Endocytosis and retrograde transport of Shiga toxin and ricin

The plant toxin ricin and the bacterial toxin Shiga toxin are not only a threat to human health but may also be used in cancer treatment and to gain new insight into the mechanisms of intracellular transport. Ricin and Shiga toxin are enzymes that exploit the endocytic pathway to kill the cells. They follow the retrograde transport route from early endosomes via the Golgi apparatus to the endoplasmic reticulum, and they are then translocated to the cytosol, where they inactivate ribosomes. This study has focused on new proteins important for transport of ricin and Shiga toxin from endosomes to the Golgi apparatus, and the sorting nexins SNX1, SNX2, and SNX4, as well as the Rab6 GTPase isoforms Rab6A and Rab6A’ have been identified as important factors in this transport. In addition, this work has revealed that Shiga toxin, by signaling through the Syk kinase, is able to increase the number of clathrin-coated pits on the cell surface

Retrograde traffic in the biosynthetic-secretory route

In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traffic of both membranes and contents. In the physiologic dynamics of cells, retrograde flow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments’ balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious effects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traffic are known; a high number of the molecular constituents have been characterized, and the complicated fine structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traffic models again are in question by contradictory results obtained with diverse cell systems, and/or different techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reflect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by different cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations

The delivery of personalised, precision medicines via synthetic proteins

Introduction: The design of advanced drug delivery systems based on synthetic and su-pramolecular chemistry has been very successful. Liposomal doxorubicin (Caelyx®), and liposomal daunorubicin (DaunoXome®), estradiol topical emulsion (EstrasorbTM) as well as soluble or erodible polymer systems such as pegaspargase (Oncaspar®) or goserelin acetate (Zoladex®) represent considerable achievements. The Problem: As deliverables have evolved from low molecular weight drugs to biologics (currently representing approximately 30% of the market), so too have the demands made of advanced drug delivery technology. In parallel, the field of membrane trafficking (and endocytosis) has also matured. The trafficking of specific receptors i.e. material to be recycled or destroyed, as well as the trafficking of protein toxins has been well characterized. This, in conjunction with an ability to engineer synthetic, recombinant proteins provides several possibilities. The Solution: The first is using recombinant proteins as drugs i.e. denileukin diftitox (Ontak®) or agalsidase beta (Fabrazyme®). The second is the opportunity to use protein toxin architecture to reach targets that are not normally accessible. This may be achieved by grafting regulatory domains from multiple species to form synthetic proteins, engineered to do multiple jobs. Examples include access to the nucleocytosolic compartment. Herein the use of synthetic proteins for drug delivery has been reviewed

FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

This study shows that Rab11 and its binding protein FIP1 are required for retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN. We also demonstrate that Golgin-97 as a FIP1-binding protein and that this binding regulates the targeting of retrograde transport vesicles to the TGN

Shiga Toxin Increases Formation of Clathrin-Coated Pits through Syk Kinase

Clathrin-dependent endocytosis is a main entry mechanism for the glycolipid-binding Shiga toxin (Stx), although clathrin-independent pathways are also involved. Binding of Stx to its receptor Gb3 not only is essential for Stx retrograde transport to the endoplasmic reticulum and toxicity but also activates signaling through the tyrosine kinase Syk. We previously described that Syk activity is important for Stx entry, but it remained unclear how this kinase modulates endocytosis of Stx. Here we characterized the effects of Stx and Syk on clathrin-coated pit formation. We found that acute treatment with Stx results in an increase in the number of clathrin-coated profiles as determined by electron microscopy and on the number of structures containing the endocytic AP-2 adaptor at the plasma membrane determined by live-cell spinning disk confocal imaging. These responses to Stx require functional Syk activity. We propose that a signaling pathway mediated by Syk and modulated by Stx leads to an increased number of endocytic clathrin-coated structures, thus providing a possible mechanism by which Stx enhances its own endocytosis

Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin

<div><p>Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA₂), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA₂ activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the <em>trans</em>-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA₂.</p> </div

Stx transport in annexin A1 depleted cells is regulated by PKCδ and PLA2.

<p>(A) Golgi transport of Shiga toxin was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040429#s4" target="_blank">materials and methods</a> by quantification of sulfated ShigaB in HeLa cells transfected with siRNA against annexin A1 or non targeting siRNA, pretreated with the indicated inhibitors. Data from Stx sulfation are plotted as percentages of the value obtained for HeLa cells transfected with control siRNA and treated with DMSO. The white and black bars represent ShigaB-sulf2 sulfation for control and annexin A1 knockdown cells respectively. Data presented are the average of 3 independent experiments, each performed in parallel, error bars indicating standard error of the mean. *p<0.05 indicates statistically significant change between annexin A1 knockdown cells and the corresponding control siRNA treated cells. (B) After treatment with either 5 µM ONO-RS-082 for 30 min or 30 µM MAFP for 1 h, HeLa cells were incubated for 30 min with ShigaB before fixation and staining as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040429#s4" target="_blank">materials and methods</a> section with antibodies against TGN46 and ShigaB. Panel shows representative confocal pictures, scale bars 20 μm. Graphic shows quantification of amount of ShigaB colocalized with TGN46 in one representative experiment plotted as percentage of control condition. Data presented for one representative experiment (n = 3) are the average of at least 30 cells per condition. Quantifications were obtained with Zen 2009 software from Zeiss, error bars indicating standard error of the mean.</p

ShigaB transport to the Golgi is regulated by annexin A1 and A2.

<p>HeLa cells were transfected with control, annexin A1 or A2 siRNA for 72 h. For sulfation measurements, cells were starved in the presence of radioactive sulfate for 3 h. ShigaB-sulf2 or Ricin-sulf1 was then added and the incubation proceeded for an additional one or two hours, respectively. Cells were lyzed, ShigaB or ricin immunoprecipitated, separated by electrophoresis and analyzed by autoradiography. The protein knockdown level was investigated in total cell lysates by immunoblotting. (A) Cell lysates were analyzed by western blotting (upper panel) with the indicated antibodies demonstrating protein knockdown of annexin A1 and A2 by the indicated siRNA oligos. Hsp90 represents loading control. Autoradiography (lower panel) showing results from the corresponding sulfation experiment. (B) and (C) Quantative data from protein sulfation for ShigaB and ricin respectively, plotted as percentages of control values. Quantifications of sulfation are the average of 3–8 independent experiments, each performed in parallel, error bars indicating standard error of the mean; *p<0.05, **p<0.005 indicate statistically significant change.</p

Time course of ShigaB sulfation in annexin A1 or A2 depleted cells.

<p>(A) HeLa cells transfected with control or annexin A1 siRNA for 72 h were analyzed for the amount of ShigaB being sulfated at different time points after addition of StxB. Sulfation data are plotted as percentages of the amount of sulphated ShigaB after 30 min incubation with control siRNA treated cells. Black and white bars represent sulfation of ShigaB in control and annexin A1 siRNA treated cells, respectively. Data presented are the average of 3–4 independent experiments, each performed in parallel, error bars indicating standard error of the mean, *p<0.05; **p<0.005 indicates statistically significant change. (B) After the same treatment as in (A), cells were fixed and stained with antibodies against TGN46 and ShigaB. Top panel shows representative confocal pictures for 30 min incubation with StxB, scale bars 20 μm. Left graphic shows quantification of amount of ShigaB colocalized with TGN46. In the right graphic, mean intensity of ShigaB in the Golgi area for the same representative experiment is plotted as percentage of mean intensity in the whole cell. Data presented are the average of at least 35 cells per condition. Quantifications where obtained with ImageJ software, error bars indicating standard error of the mean.</p