Eukaryotic Stress Research Articles

Rational Tuning of the Concentration-independent Enrichment of Prion-like Domains in Stress Granules

Stress granules (SGs) are large ribonucleoprotein assemblies that form in response to acute stress in eukaryotes. SG formation is thought to be initiated by liquid–liquid phase separation (LLPS) of key proteins and RNA. These molecules serve as a scaffold for recruitment of client molecules. LLPS of scaffold proteins in vitro is highly concentration-dependent, yet biomolecular condensates in vivo contain hundreds of unique proteins, most of which are thought to be clients rather than scaffolds. Many proteins that localize to SGs contain low-complexity, prion-like domains (PrLDs) that have been implicated in LLPS and SG recruitment. The degree of enrichment of proteins in biomolecular condensates such as SGs can vary widely, but the underlying basis for these differences is not fully understood. Here, we develop a toolkit of model PrLDs to examine the factors that govern efficiency of PrLD recruitment to stress granules. Recruitment was highly sensitive to amino acid composition: enrichment in SGs could be tuned through subtle changes in hydrophobicity. By contrast, SG recruitment was largely insensitive to PrLD concentration at both a population level and single-cell level. These observations point to a model wherein PrLDs are enriched in SGs through either simple solvation effects or interactions that are effectively non-saturable even at high expression levels.

The NS2B-PP1α-eIF2α axis: Inhibiting stress granule formation and Boosting Zika virus replication.

Stress granules (SGs), formed by untranslated messenger ribonucleoproteins (mRNPs) during cellular stress in eukaryotes, have been linked to flavivirus interference without clear understanding. This study reveals the role of Zika virus (ZIKV) NS2B as a scaffold protein mediating interaction between protein phosphatase 1α (PP1α) and eukaryotic initiation factor 2α (eIF2α). This interaction promotes eIF2α dephosphorylation by PP1α, inhibiting SG formation. The NS2B-PP1α complex exhibits remarkable stability, resisting ubiquitin-induced degradation and amplifying eIF2α dephosphorylation, thus promoting ZIKV replication. In contrast, the NS2BV35A mutant, interacting exclusively with eIF2α, fails to inhibit SG formation, resulting in reduced viral replication and diminished impact on brain organoid growth. These findings reveal PP1α's dual role in ZIKV infection, inducing interferon production as an antiviral factor and suppressing SG formation as a viral promoter. Moreover, we found that NS2B also serves as a versatile mechanism employed by flaviviruses to counter host antiviral defenses, primarily by broadly inhibiting SG formation. This research advances our comprehension of the complex interplay in flavivirus-host interactions, offering potential for innovative therapeutic strategies against flavivirus infections.

Endoplasmic reticulum: Monitoring and maintaining protein and membrane homeostasis in the endoplasmic reticulum by the unfolded protein response

The endoplasmic reticulum (ER) regulates essential cellular processes, including protein folding, lipid synthesis, and calcium homeostasis. The ER homeostasis is maintained by a conserved set of signaling cascades called the Unfolded Protein Response (UPR). How the UPR senses perturbations in ER homeostasis has been the subject of active research for decades. In metazoans, the UPR consists of three ER-membrane embedded sensors: IRE1, PERK and ATF6. These sensors detect the accumulation of misfolded proteins in the ER lumen and adjust protein folding capacity according to cellular needs. Early work revealed that the ER-resident chaperone BiP binds to all three UPR sensors in higher eukaryotes and BiP binding was suggested to regulate their activity. More recent data have shown that in higher eukaryotes the interaction of the UPR sensors with a complex network of chaperones and misfolded proteins modulates their activation and deactivation dynamics. Furthermore, emerging evidence suggests that the UPR monitors ER membrane integrity beyond protein folding defects. However, the mechanistic and structural basis of UPR activation by proteotoxic and lipid bilayer stress in higher eukaryotes remains only partially understood. Here, we review the current understanding of novel protein interaction networks and the contribution of the lipid membrane environment to UPR activation.

An adaptive biomolecular condensation response is conserved across environmentally divergent species

Cells must sense and respond to sudden maladaptive environmental changes—stresses—to survive and thrive. Across eukaryotes, stresses such as heat shock trigger conserved responses: growth arrest, a specific transcriptional response, and biomolecular condensation of protein and mRNA into structures known as stress granules under severe stress. The composition, formation mechanism, adaptive significance, and even evolutionary conservation of these condensed structures remain enigmatic. Here we provide a remarkable view into stress-triggered condensation, its evolutionary conservation and tuning, and its integration into other well-studied aspects of the stress response. Using three morphologically near-identical budding yeast species adapted to different thermal environments and diverged by up to 100 million years, we show that proteome-scale biomolecular condensation is tuned to species-specific thermal niches, closely tracking corresponding growth and transcriptional responses. In each species, poly(A)-binding protein—a core marker of stress granules—condenses in isolation at species-specific temperatures, with conserved molecular features and conformational changes modulating condensation. From the ecological to the molecular scale, our results reveal previously unappreciated levels of evolutionary selection in the eukaryotic stress response, while establishing a rich, tractable system for further inquiry.

