Saarland University Faculty of Medicine
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Membrane proteins play a crucial role in cellular and physiological processes. They are essential mediators of cargo and information transfer between cells, between intracellular compartments and between organ systems. Functionally intact membrane proteins are vital to health and specific defects therein are associated with many known human diseases. Membrane proteins are the targets of a large number of pharmacologically and toxicologically active substances and are responsible, in part, for their uptake, metabolism, and clearance. Our overall goal is to identify key components of membrane-derived signal transduction pathways, to investigate their functions in the laboratory and, finally, to apply this knowledge to patient care.

 

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X-ray crystallography of proteins

(Image source: Prof. Dr. Axel Scheidig)


The KoMM members focus on the following important aspects of function and regulation of membrane proteins:

 

Biogenisis of membrane proteins and protein translocation
In order to reach their functional states, newly synthesized membrane proteins (e.g. TRP channels) need to be inserted into their target membranes, folded into their native structures and in most cases assembled with additional components into oligomeric complexes. All these processes employ many biogenetic components, which often are themselves membrane proteins. Several of these components from various systems are currently studied by a combination of various approaches in order to elucidate mechanistic principles and mechanisms of regulation. In addition to increasing knowledge about the underlying mechanisms in the last decade it has been realized that many human diseases are caused by defects in the machinery involved in the biogenesis of membrane proteins. Therefore, the goal of a functional proteomics team is to unravel the basic principles by which membrane proteins are formed in the cell and to identify and characterize the components, which mediate this intricate process. The close collaboration with different pharmacologically and clinically oriented groups is essential for this endeavour.

 

 

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3D reconstruction of a ribosome after cryo-electron microscopy

(Image source: Prof. Dr. Richard Zimmermann)

 

 

Molecular mechanisms of neuronal exocytosis, structure and structural changes of proteins involved in membrane fusion
The release of transmitter through fusion of synaptic vesicles with the plasma membrane certainly represents the most important signalling event in the central nervous system. It not only secures the communication between the 100 billion nerve cells, but its modulation also forms the basis for plasticity of our brain function as expressed in learning and memory. Our groups investigate the specific roles of the many proteins involved in this process with high-resolution methods like electrophysiology and real-time imaging. Deciphering the molecular mechanisms of neuronal exocytosis and understanding its modulation will help us to develop therapies for age-related dementias like Alzheimer and ultimately also to devise strategies for the most efficient teaching at schools and universities.

 

 

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2-photon microscopy of neurons

(Image source: Dr. Peter Blaesse)

 

Function and regulation of transient receptor potential (TRP) channels
Current research indicates that many TRP channels are involved in sensory functions like smell, taste, hearing, temperature and pheromone sensing; others do regulate Ca2+-dependent exocytosis in non-excitable cells like mast cells and lymphocytes. Another, still mysterious group of TRP channels seems to influence the cells' ability to move and to travel in the body, potentially affecting functions as diverse as wound healing, immune response, embryonic development and even the metastasis of cancers. Using a genetically-driven, systems-oriented approach ("from genes to cells to behaviour") work in our groups focuses on the cellular and molecular basis of specific TRP functions and their impact on the physiology of the whole organism and complex behaviour.

 

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3D reconstruction of ER and mitochondria in a living COS-1 cell with GFP and DsRed2 fluorescence microscopy
(Image source: Prof. Dr. Peter Lipp and Dr. Lars Kästner)

 

Decoding of intracellular Ca2+ signals
Ca2+ ions are probably the most important intracellular second messengers. Spatially and temporarily coordinated changes in cytosolic Ca2+ initiate processes as diverse as muscle contraction, sensation, motility, synaptic transmission or transcriptional regulation. Although measurements of the global, intracellular Ca2+ concentration are readily obtained by fluorescent Ca2+ indicators, uneven distribution of plasma membrane Ca2+ channels and highly efficient buffering through Ca2+-binding proteins and organelle compartmentalization lead to Ca2+ transients that are spatially restricted and brief in time. It is this complexity that appears to be imperative for many of the functions of calcium transients. We thus investigate these Ca2+ transients and their downstream signalling e.g. through Gq-coupled signalling cascades in relationship to the physiology and pathophysiology of cells, organs and the entire organism. For this we apply an array of sophisticated real-time imaging methods at all levels of organs such as heart and brain.

