Identificazione di partners di legame del distroglicano nel nucleo della cellula: analisi proteomica di interattori nucleari del beta-distroglicano

Responsabili di progetto

Andrea Brancaccio, Bulmaro Cisneros Vega

Accordo

MESSICO - CINVESTAV - Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional

Bando

CNR/CINVESTAV biennio 2019-2020 2019-2020

Dipartimento

Scienze chimiche e tecnologie dei materiali

Area tematica

Scienze chimiche e tecnologie dei materiali

Stato del progetto

Nuovo

Proposta di ricerca

Background
The dystroglycan (DG) complex is composed by two interacting subunits, the extracellular and heavily glycosylated alpha-DG and transmembrane beta-DG. The complex protrudes through the membrane towards the extracellular matrix, where alpha-DG interacts with laminins and a range of other binding partners, whilst the cytodomain of beta-DG ensures a connection with the actin cytoskeleton binding dystrophin (1).

The importance of the DG complex, the major non-integrin adhesion complex present in cells, is highlighted by its structural role in skeletal muscle (2). In fact, a number of severe neuromuscular disorders are DG-linked both indirectly, like secondary dystroglycanopathies where several glycosyltransferases responsible for the glycosylation of DG are involved, and directly, as in primary dystroglycanopathies caused by mutations within the DG gene. In addition, DG has a wide tissue distribution and has also been linked to the early stages of embryonic development, to the formation of the glial-end feet, to the maturation of the post-synaptic element of the neuromuscular junction, to the stabilization of synapses in the central nervous system as well as in other tissues and organs.

Rationale and aim
There is a strong biological, medical and translational interest in dystroglycan research. In a series of collaborations I produced the first high resolution structural results on DG (3-4) and recently in my laboratory in Rome, we have been able to expand our interest to further DG isoforms and mutants (5-6). At the same time, over the last years the laboratory of Dr. Bulmaro Cisneros has developed a strong interest in the localization of beta-DG within cellular nuclei (7-8), reporting recently about the very interesting observation of retrograde beta-DG trafficking to the nucleus (9). Beta-DG in the nucleus should interact with lamin A/C and B1 and emerin too. Such interactions imply that beta-DG might be involved somehow in nucleopathies/laminopathies making this subject very interesting both from a cell biology and biomedical perspective. That said, the exact role played by beta-dystroglycan is still unclear and it is very likely that it should have more nuclear binding partners.

Therefore, the main aim of this proposal is to investigate beta-DG binding partners in the nucleus by techniques such as mass spectrometry and super-resolution microscopy. The strong rationale is to connect two laboratories that possess the complementary expertise required on the dystroglycan system and would be able therefore to establish all the required experimental set-up in a fast and efficient way in order to ensure a positive outcome of the proposed experiments.

Experimental plan and methodology
Our laboratory will receive a series of samples from the laboratory of Dr. Cisneros (see also the proposed visits plan for further details) obtained upon pulling down beta-DG and its binding partners from nuclear fractions derived from a series of cell lines and tissues in different experimental conditions. The samples will be then submitted to proteomic analysis to characterize the beta-DG binding partners following a bottom-up or middle-down approach by mass spectrometry platforms (10-11).

With this approach we aim at univocally confirming the presence of some of the already established nuclear binding partners of beta-DG and at possibly identifying yet unknown ones. We then intend to further validate the list of possible new ligands characterised via mass-spectrometry by using techniques such as i) immunoprecipitation and Western blot and ii) immunofluorescence-based super-resolution microscopy (3D structured illumination microscopy and single molecule localization microscopy) (12).

Visits and outcome
The project will greatly benefit from a number of reciprocal visits, whose details can be found in the appropriate sections of this proposal. Visits will be crucial for strengthen the collaboration, the experimental strategies and sharing of results and knowledge on the system.

Bibliography
1) Bozzi M et al. Functional diversity of dystroglycan. Matrix Biol. (2009) 28, 179-87.
2) Adams JC & Brancaccio A. The evolution of the dystroglycan complex, a major mediator of muscle integrity. Biol. Open (2015) 4, 1163-79.
3) Brancaccio A. et al. Electron microscopic evidence for a mucin-like region in chick muscle alpha-dystroglycan. FEBS Lett. (1995) 368, 139-42.
4) Bozic D. et al. The structure of the N-terminal region of murine skeletal muscle alpha-dystroglycan discloses a modular architecture. J. Biol. Chem. (2004) 279, 44812-6.
5) Covaceuszach S. et al. Structural flexibility of human alpha-dystroglycan. FEBS Open Bio (2017) 7, 1064-77.
6) Signorino G. et al. A dystroglycan mutation (p.Cys667Phe) associated to muscle-eye-brain disease with multicystic leucodystrophy results in ER-retention of the mutant protein. Hum. Mutat. (2018) 39, 266-280.
7) Martínez-Vieyra IA et al. A role for beta-dystroglycan in the organization and structure of the nucleus in myoblasts. Biochim. Biophys Acta. (2013) 1833, 698-711.
8) Vélez-Aguilera G. et al. Control of nuclear beta-dystroglycan content is crucial for the maintenance of nuclear envelope integrity and function. Biochim Biophys Acta. (2018) 1865, 406-420.
9) Gracida-Jiménez V et al. Retrograde trafficking of beta-dystroglycan from the plasma membrane to the nucleus. Sci. Rep. (2017) 7, 9906.
10) Inserra I. et al. Proteomic study of pilocytic astrocytoma pediatric brain tumor intracystic fluid. J. Proteome Res. (2014) 13, 4594-4606.
11) Martelli C. et al. Integrated proteomic platforms for the comparative characterization of medulloblastoma and pilocytic astrocytoma pediatric brain tumors: a preliminary study. Molecular Biosystems (2015) 11, 1668-83.
12) Schermelleh, L., R. Heintzmann, and H. Leonhardt. A guide to super-resolution fluorescence microscopy. J Cell Biol, 2010. 190(2): p. 165-75.

Obiettivi della ricerca

The objective is to identify novel beta-DG ligands in the nucleus in order to better clarify the role played by dystroglycan in this subcellular compartment and assess its possible involvement in nucleopathies/laminopathies. Thus, understanding the bases of this localisation and of the interactions of dystroglycan with potential partners could have important repercussions in biomedicine, with a high translational impact. Another important objective is to initiate a fruitful collaboration between these two laboratories interested in the same area (i.e. dystroglycan distribution and trafficking within the cell) but approaching it from two different angles and also employing different methodologies.

We believe that both the scientific and cultural outcome of establishing such a connection will be very high both for CNR (and its partner collaborator at the University of Bielefeld in Germany) and CINVESTAV. We expect to obtain interesting results that will be submitted to international scientific journals in a series of jointly prepared manuscripts.

Ultimo aggiornamento: 23/05/2025