Subcellular trafficking of proteolipid protein (PLP/DM20) and novel mechanisms of ER retention in Pelizaeus-Merzbacher disease

Subcellular trafficking of proteolipid protein (PLP/DM20) and novel mechanisms of ER retention in Pelizaeus-Merzbacher disease

by Ajit Singh Dhaunchak
Doctoral thesis
Date of Examination:2006-06-26
Date of issue:2006-11-22
Advisor:Prof. Dr. Klaus-Armin Nave
Referee:Prof. Dr. Klaus-Armin Nave
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Harald Neumann
Persistent Address: http://dx.doi.org/10.53846/goediss-3205

 

 

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Abstract

English

Missense mutations that predict the misfolding of membrane proteins have been associated with a number of neurogenetic diseases. However, it is not known how apparently minor changes in the amino acid sequence of an extracellular loop or a transmembrane domain lead to complete ER retention with complex loss- and gain-of-function effects. I have chosen PLP/DM20, a highly conserved and abundant tetraspan myelin protein, associated with Pelizaeus-Merzbacher disease (PMD), as a model system. By expressing wildtype and mutant PLP isoforms in glial cells, surprising molecular properties became apparent, including the ability to self-assemble from two truncated PLP polypeptides, and to form conformation sensitive epitope that become masked as the protein matures in the ER. With respect to human disease, it was possible to identify a novel molecular mechanism by which missense mutations cause ER retention of misfolded PLP. Unexpectedly, pairs of cysteines within an extracellular loop of PLP/DM20 play a critical role. Multiple disease-causing mutations require the presence of cysteines such that misfolded PLP/DM20 is efficiently retained in the ER. Replacing cysteines by serine completely prevents ER retention and restores normal trafficking of mutant PLP/DM20. This demonstrates a novel pathological mechanism by which missense mutations greatly reduce the efficiency of intramolecular disulfide bridging. When exposed by misfolding to the ER lumen, unpaired cysteines engage in alternative oxidations that lead to abnormal intermolecular crosslinks. Since extracellular cysteines are a feature of many membrane proteins, this novel pathomechanism is likely to contribute to a diverse group of genetic diseases. To monitor the expression and subcellular trafficking of PLP in vivo, a transgenic knock-in mouse in being generated that will express a PLP-EGFP fusion protein under control of the endogenous promoter. In an attempt to develop a cure for Pelizaeus-Merzbacher disease (PMD), we treated a genuine animal model (rumpshaker mice) with Turmeric. The active constituent of this herbal drug (Curcumin) is a non-toxic Ca2+ adenosine triphosphatase pump inhibitor, and known to release membrane proteins from ER retention. In a pilot experiment, we extended the lifespan of rumpshaker mice from 20 to 60 days. These promising data suggest that a therapeutic strategy should be developed for PMD, using turmeric and our in vitro and in vivo models.
Keywords: Myelin; proteolipid protein; PLP; PLP/DM20; ER retention; chaperons; protein folding; myelin diseases; Pelizaeus-Merzbacher disease; conformation disease; protein traffiking; exosomes; live cell imaging; cell biology; oli-neu; oligodendrocyte precursor; disulfide bond
Schlagwörter: N.A
 
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