Involvement of a Chaperone Regulator, Bcl2-associated Athanogene-4, in Apolipoprotein B mRNA Editing*

Apobec-1 is the catalytic subunit of a multicomponent editosome complex that mediates apolipoprotein B (apoB) mRNA editing. We isolated a novel apobec-1-interacting protein by yeast two-hybrid cloning and identified the protein as BAG-4. BAG-4, a chaperone-regulating protein, also known as SODD (silencer of death domains), is a member of the BAG family of proteins. In this report, we found that apobec-1 is localized in the perinucleolar compartment in HepG2 cells and rat liver MCR-RH7777 cells. BAG-4 binds to apobec-1 via its N-terminal region independent of the BAG domain. It is ubiquitously expressed with predominant occurrence in human pancreas, heart, brain, and placenta. Immunoprecipitation experiments confirmed that BAG-4 interacts with Hsc70/Hsp90 in HepG2 cells. BAG-4 tagged with green fluorescent protein (GFP) or FLAG was localized both in cytoplasm of mouse BNLCL.2 liver cells and human liver hepatoma HepG2 cells. After heat shock, GFP-BAG-4 co-localizes with Hsc70 in the nucleus in HepG2 cells, whereas GFP-BAG-4 mutants lacking the BAG domain remain perinuclear. BAG-4 has no effects on apoB mRNA editing in vitro. However, unlike other apobec-1 complementation factors studied to date, antisense knockdown of BAG-4 in BNLCL.2 cells and in MCR-RH7777 cells increases rather than decreases endogenous apoB mRNA editing. Overexpression of BAG-4 in MCR-RH7777 cells also suppresses apoB mRNA editing. ApoB-48 production also increases with antisense BAG-4 expression in MCR-RH7777 cells. We previously showed that apoB mRNA editing is an intranuclear event (Lau, P. P., Xiong, W. J., Zhu, H. J., Chen, S. H., and Chan, L. (1991) J. Biol. Chem. 266, 20550-20554). Thus, BAG-4 overexpression down-regulates apoB mRNA editing by shuttling apobec-1 from the intranuclear perinucleolar compartment to the cytoplasm. We propose that BAG-4 functions as a negative regulator for apobec-1-mediated apoB mRNA editing through its ability to suppress the Hsp/Hsc70 chaperone activity and thereby editosome formation and, as a consequence, prevents nuclear localization of the apobec-1 editosome.

in the primary structure of apobec-1 that have been postulated to be important in its distribution in both the nucleus and the cytoplasm. However, none of these experimentally meet the functional criteria of nuclear localization or nuclear export signals. Residues 97-172 in the middle of apobec-1 together with its N-terminal 56 residues modulate nuclear localization. Mutagenesis experiments, however, revealed no discrete nuclear localization signal in apobec-1. Fifteen amino acids (Leu 173 -Leu 187 ) within the C-terminal domain of apobec-1 were sufficient as a determinant for cytoplasmic distribution. These residues failed to demonstrate nuclear export function in a reporter assay. Leptomycin B, an inhibitor for substrate recognition by CRM1, did not abolish the dual nuclear/cytoplasmic distribution of apobec-1 in MCR-RH7777 cells. Thus, it was proposed that interactions with chaperones play a key role in the intracellular distribution of apobec-1 and in nuclear editing (3).
