Construction and characterization of protein libraries composed of secondary structure modules

Only a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all alpha-helix, all beta-strand, and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand, and beta-turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing alpha-helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly beta-strand modules tended to form amyloid-like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non-beta-strand-containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native-like properties despite lack of restriction in topology. Because of the intrinsic tendency of beta-strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.     

LIF receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival

Multiple sclerosis (MS) is a disabling inflammatory demyelinating disease of the central nervous system (CNS) that primarily affects young adults. Available therapies can inhibit the inflammatory component of MS but do not suppress progressive clinical disability. An alternative approach would be to inhibit mechanisms that drive the neuropathology of MS, which often includes the death of oligodendrocytes, the cells responsible for myelinating the CNS. Identification of molecular mechanisms that mediate the stress response of oligodendrocytes to optimize their survival would serve this need. This study shows that the neurotrophic cytokine leukemia inhibitory factor (LIF) directly prevents oligodendrocyte death in animal models of MS. We also demonstrate that this therapeutic effect complements endogenous LIF receptor signaling, which already serves to limit oligodendrocyte loss during immune attack. Our results provide a novel approach for the treatment of MS.     

Metal-mediated reactions modeled after nature

The Collaborative Research Center (CRC) 436 'Metal-Mediated Reactions Modeled after Nature' was founded for the express purpose of analyzing the catalytic principles of metallo-enzymes in order to construct efficient catalysts on a chemical basis. The structure of the active center and neighboring chemical environment in enzymes serves as a focal point for developing reactivity models for the chemical redesign of catalysts. Instead of simply copying enzyme construction, we strive to achieve new chemical intuition based on the results of long-lasting natural evolution. We hope for success, since nature uses a limited set of building blocks, whereas we can apply the full repertoire of chemistry. Key substrates in this approach are small molecules, such as CO2, O2 NO3- and N2. Nature complexes these substrates, activates them and performs chemical transformations--all within the active center of a metalloenzyme. In this article, we report on some aspects and first results of the Collaborative Research Center (CRC) 436, such as nitrate reductase, sphingolipid desaturase, carbonic anhydrase, leucine aminopeptidase and dopamine beta-monooxygenase.     

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.     

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.     

A study of associations of the H-FABP genotypes with fat and meat production of pigs

Although the content of intramuscular fat (IMF) influences significantly meat quality, it can be estimated only after the slaughter of animals. Variants of the H-FABP gene were suggested as candidate genes influencing the variability of IMF. The effect of H-FABP - HinfI polymorphism on the content of IMF, backfat thickness, the weight and percentages of major meat parts and of the leg in carcass weight was studied in a group of 97 pigs (46 gilts and 51 barrows) of Large White and Landrace breeds using the test of fattening capacity and carcass value. In the set of experimental animals, the frequencies of genes were H = 0.75 +/- 0.03 and h = 0.25 +/- 0.03. Biometric analyses did not corroborate differences among different H-FABP - HinfI genotypes and all the traits under study. Only in genotypes HH and Hh the differences between least-square means of phenotypic IMF values under study were close to the limit of significance (P = 0.06).     

Extracellular matrix proteins influence phenotype and cytokine expression in human breast cancer cell lines

Tumor growth and invasion are not only the result of malignant transformation but are also dependent on environmental influences from surrounding stroma, extracellular matrix (ECM), local cytokines and systemic hormones. We have investigated the influence of ECM components on three human breast cancer cell lines of different malignant potential: MCF-7, T47D and MDA-MB-231 were cultured on collagen I, collagen IV, laminin, fibronectin or poly-D-lysine, and we analyzed the proliferation rate and cytokine expression pattern. Among the three cell lines investigated we observed a distinct response to each ECM component. We hypothesize that ECM may have a significant modulatory effect on malignant behavior in vivo which might depend on individual responses and on the differentiation state of tumor cells. This study also shows that the surface on which cells are cultured greatly influences cell kinetics and the cytokine expression pattern.     

BiP binding keeps ATF6 at bay

The erythropoietin receptor transduces signals leading to the growth, differentiation, and survival of red blood cell precursors via interaction with Janus kinase 2 (JAK2). This interaction was thought to occur only at the plasma membrane. Recent evidence, however, shows that JAK2 assembles with newly synthesized erythropoietin receptors in the endoplasmic reticulum, and that this assembly is essential for efficient expression of the receptors at the cell surface.