Interactome screening implicates BAG6 as a suppressor of UBQLN2 misfolding in ALS/FTD

Abstract

Ubiquilin-2 (UBQLN2) is a ubiquitin (Ub)-binding shuttle protein that is mutated in X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS/FTD-linked mutations in UBQLN2 disrupt its conformation, increasing its tendency to form cytoplasmic aggregates that may disrupt cellular regulation through loss-of-function (LOF) and gain-of-function (GOF) effects. Here, we performed quantitative mass spectrometry (MS)-based interactome analysis of wild-type (UBQLN2^WT) and ALS-mutant UBQLN2 (UBQLN2^ALS) proteins using inducible pluripotent stem cells (iPSCs) and induced motor neurons (iMNs). Proteins showing enhanced association with UBQLN2^ALS proteins included PEG10, a known degradation target of UBQLN2, and BAG6, a chaperone involved in the triage of mislocalized proteins (MLPs). BAG6 knockdown inhibited the solubility recovery of both UBQLN2^WT and UBQLN2^ALS proteins following heat stress (HS), suggesting it functions as a UBQLN2 holdase. In addition, knockdown of BAG6 or knockout of UBQLN2 led to PEG10 accumulation, implicating both in PEG10 turnover; however, neither BAG6 nor UBQLN2 was required for PEG10 degradation in response to HS. A highly aggregation-prone UBQLN2^4XALS mutant harboring four different ALS-associated mutations showed increased PEG10 binding and modestly delayed PEG10 turnover while PEG10 degradation was not significantly different between UBQLN2^WT and iPSCs expressing a UBQLN2^P497H clinical mutant. The combined findings implicate BAG6 as a UBQLN2 holdase and identify a suite of proteins whose altered binding may contribute to pathologic changes in UBQLN2-associated ALS/FTD.

Introduction

The intrinsic toxicity of misfolded, aggregation-prone proteins is normally counteracted by evolutionarily conserved proteostasis networks, which oversee the entire lifecycle of a protein—from synthesis and folding to trafficking and degradation. In aging-related neurodegenerative diseases (NDs) such as Alzheimer’s Disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), this delicate balance is progressively disrupted, leading to pathologic protein accumulation and neuron demise (Labbadia and Morimoto, 2015).

ALS is a fatal neurodegenerative condition marked by progressive degeneration of upper and lower motor neurons (MNs), resulting in muscle weakness, paralysis, and death within 3–5 years of symptom onset in the absence of mechanical ventilation (Cicardi et al., 2021; Cook and Petrucelli, 2019; Brown and Al-Chalabi, 2017). While most ALS cases are sporadic (sALS), approximately 10–15% are familial (fALS), involving mutations in more than 40 genes. The signature histopathologic feature of ALS is the accumulation of misfolded, cytosolic TDP-43 (43 kDa TAR DNA-binding protein) which is seen in virtually all instances of ALS except those caused by mutations in superoxide dismutase 1 (SOD1) or fused in sarcoma (FUS) (Babazadeh et al., 2023; Neumann et al., 2006). Cytosolic aggregation and associated nuclear depletion of TDP-43 leads to cryptic exon inclusion and other RNA splicing changes in functionally important genes, triggering axonal and synaptic failure of motor neurons (Ma et al., 2022; Brown et al., 2022; Melamed et al., 2019; Klim et al., 2019; Ling et al., 2015; Lagier-Tourenne et al., 2010; Polymenidou et al., 2011). The upstream events leading to TDP-43 aggregation are poorly understood but may involve genetic susceptibility, environmental exposure, and aging-dependent reductions in proteostasis and nucleocytoplasmic transport (Cook and Petrucelli, 2019; Rummens and Da Cruz, 2025; Khalil et al., 2024; Portz et al., 2021).

X-linked mutations in UBQLN2 are a rare cause of ALS, FTD, and hereditary spastic paraplegia (Deng et al., 2011; Gellera et al., 2013; Gkazi et al., 2019). UBQLN2 is a Ub-binding shuttle protein that plays a critical role in cellular proteostasis by facilitating the degradation of misfolded or ubiquitinated proteins via the proteasome and autophagy pathways. Structurally, UBQLN2 contains an N-terminal ubiquitin-like (UBL) domain and a C-terminal ubiquitin-associated (UBA) domain, connected by a methionine-rich central region that includes stress-inducible protein 1 (STI1)-like motifs (Fry et al., 2021; Zientara-Rytter and Subramani, 2019; Howe et al., 2020; Schmid et al., 2012). Unlike other members of the ubiquilin family, UBQLN2 is found only in eutherian mammals, suggesting a lineage-specific role, possibly including functions in placental biology (Black et al., 2023).

ALS-associated UBQLN2 mutations cluster within a proline-rich repeat (PRR) region and are known to decrease UBQLN2 solubility, promoting cytoplasmic aggregation and interfering with its normal function in protein quality control (Sharkey et al., 2018; Dao et al., 2018). These mutations may also perturb UBQLN2 phase separation dynamics, further contributing to disease pathology. Notably, UBQLN2-positive aggregates are not restricted to genetically linked ALS cases—they are also frequently found in sALS and in ALS cases associated with C9ORF72 repeat expansions, which are typically characterized by TDP-43 inclusions (Brettschneider et al., 2012). These observations suggest that UBQLN2 dysfunction may contribute to ALS pathogenesis more broadly, even in the absence of direct mutations.

