Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor

The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production.     

Algorithms for incorporating prior topological information in HMMs: application to transmembrane proteins

Background  

Hidden Markov Models (HMMs) have been extensively used in computational molecular biology, for modelling protein and nucleic acid sequences. In many applications, such as transmembrane protein topology prediction, the incorporation of limited amount of information regarding the topology, arising from biochemical experiments, has been proved a very useful strategy that increased remarkably the performance of even the top-scoring methods. However, no clear and formal explanation of the algorithms that retains the probabilistic interpretation of the models has been presented so far in the literature.     

Metabolism of polyamines and oxidative stress in the brain of cholestatic rats

In a recently published article in “Amino Acids” it was shown that obstructive jaundice of 9 days’ duration in rats induces significant alterations of polyamines’ metabolism in the brain, which might play an important pathogenetic role in cholestatic brain injury. The authors proposed that alterations of polyamines in cholestatic brain might induce neuronal toxicity through a mechanism that implicates the production of reactive oxygen species and oxidative stress, although this parameter was not evaluated in their study. This hypothesis is supported by our recent findings on brain oxidative status in rats with obstructive jaundice of 10 days’ duration. Potential interrelations of the two studies’ findings are discussed in this commentary.     

Synthesis, characterization, and evaluation as transfection reagents of ampholytic star copolymers: effect of star architecture

Five star polymers based on the positively ionizable hydrophilic 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the hydrophobic but hydrolyzable tetrahydropyranyl methacrylate (THPMA) were prepared by group-transfer polymerization (GTP) using ethylene glycol dimethacrylate (EGDMA) as the coupling agent. In particular, four isomeric star copolymers (one heteroarm, two star block, and the statistical star), all with a 3:1 DMAEMA:THPMA molar ratio, plus one star homopolymer of DMAEMA, with degrees of polymerization of the arms equal to 15, were synthesized. After star polymer preparation and preliminary characterization, the THPMA units were hydrolyzed to negatively ionizable hydrophilic methacrylic acid (MAA) untis, thus yielding star polyampholytes. All the star polyampholytes as well as the commercially available transfection reagent SuperFect were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 bearing the enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was affected by star architecture. The DMAEMA15-star-MAA5 polyampholyte presented the highest transfection efficiency of all the star polymers tested but lower than that of SuperFect at its optimum conditions. All four star copolymers showed decreased toxicity compared to the DMAEMA star homopolymer for the same amounts of star polymer tested and also compared to the SuperFect at its optimum conditions.     

Sclerotial metamorphosis in filamentous fungi is induced by oxidative stress

Sclerotium-forming filamentous fungi are of great agriculturaland biological interest because they can be viewed as modelsof simple metamorphosis. They differentiate by asexually producingsclerotia but the processes involved in sclerotial metamorphosiswere poorly understood. In 1997, it was shown for the firsttime that the sclerotial differentiation state in Sclerotiumrolfsii concurred with increasing levels of lipid peroxides.This finding prompted the development of a theory supportingthat sclerotial metamorphosis is induced by oxidative stress.Growth factors that reduce or increase oxidative stress areexpected to inhibit or promote sclerotium metamorphosis, respectively.This theory has been verified by a series of published dataon the effect of certain hydroxyl radical scavengers on sclerotialmetamorphosis, on the identification and quantification of certainendogenous antioxidants (such as ascorbic acid, ß-carotene)in relation to the fungal undifferentiated and differentiatedstates, and on their inhibiting effect on sclerotial metamorphosisas growth nutrients. In 2004–2005, we developed assaysfor the measurement of certain redox markers of oxidative stress,such as the thiol redox state, the small-sized fragmented DNA,and the superoxide radical. These new advances allowed us toinitiate studies on the exact role of glutathione, hydrogenperoxide, and superoxide radical on sclerotial metamorphosis.The emerging data, combined with similar data from other better-studiedfungi, allowed us to make some preliminary postulations on theROS-dependent biochemical signal transduction pathways in sclerotiogenicfilamentous fungi.     

An internal carbon source for improving biological nutrient removal

This study investigates the potential of mechanically disintegrated surplus activated sludge (SAS) to be used as an internal carbon source for biological nutrient removal (BNR) using two laboratory tests. In the phosphorus release test, the addition of disintegrated sludge as a carbon source was able to enhance phosphate (PO(4)-P) release by 14.9 mg l(-1) PO(4)-P when compared with acetate (7.9 mg l(-1) PO(4)-P), considering the 4.3 mg l(-1) PO(4)-P released in the control vessel, without carbon addition. Similarly, in the denitrification test, the nitrate (NO(3)-N) consumption rate was improved after the addition of disintegrated sludge (14.9 mg NO(3)-Ng(-1)VSS h(-1)) compared with acetate (7.0 mg NO(3)-Ng(-1)VSS h(-1)), taking in consideration the rate obtained in the control vessel (6.9 mg NO(3)-Ng(-1)VSS h(-1)). Two to five minutes of SAS disintegration time in the deflaker (2300-6200 kJ kg(-1) total solids) is recommended for this application.