232 research outputs found
Retrograde transport: Going against the flow
AbstractCertain protein toxins act by catalytically modifying substrates in the cytosol of mammalian cells. To reach this compartment, these proteins undergo retrograde transport from the cell surface, via the Golgi complex, to the endoplasmic reticulum
ER Dislocation: Cdc48p/p97 Gets Into the AAAct
AbstractMisfolded or unassembled proteins present in the lumen of the endoplasmic reticulum are exported to the cytosol and degraded. Recent studies have implicated a complex containing the AAA ATPase Cdc48p/p97 in the export process
Syntaxin 16 and syntaxin 5 are required for efficient retrograde transport of several exogenous and endogenous cargo proteins
Retrograde transport allows proteins and lipids to leave the endocytic pathway to reach other intracellular compartments, such as trans-Golgi network (TGN)/Golgi membranes, the endoplasmic reticulum and, in some instances, the cytosol. Here, we have used RNA interference against the SNARE proteins syntaxin 5 and syntaxin 16, combined with recently developed quantitative trafficking assays, morphological approaches and cell intoxication analysis to show that these SNARE proteins are not only required for efficient retrograde transport of Shiga toxin, but also for that of an endogenous cargo protein - the mannose 6-phosphate receptor - and for the productive trafficking into cells of cholera toxin and ricin. We have found that the function of syntaxin 16 was specifically required for, and restricted to, the retrograde pathway. Strikingly, syntaxin 5 RNA interference protected cells particularly strongly against Shiga toxin. Since our trafficking analysis showed that apart from inhibiting retrograde endosome-to-TGN transport, the silencing of syntaxin 5 had no additional effect on Shiga toxin endocytosis or trafficking from TGN/Golgi membranes to the endoplasmic reticulum, we hypothesize that syntaxin 5 also has trafficking-independent functions. In summary, our data demonstrate that several cellular and exogenous cargo proteins use elements of the same SNARE machinery for efficient retrograde transport between early/recycling endosomes and TGN/Golgi membranes
Expression, Purification and Characterization of Ricin vectors used for exogenous antigen delivery into the MHC Class I presentation pathway
Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smith et al. J Immunol 2002; 169:99-107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression in E. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterized in vitro, via an N-glycosidase assay, and in vivo, by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass
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The N-terminal ricin propeptide influences the fate of ricin A-chain in tobacco protoplasts.
The plant toxin ricin is synthesized in castor bean seeds as an endoplasmic reticulum (ER)-targeted precursor. Removal of the signal peptide generates proricin in which the mature A- and B-chains are joined by an intervening propeptide and a 9-residue propeptide persists at the N terminus. The two propeptides are ultimately removed in protein storage vacuoles, where ricin accumulates. Here we have demonstrated that the N-terminal propeptide of proricin acts as a nonspecific spacer to ensure efficient ER import and glycosylation. Indeed, when absent from the N terminus of ricin A-chain, the non-imported material remained tethered to the cytosolic face of the ER membrane, presumably by the signal peptide. This species appeared toxic to ribosomes. The propeptide does not, however, influence catalytic activity per se or the vacuolar targeting of proricin or the rate of retrotranslocation/degradation of A-chain in the cytosol. The likely implications of these findings to the survival of the toxin-producing tissue are discussed
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
Evaluation of Coach-Based Technical Assistance: An Evolving Focus on Coachability and Goal Setting
In 2013, the National Institute of Food and Agriculture supported the creation of a professional development and technical assistance center to promote strong implementation and evaluation of University-led, community-based projects serving low-resource populations. Within this center, a coaching cadre was established to provide proactive and responsive technical assistance. Formative evaluation involving coaches and their primary contacts was used for refinement of coaching practices. Initially, coaches were encouraged to build strong interpersonal rapport. This set the stage for trusting, reciprocal interactions, but coaches recognized a need for targeted support and more tools for quality programming, evaluation, and sustainability. Greater emphasis was placed on goal-focused collaboration. Coaches received training and resources on topics such as goal setting, program quality, reduction of barriers (e.g., participant recruitment), and sustainability strategies. To assess coaching model enhancements, a survey of projects was expanded to gauge logic model usage, goal setting, strength of coaching relationships, and project implementation and sustainability progress. Overall, coaching was rated more favorably and effective when contact was consistent, inclusive of face-to-face interaction, met technical needs, and involved collaborative brainstorming and planning. Findings indicate coaching relationships strengthen over time and demand a collaborative, action-orientation to set goals, reduce barriers, and drive stronger outcomes
The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain
The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the endoplasmic reticulum (ER) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show here that the proteasome has a more complex role in ricin intoxication than previously recognised, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. Our results implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated
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