Recombinant Human Large proline-rich protein BAG6 (BAG6), partial

What is the basic structure and localization of BAG6?

BAG6 is a large proline-rich protein originally identified as a product of a gene located within the human major histocompatibility complex. It contains an N-terminal ubiquitin-like (UBL) domain (residues 17-92) and forms a heterotrimeric complex with transmembrane domain recognition complex 35 (TRC35) and ubiquitin-like 4A (Ubl4A) . Endogenous BAG6 protein is primarily expressed in brain and lymphoid tissues, as revealed by specific antibody detection . Intracellularly, BAG6 exists in a complex of approximately 450 kD under normal conditions, but treatment with proteasome inhibitors like MG132 stimulates the formation of a larger complex that comigrates with 26S proteasomes .

What are the main cellular functions of BAG6?

BAG6 functions as a central hub in multiple cellular pathways:

• Protein Quality Control: BAG6 is essential for selective elimination of defective proteasomal substrates, particularly newly synthesized misfolded proteins .

• Transmembrane Protein Targeting: The BAG6 complex plays a critical role in transmembrane domain recognition, directing tail-anchored proteins either to the endoplasmic reticulum for proper insertion or to the degradation pathway when mislocalized .

• Cytoskeletal Regulation: BAG6 supports stress fiber formation by preventing the ubiquitination of RhoA, a critical Rho family protein involved in F-actin polymerization .

• Antiviral Defense: BAG6 inhibits influenza A virus replication by targeting viral polymerase subunit PB2 for degradation and disrupting the assembly of the viral RNA-dependent RNA polymerase (RdRp) complex .

• Immunoregulation: BAG6 is implicated in immune responses, including Th1 cell survival, natural killer cell cytotoxicity, and MHC class II molecule presentation .

How does BAG6 interact with the proteasomal degradation system?

BAG6 forms a physical association with the 26S proteasome that is strengthened by proteasome inhibitor treatment (e.g., MG132) . The protein co-immunoprecipitates with 26S proteasome components, particularly the Rpt6 subunit . BAG6 does not appear to be directly polyubiquitinated itself but rather interacts with polyubiquitinated proteins destined for degradation . This interaction capability is central to BAG6's role in directing defective newly synthesized proteins to the proteasome. When the proteasome is inhibited, BAG6 associates with a larger amount of polyubiquitinated proteins, suggesting its function as an adaptor linking substrates to the degradation machinery .

What experimental approaches can be used to assess BAG6 interactions with newly synthesized defective polypeptides?

To investigate BAG6's interactions with newly synthesized defective polypeptides, researchers can employ the following methodological approaches:

• Puromycin-based assays: Puromycin incorporation into nascent polypeptides can be used to generate and track defective translation products. Co-immunoprecipitation experiments using anti-puromycin antibodies have demonstrated physical interactions between BAG6 and puromycin-labeled nascent chain polypeptides . The experimental protocol involves:

  • Treating cells with puromycin (typically 5-10 μg/ml for 15-30 minutes)

  • Cell lysis under non-denaturing conditions

  • Immunoprecipitation with anti-puromycin antibodies

  • Western blotting for BAG6

• Proteasome inhibition studies: Treatment with MG132 followed by BAG6 immunoprecipitation can reveal the accumulation of polyubiquitinated substrates bound to BAG6 . This approach helps distinguish between BAG6's role in normal protein turnover versus its specific function in handling defective proteins.

• Cycloheximide chase assays: Combined use of proteasome inhibitors and translation inhibitors (cycloheximide) can help determine whether the accumulated polyubiquitinated proteins associated with BAG6 are newly synthesized or pre-existing proteins .

How does the structure of BAG6 complex relate to its function in tail-anchored protein targeting?

The BAG6 complex has a specific molecular architecture that enables its function in tail-anchored (TA) protein targeting:

• Distinct binding domains: BAG6 contains separate binding sites for TRC35 and Ubl4A at its C-terminus, forming a minimal functional complex . Structural analysis has revealed that:

  • Ubl4A binds to BAG6 through a specific C-terminal interaction

  • TRC35 has a distinct binding site on BAG6

• Minimal targeting module: The truncated BAG6 complex containing just these C-terminal interaction domains is sufficient to facilitate substrate transfer from small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) to TRC40 . This minimal complex functions as an independent TA-targeting module.

