Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Research Program ”Systems of Life-Systems Biology” A Systems Biology Approach to Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Coordination: Matthias Reuss Institute of Biochemical Engineering University of Stuttgart
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Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Research Program ”Systems of Life-Systems Biology” A Systems Biology.
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Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Research Program ”Systems of Life-Systems Biology”
A Systems Biology Approach to Detoxification Pathways and their
Cellular and Structural Requirementsin Hepatocytes
Coordination: Matthias ReussInstitute of Biochemical Engineering University of Stuttgart
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Project Section A
Xenobiotic Metabolism and Transport:
Analysis, mathematical modeling andsimulation of the xenobiotic-metabolizing
system of the liver
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Quantitative experimental analysis of metabolism using model substrates to determine kinetic parameters in recombinant systems (ITB), human liver tissue (IKP) and in primary hepatocytes (BBZ)
Structure modeling at the molecular level including chemicals (CCC)as well as proteins (ITB) and their interactions
Mathematical modeling of the biotransformation system by integrating experimental data and structure models (IBVT)
Dynamic simulation of the most important metabolic reactionsfor functionalization and conjugation
Simulations of different individual situations regarding bothquantitative (enzyme expression levels) and qualitative differences(polymorphism)
Simulation of regulation processes (induction)
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Biological Model Systems
• Human Liver Tissue Bank with Clinical Documentation (N>300)
quantitative data on variability of function, protein, mRNA,
polymorphisms
• Human Hepatocytes in Primary Culture
dynamics of metabolism and transport, all aspects of
regulation
• Recombinant Proteins
substrate selectivities and kinetic parameters of individual
components
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
• Metabolic Pathways• Identification of Metabolites and
Responsible Enzymes• Variability of Expression• Genotype-Phenotype Relationships• Regulatory Networks
• Metabolic Pathways• Identification of Metabolites and
Responsible Enzymes• Variability of Expression• Genotype-Phenotype Relationships• Regulatory Networks
• Diagnosis• Demogr. Data • Drugs• Nic & Alc• Life Style
• Diagnosis• Demogr. Data • Drugs• Nic & Alc• Life StyleN > 300
RNA:transkripts
splicing variants
DNA:polymorphisms
genotypes
Protein Fractions:expression function, kinetics
t [min]
µV
N
O C H3
O
O C H3
C H3
O
C N
C H3
C H3
Human Liver Bank as a Tool
Clinical Documentation:
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
• Kinetics of overall biotransformation (model substrates)
• Dynamics of regulation processes (e.g. induction)
• Global analysis of cellular changes associated with regulation processes
Human Hepatocyte Models
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Recombinant Expression Systems
NADPH + H+ NADPH-P450-
reductase
FADFMN Fe+++
NADP+
2x1e-
O2 H2O OH
CytochromeP450
endoplasmic reticulum membrane lumen
cytoplasm
The Cytochrome P450 Monooxygenase System
2e-
• Various yeast strains (Schmid, ITB)
• Baculovirus / insect cells (Zanger, IKP)
Objectives:
• Kinetic parameters of individual proteins by coexpressing P450-reductase and cytochrome b5
• Analysis and modeling of protein-protein interactions by reconstitution of purified components (cooperation with Rebecca Wade, EML Heidelberg)
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Project Section B
Hepatic Differentiation and Dedifferentiation Processes:
Holistic analysis of metabolic networks, regulatory networks, signalling networks
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
• Global transcriptome analysis • Metabolite measurements (LC-MS)• Flux analysis based on labeling experiments (GC-MS)• Bioreactor cultivation• Modeling and simulation platform INSILICO discovery
INSILICO Biotechnology GmbH, Stuttgart
• Proteome analysis (automated global protein analysis)• Free-flow-electrophoresis for enrichment of rare proteins• Membrane proteins, phosphorylation patterns etc.
TECAN Proteomics GmbH, München
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
SYSTEMS BIOLOGY
Biological Information/Knowledge
Inductive
Deductive
Projektbereich B
Projektbereich A
Flussverteilungen
(Genomweite Zellmodelle)
(Reverse Engineering)
Geplante Aktivitäten für
die 2. und weitere Förderphasen Anbindung DatenbankGenetische Polymorphismen
Proteinmodellierung und Docking
Kinetik derDetoxifikationsschritte
Untersuchungen zur Regulation
der Genexpression
Anbindung Reaktionsdatenbank
und Modell zur Vorhersage
des Metabolismus
Mathematische Modellierung und
dynamische Simulation des
Fremdstoffabbaus (Aggregation
der Einzelschritte)
DNA-Arrays
Proteomics
Metabolomics
Projektbereich Z
Primäre Zellkulturen
Leberbank
Zellbiologie
Modellierungswerkzeug undDatenbanken
Projektkoordination
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
Phase I
Phase II
lipophilic polar
Transport
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
Phase I
Phase II
lipophilic polar
Transport
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
Phase I
Phase II
lipophilic polar
Transport
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
Phase I
Phase II
lipophilic polar
Transport
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes