BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

Summary Neurodegeneration is associated with the aggregation of proteins bearing solvent-exposed hydrophobicity as a result of their misfolding and/or proteolytic cleavage. An understanding of the cellular protein quality control mechanisms which prevent protein aggregation is fundamental to understanding the etiology of neurodegeneration. By examining the metabolism of disease-linked C-terminal fragments of the TAR DNA-binding protein 43 (TDP43), we found that the Bcl-2 associated athanogene 6 (BAG6) functions as a sensor of proteolytic fragments bearing exposed hydrophobicity and prevents their intracellular aggregation. In addition, BAG6 facilitates the ubiquitylation of TDP43 fragments by recruiting the Ub-ligase, Ring finger protein 126 (RNF126). Authenticating its role in preventing aggregation, we found that TDP43 fragments form intracellular aggregates in the absence of BAG6. Finally, we found that BAG6 could interact with and solubilize additional neurodegeneration-associated proteolytic fragments. Therefore, BAG6 plays a general role in preventing intracellular aggregation associated with neurodegeneration.


INTRODUCTION
Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal degeneration (FTD) are associated with the accumulation and aggregation of specific neuronal proteins. As a consequence of their misfolding, normally obscured hydrophobic portions of proteins become solvent-exposed, leading to their self-association. Proteolytic cleavages generate polypeptide fragments which can also self-interact to form a spectrum of species ranging from soluble oligomers to large insoluble proteinaceous aggregates (Ross and Poirier, 2004;Ciechanover and Kwon, 2017;Mathieu et al., 2020). Protein aggregation and its associated toxicity is typically mitigated through various protein quality control (PQC) mechanisms that prevent aggregation by facilitating protein removal through the autophagy-lysosomal system or the ubiquitin proteasome system (UPS) (Rubinsztein, 2006;Douglas and Dillin, 2010;Ciechanover and Kwon, 2015;Dubnikov et al., 2017). Indeed a number of aging-related neurodegenerative disorders have been associated with defects in autophagy or UPS activity (Komatsu et al., 2006;Kourtis and Tavernarakis, 2011;Harris and Rubinsztein, 2012;Tashiro et al., 2012).
During pathological conditions, the human TAR DNA-Binding Protein 43 (TDP43) protein undergoes proteolytic cleavage at a number of locations giving rise to a variety of proteolytic fragments susceptible to intracellular aggregation (Zhang et al., 2007;Nonaka et al., 2009b;Igaz et al., 2009;Yamashita et al., 2012;Chang et al., 2013;Cohen et al., 2015;Li et al., 2015;Kitamura et al., 2016;Kametani et al., 2016;Rabdano et al., 2017;Kasu et al., 2018;Chhangani et al., 2021). In particular, owing to a C-terminal prion-like domain, C-terminal fragments of TDP43 are the major constituents of proteinaceous aggregates found in cytoplasm of neurons of ALS and FTLD patients (Neumann et al., 2006;Zhang et al., 2007Zhang et al., , 2009Igaz et al., 2009;Nonaka et al., 2009aNonaka et al., , 2009bBudini et al., 2012;Yamashita et al., 2012;Li et al., 2015;Kametani et al., 2016;Kitamura et al., 2016). Such aggregates were shown to be associated with ubiquitin suggesting that defects in their UPS-mediated degradation may play a contributing role (Kasu et al., 2018;Nonaka et al., 2009a;Li et al., 2011;Braak et al., 2010). In previous work, we found that differences in the N-termini of otherwise identical C-terminal fragments can influence their metabolism and aggregation dynamics (Kasu et al., 2018). Of note, we found that degradation by the Arg/N-degron pathway precludes the aggregation of proteolytic fragments bearing N-degrons (N-terminal degradation signals) consisting of a basic (e.g., Arg, Lys, and His) or bulky hydrophobic (e.g., Phe, Lue, Trp, Tyr, and Ile) N-terminal amino acid (Brower et al., 2013;Kasu et al., 2018;Varshavsky, 2011). However, N-degron formation is not a requisite outcome of proteolytic cleavage. As such, not all proteolytic fragments are substrates of the Arg/N-degron pathway. Furthermore, defects in the N-degron pathway, e.g., as a result of age-related decline in activity or exhaustion because of substrate overproduction, allow many substrates to escape degradation by the Arg/N-degron pathway. To determine the fate of proteolytic fragments that escape the N-degron mediated degradation, we inactivated the N-degrons of TDP43 219 and TDP43 247 , two specific disease-linked fragments of human TDP43 consisting of amino acids 219 -414 and 247 -414, respectively. These fragments are 85% identical and differ by an extended hydrophobic N-terminal 28 residue in TDP43 219 absent in TDP43 247 (Brower et al., 2013;Kasu et al., 2018). Whereas TDP43 247 accumulates and forms abundant, large and morphologically distinct aggregates in the absence of the Arg/N-degron pathway, TDP43 219 forms sparse, tiny aggregates (Kasu et al., 2018). This indicates that an additional PQC mechanism participates in the metabolism of TDP43 219 and likely discriminates against differences in hydrophobic content. Here, we found that BAG6 prevents protein aggregation by functioning as a sensor of solvent-exposed hydrophobicity in proteolytic fragments. Whereas BAG6 does not recognize full-length TDP43, it binds strongly to TDP43 219 because of its exposed hydrophobic N-terminus and prevents its intracellular aggregation both by increasing its solubility and by facilitating its RNF126-mediated ubiquitylation. We also provide evidence that BAG6 effects are not limited to fragments of TDP43 but can interact with and solubilize fragments of the amyloid precursor protein. Therefore, BAG6 plays a general role in preventing intracellular aggregation associated with neurodegeneration.

BAG6 associates with TDP43 219 and TDP43 247
To examine proteolytic TDP43 fragments bearing their natural, cleavage-exposed, N-terminus, we use the ubiquitin (Ub)-reference technique (URT) which involves fusing Ub between a downstream test protein and an upstream, long-lived reference protein such as dihydrofolate reductase (DHFR) ( Figure 1A). Rapid (cotranslational) cleavage by intracellular deubiquitylases (DUBs) after the last residue of Ub enables the initial equimolar expression of a fragment with a specified N-terminal amino acid and an internal reference protein from a single RNA transcript (Varshavsky, 2005). Through immunolabeling, we have been unable to detect full-length URT fusion products, suggesting that DUB cleavage is highly efficient. Nonetheless, inefficient cleavage would lead to confounding results particularly in downstream aggregation studies. Therefore, to confirm efficient and rapid DUB cleavage of URT fusions, we examined their expression using metabolic labeling. For this, we labeled flag-DHFR-Ub-TDP43 219 -flag expressing cells with [ 35 S] Met/Cys, blocked translation with cycloheximide, and immediately carried out denaturing anti-FLAG immunoprecipitation to isolate URT products in conditions that prevent further processing (Tansey, 2007 iScience Article conditions facilitate highly sensitive detection of newly-formed URT products. Using this approach, we detected both flag-DHFR-Ub and TDP43 219 -flag, which migrate through SDS-PAGE at the expected sizes of $33 and $21 kDa, respectively. On the other hand, the uncleaved flag-DHFR-Ub-TDP43 219 -flag fusion (calculated molecular mass of 54.6 kDa) was only slightly detected (if at all) above background ( Figure 1A, compare lanes 1 and 2 with 3 and 4). These results indicate that DUB cleavage of URT fusion products is highly efficient and occurs co-translationally.
Previously, we found that TDP43 247 is degraded exclusively by the Arg/N-degron pathway; whereas TDP43 219 was degraded even in its absence (Kasu et al., 2018). To identify additional PQC mechanisms that prevent the aggregation of proteolytic fragments, we found that BAG6 interacts with soluble forms of TDP43 219 and TDP43 247 . To confirm this interaction, we used the URT to express TDP43 fragments bearing N-terminal Val in HEK293T cells treated with the proteasome inhibitor, MG132 to prevent their recognition by the Arg/N-degron pathway and degradation by the proteasome. C-terminally single FLAG-tagged TDP43 219 and TDP43 247 were immunoprecipitated from detergent-soluble and -insoluble (urea solubilized) fractions using an anti-FLAG antibody. Although the bulk of TDP43 fragments were detected in the insoluble fractions (consistent with their tendency to aggregate in the absence of their degradation (Brower et al., 2013)), endogenous BAG6 was co-immunoprecipitated with TDP43 247 , and especially with TDP43 219 but not with DHFR-Ub, in the soluble fractions ( Figure 1B).
To further validate the BAG6 interaction with TDP43 fragments, we performed a reciprocal co-IP with an anti-BAG6 antibody from the soluble fractions of cells overexpressing TDP43 fragments and wild type BAG6 or BAG6 lacking its N-terminal UBL domain (BAG6DUBL). Despite similar expression levels, BAG6 and BAG6DUBL interact with TDP43 219 and TDP43 247 , albeit less strongly to TDP43 247 ( Figure 1C, compare IP lanes 4 and 5, and 8 and 9). This result indicates that the N-terminal UBL domain of BAG6 is dispensable for this interaction with proteolytic fragments of TDP43. Densitometry of TDP43 proteolytic fragments interacting with BAG6 revealed a $5-fold higher affinity of BAG6 for TDP43 219 relative to TDP43 247 (Figure 1D). This is consistent with an extended hydrophobic N-terminus of TDP43 219 that is not present in TDP43 247 ( Figure 1E). Interestingly, BAG6 did not interact with endogenous full-length TDP43 harboring the same and additional regions of hydrophobicity ( Figures 1C and 1E), owing to the absence of their solvent-exposure in the correctly folded protein. Collectively, these results indicate that BAG6 functions as a sensor of proteolytic fragments bearing solvent-exposed hydrophobicity.

