integrated stress response
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JCI Insight ◽  
2022 ◽  
Author(s):  
Yae-Huei Liou ◽  
Jean Personnaz ◽  
David Jacobi ◽  
Nelson H. Knudsen ◽  
Mayer M. Chalom ◽  
...  

2022 ◽  
Author(s):  
Stefanie Krug ◽  
Pankaj Prasad ◽  
Shiqi Xiao ◽  
Shichun Lun ◽  
Camilo A. Ruiz-Bedoya ◽  
...  

Tuberculosis (TB) is a devastating infectious disease that continues to cause millions of human deaths every year. Even though most cases of TB can be cured with a 6-month antibiotic combination therapy, these long treatment durations have led to the emergence of multi-drug resistance and pose a major hurdle to global TB control. Despite numerous advances in TB drug development, a substantially shortened treatment time has yet to be achieved. Given the rise in antibiotic resistance, an alternative strategy to the direct targeting of M. tuberculosis (M.tb) is the development of host-directed therapies (HDTs) that promote bacterial clearance and/or lung health when given adjunctive to standard TB antibiotics. We recently discovered that a small molecule inhibitor of the Integrated Stress Response (ISR), which is abnormally activated in TB and associated with the formation of necrotic granulomas, reduced M.tb numbers and lung inflammation in mice. Here, we evaluated the therapeutic potential of adjunctive ISR inhibition in the context of standard TB therapy. Throughout the course of treatment, ISR inhibition robustly lowered bacterial burdens compared to standard TB therapy alone and accelerated the time-to-sterility in mice, as demonstrated by significantly reduced relapse rates after 4 months of treatment. In addition, mice receiving adjunctive ISR inhibition tended to have reduced lung necrosis and inflammation. Together, our findings identify the ISR pathway as a promising therapeutic target with the potential of shortening TB treatment durations and improving lung health.


2022 ◽  
Author(s):  
Philipp Klein ◽  
Stefan M. Kallenberger ◽  
Hanna Roth ◽  
Karsten Roth ◽  
Thi Bach Nga Ly-Hartig ◽  
...  

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response, by integrating quantitative experiments with mathematical modeling, and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2alpha, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 mRNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.


RNA ◽  
2021 ◽  
pp. rna.078964.121
Author(s):  
Lisa Houston ◽  
Evan M Platten ◽  
Sara M Connelly ◽  
Jiyu Wang ◽  
Elizabeth J Grayhack

Ribosome stalls can result in ribosome collisions that elicit quality control responses, one function of which is to prevent ribosome frameshifting, an activity that entails interaction of the conserved yeast protein Mbf1 with uS3 on colliding ribosomes. However, the full spectrum of factors that mediate frameshifting during ribosome collisions is unknown. To delineate such factors in the yeast Saccharomyces cerevisiae, we used genetic selections for mutants that affect frameshifting from a known ribosome stall site, CGA codon repeats. We show that the general translation elongation factor eEF3 and the Integrated Stress Response (ISR) pathway components Gcn1 and Gcn20 modulate frameshifting in opposing manners. We found a mutant form of eEF3 that specifically suppressed frameshifting, but not translation inhibition by CGA codons. Thus, we infer that frameshifting at collided ribosomes requires eEF3, which facilitates tRNA-mRNA translocation and E-site tRNA release in yeast and other single cell organisms. By contrast, we found that removal of either Gcn1 or Gcn20, which bind collided ribosomes with Mbf1, increased frameshifting. Thus, we conclude that frameshifting is suppressed by Gcn1 and Gcn20, although these effects are not mediated primarily through activation of the ISR. Furthermore, we examined the relationship between eEF3-mediated frameshifting and other quality control mechanisms, finding that Mbf1 requires either Hel2 or Gcn1 to suppress frameshifting with wild type eEF3. Thus, these results provide evidence of a direct link between translation elongation and frameshifting at collided ribosomes, as well as evidence that frameshifting is constrained by quality control mechanisms that act on collided ribosomes.


