Identification and quantification of drug-albumin adducts in serum samples from a drug exposure study in mice.

The formation of drug-protein adducts following the bioactivation of drugs to reactive metabolites has been linked to adverse drug reactions (ADRs) and is a major complication in drug discovery and development. Identification and quantification of drug-protein adducts in vivo may lead to a better understanding of drug toxicity, but is challenging due to their low abundance in the complex biological samples. Human serum albumin (HSA) is a well-known target of reactive drug metabolites due to the free cysteine on position 34 and is often the first target to be investigated in covalent drug binding studies. Presented here is an optimized strategy for targeted analysis of low-level drug-albumin adducts in serum. This strategy is based on selective extraction of albumin from serum through affinity chromatography, efficient sample treatment and clean-up using gel filtration chromatography followed by tryptic digestion and LC-MS analysis. Quantification of the level of albumin modification was performed through a comparison of non-modified and drug-modified protein based on the relative peak area of the tryptic peptide containing the free cysteine residue. The analysis strategy was applied to serum samples resulting from a drug exposure experiment in mice, which was designed to study the effects of different acetaminophen (APAP) treatments on drug toxicity. APAP is bioactivated to N-acetyl-p-benzoquinoneimine (NAPQI) in both humans and mice and is known to bind to cysteine 34 (cys34) of HSA. Analysis of the mouse serum samples revealed the presence of extremely low-level NAPQI-albumin adducts of approximately 0.2% of the total mouse serum albumin (MSA), regardless of the length of drug exposure. Due to the targeted nature of the strategy, the NAPQI-adduct formation on cys34 could be confirmed while adducts to the second free cysteine on position 579 of MSA were not detected.

Characterization of transgenic mice that secrete functional human protein C inhibitor into the circulation.

Protein C inhibitor (PCI) is a heparin binding serine protease inhibitor in plasma, which exerts procoagulant activity by inhibiting thrombomodulin-bound thrombin or activated protein C (APC). Since the role of PCI in vivo is largely unknown we generated genetically modified mice with expression of human PCI mRNA in hepatocytes only. Three transgenic lines have been characterized. Transgenic mice did not show gross developmental abnormalities. Two lines showed a pericentral and one line showed a periportal expression pattern of human PCI mRNA in the liver. Genetically modified mice secreted a functional transgenic protein into the circulation (3-5 microg/ml plasma in heterozygous mice and 10 microg/ml in homozygous mice), which inhibited human APC activity in the presence of heparin. Interestingly, transgenic mice in which human PCI was expressed periportally in the liver had the highest specific activity. Endogenous mouse PCI mRNA could only be detected in the male and female reproductive system, but not in the liver, indicating that endogenous PCI levels in the circulation are low or even absent in mice. These results demonstrate that the human PCI transgenic mice are a suitable model for studying the in vivo role of PCI in blood coagulation.

Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane.

We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae. pex15Delta cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants. PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p). Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail-anchored type II (Ncyt-Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy-terminus. Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane. The lumenal carboxy-terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O-glycosylation. Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N-terminus of mature invertase. This resulted in core N-glycosylation of the hybrid protein. The lumenal C-terminal tail of Pex15p is essential for targeting to the peroxisomal membrane. Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein. These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.

The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins.

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.