BAG6 solubilizes TDP43 protein fragments
To ablate BAG6 function in cells, we targeted exon four of the human BAG6 gene in HEK293T cells using CRISPR-Cas9 and clonally expanded cells lacking BAG6 (BAG6-KO; Figure 2A). To examine TDP43 219 solubility in the presence and absence of BAG6, we expressed TDP43 219 (and DHFR-Ub as a control) using the URT ( Figure 1A) in BAG6-KO cells in the presence of increasing amounts of plasmid expressing BAG6. In the absence of BAG6, the bulk of TDP43 219 was detected in the insoluble fraction ( Figures 2B and 2C). In contrast, we observed a dose-dependent increase in the levels of soluble TDP43 219 concomitant with decreased insoluble TDP43 219 upon titration of exogenous BAG6-expressing plasmids ( Figure 2B, lanes 7-9 and 2D). Notably, BAG6 had no effect on the levels of endogenous TDP43, consistent with its lack of affinity for the full-length correctly folded TDP43. Because the N-terminal UBL domain of BAG6 is dispensable for its interaction with TDP43 219 ( Figure 1C), we asked if it was required for BAG6 effects on TDP43 219 solubility. BAG6DUBL had similar effects on TDP43 219 solubility as wild type BAG6 (Figures 2C and 2D). These data indicate that TDP43 219 is largely insoluble in the absence of BAG6 and that BAG6 increases its solubility in a manner that does not require its N-terminal UBL domain.

BAG6 prevents the oligomerization of TDP43 fragments
To determine if BAG6 overexpression prevents the formation of TDP43 oligomers and higher-ordered aggregates, we expressed TDP43 219 and TDP43 247 in the presence and absence of exogenously iScience Article overexpressed BAG6 and treated cell pellets with the cross-linking agent, disuccinimidyl glutarate (DSG), to capture and preserve oligomeric species formed within cells. Monomeric and oligomeric species of TDP43 were then detected in lysates by immunoblot using an anti-TDP43 antibody. Consistent with BAG6 interaction with TDP43 fragments and its effects on TDP43 219 solubilization, the overexpression of BAG6 resulted in higher levels of monomeric TDP43 219 and TDP43 247 in the soluble fraction ( Figure 3, compare lanes 2 and 3 to 8 and 9). Treatment with DSG captured oligomeric species of both TDP43 219 and TDP43 247 as detected by a ''ladder'' of higher molecular weight species in the insoluble fractions ( Figure 3, compare lanes 2 and 3 to 5 and 6). Strikingly, overexpression of BAG6 resulted in far fewer oligomeric TDP43 219 and TDP43 247 species captured in the presence of DSG ( Figure 3, compare lanes 5 and 6 to 11 and 12). Consistent with a greater affinity to TDP43 219 , the reduction of insoluble oligomeric species in the presence of BAG6 was greater for TDP43 219 than for TDP43 247 (Figure 3, compare lanes 5 and 11 to 6 and 12).
The loss of oligomeric species in the presence of overexpressed BAG6 indicates that BAG6 prevents the self-assembly and oligomerization of neurodegeneration-associated proteolytic fragments of TDP43.

