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. 2013 Oct;12(10):2804-19.
doi: 10.1074/mcp.M112.025882. Epub 2013 Jul 3.

Bcl2-associated athanogene 3 interactome analysis reveals a new role in modulating proteasome activity

Ying Chen  1 , Li-Na Yang, Li Cheng, Shun Tu, Shu-Juan Guo, Huang-Ying Le, Qian Xiong, Ran Mo, Chong-Yang Li, Jun-Seop Jeong, Lizhi Jiang, Seth Blackshaw, Li-Jun Bi, Heng Zhu, Sheng-Ce Tao, Feng Ge
Affiliations

Bcl2-associated athanogene 3 interactome analysis reveals a new role in modulating proteasome activity

Ying Chen et al. Mol Cell Proteomics. 2013 Oct.

Abstract

Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach.

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Figures

Fig. 1.
Fig. 1.
Identification of BAG3 interacting proteins with QUICK. A, overall workflow of the QUICK method. B, expression of BAG isoforms in BAG3 knockdown HeLa cells (siBAG3) and negative control cells (siNC). Relative to control cells, BAG3 knockdown HeLa cells showed diminished BAG3 protein levels, as expected. Depletion of BAG3 showed no obvious effect on the expression of the other BAG isoforms. Actin blotting was performed to ensure equal loading.
Fig. 2.
Fig. 2.
Identification of BAG3 interacting proteins with a human proteome microarray. A, workflow of the proteome microarray strategy for the identification of protein–protein interactions. B, human proteome microarrays were probed with BAG3 (left), followed by a BAG3 antibody and a Cy5-labeled secondary antibody. A control experiment was carried out without BAG3 (right). Many stronger signals can be observed from the microarray with BAG3 than from the microarray without BAG3. Enlarged areas from the same locations of the two micorarrays with and without BAG3 are illustrated in the middle. When the signals from the two microarrays were compared, 289 candidate BAG3 interacting proteins were identified. C, representative BAG3 interacting proteins.
Fig. 3.
Fig. 3.
Comparison of BAG3 interacting proteins identified via the QUICK and proteome microarray methods. A, Venn diagrams showing the degree of overlap between the QUICK and proteome microarray data sets. B, functional diversity among proteins present in two datasets. The relative distribution of all proteins from Homo sapiens, annotated according to the PANTHER functional classes, is shown by the line labeled “Expected.” This denotes the relative fraction (coverage) of all human proteins that belong to a given category. Deviations of the QUICK and proteome microarray datasets from this distribution are shown by different colored bars; for example, proteins labeled “transcription factor” are relatively underrepresented in the two datasets.
Fig. 4.
Fig. 4.
Protein interaction network of the BAG3 interactome. A, the complete BAG3 interaction network obtained from STRING with a confidence score greater than 0.4. The network contains 283 nodes and 2,863 edges. B–E, the four top-ranked and tightly connected network clusters obtained with MCODE are color-coded and rendered as separate modules.
Fig. 5.
Fig. 5.
Validation of newly identified BAG3 interactors by co-immunoprecipitation. Immunoprecipitation assays of 14-3–3ζ, ATG3, Hsp70, PSMB1, MAPK7, HSF1, and BAG3 proteins were carried out as described under “Experimental Procedures.” A, immunoblotting demonstrated that BAG3 protein binds with 14-3–3ζ, ATG3, PSMB1, MAPK7, HSF1, and Hsp70 proteins. No BAG3-interacting bands were observed in the negative control (IgG). “Input” stands for the total cell lysate extracted from HeLa cells. Similarly, reverse immunoprecipitation assays confirmed the binding of (B) 14-3–3ζ, (C) ATG3, (D) PSMB1, (E) Hsp70, (F) MAPK7, and (G) HSF1 protein with BAG3.
Fig. 6.
Fig. 6.
The effect of knockdown of BAG3 on proteasome activity and MG-132 sensitivity in HeLa cells. A, knockdown of BAG3 in HeLa cells was confirmed by Western blotting. B, quantitative analysis of BAG3 knockdown on apoptosis in HeLa cells. Data are expressed as the mean ± S.D. of the fractions of apoptotic cells from at least three experiments. C, knockdown of BAG3 resulted in a marked decrease in proteasome activity. D, knockdown of BAG3 increased the ubiquitin-tagged proteins in HeLa cells. Western blotting was carried out with anti-ubiquitin antibody using total cell lysates extracted from HeLa cells. Actin served as a loading control. Effect of knockdown of BAG3 on the chymotryptic, tryptic, and caspase-like activities of the (E) 26S or (F) 20S proteasome in HeLa cells, as measured using fluorogenic substrates. Experiments were repeated a minimum of three times. *p < 0.05 when compared with the control group. 1: untreated HeLa cells; 2: HeLa cells treated with control siRNA; 3: HeLa cells treated with BAG3 siRNA; 4: HeLa cells treated with MG-132; 5: HeLa cells treated with control siRNA and MG-132; 6: HeLa cells treated with BAG3 siRNA and MG-132.
Fig. 7.
Fig. 7.
The effect of overexpression of BAG3 on proteasome activity and MG-132 sensitivity in HeLa cells. A, overexpression of BAG3 in HeLa cells was confirmed by Western blotting. B, overexpression of BAG3 reduces MG-132 sensitivity in HeLa cells. C, overexpression of BAG3 resulted in a marked increase in proteasome activity in MG-132-treated cells. D, overexpression of BAG3 decreased the ubiquitin-tagged proteins in MG-132-treated HeLa cells. Effect of overexpression of BAG3 on the chymotryptic, tryptic, and caspase-like activities of the (E) 26S or (F) 20S proteasome in HeLa cells, as measured using fluorogenic substrates. Experiments were repeated a minimum of three times. *p < 0.05 when compared with the control group. 1: untreated HeLa cells; 2: HeLa cells treated with blank vector; 3: HeLa cells treated with BAG3 plasmid; 4: HeLa cells treated with MG-132; 5: HeLa cells treated with blank vector and MG-132; 6: HeLa cells treated with BAG3 plasmid and MG-132.
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