Researchers have uncovered an immune mechanism by which host cells combat bacterial infection


Researchers at the University of Toronto have uncovered an immune mechanism by which host cells combat bacterial infection, and at the same time found that a protein crucial to that process can sense and respond to misfolded proteins in all mammalian cells.

The protein is called heme-regulated inhibitor or HRI, and the researchers showed that during bacterial infection it triggers and coordinates a chain reaction among other proteins that form a larger complex.

That larger group or ‘signalosome’ amplifies inflammation and leads to an anti-bacterial response.

But HRI can also regulate protein folding in other cell types, the researchers showed.

Protein folding, which helps determine the 3-D shape of a protein and is essential for its function, is implicated in non-infectious diseases including the neurodegenerative disorders Parkinson’s, Alzheimer’s and ALS.

“The innate immune function that we discovered is essentially a mechanism of protein scaffolding, which is important because you want a quick and orderly response to bacterial infection,” says Stephen Girardin, a professor of laboratory medicine and pathobiology and of immunology at U of T.

“But we also found that same pathway is important for protein scaffolding and aggregation in other cells, which opens promising research angles for neurodegenerative and other diseases.”

The journal Science published the findings today.

Researchers have studied HRI for over three decades, but mostly in the context of red blood cell disorders.

“This protein appears in all cells in the mammalian body and was recognized as a broad or promiscuous sensor,” says Mena Abdel-Nour, lead author on the paper who completed his doctorate in Girardin’s lab earlier this year.

“But it was overlooked relative to pattern recognition molecules and the formation of amyloid-like structures.

We had to test its role in several different pathways before we believed what we saw.”

Abdel-Nour and his colleagues developed a novel technique to study the effects of HRI.

They adapted a biochemistry assay from the lab of Jeffrey Lee – a professor of laboratory medicine and pathobiology at U of T whose team works beside their own – which helped them distinguish between folded and misfolded proteins when looking at protein aggregates.

Scientists have struggled to make that distinction in part because most available tests only work in test tubes and are not adaptable to cells.

The researchers have early pre-clinical data that shows HRI could protect against the type of neurodegeneration seen in Parkinson’s.

“Speculatively, it might be possible to find molecules that produce HRI’s protective effects, which could lead to a bona fide therapy,” says Abdel-Nour, who plans to pursue a career as a biotechnology and health-care consultant.

Current therapies for Parkinson’s focus on finding and clearing out protein aggregates, rather than fixing cellular defects before those clusters accumulate.

Girardin says he is committed to pursuing that research in collaboration with neuroscientists, and he just received funding to support that work.

“We are focused on Parkinson’s because it’s a very important disease for human health, and because its hallmark is protein aggregation inside cells, so it may be a perfect model to test this new pathway.”

Next steps include biochemical investigations of HRI and related complexes during protein misfolding, and animal studies of neurodegenerative disease to further validate the new pathway, which shares many features with a similar pathway in humans.

Protein synthesis is a complex multistep process, which includes peptide chain initiation, elongation, termination, and recycling [1,2].

Translation initiation is often rate limiting for protein synthesis and requires the formation and recycling of the ternary complex formed by eIF2, GTP, and Met-tRNAi.

The eIF2.GTP.Met-tRNAi ternary complex is required for recognition of the translation start codon and 80S assembly. eIF2 is a trimeric guanine nucleotide-binding protein complex that recycles between GTP-bound active and GDP-bound inactive forms.

The complex has higher affinity for GDP, which coupled with high intracellular GDP to GTP ratio necessitates activity of a guanine nucleotide exchange factor for successful recycling of eIF2.GDP to eIF2.GTP complex.

This guanine nucleotide exchange is catalyzed by a five subunit eIF2B complex whose activity is essential for formation of the ternary complex. Phosphorylation of eIF2α simultaneously increases affinity of eIF2 for eIF2B and blocks its guanine nucleotide exchange activity. This reduces ternary complex abundance and attenuates translation initiation (Figure 1)[3].

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Figure 1
Phosphorylation of eIF2α modulates protein synthesis. Phosphorylation of eIF2α on serine 51 may result from physiologic or pharmacologic activation of eIF2α kinases. There are four well-described eIF2α kinases: PERK, PKR, HRI, and GCN2 (see text for full names) each activated by a diverse set of cellular stressors. Phosphorylation of eIF2α results in attenuated guanine nucleotide (GDP/GTP) exchange activity as a result of inhibitory effects on the guanine nucleotide exchange factor (eIF2B). As a result, less eIF2-GTP-met-tRNAi ternary complex (TC) is able to be formed. Since ternary complexes are required for translation initiation, the end result is a general repression of protein synthesis. mRNAs with long or structured 5′ untranslated regions (UTRs), including many oncogenic mRNAs (e.g. growth factors, transcription factors) may be especially vulnerable to reduced TC abundance (weak 5′UTRs), compared to housekeeping mRNAs with more efficient 5′UTR sequences (strong 5′UTRs). 40S = 40S small ribosomal subunit.

Phosphorylation of eIF2α on serine 51 is mediated by four upstream eIF2 kinases: PKR-like ER kinase (PERK), protein kinase RNA-activated (PKR), general control non-derepressible 2 (GCN2), and heme-regulated inhibitor (HRI).

Though these kinases converge on eIF2α phosphorylation, they may be activated by distinct cellular stresses and phosphorylate a diverging set of additional substrates.

For example, PERK is activated by endoplasmic reticulum (ER) stress, while PKR is activated in response to viral infection, and GCN2 via nutrient (amino acid) depletion.

In contrast, HRI acts primarily as a heme sensor [4]. Heme interacts directly with and inactivates HRI.

In the setting of heme deficiency, HRI is activated to attenuate globin translation in erythroid precursors [5].

Certain stressors (such as oxidative stress or proteasome inhibition) may activate different eIF2 kinases depending on the cellular and experimental context [69].

In addition, when specific eIF2 kinases are deleted experimentally, an alternative eIF2 kinase may be activated, though usually to a lesser degree, demonstrating potential for redundant functions [10].

While phosphorylation of eIF2α results in a general repression of mRNA translation, some mRNAs are more sensitive to reduced ternary complex formation.

mRNAs containing long or highly structured 5′ untranslated region (UTR) sequences, which includes growth factors and cell cycle proteins, may be particularly affected [11].

Reduced ternary complex formation may also promote increased translation of a subset of mRNAs.

This group of mRNAs incudes those regulated by multiple upstream open reading frames (uORFs) in the 5′ UTR [12].

In the setting of reduced ternary complex availability, the scanning ribosome may bypass inhibitory uORFs upstream of the bonafide start codon, resulting in a de-repression of mRNA translation (Figure 2).

Among these mRNAs, the most well characterized of which is activating transcription factor 4 (ATF4), many are involved in mediating cellular stress responses as part of an integrative stress response [13].

An example of this coordinated response is seen in the setting of HRI activation in erythroid cells, where the attenuation of globin translation is thus paired with upregulation of ATF4, which helps reduce oxidative stress and promote erythroid survival [7,14].

Although this integrated stress response can aid in protecting cells from various stressors, phosphorylation of eIF2α may also lead to proapoptotic outcomes [15].

A notable example is seen with the transcription factor C/EBP homologous protein (CHOP), which is upregulated in response to eIF2α phosphorylation [16].

In early erythroid cells, the induction of CHOP in response to oxidative stress may serve a protective role, possibly by aiding translation recovery via feedback inhibition of eIF2α phosphorylation [7,17].

However, the induction of CHOP can also act to promote apoptosis [8,18].

The intensity and duration of stress likely plays a role in facilitating a switch toward a proapoptotic response, though the precise mechanisms remain to be elucidated [19].

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Figure 2
eIF2α phosphorylation results in translational de-repression of select mRNAs. When eIF2α is in a non-phosphorylated state, as is seen during robust translational periods, there is abundant ternary complex (TC) availability for protein synthesis. For specific mRNAs with multiple upstream open reading frames (uORFs), ribosomal occupancy of inhibitory uORFs upstream of the start codon (main ORF) can lead to low levels of protein expression. In the setting of phosphorylated eIF2α, reduced availability of ternary complexes can lead to ribosomal bypass of inhibitory uORFs leading to translational de-repression and increased protein expression of select mRNAS. Notable examples of mRNAs that are regulated in this manner include activating transcription factor 4 (ATF4) and BRCA1. A simplified schematic is demonstrated for illustrative purposes. 60S = large ribosomal subunit. 40S = small ribosomal subunit. 80S = joined 80S ribosome.