BAG6 prevents aggregation of TDP43 219 fragments in cells
Previously, we reported that in the absence of degradation by the Arg/N-degron pathway, the majority of TDP43 247 expressing cells contain cytoplasmic, perinuclear aggregates of various morphologies; whereas   Figure 4A). Co-translational cleavage of this fusion construct yields a stable mCherry-Ub R48 which ''marks'' transfected cells, and a C-terminally FLAG epitope-tagged TDP43 fragment whose fate can be monitored using indirect immunofluorescence with an anti-FLAG primary antibody and a fluorescein-conjugated secondary antibody ( Figure 4B). Consistent with earlier published results, aggregates of TDP43 219 (quantified as the number of mCherry expressing cells that also contain aggregates) could be detected in only $12% of wild type (HEK293T) cells ( Figure 4C). As evidence that the UPS is largely responsible for preventing intracellular aggregation of TDP43 219 , aggregates could be detected in $80% of treated with the proteasome inhibitor, MG132 ( Figure 4C). Remarkably, cytosolic TDP43 219 aggregates were detected in greater than 50% of BAG6-lacking cells ( Figures 4B and 4C). To establish that BAG6 mitigates aggregation, we measured the levels of TDP43 219 aggregates in BAG6-KO cells following transfection with a BAG6-expressing plasmid. Exogenous expression of BAG6 reduced the levels of TDP43 219 aggregates in BAG6-KO cells from $50% to $24% ( Figures 4B and 4C). These results indicate that BAG6 prevents the intracellular aggregation of TDP43 219 . Figure 3. BAG6 prevents the oligomerization of TDP43 proteolytic fragments HEK293T cells were either mock transfected (À) or transfected with plasmids expressing either TDP43 219 or TDP43 247 in the presence or absence of exogenously overexpressed BAG6. To detect oligomers, cell pellets were treated with 1 mM disuccinimidyl glutarate (DSG) and lysates were fractionated into detergent-soluble and -insoluble (urea-soluble) factions. TDP43 fragments were detected in the soluble and insoluble fractions by immunoblotting using an anti-TDP43 antibody. Endogenous and exogenous BAG6 was detected by immunoblotting using an anti-BAG6 antibody. Note that dimers of TDP43 247 overlap with endogenous nonspecific bands denoted by asterisks (lanes 6 and 12). To function in the GET pathway, BAG6 assembles into a trimeric complex with TRC35 and UBL4A (Mariappan et al., 2010;Mock et al., 2015Mock et al., , 2017Chio et al., 2017;Kuwabara et al., 2015;Krenciute et al., 2013;Shao et al., 2017;Leznicki et al., 2013). To function in PQC, BAG6 also interacts with various E3 Ub-ligases (Hessa et al., 2011;Rodrigo-Brenni et al., 2014;Krysztofinska et al., 2016;Yau et al., 2017;Hu et al., 2020). To determine which, if any, BAG6 partners are associated with quality control of TDP43 219 , we co-expressed FLAG-tagged TDP43 219 , BAG6, TRC35, Ubl4a, and RNF126 in BAG6-KO cells, and carried out co-immunoprecipitation from the soluble fraction using an anti-TDP43 antibody followed by anti-FLAG immunoblot ( Figure 5A). TDP43 219 is associated strongly with BAG6 and TRC35 (Figure 5A, lane 4). In addition, the E3 Ub-ligase, RNF126, was co-immunoprecipitated with TDP43 219 along with BAG6 and TRC35 ( Figure 5A, lane 5). Interestingly, UBL4a was noticeably absent from the complex regardless of the association of RNF126 ( Figure 5A, lane 4 and 5). This indicates that UBL4a, which is required for the transfer of TA clients from SGTA to TRC40, is dispensable when BAG6 is bound to clients not destined to the ER.
To determine if TDP43 219 can interact with RNF126 independently of BAG6, we carried out similar co-immunoprecipitations in BAG6-KO cells in the presence and absence of exogenously added BAG6. Remarkably, both TDP43 219 and TDP43 247 (albeit at levels near the detection limit) could associate with RNF126 independently, or along with BAG6 ( Figure 5B). A direct interaction between TDP43 fragments and RNF126 was not expected. However, there are reports of BAG6-independent RNF126 ubiquitin-ligase activity (Zhi et al., 2013;Benini et al., 2017). Collectively, these results indicate that TDP43 219 associates with BAG6 and TRC35 (but not UBL4a), and can associate with the Ub-ligase, RNF126, independently or in a complex with BAG6. To determine if RNF126 is capable of ubiquitylating TDP43 219 , we carried out in vitro ubiquitylation reactions containing purified HA-tagged Ub, the E1 Ub-activating enzyme (UBE1), the E2 Ub-conjugating enzyme (UBE2), bacterially expressed FLAG-tagged TDP43 219 and bacterial cell lysates expressing recombinant RNF126, or its catalytically inactive mutant, RNF126 C231A . Polyubiquitylation, detected by anti-Ub immunoblot, was formed only in reactions that included bacterial lysate containing wild type RNF126 (Figure 5C, lanes 1 and 8) and not from those containing non-transformed bacterial lysate or lysate containing RNF126 C231A ( Figure 5C, lane 2 and 3). This indicates that Ub-ligase activity is derived from catalytically active RNF126 and not from another component in the bacterial cell lysate or the reaction mixture. Because polyubiquitylation was also detected in reactions lacking TDP43 219 ( Figure 5C, lane 8), its association with TDP43 219 could not be determined by anti-Ub immunoblot of reaction mixtures. To distinguish TDP43 219specific from nonspecific ubiquitylation, we carried out anti-FLAG immunoprecipitation of the in vitro reaction mixtures to isolate TDP43 219 and its associated modifications. Interestingly, polyubiquitylation A B C Figure 5. TDP43 219 interacts BAG6, TRC35, and RNF126 and is associated with RNF126-catalyzed ubiquitylation (A) BAG6-KO cells were transiently transfected with FLAG epitope-tagged TDP43 219 , BAG6, TRC35, UBL4a and RNF126. Upper panels, Proteins interacting with TDP43 219 were detected by IP using anti-TDP43, followed by immunoblot using anti-FLAG. Asterisk, antibody heavy chain. Lower panels, anti-FLAG immunoblot of lysates.
(C) In vitro ubiquitylation reactions containing the indicated components. Total ubiquitylation was detected in reaction mixtures using an anti-ubiquitin antibody. TDP43 219 -specific ubiquitylation was detected by anti-ubiquitin immunoblot of anti-FLAG IP samples. TDP43 219 was detected using an anti-TDP43 antibody. Additional reaction components were identified through coomassie staining of lysate membrane. iScience Article formed in reaction mixtures lacking TDP43 219 were not co-immunoprecipitated indicating that it was nonspecific ( Figure 5C, lane 8). In contrast, significant TDP43 219 -specific polyubiquitylation was detected in immunoprecipitates of reactions containing TDP43 219 and all other reaction components (Figure 6, lanes 1). Collectively, these results indicate that RNF126 can bind TDP43 219 , either directly or can be recruited by BAG6, to catalyze the polyubiquitylation.

