Periodic Reporting for period 1 - MuRe (Sorting and export of mucins.)
Periodo di rendicontazione: 2023-02-01 al 2025-01-31
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.
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.
1.2 Explanation of the work carried out per WP
1.2.1 Work Package 1 - [Characterizing mucin sorting in the Golgi]
• Progress: Good progress.
• Brief summary of the work performed: This objective was achieved, but we had to change the methodology employed after encountering technical problems. The fusion of mutant biotin-protein ligase in the soluble and transmembrane mucin sequences presented unknown issues. When we were able to express it, the distribution of mucins was altered, and we believe that it could also modify intra-Golgi glycosylation. Therefore, we cannot rely on close interactors, since the normal localization of mucins is erroneous. However, on the one hand, we have established a connection with experts in the Rush system, to try to follow the localization of mucins from the endoplasmic reticulum (ER) in a time-dependent manner and thus trigger the Bio-ID system before the alteration in localization. On the other hand, to remedy this issue, we expressed each mucin separately (MUC13 and MUC5B) with a GFP tag and did a pulldown using GFP beads. We then classified common interactors and compared them with known resident proteins from the ER, cis-Golgi, trans-Golgi, and other cytoplasmic compartments. In this way, we refined a list of potential interactors involved in sorting these mucins within the Golgi. While we identified some potentially meaningful interactors in this task, we are developing cell lines tagged with fluorescent or knockout (KO) proteins stable by CRISPR-Cas9 editing against these proteins, to determine the molecular role in mucin sorting in the Golgi. In addition, this methodology allowed us to identify an interactor involved in the altered secretion of mucins in a rare disease of genetic origin (TANGO2 deficiency).
• Outcome and results: We have identified interactors in the pulldown of soluble and transmembrane mucins that are stable residents of the ER, cis-Golgi, and trans-Golgi. We are studying the molecular mechanisms by which the interaction of these mucin partners can sort granules from the ER to the trans-Golgi. These results are being prepared in a manuscript we hope to publish soon.
• Achievement of scientific deliverables and milestones (fully/partly achieved, not achieved).
- D1: Partly achieved. We are working to fix the bio-ID problems before sharing the curated list of mucin interactors in the cell.
- D2: Partly achieved. We are finishing a few experiments to publish and share our findings on mucin-interactors in the Golgi.
- M1: This has not been achieved yet; there is a technical problem with the stable co-expression of the mucin selected in the study. We will figure it out soon.
1.2.2 Work Package 2 - [Characterizing transmembrane and secreted mucins post-Golgi granules in the secretory pathway]
• Progress: Limited progress.
• Brief summary of the work performed. The implementation of the FAOS system for the detection of various mucin granule populations was not as expected. The analysis of mucin-labelled granule populations by cytometry did not allow the granules to be differentiated from the cell debris for collection, and the results were not reproducible. Alternatively, we labelled the transmembrane mucin MUC13 with GFP in the cytoplasmic portion. This technical modification allows us to purify MUC13-positive granules using GFP resins. We are working to improve the collection of granules for analysis by mass spectrometry. The limitation we found is that if the granules containing transmembrane mucins do not contain soluble mucins, we will be unable to identify the post-Golgi interactors of mucins that do not have transmembrane domains. However, we continue to work with the flow cytometry unit to improve the results when applying the FAOS technique.
• Outcome and results. We have succeeded in purifying granules of transmembrane mucins labelled on the cytoplasmic side. Applying drugs that limit the localization of the granules within the cell allows us to study the content and interactors of these granules.
• Achievement of scientific deliverables and milestones.
- D3: Fully achieved. We presented the progress of our results in internal laboratory meetings, CRG quantitative cell biology meetings, and PRBB inter-program meetings. We found ways to overcome the technical problems presented.
- D4: Partly achieved. Some interactors analyzed so far were shared on GitHub (Wojnacki J et al., Nat. Comm., 2023).
- D5: Fully achieved. I shared training activities and courses, a communication and dissemination plan, and a career development plan.
- M2: Not fully achieved yet; we are still working on implementing the FAOS system. In between, we are working to share the results of the MUC13-GFP-granules mass spectrometry using GitHub.
1.2.3 Work Package 3 - [Describing molecular partners involved in transmembrane and soluble mucins release]
• Progress: Satisfactory progress.
