Potential of Fragment Recombination for Rational Design of Proteins

It is hypothesized that protein domains evolved from smaller intrinsically stable subunits via combinatorial assembly. Illegitimate recombination of fragments that encode protein subunits could have quickly led to diversification of protein folds and their functionality. This evolutionary concept presents an attractive strategy to protein engineering, e.g., to create new scaffolds for enzyme design. We previously combined structurally similar parts from two ancient protein folds, the (βα)(8)-barrel and the flavodoxin-like fold. The resulting "hopeful monster" differed significantly from the intended (βα)(8)-barrel fold by an extra β-strand in the core. In this study, we ask what modifications are necessary to form the intended structure and what potential this approach has for the rational design of functional proteins. Guided by computational design, we optimized the interface between the fragments with five targeted mutations yielding a stable, monomeric protein whose predicted structure was verified experimentally. We further tested binding of a phosphorylated compound and detected that some affinity was already present due to an intact phosphate-binding site provided by one fragment. The affinity could be improved quickly to the level of natural proteins by introducing two additional mutations. The study illustrates the potential of recombining protein fragments with unique properties to design new and functional proteins, offering both a possible pathway of protein evolution and a protocol to rapidly engineer proteins for new applications

Influence of ascorbate-recycling, light and temperature during tomato fruit ripening on ascorbate pool and ascorbate-degradation

International audienc

Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition

The LINE-1 (L1) retrotransposon emerges as a major source of human interindividual genetic variation, with important implications for evolution and disease. L1 retrotransposition is poorly understood at the molecular level, and the mechanistic details and evolutionary origin of the L1-encoded L1ORF1 protein (L1ORF1p) are particularly obscure. Here three crystal structures of trimeric L1ORF1p and NMR solution structures of individual domains reveal a sophisticated and highly structured, yet remarkably flexible, RNA-packaging protein. It trimerizes via an N-terminal, ion-containing coiled coil that serves as scaffold for the flexible attachment of the central RRM and the C-terminal CTD domains. The structures explain the specificity for single-stranded RNA substrates, and a mutational analysis indicates that the precise control of domain flexibility is critical for retrotransposition. Although the evolutionary origin of L1ORF1p remains unclear, our data reveal previously undetected structural and functional parallels to viral proteins

Structural basis for the multiple roles Edc3 plays in mRNA degradation

The Dcp1:Dcp2 decapping complex catalyzes the removal of the protecting 5’ cap structure from mRNA. Adaptor proteins, including Edc3 (enhancer of decapping 3), modulate this decapping process through multiple mechanisms. First, the Edc3 protein enhances the activity of the Dcp2 enzyme directly. Secondly, Edc3 is involved in the formation of cellular processing bodies. Finally, Edc3 is important for the deadenylation independent degradation of the Rps28b mRNA. In the latter case, cellular Rps28b protein levels are regulated through a unique auto-regulatory pathway, where Rps28b that is not in complex with the ribosome binds to a specific stem-loop in the 3’-UTR of its own messenger-RNA. To understand how the Edc3 protein is able to preform these multiple functions, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. Based on that structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. We show that these multiple HLMs in Dcp2, together with the dimeric nature of Edc3, can lead to the formation of a network of intermolecular interaction. Our experiments thus provide initial insights into one of the mechanisms that underlie processing body formation. Finally, we solved the structure of the Edc3 LSm domain in complex with the Rps28b protein. These data display how the dimeric Edc3 protein is able to bring the Rps28b mRNA and the Dcp1:Dcp2 decapping complex together, thereby targeting the mRNA for degradation. In summary, we show that the Edc3 LSm domain is a plastic platform for multiple protein:protein interactions that are important for the regulation of mRNA degradation

