Alexandra Thiele

Vitamin D Receptor Deficiency and Low Vitamin D Diet Stimulate Aortic Calcification and Osteogenic Key Factor Expression in Mice

Low levels of 25-hydroxy vitamin D (25(OH)D) are associated with cardiovascular diseases. Herein, we tested the hypothesis that vitamin D deficiency could be a causal factor in atherosclerotic vascular changes and vascular calcification. Aortic root sections of vitamin D receptor knockout (VDR−/−) mice that were stained for vascular calcification and immunostained for osteoblastic differentiation factors showed more calcified areas and a higher expression of the osteogenic key factors Msx2, Bmp2, and Runx2 than the wild-type mice (P<0.01). Data from LDL receptor knockout (LDLR−/−) mice that were fed western diet with either low (50 IU/kg), recommended (1,000 IU/kg), or high (10,000 IU/kg) amounts of vitamin D3 over 16 weeks revealed increasing plasma concentrations of 25(OH)D (P<0.001) with increasing intake of vitamin D, whereas levels of calcium and phosphorus in plasma and femur were not influenced by the dietary treatment. Mice treated with the low vitamin D diet had more calcified lesions and a higher expression of Msx2, Bmp2, and Runx2 in aortic roots than mice fed recommended or high amounts of vitamin D (P<0.001). Taken together, these findings indicate vitamin D deficiency as a risk factor for aortic valve and aortic vessel calcification and a stimulator of osteogenic key factor expression in these vascular areas.

Deciphering Enzyme Function Using Peptide Arrays

Enzymes are key molecules in signal-transduction pathways. However, only a small fraction of more than 500 human kinases, 300 human proteases and 200 human phosphatases is characterised so far. Peptide microarray based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimisation of enzyme substrates. Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases. Additionally, several examples for reliable identification of substrates for lysine methyl-transferases, histone deacetylases and SUMO-transferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples like cell lysates or lysates of complete organisms is described. All published examples of peptide arrays used for enzyme profiling are summarised comprehensively.

Peptide Arrays for Enzyme Profiling

Enzymes are key molecules in signal transduction pathways. However, only a small fraction of more than 500 predicted human kinases, 250 proteases and 250 phosphatases is characterized so far. Peptide microarray-based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. Additionally, patterns of enzymatic activities could be used to fingerprint the status of cells or organisms. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimization of enzyme substrates. A comprehensive overview regarding enzyme profiling using peptide microarrays is presented with special focus on assay principles.

Collagen IV-derived peptide binds hydrophobic cavity of Legionella pneumophila Mip and interferes with bacterial epithelial transmigration.

The Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to bacterial dissemination within infected lung tissue. The Mip protein, which belongs to the enzyme family of FK506‐binding proteins (FKBP), binds specifically to collagen IV. We identified a surface‐exposed Mip‐binding sequence in the NC1 domain of human collagen IV α1. The corresponding collagen IV‐derived peptide (P290) co‐precipitated with Mip and competitively inhibited the Mip–collagen IV binding. Transmigration of Legionella pneumophila across a barrier of NCI‐H292 lung epithelial cells and extracellular matrix was efficiently inhibited by P290. This significantly reduced transmigration was comparable to the inefficient transmigration of PPIase‐negative Mip mutant or rapamycin‐treated L. pneumophila. Based on NMR data and docking studies a model for the mode of interaction of P290 and Mip was developed. The amino acids of the hydrophobic cavity of Mip, D142 and to a lesser extent Y185 were identified to be part of the interaction surface. In the complex structure of Mip77–213 and P290, both amino acid residues form hydrogen bonds to P290. Utilizing modelling, molecular dynamics (MD) simulations and structural data of human PPIase FKBP12, the most related human orthologue of Mip, we were able to propose optimized P290 variants with increased binding specificity and selectivity for the putative bacterial drug target Mip.

