Interaction of BAG1 and Hsp70 Mediates Neuroprotectivity and Increases Chaperone Activity

Abstract

It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAG⌬C) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAG⌬C compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAG⌬C was expressed exclusively in the cytosol. Furthermore, cells expressing BAG⌬C were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.

Bag-1L is a Stress-withstand Molecule Prevents the Downregulation of Mcl-1 and c-Raf Under Control of Heat Shock Proteins in Cisplatin Treated HeLa Cervix Cancer Cells

“…It was suggested that Bag1 formed a stoichiometric complex with Hsp70 and inhibited completely Hsp70-dependent in vitro protein refolding of an unfolded polypeptide (Nollen et al, 2000). Contrary to this finding, neuroprotective role of forced Bag-1 expression was found associated with the activation of Hsp70 to proceed cell survival (Liman et al, 2005). Similar to this finding, we concluded that although Cisplatin did not reduce Hsp70 expression level, Bag-1L stable transfection might increase chaperone activity of Hsp70 and prevented c-Raf downregulation after Cisplatin treatment.…”

Mutant SOD1 detoxification mechanisms in intact single cells

“…The cdYFP vector was produced by random mutagenesis as described previously. 21 EGFP-SOD1 constructs were cloned by PCR amplification of the human wtSOD1 sequence and subsequent subcloning into a pcDNA3.1 expression vector (Invitrogen, Carlsbad, USA) C-terminally of the EGFP insert with KpnI/NotI. Respective SOD1 mutant expression vectors were generated by PCR mutagenesis.…”

“…21 Folding ratio frequency histograms were analyzed with the Igor Pro analysis package (Wavemetrics Inc., Portland, OR). Individual histograms were summed and cumulative histograms were normalized to the integrated counts, that is, to the number of observed pixels per experimental data set.…”

“…Using a recently developed FRET assay with improved sensitivity, 20 we visualized SOD1 ubiquitination and chaperone interaction in intact single cells, and uncovered profound differences between wild-type and mtSOD1. Using innovative fluorescence-imaging-based biosensors, 21 we also demonstrate in a cell-by-cell investigation differences with regard to chaperone activity between wt and mtSOD1-transfected cells. Our findings substantially advance the understanding of cellular alterations by mtSOD1 and provide support for the involvement of altered chaperone function, rather than proteasomal deficits, in the mechanism of mtSOD1 toxicity.…”

“…As we found increased chaperone binding by mtSOD1, we investigated its functional influence for cellular chaperone activity in the single cell using a recently developed fluorescence imaging-based, broad-specificity chaperone activity biosensor. 21 This biosensor consists of a folding-impaired mutant of yellow fluorescent protein (cdYFP) tagged with an HA epitope. Its folding and maturation that is necessary for the generation of the chromophore is impaired and thus only minor fluorescence is generated.…”