Rodent models overexpressing UBQLN2^ALS mutants recapitulate the formation of UBQLN2 inclusions seen in ALS/FTD patients, although behavioral and neurodegenerative phenotypes vary. Transgenic mice expressing the UBQLN2^P497H mutation exhibit mild cognitive deficits without overt neurodegeneration, while other models show more pronounced motor neuron disease and reduced survival (Gorrie et al., 2014). Interestingly, even overexpression of wild-type UBQLN2 can result in toxicity in flies and rodents, suggesting that precise regulation of UBQLN2 levels and interactions is crucial for neuronal health (Kim et al., 2018; Sharkey et al., 2020; Huang et al., 2016).

The mechanisms underlying UBQLN2-linked neurodegeneration remain incompletely understood but likely involve both LOF and GOF mechanisms driven by altered interactions and protein aggregation. To investigate this further, we conducted a quantitative proteomic screen comparing the interactomes of wild-type and ALS-associated mutant UBQLN2 in iPSC iMNs. We identified enhanced interactions of mutant UBQLN2 with PEG10—a retroelement-derived protein and established UBQLN2 degradation target (Black et al., 2023)—and with BAG6, a molecular chaperone involved in the triage of MLPs.

Our functional studies revealed that UBQLN2 and BAG6 cooperatively regulate PEG10 levels, and that BAG6 preferentially associates with aggregation prone UBQLN2, acting as a holdase chaperone. Knockdown of BAG6 exacerbated UBQLN2 aggregation under proteotoxic conditions, while BAG6 overexpression suppressed UBQLN2 accumulation and toxicity. These findings establish BAG6 as a key modifier of UBQLN2 proteostasis and highlight the role of chaperone networks in buffering against UBQLN2-driven neurodegeneration.

Materials and methods

iPSC culture and iMN differentiation

A control human iPSC line (WC031i-5907-6; derived from neonatal male fibroblasts) was obtained from the WiCell Research Institute (Yin et al., 2019). ALS-associated mutations were introduced into this line using CRISPR/Cas9, generating the following genotypes: P497H, 4XALS (P497H, P506T, P509S, P525S), and I498X (a 1-nt deletion at codon 497 causing a frameshift and premature stop at codon 498). All iPSC lines exhibited normal karyotypes, expressed pluripotency markers, demonstrated trilineage differentiation capacity, and were confirmed to be mycoplasma-free (Yin et al., 2019; Kim et al., 2023). Short tandem repeat (STR) analysis confirmed the identity, clonality, and purity of each line (95–98% confidence across 15 loci).

iPSCs were cultured on Matrigel-coated plates (Corning, 47,743–720) in mTeSR1 Plus medium (STEMCELL Technologies,100–0276) and passaged every 3–4 days at a 1:3 to 1:6 split ratio using 0.5 mM EDTA and maintained at 37 °C in a humidified incubator with 5% CO₂. For the heat stress and recovery test, cells were incubated at 42 °C in 5% CO₂ for 2 h (heat stress), followed by incubation at 37 °C in 5% CO₂ for 2 h (recovery).

Differentiation into iMNs was performed as previously described (Kim et al., 2023; Du et al., 2015). Briefly, iPSCs were dissociated and plated on Matrigel-coated plates. The following day, medium was replaced with a 1:1 mixture of DMEM/F12 and Neurobasal medium supplemented with 0.5 × N2, 0.5 × B27, and 1 × GlutaMAX (all from Invitrogen). Small molecules were added to the medium: CHIR99021 (3 μM; Tocris), DMH-1 (2 μM; Tocris), and SB431542 (2 μM; Stemgent). Medium was refreshed every other day. After 7 days, cells differentiated into neuroepithelial progenitors (NEPs). NEPs were dissociated using 1 mg/mL Dispase and replated at a 1:6 ratio in the same neural differentiation medium supplemented with retinoic acid (RA; 0.1 μM; Stemgent), purmorphamine (0.5 μM; Stemgent), CHIR99021 (1 μM), DMH-1 (2 μM), and SB431542 (2 μM). After 7 days, cells differentiated into OLIG2⁺ motor neuron progenitors (MNPs). To initiate motor neuron differentiation, OLIG2⁺ MNPs were dissociated using 0.5 mM EDTA and cultured in suspension in neural differentiation medium supplemented with RA (0.1 μM) and purmorphamine (0.1 μM). After 6 days, cells differentiated into HB9⁺ neural motor precursors (NMPs). HB9⁺ NMPs were dissociated using Accutase (Invitrogen) and plated on Matrigel-coated plates. Cells were cultured in neural differentiation medium containing RA (0.1 μM), purmorphamine (0.1 μM), and Compound E (0.1 μM; Millipore) for 6 days to promote maturation into CHAT⁺ iMNs.

UBQLN2 purification and mass spectrometry (MS)

To collect UBQLN2 interacting proteins, iPSCs or iMNs resuspended in a lysis buffer containing 20 mM Tris–HCl (pH 8.0), 138 mM NaCl, 10 mM KCl, 1 mM MgCl₂, 1 mM EDTA, and 1% CHAPS (vol/vol), supplemented with 20 mM NaF, 20 mM β-glycerophosphate, and protease inhibitor cocktail. The cells were incubated on ice for 10 min, then centrifuged at 15,000 × g for 10 min at 4 °C to separate soluble and insoluble fractions. The soluble fractions were immunoprecipitated with UBQLN2 antibodies with Dynabeads Protein G beads. The co-immunoprecipitated proteins were eluted with elution buffer (0.2 M Glycine, pH 2.5) and neutralized with 1 M Tris–HCl, pH 9.0. Prior to tryptic digestion, dithiothreitol (DTT) was added to a final concentration of 10 mM to reduce disulfide bonds.

