Ancient but Not Forgotten: New Insights Into MPEG1, a Macrophage Perforin-Like Immune Effector

Abstract

Macrophage-expressed gene 1 [MPEG1/Perforin-2 (PRF2)] is an ancient metazoan protein belonging to the Membrane Attack Complex/Perforin (MACPF) branch of the MACPF/Cholesterol Dependent Cytolysin (CDC) superfamily of pore-forming proteins (PFPs). MACPF/CDC proteins are a large and extremely diverse superfamily that forms large transmembrane aqueous channels in target membranes. In humans, MACPFs have known roles in immunity and development. Like perforin (PRF) and the membrane attack complex (MAC), MPEG1 is also postulated to perform a role in immunity. Indeed, bioinformatic studies suggest that gene duplications of MPEG1 likely gave rise to PRF and MAC components. Studies reveal partial or complete loss of MPEG1 causes an increased susceptibility to microbial infection in both cells and animals. To this end, MPEG1 expression is upregulated in response to proinflammatory signals such as tumor necrosis factor α (TNFα) and lipopolysaccharides (LPS). Furthermore, germline mutations in MPEG1 have been identified in connection with recurrent pulmonary mycobacterial infections in humans. Structural studies on MPEG1 revealed that it can form oligomeric pre-pores and pores. Strikingly, the unusual domain arrangement within the MPEG1 architecture suggests a novel mechanism of pore formation that may have evolved to guard against unwanted lysis of the host cell. Collectively, the available data suggest that MPEG1 likely functions as an intracellular pore-forming immune effector. Herein, we review the current understanding of MPEG1 evolution, regulation, and function. Furthermore, recent structural studies of MPEG1 are discussed, including the proposed mechanisms of action for MPEG1 bactericidal activity. Lastly limitations, outstanding questions, and implications of MPEG1 models are explored in the context of the broader literature and in light of newly available structural data.

Keywords: MACPF domain; MACPF/CDC; MPEG1; PRF2; immune effector; immunology; pore-forming protein.

Figure 1

Domain schematic of MPEG1 in two isoforms. (A) The majority of MPEG1 consists of a MACPF (blue) and MABP (yellow) domain. The MACPF domain contains functional motifs, namely the TMH regions (tan/red) that unfurl to form a β-barrel upon pore formation. The MABP domain contains a β-hairpin motif (gray) that recognizes and binds to negatively charged phospholipids. A small EGF-like motif (pink) is located between the MACPF and MABP domains. The MABP domain is followed by a small linker region (purple) and conformationally labile motif, denoted the L-domain (green). These directly precede the transmembrane helix (dark gray) and cytosolic (red) regions. The cytosolic region contains a lysine rich motif that is monoubiquitinated during immune response. Scissors depict the putative cleavage site of MPEG1. The majority of MPEG1 constitutes the ectodomain and is postulated to be proteolytically shed from the bilayer. (B) MPEG1b is a shorter secreted isoform that is truncated in the MABP domain at K511 (arrow). Both MPEG1 and MPEG1b are directed to the ER by a signal peptide (SP; copper rose) that is cleaved (arrow) shortly after translation.

Figure 2

Illustration of the cellular context and key processes in which MPEG1 is implicated. (i) MPEG1 is constitutively expressed in macrophages and phagocytes. (ii, iii, iv) Type II IFN, TNFα and LPS signaling are involved in the regulation of MPEG1 expression. (v) Surface bound MPEG1 is essential for the correct assembly and signaling of the type I IFN pathway. (vi) MPEG1 is expressed either as a membrane tethered isoform (MPEG1) or as a secreted, truncated form (MPEG1b). (vii) LPS and IFNγ signaling are important for triggering monoubiquitination of MPEG1 in EEA1⁺ vesicles. Some pathogens produce CIF which inhibits the monoubiquitination of MPEG1 and confers resistance. (viii) Once monoubiquitinated, EEA1⁺/MPEG1⁺ vesicles traffic to and fuse with the early phagosome. The phagocytic and secretory pathways cooperate to enrich intracellular compartments with MPEG1 to aid in the disruption and killing of engulfed microbes. Proteases (scissors) may be important for MPEG1 function. (ix) As phagosomal and endosomal vesicles become acidified, MPEG1 is activated to form lytic pores. Figure produced with BioRender.

Figure 3

The canonical pathway of pore formation. A generic pore forming protein is shown, with a green ancillary (or receptor binding) domain and blue pore forming domain. Freely diffusing monomers (i) bind to the target bilayer (gray) (ii) via target recognition domains (green) that are ancillary to the pore forming machinery (blue). The target receptor can be proteins (yellow), glycans or lipids (ii; inset). Membrane-bound monomers undergo two-dimensional diffusion, colliding and eventually oligomerizing (iii). Maturation via the prepore-to-pore conformational change may occur at different stages. For example, incomplete oligomers may transition into arc-pores (iv). Other smaller arcs or monomers may also be recruited to a growing arc pore (v). Ultimately complete pores are formed upon the closure of the oligomeric ring (vi). In this context pore growth can occur in a continuous mechanism. Completed pores define large aqueous channels, capable of facilitating the passive diffusion of additional effector molecules (not shown) via the membrane channel (vi; inset). Alternatively, arc prepores may continue to grow without inserting into the membrane (vii) by recruiting additional monomers or other smaller arcs (viii). These can ultimately form complete prepores that have yet to punch into the lipid bilayer (ix). Fully formed prepores are most commonly observed for CDCs (ix). The prepore-to-pore transition is triggered resulting in a conformational change of the MACPF core machinery that unfurls into a giant β-barrel (ix goes to vi). These inserted pores possess an amphipathic region that is fully inserted into the lipid membrane (not shown). Insets (ix, vi) show top-down views.

