Short loop-targeting oligoribonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of cell growth in let-7-deficient cancer cells

MicroRNAs (miRNAs) originate from stem-loop-containing precursors (pre-miRNAs, pri-miRNAs) and mature by means of the Drosha and Dicer endonucleases and their associated factors. The let-7 miRNAs have prominent roles in developmental differentiation and in regulating cell proliferation. In cancer, the tumor suppressor function of let-7 is abrogated by overexpression of Lin28, one of several RNA-binding proteins that regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site. Using in vitro assays, we have identified a binding site for short modified oligoribonucleotides (‘looptomirs’) overlapping that of Lin28 in pre-let-7a-2. These looptomirs selectively antagonize the docking of Lin28, but still permit processing of pre-let-7a-2 by Dicer. Looptomirs restored synthesis of mature let-7 and inhibited growth and clonogenic potential in Lin28 overexpressing hepatocarcinoma cells, thereby demonstrating a promising new means to rescue defective miRNA biogenesis in Lin28-dependent cancers.     

A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development

Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system.Posttranslational modification of proteins by ubiquitin is involved in a wide range of cellular processes (1). Ubiquitination is linked to the turnover of an ever growing number of proteins; it regulates protein trafficking and is also widely used to transiently facilitate protein-protein interactions (2, 3). As the number of known ubiquitinated proteins keeps growing, the focus is turning toward identifying when, where, and how those proteins are ubiquitinated in vivo with the aim of understanding how protein function is being regulated within the context of a whole organism. The ubiquitin pathway is essential for brain development and function, and its failure is associated with a number of neurodegenerative diseases, including Parkinson and Alzheimer diseases (4–6). Ubiquitin conjugation is carried out by the sequential action of ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and can be reversed by deubiquitinating enzyme (DUB)1 proteases. The involvement of a number of those enzymes in synaptogenesis has been documented in several model systems (7–12). In Drosophila, for example, synaptogenesis is dependent on the E3 ligase Highwire and on the DUB fat facets (13). A few proteins involved in synaptogenesis have been shown to be ubiquitin substrates, including the postsynaptic proteins Shank, GKAP, and AKAP79/150 in cultured neurons (14) and the Caenorhabditis elegans synaptic protein DLK-1 kinase, which was shown to be ubiquitinated when overexpressed in HEK293T kidney cells (9). Most neuronal targets of the ubiquitin pathway, however, remain undiscovered. Yeast and HeLa cell-based proteomics approaches have failed to provide significant insights into the neuronal mechanisms regulated by ubiquitination. With the exception of a polyubiquitin affinity-based purification that successfully identified by Western blotting three ubiquitin substrates in cultured neurons (14), no proteomics approach has been described that can identify ubiquitinated neuronal proteins. Because neuronal function and activity are highly context-dependent, rather than working on neuronal culture, we have aimed to identify which proteins are ubiquitinated in vivo within the neurons of a living organism.Herein, we describe a novel strategy for the efficient isolation of neuronal ubiquitin conjugates from flies. The approach is based on the in vivo biotinylation of ubiquitin by ectopically expressing the Escherichia coli BirA enzyme to attach a biotin molecule to a specific BirA recognition sequence (15, 16) added at the N terminus of each ubiquitin chain. With the purpose of isolating ubiquitin conjugates uniquely from the nervous system of Drosophila melanogaster, we used the GAL4/UAS system for tissue-targeted expression (17). To increase the biotinylation efficiency, we took advantage of the processing activity of endogenous DUBs to digest a linear polypeptide precursor containing six copies of the tagged ubiquitin and the BirA enzyme, which are then present in the same cellular microenvironment. Because of the strength and the specificity of the avidin-biotin interaction, we were able to isolate and enrich the neuronal ubiquitinated proteins from a multicellular organism up to levels not achieved previously by any other approach. This allowed us to identify by mass spectrometry those neuronal proteins that are ubiquitinated and to resolve by Western blotting whether they are mono- or polyubiquitinated. This was achieved in the absence of proteasome inhibitors; therefore, physiological ubiquitination levels are reported. We focused on identifying the proteins that are ubiquitinated within the neurons in the period from neurite outgrowth and axonal pathfinding to target recognition and synapse formation (18). For that purpose, we applied our strategy on postmitotic neurons during embryonic stages 13–17 (19), a 12-h period during which embryos undergo synaptogenesis. Our strategy could be used to isolate ubiquitin conjugates from other tissues from the fruit flies, from different developmental stages, and in different mutant backgrounds, and it is likely to be applicable to other model organisms.     