2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jennifer Hartman ◽  
Miguel Barriera Diaz ◽  
Ronald C. Wek ◽  
Dan F. Spandau

Background and Hypothesis: Cutaneous wound healing involves: hemostatic, inflammatory, proliferative, and tissue remodeling phases. Re-epithelialization can be modeled in vitro using human keratinocytes and artificial wounds. Previous work showed undifferentiated keratinocytes closing wounds in vitro using individual cell migration (ICM), whilst differentiated keratinocytes utilize collective cell migration (KCCM). Therefore, we hypothesize that ICM in vitro is equivalent to keratinocyte migration during squamous cell carcinoma metastasis in vivo and KCCM is a model for wound re-epithelialization. Furthermore, we hypothesize that the integrated stress response (ISR) is important in ICM and KCCM. The ISR is activated by environmental stresses that protein kinases (GCN2 and PERK) can detect and phosphorylate translation factor, eIF2a. Our goal is to define how the ISR, specifically GCN2 and PERK, influence keratinocyte migration. Methods: We will evaluate in vitro wound healing and kinetic variation in protein expression and cytoskeleton remodeling. We will utilize four keratinocyte cell lines, control human keratinocyte NTERTs, and CRISPR-derived gene knockouts of GCN2, PERK, and ISR effector gene ATF4. Quantitative analysis of wound healing is accomplished using an IncuCyte ZOOM instrument. Protein expression is measured via immunoblots following high density wounding. Cytoskeletal analyses was done by immunofluorescence. Results: Preliminary results show PERK-KO and GCN2-KO cells have reduced expression of F-actin. Immunoblots showed actin-binding protein, phospho-cofilin, at lower levels in PERK-KO and GCN2-KO cells than in NTERT cells. Wound healing assays showed differentiated keratinocytes healing faster than undifferentiated in all cells, except GCN2-KO. GCN2-KO cells healed significantly slower than other differentiated cells and undifferentiated GCN2-KO cells. Wound healing assays showed undifferentiated PERK-KO cells healing slower than other undifferentiated cell lines. Conclusion/Potential Impact: The results indicate PERK and GCN2 could be key components in ICM and CCM respectfully. GCN2 and PERK could thus be potential therapeutic targets to provide cost-effective therapeutics to enhance/inhibit keratinocyte migration.


Biomedicines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1869
Author(s):  
Yu-Hsuan Fu ◽  
Chi-Yang Tseng ◽  
Jeng-Wei Lu ◽  
Wen-Hui Lu ◽  
Pei-Qi Lan ◽  
...  

Pyrvinium pamoate, a widely-used anthelmintic agent, reportedly exhibits significant anti-tumor effects in several cancers. However, the efficacy and mechanisms of pyrvinium against myeloid leukemia remain unclear. The growth inhibitory effects of pyrvinium were tested in human AML cell lines. Transcriptome analysis of Molm13 myeloid leukemia cells suggested that pyrvinium pamoate could trigger an unfolded protein response (UPR)-like pathway, including responses to extracellular stimulus [p-value = 2.78 × 10−6] and to endoplasmic reticulum stress [p-value = 8.67 × 10−7], as well as elicit metabolic reprogramming, including sulfur compound catabolic processes [p-value = 2.58 × 10−8], and responses to a redox state [p-value = 5.80 × 10−5]; on the other hand, it could elicit a pyrvinium blunted protein folding function, including protein folding [p-value = 2.10 × 10−8] and an ATP metabolic process [p-value = 3.95 × 10−4]. Subsequently, pyrvinium was verified to induce an integrated stress response (ISR), demonstrated by activation of the eIF2α-ATF4 pathway and inhibition of mTORC1 signaling, in a dose- and time-dependent manner. Additionally, pyrvinium could co-localize with mitochondria and then decrease the mitochondrial basal oxidative consumption rate, ultimately dysregulating the mitochondrial function. Similar effects were observed in cabozantinib-resistant Molm13-XR cell lines. Furthermore, pyrvinium treatment retarded Molm13 and Molm13-XR xenograft tumor growth. Thus, we concluded that pyrvinium exerts anti-tumor activity, at least, via the modulation of the mitochondrial function and by triggering ISR.


2021 ◽  
Vol 13 (623) ◽  
Author(s):  
Stefania Faletti ◽  
Daniela Osti ◽  
Elena Ceccacci ◽  
Cristina Richichi ◽  
Brunella Costanza ◽  
...  

2021 ◽  
Author(s):  
Lan Wang ◽  
Morgane Boone ◽  
Rosalie E Lawrence ◽  
Adam Frost ◽  
Peter Walter ◽  
...  

AbstractIn eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2. Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed (Schoof et al. 2021) A/I-State model of allosteric ISR regulation.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Schoof ◽  
Lan Wang ◽  
J. Zachery Cogan ◽  
Rosalie E. Lawrence ◽  
Morgane Boone ◽  
...  

AbstractViral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2’s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five ‘aromatic fingers’. NSs binding preserves eIF2B activity by maintaining eIF2B’s conformation in its active A-State.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kazuhiro Kashiwagi ◽  
Yuichi Shichino ◽  
Tatsuya Osaki ◽  
Ayako Sakamoto ◽  
Madoka Nishimoto ◽  
...  

AbstractVarious stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.


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