BAG6 interacts with additional neurodegeneration-associated protein fragments
To determine if BAG6 is specific to TDP43 219 and TDP43 247 or if it plays a general role in preventing intracellular aggregation of proteolytic fragments with solvent-exposed hydrophobicity, we examined its capacity to interact with TDP43 208 (amino acids 208-247) and proteolytic fragments linked to AD (Lauritzen et al., 2016;Nunan et al., 2001Nunan et al., , 2003. For this, we used the URT ( Figure 1A) to co-express BAG6 with FLAG-tagged TDP43 208 , Tau 3-24 , bCTF, or C-terminally 13Myc-tagged Ab in HEK293T cells. We then Figure 6. BAG6 has a general role in preventing aggregation of neurodegeneration-associated proteolytic fragments (A) FLAG-tagged DHFR-Ub (as a control), TDP43 208 , Tau, bCTF, and 13myc-tagged Ab were expressed in the presence of overexpressed BAG6 in HEK293T cells. Upper panels, Proteins interacting with BAG6 were detected using an anti-BAG6 co-IP, followed by immunoblot using the indicated antibodies. Lower panels, immunoblot of lysates using the indicated antibodies. Mock, mock-transfected. LC, antibody light chain. (B) IP and lysate fractions from panel A (lanes 7 through 12) immunoblotted with an anti-TDP43 antibody. Asterisk, endogenous, cross-reacting band.
(C and D) BAG6-KO cells expressing either TDP43 208 or bCTF in the presence or absence of BAG6. Cells were lysed and fractionated into detergent-soluble (Sol) and -insoluble (Ins) fractions. BAG6, TDP43 208 and bCTF were detected using an anti-FLAG antibody. Anti-b-actin and anti-fibrillarin was used as loading controls for the soluble and insoluble fractions, respectively (D) Model of BAG6 role in preventing the aggregation of proteolytic fragments. Limited proteolysis (indicated by scissors) of various cellular proteins generates misfolded proteolytic fragments with solvent-exposed hydrophobic regions (red portion of protein). In the absence of their degradation, these fragments self-associate to form oligomers and insoluble aggregates that are associated with neurodegeneration. Alternatively, hydrophobicity is bound by BAG6 which prevents fragment oligomerization and aggregation. The BAG6 complex can recruit various E3 Ub-ligases-e.g., RNF126-to facilitate client ubiqiutylation and proteasome-mediated degradation.

OPEN ACCESS
iScience 25, 104273, May 20, 2022 9 iScience Article carried out co-immunoprecipitation using an anti-BAG6 antibody followed by immunoblot with indicated antibodies to detect association. We were unable to detect expression of Tau 3-24 of its expected size ($14 kDa) in soluble fractions; however an interaction between BAG6 and TDP43 208 , bCTF, and Ab was detected ( Figure 6A). Notably, TDP43 208 is identical to TDP43 219 except that it contains additional hydrophobic N-terminal amino acids. bCTF and Ab share a significant region of hydrophobicity that contributes to their propensity to form amyloid deposits. Of note, the highly hydrophilic C-terminal 13Myc-tag, which was added to solubilize the highly amyloidogenic Ab, did not prevent its interaction with BAG6 ( Figure 6A, lane 12). We also carried out immunoblot of the lysate and IP fractions using an anti-TDP43 antibody and again found that BAG6 did not interact with endogenous full-length TDP43 protein, presumably because of the lack of exposed hydrophobicity in the correctly folded full-length protein ( Figure 6B). To assess the functional significance of these interactions, we examined the levels of TDP43 208 and bCTF in the soluble and insoluble (urea-solubilized) fractions in the presence and absence of BAG6 expressed in BAG6-KO cells (13Myc-tagged Ab was not examined as it is not a naturally produced product). Similar to the results obtained with TDP43 219 , the bulk of TDP43 208 and bCTF was isolated in insoluble fractions in the absence of BAG6. However, co-expression of BAG6 increased the solubility of these polypeptides as seen by their increased partitioning to the soluble fraction ( Figure 6C). These results indicate that BAG6 can interact with a variety of aggregation-prone proteins derived from structurally and functionally distinct precursors and can enhance their solubility. Thus, BAG6 plays a general role in preventing the aggregation of neurodegeneration-associated proteolytic fragments.

