Elucidation of xenobiotic metabolism pathways in human skin and human skin models by proteomic profiling

Human skin has the capacity to metabolise foreign chemicals (xenobiotics), but knowledge of the various enzymes involved is incomplete. A broad-based unbiased proteomics approach was used to describe the profile of xenobiotic metabolising enzymes present in human skin and hence indicate principal routes of metabolism of xenobiotic compounds. Several in vitro models of human skin have been developed for the purpose of safety assessment of chemicals. The suitability of these epidermal models for studies involving biotransformation was assessed by comparing their profiles of xenobiotic metabolising enzymes with those of human skin

FVa inactivation by APC and protein S

Factor Va enhances thrombin generation by several orders of magnitude. Its function is controlled by activated protein C (APC)-mediated and protein S enhanced proteolysis. The aim of my thesis was to clarify the molecular mechanisms underlying FVa inactivation. For this, all three proteins were recombinantly produced and characterised. As APC-mediated FVa inactivation is phospholipid dependent, assays were optimised to investigate interactions in the presence of phospholipids. I have shown that protein S and FVa act together to enhance APC association with phospholipid membranes. The presence of protein S is mandatory, as FVa by itself does not increase binding of APC to phospholipids. These findings strongly suggest that APC, protein S and FVa together form an inactivation complex on phospholipid surfaces. Unlike FVa, FVIIIa does not enhance APC binding to phospholipids, indicating that FVIIIa does not form a similar complex with APC and protein S. C4BP-bound protein S did not efficiently enhance APC-phospholipid binding, indicating that C4BP interfers with complex formation. Results I obtained with protein S variants with impared APC cofactor function, and FV Nara, associated with strong APC resistance, indicate that their mutations essentially abolished their ability to assemble into the tri-molecular complex. FV-810, with partial B-domain deletion, assembled the complex as efficiently as FVa. Protein S was required for complex enhancement by FV-810. However, results for FV-810 obtained with a FVa inactivation assay indicated that effective inactivation can be achieved in the absence of protein S. These findings suggest that there must be an alternative mechanism involved in APC cofactor function which is not mediated solely by increased binding of APC to phospholipids. In conclusion, my findings demonstrate that FVa promotes its own APC-mediated degradation by enhancing APC binding to phospholipids together with protein S, but also suggest that more than a single mechanism controls FVa inactivation.Open Acces

Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction

Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50–60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.This work was supported by the Swedish Research Council (grant 71430), grants from the Swedish Heart and Lung Foundation, Söderberg’s foundation, the Alfred Österlund’s Foundation, research funds from the University Hospital in Malmö and the British Heart Foundation (FS/12/60/29874).Peer Reviewe

Elucidation of Toxicity Pathways in Lung Epithelial Cells Induced by Silicon Dioxide Nanoparticles

<div><p>A study into the effects of amorphous nano-SiO₂ particles on A549 lung epithelial cells was undertaken using proteomics to understand the interactions that occur and the biological consequences of exposure of lung to nanoparticles. Suitable conditions for treatment, where A549 cells remained viable for the exposure period, were established by following changes in cell morphology, flow cytometry, and MTT reduction. Label-free proteomics was used to estimate the relative level of proteins from their component tryptic peptides detected by mass spectrometry. It was found that A549 cells tolerated treatment with 100 µg/ml nano-SiO₂ in the presence of 1.25% serum for at least 4 h. After this time detrimental changes in cell morphology, flow cytometry, and MTT reduction were evident. Proteomics performed after 4 h indicated changes in the expression of 47 proteins. Most of the proteins affected fell into four functional groups, indicating that the most prominent cellular changes were those that affected apoptosis regulation (<i>e.g.</i> UCP2 and calpain-12), structural reorganisation and regulation of actin cytoskeleton (<i>e.g.</i> PHACTR1), the unfolded protein response (<i>e.g.</i> HSP 90), and proteins involved in protein synthesis (<i>e.g.</i> ribosomal proteins). Treatment with just 10 µg/ml nano-SiO₂ particles in serum-free medium resulted in a rapid deterioration of the cells and in medium containing 10% serum the cells were resistant to up to 1000 µg/ml nano-SiO₂ particles, suggesting interaction of serum components with the nanoparticles. A variety of serum proteins were found which bound to nano-SiO₂ particles, the most prominent of which were albumin, apolipoprotein A-I, hemoglobin, vitronectin and fibronectin. The use of a proteomics platform, with appropriately designed experimental conditions, enabled the early biological perturbations induced by nano-SiO₂ in a model target cell system to be identified. The approach facilitates the design of more focused test systems for use in tiered evaluations of nanomaterials.</p></div

XMEs detected in whole skin. Protein identification was based on the presence of ≥2 different tryptic peptides in at least two donors.

<p>The proteins identified have been classified into functional groups as indicated. The corresponding NCBI numbers are indicated for each protein and for all members of groups of related proteins. The sub-cellular fraction in which each protein was principally detected is shown. The proportion of donor samples (skin n = 10, liver n = 5) in which each protein was identified is indicated. Fold difference was calculated by summing the intensity values of all detected peptides for a protein and comparing the values obtained for skin and liver. Where no peptides were detected, an intensity value equivalent to the limit of detection was used. Statistical significance was assessed using the Mann-Whitney U test.</p

Analysis of CYP expression in skin by immunoblotting.

<p>Samples of human whole skin microsomal fraction (75 µg) prepared from 5 donors were separated by SDS-PAGE, transferred to nitrocellulose filters and the presence of CYP1A1, CYP1A2, CYP2E1 and CYP3A4 detected using antibodies specific to each form. The lane on the left hand side contained either 25 µg lymphoblast cell microsomes containing recombinant human CYP1A1 (∼2 pmol), or a sample of human liver loaded with 25, 35 or 5 µg microsomal fraction for detection of CYP1A2, CYP2E1, or CYP3A4, respectively. Immunoreactive bands were developed using goat anti-rabbit-horseradish peroxidase and ECL detection.</p

Comparison of XME profiles from <i>in vitro</i> skin models and whole skin.

<p>The relative amount of each protein is represented by the number of different tryptic peptides specific to each protein or protein family that were detected. Details of the protein accession numbers and their subcellular location are shown in Table S1. Shading indicates different enzyme classes: oxidoreductase (black), hydrolase (magenta), transferase (red), antioxidant (green), and other (blue).</p

Detection of CYP proteins in skin and liver microsomal fraction by LC-MS/MS.

<p>Samples of skin microsomal fraction were spiked with a range of quantities of either recombinant CYP1A1 (expressed in lymphoblast cells) or with human liver microsomal fraction that contains known amounts of CYP1A2, CYP2E1, CYP3A4, and CYP3A5. The normal proteomics workflow was followed to identify peptides corresponding to the CYP proteins. Limits of detection based on the use of at least 2 tryptic peptides were established and based on these values the minimum level detectable by this technique was calculated for skin and compared with the mean level measured in liver. From these values the minimum comparative level in skin was calculated. CYP1A1 was not detected in either skin or liver making any comparison redundant (n/a; not applicable).</p