• Brief summary of the work performed. We developed stable cell lines to study the behaviour of four selected interactors (TSPAN-8, syntaxin-2, syntaxin-3, and TANGO2). We studied these proteins' phenotypes and molecular mechanisms in mucin synthesis, trafficking, and secretion. This objective allowed us to describe in detail the function of TSPAN-8, Syntaxin-2 and Syntaxi-3 in post-Golgi trafficking and mucin secretion related to pathophysiological processes of the airway and colon tissues. In addition, we demonstrated that these interactors' action is not exclusive to mucins since it is also relevant for the secretion of other proteins, such as insulin, in pancreatic beta cells. On the other hand, we found that mutations in one of the interactors of interest (TANGO2) generate a rare disease in humans. Upon further investigation, we observed that patients with TANGO2 deficiency commonly present constipation. This observation led us to study further TANGO2 related to mucins and to observe that the absence of this interactor increases the expression of the sodium channel TRPM4, which was described as a key interactor in mucin secretion by the Malhotra laboratory (Cantero-Recasens G et al., JCB, 2018). We continue to study this pathology to understand the molecular mechanisms that govern the classification and secretion of certain mucins and to contribute knowledge to a rare disease of great importance to the community to improve patients' quality of life.
• Outcome and results. We have identified several components of mucin secretion between the Golgi and the plasma membrane. This approach has allowed us to describe fundamental mechanisms in conventional protein secretion and contribute knowledge to the community of patients suffering TANGO2 deficiency. Part of this objective has been presented at international congresses (Annual EMBO Meeting, TANGO2 Research Foundation Meeting) and published in prestigious journals (Lujan AL et al., eLife 2023; Wojnacki J et al., Nat. Comm. 2023; Lujan AL et al., JCB 2025).
• Achievement of scientific deliverables and milestones (fully/partly achieved, not achieved).
- D6: Fully achieved. We presented the progress of our results in internal laboratory meetings, CRG quantitative cell biology area, and PRBB inter-program meetings.
- D7: Partly achieved. We published a list of interactors on Git Hub and a detailed description of the function of four interactors of interest. We are working on a fifth interactor belonging to the GPCR proteins family.
- D8: Fully achieved.
- D9: Fully achieved.
- M3: Partly achieved. We are still generating cell lines for the new interactors we are working on.
- M4: Partly achieved. We are collaborating with experts in the field to block TSPAN-8 functions with small-targeted peptides.
1.2.1 Work Package 1 - [Characterizing mucin sorting in the Golgi]
• Progress: Good progress.
• Brief summary of the work performed: This objective was achieved, but we had to change the methodology employed after encountering technical problems. The fusion of mutant biotin-protein ligase in the soluble and transmembrane mucin sequences presented unknown issues. When we were able to express it, the distribution of mucins was altered, and we believe that it could also modify intra-Golgi glycosylation. Therefore, we cannot rely on close interactors, since the normal localization of mucins is erroneous. However, on the one hand, we have established a connection with experts in the Rush system, to try to follow the localization of mucins from the endoplasmic reticulum (ER) in a time-dependent manner and thus trigger the Bio-ID system before the alteration in localization. On the other hand, to remedy this issue, we expressed each mucin separately (MUC13 and MUC5B) with a GFP tag and did a pulldown using GFP beads. We then classified common interactors and compared them with known resident proteins from the ER, cis-Golgi, trans-Golgi, and other cytoplasmic compartments. In this way, we refined a list of potential interactors involved in sorting these mucins within the Golgi. While we identified some potentially meaningful interactors in this task, we are developing cell lines tagged with fluorescent or knockout (KO) proteins stable by CRISPR-Cas9 editing against these proteins, to determine the molecular role in mucin sorting in the Golgi. In addition, this methodology allowed us to identify an interactor involved in the altered secretion of mucins in a rare disease of genetic origin (TANGO2 deficiency).
• Outcome and results: We have identified interactors in the pulldown of soluble and transmembrane mucins that are stable residents of the ER, cis-Golgi, and trans-Golgi. We are studying the molecular mechanisms by which the interaction of these mucin partners can sort granules from the ER to the trans-Golgi. These results are being prepared in a manuscript we hope to publish soon.
• Achievement of scientific deliverables and milestones (fully/partly achieved, not achieved).
- D1: Partly achieved. We are working to fix the bio-ID problems before sharing the curated list of mucin interactors in the cell.