UBQLN2 immunoprecipitates (IPs) were subjected to tryptic digestion using the filter aided sample preparation (FASP) method (Wisniewski et al., 2009) and HPLC-ESI-MS/MS analysis using an Orbitrap mass spectrometer. The tryptic digest solution was desalted/concentrated using an Omix 100 μL (80 μg capacity) C18 tip. The solution was pipetted over the C18 bed 5 times, and rinsed 3 times with H₂O, 0.1% trifluoroacetic acid (TFA) to desalt. The peptides were eluted from the C18 resin into 150 μL 70% acetonitrile, 0.1% TFA and lyophilized. The peptides were re-suspended in 95:5 H₂O:acetonitrile, 0.2% formic acid and analyzed in duplicate as described below.

Peptides from the iPSC IPs were separated and analyzed using a UPLC-ESI-MS/MS system consisting of a NanoAcquity ultra-high-pressure liquid chromatography system (Waters) and an Orbitrap Q Exactive HF mass spectrometer (Thermo Fisher Scientific). UPLC separation employed an in-house constructed 100 × 365 μm fused silica capillary micro-column packed with 20 cm of 1.7 μm-diameter, 130 Angstrom pore size, C18 beads (Waters BEH), with an emitter tip pulled to approximately 1 μm using a laser puller (Sutter instruments). Peptides were loaded in buffer A (H₂O, 0.2% formic acid) at a flow-rate of 400 nL/min for 30 min and eluted over 120 min at a flow-rate of 300 nL/min with a gradient of 5 to 35% acetonitrile, in 0.1% formic acid. The nano-column was held at 60 °C using a column heater (in-house constructed). The nanospray source voltage was set to 2,200 V. Full-mass profile scans were performed in the orbitrap between 375–1,500 m/z at a resolution of 120,000, followed by MS/MS HCD scans of the 10 highest intensity parent ions at 30% relative collision energy and 15,000 resolution, with a 2.5 m/z isolation window and a mass range starting at 100 m/z. Charge states 2–6 were included and dynamic exclusion was enabled with a repeat count of one over a duration of 15 s.

Peptides from the iMN IPs were separated and analyzed using a UPLC-ESI-MS/MS system consisting of an Easy-nLC 1,200 ultra-high-pressure liquid chromatography system and an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). UPLC separation employed an in-house constructed 100 × 365 μm fused silica capillary micro-column packed with 20 cm of 1.7 μm-diameter, 130 Angstrom pore size, C18 beads (Waters BEH), with an emitter tip pulled to approximately 1 μm using a laser puller (Sutter instruments). Peptides were loaded in buffer A (H₂O, 0.2% formic acid) at a pressure of 300 Bar and eluted over 120 min at a flow rate of 350 nL/min with the following gradient established by buffer A (H₂O, 0.2% formic acid) and buffer B (80% acetonitrile (ACN), 0.2% formic acid): Time/T = 1 min, 5% buffer B; T = 52 min, 30% buffer B; T = 80 min, 42% buffer B; T = 90 min, 55% ACN; T = 95 to 100 min, 85% buffer B; T = 101 to 120 min, equilibrate at 0% buffer B. The nano-column was held at 60 °C using a column heater (in-house constructed). The nanospray source voltage was set to 2,200 V. Full-mass profile scans were performed in the orbitrap between 375 and 1,500 m/z at a resolution of 120,000, followed by MS/MS HCD scans in the orbitrap of the highest intensity parent ions in a 3 s cycle time at 30% relative collision energy and 15,000 resolution, with a 2.5 m/z isolation window. Charge states 2–6 were included and dynamic exclusion was enabled with a repeat count of one over a duration of 30 s and a 10 ppm exclusion width both low and high. The AGC target was set to “standard,” maximum inject time was set to “auto,” and 1 μscan was collected for the MS/MS orbitrap HCD scans.

Bioinformatic analyses of mass spectrometric data

The MetaMorpheus software program (version 1.05) was used to identify peptides and proteins in the samples (Shortreed et al., 2015; Solntsev et al., 2018). Protein fold changes were quantified with FlashLFQ with match between runs enabled (Millikin et al., 2018; Millikin et al., 2020). The UniProt, reviewed human database (downloaded on 04/03/2024 20:34:31) as well as the MetaMorpheus contaminant database was used for calibration, global post translational modification discovery (G-PTM-D), and search. Default parameters were used including a maximum of 2 missed cleavages and minimum peptide length of 7 amino acids for a tryptic digest. Carbamidomethylation of cysteine was set as a fixed modification while oxidation of methionine was variable with 2 maximum modifications per peptide. Differential expression analysis of the quantified protein groups was performed in Perseus (version 1.6.15.0) (Tyanova et al., 2016). Contaminant and decoy proteins were filtered out as well as protein groups above a protein Q-value threshold of 0.01 for the iPSC samples and 0.05 for the iMN samples. The quantified intensities were log2 transformed and normalized by Z-score for each column. A minimum of 2 valid values in the iPSC and 3 valid values in the iMN samples were required. Imputation was performed for the iPSC samples, but not the iMN samples due to having 5 biological replicates and two technical replicates for the motor neuron data compared to only 3 biological replicates in the iPSC data. Imputation involved replacing missing values from a normal distribution with the default Perseus parameters. Two sample student’s t-tests were performed with a p-value cutoff of 0.05 for significance.

Antibodies

The following primary antibodies were used for immunoblotting and immunoprecipitation: anti-UBQLN2 (ab190283, Abcam; 85509S, Cell Signaling Technology), anti-BAG6 (sc-365928, Santa Cruz Biotechnology), anti-PEG10 (14412-1-AP, Proteintech), and anti-β-Actin (4967S, Cell Signaling Technology). The following secondary antibodies were used for immunoblotting: IRDye 680RD goat anti-mouse (926–68,070) and IRDye 800CW goat anti-rabbit (926–32,211), both from LI-COR.

