c-Cbl targets active Src for autophagy

Autophagy can promote both cancer cell survival and death, and the mechanisms by which it mediates these disparate processes are under intense investigation. Autophagosomes are now shown to entrap and promote degradation of the active tyrosine kinase Src, enabling tumour cell survival. The E3 ubiquitin ligase c-Cbl acts as an autophagosome cargo receptor for Src.     

Functional human insulin-degrading enzyme can be expressed in bacteria

Insulin-degrading enzyme (IDE) has been shown to degrade a number of biologically important peptides, including insulin and the amyloid-beta protein implicated in Alzheimer's disease. However, lack of a facile method to generate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides. Therefore, we determined whether recombinant wild-type and mutant human IDEs can be overexpressed as functional enzymes in bacteria. Three vectors carrying cDNAs encoding N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximately 8 mg per liter of culture). The recombinant IDEs, like the endogenous mammalian enzyme, migrate with 110-kDa apparent molecular masses in SDS-polyacrylamide gels and as a approximately 200-kDa species in gel filtration. Further analysis by native PAGE indicates that IDE can form multimers of different complexities. The wild-type recombinant endopeptidase degrades insulin with an efficiency similar to that of the enzyme purified from mammalian tissues. Purified IDEs are stable at 4 degrees C for at least 1 month. Purified recombinant protein was used to raise specific polyclonal antibodies that can immunoprecipitate native mammalian IDE. Thus, the procedure described allows the rapid production of large amounts of purified IDE and demonstrates that IDE can be produced in an active form in the absence of other potential interacting mammalian proteins.     

Colicin V can be produced by lactic acid bacteria

Colicin V is a small, proteinaceous bacterial toxin, produced by many strains of Escherichia coli and other members of the Enterobacteriaceae, that fits the definition of class II bacteriocins of Gram-positive bacteria. Export of colicin V is dependent on specific ABC (ATP-binding cassette) secretion proteins which recognize a double-glycine-type leader peptide on the immature colicin V bacteriocin. Replacement of the colicin V leader peptide by a signal peptide from the signal sequence-dependent bacteriocin divergicin A allowed expression of colicin V in lactic acid bacteria. This system may serve as a model for the heterologous expression of other small bacteriocins active against Gram-negative bacteria and other antibacterial peptides from lactic acid bacteria.     

Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy

Cisplatin (CDDP) is commonly used to treat a multitude of tumors including sarcomas, ovarian and cervical cancers. Despite recent investigations allowed to improve chemotherapy effectiveness, the molecular mechanisms underlying the development of CDDP resistance remain a major goal in cancer research. Here, we show that mitochondrial morphology and autophagy are altered in different CDDP resistant cancer cell lines. In CDDP resistant osteosarcoma and ovarian carcinoma, mitochondria are fragmented and closely juxtaposed to the endoplasmic reticulum; rates of mitophagy are also increased. Specifically, levels of the mitophagy receptor BNIP3 are higher both in resistant cells and in ovarian cancer patient samples resistant to platinum-based treatments. Genetic BNIP3 silencing or pharmacological inhibition of autophagosome formation re-sensitizes these cells to CDDP. Our study identifies inhibition of BNIP3-driven mitophagy as a potential therapeutic strategy to counteract CDDP resistance in ovarian carcinoma and osteosarcoma.Subject terms: Cancer therapeutic resistance, Mitophagy     

Molecules in the signaling pathway activated by gangliosides can be targets of therapeutics for malignant melanomas

There have been a number of studies on tumor-specific glycolipid antigens. In particular, neuroectoderm-derived cancers express characteristic ganglioside antigens, some of them have been used as tumor markers and/or target of immunotherapy. Molecules in the signaling pathway activated by gangliosides have been analyzed. Here, we reported results on the functions of molecules involved in the signaling pathway to enhance malignant properties of human melanomas under GD3 expression, and emphasized that those molecules including tumor-associated antigens can be targets of therapeutics for malignant melanomas.     

Spatial sensing of stimulus gradients can be superior to temporal sensing for free-swimming bacteria.

Predictions of the minimal size an organism must have to swim along stimulus gradients were used to compare the relative advantages of sensory systems employing spatial (simultaneous) and temporal (sequential) gradient detection mechanisms for small free-swimming bacteria, leading to the following conclusions: 1) there are environmental conditions where spatial detection mechanisms can function for smaller organisms than can temporal mechanisms, 2) temporal mechanisms are superior (have a smaller size limit) for the difficult conditions of low concentration and shallow gradients, but 3) observed bacterial chemotaxis occurs mostly under conditions where spatial mechanisms have a smaller size limit, and 4) relevant conditions in the natural environment favor temporal mechanisms in some cases and spatial mechanisms in others. Thus, sensory ecology considerations do not preclude free-swimming bacteria from employing spatial detection mechanisms, as has been thought, and microbiologists should be on the lookout for them. If spatial mechanisms do not occur, the explanation should be sought elsewhere.     