DISCUSSION
Previously, we reported that the N-termini of otherwise similar aggregation-prone protein fragments influence their degradation, aggregation dynamics, and potentially the clinical outcomes of neurodegenerative disorders associated with proteinaceous aggregates. For example, TDP43 247 is degraded exclusively by the Arg/N-degron pathway; but an additional pathway(s) is capable of degrading TDP43 219 (Kasu et al., 2018). In efforts to identify additional PQC mechanisms that participate in protecting cells from proteinopathies, we found that the molecular chaperone, BAG6, functions as an intracellular sensor of solventexposed hydrophobicity in proteolytic fragments. Chaperone-assisted clearance of aggregation-prone proteins and their involvement in the prevention of neurodegenerative diseases is well established and impairment of PQC results in a variety of disease-linked proteinopathies (Ciechanover and Kwon, 2017). We provide a number of independent lines of evidence that BAG6 prevents the intracellular aggregation of neurodegeneration-associated proteolytic fragments. First, using co-immunoprecipitation, we found that BAG6 interacts with specific aggregation-prone fragments of TDP43 (TDP43 208 , TDP43 219 , and TDP43 247 ) as well as other proteins associated with neurodegeneration (bCTF and Ab) (Figure 1 and Figure 6). Second, through biochemical fractionation of CRISPR-Cas9-mediated BAG6-lacking cells, we found that TDP43 219 is insoluble in the absence of BAG6 but its solubilization is directly correlated to BAG6 levels (Figures 2 and 6). Third, using chemical cross-linking to capture and maintain oligomeric species, we found that BAG6 prevents the self-association and oligomerization of TDP43 219 and TDP43 247 (Figure 3). Fourth, using immunocytochemistry, we show that TDP43 219 forms $5-fold more aggregates in BAG6-lacking cells compared to BAG6-containing cells (Figure 4). Lastly, we show that TDP43 219 can interact with the BAG6recruited Ub-ligase, RNF126 and is associated with RNF126-catalyzed ubiquitylation ( Figure 5). In support of a general function in preventing the aggregation of neurodegeneration-associated fragments, we found that BAG6 also interacts with and solubilizes the amyloidogenic Ab peptide and the b-secretase generated C-terminal fragment of the amyloid precursor protein, bCTF, involved in AD pathology (Pulina et al., 2020;Checler et al., 2021). Our data support a model ( Figure 6D) whereby limited proteolysis during pathological conditions generate proteolytic fragments containing exposed hydrophobicity. A number of specific UPS pathways (e.g., the Arg/N-degron pathway) have been reported to play a role in the clearance of proteolytic fragments associated with neurodegeneration (Hebron et al., 2013;Brower et al., 2013;Uchida et al., 2016;Kasu et al., 2018;Watabe et al., 2020). However those that escape degradation can self-associate, forming higher order oligomers and ultimately insoluble aggregates in attempts to shield exposed hydrophobicity. Alternatively, they are bound by BAG6, maintained in a soluble state to prevent aggregation, and targeted for UPS-mediated degradation through ubiquitylation catalyzed by recruited Ub-ligases, such as RNF126.
Although our data suggests that RNF126 is recruited to TDP43 proteolytic fragments to facilitate their UPSmediated degradation, it does not preclude the involvement of alternative Ub-ligases as well. BAG6 has been shown to recruit a number of distinct Ub-ligases. For example, the ER-associated Ub-ligase, gp78 is ll OPEN ACCESS iScience Article recruited by BAG6 during ERAD of misfolded ER substrates (Wang et al., 2011). In a clever study employing the use of bispecific antibodies to detect K11/K48-linked ubiquitin chains, the BAG6 complex was shown to recruit HUWE1, UBR4, and UBR5 to catalyze the attachment of heterotypic ubiquitin to BAG6-bound substrates (Yau et al., 2017). Interestingly, UBR4 and UBR5 are both Ub-ligases of Arg/N-degron pathway (referred to as ''N-recognins'' by their ability to recognize N-degrons) (Dougan et al., 2012;Cha-Molstad et al., 2017;Melnykov et al., 2019;Varshavsky, 2019). As such, one possibility is that BAG6 cooperates with the Arg/N-degron pathway in the degradation of proteolytic fragments not only by solubilizing substrates but also through recruitment of UBR4 and UBR5. This is an interesting direction of future study.
