Final Report Summary - PROFITS (Bridging the world of fungi and dementia)
The PROFITS project aims at elucidating failures in PROtein Folding, Intracellular Transport and Secretion (PROFITS). Such failures in PROFITS are the major cellular hallmark of the neurodegenerative Alzheimer’s disease (AD) but have been described as well for the industrially important filamentous fungus Aspergillus niger, e.g. when forced to express homologous or heterologous proteins. Both man and fungus share similar defense strategies to dispose of misfolded proteins. Hence, the aim of this project is the establishment and exploitation of A. niger as a model system to study failures in PROFITS for gaining further insights into the onset and progression of AD. Secretion stress and failures in PROFITS can be induced by various treatments of A. niger including forced expression of homologous proteins at high rates, expression of heterologous proteins, treatment with chemicals or constitutive activation of transcription factors involved in the induction of secretion stress responses. We have collected 50 published and unpublished A. niger microarray samples from six independent experiments that followed different approaches to induce failures in PROFITS. Their analysis has led to the identification of 40 genes that show condition-independent differential expression.
We also performed a gene co-expression network analysis for A. niger to functionally annotate uncharacterized genes of A. niger. Although its genome sequence is known since almost 10 years, only 2% of the predicted 14,000 genes of A. niger are characterized. Gene co-expression network analysis is based on the assumption that genes with shared expression profiles are tightly connected and are predicted to function in the same regulatory and/or functional pathway (“guilt-by-association” approach). We thus identified genes with a consistent, correlated expression pattern across ~280 microarray data covering 155 phenotypically diverse samples or experimental conditions for A. niger. The functional modules predicted by the gene co-expression network for the 14,000 genes of A. niger were investigated for gene content and validated based on published data for the function of known secretory pathway genes. These analyses supported the biological relevance of these modules, suggesting that the co-expression network obtained presents a valuable predictive tool for functional annotation of A. niger genes.
In addition to this in silico meta-analysis of trancriptomic data for A. niger, the PROFITS projects aimed at establishing and analyzing specific secretion stress transcriptome data by forced expression of AD related proteins and peptides in A. niger. In doing so, the tetracycline/doxycycline-inducible TetON promoter system for A. niger was exploited and used to express the fibrillogenic amyloid peptide Aß, its precursor protein APP and the intracellular tangle forming Tau protein in A. niger. To ensure entry of Aß peptides into the secretory pathway, expression constructs were designed as such, that the Aß peptide was N-terminally fused to the glucoamylase A, serving as a carrier protein, which is a strongly secreted homologous protein of A. niger. APP and Tau proteins have not been fused to intend intracellular expression.
We also performed a gene co-expression network analysis for A. niger to functionally annotate uncharacterized genes of A. niger. Although its genome sequence is known since almost 10 years, only 2% of the predicted 14,000 genes of A. niger are characterized. Gene co-expression network analysis is based on the assumption that genes with shared expression profiles are tightly connected and are predicted to function in the same regulatory and/or functional pathway (“guilt-by-association” approach). We thus identified genes with a consistent, correlated expression pattern across ~280 microarray data covering 155 phenotypically diverse samples or experimental conditions for A. niger. The functional modules predicted by the gene co-expression network for the 14,000 genes of A. niger were investigated for gene content and validated based on published data for the function of known secretory pathway genes. These analyses supported the biological relevance of these modules, suggesting that the co-expression network obtained presents a valuable predictive tool for functional annotation of A. niger genes.
In addition to this in silico meta-analysis of trancriptomic data for A. niger, the PROFITS projects aimed at establishing and analyzing specific secretion stress transcriptome data by forced expression of AD related proteins and peptides in A. niger. In doing so, the tetracycline/doxycycline-inducible TetON promoter system for A. niger was exploited and used to express the fibrillogenic amyloid peptide Aß, its precursor protein APP and the intracellular tangle forming Tau protein in A. niger. To ensure entry of Aß peptides into the secretory pathway, expression constructs were designed as such, that the Aß peptide was N-terminally fused to the glucoamylase A, serving as a carrier protein, which is a strongly secreted homologous protein of A. niger. APP and Tau proteins have not been fused to intend intracellular expression.