Patrick Schindler

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, THE PUTATIVE TARGET OF THE ANTIAPOPTOTIC COMPOUNDS CGP 3466 AND R-(-)-DEPRENYL

R-(−)-Deprenyl (Selegiline) represents one of the drugs currently used for the treatment of Parkinson’s disease. This compound was shown to protect neurons or glias from programmed cell death in a variety of models. The mechanism of action of neuroprotection as well as inhibition of apoptosis remains elusive. CGP 3466 is a structurally related analog ofR-(−)-deprenyl that exhibits virtually no monoamine oxidase type B inhibiting activity but is neuroprotective in the picomolar concentration range. We showed specific binding of CGP 3466 to glyceraldehyde-3-phosphate dehydrogenase by affinity binding, by affinity labeling, and by means of BIAcore® technology. Apoptosis assays based on the human neuroblastoma cell line PAJU established the importance of this interaction for mediating drug-induced inhibition of programmed cell death.

Fluoroaluminate stimulates phosphorylation of p130 Cas and Fak and increases attachment and spreading of preosteoblastic MC3T3-E1 cells.

Fluoroaluminate is a G-protein activator, it stimulates osteoblastic cells in culture, and is a bone-forming agent in vivo. To elucidate the mechanisms of G-protein-mediated action of fluoroaluminate in osteoblasts, we studied protein tyrosine phosphorylation in the preosteoblastic cell line MC3T3-E1. Fluoroaluminate, lysophosphatidic acid (LPA; an agonist for G-protein-coupled receptor), or adhesion to type I collagen all stimulated phosphorylation of a similar set of proteins, including p130, p120, p110 (previously identified as proline-rich tyrosine kinase 2, Pyk2), and p70. The phosphorylation of these proteins was sensitive to an Src inhibitor, but not to a Gi-protein inactivator, pertussis toxin. By purification/mass spectrometry and by immunodepletion, p130 protein was identified as p130 Cas (Crk-associated protein), a Src substrate and a protein involved in signaling by cell-adhesion receptors, integrins. Phosphorylation of immunoprecipitated p130 Cas increased upon stimulation with fluoroaluminate and with agonists of G-protein-coupled receptors, but not with growth factors. By immunodepletion, the p120 protein was identified as focal adhesion kinase, Fak. The addition of fluoroaluminate during cell attachment to type I collagen further stimulated phosphorylation of p130 Cas and of Fak. Simultaneously, fluoroaluminate increased the number of attached MC3T3-E1 cells and their spreading. These novel aspects of fluoroaluminate action in cell culture may be important for the bone-forming action of fluoroaluminate in vivo.

Post-translational modification detection using metastable ions in reflector matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

In addition to protein identification, characterization of post‐translational modifications (PTMs) is an essential task in proteomics. PTMs represent the major reason for the variety of protein isoforms and they can influence protein structure and function. Upon matrix‐assisted laser desorption/ionization (MALDI) most post‐translationally modified peptides form a fraction of labile molecular ions, which lose PTM‐specific residues only after acceleration. Compared to fully accelerated ions these fragment ions are defocused and show in reflector mass spectra reduced resolution. A short time Fourier transform using a Hanning window function now uses this difference in resolution to detect the metastable fragments. Its application over the whole mass range yields frequency distributions and amplitudes as a function of mass, where an increased low frequency proportion is highly indicative for metastable fragments. Applications on the detection of metastable losses originating from carboxamidomethylated cysteines, oxidized methionines, phosphorylated and glycosylated amino acid residues are presented. The metastable loss of mercaptoacetamide detected with this procedure represents a new feature and its integration in search algorithms will improve the specificity of MALDI peptide mass fingerprinting.

Automated harvesting and 2-step purification of unclarified mammalian cell-culture broths containing antibodies.

Therapeutic monoclonal antibodies represent one of the fastest growing segments in the pharmaceutical market. The growth of the segment has necessitated development of new efficient and cost saving platforms for the preparation and analysis of early candidates for faster and better antibody selection and characterization. We report on a new integrated platform for automated harvesting of whole unclarified cell-culture broths, followed by in-line tandem affinity-capture, pH neutralization and size-exclusion chromatography of recombinant antibodies expressed transiently in mammalian human embryonic kidney 293T-cells at the 1-L scale. The system consists of two bench-top chromatography instruments connected to a central unit with eight disposable filtration devices used for loading and filtering the cell cultures. The staggered parallel multi-step configuration of the system allows unattended processing of eight samples in less than 24h. The system was validated with a random panel of 45 whole-cell culture broths containing recombinant antibodies in the early profiling phase. The results showed that the overall performances of the preparative automated system were higher compared to the conventional downstream process including manual harvesting and purification. The mean recovery of purified material from the culture-broth was 66.7%, representing a 20% increase compared to that of the manual process. Moreover, the automated process reduced by 3-fold the amount of residual aggregates in the purified antibody fractions, indicating that the automated system allows the cost-efficient and timely preparation of antibodies in the 20-200mg range, and covers the requirements for early in vitro and in vivo profiling and formulation of these drug candidates.

ADAM17 is the main sheddase for the generation of human triggering receptor expressed in myeloid cells (hTREM2) ectodomain and cleaves TREM2 after Histidine 157

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Here, the question was addressed at which position sheddases cleave TREM2 and what are the proteases involved in this process. Using both pharmacological and genetic approaches we report that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.

Biophotonics Applied to Proteomics

Since the completion of the human genome sequencing, our understanding of gene and protein function and their involvement in physiopathological states has increased dramatically, partly due to technological developments in photonics. Photonics is a very active area where new developments occur on a weekly basis, while established tools are adapted to fulfill the needs of other disciplines like genomics and proteomics. Biophotonics emerged at the interface of photonics and biology as a very straightforward and efficient approach to observe and manipulate living systems. In this chapter, we review the current applications of photonics and imaging to proteomics from 2D gels analysis to molecular imaging.

Protein Characterization by MS in the Pharmaceutical Industry

LC-MS based methodology for the rapid identification of congeners in preparations of the protein r-hirudin sequence variant 1 (rHV1, structure I) was developed. On-line coupling of reversed phase HPLC with electrospray ionization mass spectrometry (LC-ESMS) was found sufficiently sensitive to allow direct detection of such congeners at low concentrations in batch samples despite the presence of the large bulk of r-HV1 and the relatively high concentration of ammonium acetate necessary as a buffer system.