Musculoskeletal Pathology Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing -Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing -, -, and -Sarcoglycan Stefano Gastaldello,* Simona D’Angelo,* Susanna Franzoso,* Marina Fanin, † Corrado Angelini, † Romeo Betto, ‡§ and Dorianna Sandona `,* From the Department of Biomedical Sciences,* and Department of Neurological Sciences, † University of Padova; Consiglio Nazionale delle Richerche Institute of Neuroscience, Neuromuscular Biology and Physiopathology; ‡ and Interuniversity Institute of Myology, § Padova, Italy Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins , -, -, -, and -sarcoglycan , which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex repre- sents a critical issue of sarcoglycanopathies , as sev- eral mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell’s quality-control system; however , this can also lead to the removal of some functional polypep- tides. To explore whether it is possible to rescue sar- coglycan mutants by preventing their degradation , we generated a heterologous cell system , based on human embryonic kidney (HEK) 293 cells , constitu- tively expressing three ( ,  , and ) of the four sar- coglycans. In these -HEK cells, the lack of -sar- coglycan prevented complex formation and cell surface localization , wheras the presence of -sarco- glycan allowed maturation and targeting of the tet- ramer. As in muscles of sarcoglycanopathy patients , transfection of -HEK cells with disease-causing -sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assem- bly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important in- sights for the potential development of pharmacolog- ical therapies for sarcoglycanopathies. (Am J Pathol 2008, 173:170 –181; DOI: 10.2353/ajpath.2008.071146) Mutations in sarcoglycans are responsible of autosomal recessive Limb-Girdle Muscular Dystrophy (LGMD) type 2C (-sarcoglycan), 2D (-sarcoglycan), 2E (-sarcogly- can), and 2F (-sarcoglycan), collectively named sarco- glycanopathies. 1–4 These disorders are characterized by the progressive wasting of skeletal muscle with predom- inant involvement of the pelvic and shoulder girdle mus- culature. 5 In muscle membrane, the four sarcoglycans form a subcomplex closely associated to a major com- plex comprising dystrophin, the gene product of Duch- enne and Becker Muscular Dystrophy, dystroglycans ( and ), dystrobrevins, syntrophins, and sarcospan. 6 This multimeric complex, known as the dystrophin glycopro- teins complex (DGC), provides a physical linkage be- tween the actin cytoskeleton and the extracellular matrix 7 and is essential to protect muscle membrane integrity during contraction. In addition, recent evidence shows that the DGC also holds signal transduction properties. 8 Studies on LGMD-2C/F patients and animal models demonstrated that loss of one sarcoglycan subunit re- sults in the absence or severe reduction in the other sarcoglycans at the sarcolemma. A mild disease pheno- type is usually associated with a moderate reduction of the sarcoglycan complex. 9 –12 In sarcoglycanopathy pa- Supported by the Association Franc ¸ aise contre les Myophaties (grant 12988, to R.B.), University of Padova Athenaeum Project (to D.S.), Italian Space Agency (grant 1B51–5, to D.S. and R.B.), and Italian National Research Council (to R.B.). Accepted for publication March 27, 2008. Supplemental material for this article can be found on http://ajp. amjpathol.org. Address reprint requests to Romeo Betto, C.N.R. Neuroscience Insti- tute, Neuromuscular Biology and Physiopathology, Viale G. Colombo 3, 35121 Padova, Italy, or to Dorianna Sandona ` , Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy. E-mail: romeo.betto@bio.unipd.it, or dorianna.sandona@unipd.it. The American Journal of Pathology, Vol. 173, No. 1, July 2008 Copyright © American Society for Investigative Pathology DOI: 10.2353/ajpath.2008.071146 170