BAG6 knockdowns

The shRNA lentiviral vector targeting BAG6 was purchased from Sigma (TRCN0000007353). 293FT (Invitrogen, R70007) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 1% Penicillin–Streptomycin (Corning, 30-002-CI) and maintained at 37 °C in a humidified incubator with 5% CO₂. Lentiviral particles were produced by transient transfection of 293FT cells with shRNA vector, psPAX2 (Addgene, plasmid #12260) and pCMV-VSV-G (Addgene, plasmid #8454) in a ratio of 6:4:1 as described (Jia et al., 2021; Mastrocola et al., 2013). Viral supernatants harvested at 24 h and 48 h post-transfection were incubated with iPSCs for 24 h followed by selection in media containing 2 μg/mL puromycin for 72 h.

Protein extraction, cell fractionation and immunoblotting

For subcellular fractionation, cells were resuspended in a lysis buffer containing 20 mM Tris–HCl (pH 8.0), 138 mM NaCl, 10 mM KCl, 1 mM MgCl₂, 1 mM EDTA, and 1% CHAPS (vol/vol), supplemented with 20 mM NaF, 20 mM β-glycerophosphate, and protease inhibitor cocktail (Sigma, P8340-5 mL). The cells were incubated on ice for 10 min, then centrifuged at 15,000 × g for 10 min at 4 °C to separate soluble and insoluble fractions. Western blot images were acquired using the Odyssey XF imaging system (LI-COR Biosciences) and analyzed with ImageStudio software (v5.2, LI-COR Biosciences).

Proximity-ligation assay (PLA)

iPSCs expressing UBQLN2^WT, UBQLN2^P497H, UBQLN2^4XALS, or UBQLN2^I498X were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2% Triton X-100 in PBS. The cells were incubated with primary antibodies (α-UBQLN2, 85509S, CST, 1:500; α-BAG6, sc-365928, SC, 1:500) overnight. Duolink® Proximity Ligation Assay (PLA) kits (Sigma) were used according to the manufacturer’s instructions. Briefly, PLA probes were incubated for 1 h, followed by 30 min of ligation and 100 min of signal amplification. Images were acquired using a Nikon AXR confocal microscope using a 60 × oil lens. PLA puncta were quantified using Fiji (ImageJ) automatic particle analysis. Puncta counts were normalized to the number of DAPI-stained nuclei to calculate PLA puncta per cell.

Statistical analysis

Statistical analysis information including individual biological and technical replicates number, mean or median, and error bars are explained in the figure legends. Statistical tests were performed in Prism (v10, GraphPad). The tests performed and the resulting p values are listed in the figure legends.

Results

UBQLN2^ALS mutations alter protein–protein interaction networks in iPSCs

To explore how ALS-associated UBQLN2 mutations influence UBQLN2 function through altered protein interactions, we performed label-free quantitative MS on immunoprecipitated UBQLN2 complexes from human iPSCs. We compared wild-type UBQLN2 (UBQLN2^WT) to three ALS-associated UBQLN2 alleles: a common clinical point mutation (UBQLN2^P497H), a 4XALS mutant carrying four clinical mutations (UBQLN2^4XALS: P497H, P506T, P509S, P525S) previously shown to exhibit increased aggregation and toxicity (Kim et al., 2018; Kim et al., 2023), and a truncation mutant (UBQLN2^I498X) that is undetectable by Western blotting due to its instability (Kim et al., 2018; Kim et al., 2023).

When comparing UBQLN2^WT with UBQLN2^I498X, we identified 24 proteins that were significantly enriched in UBQLN2^WT IPs (Figures 1A,B). As expected, UBQLN2 was the most enriched interactor (~6-fold). Additional interactors included UBQLN1, the ferroptosis-related enzymes VKORC1L1 and VKORC1, and the HSP70 family member HYOU1 (also known as GRP170), a chaperone upregulated by hypoxia. The presence of these interactors is consistent with prior reports of ubiquilin association with chaperone and redox-regulatory proteins (Yang et al., 2023; Hjerpe et al., 2016).

Figure 1

We next examined differences in interactome profiles across ALS-associated UBQLN2 alleles. Compared to UBQLN2^WT, UBQLN2^P497H showed differential enrichment of 67 proteins (30 enriched, 37 depleted), while UBQLN2^4XALS showed 59 differentially enriched proteins, of which 53 were uniquely enriched in the UBQLN2^4XALS background (Figures 1C,D). Notably, only four proteins overlapped between the UBQLN2^P497H and UBQLN2^4XALS datasets, suggesting that the 4XALS mutation elicits broader and more distinct interaction changes. Among the most strongly enriched interactors in the 4XALS IPs was PEG10, a retroelement-derived protein previously identified as a UBQLN2 degradation target with important roles in placental biology (Black et al., 2023; Abed et al., 2019; Pandya et al., 2021). Also enriched was ANK2, a cytoskeletal scaffolding protein. Interestingly, the redox regulator and apoptosis suppressor VKORC1L1 (Yang et al., 2023) was significantly depleted in UBQLN2^4XALS IPs compared to all other UBQLN2 variants, indicating a potential LOF interaction specific to the 4XALS mutant. These findings suggest that while UBQLN2^4XALS gains aberrant associations (e.g., PEG10, ANK2), it simultaneously loses physiologic interactions such as with VKORC1L1.

