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

A human embryonic kidney 293T cell line mutated at the Golgi -mannosidase II locus

Disruption of Golgi -mannosidase II activity can result in type II congenital dyserythropoietic anemia and can induce lupus-like autoimmunity in mice. Here, we isolate a mutant human embryonic kidney (HEK) 293T cell line, called Lec36, that displays sensitivity to ricin that lies between the parental HEK 293T cells, whose secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which only produce oligomannose-type N-linked glycans. The stem cell marker, 19A, was transiently expressed in the HEK 293T Lec36 cells, and in parental HEK 293T cells with and without the potent Golgi -mannosidase II inhibitor, swainsonine. Negative-ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi -mannosidase II: a point mutation in one allele mapping to the active site and an in-frame deletion of twelve-nucleotides in the other. Expression of wild-type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and provides a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia

Fine tuning Exo2, a small molecule inhibitor of secretion and retrograde trafficking pathways in mammalian cells

The small molecule 4-hydroxy-3-methoxybenzaldehyde (5,6,7,8-tetrahydro[1]benzothieno[2,3- d]pyrimidin-4-yl)hydrazone (Exo2) stimulates morphological changes at the mammalian Golgi and trans-Golgi network that are virtually indistinguishable from those induced by brefeldin A. Both brefeldin A and Exo2 protect cells from intoxication by Shiga(-like) toxins by acting on other targets that operate at the early endosome, but do so at the cost of high toxicity to target cells. The advantage of Exo2 is that it is much more amenable to chemical modification and here we report a range of Exo2 analogues produced by modifying the tetrahydrobenzothienopyrimidine core, the vanillin moiety and the hydrazone bond that links these two. These compounds were examined for the morphological changes they stimulated at the Golgi stack, the trans Golgi network and the transferrin receptor-positive early endosomes and this activity correlated with their inherent toxicity towards the protein manufacturing ability of the cell and their protective effect against toxin challenge. We have developed derivatives that can separate organelle morphology, target specificity, innate toxicity and toxin protection. Our results provide unique compounds with low toxicity and enhanced specificity to unpick the complexity of membrane trafficking networks

The Folliculin Tumor Suppressor Is a GAP for the RagC/D GTPases That Signal Amino Acid Levels to mTORC1

The mTORC1 kinase is a master growth regulator that senses numerous environmental cues, including amino acids. The Rag GTPases interact with mTORC1 and signal amino acid sufficiency by promoting the translocation of mTORC1 to the lysosomal surface, its site of activation. The Rags are unusual GTPases in that they function as obligate heterodimers, which consist of RagA or B bound to RagC or D. While the loading of RagA/B with GTP initiates amino acid signaling to mTORC1, the role of RagC/D is unknown. Here, we show that RagC/D is a key regulator of the interaction of mTORC1 with the Rag heterodimer and that, unexpectedly, RagC/D must be GDP bound for the interaction to occur. We identify FLCN and its binding partners, FNIP1/2, as Rag-interacting proteins with GAP activity for RagC/D, but not RagA/B. Thus, we reveal a role for RagC/D in mTORC1 activation and a molecular function for the FLCN tumor suppressor.United States. National Institutes of Health (CA103866)United States. National Institutes of Health (AI47389)United States. Department of Defense (W81XWH-07-0448)National Cancer Institute (U.S.) (F30CA180754

The secretion inhibitor Exo2 perturbs trafficking of Shiga toxin between endosomes and the trans-Golgi network

The small-molecule inhibitor Exo2 {4-hydroxy-3-methoxy-(5,6,7,8-tetrahydrol[1]benzothieno[2,3-d]pyrimidin-4-yl)hydraz-one benzaldehyde} has been reported to disrupt the Golgi apparatus completely and to stimulate Golgi–ER (endoplasmic reticulum) fusion in mammalian cells, akin to the well-characterized fungal toxin BFA (brefeldin A). It has also been reported that Exo2 does not affect the integrity of the TGN (trans-Golgi network), or the direct retrograde trafficking of the glycolipid-binding cholera toxin from the TGN to the ER lumen. We have examined the effects of BFA and Exo2, and found that both compounds are indistinguishable in their inhibition of anterograde transport and that both reagents significantly disrupt the morphology of the TGN in HeLa and in BS-C-1 cells. However, Exo2, unlike BFA, does not induce tubulation and merging of the TGN and endosomal compartments. Furthermore, and in contrast with its effects on cholera toxin, Exo2 significantly perturbs the delivery of Shiga toxin to the ER. Together, these results suggest that the likely target(s) of Exo2 operate at the level of the TGN, the Golgi and a subset of early endosomes, and thus Exo2 provides a more selective tool than BFA for examining membrane trafficking in mammalian cells

