Cellular Stress: Modulator of Regulated Cell Death

Abstract

Cellular stress response activates a complex program of an adaptive response called integrated stress response (ISR) that can allow a cell to survive in the presence of stressors. ISR reprograms gene expression to increase the transcription and translation of stress response genes while repressing the translation of most proteins to reduce the metabolic burden. In some cases, ISR activation can lead to the assembly of a cytoplasmic membraneless compartment called stress granules (SGs). ISR and SGs can inhibit apoptosis, pyroptosis, and necroptosis, suggesting that they guard against uncontrolled regulated cell death (RCD) to promote organismal homeostasis. However, ISR and SGs also allow cancer cells to survive in stressful environments, including hypoxia and during chemotherapy. Therefore, there is a great need to understand the molecular mechanism of the crosstalk between ISR and RCD. This is an active area of research and is expected to be relevant to a range of human diseases. In this review, we provided an overview of the interplay between different cellular stress responses and RCD pathways and their modulation in health and disease.

Keywords: GCN2; HRI; PERK; PKR; apoptosis; integrated stress response; necroptosis; programmed cell death; pyroptosis; stress granules.

Figure 1

Regulation of stress kinases: (A) Heme deficiency, oxidative stress, heat shock, starvation, viral infection, and ER stressors activate HRI, GCN2, PKR and PERK kinases, respectively. Kinase activation phosphorylates eIF2α leading to attenuation of cap dependent translation [23,24,25,26]. (B) Cellular stress leads to an increase in local concentration of mRNAs and ribonucleoproteins. RNA binding proteins such as T-cell intracellular antigen 1 (TIA1), GTPase-activating protein-(SH3 domain)-binding protein1 (G3BP1) and poly(A) binding proteins (PABPs) act as nucleation factors for stress granule (SG) assembly [36,37,38]. SGs can sequester proteins involved in apoptosis [4,10] and pyroptosis [9] to inhibit them. ISR can inhibit necroptosis by reducing RIPK3 mediated phosphorylation of MLKL through an unknown mechanism. ER stress activates stress kinase PERK [11] and inhibits phosphorylation of RIPK1 at S166, which inhibits downstream signal transduction to RIPK3 and MLKL, restricting necroptosis [11].

Figure 2

Regulated cell death pathways: (A) Apoptosis. Ligand binding induces oligomerization of death receptors activating the extrinsic apoptosis cascade. Tumor necrosis factor receptor 1 (TNFR1), TNFR1/2-associated death domain protein (TRADD), Fas-associated death domain protein (FADD) and pro-caspase-8 are recruited to the oligomerized death receptors forming death-inducing signaling cascade (DISC). Autoproteolytic processing of pro-caspase 8 generates catalytically active initiator caspase 8, which cleaves executioner caspases, caspase-3 and caspase-7, completing extrinsic apoptosis [41,42,43]. Intrinsic apoptosis is triggered by various intracellular danger signals that induce an increase in mitochondrial outer membrane permeabilization (MOMPs). An increase in MOMPs releases cytochrome c in the cytosol. Cytochrome c binds with APAF1 and pro-caspase-9 forming an apoptosome signaling complex. Autoproteolytic processing of pro-caspase-9 in the apoptosome generates catalytically active initiator caspase-9, which cleaves executioner caspases, caspase-3 and caspase-7, leading to intrinsic apoptosis [41,42,43]. (B) Pyroptosis. Diverse PAMPs and DAMPs are sensed by different inflammasome sensors, NLRP1 [44], NLRP3 [45,46,47,48,49,50,51], NLRC4 [52,53,54], PYRIN [55,56], and AIM2 [57,58,59,60]. Activated inflammasome sensors mediate ASC (apoptosis-associated speck-like protein containing CARD) dependent recruitment of pro-Caspase 1 and subsequent autoprocessing generating active caspase-1 [61,62,63,64]. Active caspase-1 cleaves pro-interleukin-1β (proIL-1β) [65,66,67], pro-IL-18 [68,69] and Gasdermin D (GSDMD) [70,71]. The N-terminal fragment of GSDMD forms pyroptotic pores through which IL-1β and IL-18, along with intracellular content, are released [70,72]. (C) Necroptosis. TNFR1 signaling in the absence of caspase-8 recruits RIPK1 (receptor-interacting protein kinase 1) and RIPK3 forming a necroptosis-inducing complex called the necrosome [42,73]. MLKL (mixed lineage kinase domain-like pseudokinase) is phosphorylated at the necrosome and translocates to the cell membrane to form pores that cause osmotic lysis of cells [74,75,76].

Figure 3

Domain organization of stress kinases. The domain architecture of the four mammalian eIF2α kinases is represented as bars. GCN2 is 1649 amino acids long and has an N-terminal RWD domain (RING finger-containing protein, WD repeat-containing protein and yeast DEAD-like helicase), pseudokinase domain, catalytically active kinase domain (KD), histidyl-tRNA synthetase like (HisRS) domain and C-terminal domain (CTD) [80,81]. PKR is 551 amino acids long and has an N-terminal regulatory domain joined by a linker domain to the catalytic C-terminal kinase domain. The regulatory domain has two double-stranded RNA (dsRNA) binding motifs; dRBM1 and dRBM2 [28,85]. PERK is 1116 amino acids long and consists of an N-terminal regulatory lumenal domain and a C-terminal cytosolic kinase domain [86]. HRI is 630 amino acids in length and has an N-terminal domain, a central regulatory kinase insertion (KI) domain flanked by two kinase domains (Kinase I and Kinase II) and the C-terminal domain [86,87].