To determine whether proteotoxic stress further modulates the UBQLN2 interactome, we repeated the UBQLN2 IP-MS analysis after exposing UBQLN2^WT, UBQLN2^P497H, UBQLN2^4XALS and UBQLN2^I498X iPSCs to heat stress (HS, 42 °C, 1 h). In UBQLN2^WT IPs, 42 proteins exhibited significant interaction changes post-HS (10 enriched, 32 depleted), reflecting a stress-induced remodeling of the interactome (Figures 1E–G). Similarly, UBQLN2^4XALS displayed heat stress-responsive changes in 36 proteins relative to UBQLN2^WT (Figures 1G–I). Gene Ontology (GO) enrichment of proteins differentially interacting with UBQLN2^WT and UBQLN2^4XALS revealed context-dependent effects across cell types and conditions. In iPSCs at 37 °C, enriched terms included structural molecular activity, RNA binding, and actin filament binding, suggesting early disruptions in cytoskeletal organization and RNA metabolism (Supplementary Figure S1). Under heat stress (42 °C), proteins involved in sequence-specific mRNA binding and extracellular exosomes were enriched, indicating stress-induced exacerbation of RNA regulatory defects and altered intercellular communication (Supplementary Figure S2). In iMNs, enriched categories included proteasome core complex, spliceosomal complex, cytoplasmic ribonucleoprotein granules, postsynaptic cytoskeleton, and lysosomal lumen. These terms are consistent with impairment of protein homeostasis RNA splicing, and synaptic integrity in UBQLN2^4XALS neurons (Supplementary Figure S3). Collectively, UBQLN2^4XALS mutations perturb pathways critical for RNA metabolism, cytoskeletal organization, and protein quality control, with effects amplified under stress and in differentiated neurons.

Notably, PEG10 remained associated with UBQLN2^4XALS even after HS. Another significant finding was that HYOU1, which lost interaction with UBQLN2^WT after HS, gained association with UBQLN2^4XALS under HS, indicating altered recruitment of stress-responsive chaperones (Figures 1H,I). Additionally, BAG6—a ubiquitin-binding holdase chaperone involved in triage of mislocalized proteins (Chio et al., 2017; Payapilly and High, 2014; Cisneros-Aguirre et al., 2024)—was enriched in UBQLN2^4XALS versus UBQLN2^WT IPs. This finding raised the possibility that BAG6 chaperones aggregation prone UBQLN2.

BAG6 is a common interactor of UBQLN2^ALS mutants in iPSCs and iMNs

To determine whether UBQLN2 interaction changes extend to neurons, we performed analogous UBQLN2 IP-MS analyses using iMNs expressing wild-type and ALS-mutant UBQLN2 alleles. Pairwise comparisons revealed that 21 proteins were differentially associated between UBQLN2^WT and UBQLN2^P497H, while 28 proteins showed differential enrichment between UBQLN2^WT and UBQLN2^4XALS (Figure 2A). Notably, the vast majority of these interactions were enhanced in UBQLN2^4XALS IPs, suggesting that, as was seen in iPSCs, the UBQLN2^4XALS mutant acquires aberrant protein associations in iMNs. Similar to what was observed in heat-stressed iPSCs, BAG6 was enriched in UBQLN2^4XALS IPs prepared from iMNs, suggesting it is a common ALS-linked interactor (Figures 2B,C). Other proteins enriched in UBQLN2^4XALS IPs included BTBD2, which encodes a Ub E3 ligase involved in cytoskeletal dynamics and Top1-mediated chromatin relaxation (Xu et al., 2003; Xu et al., 2002; Bagattin et al., 2024), and phospholipase B D2 (PLBD2), a lysosomal phospholipase that has been implicated in the lysosomal storage disorder, Batten’s Disease (Nyame et al., 2024). Conversely, several proteins showed reduced association with UBQLN2^4XALS, including FAM168A/TCRP1, implicated in Polβ stabilization and base excision repair (Liu et al., 2015) and BAX, a pro-apoptotic factor involved in mitochondrial stress responses (Walensky and Gavathiotis, 2011) that was deenriched in UBQLN2^P497H and UBQLN2^4XALS IPs relative to wild-type, suggesting potential LOF interactions shared across ALS alleles. PEG10, which was strongly enriched in UBQLN2^4XALS IPs in iPSCs, was not detected in iMN IPs, likely reflecting its low expression level in this cell type. Altogether, these results demonstrate that ALS-associated UBQLN2 mutations—particularly the aggregation-prone 4XALS allele—remodel the UBQLN2 interactome in a cell type- and stress-dependent manner. This remodeling involves both aberrant gains of interaction with stress-responsive proteins (e.g., PEG10, BAG6, HYOU1) and loss of interactions (e.g., VKORC1L1, BAX), pointing to a complex interplay of gain- and loss-of-function mechanisms in UBQLN2-linked ALS.

Figure 2

Given the consistent enrichment of BAG6 in UBQLN2^4XALS IP from both iPSCs and iMNs, and its role as an important regulator of proteostasis (Hegde and Zavodszky, 2019) (Figures 1G, 2C), we investigated this interaction more closely. BAG6 is a multifunctional holdase chaperone best known for its role in the guided entry of tail-anchored (GET) protein pathway, where it facilitates the insertion of tail-anchored (TA) proteins into the endoplasmic reticulum (ER), nuclear, and mitochondrial membranes (Chio et al., 2017; Rodrigo-Brenni et al., 2014; Hessa et al., 2011). In this context, BAG6 forms a cytosolic complex with GET4 (TRC35) and UBL4A to mediate the transfer of tail-anchored (TA) proteins from the ribosome-associated co-chaperone SGTA to TRC40, which then delivers them to the WRB–CAML membrane receptor complex. Outside the canonical GET pathway, BAG6 also plays an important role in triaging MLPs by recognizing exposed hydrophobic regions and directing aberrant proteins for degradation (Shan, 2019).

To validate these MS findings, we performed co-IP followed by Western blotting in UBQLN2 iPSCs. These experiments confirmed that UBQLN2^4XALS exhibits a markedly stronger interaction with BAG6 compared to UBQLN2^WT (Figure 3A). To further confirm this association in situ, we employed a proximity ligation assay (PLA), which revealed a significantly increased UBQLN2–BAG6 proximity (<40 nm) in UBQLN2^4XALS cells compared with UBQLN2^WT or UBQLN2^P497H cells (Figures 3B,C). These data support BAG6 as a physiologically relevant interactor of ALS-associated UBQLN2 mutants.