Glycosylation of gp41 of simian immunodeficiency virus shields epitopes that can be targets for neutralizing antibodies

Human immunodeficiency virus type 1 and simian immunodeficiency virus possess three closely spaced, highly conserved sites for N-linked carbohydrate attachment in the extracellular domain of the transmembrane protein gp41. We infected rhesus monkeys with a variant of cloned SIVmac239 lacking the second and third sites or with a variant strain lacking all three of SIVmac239's glycosylation sites in gp41. For each mutation, asparagine (N) in the canonical N-X-S/T recognition sequence for carbohydrate attachment was changed to the structurally similar glutamine such that two nucleotide changes would be required for a reversion of the mutated codon. By 16 weeks, experimentally infected monkeys made antibodies that neutralized the mutant viruses to high titers. Such antibodies were not observed in monkeys infected with the parental virus. Thus, new specificities were revealed as a result of the carbohydrate attachment mutations, and antibodies of these specificities had neutralizing activity. Unlike monkeys infected with the parental virus, monkeys infected with the mutant viruses made antibodies that reacted with peptides corresponding to the sequences in this region. Furthermore, there was strong selective pressure for the emergence of variant sequences in this region during the course of infection. By analyzing the neutralization profiles of sequence variants, we were able to define three mutations (Q625R, K631N, and Q634H) in the region of the glycosylation site mutations that conferred resistance to neutralization by plasma from the monkeys infected with mutant virus. Based on the reactivity of antibodies to peptides in this region and the colocalization of neutralization escape mutations, we conclude that N-linked carbohydrates in the ectodomain of the transmembrane protein shield underlying epitopes that would otherwise be the direct targets of neutralizing antibodies.     

Cytoplasmic bacteria and the autophagic pathway

Cytoplasmic bacteria may assist in our study of the autophagic pathway. This review highlights the use of Listeria monocytogenes for examining the assembly of autophagic vacuoles in mammalian cells. Inhibiting protein synthesis of cytoplasmic L. monocytogenes results in their being sequestered into the autophagic pathway. Autophagic vacuoles form around the easily identified bacterial particles making the assembly process easy to study using morphological and biochemical methods. L. monocytogenes, which appears to be ideally adapted to life in the cell cytoplasm, does not normally become a target of autophagy. In model systems the bacteria thrive within host cell cytoplasm, indicating the importance of de novo protein synthesis in avoiding the autophagic pathway. This observation indicates an interesting opportunity for identifying the bacterial mechanisms that are mobilized to avoid the autophagic pathway.     

Ubiquitination-mediated autophagy against invading bacteria

Autophagy is primarily a non-selective intracellular bulk degradation process. However, it was recently shown that ubiquitin-positive substrates, such as protein aggregates, mitochondria, peroxisomes, and invading bacteria, are selectively targeted to lysosomes via autophagy. Thus, ubiquitination seems to function as a general tag for selective autophagy in mammalian cells. This review discusses the present model of how autophagy sequesters invading bacteria through ubiquitination.     

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.     

Escherichia coli molybdoenzymes can be activated by protein FA from several gram-negative bacteria

Six Gram-negative bacteria (Klebsiella pneumoniae, Erwinia chrysanthemi, Proteus vulgaris, Serratia marescens, Salmonella typhimurium, and Pseudomonas aeruginosa) were shown to contain an FA-type protein capable of activating aponitrate reductase, apotrimethylamine N-oxide reductase and apoformate dehydrogenase of Escherichia coli. Protein FA activity was highest in Erwinia chrysanthemi and lowest in Pseudomonas aeruginosa. All the species also contained the low-Mr (less than or equal to 1500) heat-resistant material previously reported to be necessary for the protein-FA-dependent activation of E. coli chlB nitrate reductase.     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

Ribavirin can be mutagenic for arenaviruses

Arenaviruses include several important human pathogens, and there are very limited options of preventive or therapeutic interventions to combat these viruses. An off-label use of the purine nucleoside analogue ribavirin (1-β-d-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is the only antiviral treatment currently available for arenavirus infections. However, the ribavirin antiviral mechanism action against arenaviruses remains unknown. Here we document that ribavirin is mutagenic for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) in cell culture. The mutagenic activity of ribavirin on LCMV was observed under single- and multiple-passage regimes and could not be accounted for by a decrease of the intracellular GTP pool promoted by ribavirin-mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). Our findings suggest that the antiviral activity of ribavirin on arenaviruses might be exerted, at least partially, by lethal mutagenesis. Implications for antiarenavirus therapy are discussed.     

Oligomerization of paramagnetic substrates result in signal amplification and can be used for MR imaging of molecular targets

Magnetic resonance imaging (MRI) has evolved into a sophisticated, noninvasive imaging modality capable of high-resolution anatomical and functional characterization of transgenic animals. To expand the capabilities MRI, we have developed a novel MR signal amplification (MRamp) strategy based on enzyme-mediated polymerization of paramagnetic substrates into oligomers of higher magnetic relaxtivity. The substrates consist of chelated gadolinium covalently bound to phenols, which then serve as electron donors during enzymatic hydrogen peroxide reduction by peroxidase. The converted monomers undergo rapid condensation into paramagnetic oligomers leading to a threefold increase in atomic relaxtivity (R1/Gd). The observed relaxtivity changes are largely due to an increase in the rotational correlation time tau r of the lanthanide. Three applications of the developed system are demonstrated: (1) imaging of nanomolar amounts of an oxidoreductase (peroxidase); (2) detection of a model ligand using an enzyme-linked immunoadsorbent assay format; and (3) imaging of E-selectin on the surface of endothelial cells probed for with an anti-E-selectin-peroxidase conjugate. The development of "enzyme sensing" probes is expected to have utility for a number of applications including in vivo detection of specific molecular targets. One particular advantage of the MRamp technique is that the same paramagnetic substrate can be potentially used to identify different molecular targets by attaching enzymes to various antibodies or other target-seeking molecules.