Plant, Cell and Environment

Ascorbate is oxidized into the radical monodehydroascorbate (MDHA) through ascorbate oxidase or peroxidase activity or non-enzymatically by reactive oxygen species. Regeneration of ascorbate from MDHA is ensured by the enzyme monodehydroascorbate reductase (MDHAR). Previous work has shown that growth processes and yield can be altered by modifying the activity of enzymes that recycle ascorbate, therefore we have studied similar processes in cherry tomato (Solanum lycopersium L.) under- or overexpressing MDHAR. Physiological and metabolic characterization of these lines was carried out under different light conditions or by manipulating the source-sink ratio. Independently of the light regime, slower early growth of all organs was observed in MDHAR silenced lines, decreasing final fruit yield. Stomatal conductance and photosynthesis were altered as was the accumulation of hexoses and sucrose in a light-dependent manner in plantlets. Sucrose accumulation was also repressed in young fruits and final yield of MDHAR silenced lines showed a stronger decrease under carbon limitation. Ascorbate and monodehydroascorbate appear to be involved in control of growth and sugar metabolism in cherry tomato and these enzymes could be potential targets for yield improvemen

Circulating mi RNA

OBJECTIVE: Myasthenia gravis (MG) is a chronic autoimmune disorder where autoantibodies target the nicotinic acetylcholine receptors (AChR+) in about 85% of cases, in which the thymus is considered to play a pathogenic role. As there are no reliable biomarkers to monitor disease status in MG, we analyzed circulating miRNAs in sera of MG patients to find disease-specific miRNAs. METHODS: Overall, 168 miRNAs were analyzed in serum samples from four AChR+ MG patients and four healthy controls using Exiqon Focus miRNA polymerase chain reaction (PCR) Panel I + II. Specific accumulation pattern of 13 miRNAs from the discovery set was subsequently investigated in the sera of 16 AChR+ MG patients and 16 healthy controls. All patients were without immunosuppressive treatment. Selected specific miRNAs were further analyzed in the serum of nine MG patients before and after thymectomy to assess the effect of thymus removal on the accumulation of the candidate miRNAs in patient sera. RESULTS: Three miRNAs were specifically dysregulated in AChR+ MG patient sera samples. Hsa-miR150-5p, which induces T-cell differentiation, as well as hsa-miR21-5p, a regulator of Th1 versus Th2 cell responses, were specifically elevated in MG sera. Additionally, hsa-miR27a-3p, involved in natural killer (NK) cell cytotoxicity, was decreased in MG. Hsa-miR150-5p levels had the highest association with MG and were significantly reduced after thymus removal in correlation with disease improvement. INTERPRETATION: We propose that the validated miRNAs: hsa-miR150-5p, hsa-miR21-5p, and hsa-miR27a-3p can serve as novel serum biomarkers in AChR+ MG. Hsa-miR-150-5p could be a helpful marker to monitor disease severity

Control of eukaryotic phosphate homeostasis by SPX inositol polyphosphate sensor domains

Phosphate is an important macronutrient and thus eukaryotic cells tightly regulate their intracellular phosphate (Pi) levels. Pi homeostasis can be maintained by adapting phosphate uptake, storage and transport. However, it is poorly understood how cells sense and signal their phosphate status.SPX domains are found at the N-terminus of eukaryotic phosphate transporters, inorganic polyphosphate polymerases and signaling proteins. I will present structural, biochemical and genetic evidence that SPX domains are sensors for inositol polyphosphate (InsP) ligands, signaling molecules whose concentration change in response to phosphate availability1. Mutations in the SPX InsP binding pocket impair InsP binding in biochemical binding assays, down-regulate synthesis of inorganic polyphosphate in yeast and reduce phosphate transport in Arabidopsis. Further, InPs trigger the interaction between stand-alone plant SPX proteins with a family of phosphate-starvation responsive transcription factors, thereby controlling the induction of phosphate starvation responses under low Pi.Taking together, we suggest that InsPs act as novel signaling molecules in fungi, plants and animals. Binding of InsPs allows SPX domains to bind and regulate their downstream signaling partners and thus to regulate phosphate homeostasis in eukaryotes