly and cellular trafficking. In contrast to dicots, xylan synthase activity was recently immunopurified from etiolated wheat (Triticum aestivum) microsomes (Zeng et al., 2010). This purified wheat XS activity was shown to catalyze three activities, xylan-glucuronosyltransferase (XGlcAT), xylan-xylosyltransferase (XXylT), and xylan-arabinofuranosyltranferase (XAT), which work synergistically to synthesize xylan-type polymers in vitro (Zeng et al., 2008, 2010). This work focuses on describing protein composition, assembly, and trafficking of this purified wheat XS activity.In all eukaryotes, proteins of the secretory pathway (including GTs) are synthesized in the endoplasmic reticulum (ER) and modified as they go through the Golgi cisternae. Most proteins exit the ER from ER export sites (ERESs; Hanton et al., 2009) and use a signal-based sorting mechanism that allows them to be selectively recruited into vesicles coated by coat protein II complexes (Barlowe, 2003; Beck et al., 2008). For many Golgi-resident type II membrane proteins, di-Arg motifs, such as RR, RXR, and RRR located in their cytosolic NH₂-terminal ends, have been shown to be required for their ER export (Giraudo et al., 2003; Czlapinski and Bertozzi, 2006; Schoberer et al., 2009; Tu and Banfield, 2010). Interestingly, di-Arg motifs located ∼40 amino acids from the membrane on the cytosolic side can also be used to retrieve some type II ER-resident proteins from cis-Golgi (Schutze et al., 1994; Hardt et al., 2003; Boulaflous et al., 2009). In contrast to the signal-based sorting mechanism involved in trafficking between the ER and Golgi, the steady-state localization/retention of proteins (including GTs) in the Golgi is thought to occur through vesicular cycling. Cycling is influenced by various mechanisms, including the length and composition of the transmembrane domain (TMD) of type II GTs (Bretscher and Munro, 1993; Colley, 1997; van Vliet et al., 2003; Sousa et al., 2003; Sharpe et al., 2010), and the oligomerization/aggregation of GTs (kin hypothesis), which suggests that formation of homo- or heterooligomers of GTs in the Golgi may prevent their recruitment into clathrin-coated vesicles (Machamer, 1991; Nilsson et al., 1993; Weisz et al., 1993; Cole et al., 1996). Some Golgi-resident GTs are predicted to have a cleavable NH₂-terminal secretion signal peptide (SP) and would therefore exist as soluble proteins in the Golgi lumen. To maintain their proper Golgi localization, these processed GTs are likely part of multiprotein complexes anchored to integral membrane proteins. The fact that homologs of many of the trafficking proteins from mammalian and yeast cells are found in plants indicates that trafficking machineries of the plant secretory pathway are likely conserved (d’Enfert et al., 1992; Bar-Peled and Raikhel, 1997; Batoko et al., 2000; Pimpl et al., 2000; Phillipson et al., 2001; Hawes et al., 2008).It is becoming increasingly evident that understanding the mechanisms controlling protein-protein interaction, sorting, and trafficking of polysaccharide synthases (including XSCs) will help elucidate how plants regulate cell wall synthesis and deposition during their development. To this end, we believe that the purified wheat XS activity (Zeng et al., 2010) is an excellent model for this type of study. In this work, proteomics was used to determine the protein composition of the purified XS activity. Confocal microscopy and immunogold transmission electron microscopy (TEM) were used to investigate the assembly and trafficking of the complex. Our proteomics data showed that the purified activity contains two GTs, TaGT43-4 and TaGT47-13, two putative mutases, TaGT75-3 and TaGT75-4, and two non-GT proteins: a germin-like protein (TaGLP) belonging to cupin superfamily and a protein specific to monocots annotated as wheat vernalization-related protein 2 (TaVER2). Microscopy analyses revealed that all these proteins interact in the ER, but the assembled complexes accumulate in the Golgi. Export of these complexes from the ER is controlled by the interaction between TaGT43-4 and TaGT47-13. Characterization of the wheat XSC and its trafficking furthers our understanding of xylan biosynthesis in grasses and helps elucidate how polysaccharide synthase complexes are assembled, sorted, and maintained in different compartments of the secretory pathway.     

Modification of the plasmid initiator protein RepC active site during replication

Abstract pT181 is a Staphylococcus aureus rolling circle replicating plasmid whose copy number is controlled by regulating the synthesis and activity of the initiator protein, RepC. The RepC dimer is modified during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. To purify RepC*, RepC was expressed in S. aureus as a fusion protein with a polyhistidine tail. The histidine-tagged RepC retains its initiation and topoisomerase activities in vitro. Histagged RepC/RepC and RepC/RepC* were purified in a two-step procedure. Peptide mapping, mass spectrometric analysis and protein sequencing of purified RepC and RepC* were carried out, and both proteins appeared identical, except that the peptide containing the RepC active site tyrosine used in nicking activity was absent when the purified RepC* sample was analyzed. The absence of the active site in RepC* suggests that this site was modified during replication. The results provide the first direct biochemical evidence that RepC* is a modified form of RepC, and support a model in which RepC replication of pT181 leaves RepC with an oligonucleotide blocking the active site of one of its subunits.