The Future and Praxis of Decent Work

The two contributions to this Global Issues piece are based on keynote addresses to the ‘Future and Praxis of Decent Work’ Conference held at the University of Kassel, Germany, on 14 February 2013

Friends matter but so does their substance use: The impact of social networks on substance use, offending and wellbeing among young people attending specialist alcohol and drug treatment services

Aims: The current study assesses the impact of youth drug treatment on substance use, offending and wellbeing in a sample of young people recruited from specialist youth alcohol and drug treatment. The paper examines the impact of treatment engagement on the size and substance use profile of the young person's social network and hypothesises that the best treatment outcomes are associated with maintaining the size of the young person's social network but changing its composition to reduce the representation of substance use in social networks. Methods: A cohort study of 112 young people (aged 16–21) engaged in specialist youth alcohol and drug treatment services in Victoria, Australia, were recruited at the beginning of treatment and re-interviewed six months later using a structured questionnaire. Findings: There were improvements in substance use, social functioning, mental health and life satisfaction from baseline to follow-up. While network size was associated with mental health and quality of life markers, only having a lower proportion of substance users in the social network was associated with lower substance use and offending at follow-up. Conclusions: Social networks are a key component of wellbeing in adolescence. This study suggests that through independent analysis of network size and network composition, both the size and the composition of social networks have an important role to play in developing interventions for adolescent substance users that will sustain behaviour changes achieved in specialist treatment

Classification of wild tomatoes: a review

Wild tomatoes are native to western South America. The generic status of wild tomatoes within the Solanaceae has been controversial since the eighteen century. Linnaeus in 1753 placed tomatoes in Solanum while Miller, a contemporary of Linnaeus, classified tomatoes in a new genus. The majority of later botanists have followed Miller. Differing numbers of species and conflicting supraspecific classifications have been proposed, based on morphology or crossing studies. Two major crossability groups have been identified, one that includes mainly self-compatible species that easily cross with the cultivated tomato, and another that comprises self-incompatible species not easily cross with the cultivated tomato. Recent molecular investigations using appropriate outgroups have shown that tomatoes and potatoes are close related phylogenetically, and support the inclusion of tomatoes within Solanum, the classification advocated here. We discuss the conflicting goals of classifications based on predictivity versus stability, a continuing controversy in systematics.Las especies de tomates silvestres son nativas del oeste de América de Sur. Su posición genérica dentro de las Solanáceas ha sido controvertida desde el Siglo XVIII. Linneo en 1753 ubico a los tomates en Solanum mientras que Miller, un contemponineo de Linneo, los incluyo dentro del nuevo genero Lycopersicon. Posteriormente, la mayoría de los botánicos siguieron la clasificación de Miller. Las clasificaciones basadas en morfología 0 en estudios de cruzamientos han propuesto diferente numero de especies 0 categorías supraespecíficas. Sobre la base de la cruzamientos entre especies se han identificado dos grupos; uno de ellos incluye especies autocompatibles que pueden cruzarse fácilmente con el tomate cultivado, el otro comprende especies autoincompatibles que no pueden cruzase fácilmente con esta especie. Recientes investigaciones moleculares, utilizando grupos externos adecuados, han mostrado que los tomates y las papas están muy relacionados filogenéticamente y apoyan la inclusión de los tomates dentro de Solanum, clasificación que hemos adoptado aquí. Se discute acerca del conflicto de los objetivos de las clasificaciones basados en la predicción 0 la estabilidad, una continua controversia en sistemática.Fil: Peralta, Iris Edith. University of Wisconsin; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Provincia de Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Universidad Nacional de Cuyo. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: Spooner, David M.. University of Wisconsin; Estados Unido