dystrophy
Morrée, A. de
Citation
Morrée, A. de. (2011, January 12). Functional protein networks unifying limb girdle muscular dystrophy. Retrieved from https://hdl.handle.net/1887/16329
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Muscular dystrophies comprise a genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and weakness.
Limb Girdle Muscular Dystrophy (LGMD) is a rare disorder that can be caused by mutations in at least 21 different genes. These genes are often widely expressed and encode proteins with highly diverse functions. And yet mutations in all of them give rise to a similar clinical presentation: adult onset muscle weakness, with muscles of the pelvic and shoulder girdle as predominantly affected muscle groups (Chapter 1).
This thesis explores a potential molecular mechanism that unifies the different genetic defects, which individually can cause a limb girdle muscular dystrophy, starting with LGMD2B.
LGMD2B is caused by mutations in Dysferlin. Dysferlin is critical for calcium- dependent muscle membrane repair upon mechanically- or laser-inflicted membrane damage. However, Dysferlin is widely expressed and a muscle membrane repair defect cannot fully explain the LGMD phenotype. This suggests additional roles for Dysferlin. The identification of complex partners helps elucidate protein functions.
In literature, <10 Dysferlin interaction partners were described thus far. Therefore we analyzed the composition of Dysferlin protein complexes in cultured myoblasts, myotubes and skeletal muscle tissue by immunoprecipitation followed by mass spectrometry. We subsequently employed bioinformatics analyses to infer potential protein functions (Chapter 2). We identified >100 novel complex partners, that confirm existing hypotheses (endocytosis and vesicle trafficking) and point to new putative functionalities (adhesion, energy metabolism) of Dysferlin. We conclude that Dysferlin is a key player in the spatiotemporal dependent maintenance of skeletal muscle membrane integrity.
We were interested whether Dysferlin function is conserved between Dysferlin expressing tissues. We therefore investigated Dysferlin function in monocytes (Chapter 7). Monocytes were described to express high levels of Dysferlin.
Moreover, LGMD2B skeletal muscle tissue is characterized by strong monocyte and macrophage infiltrates, and LGMD2B monocytes exhibit impaired phagocytotic behavior. We found that, like in muscle, Dysferlin expression increases with differentiation in primary monocytes and the THP1 monocyte cell model. Both LGMD2B monocytes and Dysferlin depleted THP1 cells showed deregulated expression of Fibronectin and Fibronectin-binding Integrins α5, αV, β1 and β3. In the absence of Dysferlin, THP1 cells displayed a differentiation defect and adhered less efficiently to a plastic or glass surface. These observations suggest additional parallels between Dysferlin function in muscle and monocytes, which involve cell adhesion. As in muscle, monocyte Dysferlin forms a complex with adhesion proteins, including Integrin β3, and both proteins were rapidly endocytosed in response to Integrin β3 inhibition. These findings yield new insights into Dysferlin function in inflammatory cells, provide an explanation for the impaired immune response in LGMD2B, and support a function for Dysferlin beyond muscle membrane repair.
We wondered how Dysferlin is regulated. Two proteins that were identified in the Dysferlin protein complex allowed us to investigate this question. Calpain 3 is a cysteine protease mutated in LGMD2A, and AHNAK is a ubiquitously expressed 700 kDa protein involved in cell-matrix adhesion and cytoskeleton remodeling.
We could demonstrate that AHNAK is cleaved by Calpain 3, and AHNAK fragments cleaved by CAPN3 have lost their affinity for Dysferlin (Chapter 3). In skeletal muscle of LGMD patients, AHNAK was lost in the absence of Dysferlin (LGMD2B), while it accumulated when Calpain 3 was defective (LGMD2A). Thus, our findings suggest that Calpain 3 proteolytically regulates the Dysferlin protein complex, and suggest interconnectivity between two LGMD variants.
Calpain 3 is the only described Calpain family member that genetically causes a disease. The protein autolytically activates and inactivates itself in a short time-span. Due to this inherent instability little is known of its substrates or its mechanism of activity and pathogenicity. With a novel multi-disciplinary approach that combines bioinformatics with biochemistry, cell biology and structural modeling we were able to define a tertiary folded primary sequence motif underlying Calpain 3 substrate cleavage (Chapter 4). This motif could transform non-related proteins into substrates, and identified >300 new Calpain 3 targets.
Bioinformatics analyses of these targets demonstrated a critical role in muscle cytoskeleton remodeling and identified novel Calpain 3 functions. Among the new Calpain 3 substrates were three E3 SUMO ligases of the Protein Inhibitor of Activated Stats (PIAS) family. Calpain 3 could cleave PIAS proteins and negatively regulate PIAS3 sumoylase activity. Consequently, SUMO2 was deregulated in LGMD2A patient muscle tissue. Our study thus uncovered unexpected crosstalk between Calpain 3 proteolysis and protein sumoylation and provides a molecular
model for Calpain 3 (dys)function, with strong implications for muscle remodeling.
We hypothesized that AHNAK is important for muscle cyto-architecture and adhesion. However, little was known of AHNAK regulation. We investigated the AHNAK gene and its expression in skeletal muscle and identified a novel mechanism of self-regulation through alternative mRNA splicing (Chapter 5). We found that the AHNAK gene is part of a larger family that includes AHNAK2 and Periaxin, and is characterized by a giant exon that is flanked by multiple small exons. These exons allow for alternative splicing. AHNAK, like Periaxin, expresses two isoforms, a large 700 kDa and small 17 kDa protein. These proteins interact in the cytoplasm, but the small AHNAK is also present in the nucleus. During muscle differentiation the small AHNAK is strongly increased thereby establishing a positive feedback loop to regulate splicing of mRNA transcripts that emanate from its own locus.
To investigate a potential role for the large AHNAK in cell adhesion we constructed a miniAHNAK protein (Chapter 6), which retains described AHNAK functionalities. Using clonal IM2-miniAHNAK myoblast cells we could show that miniAHNAK rapidly redistributes in response to Integrin inhibition (as Dysferlin), supporting the hypothesis that in muscle cells AHNAK functions as a structural sensor.
In summary, both muscle and monocyte cells require proper maintenance of cell-cell contacts and timely restructuring of membrane/cytoskeleton architecture.
We propose that the triad of Dysferlin, AHNAK and Calpain 3 is important for maintenance of Integrin based cell-cell contacts (Chapter 8). Dysfunction of any of the three proteins results in impaired maintenance at the level of molecular cell contact points. This indicates that LGMD is the result of impaired muscle maintenance systems.
