Bernaleau et al. (https://doi.org/10.1084/jem.20240825) show that CCDC134 located in the ER is required for TLR biogenesis by controlling the N-glycosylation, folding, and stabilization of the ER chaperone Gp96.
TLRs are key innate immune sensors whose localization to the plasma membrane or endosomal compartments facilitates cellular recognition of distal and proximal threats of microbial or host origin. Tight regulation of TLR functions is essential, with failure promoting inflammatory and autoimmune disorders. One critical step of TLR regulation occurs in the ER, where chaperones aid protein folding, export from the ER, and sorting for directed transport to either the plasma membrane (TLR1, 2, 4–6, 10) or endosomal compartments (TLR3, 7–9, 11–13) (Kawai et al., 2024).
Antje Blumenthal and Leslie C. Domínguez Cadena.
Folding and subsequent trafficking of all TLRs, except TLR3, requires the ER-resident chaperone glycoprotein 96 (Gp96) (also known as Hsp90b1, Gpr94, Hspc4), assisted by the Gp96 co-chaperone canopy (CNPY) 3 (also known as protein associated with TLR4; PRAT4A) and/or CNPY4 (PRAT4B) in some cases (Kawai et al., 2024). The chaperone protein unc-93 homolog B1 (UNC93B1) promotes ER exit and trafficking of TLR5 and endosomal TLRs. UNC93B1 also directs post-Golgi sorting, proteolytic activation, as well as degradation of endosomal TLRs (Kawai et al., 2024). In addition to TLRs, Gp96 client proteins include TLR-related and accessory molecules (e.g., CD180/RP105 and MD-2), some integrins (e.g., CD18 and CD11a), and growth factors (e.g., insulin-like growth factors) (Weekes et al., 2012). In the absence of functional Gp96, client proteins are targeted for ER-associated degradation (ERAD).
Model of how ER-resident CCDC134 stabilizes the chaperone Gp96 for TLR biogenesis. During translation, a pseudosubstrate sequon in the pre-N segment of the Gp96 nascent chain interacts with the STTA3 subunit of the OST-A complex. This prevents N-glycosylation of downstream sequons in Gp96 and enables co-translational recruitment of CCDC134 to the nascent Gp96 chain and Gp96 protein folding. Functional Gp96, in complex with CCDC134, promotes folding of TLRs and other client proteins. The Gp96 co-chaperone, CNPY3, contributes to TLR folding. TLRs traffic from the ER to the Golgi complex, a process that requires UNC93B1 for endosomal TLRs and TLR5. Absence of CCDC134 results in Gp96 hyperglycosylation, which prevents Gp96 from folding into a functional protein and targets it for ERAD. In the absence of Gp96, its client proteins are not folded appropriately. (Figure integrates models proposed by Bernaleau et al. [2024] and Ma et al. [2024].)
In their recent study, Bernaleau et al. undertook a genome-wide loss-of-function screen for regulators of TLR7-mediated cellular activation and discovered coiled-coil domain containing 134 (CCDC134) as a high-confidence hit (Bernaleau et al., 2024). CCDC134 is poorly characterized but some described roles include the regulation of MAPK activation and T cell functions (Huang et al., 2014, 2008). Through a combination of genetic and biochemical approaches, Bernaleau et al. identified that CCDC134 is crucial for the processing, trafficking, proteolytic activation, and function of endosome- (TLR7–9) and plasma membrane–localized TLRs (TLR1, TLR2, and TLR4–6). Functional assays across multiple human and mouse cell lines confirmed that CCDC134 was essential for TLR-mediated intracellular signaling (IRF5, NF-κB, and MAPK) and cytokine production. Comparative studies established that the absence of CCDC134 phenocopied cellular deficiency in the TLR chaperones Gp96 and CNPY3. Thus, Bernaleau et al. identified CCDC134 as a central regulator of TLR biogenesis (Bernaleau et al., 2024).
