CCT2, a newly identified aggrephagy receptor in mammals, specifically mediates the autophagic clearance of solid protein aggregates

ABSTRACT

Protein aggregates have a strong correlation with the pathogenesis of multiple human pathologies represented by neurodegenerative diseases. One type of selective autophagy, known as aggrephagy, can selectively degrade protein aggregates. A recent study from Ge lab reported the TRiC subunit CCT2 (chaperonin containing TCP1 subunit 2) as the first identified specific aggrephagy receptor in mammals. The switch of CCT2ʹs role from a chaperonin to a specific aggrephagy receptor is achieved by CCT2 monomer formation. CCT2 functions independently of ubiquitin and the TRiC complex to facilitate the autophagic clearance of solid protein aggregates. This study provides the intriguing possibility that CCT2, as a specific aggrephagy receptor, might be an important target for the treatment of various diseases associated with protein aggregation.

KEYWORDS:

• Aggrephagy
• autophagy
• CCT2
• chaperonin TRiC subunit
• protein aggregation
• solid protein aggregates

Partially resulting from the increase of the elderly population, age-related neurodegenerative diseases are becoming more prevalent and posing a major threat to human health [1,2]. One critical cause and hallmark for neurodegenerative diseases is the accumulation of toxic protein aggregates [3]. Therefore, an effective removal of protein aggregates has always been a potential therapeutic target for neurodegenerative disease treatments [4].

In addition to the ubiquitin-proteasome system, the macroautophagy/autophagy-lysosome pathway is another major mechanism to degrade protein aggregates in eukaryotes. A type of selective autophagy, aggrephagy, engulfs protein aggregates by forming a phagophore, which matures into a double-membrane compartment called an autophagosome; the protein aggregates are then degraded after fusion of the autophagosome with a lysosome. Previously known receptors involved in aggrephagy in mammals are all ubiquitin-binding receptors (including NBR1, OPTN, SQSTM1/p62, TAX1BP1, and TOLLIP). These ubiquitin-binding receptors mediate aggrephagy by binding to Atg8-family proteins on the phagophore membrane with their LC3-interacting region (LIR) as well as binding to ubiquitin chains on the ubiquitinated protein aggregates via their ubiquitin-binding domain (UBA). Thus, these receptors recognize and bind to ubiquitin chains, but they do not specifically bind directly to protein aggregates. In addition, some receptors are also involved in other types of selective autophagy such as mitophagy. Nevertheless, receptors specifically regulating aggrephagy do exist and have been identified in other organism such as C. elegans (SEPA-1 and EPG-7) while there was, until now, no specific aggrephagy receptor identified in mammals.

The study recently published by the Ge lab in Cell and highlighted here revealed that the chaperonin TRiC subunit CCT2 switches its function from chaperonin component to aggrephagy receptor when it acts as a monomer and specifically promotes the autophagic clearance of solid protein aggregates in a manner independent of ubiquitin as well as other TRiC subunits [5].

How was CCT2 identified as a specific aggrephagy receptor? Ma and colleagues successfully constructed an in vitro system to mimic the recruitment of LC3 by protein aggregates. In this approach the LC3 proteins are fluorescently labeled and protein aggregates are sorted into two categories according to LC3 density: high-LC3- and low-LC3-containing aggregates. With further unlabeled quantitative mass spectrometry analyses, many chaperones and co-chaperones were surprisingly enriched in high-LC3-containing aggregates. Among them, CCT2 was highly enriched. Subsequently, CCT2 was found to promote the in vitro association of LC3 and protein aggregates, whereas the knockdown of CCT2 decreases this association. Moreover, the study also found that CCT2 binds to and mediates the autophagic degradation of other toxic protein aggregates implicated in neurodegenerative diseases, such as MAPT/Tau^P301L in Alzheimer disease, and SOD1^G93A in amyotrophic lateral sclerosis.

Given the fact that CCT2 lacks a classical LIR motif, it is of interest to dissect the mechanism of the interaction between CCT2 and LC3. By applying peptide affinity-isolation and co-immunoprecipitation approaches, Ma et al. discovered that the CCT2-LC3 interaction is dependent on two consecutive residues (VLL and VIL), which echoes the CALCOCO2/NDP52 CLIR-motif [6]. These two residues are named the V-LC3-interacting region (VLIR) and its aggrephagy function (association with LC3/Atg8-family proteins) was further verified in yeast.