Recent manuscripts have reviewed the structure and function of eIF2 kinases and the role of HRI in erythroid cells [4,5]. This review will highlight data on targeting HRI as an anticancer strategy.

1.1. HRI background

HRI (EIF2AK1) is best known for its role in coupling heme availability to globin synthesis in early erythroid cells [5].

Indeed, initial studies characterizing HRI tissue distribution in rabbits noted a restricted expression pattern in reticulocytes and bone marrow but not in other tissues [20,21].

Subsequently, an eIF2α kinase was isolated from rat brain and mouse liver with 82–83% sequence homology to rabbit reticulocyte HRI and similarly containing conserved heme regulatory motifs [22,23].

Using a cDNA probe, mRNA expression for rat and mouse HRI was identified across a wide range of tissues suggesting ubiquitous expression.

Nonetheless, Berlanga et al. noted that HRI expression was variable, with higher levels in the liver and spleen, and lower levels in the kidney, brain, and lung [23].

While it was convincingly demonstrated that non-erythroid HRI demonstrates heme-sensitivity [23], it is also known that HRI can be activated by heme-independent mechanisms.

For example, HRI from mouse reticulocytes can be activated in response to arsenite, heat shock, or osmotic stress [24].

While reactive oxygen species are important for arsenite-induced activation of HRI, the chaperone molecules Hsp90 and Hsc70 are required for HRI activation in response to multiple stressors, as blockade of either molecule disrupts HRI activation in intact reticulocytes [24].

Modulation of HRI also results in physiologic changes in tissues beyond the erythroid lineage.

For example, in mouse hepatocytes, knockout of HRI results in increased ER stress [25], while pharmacologic activation of HRI reduces ER-stress-induced hepatic steatosis and glucose intolerance in mouse models [26].

HRI activation is associated with increased hepatic expression of fibroblast growth factor 21 (FGF21), an important protein for attenuating ER stress [27]. Deletion of HRI in mouse models also results in reduced expression of hepcidin in the liver, impaired macrophage maturation, and reduced erythrophagocytosis [28].

In neuronal cells, HRI has been found to mediate the translation of GluN2B [29], a subunit for the N-methyl-D-aspartate (NMDA) receptor important for neuronal activity [30].

Inhibition of HRI in mice impairs memory retrieval [29], while HRI-mediated translation of β-site APP cleaving enzyme-1 (BACE1) may have a physiologic role in memory consolidation [31].

The precise role of HRI, distinct from other eIF2 kinases, in tissues beyond the erythroid lineage continues to be an area of active investigation.

In summary, HRI has demonstrated the capacity for heme-dependent and heme-independent activation in erythroid and non-erythroid tissues (Table 1).

Table 1

Modulation of HRI across tissue types.

TissueHRI Deletion/InhibitionHRI Activation
ErythroidIncreased ROS; Impaired erythroid differentiation and survival [7,14]Reduced globin translation [7,14], Increased ratio of fetal to adult globin [71]
MacrophageImpaired maturation and function [28]Enhanced inflammatory response [28]
HepaticIncreased ER stress [25] Decreased hepcidin [28]Increased FGF21/Decreased fatty liver changes [26]
NeuronalImpaired memory retrieval [29]Increased BACEl [31]
CancerNot reportedInhibition of tumor growth; apoptosis [3234]

The deletion/inhibition or activation of HRI results in tissue-specific effects.

The consequence of HRI modulation in each cell type (where known) is reviewed with the relative references. HRI: heme regulated inhibitor; ROS: reactive oxygen species; ER: endoplasmic reticulum; FGF: fibroblast growth factor; BACE: beta-site amyloid precursor protein cleaving enzyme 1.

More information: Mena Abdel-Nour et al. The heme-regulated inhibitor is a cytosolic sensor of protein misfolding that controls innate immune signaling, Science (2019). DOI: 10.1126/science.aaw4144

Journal information: Science
Provided by University of Toronto


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