Nuclear destruction: A suicide mission by AKIRIN2 brings intact proteasomes into the nucleus

Abstract

de Almeida et al. (2021) developed a temporally controlled CRISPR-Cas9 screen to identify mechanisms controlling MYC levels and discovered that intact proteasomes are imported into the nucleus by AKIRIN2 binding to proteasomes at one end and a nuclear import receptor at the other.

The proteasome acts as a grim reaper for proteins, shutting down their cellular activity by digesting them into peptides. Many short-lived proteins function and are proteolyzed in the nucleus, yet few proteasome subunits contain nuclear localization signals (Wendler et al., 2004). Experiments in yeast suggest that the 26S proteasome is assembled in the cytosol prior to translocation into the nucleus (Pack et al., 2014), with essential protein Sts1 driving proteasome import (Budenholzer et al., 2020; Chen et al., 2011). Sts1 is not evolutionarily conserved however, furthering curiosity of how proteasomes arrive within the nucleus in higher eukaryotes and whether an analogous mechanism exists. By enabling the study of essential proteins with a temporally controlled CRISPR-Cas9 screen to probe determinants of MYC protein levels, de Almeida et al. made a transformative advancement in resolving how nuclear proteins are degraded. They find that AKIRIN2, an essential but understudied protein, binds to proteasomes in the cytosol and by also interacting with a nuclear import factor, transports the intact proteasomes into the nucleus (de Almeida et al., 2021).

Proteolysis by proteasomes is catalyzed within the hollow interior of a cylindrical 4-ring complex known as the core particle (CP), with three centrally located enzymatic subunits that vary based on cell type or cytokine induction to influence resulting peptide products. Two inner β-rings hold the proteolytically active subunits while the N termini of outer α-rings form a dynamic gate. Accessibility through this gate is modulated by interactions with specific activator complexes that dock into and/or against the α-rings to expand or specialize proteasome activity. For example, the 19S regulatory particle (RP) allows tightly controlled degradation of ubiquitinated proteins (Chen et al., 2021); 11Sαβ promotes production of peptides optimal for MHC class I antigen presentation; and 11Sγ is abundant in the nucleus and contributes to degradation of cell-cycle regulators and transcription factors, including MYC (Li et al., 2015). Like these and other proteasome activators, AKIRIN2 binds to the CP apical surface (de Almeida et al., 2021). The extent to which the CP is capped at both ends and whether it tends to be capped by the same or different activator complexes is unknown. de Almeida et al. find that AKIRIN2 transports RP-capped proteasomes into the nucleus, indicating its binding to one end as the RP binds the other.

de Almeida et al. (2021) use cryo-electron microscopy to solve the structure of the C-terminal region of AKIRIN2 complexed with the CP, revealing that an AKIRIN2 coiled-coil homodimer extends across the CP gate with the two C termini docked into pockets at the α2/α3 and α3/α4 interfaces. Interactions between the coiled-coil and the CP N-terminal gating residues are expected to prevent proteolysis by the proteasome at the AKIRIN2-bound end. Whether AKIRIN2 binding at one CP end allosterically influences the activity of the RP at the other remains to be explored. Further study is also required to resolve the extent to which AKIRIN2 transports CP lacking the RP into the nucleus, whether it can bind to both ends of the CP simultaneously, and/or whether proteasomes with one surface of the CP uncapped are transported into the nucleus. The AKIRIN2 binding mechanism however is expected to prevent doubly capped RP from being translocated into the nucleus. This restriction may have been selected for as a mechanism to preserve one CP face for interaction with a nuclear activator complex, such as 11Sγ (Figure 1). Consistent with this model, AKIRIN2 is degraded by the proteasome soon after nuclear import (de Almeida etal., 2021).

Figure 1. Model of AKIRIN2-mediated proteasome import into the nucleus.

In the cytosol, the C-terminal regions of an AKIRIN2 dimer (orange) bind to a proteasome CP (gray) capped at the other end by the RP (blue). IPO9 (green) binds to the N-terminal region of AKIRIN2 and transports the proteasome assembly through the nuclear pore complex (NPC, beige, orange, and green). Once in the nucleus, IPO9 binds to GTP-bound RAN (purple), releasing it from AKIRIN2 and driving its nuclear export. AKIRIN2 is in turn degraded by the CP, providing a binding site for 11Sγ (pink). In the lower panel, key interactions within AKIRIN2 and to the CP are highlighted with hydrogen bonds displayed as black dashed lines. This figure was generated by using PDB entries 7NHT, 5VFS, 6N1Z, 3ZJY, 3GJ0, EMD-3103, and AlphaFold2-predicted AKIRIN2, and a model of human 11Sγ generated by the SWISS-MODEL server.

AKIRIN2 forms a ternary complex with the proteasome and Importin-9 (IPO9), which most likely interacts with an N-terminal nuclear localization signal present in AKIRIN2. A compelling model is that AKIRIN2 degradation by the proteasome following nuclear import is coupled to release of IPO9 and coordinated docking of 11Sγ (Figure 1). A trigger for IPO9 release from AKIRIN2 may be GTP-binding by the RAN GTPase, an established signaling mechanism for transport of importins back to the cytosol. RAN is present in an AKIRIN2 complex (de Almeida et al., 2021) and IPO9 is required for nuclear localization of proteasomes (Palacios et al., 2021).

The paramount role of AKIRIN2 in defining the abundance of MYC and other nuclear proteins by its import of proteasomes into the nucleus redefines previous studies reporting its importance for innate immunity, chromosome remodeling, transcriptional regulation, and development. Humans express two AKIRIN proteins with 57% sequence identity, but only AKIRIN2 was found to interact with proteasomes (de Almeida et al., 2021). Loss of AKIRIN2 and not AKIRIN1 leads to embryonic lethality in mice (Goto et al., 2008), perhaps due to defective proteasome import into the nucleus. The extreme C terminus of both AKIRIN proteins contains a conserved SYVS motif required for AKIRIN2:CP interactions that dock into α2/α3 and α3/α4 pockets (de Almeida et al., 2021). AKIRIN2 A199 packs against this motif’s Y201, promoting a compact structure that facilitates intermolecular interactions, while AKIRIN2 E196 defines the orientation of the coiled-coil relative to the C-terminal region by hydrogen bonds to AKIRIN2 R193 and α2 K17 (Figure 1, insert). Each of these key residues are replaced with threonine in AKIRIN1.

Inhibition of the proteasome for the treatment of disease is an active area of research (Chen et al., 2021), with CP inhibitors used to treat hematological cancers and an inhibitor specific to the immunoproteasome approved for treatment of polymyositis and dermatomyositis (Johnson et al., 2018). The binding mechanism of AKIRIN2 to the α-ring suggests that it is likely to interact with all forms of the CP, including the immunoproteasome. As de Almeida et al. (2021) propose, AKIRIN2 presents new therapeutic opportunities, with proliferating cells expected to be particularly sensitive to defective import of proteasomes into the nucleus. The discovery of this new pathway is a transformative step forward in defining the activity and regulation of the proteasome in cells.