BAG6 has been described as a ''holdase'' or ''sortase'' chaperone widely studied in the context of posttranslational ER-targeting of tail-anchored (TA) proteins bearing hydrophobic C-terminal transmembrane domains. It functions within a trimeric complex containing TRC35 and UBL4a to facilitate client transfer from the upstream co-chaperone, SGTA to the downstream cytosolic ATPase, TRC40 (Mariappan et al., 2010;Mock et al., 2015Mock et al., , 2017Chio et al., 2017;Kuwabara et al., 2015;Krenciute et al., 2013;Shao et al., 2017;Leznicki et al., 2013). The BAG6 complex also participates in PQC by recruiting Ub-ligases to facilitate client ubiquitylation (Hessa et al., 2011;Rodrigo-Brenni et al., 2014;Krysztofinska et al., 2016;Yau et al., 2017;Hu et al., 2020). Although it does not preclude ubiquitylation by alternative Ub-ligases, we found that RNF126 is recruited to the TDP43 219 -bound BAG6 complex and catalyzes TDP43 219 -associated polyubiquitylation. Interestingly, the TDP43 219 -associated BAG6 complex lacked UBL4a. The reason for UBL4a exclusion is unclear. However there is precedence for BAG6 functions in the absence of UBL4a (Thress et al., 1998;Krenciute et al., 2013). An interesting possibility is that the trimeric BAG6 complex (BAG6/TRC35/ UBL4a) undergoes remodeling to accommodate the triage needs of specific client proteins. To maintain proteostasis, the BAG6 complex drives either biosynthetic or PQC agendas determined largely by client dissociation from SGTA and capture by BAG6 (Shao et al., 2017). However, SGTA competes for clients and antagonizes BAG6-mediated degradation by promoting de-ubiquitylation (Leznicki and High, 2012;Wunderley et al., 2014). Structural studies have shown that RNF126 and SGTA compete for the N-terminal UBL domain of BAG6 (Krysztofinska et al., 2016). However, the UBL domain of UBL4a also competes for SGTA binding (Leznicki et al., 2013). As such, BAG6 commitment to PQC could be accomplished by eliminating SGTA antagonizing effects through the recruitment of Ub-ligases and disengagement with UBL4a. This may explain why TDP43 219 -bound BAG6 is not associated with UBL4a ( Figure 5A). Further studies are needed to confirm this conjecture.
Although TDP43 219 and TDP43 247 are $85% identical, TDP43 219 contains an additional N-terminal 28amino acids that contribute a significant hydrophobic ''handle'' not included in TDP43 247 . Consistent with an exposed hydrophobicity sensing function, BAG6 interacts with $5-fold affinity for TDP43 219 than for TDP43 247 ( Figure 1D). This suggests that BAG6 has a graded response in cellular protection as it has the highest affinity for the most aggregation-prone fragments. As such, BAG6 may prioritize removal of the most toxic proteins first.
Although many chaperones are upregulated during stress, BAG6 does not appear to be upregulated in response to the expression of TDP43 fragments (Figure 3, lanes 1 to 3). As such, the overproduction of toxic proteolytic fragments may not only exhaust PQC functions but also compete with TA proteins otherwise destined for the ER. Interestingly, a recent study showed that SGTA, which senses hydrophobic transmembrane domains in TA proteins also associates with intracellular aggregates associated with neurodegenerative diseases (Kubota et al., 2021). Consequently, disruption of the GET pathway could be involved in the etiology of neurodegeneration.
In sum, we show that the molecular chaperone, BAG6, plays a role in preventing the intracellular aggregation of proteolytic fragments bearing solvent-exposed hydrophobicity by maintaining their solubility and preventing their self-association. In addition, BAG6 facilitates their ubiquitylation and subsequent degradation by recruiting Ub-ligases such as RNF126. Therefore, BAG6 plays a protective role by preventing intracellular aggregation associated with neurodegeneration. However, as only one of multiple Ub-ligases recruited by BAG6 to facilitate UPS-mediated degradation, our work does not preclude the recruitment of alternative Ub-ligases. In addition, BAG6 may also play a redundant role in protein quality control. For example, chaperones of the Hsp70 family and Hsp90 also interact with the Ub-ligase CHIP (carboxy-terminus of Hsc70 interacting protein) to triage proteins with small hydrophobic regions (Connell et al., 2001).

