新発見は感染症や癌治療の未来を「解き明かす」かもしれない(New discovery may ‘unlock’ the future of infectious disease and cancer treatment)


2023-10-06 バーミンガム大学



PIM1がGBP1の活性を制御して自己損傷を抑制し、病原体感染を防御することを発見 PIM1 controls GBP1 activity to limit self-damage and to guard against pathogen infection

Daniel Fisch,Moritz M. Pfleiderer,Eleni Anastasakou,Gillian M. Mackie,Fabian Wendt,Xiangyang Liu,Barbara Clough,Samuel Lara-Reyna,Vesela Encheva,Ambrosius P. Snijders,Hironori Bando,Masahiro Yamamoto,Andrew D. Beggs,Jason Mercer,Avinash R. Shenoy,Bernd Wollscheid,Kendle M. Maslowski,Wojtek P. Galej,and Eva-Maria Frickel
Science  Published:6 Oct 2023

Editor’s summary

Mammalian cells use guard mechanisms to monitor their functional pathways for interference by pathogens. Infection causes the production of the inflammatory cytokine interferon-γ (IFN-γ), which triggers the expression of hundreds of IFN-stimulated-genes, including the kinase PIM1 and GBP1, a membrane-perturbing GTPase. Fisch et al. identified a guard mechanism whereby PIM1 phosphorylates GBP1 and subjects it to sequestration by a 14-3-3 protein. In human macrophages, this mechanism was found to prevent GBP1 activity from causing Golgi fragmentation and cell death. Pathogens can interfere with IFN-γ signaling and thereby potentially escape immune detection. However, when this signaling is inhibited, short-lived PIM1 is degraded, which allows GBP1 to control pathogen growth. These findings suggest a model of IFN-γ–dependent protection of uninfected bystander cells against self-inflicted innate immune damage. —Stella M. Hurtley

Structured Abstract

Cells in infected tissues are exposed to inflammatory stimuli, including the innate and adaptive immunity–stimulating cytokine interferon-γ (IFN-γ). Although most tissue-resident and infiltrating cells are not infected, when exposed to IFN-γ, these bystander cells preemptively express a repertoire of interferon-stimulated genes (ISGs) with robust antimicrobial activities and the potential for self-harm. ISGs of the guanylate-binding protein (GBP) family are large, membrane-active guanosine triphosphatases (GTPases). GBPs can control intracellular microbes in various ways, most importantly by promoting membrane rupture and the release of microbial ligands and by the induction of programmed cell death, including pyroptosis and apoptosis. How uninfected cells protect themselves from the potentially self­-destructive actions of GBPs while keeping these proteins readily available to combat infection is unknown.

Cells need to tightly control the activity of antimicrobial proteins but rapidly deploy them upon infection. How is this achieved in human cells? Posttranslational modifications, such as phosphorylation by protein kinases, enable rapid and precise control of protein activities. We studied the phosphorylation of GBP1, a typical ISG, and how this modification affects its function, activity, and localization in human macrophages.

Ectopic expression of GBP1 in human macrophages led to changes in cell morphology, GBP1 accumulation at the Golgi apparatus, Golgi fragmentation, and uncontrolled cellular necrosis. These findings illustrate GBP1’s potential to inflict self-damage. This phenotype was mitigated by IFN-γ treatment, suggesting that another IFN-γ–inducible factor limited GBP1 activity. We identified the kinase PIM1 as being this factor. We generated a phosphorylation-specific antibody and used high-resolution mass spectrometry to demonstrate GBP1 phosphorylation at serine-156 (Ser156), which was guided by a basophilic PIM1 recognition motif. Ser156 is the central residue of a 14-3-3 protein binding motif, which suggests a switch-like function for its phosphorylation. Indeed, 14-3-3 proteins, especially 14-3-3σ, interacted with phosphorylated GBP1. In vitro reconstitution of this complex followed by single-particle cryo–electron microscopy confirmed a 14-3-3σ dimer grabbing onto the GBP1 GTPase domain. This binding locked GBP1 in a GTPase-inactive, monomeric state and restrained its activity in the macrophage cytosol. Expressing phosphorylation-deficient GBP1 mutants or mutants that could not be recognized by the kinase PIM1 or bound by 14-3-3σ led to uncontrolled GBP1 activation and subsequent cell death. Genetic depletion of either PIM1 or 14-3-3σ had similar outcomes, as did treatment with the GBP1:PIM1 interaction inhibitor NSC756093. Using the inhibitor in IFN-γ–activated patient-derived tumor organoids increased organoid death and prevented organoid reformation. Thus, we found that PIM1 and 14-3-3σ together controlled the activity of GBP1 in human cells. Disrupting PIM1-driven control of GBP1 has potential therapeutic implications for cancer therapy and innate immunity.

We observed that PIM1 mRNA and protein were extremely short-lived. Infection with the apicomplexan parasite Toxoplasma gondii, a pathogen that resides within intracellular vacuoles and blocks IFN-γ signaling by means of the effector protein TgIST, led to fast depletion of PIM1. This in turn reduced GBP1 Ser156 phosphorylation levels and liberated GBP1 from 14-3-3σ sequestration. High-throughput imaging revealed that GBP1 then rapidly targeted Toxoplasma-containing vacuoles to improve control of the infection.

The IFN-γ–induced, short-lived kinase PIM1 guards the integrity of IFN-γ signaling and protects self-membranes by regulating the activity of the potent antimicrobial effector GBP1. Pathogens that block IFN-γ signaling, thereby reducing the levels of PIM1, are then exposed to GBP1-driven innate immune control. The phosphoregulation of GBP1 by PIM1 reveals an IFN-γ–dependent control mechanism that protects uninfected bystander cells from self-inflicted innate immune damage during the process of pathogen elimination.

The protein kinase PIM1 controls GBP1 activity.

In uninfected bystander cells, IFN-γ signaling induces expression of PIM1, which phosphorylates GBP1, subjecting it to sequestration by 14-3-3σ. This shunts aberrant GBP1 activity and ensures cell survival (left). Perturbation of this homeostasis leads to GBP1 dephosphorylation and Golgi targeting and disruption, culminating in necrosis (middle). Intracellular microbes that disrupt IFN-γ signaling, e.g., T. gondii, deplete PIM1 and liberate GBP1, which then attacks and controls the infection (right).


Disruption of cellular activities by pathogen virulence factors can trigger innate immune responses. Interferon-γ (IFN-γ)–inducible antimicrobial factors, such as the guanylate binding proteins (GBPs), promote cell-intrinsic defense by attacking intracellular pathogens and by inducing programmed cell death. Working in human macrophages, we discovered that GBP1 expression in the absence of IFN-γ killed the cells and induced Golgi fragmentation. IFN-γ exposure improved macrophage survival through the activity of the kinase PIM1. PIM1 phosphorylated GBP1, leading to its sequestration by 14-3-3σ, which thereby prevented GBP1 membrane association. During Toxoplasma gondii infection, the virulence protein TgIST interfered with IFN-γ signaling and depleted PIM1, thereby increasing GBP1 activity. Although infected cells can restrain pathogens in a GBP1-dependent manner, this mechanism can protect uninfected bystander cells. Thus, PIM1 can provide a bait for pathogen virulence factors, guarding the integrity of IFN-γ signaling.