A TSC22-like motif defines a novel antiapoptotic protein family

The apoptotic programme is evolutionarily conserved between yeast and metazoan organisms. We have previously identified a number of mammalian cDNAs capable of suppressing the deleterious effects of Bax expression in yeast. We herein report that one such suppressor, named Tsc22(86), represents the C-terminal 86 amino acids of the previously characterized leucine zipper (LZ) motif-containing transcriptional regulator Tsc22. Employing a genome-wide two-hybrid screen, functional genomics, and deletion mutagenesis approaches, we conclude that Tsc22(86)-mediated antiapoptosis is independent of the LZ motif and is likely independent of effects on gene transcription. Rather, a 16-residue sequence within the conserved 56-residue TSC22 domain is necessary for antiapoptosis. The presence of a similar sequence was used to predict an antiapoptotic role for two yeast proteins, Sno1p and Fyv10p. Overexpression and knock-out experiments were used to validate this prediction. These findings demonstrate the potential of studying heterologous proteins in yeast to uncover novel biological insights into the regulation of apoptosis

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

ON TREMATOSPHAERIA (LOCULOASCOMYCETES, FUNGI) AND DISPOSITION OF HETEROGENEOUS ELEMENTS

This taxonomic study on the disparate species that have been named in Trematosphaeria Fuckel, begins by selecting a generitype specimen and determining that by Barr\u27s classification of the Loculoascomycetes Trematosphaeria belongs to the Melanommataceae, Melanommatales. A survey of eight-two species that have been named in Trematosphaeria, or are reported synonyms of Trematosphaeria species, or are reported to be Trematosphaeria-like, reveals that most of these species do not meet the fundamental criteria for membership in Trematosphaeria that can be inferred from the higher taxa. Instead, they represent two classes, six orders, eleven families, thirty genera and fifty-one species in the Ascomycota. The species are described and illustrated. To accommodate the species excluded from Trematosphaeria where no legitimate name is available for synonymy, two new genera and thirty-eight new combinations are proposed. Five species are accepted in Trematosphaeria: T. pertusa (lectotype), T. cariosa, T. confusa, T. hydrela, T. mangrovis. Species of Trematosphaeria grow on wood or bark of dicotyledonous angiosperms as hemibiotrophs or saprobes. They are little collected, but appear to be distributed worldwide in the temperate to tropical climatic regions. No anamorph is known in species of Trematosphaeria. Astrosphaeriella and Caryospora are the genera that demonstrate close relationships with Trematosphaeria

The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation

We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration. The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity. Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis. These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells