3 research outputs found
An investigation of the role of CreB deubiquitinating enzyme in the regulation of carbon metabolism in Aspergillus nidulans
In A. nidulans, carbon catabolite repression is regulated by the global repressor protein
CreA which, in the presence of repressing carbon sources, represses those genes that are
required to utilize less preferable carbon sources. Mutational analyses suggested that
ubiquitination, mediated by CreD together with ubiquitin ligase HulA, and
deubiquitination, mediated by the deubiquitinating enzyme CreB, are involved in the
regulatory pathway in A. nidulans. However, the molecular mechanisms are still
unknown.
Previously, partial loss-of-function alleles of creA and creB indicated genetic
interaction, and this was extended to analysis of complete loss-of-function alleles. Both
morphological and phenotypic analyses of the double null mutant confirmed genetic
interactions between the genes. Moreover, RT-qPCR and enzyme assays also validated
genetic interactions as the double null mutant showed synergistic effects for transcript
levels and enzyme activity. Co-purifications of CreA and CreB expressed from their
native promoters were used to determine whether CreA, or a protein in a complex with
it, is a direct target of the CreB deubiquitinating enzyme, and no direct or indirect
physical interactions were identified. The Phos-tag system was used to show that CreA
is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin
antibodies and Western analysis. These findings were confirmed using mass
spectrometry, which confirmed that CreA was differentially phosphorylated but not
ubiquitinated. These results open up new questions regarding the molecular mechanism
of CreA action, and how the ubiquitinating pathway involving CreB interacts with this
regulatory network.
To identify any possible protein(s) that may form a bridge between CreA and CreB,
independently purified lysates were analysed by mass spectrometry and, for both CreA
and CreB, proteins were identified in repressing and derepressing conditions.
Orthologues of the co-purified proteins were identified in S. cerevisiae and humans.
Functional annotation analysis revealed that proteins that preferentially interact with
CreA in repressing conditions include histones and the histone transcription regulator 3,
Hir3. Proteins interacting with CreB were involved in cellular transportation and
organization. Similar findings were obtained using yeast and human orthologues,
although the yeast background generated a number of other biological processes
involving Mig1p which were not present in the A. nidulans or human background analyses. Hir3 was present in repressing conditions for CreA, and in both growth
conditions for CreB, suggesting that Hir3, or proteins interacting with Hir3, could be a
possible target of CreB.
Earlier, genome-wide microarray analysis showed that CreA was involved in the
transcriptional regulation of a significant number of genes in A. nidulans, however, this
approach cannot show whether the targets are directly or indirectly regulated. To
identify the direct targets, and whether CreA binds in derepressing conditions, ChIP-seq
analyses were performed. CreA constitutively bound to the promoters of target genes in
both growth conditions, indicating that the function of CreA may be controlled on the
chromatin by post-translational modifications. A total of 1946 unique targets were
identified for both strains in repressing and derepressing conditions including genes that
are involved in carbohydrate metabolic/catabolic processes, alcohol biosynthetic
processes, secondary metabolism, and sugar and amino acid transporters.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 201