Figure 4

Exemplar structures of MACPF pore forming proteins in the monomeric and pore states. (A) Crystal structure of lymphocyte PRF in the monomeric state [PDB: 3NSJ] (43). The ancillary domain is colored gray and omitted from the topology diagram for clarity. (B) The cryoEM pore structure of the fungal MACPF protein, pleurotolysin (PlyB)[PDB: 4V2T] (13). Pleurotolysin is a homolog of PRF found in oyster mushroom. The β-trefoil domain of PlyB is not shown for clarity. (C) A dimer of the polyC9 cryoEM reconstruction (bottom right) is shown to illustrate the intra-subunit contacts at the MACPF interface [PDB: 6DLW] (22, 23). (D) The cryoEM pore structure of the intracellular GSDMA3-NT shows structural and topological similarity to the MACPF domain [PDB: 6CB8] (103). HTH, helix-turn-helix (purple); TMH, transmembrane β-hairpin (pale brown). Topology diagrams are colored consistently with the PDB coordinates.

Figure 5

The various structural states of MPEG1. (A) When incubated with liposomes (gray), MPEG1 binds the lipid bilayer as a single prepore ring, via the MABP β-hairpin (yellow), orienting the MACPF domain away from the lipid bilayer [PDB: 6U2W] (82). Lipids are illustrated with a cartoon model. Both the unsharpened (gray) and sharpened (alternating color) maps are superimposed to illustrate the lipid density (gray). (B) In solution, recombinant MPEG1 (truncated between the L-domain [green] and TM region [not shown]) forms a loosely associated ring–ring dimer whereby the helix of the L-domain mediates interactions between rings (termed the α-conformation) [PDB: 6U2J, 6U2K] (82). (C) A second, tightly associated ring–ring dimer is also possible; this conformation is defined by inter-ring strand swapping (termed the β-conformation) [PDB: 6U2L] (82). This is achieved by the L-domain which adopts an extended β-sheet conformation. (D) Murine MPEG1 truncated at a similar position to (A, B), forms single ring structures after prolonged incubation in acidic conditions [PDB: 6SB3] (83). These rings were observed in the α-conformation (with respect to the L-domain). (E) A view of an MPEG1 dimer is shown from the periphery of the complex in both the membrane-bound (left) and soluble prepore (right) states. Upon interchanging between these states, the L-domain and β-hairpin undergo conformational change. Inset shows a magnified view of the interaction. (F) Incubation of murine MPEG1 at low pH and in the presence of the detergent CYMAL6 results in MPEG1 pores [PDB: 6SB5] (83) where the MABP domain is flipped relative to (A–D). This conformational change re-orients the MACPF and MABP domains into the same direction. The extremity of the β-barrel forms an amphipathic region (illustrated by a cartoon micelle). Top row: CryoEM reconstructions of MPEG1 (alternating colors show individual subunits) of the overall quaternary structure. Both the full reconstruction (left) and a partial cross section (right) are shown for each panel (A–D, F). The cross section enables visualization of the inner structure of the complex. Second row: Exemplar 2D class averages are shown below each reconstruction [reproduced from (82, 83)]. The atomic coordinates for the full reconstruction are shown next to the corresponding 2D class average (alternating colours show individual subunits). Third row: A single magnified subunit from each complex is shown. MACPF β-sheet (red), TMH regions (tan), MACPF core (blue), MABP/β-hairpin (yellow), EGF-like (pink), linker region (purple), L-domain (green).

Figure 6

Schematic comparison between current models of PRF and MPEG1 assembly. (A) At the T-cell immune synapse, vesicles (i) containing PRF [only the MAPCF (blue) and C2 domains (green) are shown] and granzymes (purple) fuse with the plasma membrane releasing their contents onto the target cell (ii). Within these cytotoxic granules (i), PRF is kept in a Ca²⁺-deficient environment at low pH, therefore PRF is unable to bind membranes. Upon being released into the immune synapse (ii), PRF encounters Ca²⁺ (zoomed inset; red spheres) and therefore, binds the lipid bilayer via a target recognition C2 domain (iii). PRF begins to oligomerize into arcs (iv) and, later, fully formed pores (vi). In the PRF mechanism, early arc intermediates can puncture the lipid membrane (v); these can continue to grow in a continuous manner by recruiting monomers or other arcs. Functional arcs that have punctured into the lipid bilayer are depicted with a white membrane lesion (v). The final PRF pore enables granzyme B (GrB; purple) to diffuse into the target cell (vii). (B) EEA1⁺ vesicles containing MPEG1 (i) are triggered to traffic toward and fuse with the phagosomal membrane by monoubiquitination (blue diamond) (ii). Tethered MPEG1 [only the MACPF (red) and MABP domains (yellow) are shown] is proteolyzed from the lipid bilayer [transmembrane region and cytosolic tail are shown as a line (green)] (iii). Cleaved MPEG1 oligomerizes into a prepore (v). Upon strong acidification (pH < 5), MPEG1 is activated and transitions into a pore (vi or ix). MPEG1 may follow two proposed pathways (iv or vii). In the trans-pore model, oligomerization occurs on the host bilayer (iv to v) and trans-pores breach the bilayer of target membranes in close proximity (vi) (82). Other receptor complexes may be required to drive the formation of a close membrane–membrane junction (blue/orange receptor complex; asterisk). Alternatively, MPEG1 monomers diffuse within the synapse (vii) and oligomerize on microbial bilayers (vii to viii). The MACPF or MABP domains rotate, to re-orient the MACPF machinery toward the microbial bilayer [vii or viii; unclear (83)]. A cis-pore breaches the microbial bilayer (ix). The stage of MACPF or MABP domain rotation is unclear. After either a trans- or cis-pore has formed, effector molecules enter the target cell via the MPEG1 pore (x).