Research Group BRAAKMAN

Dept. Bio-Organic Chemistry
Contact: Prof. Ineke Braakman
E-mail: i.braakman@chem.uu.nl
Website: http://www.bijvoet-center.nl/cpc

General research focus: Protein folding in the ER

The endoplasmic reticulum (ER) is specialized in the folding of newly synthesized proteins and is loaded with molecular chaperones and folding enzymes. They assist the folding process and check the quality of folding proteins. Young proteins undergo various modifications essential for proper folding, such as glycosylation of asparagine residues, cis-trans isomerization of prolines, and oxidation of cysteines to disulfide bonds. The quantity of ER in a cell (i.e. the amount of membrane as well as the amount of chaperones and folding enzymes) is adapted to the need, to the circumstances. If the ER fills up with aggregated misfolded proteins, or when a large amount of protein needs to be synthesized and folded, the compartment increases its size.
The research lines below are all subject for stages. Topics will be determined depending on available support and on the desires of the student.

Research line 1: Characterizing the folding pathway of our model proteins in the ER.
These model proteins are the Influenza virus hemagglutinin (HA), HIV-1 Envelope glycoprotein gp160, the low density lipoprotein receptor (LDL-receptor), and CFTR. The latter two proteins contain mutations in patients with FH (familial hypercholesterolemia) and cystic fibrosis, respectively.

Techniques: This multidisciplinary research uses many different techniques: cell culture, (recombinant) virus infection, radioactive pulse-chase, immunoprecipitation, SDS-PAGE, immunofluorescence, DNA cloning, 2D-gel electrophoresis, yeast genetics, in vitro translations, and a recently developed in vitro folding assay.

Research line 2: Determine the role cellular factors play in protein folding and quality control.
These factors include ATP, calcium, redox-milieu, but also molecular chaperones and folding enzymes. We characterize the known folding factors and identify and study new ones.

Techniques: Cell culture, (recombinant) virus infection, radioactive pulse-chase, (co-)immuno-precipitation, SDS-PAGE, immunofluorescence, DNA cloning, proteomics, 2D-gel electrophoresis, yeast genetics, in vitro translations, and a recently developed in vitro folding assay. Both mammalian cells and the yeast Saccharomyces cerevisiae are used.

Research line 3: This line does not focus on the ER but on another cellular organelle, the peroxisome. We study the role of cytosolic chaperones and associated proteins in the maturation and import of proteins into the peroxisome. Since peroxisomal membranes most likely originate from the ER, we plan to examine an intermediate compartment between these two organelles.

Techniques: Yeast and mammalian cell culture, radioactive pulse-chase, (co)-immunoprecipitation, SDS-PAGE, immunofluorescence, DNA cloning, proteomics, 2D-gel electrophoresis, yeast genetics, in vitro translations. Both mammalian cells and the yeast Saccharomyces cerevisiae are used.

Back