A bioluminescent assay for the sensitive detection of proteases

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.     

The use of the orthotopic model to validate antivascular therapies for cancer

The orthotopic model reproduces aspects of the tumour microenvironment and emulates a number of important biological features of cancer progression, angiogenesis, metastasis and resistance. Due to its parallels with human cancer, the model can be used to evaluate therapeutic responses to various therapies. This review outlines the importance of using the orthotopic implantation of tumour cells in mice models for evaluating the effectiveness of antivascular therapies.     

The evolution of glycogen and starch metabolism in eukaryotes gives molecular clues to understand the establishment of plastid endosymbiosis

Solid semi-crystalline starch and hydrosoluble glycogen define two distinct physical states of the same type of storage polysaccharide. Appearance of semi-crystalline storage polysaccharides appears linked to the requirement of unicellular diazotrophic cyanobacteria to fuel nitrogenase and protect it from oxygen through respiration of vast amounts of stored carbon. Starch metabolism itself resulted from the merging of the bacterial and eukaryote pathways of storage polysaccharide metabolism after endosymbiosis of the plastid. This generated the three Archaeplastida lineages: the green algae and land plants (Chloroplastida), the red algae (Rhodophyceae), and the glaucophytes (Glaucophyta). Reconstruction of starch metabolism in the common ancestor of Archaeplastida suggests that polysaccharide synthesis was ancestrally cytosolic. In addition, the synthesis of cytosolic starch from the ADP-glucose exported from the cyanobacterial symbiont possibly defined the original metabolic flux by which the cyanobiont provided photosynthate to its host. Additional evidence supporting this scenario include the monophyletic origin of the major carbon translocators of the inner membrane of eukaryote plastids which are sisters to nucleotide-sugar transporters of the eukaryote endomembrane system. It also includes the extent of enzyme subfunctionalization that came as a consequence of the rewiring of this pathway to the chloroplasts in the green algae. Recent evidence suggests that, at the time of endosymbiosis, obligate intracellular energy parasites related to extant Chlamydia have donated important genes to the ancestral starch metabolism network.     

Distinct roles of MK2 and MK5 in cAMP/PKA- and stress/p38MAPK-induced heat shock protein 27 phosphorylation

Background

Classical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three successive phosphorylation events resulting in the phosphorylation of a variety of substrates, including another class of protein kinases referred to as MAPK-activating protein kinases (MAPKAPKs). The MAPKAPKs MK2, MK3 and MK5 are closely related, but MK2 and MK3 are the major downstream targets of the p38^MAPK pathway, while MK5 can be activated by the atypical MAPK ERK3 and ERK4, protein kinase A (PKA), and maybe p38^MAPK. MK2, MK3, and MK5 can phosphorylate the common substrate small heat shock protein 27 (HSP27), a modification that regulates the role of HSP27 in actin polymerization. Both stress and cAMP elevating stimuli can cause F-actin remodeling, but whereas the in vivo role of p38^MAPK-MK2 in stress-triggered HSP27 phosphorylation and actin reorganization is well established, it is not known whether MK2 is involved in cAMP/PKA-induced F-actin rearrangements. On the other hand, MK5 can phosphorylate HSP27 and cause cytoskeletal changes in a cAMP/PKA-dependent manner, but its role as HSP27 kinase in stress-induced F-actin remodeling is disputed. Therefore, we wanted to investigate the implication of MK2 and MK5 in stress- and PKA-induced HSP27 phosphorylation.

Results

Using HEK293 cells, we show that MK2, MK3, and MK5 are expressed in these cells, but MK3 protein levels are very moderate. Stress- and cAMP-elevating stimuli, as well as ectopic expression of active MKK6 plus p38^MAPK or the catalytic subunit of PKA trigger HSP27 phosphorylation, and specific inhibitors of p38^MAPK and PKA prevent this phosphorylation. Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels. Stimulation of the p38^MAPK, but not the PKA pathway, caused activation of MK2.

Conclusion

Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway. Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization.     

Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation

The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation–deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine–haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO₂ thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane.