In contrast, overexpression of the BAG family member, BAG5, antagonizes CHIP-mediated [alpha]-synuclein ubiquitination, which prevents the ability of CHIP to suppress oligomer formation  and also enhances [alpha]-synuclein-mediated toxicity .
The pathogenic G2019S, R1441C, and Y1699C LRRK2 mutations were shown to enhance the clearance of the trans-Golgi network (TGN) via a protein complex including the chaperone proteins Hsp70 and BAG5 plus Rab7L1 and Cyclin G Associated Kinase (GAK), which are both located in risk loci for sporadic
In contrast, the cochaperone BAG5 inhibits Parkin E3 activity, which may provide a mechanistic explanation as to how BAG5 enhances dopaminergic neurodegeneration .
Furthermore, a recent study demonstrated that the cochaperone BAG5 interacts with DJ-1 and decreases its stability .
Ohsawa et al., "The C-terminal BAG domain of BAG5 induces conformational changes of the Hsp70 nucleotide- binding domain for ADP-ATP exchange," Structure, vol.
In previous studies, BAG5 (GRMZM2G097135) was considered to be a control of endoplasmic reticulum protein quality and was also closely related to environmental stress responses in plants (Liu and Howell, 2010).
For example, BAG5 and BAG2 were closely related to environmental stress responses and meiosis processes in maize, respectively (Liu and Howell,2010; Nan et al., 2011).
Here, by Co-IP and immunofluorescence methods, we investigate whether BAG5 interacts with DJ-1 in mammalian cells.
Full length BAG5 cDNA amplified from a human fetal brain library was cloned into the pEGFP-N1 vector and pcDNA3.1 vector (Clontech), respectively.
The antibodies against different tags and proteins used for immunoprecipitation and immunoblotting were as follows: GFP antibodies (rabbit polyclonal, ab290; mouse monoclonal ab1218, Abcam); DJ-1 antibodies (rabbit monoclonal, #5933, Cell Signaling; mouse monoclonal, ab11251, Abcam); mouse monoclonal Bag5 antibody (ab56738, Abcam); Myc antibodies (rabbit polyclonal, #2272, Cell Signaling; mouse monoclonal, #2276, Cell Signaling); HA-Tag rabbit monoclonal antibody (#3724, Cell Signaling); Hsp70 rabbit monoclonal antibody (#4876, Cell Signaling); mouse monoclonal ANTI-FLAG[R] M2 antibody (F1804, Sigma-Aldrich).
To further confirm the subcellular localization of DJ-1 and BAG5, immunofluorescence colocalization analysis was performed.
We further investigated whether BAG5 regulates the degradation of exogenous DJ-1.