- D2: Partly achieved. We are finishing a few experiments to publish and share our findings on mucin-interactors in the Golgi.
- M1: This has not been achieved yet; there is a technical problem with the stable co-expression of the mucin selected in the study. We will figure it out soon.
1.2.2 Work Package 2 - [Characterizing transmembrane and secreted mucins post-Golgi granules in the secretory pathway]
• Progress: Limited progress.
• Brief summary of the work performed. The implementation of the FAOS system for the detection of various mucin granule populations was not as expected. The analysis of mucin-labelled granule populations by cytometry did not allow the granules to be differentiated from the cell debris for collection, and the results were not reproducible. Alternatively, we labelled the transmembrane mucin MUC13 with GFP in the cytoplasmic portion. This technical modification allows us to purify MUC13-positive granules using GFP resins. We are working to improve the collection of granules for analysis by mass spectrometry. The limitation we found is that if the granules containing transmembrane mucins do not contain soluble mucins, we will be unable to identify the post-Golgi interactors of mucins that do not have transmembrane domains. However, we continue to work with the flow cytometry unit to improve the results when applying the FAOS technique.
• Outcome and results. We have succeeded in purifying granules of transmembrane mucins labelled on the cytoplasmic side. Applying drugs that limit the localization of the granules within the cell allows us to study the content and interactors of these granules.
• Achievement of scientific deliverables and milestones.
- D3: Fully achieved. We presented the progress of our results in internal laboratory meetings, CRG quantitative cell biology meetings, and PRBB inter-program meetings. We found ways to overcome the technical problems presented.
- D4: Partly achieved. Some interactors analyzed so far were shared on GitHub (Wojnacki J et al., Nat. Comm., 2023).
- D5: Fully achieved. I shared training activities and courses, a communication and dissemination plan, and a career development plan.
- M2: Not fully achieved yet; we are still working on implementing the FAOS system. In between, we are working to share the results of the MUC13-GFP-granules mass spectrometry using GitHub.
1.2.3 Work Package 3 - [Describing molecular partners involved in transmembrane and soluble mucins release]
• Progress: Satisfactory progress.
• Brief summary of the work performed. We developed stable cell lines to study the behaviour of four selected interactors (TSPAN-8, syntaxin-2, syntaxin-3, and TANGO2). We studied these proteins' phenotypes and molecular mechanisms in mucin synthesis, trafficking, and secretion. This objective allowed us to describe in detail the function of TSPAN-8, Syntaxin-2 and Syntaxi-3 in post-Golgi trafficking and mucin secretion related to pathophysiological processes of the airway and colon tissues. In addition, we demonstrated that these interactors' action is not exclusive to mucins since it is also relevant for the secretion of other proteins, such as insulin, in pancreatic beta cells. On the other hand, we found that mutations in one of the interactors of interest (TANGO2) generate a rare disease in humans. Upon further investigation, we observed that patients with TANGO2 deficiency commonly present constipation. This observation led us to study further TANGO2 related to mucins and to observe that the absence of this interactor increases the expression of the sodium channel TRPM4, which was described as a key interactor in mucin secretion by the Malhotra laboratory (Cantero-Recasens G et al., JCB, 2018). We continue to study this pathology to understand the molecular mechanisms that govern the classification and secretion of certain mucins and to contribute knowledge to a rare disease of great importance to the community to improve patients' quality of life.
• Outcome and results. We have identified several components of mucin secretion between the Golgi and the plasma membrane. This approach has allowed us to describe fundamental mechanisms in conventional protein secretion and contribute knowledge to the community of patients suffering TANGO2 deficiency. Part of this objective has been presented at international congresses (Annual EMBO Meeting, TANGO2 Research Foundation Meeting) and published in prestigious journals (Lujan AL et al., eLife 2023; Wojnacki J et al., Nat. Comm. 2023; Lujan AL et al., JCB 2025).
• Achievement of scientific deliverables and milestones (fully/partly achieved, not achieved).
- D6: Fully achieved. We presented the progress of our results in internal laboratory meetings, CRG quantitative cell biology area, and PRBB inter-program meetings.
- D7: Partly achieved. We published a list of interactors on Git Hub and a detailed description of the function of four interactors of interest. We are working on a fifth interactor belonging to the GPCR proteins family.
- D8: Fully achieved.
- D9: Fully achieved.
