Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adapter ligation, to comprehensively interrogate the human transcriptome at single-molecule and -nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on deadenylation or decapping. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome while inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 rescues RNA length. Our findings reveal RNA decay as a key component of RNA metabolism upon cellular stress that is dependent on stress granule formation.
- Klíčová slova
- RNA decay, cell biology, cell line, genetics, genomics, human, mouse, stress response,
- MeSH
- adaptorové proteiny signální transdukční metabolismus genetika MeSH
- DNA-helikasy metabolismus genetika MeSH
- exoribonukleasy * metabolismus genetika MeSH
- fyziologický stres * genetika MeSH
- lidé MeSH
- proteiny asociované s mikrotubuly MeSH
- proteiny vázající poly-ADP-ribosu * metabolismus genetika MeSH
- proteiny vázající RNA MeSH
- ribozomy metabolismus MeSH
- RNA-helikasy metabolismus genetika MeSH
- RRM proteiny * metabolismus genetika MeSH
- sekvenční analýza RNA * MeSH
- stabilita RNA * genetika MeSH
- stresová tělíska metabolismus genetika MeSH
- transkriptom MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adaptorové proteiny signální transdukční MeSH
- DNA-helikasy MeSH
- exoribonukleasy * MeSH
- G3BP1 protein, human MeSH Prohlížeč
- G3BP2 protein, human MeSH Prohlížeč
- proteiny asociované s mikrotubuly MeSH
- proteiny vázající poly-ADP-ribosu * MeSH
- proteiny vázající RNA MeSH
- RNA-helikasy MeSH
- RRM proteiny * MeSH
- XRN1 protein, human MeSH Prohlížeč
Nearly all aerobic organisms are equipped with catalases, powerful enzymes scavenging hydrogen peroxide and facilitating defense against harmful reactive oxygen species. In trypanosomatids, this enzyme was not present in the common ancestor, yet it had been independently acquired by different lineages of monoxenous trypanosomatids from different bacteria at least three times. This observation posited an obvious question: why was catalase so "sought after" if many trypanosomatid groups do just fine without it? In this work, we analyzed subcellular localization and function of catalase in Leptomonas seymouri. We demonstrated that this enzyme is present in the cytoplasm and a subset of glycosomes, and that its cytoplasmic retention is H2O2-dependent. The ablation of catalase in this parasite is not detrimental in vivo, while its overexpression resulted in a substantially higher parasite load in the experimental infection of Dysdercus peruvianus. We propose that the capacity of studied flagellates to modulate the catalase activity in the midgut of its insect host facilitates their development and protects them from oxidative damage at elevated temperatures.
- Klíčová slova
- Catalase, Hydrogen peroxide, Trypanosomatids,
- MeSH
- cytoplazma MeSH
- katalasa * metabolismus MeSH
- mikrotělíska metabolismus MeSH
- peroxid vodíku * metabolismus MeSH
- Trypanosomatina * enzymologie genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- katalasa * MeSH
- peroxid vodíku * MeSH
The ectoparasitic mite Varroa destructor transmits and triggers viral infections that have deleterious effects on honey bee colonies worldwide. We performed a manipulative experiment in which worker bees collected at emergence were exposed to Varroa for 72 h, and their proteomes were compared with those of untreated control bees. Label-free quantitative proteomics identified 77 differentially expressed A. mellifera proteins (DEPs). In addition, viral proteins were identified by orthogonal analysis, and most importantly, Deformed wing virus (DWV) was found at high levels/intensity in Varroa-exposed bees. Pathway enrichment analysis suggested that the main pathways affected included peroxisomal metabolism, cyto-/exoskeleton reorganization, and cuticular proteins. Detailed examination of individual DEPs revealed that additional changes in DEPs were associated with peroxisomal function. In addition, the proteome data support the importance of TGF-β signaling in Varroa-DWV interaction and the involvement of the mTORC1 and Hippo pathways. These results suggest that the effect of DWV on bees associated with Varroa feeding results in aberrant autophagy. In particular, autophagy is selectively modulated by peroxisomes, to which the observed proteome changes strongly corresponded. This study complements previous research with different study designs and suggests the importance of the peroxisome, which plays a key role in viral infections.