Dynamic DNA N 6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus.

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N 6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

Characterization of GshAB of Tetragenococcus halophilus: a two-domain glutathione synthetase.

The γ-glutamyl tripeptide glutathione (γ-Glu-Cys-Gly) is a low molecular thiol that acts as antioxidant in response to oxidative stress in eukaryotes and prokaryotes. γ-Glutamyl dipeptides including γ-Glu-Cys, γ-Glu-Glu, and γ-Glu-Gly also have kokumi activity. Glutathione is synthesized by first ligating Glu with Cys by γ-glutamylcysteine ligase (Gcl/GshA), and then the resulting dipeptide γ-glutamylcysteine is ligated with Gly by glutathione synthetase (Gs/GshB). GshAB/GshF enzymes that contain both Gcl and Gs domains are capable of catalyzing both reactions. The current study aimed to characterize GshAB from Tetragenococcus halophilus after heterologous expression in Escherichia coli. The optimal conditions for GshAB from T. halophilus were pH 8.0 and 25 °C. The substrate specificity of the Gcl reaction of GshAB was also determined. GshAB has a high affinity to Cys. γ-Glu-Cys was the only dipeptide generated when Glu, Cys, Gly, and other amino acids were present in the reaction system. This specificity differentiates GshAB from T. halophilus from Gcl of heterofermentative lactobacilli and GshAB of Streptococcus agalactiae, which also use amino acids other than Cys as glutamyl-acceptor. Quantification of gshAB in cDNA libraries from T. halophilus revealed that gshAB was overexpressed in response to oxidative stress but not in response to acid, osmotic, or cold stress. In conclusion, GshAB in T. halophilus served as part of the oxidative stress response but this study did not provide any evidence for a contribution to the resistance to other stressors.Key points Glutathione synthesis in Tetragenococcus halophilus is carried out by the two-domain enzyme GshAB. GshAB is inhibited by glutathione and is highly specific for Cys as acceptor. T. halophilus synthesizes glutathione in response to oxidative stress.

Modification of Phosphorylation Sites in the Yeast Lysine Methyltransferase Set5 Exerts Influences on the Mitogen-Activated Protein Kinase Hog1 under Prolonged Acetic Acid Stress.

Responses to acetic acid toxicity in the budding yeast Saccharomyces cerevisiae have widespread implications in the biorefinery of lignocellulosic biomass and food preservation. Our previous studies revealed that Set5, the yeast lysine methyltransferase and histone H4 methyltransferase, was involved in acetic acid stress tolerance. However, it is still mysterious how Set5 functions and interacts with the known stress signaling network. Here, we revealed that elevated phosphorylation of Set5 during acetic acid stress is accompanied by enhanced expression of the mitogen-activated protein kinase (MAPK) Hog1. Further experiments uncovered that the phosphomimetic mutation of Set5 endowed yeast cells with improved growth and fermentation performance and altered transcription of specific stress-responsive genes. Intriguingly, Set5 was found to bind the coding region of HOG1 and regulate its transcription, along with increased expression and phosphorylation of Hog1. A protein-protein interaction between Set5 and Hog1 was also revealed. In addition, modification of Set5 phosphosites was shown to regulate reactive oxygen species (ROS) accumulation, which is known to affect yeast acetic acid stress tolerance. The findings in this study imply that Set5 may function together with the central kinase Hog1 to coordinate cell growth and metabolism in response to stress. IMPORTANCE Hog1 is the yeast homolog of p38 MAPK in mammals that is conserved across eukaryotes, and it plays crucial roles in stress tolerance, fungal pathogenesis, and disease treatments. Here, we provide evidence that modification of Set5 phosphorylation sites regulates the expression and phosphorylation of Hog1, which expands current knowledge on upstream regulation of the Hog1 stress signaling network. Set5 and its homologous proteins are present in humans and various eukaryotes. The newly identified effects of Set5 phosphorylation site modifications in this study benefit an in-depth understanding of eukaryotic stress signaling, as well as the treatment of human diseases.

