Periodic Reporting for period 1 - LIPMETIN-sURFing (Small open reading frames (smORF) as novel modulators of disorders of dietary excess)
Reporting period: 2018-01-15 to 2020-01-14
The first aim of the ongoing project is focused on the use combined of MS peptidomics, ribosome profiling, and bioinformatics to screen for intestinal-specific microproteins modulated by dietary lipids.
Caco-2 cell model is the gold standard of intestinal in vitro models. This cell line spontaneously differentiates into polarized enterocytes expressing high levels of brush border enzymes typical of small intestinal epithelial cells such as alkaline phosphatase (ALP). As shown in figure 1, the maximum ALP activity was observed at 2 weeks which reflects that the differentiation of Caco-2 was reached at 2 weeks. In this point, it´s important to indicate that due to the differentiation of Caco-2 is prolonged in the time and in order to obtain the best possible project performance, it was performed experiments initially not described such as characterization of smORFs in undifferentiated Caco-2 and liver cells (Hep-G2).
Regarding proteomics analysis, a total of 12 smORFs were detected. Three of them were detected in all the samples. The next step was RNA-Seq and Ribo-Seq, both techniques are more sensitive than MS-proteomic. In relation to gene expression genetic in Caco-2 cells, the results showed a significant difference between undifferentiated and differentiated cells (Figure 2). Regarding differentiated Caco-2 cells, these cells were clustered in two groups: cells treated and not with lipid micelles. A total of 1,957 smORFs were identified in both sorts of differentiated Caco-2 cells, being modified the expression of more 100 of these smORFs in the presence of lipid micelles. Furthermore, BLAST analysis determined that some of the smORFs that exhibited differential expression in the presence of lipid micelles were conserved in other species like the mouse. With regard to RNA-Seq and Ribo-Seq of Hep-G2 data, total of 5,051 unique microproteins were detected, of which 911 SEP were identified in Hep-G2 cells treated and not treated with lipid micelles.
The second aim of the project is the characterization of selected novel intestinal-specific microproteins.
Currently, the researcher is performing the validation of the selected microproteins. This assay was programmed for the second year of the project, but due to that the optimization period of the experiments included in the WP 1 has taken longer than estimated, the initiation of this WP has been delayed.
In addition to the experiments described in the project, the researcher has carried out the search of smORFs involved in the regulation of cytoplasmic lipid droplet (CLD).
For this aim, differentiated Caco-2 and Hep-G2 treated with lipid micelles were selected. The proteomic analysis of both cell lines was in accordance with the CLD proteome previously reported in the literature. Regarding microproteins, two smORFs were identified, but possibly they were incorrectly identified because they didn't have a high quality. In a second analysis (only Hep-G2), to avoid that the larger peptides could mask the SEP, a pre-step of concentration of micropeptide was added to the protocol. Currently, the samples are being analyzed by mass spectrometer.
The data obtained until now reports for the first time the presence of microproteins in the intestine and liver. The next step will be to evaluate the biological processes in which these microproteins are implicated, especially related to the lipid metabolism. In this line, the results obtained in this ongoing project will open up a completely new field of research in the area of nutrition and health that could eventually help us to combat the devastating consequences of our modern human diseases associated with dietary excess. Overall, our results will contribute to gain insight into disease mechanisms and innovative therapeutic strategies to combat dyslipidemia and CVD associated with dietary excess. Finally, innovative results are intended to be transferred to small companies in the area of peptide therapy and CVD field and to lead to the development of several patent applications.