Molecular chaperones of the Hsp70 family play an important role in the folding, refolding, translocation, and degradation of proteins in eukaryotic cells (4). The mammalian chaperone proteins comprise multiple members; Hsp72 is the inducible form, and Hsp73 (or cognate) is the constitutive form. Earlier we reported that an Hsp40/DnaJ protein, ABBP-2, also called DnaJb11, is involved in eukaryotic mRNA editing (5). A major role of the J domain of DnaJ proteins appears to be the recruitment of Hsp70 to a specific polypeptide substrate for cellular functions. We found that down-regulation of ABBP-2 abolished endogenous apoB mRNA editing in apobec-1-expressing HepG2 cells and mouse liver BNLCL.2 cells and that ABBP-2 works in concert with Hsc70 (5). In this report, we identify BAG-4 as a regulator of apoB mRNA editing by its interaction with apobec-1 in association with the chaperone, Hsc70. BAG-4 belongs to a family of BAG proteins, which share a common BAG domain, a conserved 45-amino acid region near their C termini that binds to Hsc70/Hsp70. Using Hsc70 as the bait, BAG-1 was the first BAG (Bcl2-associated athanogene) protein cloned from two-hybrid screening as a Bcl2-associated protein and was shown to regulate Hsp70 nucleotide exchange and ATPase activity in the same fashion as GrpE does in Escherichia coli (6 -8). Other BAG family proteins, BAG-2, BAG-3, and BAG-4, were subsequently cloned by Hsc70 screening using the twohybrid method. BAG-1, BAG-2, and BAG-3 also bind the ATPase domain of Hsc70 with high affinity and suppress Hsp70/Hsc70 chaperone activity in vitro (9). BAG-3 and BAG-4 both bind to Hsp70 and Bcl2 (10). BAG-4, also known as SODD (silencer of death domains), binds to and inhibits signaling by tumor necrosis factor receptor 1 (11). Moderate to high levels of BAG-4 mRNA transcript were detected in a variety of pancreatic cancer patient samples and cell lines (12), conferring to these cells resistance to tumor necrosis factor receptor 1. The antiapoptotic activities of BAG family proteins may be a result of their interactions with Hsc70/Hsp70 and/or binding to Bcl-2 (13). In addition to these cellular functions, here we identify BAG-4/SODD as a negative regulator of apobec-1 function exerting an inhibitory effect on apoB mRNA editing. We demonstrate how BAG-4 interacts with Hsc70 to modulate apoB mRNA editing.

EXPERIMENTAL PROCEDURES
Two-hybrid Cloning-Three of the five positive apobec-1-interacting clones that successfully passed the mating assay by failing to interact with nonspecific diploids were reported as two RNA-binding proteins, ABBP-1 (14) and GRY-RBP (15). These two RNA-binding proteins were isolated from a human placenta pGAD cDNA library, when human apobec-1 was used as bait. Subsequently, a DnaJ type of co-chaperone, ABBP-2, was isolated from a human liver library (5). Besides ABBP-2, another clone that was obtained from the human liver library (Clontech) contains a nearly full-length cDNA of a known chaperone regulator, named SODD or BAG-4 (11). Three codons were missing from the 5Ј end of this BAG-4 cDNA clone, which were recovered by PCR subcloning, and the authenticity of the full-length BAG-4 cDNA was verified by sequencing.
Biochemical Methods-Polyclonal antibodies to glutathione S-transferase (GST) 1 -BAG-4 fusion protein and human apobec-1 were raised in New Zealand White rabbits. These BAG-4 antibodies recognized a predominant band on Western blots of mouse S-100 extracts (100,000 ϫ g supernatant). Northern blots of human tissues were purchased from Clontech. Hybridization was performed as previously described (15) in ExpressHyb hybridization solution (Clontech). After immobilization to glutathione beads, binding of GST-BAG-4 fusion protein to [ 35 S]methionine-labeled apobec-1 was performed in phosphate-buffered saline, 0.2% Nonidet P-40 solution at 4°C for 1 h in a rotary shaker as described previously (5). Truncated GST-BAG-4 cDNAs were constructed into pGEX-4T-2 (Amersham Biosciences) by PCR subcloning, and their sequences were verified. Similarly, E. coli expressed truncated GST proteins were purified and immobilized onto the glutathione beads and used for in vitro binding to apobec-1.