Figure 3

Effect of BAG6 silencing on UBQLN2 solubility

We next investigated whether BAG6 modulates UBQLN2 proteostasis by assessing protein solubility under HS. iPSCs expressing UBQLN2^WT, UBQLN2^P497H, or UBQLN2^4XALS, along with either control or BAG6-targeting shRNAs, were subjected to a 42 °C HS for 2 h followed by a 2 h recovery period. Under basal conditions, the solubility of all three UBQLN2 variants was comparable (Figures 4A,B). Following HS, UBQLN2^WT and UBQLN2^P497H became less soluble, possibly reflecting their association with insoluble proteins, but their solubility partially recovered after 2 h. In contrast, UBQLN2^4XALS exhibited a more pronounced loss of solubility that failed to recover during the post-stress period (Figures 4A,B). These results indicate that the UBQLN2^4XALS mutant is more vulnerable to proteotoxic stress and has a diminished capacity to regain solubility. BAG6 knockdown impaired the solubility recovery of UBQLN2^WT and UBQLN2^P497H following HS but did further reduce the severe solubility defect of UBQLN2^4XALS (Figures 4A–C). There was also a trend toward increased UBQLN2 expression in shBAG6 cells, however, the differences were not statistically significant (Figure 4C). Taken together, these findings suggest that BAG6 suppresses UBQLN2 misfolding under conditions of HS, but that its endogenous activity is insufficient to fully rescue the HS-induced aggregation of the UBQLN2^4XALS mutant.

Figure 4

UBQLN2 and BAG6 coregulate PEG10 degradation

An enhanced interaction between PEG10 and UBQLN2^4XALS relative to UBQLN2^WT was also observed in iPSC MS experiments (Figures 1D,G,I). PEG10 is a domesticated retroviral gene that undergoes programmed −1 ribosomal frameshifting after codon 319 to produce two functionally distinct isoforms (Figure 5A) (Clark et al., 2007). PEG10-reading frame 1 (RF1) encodes a ~ 50 kDa protein containing Zn finger, Gag, and Pol domains while PEG10-RF1/2 produces a ~ 100 kDa protein notable for the additional presence of a proline-rich region and aspartyl protease domain (Clark et al., 2007). Interestingly, BAG6 was previously found to associate with PEG10, raising the possibility that BAG6, UBQLN2, and PEG10 form a ternary complex (Abed et al., 2019). To investigate this hypothesis, we measured PEG10-UBQLN2 interactions in UBQLN2^WT, UBQLN2^P497H, and UBQLN2^4XALS iPSCs in the absence or presence of BAG6 knockdown. The amount of PEG10 that coimmunoprecipitated with UBQLN2 did not significantly differ between UBQLN2^WT, UBQLN2^P497H, and UBQLN2^4XALS iPSCs expressing a control shRNA (shControl), though there was a trend for higher co-IP of PEG10 with UBQLN2^P497H and UBQLN2^4XALS (Figures 5A,B). BAG6 knockdown caused a significant increase in the level of PEG10 that coimmunoprecipitated with UBQLN2^4XALS when compared to UBQLN2^4XALS:shControl, UBQLN2^WT:shControl, or UBQLN2^WT:shBAG6 iPSCs. The co-IP between UBQLN2^P497H and PEG10 also trended higher in shBAG6 cells, but was not significantly different from UBQLN2^WT iPSCs expressing control or BAG6 shRNA (Figures 5A,B).

Figure 5

Reciprocal coimmunoprecipitation (co-IP)-WB experiments revealed that levels of immunoprecipitated PEG10 were comparable between UBQLN2^WT, UBQLN2^P497H, and UBQLN2^4XALS iPSCs but higher in UBQLN2^I498X iPSCs, which is consistent with higher expression of PEG10 in UBQLN2-deficient cell lines (Figures 5C,E) (Black et al., 2023; Whiteley et al., 2021). Similarly, BAG6 knockdown increased PEG10 immunoprecipitation across all UBQLN2 genotypes, with progressively higher levels seen in UBQLN2^WT, UBQLN2^P497H, UBQLN2^4XALS and UBQLN2^I498X iPSCs (Figures 5C,D). Levels of coimmunoprecipitated UBQLN2 were significantly higher in PEG10 IPs prepared from UBQLN2^4XALS:shBAG6 iPSCs versus UBQLN2^4XALS:shControl iPSCs. The co-IP of UBQLN2^WT with PEG10 antibodies was not detected either in the absence or presence of BAG6 knockdown while co-IP of UBQLN2^P497H was only detectable in UBQLN2^P497H: shBAG6 iPSCs (Figures 5C,D). PEG10 expression levels were slightly increased in UBQLN2^P497H:shBAG6 and UBQLN2^4XALS:shBAG6 iPSCs relative to UBQLN2^WT:shBAG6 iPSCs, which may contribute to the enhanced UBQLN2-PEG10 co-IP seen in these lines.

We also noted that the relative abundance of PEG10 isoforms—RF1 (short) and RF1/2 (long)—was differentially affected by UBQLN2 and BAG6 status. UBQLN2^I498X iPSCs exhibited a selective increase in the longer RF1/2 isoform, resulting in an increased RF1/2 to RF1 ratio while shBAG6 cells exhibited increases in both RF1 and RF1/2 species and an unchanged RF1/2 to RF1 ratio (Figure 5E). The isoform shift to RF1/2 persisted in cells with both UBQLN2 deletion and BAG6 knockdown despite the overall increase in total PEG10 levels (Figures 5A,B). Across all genotypes, PEG10 mRNA levels remained unchanged (Supplementary Figure S4). These results indicate that pan-PEG10 abundance is primarily controlled by BAG6, while UBQLN2 more specifically regulates PEG10 RF1/2 abundance.