Stress granule homeostasis is modulated by TRIM21-mediated ubiquitination of G3BP1 and autophagy-dependent elimination of stress granules

ABSTRACT Eukaryotic stress granules (SGs) are highly dynamic assemblies of untranslated mRNAs and proteins that form through liquid-liquid phase separation (LLPS) under cellular stress. SG formation and elimination process is a conserved cellular strategy to promote cell survival, although the precise regulation of this process is poorly understood. Here, we screened six E3 ubiquitin ligases present in SGs and identified TRIM21 (tripartite motif containing 21) as a central regulator of SG homeostasis that is highly enriched in SGs of cells under arsenite-induced oxidative stress. Knockdown of TRIM21 promotes SG formation whereas overexpression of TRIM21 inhibits the formation of physiological and pathological SGs associated with neurodegenerative diseases. TRIM21 catalyzes K63-linked ubiquitination of the SG core protein, G3BP1 (G3BP stress granule assembly factor 1), and G3BP1 ubiquitination can effectively inhibit LLPS, in vitro. Recent reports suggested the involvement of macroautophagy/autophagy, as a stress response pathway, in the regulation of SG homeostasis. We systematically investigated well-defined autophagy receptors and identified SQSTM1/p62 (sequestosome 1) and CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2) as the primary receptors that directly interact with G3BP1 during arsenite-induced stress. Endogenous SQSTM1 and CALCOCO2 localize to the periphery of SGs under oxidative stress and mediate SG elimination, as single knockout of each receptor causes accumulation of physiological and pathological SGs. Collectively, our study broadens the understanding in the regulation of SG homeostasis by showing that TRIM21 and autophagy receptors modulate SG formation and elimination respectively, suggesting the possibility of clinical targeting of these molecules in therapeutic strategies for neurodegenerative diseases. Abbreviations: ACTB: actin beta; ALS: amyotrophic lateral sclerosis; BafA1: bafilomycin A1; BECN1: beclin 1; C9orf72: C9orf72-SMCR8 complex subunit; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Co-IP: co-immunoprecipitation; DAPI: 4’,6-diamidino-2-phenylindole; FTD: frontotemporal dementia; FUS: FUS RNA binding protein; G3BP1: G3BP stress granule assembly factor 1; GFP: green fluorescent protein; LLPS: liquid-liquid phase separation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; NES: nuclear export signal; OPTN: optineurin; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; SG: stress granule; TAX1BP1: Tax1 binding protein 1; TOLLIP: toll interacting protein; TRIM21: tripartite motif containing 21; TRIM56: tripartite motif containing 56; UB: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Methylarginine efflux in nutrient-deprived yeast mitigates disruption of nitric oxide synthesis.

Protein arginine N-methyltransferases (PRMTs) have emerged as important actors in the eukaryotic stress response with implications in human disease, aging, and cell signaling. Intracellular free methylarginines contribute to cellular stress through their interaction with nitric oxide synthase (NOS). The arginine-dependent production of nitric oxide (NO), which is strongly inhibited by methylarginines, serves as a protective small molecule against oxidative stress in eukaryotic cells. NO signaling is highly conserved between higher and lower eukaryotes, although a canonical NOS homologue has yet to be identified in yeast. Since stress signaling pathways are well conserved among eukaryotes, yeast is an ideal model organism to study the implications of PRMTs and methylarginines during stress. We sought to explore the roles and fates of methylarginines in Saccharomyces cerevisiae. We starved methyltransferase-, autophagy-, and permease-related yeast knockouts by incubating them in water and monitored methylarginine production. We found that under starvation, methylarginines are expelled from yeast cells. We found that autophagy-deficient cells have an impaired ability to efflux methylarginines, which suggests that methylarginine-containing proteins are degraded via autophagy. For the first time, we determine that yeast take up methylarginines less readily than arginine, and we show that methylarginines impact yeast NO production. This study reveals that yeast circumvent a potential methylarginine toxicity by expelling them after autophagic degradation of arginine-modified proteins.

Eukaryotic stress-induced mutagenesis is limited by a local control of translesion synthesis.

The DNA damage response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this work, we raise the question whether TLS is controlled locally or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that (i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, (ii) a single lesion is enough to induce PCNA ubiquitination and (iii) PCNA ubiquitination is imperative for TLS to occur. More importantly, we show that the activation of the DDR that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.

A comprehensive overview of PPM1A: From structure to disease.

PPM1A (magnesium-dependent phosphatase 1 A, also known as PP2Cα) is a member of the Ser/Thr protein phosphatase family. Protein phosphatases catalyze the removal of phosphate groups from proteins via hydrolysis, thus opposing the role of protein kinases. The PP2C family is generally considered a negative regulator in the eukaryotic stress response pathway. PPM1A can bind and dephosphorylate various proteins and is therefore involved in the regulation of a wide range of physiological processes. It plays a crucial role in transcriptional regulation, cell proliferation, and apoptosis and has been suggested to be closely related to the occurrence and development of cancers of the lung, bladder, and breast, amongst others. Moreover, it is closely related to certain autoimmune diseases and neurodegenerative diseases. In this review, we provide an insight into currently available knowledge of PPM1A, including its structure, biological function, involvement in signaling pathways, and association with diseases. Lastly, we discuss whether PPM1A could be targeted for therapy of certain human conditions.