Plasmids and Cell Culture Transfection-Full-length cDNAs of BAG-4 and human apobec-1 (17) were subcloned in-frame into pFLAG-CMV-2 expression vector (Eastman Kodak Co.). BAG-4 and truncated BAG-4 mutants were cloned into pcDNA3.1/NT-GFP-TOPO (Invitrogen). Antisense DNAs of BAG-4, apobec-1 complementation factor (ACF), and ABBP-2 were subcloned into pHook-3 (Invitrogen). All subclones were verified by direct sequencing. A mouse liver BNLCL.2 cell line that stably expressed mouse apobec-1 and human hepatoma cell line HepG2 cells that stably expressed human apobec-1 (5) were used for transfection. Transient transfection was performed with electroporation or LipofectAMINE. For pHook-3 transfection, the transfected cells were collected by hapten-coated magnetic beads. Besides the single chain antibody against a specific hapten, hemagglutinin A epitope tag and Myc epitope tag were preceded by a signal peptide and co-expressed by the Rous sarcoma virus promoter contained in pHook-3; a CMV promoter was used for expressing the inserted DNA. 12-mm poly-Llysine coverslips (Becton Dickinson) or collagen-coated cover slips were used for BNLCL.2 cells and HepG2 cells, respectively. After 48 h, cells were fixed by paraformaldehyde, and antibody staining was done at room temperature for 30 min and detected by Rhodamine Red-conjugated second rabbit antibody or by MOM fluorescence kit (Vector Laboratories Inc.) In parallel experiments after 48 h, cells were harvested, and RNA was extracted for reverse transcriptase-PCR amplification for apoB mRNA. Gel-purified fragments of apoB were used for dideoxy-G reverse transcriptase editing assays as described (5).

RESULTS
Structure of BAG-4 -Using human apobec-1 cDNA as the bait in a yeast two-hybrid screen, we identified a cloned cDNA for another interacting protein besides ABBP-2 from a human liver library; both clones successfully passed the mating assay. The clone obtained was searched with BLAST and revealed a previously published sequence annotated as SODD or BAG-4. The two-hybrid clone contained all but the first three codons. The predicted protein structure of BAG-4 has a BAG domain in the carboxyl-terminal region ( Fig. 1A) but lacks the ubiquitin domain present in BAG-1 or the WW domain present in BAG-3. It contains 458 amino acid residues and has a predicted subunit molecular mass of 49.59 kDa.
The secondary structure of BAG-4 and the BAG-Hsc70 complex is well characterized (18). In the crystal structure of a complex with the ATPase of Hsc70, the BAG domain forms a three-helix bundle, inducing a conformational switch in the ATPase that is incompatible with nucleotide binding in the same fashion as observed in bacterial Hsp70 homologue, DnaK, upon binding of the nucleotide exchange factor GrpE. The tertiary structure of the three helices of the BAG domain as determined by NMR in solution was obtained from a VAST search (Fig. 1A). The second and third helices interact with the ATP-binding pocket of Hsc70/Hsp70. The BAG domain and GrpE of E. coli are structurally unrelated (18). However, they both interact with the same ATPase subdomains of their respective Hsp70 or DnaK.
Tissue Distribution-One criterion for identifying candidate proteins involved in the apoB mRNA editing process is their presence in the tissues actively expressing editing activity. BAG-4 mRNA is present in multiple human tissues including the liver and small intestine as shown on the poly(A) mRNA Northern blots (Fig. 1B). In humans, apoB mRNA editing occurs exclusively in the small intestine, which is also the exclusive tissue for the existence of apobec-1 (17). BAG-4 is a molecular chaperone regulator and participates in various cellular functions, and its universal existence is not unexpected.
BAG-4 Interacts with Apobec-1 at the N Terminus-The fulllength human BAG-4 cDNA and different deletion mutants were subcloned into pGEX-4T2. The GST fusion proteins of BAG-4 of different lengths were used for an in vitro binding assay ( Fig. 2A) to verify the in vivo interaction of BAG-4 and apobec-1 observed in the two-hybrid screening. Nine mutants of progressive deletion of the C-terminal region and the Nterminal region were used to examine the role of the different parts of BAG-4 that are involved in apobec-1 binding. We found that mutants (mu1-5) lacking the BAG domain still bind to apobec-1 essentially as well as the full-length BAG-4. Only when the P/G-rich region of the N terminus was deleted was there a significant attenuation of apobec-1 binding. The apobec-1 binding region of ABBP-1 is proline-rich (14). Proline-rich motifs are commonly involved in protein-protein interaction, and proline-rich sequences are commonly found in situations requiring the rapid recruitment or interchange of several proteins, such as during initiation of transcription and signal cascades (19). It was postulated that the nonconserved N-terminal regions of BAG proteins target proteins to their right partners. The C-terminal BAG domain is used for Hsc70/Hsp70 binding in the presence of ATP and its co-chaperone, ABBP-2, and subsequently releases Hsc70-bound substrate and ADP.
Apobec-1 Editosome Is Localized in the Perinucleolar Compartment-Human liver HepG2 cells contain no apobec-1 and are incapable of apoB mRNA editing. We introduced apobec-1 into HepG2 cells by transient transfection of pCMV-FLAGapobec-1 plasmid and examined hundreds of cells. In all cells examined, the presence of apobec-1 was detected by rabbit polyclonal anti-apobec-1 antibodies coupled with Rhodamine Red anti-rabbit IgG second antibodies (Fig. 3A). The intracellular localization of the protein is identical to that detected by the mouse monoclonal anti-FLAG (M 5 ) antibodies as visualized by fluorescence anti-mouse IgG antibodies. The two images completely overlapped. Thus, by using these two different antibodies, we determined that apobec-1 was predominantly nuclear (Fig. 3A). Apobec-1 was also found similarly located predominantly in the nucleus in (rat liver) MCR-RH7777 cells and apobec-1-transfected (mouse liver) BNLCL.2 cells. This finding is consistent with our earlier observation that apoB mRNA editing is a predominantly intranuclear event (1). Upon close observation of the apobec-1-expressing HepG2 cells, we found that apobec-1 is absent from the nucleoli but is localized exclusively in the nucleoplasm with enrichment in the perinucleolar region (Fig. 3B). A similar observation was obtained in MCR-RH7777 cells (Fig. 3B). The perinucleolar compartment is involved in RNA metabolism including splicing and polyadenylation (20); it is also where other apobec-1 auxiliary factors, such as KSRP, CUG-BP, and heterogeneous nuclear ribo- nucleoproteins, are located predominantly (20,21). KSRP is a splicing regulatory protein. CUG-BP is a poly(A)-binding protein. Deletion mutagenesis studies showed that at least three RNA recognition motifs at either the carboxyl or amino end of the polypyrimidine binding proteins are required to target the protein to the perinucleolar compartment, suggesting that RNA binding is needed for such intracellular localization. These RNA-binding proteins were previously found to be involved in apoB mRNA editing (22,23), and apoB mRNA editing was coincidental with splicing and polyadenylation (1). Besides apobec-1, it is of interest to localize the ACF, the only RNAbinding protein that was reported to enhance apoB RNA editing in vitro (16). A polyclonal rabbit antibody against ACF (a gift from Dr. Donna Driscoll) was used to detect its intracellular localization in HepG2 cells, BNLCL.2 cells, and MCR-RH7777 cells; in contrast to apobec-1, which is predominantly in the nucleus, ACF is localized in both the nuclear and cytoplasmic compartments (Fig. 3C, top panel) in HepG2 cells and BNLCL.2 cells but predominantly nuclear and nonnucleolar (Fig. 3C, bottom panel) in MCR-RH7777 cells. According to observations in other laboratories, ACF intracellular localization varies from cytoplasmic to nuclear in different cell lines and varies according to the antibodies used and whether or not it is tagged with epitopes (24 -27, 28). When ACF was tagged with c-Myc epitope in CCL13 (a HeLa cell derivative), ACF was FIG. 5. Association of BAG-4, Hsc70, Hsp90, and apobec-1 in HepG2 S-100. A, co-immunoprecipitation of BAG-4, Hsc70, and apobec-1. Immunoprecipitation (IP) was performed using anti-FLAG beads on S-100 prepared from apobec-1 stably transfected HepG2 cells, which was also transiently transfected with pFLAG-BAG-4 DNA. The immunoprecipitates were separated on SDS-PAGE and Western blotting was performed using anti-Hsc70 antibody. Reciprocally, a polyclonal antibody against human apobec-1 was used to immunoprecipitate the S-100 extracts followed by GammaBindG beads (bd) pull-down; the control lane has GammaBindG beads only. The Western blot was probed by anti-Hsc70 antibody (␣ Hsc70) or by a monoclonal antibody to FLAG epitope (M2) (right). B, immunoidentification of Hsp70 and Hsp90 in anti-BAG-4 immunoprecipitate. Reciprocate precipitation was performed using polyclonal antibodies against BAG-4; the Western blots were probed by monoclonal antibodies against Hsp70 (left) or Hsp90 predominately localized in the cytoplasm (28). When ACF is nuclearly localized, the putative nuclear localization signal motif in ACF may not be responsible for its nucleo-cytoplasmic trafficking, since the nuclear import of apobec-1 may depend on protein-protein interaction (26).