 

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"Real-time" imaging of Ca2+-concentrations in cells

(Image source: Prof. Dr. Peter Lipp and Dr. Lars Kästner)

 

Deciphering membrane-protein regulated adaptive and mal-adaptive remodelling processes in the heart
Deciphering membrane-protein regulated adaptive and mal-adaptive remodelling processes in the heart Heart failure is a common clinical disorder, which accounts for much of the morbidity and mortality in the Western society and which remains a major public health threat. Our groups are actively engaged in attempting to understand the molecular events that affect maladaptive cardiac remodelling in heart failure in response to pathologic stimuli such as pressure overload (hypertension) or ischemic injury at the whole organ level, at the cellular level, and at the molecular level. The underlying signal transduction pathways are activated at the cell membrane and involve the orchestration of G-protein-coupled receptors, receptor tyrosine kinases, various traditional G-proteins as well as the low molecular weight G-proteins of the Rho family, ion channels (including TRPs) and transporters, adaptor signaling complexes, cascades of kinases and phosphatases, and ultimately transcription factors. We rely heavily on generating gene targeted (knock-out and knock-in) transgenic mice as models for disease to assess the malfunction of specific signaling factors within the intact myocardium.

 

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Transgenic mice as models for disease

(Image source: Prof. Dr. Marc Freichel)

 

Role of membrane microdomains in neurodegenerative diseases
Amyloid precursor protein (APP), β-secretase and presenilin 1 all reside in membrane micro domains, also referred to as lipid rafts. We have shown that certain lipids drastically increase Aβ peptide production, whereas lowering cholesterol levels in animals reduces brain Aβ42 levels. These findings were followed by the first clinical pilot study in 2002, which indicated that lowering cholesterol in Alzheimer disease patients decreased brain Aβ42 levels and that this may have a beneficial effect on disease regression. Current work is focussed on the possible interactions of lipid metabolism and lipid trafficking with Alzheimer disease proteins (Aβ, presenilins) and processing of these proteins using in vitro systems as well as various transgenic mice designed to model various aspects of the disease.

 

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Analysis of cellular ultra structure via electron microscopy

(Image source: Prof. Dr. Frank Schmitz)

 

Membrane proteins linked to the development and progression of cancer
Deregulated signalling across membranes and changes in dynamic membrane processes (e.g. altered expression of ion-channel proteins or receptors) account for or are the consequence of oncogenic transformation and can be utilized for diagnostic tumour profiling or novel therapeutic strategies. Moreover, the genetic and epigenetic reprogramming during oncogenic transformation leads to expression of immunogenic antigens, which are localized in the cellular membrane. We investigate antigens that have some causal relation to cancer aetiology or cancer phenotype although it is a daunting task to distinguish them from antigens, which have no direct relevance to cancer (e.g. antigens detected by pre-existing auto antibodies or antibodies elicited by antigens related to necrotic tumour products). This investigation requires novel methodological and interdisciplinary approaches including functional genetics and epigenetics to yield a profound understanding of the complex crosstalk between genetics, epigenetics and cellular physiology.

 

 

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Electrophysiology at the level of a single cell with the patch clamp technique

(Image source: Prof. Jens Rettig)

 

Function and regulation of membrane proteins in angiogenesis and microvascular inosculation in vivo
Endogenous regeneration of diseased organs and in vivo integration of ex vivo engineered tissue depend on new vessel formation and microvascular inosculation, involving distinct dynamic membrane processes. We investigate several molecular systems in vivo, i.e. VEGF-VEGFR2, sdf-1-CXCR4, and Ang-2-Tie2 for angiogenesis and revascularization and the interaction of beta integrins and intercellular adhesion molecules of the immunoglobulin gene superfamily for recruitment and homing of endothelial progenitor cells. These molecular interactions contribute to the development of microvascular networks in three-dimensional ex vivo cultures and in vivo. A major goal of the present work is to modulate the regulation and function of these dynamic membrane processes in order to enhance functionality of the newly created vascular systems and to fuse microvascular and parenchymal constructs in architecture appropriate for transplantation and inosculation. High resolution multifluorescence real-time imaging of molecular, cellular and microhemodynamic functionality in chronicle animal models aims at reconstructing and replacing endocrine pancreas, bone, soft tissue and skin.

 

 

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Total-Internal-Reflection-Fluorescence (TIRF) microscopy

(Image source: Dr. Ute Becherer)

 

Bridging the gaps between in vivo signaling pathways, molecular drug design, in vitro testing and therapy in vivo
Membrane proteins account for up to two thirds of known drugable targets, and our groups developed a platform to address target-oriented development of novel drugs which affect membrane proteins and membrane-associated proteins. Based on available X-ray structures we apply molecular modelling approaches combined with virtual screening procedures, followed by chemical synthesis of structurally notable and promising novel compounds. Alternatively, such compounds are identified by fluorescence-based high-throughput screens. These compounds are subsequently tested for biological activity in various cellular and cell-free systems including purified recombinant target proteins. Following this approach we are currently pursuing development of antiviral compounds interfering with binding of hepatitis C virus with the extracellular loops of CD81 in hepatocytes as well as of antiproliferative steroidomimetic drugs inhibiting membrane-bound enzymes of steroid hormone biosynthesis.

 

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Measurements of Protein-Protein-Interactions: Plasmon-resonance-spectroscopy (BIAcore)
(Image source: Prof. Dr. Richard Zimmermann)

 


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