Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still ...unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8
T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.
In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of ...the transcription factor ATF4
. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.
Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to ...as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments in senescent cells. The activation of cGAS, in turn, triggers the production of SASP factors via stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence following irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.
Monitoring of the cytosolic compartment by the innate immune system for pathogen-encoded products or pathogen activities often enables the activation of a subset of caspases. In most cases, the ...cytosolic surveillance pathways are coupled to activation of caspase-1 via canonical inflammasome complexes. A related set of caspases, caspase-11 in rodents and caspase-4 and caspase-5 in humans, monitors the cytosol for bacterial lipopolysaccharide (LPS). Direct activation of caspase-11, caspase-4 and caspase-5 by intracellular LPS elicits the lytic cell death called 'pyroptosis', which occurs in multiple cell types. The pyroptosis is executed by the pore-forming protein GSDMD, which is activated by cleavage mediated by caspase-11, caspase-4 or caspase-5. In monocytes, formation of GSDMD pores can induce activation of the NLRP3 inflammasome for maturation of the cytokines IL-1β and IL-18. Caspase-11-mediated pyroptosis in response to cytosolic LPS is critical for antibacterial defense and septic shock. Here we review the emerging literature on the sensing of cytosolic LPS and its regulation and pathophysiological functions.
Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies
. Mitochondrial malfunction needs to be relayed to the cytosol, where an ...integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells
. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress
. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown
. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.
Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its ...role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.
Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and the maturation of interleukin-1β. Among various inflammasome complexes, the NLRP3 ...inflammasome is best characterized and has been linked with various human autoinflammatory and autoimmune diseases. Thus, the NLRP3 inflammasome may be a promising target for anti-inflammatory therapies. In this review, we summarize the current understanding of the mechanisms by which the NLRP3 inflammasome is activated in the cytosol. We also describe the binding partners of NLRP3 inftammasome complexes activating or inhibiting the inflammasome assembly. Our knowledge of the mechanisms regulating NLRP3 inflammasome signaling and how these influence inflammatory responses offers further insight into potential therapeutic strategies to treat inflammatory diseases associated with dysregulation of the NLRP3 inflammasome,
Hydrogen peroxide (H
O
) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H
O
notoriously hard to detect dynamically, ...specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H
O
sensing in living plants. We established H
O
monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry. We confirmed sensor oxidation by H
O
, show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H
O
, pharmacologically-induced H
O
release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H
O
dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants. We provided a well defined toolkit for H
O
monitoring in planta and showed that intracellular H
O
measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H
O
dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.
Biomacromolecules are highly promising therapeutic modalities to treat various diseases. However, they suffer from poor cellular membrane permeability, limiting their access to intracellular targets. ...Strategies to overcome this challenge often employ nanoscale carriers that can get trapped in endosomal compartments. Here we report conjugated peptides that form pH- and redox-responsive coacervate microdroplets by liquid-liquid phase separation that readily cross the cell membrane. A wide range of macromolecules can be quickly recruited within the microdroplets, including small peptides, enzymes as large as 430 kDa and messenger RNAs (mRNAs). The therapeutic-loaded coacervates bypass classical endocytic pathways to enter the cytosol, where they undergo glutathione-mediated release of payload, the bioactivity of which is retained in the cell, while mRNAs exhibit a high transfection efficiency. These peptide coacervates represent a promising platform for the intracellular delivery of a large palette of macromolecular therapeutics that have potential for treating various pathologies (for example, cancers and metabolic diseases) or as carriers for mRNA-based vaccines.
Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthase (cGAS) ...is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2′3′-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells.
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