Mammalian integrated stress responses in stressed organelles and their functions

Abstract

The integrated stress response (ISR) triggered in response to various cellular stress enables mammalian cells to effectively cope with diverse stressful conditions while maintaining their normal functions. Four kinases (PERK, PKR, GCN2, and HRI) of ISR regulate ISR signaling and intracellular protein translation via mediating the phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α) at Ser51. Early ISR creates an opportunity for cells to repair themselves and restore homeostasis. This effect, however, is reversed in the late stages of ISR. Currently, some studies have shown the non-negligible impact of ISR on diseases such as ischemic diseases, cognitive impairment, metabolic syndrome, cancer, vanishing white matter, etc. Hence, artificial regulation of ISR and its signaling with ISR modulators becomes a promising therapeutic strategy for relieving disease symptoms and improving clinical outcomes. Here, we provide an overview of the essential mechanisms of ISR and describe the ISR-related pathways in organelles including mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes. Meanwhile, the regulatory effects of ISR modulators and their potential application in various diseases are also enumerated.

Keywords: activating transcription factor 4; eukaryotic translation initiation factor 2; integrated stress response; organelles; protein homeostasis.

Fig. 1. PERK, GCN2, PKR, HRI are the four kinases of ISR.

In particular, PERK, located on the endoplasmic reticulum (ER) membrane, plays an irreplaceable role in ISR. External stimuli (e.g., UV light, nutrient deprivation, viral infection, proteasome inhibitors, ischemia-reperfusion, etc.) or intracellular stimuli (e.g., reactive oxygen species, heme deprivation, unfolded protein, DNA damage, etc.) activate the four phosphokinases and promote phosphorylation of eIF2α at the Ser51 position, thereby activating the ISR related signaling pathway.

Fig. 2. The eIF2B-mediated ternary complex (TC) cycle and PP1-mediated negative feedback regulatory pathway.

The eIF2-GTP-Met-tRNAi ternary complex (TC), consisting of eIF2-GTP and methionine transfer initiator RNA (Met-tRNAi), identifies the "AUG" codon in the ribosome and initiates protein translation. After the new translation program is initiated, eIF2-GDP is re-dissociated from the ribosome after codon selection and hydrolysis of GTP. And then eIF2-GDP can be converted back into eIF2-GTP through eIF2B to re-enter the assembly program of TC. The four kinases of ISR (PERK, GCN2, PKR, and HRI) promote the phosphorylation of eIF2α. P-eIF2α is a potent non-competitive inhibitor of eIF2B that inhibits GEF activity of eIF2B, thereby slowing protein synthesis. PP1 and its subunits (GADD34 and CREP) mediate the dephosphorylation of P-eIF2α, thereby inhibiting ISR-related signaling pathways. GEF guanine nucleotide exchange factor, TC, eIF2-GTP-Met-tRNAi ternary complex; PP1 protein Ser/Thr phosphatase type 1.

Fig. 3. Translation translocation of ATF4 under ISR regulation.

In the absence of eIF2α phosphorylation, the ribosome restarts the translation at uORF2 to recognize the "AUG" start codon and inhibits the ATF4 translation after translating uORF1. Activation of the ISR inhibits TC synthesis, and translation of uORF2 is inhibited by TCs deficiency, allowing the ribosome to bypass uORF2 and promote ATF4-associated mRNA transcription. TC eIF2-GTP-Met-tRNA_i ternary complex, ORF open reading frame, uORF upstream open reading frame.

Fig. 4. Interaction between ISR and mTORC1.

Both mTORC1 and ISR are essential signaling pathways that regulate protein synthesis in response to stress. The mTORC1-ATF4 pathway stimulates the expression of a subset of the ATF4 target genes induced by the ISR. mTORC1 is the overarching inducer of the integrated mitochondrial stress response (ISR^mt). In addition, ISR also inhibits the mTOR pathway through ATF4 or ATF4-independent pathways.

Fig. 5. Integrated stress responses within various organelles, including the endoplasmic reticulum (ER), mitochondria (MT), Golgi apparatus (GA), and lysosome (LYS).

The primary pathway of ISR involves the phosphorylation of eIF2α by four distinct phosphokinases (PERK, PKR, GCN2, HRI), which in turn promotes the transcription of ATF4-related genes (ATF3, ATF5, CHOP, BiP, GADD34, etc.). Additionally, ISR modulates a complex array of downstream signaling and cellular activities including autophagy (e.g., LC3), senescence (e.g., p53), apoptosis (e.g., Bax, Bcl-2, caspase-3), and metabolic reprogramming to determine the final destination of stressed cells. PERK, IRE1, and ATF6 collaboratively mediate the UPR signaling in the ER. In addition to serving as a UPR sensor, PERK also plays a crucial role as a phosphokinase for ISR. The ATF4-CHOParm of ISR is closely related to MSR and apoptosis. Mitochondrial LONP1 can degrade Parkin1 and block Parkin1-mediated mitophagy. Furthermore, the complex composed of LONP1, HSP60, and mtHSP70 has been identified as an important component of ER stress signal transmission to mitochondria. During mitochondrial dysfunction, the OMA1-DELE1-HRI-arm can fine-tune ISR in reverse. In addition, incomplete mitochondrial outer membrane penetration (IMOMP) leads to the production of cytochrome c by BAX, BOK, and BAK, which can also activate HRI. ClpXP not only recognizes unfolded proteins but also acts in reverse on eIF2α. TRAP1 is an important member of the HSP90 family of mitochondrial molecular chaperone proteins. Mitokines (e.g., GDF15, FGF21) are cell-non-autonomous factors released by local mitochondria upon mitochondrial dysfunction and play an important role in regulating immune response and inflammation. The Golgi stressor monensin stimulates CSE to generate a Golgi stress response through the PERK/ATF4 arm, which also induces LAMP3 formation in the lysosome. Note: black solid line - main pathway; black dashed line - anterograde auxiliary pathway; red solid line - anti-regulatory pathway; green solid line - ATF4 auxiliary pathway; Gray icon - the other two paths of the UPR.

Fig. 6. ISR modulators in diseases.

The ISR process entails eIF2α phosphorylation and downstream gene transcription. While the early stage of ISR is beneficial for maintaining cellular function, the late stage of ISR may induce cell damage. When the stress-induced damage exceeds the regulatory capacity of ISR, the effects of ISR are reversed. ISRIB, Salubrinal, Sephin1, GSK2606414, GSK2656157, ONC201, Guanabenz, CCT020312, and 2BAct are all examples of effective ISR modulators that primarily target upstream or negative feedback loops of ISR to regulate extensive and complex downstream pathways.