(modified last January 18, 2000)
Identification of factors/peptides affecting the aggregation/toxicity of the mutant huntingtin protein which causes the devastating neurodegenerative protein-conformation disorder, Huntington’s disease
Huntington’s disease (HD) is a human neurodegenerative disorder caused by expansion of a CAG repeat in the coding region of the HD gene resulting in a large protein with an expanded glutamine (gln) stretch. Both in HD mouse models and in HD patients large protein aggregates have been detected in certain regions of the brain. In several other neurodegenerative diseases including other disorders caused by poly-gln expansion, but also in Alzheimer- and Creutzfeld-Jacob- disease, protein aggregates in the brain can be detected too. These results suggest an important role for altered protein conformation in the development of these diseases. In the Department of Human and Clinical Genetics various models have been developed to study altered protein conformation in relation to the molecular mechanism of HD. Firstly models have been generated in an organism amenable to genetic analysis, the nematode C. elegans. Mammalian cell models and yeast models are also available. In all models protein aggregation of proteins with (very) long gln-repeats can be detected by various techniques, including immunofluorescence studies and dot-blot analysis.
Which proteins/peptides bind to the N-terminus of huntingtin with an expanded gln-stretch, do these proteins/peptides affect protein conformation/aggregate formation c.q. are these recruited to the aggregates and does this affect expression of cellular genes and toxicity ?
The accumulation of proteins with an expanded repeat in protein aggregates is an important feature of HD. Recently it has been found that a cellular protein with a long gln-stretch, the transcriptional co-activator CBP, can be recruited into poly-gln aggregates. A possible model is thus that the entrapment of important cellular proteins in aggregates contributes to the observed toxicity of proteins with expanded repeats. The further identification and analysis of such proteins can thus provide insight in the molecular mechanisms of poly-gln-induced toxicity. In addition, it is possible that some binding proteins/peptides influence protein conformation/aggregate formation as such and thereby alter poly-gln induced toxicity.
For the cloning of cDNAs for associating proteins/peptides yeast-based approaches will be primarily used, including an adaptation of the yeast “two-hybrid” system, the RRS system. Relevant plasmids expressing various lengths of gln-stretches in yeast are already available and have already been tested. After transformation of libraries positive clones will be analysed further. The specifity of the observed interaction will be determined in re-screens, and the remaining clones will be sequenced. Subsequently, interactions will be tested with independent biochemical approaches. When the interaction is verified, the full length cDNAs or peptide-sequences will be cloned in expression vectors and tested in the available models for their effects on aggregation or toxicity. The recently in our research school developed method of expression arrays can be used to investigate the effects on cellular gene expression. In parallel, several candidate proteins/peptides for interaction with the N-terminus of huntingtin, including proteins with long poly-gln stretches, will be tested along the same lines.
Yeast-transformation, E. coli transformation, recombinant DNA technologies, DNA sequence analysis, Polymerase Chain Reaction (PCR), in-vitro transcription-translation, co-immunoprecipitation, GST-“pull-down”, transformation of mammalian cells, dot-blot assays, cell-death assays, sub-cellular localization studies including immunofluorescence analysis, expression-array. All the techniques are routinely used in our laboratory.
Josephine Dorsman (+31 - 71 - 527 6122, J.C.Dorsman @ lumc.nl)
Johan den Dunnen (+31 - 71 - 527 6105 / 6293, ddunnen @ lumc.n)l
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