Cytoplasmic Localization of BAG-4 -We have previously constructed a HepG2 cell line stably expressing apobec-1 with an apoB mRNA editing activity of about 30% (17). BAG-4 was not readily detectable with the rabbit polyclonal antibodies in HepG2 cells, although it was faintly detected in the cytoplasmic compartment in all of the cells examined. BAG proteins (BAG-1) are generally not abundant, at least 10-fold less than HIP (Hsp-interacting protein), so as to allow Hsc70 to fulfill its expanded spectrum of cellular functions (7). In order to more clearly ascertain the localization of BAG-4, we transfected pCMV-FLAG2-BAG-4 cDNA and pcDNA3.1NT-GFP-BAG-4 into this cell line. BAG-4 was detected in the cytoplasmic compartment (Fig. 4A), although occasionally we detected GFP-BAG-4 in both nuclear and cytoplasmic compartments when it was overly expressed. We also transfected GFP-BAG-4 into a mouse liver cell line BNLCL.2 that stably expressed apobec-1, producing about 50% apoB mRNA editing (5). GFP-BAG-4 was localized predominantly in the cytoplasm with some enrichment in the perinuclear region (Fig. 4B). Similarly, BAG-1 and BAG-1M were mostly located in the cytoplasm but were occasionally found in the nucleus, depending on the cell type and whether the cells were exposed to stress conditions (13).
Association of BAG-4 with Hsp70 and Hsp90 in HepG2 Cells-It is known that BAG-4 binds to both forms of Hsp70, Hsc70 (Hsp73), the constitutive form of heat shock protein 70, and Hsp72, the inducible form (18). We have previously characterized ABBP-2 (DnaJb11) and its association with the constitutive form of the Hsp70, Hsc70, in HepG2 cells. In this study, we ascertained Hsp70 association with BAG-4 in these cells. We transfected HepG2 cells with FLAG-BAG-4 and prepared S-100 extracts from them; we then performed an immunoprecipitation using anti-FLAG monoclonal antibody beads. Indeed, we detected Hsc70 with an Hsc70-specific antibody in the immunoprecipitate (Fig. 5A). Conversely, an antibody to apobec-1 also pulled down BAG-4 (Fig. 5A). Similarly, Hsp70 was immunoprecipitated with a polyclonal antibody against BAG-4 (Fig. 5B). In the same immunoprecipitate, Hsp90 was also detected with a monoclonal antibody against Hsp90 (Fig.  5B). Thus, these pull-down experiments suggest that BAG-4 associates with Hsp70 and Hsp90 as well as with apobec-1 inside HepG2 cells.
BAG-1 regulates Hsc70 in a manner opposite to that of HIP (7), which stabilizes the ADP-bound state of Hsc70 and inhibits ATPase activity of Hsc70. BAG proteins are known to stimulate Hsc70 ATPase activity in the presence of a J-protein. Hence, we investigated the effect of BAG-4 acting as a nucleotide exchange factor. In Fig. 5C, we showed that ABBP-2 stimulates Hsc70 ATPase activity and that the addition of BAG-4 enhances the effect of ABBP-2.