- Klíčová slova
- Apis mellifera, DWV, autophagy, host‐pathogen interaction, lipid metabolism,
- MeSH
- hmyzí proteiny metabolismus MeSH
- interakce hostitele a parazita MeSH
- peroxizomy * metabolismus virologie MeSH
- proteom metabolismus analýza MeSH
- proteomika metody MeSH
- RNA-viry * fyziologie MeSH
- signální transdukce MeSH
- Varroidae * virologie MeSH
- včely virologie parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- hmyzí proteiny MeSH
- proteom MeSH
PURPOSE: Zellweger spectrum disorders (ZSDs) are known as autosomal recessive disorders caused by defective peroxisome biogenesis due to bi-allelic pathogenic variants in any of at least 13 different PEX genes. Here, we report 2 unrelated patients who present with an autosomal dominant ZSD. METHODS: We performed biochemical and genetic studies in blood and skin fibroblasts of the patients and demonstrated the pathogenicity of the identified PEX14 variants by functional cell studies. RESULTS: We identified 2 different single heterozygous de novo variants in the PEX14 genes of 2 patients diagnosed with ZSD. Both variants cause messenger RNA mis-splicing, leading to stable expression of similar C-terminally truncated PEX14 proteins. Functional studies indicated that the truncated PEX14 proteins lost their function in peroxisomal matrix protein import and cause increased degradation of peroxisomes, ie, pexophagy, thus exerting a dominant-negative effect on peroxisome functioning. Inhibition of pexophagy by different autophagy inhibitors or genetic knockdown of the peroxisomal autophagy receptor NBR1 resulted in restoration of peroxisomal functions in the patients' fibroblasts. CONCLUSION: Our finding of an autosomal dominant ZSD expands the genetic repertoire of ZSDs. Our study underscores that single heterozygous variants should not be ignored as possible genetic cause of diseases with an established autosomal recessive mode of inheritance.
- Klíčová slova
- Autophagy, Metabolic disorder, Peroxisomal disorder, Peroxisome, Peroxisome biogenesis,
- MeSH
- alely MeSH
- lidé MeSH
- peroxizomy genetika metabolismus MeSH
- proteiny genetika MeSH
- transport proteinů fyziologie MeSH
- Zellwegerův syndrom * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- PEX14 protein, human MeSH Prohlížeč
- proteiny MeSH
Actin-related protein (ARP2/3) complex is a heteroheptameric protein complex, evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. Here we demonstrate that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile spot-like structures in the cytoplasm that are associated with peroxisomes in Arabidopsis and tobacco. ARP2/3 is found at the peroxisome periphery and contains the assembled ARP2/3 complex and the WAVE/SCAR complex subunit NAP1. This ARP2/3-positive peroxisomal domain colocalizes with the autophagosome and, under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f and peroxisome-associated ARP2/3 interact in vivo with the ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than wild type, we suggest that ARP2/3 has a novel role in the process of peroxisome degradation by autophagy, called pexophagy.
- MeSH
- aktiny MeSH
- Arabidopsis * metabolismus MeSH
- komplex proteinů 2-3 souvisejících s aktinem metabolismus MeSH
- makroautofagie MeSH
- peroxizomy metabolismus MeSH
- proteiny huseníčku * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aktiny MeSH
- komplex proteinů 2-3 souvisejících s aktinem MeSH
- proteiny huseníčku * MeSH
Meiosis, at the transition between diploid and haploid life cycle phases, is accompanied by reprograming of cell division machinery and followed by a transition back to mitosis. We show that, in Arabidopsis, this transition is driven by inhibition of translation, achieved by a mechanism that involves processing bodies (P-bodies). During the second meiotic division, the meiosis-specific protein THREE-DIVISION MUTANT 1 (TDM1) is incorporated into P-bodies through interaction with SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA 7 (SMG7). TDM1 attracts eIF4F, the main translation initiation complex, temporarily sequestering it in P-bodies and inhibiting translation. The failure of tdm1 mutants to terminate meiosis can be overcome by chemical inhibition of translation. We propose that TDM1-containing P-bodies down-regulate expression of meiotic transcripts to facilitate transition of cell fates to postmeiotic gametophyte differentiation.
