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Topic 1: Protein biogenesis and degradation from the ER We study the molecular characterization of a quality control system that operates in the endoplasmic reticulum (ER) to ensure that only properly folded proteins will be released. Misfolded polypeptides are retro-translocated to the cytosol to become poly-ubiquitinated and destructed by proteasomes. ER-associated degradation (ERAD) is relevant for many genetically inherited and neurodegenerative diseases with protein folding defects.
Topic 2: Regulation of genes involved in lipid homeostasis Cardiovascular diseases are still the number 1 death cause worldwide. Atherosclerosis has been identified as an underlying etiological factor, promoted by high cholesterol levels. Here we try to understand, how lipid profiles in individuals may be influenced by certain factors, such as different diets. Beyond that we aim at deciphering, which genes involved in relevant pathways show changes in their expression when lipid patterns are modified.
Topic 1 (ERAD) We use mammalian cell culture systems to study ERAD. To analyze the role of N-glycosylation in this process, we take advantage of both pharmacological interventions and mutant cell lines. Interaction partners of ERAD substrate proteins are determined in immunoprecipitation experiments with cell lysates from cells grown in the presence of proteasome and/or glycan processing inhibitors. Positive candidates will be identified using antibodies to known ER-associated proteins, and/or by mass spectroscopy and then further characterized. Precise intracellular localization of the ERAD pathway of glycoproteins is studied by indirect immunofluorescence and confocal laser scanning microscopy.
Topic 2 (Lipid homeostasis) We initiated a study with hyperlipidemic probands that follow a so-called portfolio diet. Apart from determining their blood values for carbohydrate and lipid parameters, we analyze their expression of genes involved in pertinent pathways by qPCR and microarrays.
We have shown that a truncated form of ribophorin I, a model glycoprotein for ERAD, is degraded by the ubiquitin/proteasome system. The role of N-linked glycans in ERAD was pinpointed as temporary retention devices in the ER. Thus, interaction of N-glycosylated substrates with the calnexin cycle and perhaps other ER constituents appears to prolong their half lives.
Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein required for the assembly and secretion of very low density lipoproteins (VLDL). Active MTP is a heterodimer containing a 97 kDa catalytic subunit and a 58 kDa subunit (protein disulfide isomerase (PDI)). The MTP complex catalyzes the loading of apolipoprotein B (apoB) with lipids. In avians, the synthesis of VLDL is inducible by estrogen. We are studying the effect of estrogen treatment on MTP activity and on the regulation of VLDL secretion. The consequence of altered intracellular MTP activity on VLDL assembly and secretion is being analyzed. Another aspect is concerned with the mechanism of retention of the MTP complex in the ER.
Molecular cloning, expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein.
Ivessa, N Erwin; Rehberg, Edward; Kienzle, Bernadette; Seif, Fridolin; Hermann, Robert; Hermann, Marcela; Schneider, Wolfgang J; Gordon, David A
Expression of microsomal triglyceride transfer protein in lipoprotein-synthesizing tissues of the developing chicken embryo.
Eresheim, Christine; Plieschnig, Julia; Ivessa, N Erwin; Schneider, Wolfgang J; Hermann, Marcela
Estrogen enhances secretion of apolipoprotein B-100 containing lipoproteins by BeWo cells.
Kamper, Miriam; Manns, Clara C; Plieschnig, Julia A; Schneider, Wolfgang J; Ivessa, N Erwin; Hermann, Marcela