An interaction study in mammalian cells demonstrates weak binding of HSPB2 to BAG3, which is regulated by HSPB3 and abrogated by HSPB8

The ten mammalian small heat shock proteins (sHSPs/HSPBs) show a different expression profile, although the majority of them are abundant in skeletal and cardiac muscles. HSPBs form hetero-oligomers and homo-oligomers by interacting together and complexes containing, e.g., HSPB2/HSPB3 or HSPB1/HSPB5 have been documented in mammalian cells and muscles. Moreover, HSPB8 associates with the Hsc70/Hsp70 co-chaperone BAG3, in mammalian, skeletal, and cardiac muscle cells. Interaction of HSPB8 with BAG3 regulates its stability and function. Weak association of HSPB5 and HSPB6 with BAG3 has been also reported upon overexpression in cells, supporting the idea that BAG3 might indirectly modulate the function of several HSPBs. However, it is yet unknown whether other HSPBs highly expressed in muscles such as HSPB2 and HSPB3 also bind to BAG3. Here, we report that in mammalian cells, upon overexpression, HSPB2 binds to BAG3 with an affinity weaker than HSPB8. HSPB2 competes with HSPB8 for binding to BAG3. In contrast, HSPB3 negatively regulates HSPB2 association with BAG3. In human myoblasts that express HSPB2, HSPB3, HSPB8, and BAG3, the latter interacts selectively with HSPB8. Combining these data, it supports the interpretation that HSPB8-BAG3 is the preferred interaction

Co-chaperones are limiting in a depleted chaperone network

To probe the limiting nodes in the chaperoning network which maintains cellular proteostasis, we expressed a dominant negative mutant of heat shock factor 1 (dnHSF1), the regulator of the cytoplasmic proteotoxic stress response. Microarray analysis of non-stressed dnHSF1 cells showed a two- or more fold decrease in the transcript level of 10 genes, amongst which are the (co-)chaperone genes HSP90AA1, HSPA6, DNAJB1 and HSPB1. Glucocorticoid signaling, which requires the Hsp70 and the Hsp90 folding machines, was severely impaired by dnHSF1, but fully rescued by expression of DNAJA1 or DNAJB1, and partially by ST13. Expression of DNAJB6, DNAJB8, HSPA1A, HSPB1, HSPB8, or STIP1 had no effect while HSP90AA1 even inhibited. PTGES3 (p23) inhibited only in control cells. Our results suggest that the DNAJ co-chaperones in particular become limiting in a depleted chaperoning network. Our results also suggest a difference between the transcriptomes of cells lacking HSF1 and cells expressing dnHSF1

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

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

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 with a defined 1:3 ratio. The HSPB2/HSPB3 complex is induced during muscle differentiation and plays a role in muscle maintenance. Recently the R7S mutation in HSPB3 has been associated with distal hereditary motor neuropathy type 2C (dHMN 2C). Here we report the identification in myopathic patients of two novel mutations in HSPB3: 1) one mutation affects the R116 residue, which corresponds to a key amino acid in the alpha-crystallin domain, whose mutation in other members of the HSPB family also causes disease (it is equivalent to e.g. R120 in HSPB5, whose mutation into G causes MFM and to K141 in HSPB8, whose mutation into E or N causes dHMN); 2) the other mutation disrupts the reading frame leading to a premature stop codon at amino acid 50. Both mutations were not found in more than 400 normal alleles. Expression studies allowed us to confirm that the mutation causing a premature stop codon leads to the generation of an unstable protein that is likely immediately degraded after synthesis and cannot be detected. Also, while both expressed, the R7S mutant was more stable than the R116 one. We next characterized in cells and in vitro the ability of these HSPB3 mutants to interact with HSPB2 and form the HSPB2/HSPB3 complex. We found that while the R7S mutant of HSPB3 was still able to interact with HSPB2, the R116 mutant was not. Future studies will allow us to better characterize how these HSPB3 mutants affect HSPB3 and, indirectly, HSPB2 stability, subcellular localization and function. They will also elucidate on HSPB3 and HSPB2 function in both motor neurons and myoblasts and will shed light on how mechanistically the mutations in HSPB3 affect the function and viability of these cell types, contributing to disease

An Atypical Unfolded Protein Response in Heat Shocked Cells

Contains fulltext : 91546.pdf (publisher's version ) (Open Access

The ATF6-Met[67]Val Substitution Is Associated With Increased Plasma Cholesterol Levels

Objective-Activating transcription factor 6 (ATF6) is a sensor of the endoplasmic reticulum stress response and regulates expression of several key lipogenic genes. We used a 2-stage design to investigate whether ATF6 polymorphisms are associated with lipids in subjects at increased risk for cardiovascular disease (CVD). Methods and Results-In stage 1, 13 tag-SNPs were tested for association in Dutch samples ascertained for familial combined hyperlipidemia (FCHL) or increased risk for CVD (CVR). In stage 2, we further investigated the SNP with the strongest association from stage 1, a Methionine/Valine substitution at amino-acid 67, in Finnish FCHL families and in subjects with CVR from METSIM, a Finnish population-based cohort. The combined analysis of both stages reached region-wide significance (P=9x10(-4)), but this association was not seen in the entire METSIM cohort. Our functional analysis demonstrated that Valine at position 67 augments ATF6 protein and its targets Grp78 and Grp94 as well as increases luciferase expression through Grp78 promoter. Conclusions-A common nonsynonymous variant in ATF6 increases ATF6 protein levels and is associated with cholesterol levels in subjects at increased risk for CVD, but this association was not seen in a population-based cohort. Further replication is needed to confirm the role of this variant in lipids. (Arterioscler Thromb Vasc Biol. 2009;29:1322-1327.