BAG-4 Is an Hsp70 Co-chaperone-Previously, we showed that the Hsc70/ABBP-2 pair was involved in modulating apoB mRNA editing in vivo (5). ABBP-2 is a co-chaperone DnaJ protein. The orthodox members of DnaJ subfamily Dj2 and Dj3 have J, G/F, and zinc finger domains and are farnesylated, whereas Dj1 (Hsp40/Hdj-1) and ABBP-2 are noncanonical members that lack the zinc finger domain and the prenylation motif. The BAG-1 protein stimulates refolding of denatured proteins of Hsc70/Dj2 and Hsc70/Dj3 pairs but is not effective with Hsc70/Dj1. BAG-1, BAG-2, and BAG-3 were also reported to suppress chaperone activity of Hsc70 (7,29). It was further shown that Hsc70/Dj2 and Hsc70/Dj3 do not translocate into the nucleus and remain cytoplasmic and perinuclear after heat shock, whereas the Hsc70/Dj1 pair translocates inside the nucleus and accumulates in the nucleolus (30). The fact that apobec-1 interacts with both ABBP-2 and BAG-4 has prompted us to investigate whether BAG-4 is a co-chaperone of the Hsc70/ABBP-2 pair. After heat shock, BAG-4 enters the nucleus in HepG2 cells (Fig. 6A). Since the BAG domain is required to suppress Hsc70 chaperone activity, we transfected BAG-4 mutant (mu2) that lacks a functional BAG domain with helix 2 partially deleted and helix 3 totally deleted. The transfected cells were then subjected to heat shock. As expected, BAG-4-mu2 remained perinuclear and cytoplasmic after heat shock (Fig. 6C). In the same mu2-transfected HepG2 cells, Hsc70 was still able to enter the nucleus and nucleolus (Fig.  6B). Thus, BAG-4 is a co-chaperone of Hsc70 and is totally dependent on its BAG domain for its interaction with Hsc70 so that it can move into the nucleus with the latter after heat shock.
Down-regulation of BAG-4 Increases apoB mRNA Editing in Vivo-One criterion to qualify a candidate protein to be an auxiliary factor is that alteration in its expression changes apoB mRNA editing. In all cases studied with one exception to date, down-regulation of the auxiliary factors inhibits apoB mRNA editing in vivo. This was true for ABBP-1 (14), ABBP-2 (5), and ACF (5). In one case (GRY-RBP), 2 there was no effect. Contrary to these previous examples, we found that antisense down-regulation of BAG-4 enhances editing in BNLCL.2-apobec-1-expressing cells. In Fig. 7A, the bound fraction in the Capture-Tec pHook-3 system represents the enriched fraction of antisense BAG-4 DNA-transfected cells; in these cells, editing increased from 50% (mock control in Fig. 7A, lanes 1 and 2) to almost completion, 98% (Fig. 7A, lanes 3-6). This observation indicates that BAG-4 is an inhibitor of apoB mRNA editing in vivo. This is consistent with the hypothesis that BAG-4 inhibits the assembly of apobec-1-editosome in the presence of the Hsc70/ABBP-2 pair. In contrast and consistent with previous observations, we found that antisense DNAs to ACF and ABBP-2 knocked down the endogenous apoB mRNA editing from 55% to 2.5 and 5%, respectively, in BNLCL.2-apobec-1expressing cells (Fig. 7A, lanes 7-9). We also tested the effects of antisense BAG-4 cDNA expression in MCA-RH7777 rat hepatoma cells that naturally expressed apobec-1 and displayed about 45% apoB mRNA editing. In these cells, expression of antisense BAG-4 cDNA by transfection increased endogenous apoB mRNA editing from 45 to 96% (Fig. 7B, lanes 1 and 2). On the other hand, overexpression of sense BAG-4 DNA suppressed editing from 40% down to ϳ5% (Fig. 7B, lanes 3-5). pared with those from mock-transfected cells (Fig. 7C). This observation confirms that down-regulation of BAG-4 increases endogenous editing. Interestingly, when we co-incubated purified BAG-4 with apobec-1-expressing S-100 extract from HepG2 cells, we observed no effect on in vitro editing (Fig. 7D). The data suggest that the effect of BAG-4 on editing in vivo is indirect and probably involves the targeting of apobec-1 and other required proteins to the right partner and/or cellular compartment for editing to occur through regulation of Hsp70 or other chaperones.