- MeSH
- Arabidopsis * cytologie genetika MeSH
- buněčná diferenciace MeSH
- cykliny * genetika metabolismus MeSH
- meióza * genetika MeSH
- mitóza MeSH
- procesní tělíska * metabolismus MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- proteosyntéza MeSH
- transportní proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cykliny * MeSH
- proteiny huseníčku * MeSH
- SMG7 protein, Arabidopsis MeSH Prohlížeč
- TDM1 protein, Arabidopsis MeSH Prohlížeč
- transportní proteiny MeSH
The formation of stress granules (SGs), membrane-less organelles that are composed of mainly messenger ribonucleoprotein assemblies, is the result of a conserved evolutionary strategy to cellular stress. During their formation, which is triggered by robust environmental stress, SGs sequester translationally inactive mRNA molecules, which are either forwarded for further processing elsewhere or stored during a period of stress within SGs. Removal of mRNA molecules from active translation and their sequestration in SGs allows preferential translation of stress response transcripts. By affecting the specificity of mRNA translation, mRNA localization and stability, SGs are involved in the overall cellular reprogramming during periods of environmental stress and viral infection. Over the past two decades, we have learned which processes drive SGs assembly, how their composition varies under stress, and how they co-exist with other subcellular organelles. Yeast as a model has been instrumental in our understanding of SG biology. Despite the specific differences between the SGs of yeast and mammals, yeast have been shown to be a valuable tool to the study of SGs in translation-related stress response. This review summarizes the data surrounding SGs that are formed under different stress conditions in Saccharomyces cerevisiae and other yeast species. It offers a comprehensive and up-to-date view on these still somewhat mysterious entities.
- Klíčová slova
- P-bodies, mRNA, stress granules, stress response, translation, yeast,
- MeSH
- cytoplazmatická granula * fyziologie MeSH
- fyziologický stres MeSH
- messenger RNA genetika MeSH
- Saccharomyces cerevisiae * genetika MeSH
- savci genetika MeSH
- stresová tělíska MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- messenger RNA MeSH
BACKGROUND: Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. RESULTS: We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5'-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. CONCLUSIONS: This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.
- Klíčová slova
- Anaerobic peroxisome, Anaerobiosis, FeS cluster assembly, Hydrogenosome, Mitochondrion-related organelle, Pelomyxa, Single-cell genomics,
- MeSH
- Amoeba * genetika metabolismus MeSH
- anaerobióza MeSH
- Archamoebae * genetika metabolismus MeSH
- genomika MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- peroxizomy metabolismus MeSH
- Saccharomyces cerevisiae MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stress granules (SGs) are among the most studied membraneless organelles that form upon heat stress (HS) to sequester unfolded, misfolded, or aggregated protein, supporting protein quality control (PQC) clearance. The folding states that are primarily associated with SGs, as well as the function of the phase separated environment in adjusting the energy landscapes, remain unknown. Here, we investigate the association of superoxide dismutase 1 (SOD1) proteins with different folding stabilities and aggregation propensities with condensates in cells, in vitro and by simulation. We find that irrespective of aggregation the folding stability determines the association of SOD1 with SGs in cells. In vitro and in silico experiments however suggest that the increased flexibility of the unfolded state constitutes only a minor driving force to associate with the dynamic biomolecular network of the condensate. Specific protein-protein interactions in the cytoplasm in comparison to SGs determine the partitioning of folding states between the respective phases during HS.
- MeSH
- HeLa buňky MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- rozbalení proteinů MeSH
- stabilita proteinů MeSH
- stresová tělíska metabolismus MeSH
- superoxiddismutasa 1 metabolismus MeSH
- změna skupenství MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- SOD1 protein, human MeSH Prohlížeč
- superoxiddismutasa 1 MeSH
Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.
- MeSH
- anaerobióza MeSH
- Entamoeba histolytica metabolismus MeSH
- fylogeneze MeSH
- inositol metabolismus MeSH
- mutace * MeSH
- peroxiny metabolismus MeSH
- peroxizomální cílové signály * MeSH
- peroxizomy metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- inositol MeSH
- peroxiny MeSH
- peroxizomální cílové signály * MeSH
- protozoální proteiny MeSH
