MuRe · Sorting and export of mucins.

Sorting and export of mucins.

Internal mucosae serve a vital function in human health. The mucus that lines the luminal epithelial surfaces of the body acts as a selectively permeable biological shield. This mucous layer participates in the exchange of beneficial molecules for the cell but also interacts with the microbiota, toxic substances, and potential pathogens [1,2]. Mucus is mainly composed of a high-molecular-weight protein family known as mucins (MUC) [1-3]. Whilst the function of mucins is intensively studied, the molecular mechanisms that regulate mucin sorting, transport, and secretion have been poorly explored. Currently, millions of people worldwide are affected by conditions dependent on the expression and secretion of mucins, such as asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), and colon cancer, among many others [4-9]. Mucins are produced in all epithelial cells, so the mixture and amount of individual mucins vary in a cell and tissue type-specific manner [3]. The quantity and composition of mucins provide major rheological and biochemical characteristics of the mucus hydrogel. Several data show that tissue-specific expression of mucins has essential functions in cell homeostasis, cell adhesion, differentiation, and protection against environmental threats [3,13]. However, growing evidence reveals that disturbance in the expression of several mucins, both increasing or decreasing the quantity or quality, can predispose to pathological conditions like inflammation, infection, autoimmunity and cancer [3-8,14,15]. Particularly, the abnormal decrease in intestinal mucin secretion – known as mucin hyposecretion – is closely related to IBD, and increased predisposition to abdominal infections and colon cancer [9,15]. Notwithstanding, the regulatory mechanisms affected in the intestinal mucin secretion misbalance remains a big question in this field. In the intestine, secreted-mucins are produced mainly by differentiated epithelial cells, the Goblet cells. Mucins are glycosylated proteins characterized for sharing a region of repeating sequences of amino acids rich in proline, threonine, and serine [1-3]. Apomucins are synthesized and dimerized in the ER, then are heavily O-glycosylated, polymerized, and packaged in the Golgi apparatus for sorting and release as bulky cargoes [2,3]. Mature mucins own an estimated size of up to ~2500kDa, and the O-glycans decorating the apomucin core constitute up to ~75%. 21 mucin genes have been identified in human tissues thus far. These glycoproteins can be divided into two subfamilies: transmembrane mucins – MUC1, MUC3A, MUC3B, MUC4, MUC7-9, MUC12-17, MUC20-22 – or secreted-mucins – MUC2, MUC5AC, MUC5B, MUC6, MUC19 [10-12]. Mucins are packed into specialized granules and constitutively released (baseline level) or in response to an external agonist or acute stimuli (stimulated) [3]. Malhotra's laboratory is a world leader in understanding the process of bulky-cargo secretion [16] and has developed a clever technique to quantify secreted mucins in the media [17,18]. Moreover, to challenge the difficulties of studying bulky-cargoes like mucins, I have recently expressed by CRISPR/Cas9 gene-editing technology the green fluorescent protein(GFP) in MUC5B and the red fluorescent protein(mCherry) in MUC13 in colon cancer-derived HT29-18N2 cells. Also, I have developed a novel technique to quantify mucins expression intracellularly using flow cytometry. These approaches will allow me to follow the mucins – transmembrane and secreted – in i) static and dynamic assays; ii) presence or absence of a regulatory partner. For this reason, in this project, we will address the mechanisms that control mucins - transmembrane and secreted - secretion. This project had three main objectives: 1) To characterize mucin sorting in the Golgi. 2) To characterize transmembrane and secreted mucins post-Golgi granules in the secretory pathway. 3) To describe molecular partners involved in transmembrane and soluble mucins release. The project fulfilled several of its objectives and milestones. However, some objectives were adjusted due to 1) technical issues beyond my control and 2) the exploration of new research lines that are significant for the community.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз