Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states

Longitudinal alterations in motivational salience processing in ultra-high-risk subjects for psychosis

Impairments in the attribution of salience are thought to be fundamental to the development of psychotic symptoms and the onset of psychotic disorders. The aim of the present study was to explore longitudinal alterations in salience processing in ultra-high-risk subjects for psychosis.; A total of 23 ultra-high-risk subjects and 13 healthy controls underwent functional magnetic resonance imaging at two time points (mean interval of 17 months) while performing the Salience Attribution Test to assess neural responses to task-relevant (adaptive salience) and task-irrelevant (aberrant salience) stimulus features.; At presentation, high-risk subjects were less likely than controls to attribute salience to relevant features, and more likely to attribute salience to irrelevant stimulus features. These behavioural differences were no longer evident at follow-up. When attributing salience to relevant cue features, ultra-high-risk subjects showed less activation than controls in the ventral striatum at both baseline and follow-up. Within the high-risk sample, amelioration of abnormal beliefs over the follow-up period was correlated with an increase in right ventral striatum activation during the attribution of salience to relevant cue features.; These findings confirm that salience processing is perturbed in ultra-high-risk subjects for psychosis, that this is linked to alterations in ventral striatum function, and that clinical outcomes are related to longitudinal changes in ventral striatum function during salience processing

The impact of CACNA1C gene, and its epistasis with ZNF804A, on white matter microstructure in health, schizophrenia and bipolar disorder

Genome-wide studies have identified allele A (adenine) of single nucleotide polymorphism (SNP) rs1006737 of the calcium-channel CACNA1C gene as a risk factor for both schizophrenia (SZ) and bipolar disorder (BD) as well as allele A for rs1344706 in the zinc-finger ZNF804A gene. These illnesses have also been associated with white matter abnormalities, reflected by reductions in fractional anisotropy (FA), measured using diffusion tensor imaging (DTI). We assessed the impact of the CACNA1C psychosis risk variant on FA in SZ, BD and health. 230 individuals (with existing ZNF804A rs1344706 genotype data) were genotyped for CACNA1C rs1006737 and underwent DTI. FA data was analysed with tract-based spatial statistics and threshold-free cluster enhancement significance correction (p < 0.05) to detect effects of CACNA1C genotype on FA, and its potential interaction with ZNF804A genotype and with diagnosis, on FA. There was no significant main effect of the CACNA1C genotype on FA, nor diagnosis by genotype(s) interactions. Nevertheless, when inspecting SZ in particular, risk allele carriers had significantly lower FA than the protective genotype individuals, in portions of the left middle occipital and parahippocampal gyri, right cerebelleum, left optic radiation and left inferior and superior temporal gyri. Our data suggests a minor involvement of CACNA1C rs1006737 in psychosis via conferring susceptibility to white matter microstructural abnormalities in SZ. Put in perspective, ZNF804A rs1344706, not only had a significant main effect, but its SZ-specific effects were two orders of magnitude more widespread than that of CACNA1C rs1006737

The role of CDC48 in the retro-translocation of non-ubiquitinated toxin substrates in plant cells

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterise the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that while RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesised RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated

Numerical modelling of mechanical stresses in bulk superconductor magnets with and without mechanical reinforcement

The magnetic field trapping capability of a bulk superconductor is essentially determined by the critical current density, Jc(B, T), of the material. With state-of-the-art bulk (RE)BCO (where RE = rare earth or Y) materials it is clear that trapped fields of over 20 T are potentially achievable. However, the large Lorentz forces, FL = J × B, that develop during magnetisation of the sample lead to large mechanical stresses that can result in mechanical failure. The radial forces are tensile and the resulting stresses are not resisted well because of the brittle ceramic nature of (RE)BCO materials. Where fields of more than 17 T have been achieved, the samples were reinforced mechanically using resin impregnation and carbon-fibre wrapping or shrink-fit stainless steel. In this paper, two-dimensional (2D) axisymmetric and three-dimensional (3D) finite-element models based on the H-formulation, implemented in the commercial finite element software package COMSOL Multiphysics, are used to provide a comprehensive picture of the mechanical stresses in bulk superconductor magnets with and without mechanical reinforcement during field-cooled magnetization (FCM). The chosen modelling framework couples together electromagnetic, thermal and structural mechanics models, and is extremely flexible in allowing the inclusion of various magnetisation processes and conditions, as well as detailed and realistic properties of the materials involved. The 2D model – a faster route to parametric optimisation – is firstly used to investigate the influence of the ramp rate of the applied field and any heat generated in the bulk. Finally, the 3D model is used to investigate the influence of inhomogeneous Jc(B, T) properties around the ab-plane of the bulk superconductor on the developed mechanical stress.EPSRC Early Career Fellowship EP/P020313/1 JSPS KAKENHI Grant No. JP15K0464

Elderberry ( Sambucus Nigra ) Bark Contains two Structurally Different Neusac(Α2,6)Gal/Galnac-Binding Type 2 Ribosome-Inactivating Proteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65709/1/j.1432-1033.1997.00648.x.pd

The utility of single nucleotide DNA variations as predictors of postoperative pain

Objectives: Genetic variation is an important contributor to postsurgical pain and thereby analgesia requirements. A description of the potential predictive power of genetic variants in pain should instruct improvements in pain management postoperatively. We set out to examine whether a set of genetic variants in pain related genes would show any association with actual pain outcomes in a typical surgical population. Methods: A candidate gene study was carried out in 135 surgical patients with 12 DNA variants (single nucleotide polymorphisms or ‘SNPs’) in known or putative pain pathway genes to detect associations with postoperative pain - measured by a verbal rating score (VRS) and patient-controlled analgesia (PCA) usage rate. Standard PCR based molecular biology approaches were used. Results: At 20-24h after surgery, patients with the 1032G/1032G variant pair for the A1032G variant of the potassium channel KCNJ6 gene had a slightly higher median VRS than those with 1032A/1032A or 1032A/1032G pairs (p=0.04; dominant genetic model). This small difference was most apparent in the orthopaedic surgery patients where the 1032G/1032G pair associated with VRS (median(interquartile range)) of 5(4-6) vs. 3(0.5-4) in 1032A/1032A or 1032A/1032G groups. For PCA, patients with 3435C/3435C or 3435C/3435T pairs for ATPdependent efflux pump gene ABCB1 variant C3435T used PCA at a considerably higher rate of 0.89(0.07-1.66) mg.h-1 compared with just 0.11 (0-0.52) mg.h-1 for the 3435T/3435T pair (p=0.03; dominant model). A significantly higher usage rate was also detected for opioid receptor OPRM1 variant IVS2-691 with usage of 0.77(0.01-1.56) mg.h-1 for the IVS2C/IVS2C or IVS2C/IVS2G group vs. 0.24(0-1.26) mg.h-1 in the IVS2G/IVS2G group (p=0.04; recessive model). Conclusion: While this study has identified some significant statistical associations the potential utility of the studied DNA variants in prediction of postoperative pain and patient-controlled opioid analgesia requirements appears to be quite limited at present

Stimulation of colonic motility by oral PEG electrolyte bowel preparation assessed by MRI : comparison of split vs single dose

Peer reviewe