Parvulin 17 Promotes Microtubule Assembly by Its Peptidyl-Prolyl Cis/Trans Isomerase Activity

The parvulin-type peptidyl-prolyl cis/trans isomerases (PPIases) have been shown to be involved in tumor progression and the pathogenesis of Alzheimers disease and were therefore a subject of intense research. Here, we describe a role for parvulin 17 in microtubule assembly. Co-precipitation experiments and sedimentation assays demonstrated that parvulin 17 interacts with tubulin in a GTP-dependent manner and thereby promotes the formation of microtubules, as shown by transmission electron microscopy and a microtubule polymerization assay. The microtubule-assembly-promoting properties of parvulin 17 seem to depend on its PPIase activity. Thus, catalytic deficient variants of parvulin 17 were not able to promote microtubule formation. Accordingly, inhibitors of parvulin 17 activity also prevent parvulin-catalyzed tubulin polymerization. The analysis of tubulin interaction sites on parvulin using peptide microarrays revealed that tubulin interacts with the substrate binding pocket of parvulin. Additionally, β-tubulin peptide scan on microarrays demonstrates interaction of parvulin 17 with an Arg-Pro-Asp motif corresponding to proline residue 87 of β-tubulin. Confocal laser scanning microscopy points to a function of parvulin 17 in microtubule dynamics as well. Parvulin 17 is predominantly found in the cytosol and colocalizes with microtubules.

High-Density Peptide Microarrays for Reliable Identification of Phosphorylation Sites and Upstream Kinases

The human genome encodes about 25,000 genes. This number seems to be very small compared to the multitude of different protein functions in highly regulated pathways that are responsible for complex biochemical mechanisms like growth, metabolism, signal transduction and reproduction. Obviously, there are mechanisms creating additional protein diversity. The most important mechanism is post-translational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation and sumoylation resulting in an about 100-fold higher complexity (1, 2). This chapter presents a very efficient way to detect potential phosphorylation sites in proteins using overlapping peptide scans immobilized on glass slides. Results from 35 different human kinases using peptide microarrays displaying overlapping peptide scans through either all human cyclophilins or all human FK506-binding proteins are shown. Additionally, detection of phosphorylation sites in a proteome-wide manner is demonstrated using peptide microarrays displaying cytomegalovirus proteome in the form of more than 17,000 overlapping peptides.

High density peptide microarrays for proteome-wide fingerprinting of kinase activities in cell lysates.

Protein function is highly regulated in pathways that are responsible for complex biochemical mechanisms such as growth, metabolism, and signal transduction. One of the most important mechanisms is posttranslational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation, glycosylation, and sumoylation resulting in a more than 100-fold higher complexity (Geiss-Friedlander and Melchior, Nat Rev Mol Cell Biol 8, 947-956, 2007; Hunter, Mol Cell 28, 730-738, 2007). This chapter presents a very efficient way to detect potential phosphorylation sites in protein families using overlapping peptides covering the complete primary structures (peptide scans) immobilized on glass slides. Results of kinase activity fingerprinting of cell lysates using peptide microarrays displaying peptide scans through all human peptidyl-prolyl-cis/trans-isomerases are shown.

1H, 13C and 15N resonance assignments of human parvulin 17

A 25-residue elongation at the N-terminus endows parvulin 17 (Par17) with altered functional properties compared to parvulin 14 (Par14), such as an enhanced influence on microtubule assembly. Therefore the three-dimensional structure of this N-terminal elongation is of particular interest. Here, we report the nearly complete 1H, 13C and 15N chemical shift assignments of Par17. Subsequent chemical shift index analysis indicated that Par17 features a parvulin-type PPIase domain at the C-terminus, analogous to Par14, and an unstructured N-terminus encompassing the first 60 residues. Hence the N-terminus of Par17 apparently adopts a functionally-relevant structure only in presence of the respective interaction partner(s).

Peptide microarrays for determination of cross-reactivity.

Polyclonal antibodies raised against full-length antigens are often used for localization experiments. Exact knowledge of epitopes in the antigen recognized by the antiserum is important if the target antigen belongs to a large family of proteins which are highly conserved. We have shown that epitope mapping using peptide microarrays represents a powerful tool for determination of immunodominat regions in a proteome-wide manner. As examples we show results of epitope mapping using peptide microarrays displaying overlapping peptide scans through either all human cyclophilins or all human FK506-binding proteins.