Abstract
Nuclear envelopathies are recognized genetic disorders affecting individuals with mutations in their genes encoding members of the lamin family of nuclear envelope proteins that are responsible for maintaining the architectural structure of the nucleus. Irregularity in shape and size of the nuclei, nuclear membrane rupture, and appearance of micronuclei in the cytoplasm are among the pathological features of the syndrome. Here, we demonstrate that Bcl2-associated anthanogene-3 (BAG3), a stress-induced co-chaperone protein that by association with heat-shock protein 70 (HSP70) participates in regulation of autophagy, plays a critical role in the integrity of the nuclear membrane in cardiomyocytes. Cells subjected to proteotoxic stress or BAG3 downregulation show perinuclear accumulation of the aberrant ubiquitinated proteins that are often associated with the appearance of misshapen, enlarged, and elongated nuclei. There were dense accumulations of lamin B in the perinuclear area and distribution of lamin B-positive micronuclei in the cytoplasmic space, indicative of nuclear envelope rupture. Overexpression of BAG3 in cells under proteotoxic stress ameliorated pathological nuclear morphology and reduced cytoplasmic distribution of the micronuclei particles. Subcellular co-localization and co-immunoprecipitation demonstrated interaction of lamin B with the BAG domain of BAG3 and HSP70, suggesting the importance of BAG3 in the selective clearance of a surplus of aggregated lamin B that is generated during stress conditions. Our findings define a novel role for BAG3 in nuclear protein quality control and suggest an alternative pathogenetic pathway that contributes to the development of nuclear envelopathies.
Highlights
The integrity of the nuclear membrane is critically important for separating nuclear function from cytoplasmic function
Inhibition of proteasomal degradation pathway by the proteasome inhibitor MG132 resulted in the increased levels of Bcl2-associated anthanogene-3 (BAG3), heat-shock protein 70 (HSP70), and p62 (Fig. 1f), as well as enhanced accumulation of ubiquitinated proteins in cardiomyocytes (Fig. 1g, h)
Expression of BAG3 was determined in the total cellular fraction. b–d Soluble and insoluble fractions were prepared from the transfected cardiomyocytes and expression of p62, HSP70, and ubiquitin was determined by western blotting
Summary
The integrity of the nuclear membrane is critically important for separating nuclear function from cytoplasmic function. Several studies suggested a role for lamin B in cell senescence that is liked to chromatin reorganization, changes in gene expression, and aging[15,16,17]. A member of the BAG family of proteins, BAG3 has received recent attention in the heart due to his importance in autophagy and protein quality control (PQC)[19,20] PQC is comprised of molecular chaperones and intracellular proteases in the cytosol and endoplasmic reticulum[21]. Recent studies have demonstrated that haploinsufficiency of BAG3 was associated with diminished left ventricular function by 10–12 weeks of age that was accompanied by diminished autophagy and increased apoptosis but without evidence of myofibrillar dissary[29]
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