Reduced PEG10 degradation rate in UBQLN2^4XALS iPSCs

UBQLN2^P497H and UBQLN2^4XALS alleles showed increased interaction with PEG10 in the setting of BAG6 deficiency, and this effect was most pronounced for UBQLN2^4XALS (Figure 5C). While steady state levels of PEG10 were not as upregulated in UBQLN2^4XALS:shBAG6 iPSCs as they were in UBQLN2^I498X:shBAG6 iPSCs, this finding raised the possibility that UBQLN2^4XALS is defective for PEG10 degradation. To more closely examine this possibility, we measured the degradation rate of PEG10 in UBQLN2^WT, UBQLN2^P497H, UBQLN2^4XALS and UBQLN2^I498X iPSCs treated with the protein synthesis inhibitor, cycloheximide (CHX). The levels of PEG10-RF1/2 and PEG10-RF1 decreased with similar kinetics in CHX-treated UBQLN2^WT (Figure 6A) and UBQLN2^P497H (Figure 6B) iPSCs but failed to significantly decrease in UBQLN2^I498X (Figure 6D). UBQLN2^4XALS (Figure 6C) iPSCs exhibited a PEG10 degradation rate intermediate between UBQLN2^WT and UBQLN2^I498X while BAG6 knockdown increased steady state levels of PEG10 across all UBQLN2 genotypes but did/did not affect PEG10 degradation rate (Figure 6). These finding suggest that UBQLN2^4XALS is a partial LOF allele with respect to PEG10 degradation.

Figure 6

HS-dependent degradation of PEG10 occurs independent of UBQLN2 and BAG6

Given that BAG6 silencing increased PEG10 levels and diminished UBQLN2 solubility following HS, we examined whether UBQLN2 mutations or BAG6 expression influenced PEG10 expression and solubility under HS conditions. Levels of PEG10-RF1 and PEG10-RF1/2 declined in UBQLN2^WT, UBQLN2^P497H, and UBQLN2^4XALS iPSCs following a 42 °C, 2 h HS, with little change in PEG10 solubility (Figures 7A,B). PEG10 expression recovered to comparable levels in UBQLN2^WT and UBQLN2^4XALS iPSC within 2 h after retransfer to 37 °C, suggesting active resynthesis and/or stabilization (Figures 7C,D). Similarly, while BAG6 knockdown increased steady-state PEG10 levels, particularly in UBQLN2^4XALS iPSCs, it did not affect HS-dependent PEG10 degradation or post-HS recovery (Figures 7C,D). HS-dependent degradation of PEG10 also occurred in UBQLN2^I498X iPSCs, suggesting that neither ALS-associated mutations nor UBQLN2 functional deficiency significantly affect HS-dependent PEG10 degradation (Figures 7E,F). Thus, HS-dependent PEG10 turnover occurs through a UBQLN2 and BAG6-independent mechanism.

Figure 7

Discussion

UBQLN2 functions at the intersection of protein quality control and degradation, shuttling ubiquitinated substrates to the proteasome and engaging misfolded proteins via stress-responsive chaperone networks (Matthews and Whiteley, 2025). Here, we demonstrated that ALS-associated mutations in the UBQLN2 PRR exert GOF and LOF effects on protein–protein interactions in both iPSCs and iMNs. The gain and loss of relevant protein–protein interactions may contribute to cellular defects that trigger neurodegeneration in ALS/FTD-UBQLN2.

We previously reported that the combinatorial UBQLN2^4XALS allele exhibited heightened misfolding and toxicity profiles versus UBQLN2^ALS clinical alleles, making this allele a valuable tool for probing UBQLN2 pathomechanisms (Kim et al., 2018; Kim et al., 2023). Consistent with its more severe folding defect, UBQLN2^4XALS mutant showed pronounced changes in its interactome, with a propensity toward gained interactions that were highlighted by increased binding to BAG6, a holdase, and PEG10, a previously described degradation target of UBQLN2 (Black et al., 2023; Whiteley et al., 2021).

The enhanced interaction between BAG6 and UBQLN2^4XALS is most consistent with a role for BAG6 in the triage of aggregation prone UBQLN2. BAG6 plays an important role in triaging MLPs by binding to exposed hydrophobic regions, thereby reducing the rate of aberrant intra- and intermolecular interactions that would lead to protein aggregation (Shan, 2019). We speculate that ALS-associated mutations in the PRR cause exposure of hydrophobic regions, including the Met-rich, central region, leading to recognition by BAG6, with the more severely aggregation prone UBQLN2^4XALS allele, eliciting greater BAG6 engagement. Functional importance of the BAG6-UBQLN2 interaction was suggested by the finding that BAG6 knockdown decreased the solubility of both UBQLN2^WT and UBQLN2^P497H proteins following recovery of iPSCs from HS (Figures 4A,B). Ironically, despite increased binding, BAG6 knockdown did not exacerbate the post-HS insolubility phenotype of UBQLN2^4XALS, suggesting that its insolubility phenotype is saturated (Figures 4A,B). We also noted that UBQLN2 protein levels were slightly upregulated in shBAG6 iPSCs, suggesting that a fraction of BAG6-bound UBQLN2 is destined for degradation, as seen for other BAG6 clients (Rodrigo-Brenni et al., 2014). Because BAG6 also suppressed aggregation of C-terminal fragments of TDP-43 (Kasu et al., 2022), it may be of broad relevance to understanding pathologic protein misfolding in ALS/FTD.

