FEBS Journal

A number of recombinant proteins aggregate in bacterial host cells building insoluble inclusion bodies. We summarise recent insight in the functional and structural properties of inclusion bodies, their dynamics, and their biological role. Potential uses of aggregated proteins in biotechnology and as models to study amyloids formation are described, as well as the computational methods available to predict aggregation propensity.

In the bacterial cytoplasm a number of recombinant proteins aggregate to form inclusion bodies. This first review is focused on the cellular events leading to aggregation and describes the biophysical techniques to reveal the structure of proteins within inclusion bodies. Since aggregated proteins can retain native-like structure and biological conformation, possible application in biotechnology is also discussed.

Many eukaryotic polypeptides form inclusion bodies when producedin bacteria. The dissection of the quality control machinery in bacteria and the discovery of the amyloid-like nature of bacterial aggregates have provided an excitingand new view on their dynamic biology, suggesting thatthey may serve as models to study the mechanisms of amyloid aggregation in physiologically relevant conditions.

Proteins might aggregate into ordered or amorphous structures, utilizing relatively short sequence stretches. Here, we review available software, for the prediction of such aggregation-prone stretches from protein primary structure. The results of application of four of these programs on 23 proteins related to amyloidoses are compared. An example is given of how these algorithms might become useful tools to improve the solubility of recombinant proteins, avoiding formation of bacterial inclusion bodies.

Many tumors exhibit an impaired Pasteur effect, i.e. despite the presence of oxygen, glucose is consumed with an extraordinarily high rate – the so-called ‘Warburg effect’. We compile current knowledge on enzymes for glucose utilization in tumor cells and describe how tumor cells acquire specific enzyme-regulatory mechanisms to direct the flux of glucose to those pathways most urgently needed.

Clostridial glutamate dehydrogenase was thought to favour NAD⁺ over NADP⁺ by 300 : 1. Puzzlingly, mutations that markedly shifted specificity in favour of NADPH did not favour NADP⁺. Careful analysis reveals the 300 : 1 ratio reflects levels of NAD⁺ contamination in commercial NADP⁺. With purified coenzyme the true activity ratio is 11,000 and the mutations do indeed dramatically shift this ratio.

Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data, and identified a secondary binding site for the substrate close to the catalytic site.

Apple peel contains high levels of the triterpene ursolic acid derived from α-amyrin. We have identified three oxidosqualene cyclases (MdOSC1-3). We have shown that one (MdOSC1) is highly expressed in peel and produces α-amyrin at a ratio of 5 : 1 with β-amyrin, and therefore may be ultimately responsible for the production of ursolic acid in this tissue.

Mitochondrial DNA (mtDNA) deletion mutations play a crucial role in presbycusis. We reported that mitochondrial base excision repair (BER) capacity deficiency and increased mtDNA replication resulting from mitochondrial transcription factor A (TFAM) overexpression are involved in the accumulation of mtDNA deletions in inner ear during aging. These findings provide new insight into the development of presbycusis.

Here,AraL from Bacillus subtilisis shown to be a phosphatase displaying activity towards different sugar phosphate substrates. Furthermore, we provide evidence that production of AraL is regulated by the formation of an mRNA secondary structure, which sequesters the ribosome-binding site and consequently prevents translation. AraL is the first sugar phosphatase belonging to the family of NagD-like phosphatases characterized at the level of gene regulation.

Mammalian monothiol Grx3 is able to rescue growth defects of yeast mutant grx3grx4 cells and to protect cells against oxidative stress. Disruption of Grx3 causes early embryo death. Mouse embryonic fibroblasts from Grx3 null mice display impaired growth and cell cycle progression at the G2/M phase. The defective cell cycle progression during late mitosis may account for early embryonic lethality.

Two pathways of l-glutamate catabolism were detected in the anaerobic bacterium F. varium. The methylaspartate route (A) was prominent, but the hydroxyglutarate pathway (B) functioned when cobalt ion and coenzyme B₁₂ were absent from the culture medium. The detection of a cofactor independent glutamate racemase in cell extracts accounted for the catabolism of d-glutamate by F. varium.

We describe the production of four N-glycosylation nucleolin variants lacking the N-terminal acidic domain in a baculovirus/insect cell system. Our results demonstrate that all nucleolin-derived variants are able to self-interact and that N-glycosylation on both RBD1 and 3, or RBD3 alone, but not RBD1 alone, modifies the structure of the N-terminally truncated nucleolin and enhances its self-association properties.

The design principles behind metabolic patterns of responses are still poorly understood. We develop a modular approach to analyze large changes in branched complex systems. The method reveals the sites of modulation to obtain large variable changes, without having to perform any parameter modulations. Application to a detailed model of aspartate metabolism determines the sites where the fluxes are controlled.

Cyclic-nucleotide phosphodiesterases (PDEs) are vital proteins in signal-transduction pathways, and thus are popular targets for inhibition by drugs (e.g., Viagra^®). We simulated PDE4B enzymatic reactions by generating a 30-ns two-dimensional QM/MM free-energy profile. The activation and reaction free energies are in good agreement with experiments. From the free-energy profile, we determine the reaction mechanism is an asynchronous S_N2 type.

We identified an alternative splicing event in the 5′ region of human Disc Large (DLG1) mRNA that generates transcripts with two different 5′-UTRs. We show that the DLG1 5′-UTR containing an alternatively-spliced exon interferes with the translation of a downstream ORF. This study shows for the first time that differential expression of alternatively spliced 5′UTRs could result in changes in DLG1 abundance.