| 1. |
- Brautigam, Lars, et al.
(författare)
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Glutaredoxin regulates vascular development by reversible glutathionylation of sirtuin 1
- 2013
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:50, s. 20057-20062
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Tidskriftsartikel (refereegranskat)abstract
- Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione-disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S-glutathionylation of the NAD(+)-dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S-glutathionylation to vertebrate development and successful embryonic angiogenesis.
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| 5. |
- Huang, Mingtao, 1984, et al.
(författare)
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Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
- 2015
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:34, s. E4689-E4696
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Tidskriftsartikel (refereegranskat)abstract
- There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.
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| 6. |
- Kontijevskis, Aleksejs, et al.
(författare)
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Proteochemometric analysis of small cyclic peptides' interaction with wild-type and chimeric melanocortin receptors
- 2007
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 69:1, s. 83-96
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Tidskriftsartikel (refereegranskat)abstract
- The melanocortin (MC) system confines unique G-protein coupled receptor pathways, which include the MC1-5 receptors and their endogenous agonists and antagonists, the MCs and the agouti and agouti-related proteins. The MC4 receptor is an important target for development of drugs for treatment of obesity and cachexia. While natural MC peptides are selective for the MC1 receptor, some cyclic pentapeptides, such as the HS-129 peptide, show high selectivity for the MC4 receptor. Here we gained insight into the mechanisms for its recognition by MC receptors. To this end we correlated the interaction data of four HS peptide analogues with four wild-type and 14 multiple chimeric MC receptors to the binary and physicochemical descriptions of the studied entities by use of partial least squares regression, which resulted in highly valid proteochemometric models. Analysis of the models revealed that the recognition sites of the HS peptides are different from the earlier proteochemometrically mapped linear MSH peptides' recognitions sites, although they overlap partially. The analysis also revealed important amino acids that explain the selectivity of the HS-129 peptide for the MC4 receptor.
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| 7. |
- Lapinsh, Maris, et al.
(författare)
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Improved approach for proteochemometrics modeling : application to organic compound--amine G protein-coupled receptor interactions
- 2005
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811 .- 1460-2059. ; 21:23, s. 4289-4296
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Tidskriftsartikel (refereegranskat)abstract
- MOTIVATION: Proteochemometrics is a novel technology for the analysis of interactions of series of proteins with series of ligands. We have here customized it for analysis of large datasets and evaluated it for the modeling of the interaction of psychoactive organic amines with all the five known families of amine G protein-coupled receptors (GPCRs). RESULTS: The model exploited data for the binding of 22 compounds to 31 amine GPCRs, correlating chemical descriptions and cross-descriptions of compounds and receptors to binding affinity using a novel strategy. A highly valid model (q2 = 0.76) was obtained which was further validated by external predictions using data for 10 other entirely independent compounds, yielding the high q2ext = 0.67. Interpretation of the model reveals molecular interactions that govern psychoactive organic amines overall affinity for amine GPCRs, as well as their selectivity for particular amine GPCRs. The new modeling procedure allows us to obtain fully interpretable proteochemometrics models using essentially unlimited number of ligand and protein descriptors.
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| 9. |
- Lapinsh, Maris, et al.
(författare)
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Proteochemometric mapping of the interaction of organic compounds with melanocortin receptor subtypes
- 2005
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Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 67:1, s. 50-59
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Tidskriftsartikel (refereegranskat)abstract
- Proteochemometrics was applied in the analysis of the binding of organic compounds to wild-type and chimeric melanocortin receptors. Thirteen chimeric melanocortin receptors were designed based on statistical molecular design; each chimera contained parts from three of the MC(1,3-5) receptors. The binding affinities of 18 compounds were determined for these chimeric melanocortin receptors and the four wild-type melanocortin receptors. The data for 14 of these compounds were correlated to the physicochemical and structural descriptors of compounds, binary descriptors of receptor sequences, and cross-terms derived from ligand and receptor descriptors to obtain a proteochemometric model (correlation was performed using partial least-squares projections to latent structures; PLS). A well fitted mathematical model (R(2) = 0.92) with high predictive ability (Q(2) = 0.79) was obtained. In a further validation of the model, the predictive ability for ligands (Q(2)lig = 0.68) and receptors (Q(2)rec = 0.76) was estimated. The model was moreover validated by external prediction by using the data for the four additional compounds that had not at all been included in the proteochemometric model; the analysis yielded a Q(2)ext = 0.73. An interpretation of the results using PLS coefficients revealed the influence of particular properties of organic compounds on their affinity to melanocortin receptors. Three-dimensional models of melanocortin receptors were also created, and physicochemical properties of the amino acids inside the receptors' transmembrane cavity were correlated to the PLS modeling results. The importance of particular amino acids for selective binding of organic compounds was estimated and used to outline the ligand recognition site in the melanocortin receptors.
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