Spontaneous proton transfer to a conserved intein residue determines on-pathway protein splicing

The discovery of inteins, which are protein-splicing elements, has stimulated interest for various applications in chemical biology, bioseparations, drug delivery, and sensor development. However, for inteins to effectively contribute to these applications, an increased mechanistic understanding of cleavage and splicing reactions is required. While the multistep chemical reaction that leads to splicing is often explored and utilized, it is not clear how the intein navigates through the reaction space. The sequence of reaction steps must progress in concert in order to yield efficient splicing while minimizing off-pathway cleavage reactions. In this study, we demonstrate that formation of a previously identified branched intermediate is the critical step for determining splicing over cleavage products. By combining experimental assays and quantum mechanical simulations, we identify the electrostatic interactions that are important to the dynamics of the reaction steps. We illustrate, via an animated simulation trajectory, a proton transfer from the first C-terminal extein residue to a conserved aspartate, which synchronizes the multistep enzymatic reaction that is key to splicing. This work provides new insights into the complex interplay between critical active-site residues in the protein splicing mechanism, thereby facilitating biotechnological application while shedding light on multistep enzyme activity.     

A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos

Infection of the zebrafish with Mycobacterium marinum is regarded as a well-established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin-layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall-associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.     

Correlation between decreased reparative capability of UV lesions and higher spontaneous mutability in a mutator strain of E. coli K-12

Summary The biauxotrophic strain of E. coli K-12 (), met 1/his 7, which exhibits an 8 times higher rate of met1met ⁺-backmutation as compared with the parent strain met 1, was found to be also more sensitive to UV irradiation. In addition, the maximum UV induction of prophage occurs at lower doses, and the capacity of the strain to propagate induced prophage is reduced. The mutant strain has also lost part of the ability to repair UV-induced lesions in phage T 1.The phenotypes high mutability and UV sensitivity could not be separated by means of recombination.From these findings it is concluded that part of the spontaneously occuring changes in DNA (premutations) which lead to met ⁺ genotype are similarly repaired as some UV induced ones, and that the mutator mum ⁺ of strain met 1/his 7 causes a reduced repair of both changes.     

Une station française dePhyllocardium complanatum Korshikov

Sans résumé     

Ectopic Expression of Retrotransposon-Derived PEG11/RTL1 Contributes to the Callipyge Muscular Hypertrophy

The callipyge phenotype is an ovine muscular hypertrophy characterized by polar overdominance: only heterozygous + ^Mat /CLPG ^Pat animals receiving the CLPG mutation from their father express the phenotype. + ^Mat /CLPG ^Pat animals are characterized by postnatal, ectopic expression of Delta-like 1 homologue (DLK1) and Paternally expressed gene 11/Retrotransposon-like 1 (PEG11/RTL1) proteins in skeletal muscle. We showed previously in transgenic mice that ectopic expression of DLK1 alone induces a muscular hypertrophy, hence demonstrating a role for DLK1 in determining the callipyge hypertrophy. We herein describe newly generated transgenic mice that ectopically express PEG11 in skeletal muscle, and show that they also exhibit a muscular hypertrophy phenotype. Our data suggest that both DLK1 and PEG11 act together in causing the muscular hypertrophy of callipyge sheep.     

Proteomics of life at low temperatures: trigger factor is the primary chaperone in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

The proteomes expressed at 4°C and 18°C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two‐dimensional differential in‐gel electrophoresis, showing that translation, protein folding, membrane integrity and anti‐oxidant activities are upregulated at 4°C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl‐prolyl cis‐trans isomerase activity. This suggests that protein folding at low temperatures is a rate‐limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat‐shock proteins) are downregulated at 4°C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33°C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperature‐dependent and requires near‐zero temperature to stably bind a model‐unfolded polypeptide.