Shuttling of Apobec-1 to the Cytoplasm by BAG-4 Overexpression-We found strong evidence for the role of BAG-4 in apobec-1 trafficking (Fig. 8). When BAG-4 was overexpressed, apobec-1 was shuttled to the cytoplasmic compartment in MCR-RH7777 cells (Fig. 8A). Apobec-1 was predominantly localized in the nucleus and changed dramatically to the cytoplasmic compartment after transfection by pHook-3-BAG-4 sense DNA. Similarly, overexpression of BAG-4 in BNLCL.2-apobec-1-expressing cells facilitated apobec-1 export from the nucleus to the cytoplasm (Fig. 8B). Apobec-1 was localized in the nucleus in the extranucleolar compartment in BNLCL2-apobec-1 cells and was exported drastically to the cytoplasm after transfection with pCMV2-FLAG-BAG-4 sense DNA. The appearance of speckles in the cytoplasm may represent the apobec-1 aggregates in cells with the chaperone regulator overly expressed. DISCUSSION To date, by two-hybrid cloning using human apobec-1 as bait, our laboratory has identified two RNA-binding proteins, ABBP-1 and GRY-RBP (14,15), and two co-chaperones, ABBP-2 (5) and Bag-4. Other RNA-binding proteins, such as ACF (ASP), KSRP (16,22), and CUGBP2 (23) were cloned or identified by other laboratories as auxiliary factors associated with apobec-1 and shown to play a role in editing. In this study, we have further localized apobec-1 in the perinucleolar compartment (Fig. 3B). We note that heterogeneous nuclear ribonucleoprotein, KSRP, and CUGBP are also preferentially localized in this compartment (20). The perinucleolar compartment is a highly dynamic structure; for example, fluorescence recovery after photobleaching analysis revealed a rapid turnover of the polypyrimidine tract-binding protein within this compartment (21). There is evidence indicating that the perinucleolar compartment is actively involved in RNA metabolism (20). Deletion mutagenesis analysis showed that at least three RNA recognition motifs are required for the polypyrimidine tractbinding protein to be targeted to the perinucleolar compartment (31). The identified auxiliary proteins of the apobec-1editosome, including ABBP-1, GRY-BP, KSRP, and ACF, each contain two or three RNA recognition motifs. Many of them were identified by UV cross-linking to the AU-rich apoB mRNA. Taken together, we speculate that regulated apoB mRNA editing also occurs in the perinucleolar compartment.
Here we further showed that BAG-4 regulates apobec-1mediated editing through its interaction with Hsc70 and Hsp90. This finding is consistent with our earlier identification of ABBP-2, a DnaJ co-chaperone protein that works with Hsc70, and their involvement in regulating apoB mRNA editing through their interaction with apobec-1. ABBP-2 acts as a positive regulator by stimulating chaperone activity of Hsc70 FIG. 9. Schematic description of BAG-4 as a regulator for apoB mRNA editing. A, BAG-4 as a co-factor in Hsc70 chaperone function. The ATP-bound Hsc70 has rapid association and dissociation of the unfolded substrates. ABBP-2, a J-protein, stimulates conversion of bound ATP to ADP so that the interactions of Hsc70 with apobec-1 and auxiliary factors for apobec-1-mediated apoB mRNA editing (Auxs) are stabilized. The HIP protein binds to the Hsc70-ADP complex and slows down the release of ADP; therefore, editosome assembly can be prolonged. BAG-4 normally is underexpressed, but upon up-regulation, it can conversely regulate the other competing co-factor, HIP, and accelerates ADP release and thus shortens the half-life of the folding intermediate before it can complete its assembly process. Hsp90 and HOP normally release ABBP-2 and the assembled substrates and aid the import of the assembled editosome into the nucleus. B, domains of Hsc70-mediating interactions with chaperone co-factors. HIP and BAG-4 may bind to the ATPase domain of Hsc70 in a mutually exclusive manner, and ABBP-2 and HOP may recognize distinct binding sites within the carboxyl terminus of the Hsc70. Editosome, a hypothetical high order macromolecular protein complex that is involved in editing; Foldosome, a hypothetical folding intermediate; PNC, the perinucleolar compartment. and therefore is required for apobec-1 editosome assembly in the presence of HIP by causing a slow ADP release (Fig. 9A).