The next question is how does CCT2 specifically interact with protein aggregates? A previous study showed that the chaperonin subunits bind to misfolded proteins through their apical domain [7], which is in line with Ge lab’s finding with CCT2, indicating that the latter binds protein aggregates due to its apical domain’s intrinsic ability to interact with misfolded proteins. In contrast to SQSTM1 and other autophagy receptors possessing a ubiquitin-binding domain, Ma et al. found that CCT2 does not bind to ubiquitin chains but CCT2 binds to and promotes the clearance of aggregates whether or not they are ubiquitinated. Furthermore, because some known ubiquitin-binding receptors (SQSTM1, NBR1, and TAXBP1) were also enriched in the high-LC3-containing aggregates, and to rule out the possibility that CCT2-mediated aggrephagy is dependent on these receptors, Ma et al. triply knocked down SQSTM1, NBR1 and TAX1BP1 and found normal CCT2 functions as an aggrephagy receptor. Similarly, they also knocked down HSPA8/HSC70 (the key chaperone receptor in chaperone-mediated autophagy) and found that again CCT2 functions independently of this component.

Another significant finding is that CCT2 specifically degrades solid-state protein aggregates, and this property is also observed with yeast Cct2. Previous studies have shown that various toxic protein aggregates undergo liquid-liquid phase separation to form liquid condensates before gradually solidifying into solid aggregates [8,9]. However, even though solid-state protein aggregates are usually the ones correlated with the pathogenesis of human diseases [10], they are not an ideal target for aggrephagy because this process preferentially degrades liquid protein condensates [11]. Therefore, it is of paramount importance that this study revealed CCT2ʹs capability of specifically facilitating the clearance of mature, solid-state protein aggregates, making it more promising as a potential therapeutic target for protein aggregate-related diseases. A key question is what determines the affinity of CCT2 for solid aggregates? This selective CCT2-solid aggregates association may be influenced by the protein conformation in solid aggregates: (1) Solid aggregates contain misfolded proteins, and, as mentioned above, CCT2 is a subunit of a chaperonin complex so it has the intrinsic ability to bind misfolded proteins; (2) solid aggregates contain fibers of β-sheet conformation, and a previous study shows that the TRiC chaperonin preferentially interacts with protein aggregates harboring such a conformation [12].

Last, this study also forges a link between chaperonins and aggrephagy by the discovery of the dual role of CCT2 in protein homeostasis: as a subunit of a chaperonin complex or as an aggrephagy receptor. Under normal condition, CCT2 cooperates with other TRiC subunits to function as a chaperonin to help with proper protein folding, and eventually prevent protein aggregation. In this context, the aggrephagy function of CCT2 is blocked because its VLIR motif is embedded within the complex and thus hinders its interaction with Atg8-family proteins. When the chaperone system is overcome by the excessive accumulation of misfolded proteins (and protein aggregates form), a portion of CCT2 disassembles from the TRiC complex and acts as a monomer. In this context, the aggrephagy function of monomeric CCT2 is turned on by the exposure of its VLIR motif to render Atg8-family protein association possible. Consistent with this notion, monomeric CCT2 regulates aggrephagy in a manner independent of TRiC complex integrity. However, whether this aggrephagy function of TRiC subunits is unique to CCT2 is unclear due to the following reasons: (1) A previous study shows that the overexpression of other TRiC subunit also decreases polyQ-HTT protein aggregates [13]; (2) in this study, other TRiC subunits are also present in the high-LC3-containing aggregates fraction. Further studies could reveal whether other chaperones are involved in aggrephagy and whether they function in a similar way as CCT2.

In addition to the questions mentioned above, there are also a few more issues in this study related to autophagy that need to be addressed. First, it is unclear whether the source of monomeric CCT2 that functions in aggrephagy is solely from the disassembled complex (as observed in this study) or whether it might be partially generated from newly synthesized CCT2. Second, considering that the size of solid protein aggregates can be larger than the size of an autophagosome (0.5–1.5 μm), how does the phagophore engulf these aggregates? A previous study has reported that the phagophore can divide liquid protein condensates by the differential spontaneous curvature of the phagophore membrane, which ensures piecemeal or complete liquid condensate sequestration [14]. However, whether solid aggregates undergo a similar fragmentation process is unknown. Finally, given the fact that various chaperones regulate protein aggregate disaggregation (into fibrillar fragments) [15], and the remarkable enrichment of HSP90AB1 found in the screen in this study, it is plausible that HSP90AB1 might be involved in solid protein aggregate fragmentation. Therefore, further in-depth studies are required to understand the potential role of HSP90AB1 in aggrephagy.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Institute of General Medical Sciences [GM131919].