Characterization of the R7S mutation of Heat Shock Protein HSPB3 and of two novel mutations found in patients suffering of myopathy: understanding the mechanisms leading to disease.

HSPB3 is a poorly characterized member of the small HSP/HSPB family (HSPB1-HSPB10) that forms a complex with HSPB2. Expression of HSPB2 and HSPB3 is restricted to differentiated skeletal and cardiac muscle cells, where HSPB2-HSPB3 participates in muscle maintenance. Recently the R7S mutation in HSPB3 was associated with distal hereditary motor neuropathy type 2C (dHMN 2C). We identified in myopathic patients two novel mutations of HSPB3: 1) R116P, affecting a key amino acid in the alpha-crystallin domain, whose mutation in other HSPBs also causes neuromuscular diseases; 2) p.A33AfsX50-HSPB3, which disrupts the reading frame leading to a premature stop codon at amino acid 50. Here we first characterized in cells the subcellular distribution of HSPB2 and HSPB3. Next, we studied whether/how HSPB3 mutations affect: a) HSPB3 stability and subcellular localization; b) complex formation. While p.A33AfsX50-HSPB3 is immediately degraded after synthesis and cannot be detected, R7S and R116P are rather stable. Concerning interaction with HSPB2, R7S still interacted with HSPB2, while R116 disrupted complex formation. Concerning subcellular distribution, in two cell types (HEK293T and HeLa cells), HSPB2 and HSPB3 were enriched in the nuclei, where they formed intranuclear and perinuclear aggregates. Aggregation propensity was increased by R7S and R116P mutations. Intriguingly, nuclear aggregation and alteration of nuclear morphology were also found in the muscle biopsy from the patient with the R116P mutation. Next, we found in cells that HSPB2 and HSPB3 (wt and mutants) alter nuclear envelope (NE) integrity and lamin A/C distribution. Interestingly, DMPK, which has been shown to interact with HSPB2, also affects NE. Lamins regulate not only nuclear shape, but also gene expression, transcription and mutations in NE components cause neuromuscular and muscular diseases. Lamin A/C has been associated with intranuclear speckles, where it can colocalize with HSPB1 or HSPB5. Speckles contain splicing factors such as SC35. We thus analyzed speckles in HSPB2-B3 expressing cells. HSPB2-B3 did not inhibit Lamin A/C recruitment into speckles. Also, we did not find any major colocalization of HSPB2 and HSPB3 with speckles. Instead, HSPB2 and HSPB3 altered the shape of speckles, which became round (mimicking a treatment with the RNA transcription inhibitor actinomycin D). In summary, our results show that HSPB2-HSPB3 affect nuclear architecture, which may in turn deregulate RNA transcription. Future studies will identify the molecular mechanisms leading to the observed effects on NE and speckles. Since remodeling of nuclear lamina is required for muscle differentiation, it is tempting to speculate that HSPB2-B3 may modulate muscle differentiation/maintenance by regulating lamin functions and NE stability. Alteration of such functions due to HSPB3 mutations may be detrimental for motor neuron and muscle cells, contributing to disease progression.