CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Molecular pathogenesis and therapeutic prospects of sarcoglycanopathies

Sarcoglycanopathies are a group of autosomal recessive muscle-wasting disorders caused by genetic defects of four cell membrane glycoproteins, α-, β-, γ-, and δ-sarcoglycan. The four proteins form a sub-complex closely linked to the major dystrophin-associated protein complex, which is essential to maintain muscle membrane integrity during contraction and to provide a scaffold for important signaling molecules (Fig. 1).

 

Sarcoglycanopathies are included into the large group of limb-girdle muscular dystrophy (LGMD) because of common clinical features, as these progressive muscle disorders predominantly affect proximal muscles around the scapular and the pelvic girdles. Mutations in individual sarcoglycans are responsible for LGMD-2C (γ-sarcoglycan), 2D (α-sarcoglycan), 2E (β-sarcoglycan) and 2F (δ-sarcoglycan). The analysis of muscle samples from patients demonstrates that gene defects in each of sarcoglycan result in either the complete absence or the presence of trace amounts of the protein in the cell membrane and cause the loss or the reduction of the other subunits. Data from LGMD patients and related animal models demonstrate therefore that the four sarcoglycans have an important role at the plasma membrane, but only when they exist as complex.

 

Sarcoglycans are single-pass transmembrane proteins, with short intracellular tail and large extracellular glycosylated domain. Protein sequence analysis shows that sarcoglycans contain different putative functional domains, including an ATP-binding site in α-sarcoglycan (Sandonà et al., 2004), whose physiological role remains however undefined. The four sarcoglycans are synthesized in the endoplasmic reticulum (ER) where they undergo cotranslational glycosylation and proper conformational folding through the activity of an efficient quality control system (Fig. 2, steps a and b). Association of the four sarcoglycans occurs in a strict equimolar stoichiometry in the ER, with the β/δ-sarcoglycan core being the trigger for the assembly of the tetrameric complex (Fig. 2, step c). The complex is then transported to the plasma membrane through the Golgi system (Fig. 2, step d).

Processing, assembly, trafficking and targeting of the sarcoglycan complex represent critical issues of sarcoglycanopathies. The majority of sarcoglycanopathies are associated with missense mutations generating substitution of single residues that could lead to a misfolded protein. In general, misfolded proteins are identified by the quality control system and retro-translocated to the cytosol for proteasomal degradation through the ER-associated protein degradation (ERAD) pathway (Fig. 2, steps e and f). Reduction or absence of one sarcoglycan prevents the formation of the complex and causes the disposal even of the unused "healthy" sarcoglycans. Sequence analysis of sarcoglycans indicates however that many disease-causing missense mutations most likely do not have functional consequences, but they are intercepted by the quality control system that significantly slows their processing and results in the disposal of the mutant protein.

The pathogenetic mechanisms thus comprise 1) the processing of defective sarcoglycan subunits, 2) the inability to assemble into a complete complex, or 3) the targeting of a dysfunctional sarcoglycan complex to the cell membrane (Sandonà & Betto, 2009).

 

We have recently demonstrated, in a heterologous cell model, the involvement of the cell quality control system in the pathogenesis of sarcoglycanopathies. Different α-sarcoglycan mutants with single amino acid substitutions were ubiquitinated and rapidly degraded by proteasome. Inhibition of proteasome was able to recover the expression of α-sarcoglycan mutants and, more importantly, the mutants formed a stable sarcoglycan complex that localized at the cell membrane (Sandonà et al., 2008). Our finding demonstrates that inhibition of proteasome, the last step in the ERAD pathway (Fig. 2, step f), has a retrograde effect on mutant processing permitting the assembly of partly misfolded proteins with the other sarcoglycan components, in a condition that favors their transport to the cell membrane. Importantly, we have shown that α-sarcoglycan mutants maintain the capability to form a potentially functional complex at the cell membrane. An essential prerequisite of this salvage approach is in fact that the rescued protein mutant retains residual activity/function to be exerted at the cell membrane. Last, but not least, inhibition of proteasome with the FDA-approved inhibitor bortezomib (Velcade) successfully rescued the localization of D97G α-sarcoglycan mutant in muscle explants from an LGMD-2D patient (Sandonà et al., 2008; Fig. 3).

 

The main objective of our research group is to provide important insights for the development of pharmacological therapies for sarcoglycanopathies. To this end, the overall research plan is articulated as follows: 1) investigate the molecular mechanisms of sarcoglycan processing, assembly, and trafficking; 2) identify ERAD components involved in the processing of sarcoglycan mutants (Fig. 4); 3) investigate ERAD processing of mutants in skeletal muscle of α-sarcoglycan knockout mouse; 4) screen in our heterologous cell system novel molecules and treatments able to rescue sarcoglycan mutants; 5) test in muscle explants from LGMD-2C/F patients the efficacy of salvage maneuvers.

Publications

  • Sandonà D, Betto R (2009) Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects. 11:e28.
  • Gastaldello S, D'Angelo S, Franzoso S, Fanin M, Angelini C, Betto R, Sandonà D (2008) Inhibition of proteasome activity promotes the correct localization of disease-causing alpha-sarcoglycan mutants in HEK-293 cells constitutively expressing beta-, gamma-, and delta-sarcoglycan. Am. J. Pathol. 173:170-81.
  • Danieli-Betto D, Esposito A, Germinario E, Sandonà D, Martinello T, Jakubiec-Puka A, Biral D, Betto R (2005) Deficiency of alpha-sarcoglycan differently affects fast- and slow-twitch skeletal muscles. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289:R1328-37.
  • Jakubiec-Puka A, Biral D, Krawczyk K, Betto R (2005) Ultrastructure of diaphragm from dystrophic alpha-sarcoglycan-null mice. Acta Biochim. Pol. 52:453-60.
  • Sandonà D, Gastaldello S, Martinello T, Betto R (2004) Characterization of the ATP-hydrolysing activity of alpha-sarcoglycan. Biochem. J. 381:105-12.

Grants

Association Francaise contre les Myopathies; project "Rescue of disease-causing α-sarcoglycan mutants by interfering with the ubiquitin-proteasome system" (grant # 10288, RB)
Università di Padova; Athenaeum Project "Development and validation of a heterologous expression system to study the effects of α-sarcoglycan missense mutations on the assembly and targeting of sarcoglycan complex and to set up pharmacological protocols able to force proper cytolocalization of the mutated proteins" (DS)
Institutional funds from Consiglio Nazionale delle Ricerche (RB)

Collaborations

  • Corrado Angelini and Marina Fanin, Dipartimento di Neuroscienze, Università di Padova
  • Daniela Danieli-Betto, Dipartimento di Anatomia e Fisiologia Umana, Università di Padova
  • Anna Jakubiec-Puka, Nencki Institute, Warsa
  • Michéle Reboud-Ravaux, Département Biologie Cellulaire, Institute Jacques Monod, Paris

 

PI photo

Romeo Betto

Contact information

email  E-mail

email  049 8276027

Participating staff

Dorianna Sandonà
university researcher

Manuela Miozzo
CNR associated research fellow

Stefano Gastaldello
university technician

Elisa Bianchini
PhD student