CHARACTERIZATION OF THE R7S MUTATION OF HEAT SHOCK PROTEIN HSPB3 AND TWO NOVEL MUTATIONS FOUND IN PATIENTS SUFFERING OF MYOPATHY: UNDERSTANDING THE MECHANISMS LEADING TO DISEASE.

HSPB3 is a poorly characterized member of the small HSPB family that forms a complex with HSPB2. The complex is induced in differentiated muscle cells and might play a role in muscle maintenance. R7S mutation was associated with distal hereditary motor neuropathy type 2C. We identified in myopathic patients two novel mutations of HSPB3: a) R116P, affecting a key amino acid in the alpha-crystallin domain, whose mutation in other HSPBs causes neuromuscular diseases; b) p.A33AfsX50-HSPB3, a missense mutation, which leads to a premature stop codon. We characterized the subcellular localization of HSPB2-HSPB3 and the effects of the HSPB3 mutants on protein localization, stability and complex formation. While p.A33AfsX50-HSPB3 is degraded after synthesis, R7S and R116P are stable. Unlike of R116P, R7S still interacts with HSPB2. HSPB2 and HSPB3 are enriched in the nuclei, where they form intranuclear (IN) and perinuclear (PN) aggregates. Aggregation tendency of HSPB3 is increased by its mutants. These IN and PN aggregates influence nuclear envelope and lamin A/C distribution. Lamins regulate not only nuclear shape, but also transcription. Moreover, mutations on lamin A/C are associated with neuromuscular disease. In addition, lamin A/C is recruited in nuclear speckles that contain splicing factors such as SC35. HSPB2-HSPB3 do not colocalize with speckles and do not alter the recruitment in speckles of lamin A/C; instead, they alter speckles shape, which became round (mimicking transcription inhibition). Combined our results show that HSPB2-HSPBB3 affect nuclear structure; this in turn may deregulate remodeling of nuclear lamina and RNA transcription. Intriguingly, the muscle biopsy from patient with R116P mutation shows nuclear aggregation and morphological alterations. This further suggests that HSPB3 (in complex with HSPB2) may modulate nuclear structure/functions and that alteration thereof may contribute to disease.