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The authors declare no competing interests.

INCLUSION AND DIVERSITY
One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper received support from a program designed to increase minority representation in science.
iScience Article iScience Article RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Christopher Brower (cbrower@twu.edu).

Materials availability
Plasmids and cell lines generated in this study are available on request.
Data and code availability d All immunoblotting and microscopy data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code. We specify tools used in the quantification and statisctical analysis section.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Assays performed in this study utilized Human embryonic kidney (HEK)-293T cells originally received and authenticated from American Type Culture Collection (ATCC). BAG6-KO cells were derived from HEK293T cells.

Generation of BAG6-lacking HEK293T cells
HEK293T lacking BAG6 were generated using the CRISPR-Cas9 system (Cong et al., 2013). Briefly, pJH551, a plasmid encoding a human codon-optimized SpCas9 and a chimeric guide RNA targeting the exon 4 of human BAG6 gene, was constructed by the ligation of a double strand oligomer (made by the denaturation and renaturation of CB521F and CB522R) into BbsI digested pX330 (pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang; Addgene plasmid # 42230). Cells were transfected with pJH551 and individual clones were selected and screened for loss of the BAG6 protein by immunoblot (Figure 2A).

Protein cross linking
HEK293T cells were transfected with plasmids expressing DHFR-Ub K48R (pYK27), or TDP43 219 (pCB328) and TDP43 247 (pCB332) bearing N-terminal Val from the P CMV promoter. Forty-eight hours post-transfection, cells were resuspended in 1mM disuccinimidyl glutarate containing phosphate buffered saline and incubated at room temperature with shaking for 30mins. Then, 20mM Tris (pH 8) was added to quench the reaction and incubated at room temperature with shaking for 15mins. The cells were then centrifuged at 1000 x rpm for 3 mins and the cell pellet was resuspended in chilled TLB for protein extraction as described above.
The supernatant was applied to 0.5mL resin volume Nickel-column pretreated with IBS buffer ((40mM Tris (pH 8), 6M guanidine hydrochloride, 0.5M KCl, and 0.5mM PMSF). The columns were then washed three times with IBS-40 buffer (IBS buffer containing 40mM imidazole) and then eluted in IBS-300 buffer (IBS buffer containing 300mM imidazole). The presence of recombinant TDP43 protein fragment in eluate was detected by immunoblotting with anti-TDP43 antibody.

In vitro ubiquitylation reaction
Ubiquitylation reactions were adapted from those described in Hu et al., (2020) (Hu et al., 2020). Briefly, ubiquitylation reactions contained ubiquitylation buffer (30 mM HEPES, pH 7.5, 50 mM NaCl, 2.5 mM MgCl 2 , and 0.25 mM DTT), HA-Ubiquitin (10mM), GST-Ube1 (0.25mM), UbcH5b (0.5mM), ATP (2mM), Met 219 -TDP43 fÀ6xHis (500ng) and clarified (13,000rpm for 15 min at 4 C) BL21 (DE3) lysates expressing wild type RNF126 (transformed with GST-RNF126), RNF126 C231A (transformed with GST-RNF126 C231A ), or non-transformed BL21 (DE3). The reaction was incubated at 25 C for 1 h, then stopped by freezing in liquid nitrogen. To detect polyubiquitylation associated with TDP43 protein fragments, anti-FLAG antibody was used to immunoprecipitate Met 219 -TDP43 fÀ6xHis , followed by immunoblotting with anti-ubiquitin antibody.  (Figure 4C), aggregates were detected by immunocytochemistry using an antibody to the C-terminal flag epitope of TDP43 219 and an Alexa 488-conjugated secondary antibody. Aggregates were scored as the number of mCherry-expressing cells that also display detectable TDP43 219 aggregates. mCherry positive cells in at least ten randomly selected fields were examined. Experiments were carried out in triplicate. At least 600 transfected cells were analyzed per group. A one-way ANOVA revealed a significant effect of BAG6 knockout on protein aggregation (F(3,13) = 13.661, p = 0.001) with Fisher LSD post-hoc tests showing significant group differences. To determine the relative affinity of BAG6 for TDP43 219 versus TDP43 247 ( Figure 1D), densitometry of TDP43 219 and TDP43 247 co-immunoprecipitated from equal amounts of BAG6 was carried out using ImageJ from three independent experiments. An independent t-test yielded significant differences between TDP43 219 and TDP43 247 (t(4) = -22.9, p < 0.001). To determine relative levels of TDP43 219 in soluble fractions of BAG6-KO cells as a result of increasing amounts of BAG6 or BAG6DUBL ( Figure 2D), densitometry of soluble TDP43 219 was carried out using ImageJ and levels obtained in the presence of BAG6 were compared to the levels obtained in the absence of BAG6. A one-way ANOVA demonstrated significant between group differences in soluble TDP43 219 as a result of increasing concentrations (0, 0.