2 Work Performed
The proposal describes in the WP1, preparation of the nuclear fraction protocol isolating nuclear fractions from the samples grown in xylan and glucose plus xylan, finally, mass spectrometry using nuclear fractions should be applied and samples analyzed. The enrichment of the nuclear fraction was observed by Western blot using anti-histone H3 antibody (Abcam ab1791) rabbit polyclonal and as a positive control was used calf thymus histone purified (Roche #10223565001). The samples were divided into control (Histone H3 purified), XT (Xylan total cell lysate), XC (Xylan cytoplasm fraction), and XN (Xylan nuclear fraction), GT (Glucose total cell lysate), GC (Glucose cytoplasm fraction), and GN (Glucose nuclear fraction), as observed in Fig. 2 (attached), the fraction XN and GN showed higher concentration of histone H3 while the fractions XC didn’t show histone detection confirming that nuclei enrichment was achieved. As expected, XT and GT holding the total cell lysate fractions showed a weaker band detecting histone H3 due to the presence of both cytoplasm and nuclear fractions (Fig. 2). Additionally, the amount of ß-actin was measured by Western blot, as expected, total cell lysate and cytoplasm fractions showed the highest amount of ß-actin than the nuclear fraction in both conditions, ß-actin abundance is higher in the cytoplasm than nucleus5 confirming again the absence in the nuclear preparation.
Proteins from the cytoplasm and nuclear fractions from strains grown in xylan or xylan supplemented with glucose as a carbon source were prepared for mass spectrometry as described previously1,2. The data analysis was done using the software Proteome Discoverer 2.0 Daemon (Thermo Fisher) using Aspergillus nidulans database for peptide identification. The results were submitted to a second analysis using filtering from Microsoft Office Excel checking communal proteins identified in each replicate for consistency of the data. The results from cells grown in xylan (cytoplasm and nuclear fractions) and xylan supplemented with glucose (cytoplasm and nuclear fractions) were submitted to a third filtering process selecting communal and unique proteins for each condition. Indeed, after the addition of glucose, it was identified unique proteins in glucose treatment that moved to the nucleus (Fig. 3), among them was found the transcription factor CreA (AN6195) already described as a nuclear regulator of CCR in the presence of glucose. CreA also can be used as an experimental control showing that CCR is in place. Regarding the presence of the nuclear proteins due to posttranslational modification and movement to the nucleus or increased expression in the presence of glucose, the analysis of RNAseq using A. nidulans AGB551 in the same conditions showed lower fold-increase variation (<1) for all proteins of interest (POI) identified (Fig. 3). Moreover, the POIs have submitted to functional classification grouping the target proteins and showing the nuclear regulators highlighted in red (Fig. 4).
The results are promising and the delivery of the WP1 shown in the Gantt Chart was done, it was predicted to be executed into 6 months since the project was started, showing that the project was 2 months forward in the schedule, however, due to premature termination of the grant, the step for dissemination and exploitation could not be executed as planned. The researcher was able to acquire skills in nuclear fraction preparation and mass spectrometer device learning and analysis including mass spectrometry analysis and functional classification for the POI.
