Skip to main content

Small open reading frames (smORF) as novel modulators of disorders of dietary excess

Periodic Reporting for period 2 - LIPMETIN-sURFing (Small open reading frames (smORF) as novel modulators of disorders of dietary excess)

Reporting period: 2020-01-15 to 2021-01-14

Dietary excess is a major driver of human modern diseases, including dyslipidemia and cardiovascular (CVD) diseases, the leader in the cause of mortality worldwide. The intestine plays a unique role in the metabolic defense against energy excess through regulation of dietary lipid uptake, lipoprotein production, and cholesterol excretion. The regulation of lipid metabolism in the intestine is very complex and is mediated by different genes. Recent studies have suggested that the regulation of this metabolism in the intestine could be also modulated by non-coding RNAs, specifically by small open reading frames (smORFs)-encoded peptides (SEP). However, whether the enterocyte produces specific smORF-encoded bioactive or regulatory peptide that regulates lipid metabolism remains completely obscure. In this sense, recent advances in bioinformatics, proteomics, and high-throughput analyses of translation start sites have begun to address these challenges and identified hundreds of putative coding smORFs and occasionally widely conserved. Indeed, the recent finding suggests that some SEPs or microproteins with a diverse physiological role in different species and human cells and tissues. All these findings provide strong evidence of the unanticipated complexity of our human small proteome. However, the contribution of their potential peptide products to cellular functions, if any, remains unknown. Thus, the discovery of novel intestinal players as microproteins, and a deeper insight into its biological function may contribute significantly to the understanding of modern human diseases and provide new opportunities to treat dyslipidemia and other deadly sequelae of dietary excess. In this context, the LIPMETIN-sURFing project and training program is designed to go beyond the state-of-the-art and will provide new data to 1) screen novel microproteins from intestine, 2) validate the presence of a selected novel microprotein and search for a biological function in lipid metabolism, and 3) evaluate whether the microproteins could be modulated by a food bioactive component.
The main activities and achievements of the present project are briefly described below.

The first aim of the 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. Its differentiation is prolonged in the time, for that reason, and to obtain the best possible project performance, it was performed experiments initially not described such as characterization of smORFs in undifferentiated Caco-2. Regarding proliferative vs differentiated Caco-2 cells data, a total of 5,505 protein-coding smORFs were detected during the differentiation process of Caco-2 (Figure 1). Hundreds of smORFs showed changes in their expression during the maturation process. In this line, the down-regulated smORFs are globally involved in processes associated with cell proliferation, whereas the up-regulated smORFs are involved in the response to xenobiotic stimulus and to metal ion increased their expression, among others. Concerning differentiated Caco-2 cells: treated vs not treated with lipid micelles, 90 smORFs changes their expression after the exposition of Caco-2 cells to lipid micelles. Concretely, 54 smORFs showed an increase in their expression, while 36 decreased it.

The second aim of the project is the characterization of selected novel intestinal-specific microproteins. Currently, the researcher is performing this analysis. 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 and the COVID-19 pandemic situation the initiation of this WP has been delayed.

The third goal of the project is to evaluate the modulation of expression microproteins by dietary components. Our diet is major driver of human health and disease. Therefore, the next step in this project was to determine if food bioactive compounds, well described to affect lipid metabolism when supplemented (i.e. docosahexaenoic acid [DHA]), could modulate the expression microproteins. Unfortunately, we did not detect changes in the expression of smORFs after their exposition to lipid micelles in the presence or absence DHA.

In addition to the experiments described in the project, the researcher has carried out the search of smORFs involved in the lipid metabolism and regulation of cytoplasmic lipid droplet (CLD) from Hep-G2. Regarding lipid metabolism, a total of 6,848 unique smORFs protein-coding smORFs were detected, showing 49 of them changes their expression. Concerning CLD, 23 microproteins were detected using two preparation sample methods (14 by chloroform /methanol, 10 by acetone).
In summary, the results exposed demonstrated for the first time that the smORFs play a role in the differentiation process and lipid metabolism of Caco-2 cells, as well as in the lipid metabolism and CLD of Hep-G2.

Problems in the optimization of WP1 and the COVID-19 pandemic situation have delayed the dissemination of the data obtained in LIPMETIN-sURFing. Currently, it is being written an article whose provisional title is: “The Changing Landscape of smORF Expression in Differentiating Caco-2 Cells”.
Our diet is the major driver of human health and disease. The intestine plays an important role in energy homeostasis and is a “gatekeeper” of lipid metabolism. Although much has been learned about all molecular processes that handle and mishandle dietary lipids, including novel noncoding RNAs, little is known about all the biological processes and mechanisms that regulate intestinal lipid metabolism. The discovery of novel intestinal players in lipid metabolism (i.e. microprotein), and a deeper insight into its biological function will contribute significantly to the understanding of modern human diseases and provide new opportunities to treat dyslipidemia and other deadly sequelae of dietary excess. In this context, the ongoing project has been designed to go beyond the state-of-the-art and will provide new data in order to: 1) screen novel microproteins from intestine 2) validate and evaluate of a selected novel peptide for their role in lipid metabolism, which may open new therapeutic strategies against dyslipidemia and CVD, and 3) evaluate whether microproteins could be modulated by a food bioactive component.

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.
figure-report-final-project-746435-page-0001.jpg