A second finding of this study is that UBQLN2^ALS mutants exhibited increased interaction with PEG10, a degradation target of UBQLN2 (Black et al., 2023; Abed et al., 2019; Pandya et al., 2021; Whiteley et al., 2021). Although, the biochemical basis for the enhanced association between UBQLN2^ALS mutants and PEG10 is not clear, it may involve stabilized interactions between ubiquitylated PEG10 and the UBQLN2 UBA domain and/or aberrant association of PEG10 with exposed hydrophobic patches that are also engaged by BAG6. The fact that knockdown of BAG6 potentiated the co-IP of PEG10 with UBQLN2^P497H and UBQLN2^4XALS, but not UBQLN2^WT (Figures 5A–C), is consistent with the idea that aggregation prone UBQLN2 species form stabilized complexes with PEG10. However, because BAG6 silencing marginally increased the levels of UBQLN2 and significantly increased PEG10 expression (Figures 5A,C, 7C,D), the increased co-IP stoichiometry between PEG10 and UBQLN2^ALS mutants in BAG6 knockdown cells may be partially attributed to increased input of the target proteins. The enhanced co-IP between UBQLN2 and PEG10 in shBAG6 cells also ruled out that BAG6 bridged the PEG10-UBQLN2 complex.

A previous study identified BAG6 as a PEG10-associated protein (Abed et al., 2019). Our findings extend this observation to show that BAG6 inhibits PEG10 expression (Figure 5). However, while both UBQLN2 and BAG6 suppressed PEG10 protein levels; they appear to do so independently and via different mechanisms. Interestingly, UBQLN2 LOF selectively stabilized the RF1/2 PEG10 translation product (Figures 5C,E, 6D) (Black et al., 2023), while BAG6 knockdown stabilized both RF1 and RF1/2 PEG10 species (Figures 5C, 6). One speculative possibility is that BAG6 regulates PEG10 stability through recognition elements in the PEG10 RF1 while UBQLN2 controls RF1/2 stability by binding to elements unique to RF1/2, including the Pro-rich region and peptidase domains. It is also possible that UBQLN2 inhibits the −1 frame shift required to generate PEG10 RF1/2. Finally, the existence of a third PEG10 degradation pathway is suggested by the fact that neither UBQLN2 nor BAG6 LOF affected HS-dependent PEG10 degradation (Figures 7C,D). Further studies are needed to discern unique and shared functions of UBQLN2 and BAG6 in controlling PEG10 isoform abundance.

Prior studies reported variable effects of UBQLN2^ALS mutants on PEG10 stability (Black et al., 2023). Steady-state expression and half-life of PEG10 were marginally increased in UBQLN2^4XALS iPSCs and unchanged in UBQLN2^P497H iPSCs relative to UBQLN2^WT controls (Figures 6A–C). By contrast, the UBQLN2^I498X mutation had a strong stabilizing effect on PEG10 (Figure 6D), which is consistent with previous studies (Black et al., 2023; Whiteley et al., 2021). From this, we surmise that the ALS mutations have a modest, quantitative impact on PEG10 stability that correlated with the strength of UBQLN2-PEG10 interaction in co-IP assays. Our findings further indicate that the effects of ALS mutations—and the 4XALS mutation in particular—on PEG10 proteostasis are exacerbated by BAG6 functional insufficiency. This finding may hold relevance to understanding how age-dependent declines in proteostasis synergize with disease mutations to instigate neurodegeneration in ALS/FTD and other NDs (Labbadia and Morimoto, 2015).

The identification of BAG6 is an associated factor of UBQLN2^ALS mutants suggest a broader role for BAG6-mediated proteostasis in the suppression of neurodegeneration. Consistent with this, BAG suppressed the aggregation of TDP-43 fragments in cell culture (Kasu et al., 2022). Interestingly, the UBL4A subunit of the BAG6-TRC35-UBL4A complex was not detected as a TDP-43 interacting factor, by Kasu et al., nor was it identified in our UBQLN2^ALS MS studies, suggesting that BAG6 functions outside the canonical BAG6-TRC35-UBL4A complex to mediate triage of these neurodegeneration-associated proteins. BAG6 was also implicated in the binding and degradation of ALS-associated C9ORF72 when its obligatory subunit SMCR8 was depleted (Julg et al., 2023). Given the propensity of BAG6 to bind hydrophobic surfaces that are commonly exposed in misfolded, neurodegeneration-associated proteins, BAG6 may be of interest as a suppressor of neurodegenerative proteinopathies (Hegde and Zavodszky, 2019; Munch and Bertolotti, 2010; Casson et al., 2016; Muchowski, 2002).

Although our validation studies were primarily focused on BAG6 and PEG10, other proteins showing differential association with UBQLN2^ALS mutants may be relevant to understanding the UBQLN2 pathomechanism. In iPSCs, the ER resident HSP70 family member HYOU1 showed reduced association with UBQLN2^ALS mutants in iPSCs the absence of HS and increased association with UBQLN2^ALS mutants following HS (Figures 1D,I). Several prior studies have reported that ALS-associated mutations in UBQLN2 reduce its association with canonical HSP70 (Hjerpe et al., 2016; Zhang et al., 2021; Teyssou et al., 2017; Gavriilidis et al., 2018). Enhanced association between UBQLN2^4XALS and the Ub E3 ligase BTBD2 in iMNs may reflect a role for BTBD2 in UBQLN2 turnover while reduced association of UBQLN2^4XALS with FAM168A may impact FAM168A-dependent DNA Polβ stabilization and base-excision repair (BER) critical to neuronal integrity (Liu et al., 2015). Further studies are needed to define which altered protein–protein interactions are most central to the UBQLN2 pathomechanism in neurons.

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