As both Gp96 and CNPY3 exert their functions as TLR chaperones in the ER, it was pertinent to spatially position CCDC134 within cells. Thus far, CCDC134 has been known as a secreted and nuclear protein. In contrast, Bernaleau et al. identified that CCDC134 localizes to the ER via its N-terminal signal peptide and aided by a C-terminal domain ER retention motif (Bernaleau et al., 2024), findings independently corroborated by a recent study (Ma et al., 2024). Notably, ER-resident rather than secreted CCDC134 facilitated TLR responses, and transient CCDC134 ER localization was sufficient. Tackling the question of how CCDC134 regulates TLR processing and function, Bernaleau et al. showed interactions between CCDC134 and Gp96 through complementary co-immunoprecipitation and mass spectrometry analyses. CCDC134 interaction with Gp96 required the Gp96 middle domain (harbors catalytic activity for ATP hydrolysis) and C-terminal dimerization domain that binds client proteins, but not the N-terminal GTPase domain. As Gp96 did not interact with mutant CCDC134 lacking the signal peptide, the authors concluded that CCDC134 ER localization was required for interactions with Gp96 (Bernaleau et al., 2024).
To date, mechanisms known to regulate Gp96 functions include stress-induced increase in gene and protein expression as well as hyperglycosylation that disrupts folding of Gp96 and accelerates ERAD-mediated degradation (Dersh et al., 2014; Hoter et al., 2018). Bernaleau et al. showed that loss of CCDC134, but not CNYP3 or UNC93B1, reduced cellular Gp96 protein expression accompanied by increased Gp96 hyperglycosylation, with minimal effect on Gp96 mRNA expression. In contrast, CCD134 expression and function were not affected by loss of Gp96. Based on these observations, Bernaleau et al. concluded that CCDC134–Gp96 interactions in the ER prevented Gp96 hyperglycolsylation and degradation by the ERAD pathway, upstream of CNYP3 and UNC93B1. Stabilized functional Gp96 consequently acts as chaperone for TLR client proteins (Bernaleau et al., 2024) (see figure). These findings provided further context to the recent identification of CCDC134 requirements for TLR4 expression and cellular responses to bacterial lipopolysaccharide (Lampson et al., 2024).
More detailed insights into the potential molecular mechanisms underpinning CCDC134-mediated stabilization of Gp96 are offered by a recent study that independently identified that CCDC134 stabilized Gp96 (Ma et al., 2024). Previous work had shown that ablation of the STT3A subunit of the oligosaccharyltransferase complex A (OST-A) resulted in Gp96 hyperglycosylation at cryptic N-linked glycan acceptor sites (Cherepanova et al., 2019). The recent data by Ma et al. revealed that during Gp96 translation, a serine-arginine-threonine sequon in the pre–N-terminal segment of the Gp96 nascent chain, acted as an inhibitory pseudosubstrate for STTA3, partially inhibiting N-glycolsylation of downstream sequons in Gp96 (Ma et al., 2024). Anchoring STT3A through the pseudosubstrate facilitated co-translational recruitment of CCDC134 to the Gp96 nascent chain, preventing STTA3-mediated N-glycosylation at facultative sequons as Gp96 progressed through the Sec61 translocation channel into the ER lumen. Consequently, Gp96 was folded correctly, rendering its facultative sequons inaccessible to posttranslational N-glycosylation by ER-based OST-B. By showing that residues in STT3A critical for binding and transferring oligosaccharide chains to active asparagine side chains were not required for formation of the protective protein scaffold, the authors identified that OST-A exhibits scaffolding functions in addition to its OST activity. The protective protein scaffold is thought to be anchored to Sec61 via the OSTC subunit of OST-A, consistent with OSTC requirements for Gp96 folding and stabilization (Ma et al., 2024) (see figure). The resulting model provides a compelling explanation for the seemingly paradoxical hyperglycosylation and destabilization of Gp96 when OST-A is deleted or inhibited (Cherepanova et al., 2019; Lampson et al., 2024; Ma et al., 2024). While Ma et al. focused their analyses on consequences for the cell surface expression and functions of low-density lipoprotein receptor-related protein (LRP) 6 and LRP5, receptors for WNT proteins (Ma et al., 2024), the data provided by Bernaleau et al. and Lampson et al. indicate that this model is likely broadly applicable to Gp96 clients, including TLRs (Bernaleau et al., 2024; Lampson et al., 2024).