• Non-canonical BAG domain: Unlike other BAG family proteins, the BAG domain of BAG6 does not function as a canonical BAG domain in terms of Hsc70 regulation . Experimental evidence using β-galactosidase refolding assays has shown that:

  • Human Bag1-BAG completely inhibits Hsc70-mediated protein folding

  • In contrast, Bag6-BAG has no effect on refolding by Hsc70

This structural uniqueness may explain BAG6's specialized functions in TA protein targeting compared to other BAG family members.

What is the role of BAG6 in viral infection, and how can it be experimentally investigated?

BAG6 has recently been identified as a restriction factor for influenza A virus (IAV) replication, operating through specific mechanisms:

• Targeting viral polymerase: BAG6 specifically interacts with the N-terminus of the viral PB2 polymerase subunit, promoting its K48-linked ubiquitination at residue K189, which leads to proteasomal degradation .

• Disrupting viral polymerase complex: BAG6 competes with PB1 for binding to PB2, thereby interfering with the assembly of the viral RNA-dependent RNA polymerase (RdRp) complex .

Experimental approaches to investigate BAG6's antiviral function include:

• Overexpression and knockout studies:

  • Overexpression of BAG6 reduces viral protein expression and virus titers

  • BAG6 knockout using CRISPR/Cas9 significantly enhances virus replication, with up to 10-fold higher viral titers observed in BAG6-KO cells

• In vivo models:

  • BAG6-knockdown mice develop more severe clinical symptoms and higher viral loads upon IAV infection

  • PPMO-BAG6 (peptide-conjugated phosphorodiamidate morpholino oligomer targeting BAG6) can be used to deplete BAG6 in mouse lungs

• Domain mapping experiments:

  • The antiviral effect of BAG6 requires its N-terminal region containing the ubiquitin-like (UBL) domain (residues 17-92) and a PB2-binding domain (residues 124-186)

  • Mutational analysis can identify critical residues for the interaction between BAG6 and viral proteins

How does BAG6 regulate stress fiber formation, and what methodologies are appropriate for investigating this function?

BAG6 plays a novel role in maintaining actin cytoskeleton integrity through its regulation of RhoA:

• RhoA stabilization mechanism: BAG6 prevents the destabilization of endogenous RhoA protein by inhibiting its association with CUL3-based ubiquitin ligases, thereby preventing excessive polyubiquitination and subsequent degradation .

• Downstream effects: BAG6 deficiency leads to abrogation of stress fiber formation, defects in focal adhesion (FA) assembly, and impaired cell migration .

Experimental approaches to study this function include:

• RhoA rescue experiments: Transient overexpression of RhoA can rescue the defects in stress fiber formation induced by BAG6 depletion, bypassing the requirement for BAG6 .

• Ubiquitination assays:

  • Co-immunoprecipitation studies to assess the association between RhoA and CUL3-based ubiquitin ligases

  • In vivo ubiquitination assays to measure RhoA polyubiquitination levels in BAG6-depleted cells compared to controls

• Cytoskeletal visualization techniques:

  • Phalloidin staining for F-actin visualization

  • Immunofluorescence for focal adhesion proteins

  • Live-cell imaging to monitor stress fiber dynamics

What techniques can be used to manipulate BAG6 levels for experimental purposes?

Several approaches can be employed to modulate BAG6 expression or function:

• Gene knockout methods:

  • CRISPR/Cas9 system can be used to generate BAG6 knockout cell lines

  • Verification should include Western blotting and cell viability assays to ensure knockout does not affect cell growth (as observed in A549 cells)

• RNA interference:

  • siRNA targeting BAG6 has been successfully used to achieve approximately 80% knockdown in various cell types

  • Phenotypic effects should be carefully compared with controls, as BAG6 knockdown alone does not significantly affect cell viability and growth under normal conditions

• In vivo knockdown:

  • PPMO-BAG6 (peptide-conjugated phosphorodiamidate morpholino oligomer) has been successfully used to deplete BAG6 in mouse lungs

  • Confirmation of knockdown should be performed by Western blotting of tissue samples

• Expression vectors:

  • Transient overexpression using plasmids encoding full-length BAG6 or domain-specific constructs

  • Domain truncation experiments can help identify which regions of BAG6 are necessary for specific functions