In Fig. 9A, we depict the possible role of the various cochaperone proteins involved in apobec-1 editosome assembly and transport. In the presence of ATP, Hsc70 and DnaJ (ABBP-2) mediate the assembly of the apobec-1 editosome before it is imported into the nucleus. In the presence of Hsp90 and HOP (hsp-organizing protein), Hsp90 may assist in the import of apobec-1 into the nucleus. BAG-4 acts as a chaperone regulator by binding to the ATPase domain of Hsc70 N-terminal region, dissociating the Hsp90-editosome complex. This is analogous to the previous findings that BAG-1 regulates p53 stabilization and subcellular localization (8) and that Hsp90 may assist p53 import into the nucleus by exposing its nuclear localization signal (32). Overexpression of BAG-4 would facilitate both the stabilization and cytoplasmic sequestration of apobec-1 aggregates (Fig. 8B). BAG domain proteins stimulate the ATPase activity of Hsc70 in an Hsp40/Dnaj-dependent manner (Fig. 5C) and in this way induce the release of substrates from Hsc70. Efficient interaction of Hsc70 with a polypeptide depends on the conversion of bound ATP to ADP (7). Only the ADP-bound form confers stable substrate binding (33). By accelerating substrate release, the half-life of the Hsp70-substrate complex is decreased (7). Therefore, BAG proteins suppress Hsc70 chaperone refolding activity. Indeed, it was shown in a comparative study (including human BAG-1, BAG-2, BAG-3, BAG-4, and BAG-5) that BAG proteins, which functioned as a competitive antagonist of the co-chaperone, HIP, inhibited restoration of denatured luciferase activity by Hsc70/DnaJ in vitro (9).
Our finding is consistent with the model of BAG-4 acting as a negative regulator of the Hsc70/ABBP-2 pair in suppressing Hsc70 chaperone activity by interfering with HIP in stabilizing the Hsc70-ADP-substrate complex, and, as a consequence, BAG-4 is a negative regulator for apobec-1-editosome assembly by speeding up ADP release and subsequently inhibits apobec-1-mediated editing as shown in Fig. 7, A-C. However, the exact network of competing and cooperating cofactors of the Hsc70 or Hsp90 chaperone system that modulates the assembly of editosome and intracellular localization needs further investigation when all of the components are identified and specific antibodies against them become available. We, however, postulate that the possible roles of BAG-4 and ABBP-2 play are their competitive and noncompetitive binding with HIP and HOP to Hsc70 in its N-terminal and C-terminal binding domains, respectively (Fig. 9B). We propose this hypothesis based on the fact that the modulation of chaperone activity of Hsc70 involves cooperation with Hsp40 (a J-domain protein); BAG-1; the Hsc70-interacting protein, HIP; and the Hsc70-Hsp90-organizing protein, HOP. It is also known that HOP and Hsp40 bind to the carboxyl terminus of Hsc70 in a noncompetitive manner, whereas Bag-1 and HIP compete in binding to Hsc70 at the amino-terminal ATPase domain (34).
It is not surprising that the multitasking housekeeping chaperone system is involved in regulating the highly precise and targeted editing of a specific glutamine codon on the pre-mRNA of apoB in the nucleus so that potentially harmful, uncontrolled, promiscuous RNA editing in the cytoplasm can be prevented. Our report on the regulation of RNA editing by molecular chaperones is unique in uncovering the fact that Hsc70 and Hsp90 chaperone system modulates apoB mRNA editing by utilizing a positive regulator, the J-protein ABBP-2 (5), and a negative regulator BAG-4 (this report) to control apobec-1 editosome assembly and its subcellular localization.