Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression.

BACKGROUND: S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879-41887). RESULTS: In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ). CONCLUSION: The candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer.

Surface plasmon resonance/mass spectrometry interface.

A strategy for combining surface plasmon resonance (SPR) biomolecular interaction analysis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. Both techniques are highly complementary but need separate optimization to improve their individual specificity and sensitivity. Sensor surfaces that are optimal for kinetic analysis are not well suited for MALDI-MS and vice versa. In addition, the transfer of analyte from SPR to MS is crucial and often accompanied by sample loss. To address both of these points, a bifunctional SPR fluid cell was constructed where optimized surfaces can be used for binding studies and MS simultaneously with regard to the special need of each technique. The setup guarantees that the SPR and the loading experiment for MS are performed at identical conditions. A removable pin carries the affinity-surface-bound analyte to the mass spectrometer so that handling is minimized, avoiding analyte elution. Functionalized transfer pins can also be used independently of SPR for microaffinity capture-MS.