- M3: Partly achieved. We are still generating cell lines for the new interactors we are working on.
- M4: Partly achieved. We are collaborating with experts in the field to block TSPAN-8 functions with small-targeted peptides.
The overall result of this MSCA has exceeded the impacts proposed in this project, even having undergone modifications in its methodological approach. The scientific results of this project are reflected in three high-impact scientific publications, which have opened new lines of research and new scientific collaborations and have provided relevant knowledge to the community. For instance, Lujan et al., eLife 2023, showcased a novel approach to the TANGO2 community, emphasizing the significance of this protein in lipid metabolism. This finding has prompted the international scientific community to focus on TANGO2 energy metabolism. In Wojnacki et al., Nat. Comm. 2023, we described a key mechanism in the secretion of mucins and other cargoes such as insulin. These findings have allowed us to open a new line of research to develop therapies that regulate this mechanism's function to control those molecules' secretion. In Lujan et al., JCB 2025, we have demonstrated the primary function of TANGO2 protein. These findings enable the medical community to make more accurate therapeutic decisions for patients with TANGO2 deficiency. Collaborations with the Hospital Sant Joan de Deu in Barcelona, the Baylor College of Medicine, and the TANGO2 Foundation, along with a further study of this transport and Golgi organization protein, are facilitating our understanding of its role in mucin secretion at the colonic level and its direct impact on the gastrointestinal symptoms experienced by these patients.
In addition to the delays caused by technical problems, the unpublished results are being prepared for publication and will be available to the scientific community as soon as possible. These will include a list of key interactors in the sorting and secretion pathway of soluble and membrane mucins and images showing different mucins sharing the same container from the Golgi apparatus to the plasma membrane pathway. Although this project had a wide range of objectives with some risk of success, we believe that we will not only be able to meet the commitments made (not on time) but also that we will have made fundamental contributions to the knowledge of the mechanisms studied, with a direct impact on the medical community and affected patients.
The technical problems experienced in applying the Bio-ID and FAOS techniques caused us to lose time. We had to change our methodological strategies to achieve our overall goal of finding relevant interactors in the sorting and secretion of mucins via the conventional protein secretion pathway. To save time and the project, I relied on bioinformatic analyses of several published proteomics studies from the Malhotra Lab (Bard et al., Nature 2006; Mitrovic et al., eLife 2013) on mucin secretion and the identification of genes involved in protein transport and Golgi organization. This arrangement has allowed us to achieve our overall goal. However, in parallel, I continued to resolve the technical issues I had found in the original plan. I believe that soon, and after completing the appropriate replicates, I will also be able to achieve the original objectives outside the timeframe set. Therefore, we planned with the PI that I would stay in the lab for another 6-8 months to complete the original objectives, publish the results, and start writing a review with everything I have learned during my stay in his laboratory. Malhotra's laboratory has other national and international grants that will allow me to extend my stay to fulfil this plan.
In addition to the delays caused by technical problems, the unpublished results are being prepared for publication and will be available to the scientific community as soon as possible. These will include a list of key interactors in the sorting and secretion pathway of soluble and membrane mucins and images showing different mucins sharing the same container from the Golgi apparatus to the plasma membrane pathway. Although this project had a wide range of objectives with some risk of success, we believe that we will not only be able to meet the commitments made (not on time) but also that we will have made fundamental contributions to the knowledge of the mechanisms studied, with a direct impact on the medical community and affected patients.
The technical problems experienced in applying the Bio-ID and FAOS techniques caused us to lose time. We had to change our methodological strategies to achieve our overall goal of finding relevant interactors in the sorting and secretion of mucins via the conventional protein secretion pathway. To save time and the project, I relied on bioinformatic analyses of several published proteomics studies from the Malhotra Lab (Bard et al., Nature 2006; Mitrovic et al., eLife 2013) on mucin secretion and the identification of genes involved in protein transport and Golgi organization. This arrangement has allowed us to achieve our overall goal. However, in parallel, I continued to resolve the technical issues I had found in the original plan. I believe that soon, and after completing the appropriate replicates, I will also be able to achieve the original objectives outside the timeframe set. Therefore, we planned with the PI that I would stay in the lab for another 6-8 months to complete the original objectives, publish the results, and start writing a review with everything I have learned during my stay in his laboratory. Malhotra's laboratory has other national and international grants that will allow me to extend my stay to fulfil this plan.