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Huntington's disease: from experimental results to interaction networks, patho-pathway construction and disease hypothesis
BMC Systems Biology volume 1, Article number: P45 (2007)
Protein-protein interaction networks and mechanistic pathway models are excellent tools in the drug discovery process. They can be used to identify and select targets for a given disease hypothesis. Combining information from diverse sources, like in house experiments as well as literature, allows further development of interaction networks into detailed descriptions of cellular pathways. Computerized pathway diagrams allow integrating all relevant data regarding a project into one framework by linking the different data sources. Interaction networks analysis and pathway design tools are used to support target identification and validation activities. Experimental results are incorporated in protein interaction networks, analyzed and further developed into biomolecular pathopathways including literature findings to understand the underlying modulation mechanisms. The pathway diagrams are also used as communication tools, particularly for interdisciplinary project teams, thus ensuring a common understanding and facilitating critical interrogations about disease hypotheses. The analyses of experimental results, the initial construction of an HD pathopathway are presented and two mechanistic disease hypotheses are discussed.
Materials and methods
Differential proteomics experiments (DPEs) were performed on rat PC12 cells containing either wild-type or mutant full-length (PolyQ) Huntingtin (Htt) under control of a doxycycline-inducible promoter . Cell extracts were prepared at 0, 12, and 48 hours post-induction to identify proteins involved in pre-apoptotic intracellular events. Protein expression modulation was measured using DIGE technology  followed by statistical analysis for spot selection and automated spot picking. The protein content of each picked spot was analyzed by mass spectrometry (MS).
Three independent DPEs were performed and the modulated proteins, confirmed in at least two experiments, were analyzed in the context of protein networks. In a typical experiment, 121 differential spots were picked and MS identification produced 3671 entries grouped into 359 proteins, an expected average of about 3 proteins per spot . In the end, 48 proteins were confirmed to be modulated by the expression of PolyQ Htt, and were therefore considered for bioinformatics analysis. Since the Ubiquitin-Proteasome system is a particularly important biological process shown to be involved in neurodegeneration , the focus was put on the "Proteasome degradation" (Figure 1) class for enrichment of the mechanistic HD pathopathway. In this class of cellular function, the Ubiquitin Carboxyl-terminal Hydrolase isozyme L1 (UCHL1) protein was of particular interest, since it is known to be associated with Parkinson , Alzheimer , and was described as a genetic modifier of the age of onset of HD [7, 8]. Since modulation of UCHL1's mRNA by PolyQ Htt was confirmed by RT-PCR (data not shown), we linked UCHL1 into our HD pathopathway (Figure 2).
Divergent hypotheses can be elaborated for the role of UCHL1 in HD. First, a positive role by contributing to Ubiquitin recycling, thus maintaining normal Proteasome pathway function counteracting the accumulation of insoluble PolyQ Htt. Second, based on the recent discovery that peptide sequences can modulate the toxicity of PolyQ tracts in cis or trans, the transient interaction between UCHL1 and PolyQ Htt to recycle Ubiquitin could actually increase the toxicity of the extended PolyQ tract of mutant Htt by initiating the first step of the formation of aggregates. The latter mechanism can be envisaged, as UCHL1's structure can be superposed to a typical fibrillogenic domain (Figure 3).
DPE results were used to develop an HD pathopathway including proteins modulated by PolyQ Htt expression. In this experimental result set, the proteins from the Ubiquitin-Proteasome pathway were chosen for in depth analysis, and UCHL1 was identified as a component potentially playing opposite roles in HD.
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