Figure 4

Stress kinases in modulation of apoptosis in tumors. (A) Tumor cells have increased expression of GCN2 [90,91,92,93,94,95]. Nutrient deprivation in tumor microenvironment (TME) increases the pool of uncharged tRNAs, which binds to HisRS domain of GCN2. Activated GCN2 phosphorylates eIF2α leading to translational inhibition and promoting survival of tumor cells. In contrast, inhibition of GCN2 by anti-tumor drugs promoted tumor cell death [95,96]. (B) dsRNA generated from diverse cellular processes within the TME activates PKR. PKR activation can have tumorigenic [28,97,98,99,100,101,102] as well as tumor-suppressive roles [103,104,105,106]. How PKR promotes tumorigenesis is unknown. PKR-dependent eIF2α phosphorylation mediates apoptosis of the tumor cells [105,106,107] and promotes tumor suppression. (C) Stress in TME leads to accumulation of unfolded or misfolded proteins [108], which translocate to the endoplasmic reticulum (ER). An increase in misfolded proteins in the ER leads to dissociation of immunoglobulin binding protein (BiP) from the PERK and subsequently activates PERK [109]. Activated PERK phosphorylates eIF2α leading to formation of SGs. In addition, chemotherapeutic agents also favor eIF2α phosphorylation mediated formation of SGs [110,111,112,113]. SGs formation stalls translation at the initiation step, inhibits apoptosis and favors tumor cell survival. (D) Compounds such as N,N′-diarylureas activate HRI leading to eIF2α phosphorylation-dependent inhibition of oncogenesis [114,115,116]. Chemotherapeutic agents induce incomplete mitochondrial outer membrane permeabilization (iMOMPs), liberating suboptimal cytochrome c, which activates HRI kinase [117]. HRI kinase-mediated translational reprogramming generates drug-resistant cancer cells that promote tumorigenesis.

Figure 5

Stress kinases in modulation of viral infection outcome. (A) Virus infection activates GCN2 leading to eIF2α phosphorylation mediated translation shutdown, which inhibits virus replication [121,122]. In contrast, HIV-1 proteases cleave GCN2, abrogating eIF2α phosphorylation mediated translation inhibition and promoting virus growth [122]. Moreover, GCN2 phosphorylation during SARS coronavirus 2 infection increases expression of the ACE2 (Angiotensin-converting enzyme 2) receptor [120] that increases SARS-CoV2 infectivity. (B) dsRNA generated during virus replication activates PKR. PKR-mediated eIF2α phosphorylation inhibits translation leading to the formation of stress granules favoring virus replication through poorly explored mechanisms. Rift valley fever virus [123,124] and mouse adenovirus type I [125] routes activated PKR for proteasomal degradation to favor its replication. Viral proteins such as influenza virus nonstructural protein 1 (NS1) [126], MERS-CoV p4a protein [127], mammalian orthoreovirus σ3 protein [128], and vaccinia virus E3L protein [129] sequestered viral dsRNA away from PKR abrogating PKR activation to favor virus replication. (C) Profound virus replication generates unfolded or misfolded proteins which translocate to the endoplasmic reticulum (ER). An increase in misfolded proteins in the ER leads to dissociation of immunoglobulin binding protein (BiP) from the PERK [109]. Hepatitis C virus E2 protein [130] and human cytomegalovirus TRSI protein [131] inhibit the kinase activity of PERK, abrogating eIF2α phosphorylation and favoring virus replication. The bovine viral diarrhea virus mediates PERK-dependent inhibition of anti-apoptotic protein Bcl-2, promoting apoptosis of infected cells [132]. In contrast, Japanese encephalitis virus induces stress granules in a PERK-dependent manner to inhibit apoptosis and promote virus replication [133]. In (B,C), each number represents the unique pathway being followed (example: number 1 is one pathway while number 2 represents the other pathway).

Figure 6

RCD pathways and Stress kinases. (A) M. tuberculosis [167] and A. fumigatus [168] infection induces ER stress-mediated PERK phosphorylation. Double-stranded RNA (dsRNA) phosphorylated PKR [169]. Phosphorylated PERK and PKR can activate inflammasomes leading to pyroptosis [167,168,169]. In contrast, amino acid depletion during inflammation phosphorylates GCN2, which suppresses inflammasome activation, reducing pyroptosis [170]. Additionally, SGs formed in response to several stressors, including arsenite-induced oxidative stress sequester DDX3X and inhibit NLRP3 inflammasome activation [9]; however, whether HRI alone can suppress activation of the NLRP3 inflammasome or other stress kinases are also capable is unclear. (B) Oxidative stress phosphorylates HRI leading to translation arrest and formation of SGs. SGs act as a site for necrosome assembly that activates necroptosis [40]. (C) Selenium deficiency sensitizes cells to ferroptosis [171,172]; uncharged tRNA during selenium deficiency might activate the amino acid sensor GCN2 that possibly modulates the outcome of ferroptosis. Oxidative stress induces MESH1 (Metazoan SpoT Homologue 1), leading to cell death by ferroptosis [173,174]. In addition, MESH1 can inhibit PERK activation [174]; the crosstalk between PERK activation and ferroptosis is yet to be discovered. Cellular iron accumulation activates ferroptosis [175,176]. How stress kinases that sense oxidative stress or heme interconnect cellular stress with ferroptosis is unknown.