Collectively, the findings by Bernaleau et al. (2024) and other recent studies (Lampson et al., 2024; Ma et al., 2024) reveal translocon composition as a key regulatory mechanism for Gp96 abundance and functionality in the cell. With the identification of ER-resident CCDC134, pertinent questions arise: What are the mechanisms that determine expression of secreted, ER-resident and nuclear CCDC134 and is each form of the protein produced by expressing cells? How is ER-resident CCDC134 regulated? Is CCDC134 part of additional translocons? Preliminary insights into the proteome of CCDC134-deficient resting macrophages offered by Bernaleau et al. encourage comparison with, or exploration beyond, known Gp96 clients (Bernaleau et al., 2024; Weekes et al., 2012). Future studies will benefit from assessing cellular CCDC134 requirements under various stress conditions and overlay with requirements for Gp96 and other ER chaperones. Whether the impact of CCDC134 deficiency on the expression of interferon responsive gene products reflects perturbed tonic TLR signaling (Bernaleau et al., 2024) or a regulatory loop between interferon signaling, Gp96, and ER stress is worth further investigation (Anderson et al., 1994; Sprooten and Garg, 2020). Deeper understanding of the CCDC134–Gp96 interactions might reveal how CCDC134 controls Gp96 folding and functions. Ma et al. reported that residues in the Gp96 N-terminal domain were required for CCDC134 recruitment (Ma et al., 2024), whereas Bernaleau et al. showed requirements for the Gp96 middle and C-terminal domains for interactions with CCDC134. As CCDC134 also co-immunoprecipitated with TLRs, CCDC134–Gp96 interactions appear to reach beyond the initial recruitment to the Gp96 translocon (Bernaleau et al., 2024). This invites further exploration of the spatiotemporal positioning of the chaperones CNPY3 and UNC93B1 in TLR biogenesis. Further insights into the molecular mechanisms that regulate the hierarchy and interactions of these ER chaperones might also deliver the foundations for better understanding of differential Gp96 requirements for the folding of various groups of client proteins (e.g., TLRs versus integrins) (Randow and Seed, 2001) and clarify Gp96 contributions, or lack thereof, to TLR2 biogenesis (Bernaleau et al., 2024; Graustein et al., 2018).
Positioning CCDC134 as an ER protein that controls the key chaperone Gp96, together with investment into delineating the Gp96 client proteome, holds potential for improved molecular understanding of disease mechanisms. The discovery that CCDC134 controls Gp96 stability, thereby controling LRP5/6 folding and trafficking connects CCDC134 and wingless-type MMTV integration site 1 (WNT1) genetic variants in the pathogenesis of osteogenesis imperfecta (Jovanovic and Marini, 2024). With emerging immune functions of WNT signaling in infectious and inflammatory conditions (Ljungberg et al., 2019), LRP5/6 integrate well into the landscape of Gp96 client proteins (e.g., TLRs, integrins) that integrate extracellular cues to orchestrate immune cell functions. Exploration of these molecular links could offer new perspectives on correlations between impaired expression of CCDC134 and cancer metastasis (Zhong et al., 2013), including potential links to anti-tumor immunity and WNT-driven carcinogenesis. Delineating the mechanisms that govern formation and disassembly of the CCDC134-OST-A–decorated Gp96 translocon might also reveal whether Gp96-dependent regulation of cell surface receptor folding and expression represents a cellular coping mechanism in response to ER stress or otherwise altered functionality (Ma et al., 2024).
With new molecular insights into how a critical ER chaperone is regulated comes the hope for new therapeutic targets. Opportunities might arise through correction of disease-causing mutations (e.g., CCDC134 in osteogenesis imperfecta) or identification of gene polymorphisms that affect immune responses (Graustein et al., 2018). Targeting the functions and interactions of CCDC134, OST-A, and Gp96 with small molecules or biologics appears to be within reach (Lampson et al., 2024). Whether such approaches translate into pathways for therapeutic interventions will require detailed understanding of the expression and functions of cellular and secreted forms of CCDC134 and Gp96, and whether targeting specific client proteins is to be favored over broad perturbations to cellular receptors, antigen presentation, lymphopoiesis, and tissue homeostasis (Binder, 2014; Staron et al., 2010).
Acknowledgments
The authors are grateful to Thomas E. Schultz and Ashleigh Gordon for their feedback on the manuscript.
This work was supported by an Australian Research Council Future Fellowship (A. Blumenthal, FT220100487) and National Health and Medical Research Council of Australia (A. Blumenthal, GNT2027755).
Author contributions: A. Blumenthal: conceptualization, funding acquisition, visualization, and writing—original draft, review, and editing. L.C. Domínguez Cadena: conceptualization, investigation, methodology, project administration, software, validation, visualization, and writing—original draft, review, and editing.