A role for the ribosome-associated complex in activation of the IRE1 branch of UPR.

The ubiquitous ribosome-associated complex (RAC) is a chaperone that spans ribosomes, making contacts near both the polypeptide exit tunnel and the decoding center, a position prime for sensing and coordinating translation and folding. Loss of RAC is known to result in growth defects and sensitization to translational and osmotic stresses. However, the physiological substrates of RAC and the mechanism(s) by which RAC is involved in responding to specific stresses in higher eukaryotes remain obscure. The data presented here uncover an essential function of mammalian RAC in the unfolded protein response (UPR). Knockdown of RAC sensitizes mammalian cells to endoplasmic reticulum (ER) stress and selectively interferes with IRE1 branch activation. Higher-order oligomerization of the inositol-requiring enzyme 1α (IRE1α) kinase/endoribonuclease depends upon RAC. These results reveal a surveillance function for RAC in the UPR, as follows: modulating IRE1α clustering as required for endonuclease activation and splicing of the substrate Xbp1 mRNA.

Arthropods Under Pressure: Stress Responses and Immunity at the Pathogen-Vector Interface.

Understanding what influences the ability of some arthropods to harbor and transmit pathogens may be key for controlling the spread of vector-borne diseases. Arthropod immunity has a central role in dictating vector competence for pathogen acquisition and transmission. Microbial infection elicits immune responses and imparts stress on the host by causing physical damage and nutrient deprivation, which triggers evolutionarily conserved stress response pathways aimed at restoring cellular homeostasis. Recent studies increasingly recognize that eukaryotic stress responses and innate immunity are closely intertwined. Herein, we describe two well-characterized and evolutionarily conserved mechanisms, the Unfolded Protein Response (UPR) and the Integrated Stress Response (ISR), and examine evidence that these stress responses impact immune signaling. We then describe how multiple pathogens, including vector-borne microbes, interface with stress responses in mammals. Owing to the well-conserved nature of the UPR and ISR, we speculate that similar mechanisms may be occurring in arthropod vectors and ultimately impacting vector competence. We conclude this Perspective by positing that novel insights into vector competence will emerge when considering that stress-signaling pathways may be influencing the arthropod immune network.

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a "prion-related domain" or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

Elucidation of the Metabolic Pathways of Methylarginines in Saccharomyces cerevisiae

BACKGROUNDThe methylation of arginine residues in proteins is a ubiquitous post‐translational modification that is predominantly metabolized during protein catabolism. It has been postulated that methylarginines play a functional role in the mammalian stress response due to their presence in stress granules, although this has not been largely explored. Saccharomyces cerevisiae is a suitable model organism to study the role of methylarginines in the eukaryotic stress response due to the high degree of similarity between the yeast and mammalian stress pathways and methyltransferasomes. Free methylarginines need to be tightly regulated because they inhibit the production of nitric oxide, a cell signalling molecule that is crucial in eukaryotic stress responses. Mammals metabolize free methylarginine into citrulline and methylamine by dimethylarginine dimethylaminohydrolase (DDAH) to circumvent nitric oxide synthase inhibition. Conversely, yeast lack classical DDAH activity and therefore cannot counteract this inhibition in the same way. The purpose of this research is to explore how yeast cope with free methylarginine in response to stress.METHODSWe applied different stress conditions to yeast cells, including heat shock, osmotic shock, rapamycin treatment, nutrient deprivation, and stationary phase and observed the effect on methylarginine concentration in proteins and in spent culture media. Methylarginine species were quantified using a tandem mass spectrometry assay. To explore possible metabolic pathways of yeast methylarginines, we also monitored the expression levels and subcellular localization of a constitutively expressed autophagy marker GFP‐Atg8, a key component in the formation of the phagophore assembly site, via Western Blot and fluorescence microscopy. We also observed the effect of gene deletions involved in autophagy and the proteasome on methylarginine processing.RESULTSThe condition that elicited the most significant change in methylarginine concentration response was stationary phase, a nutrient‐depriving growth condition. Specifically, we observed a substantial release of free methylarginine species into the culture media, accompanied by cleavage of GFP‐Atg8 and localization of GFP‐Atg8 to the vacuole, consistent with functional autophagy. In yeast mutants lacking functional autophagy, we fail to see the expelled free methylarginine species. However, in proteasome‐deficient yeast, methylarginine efflux is still observed.CONCLUSIONSWe postulate that autophagy serves as the major metabolic pathway for degradation of methylarginine‐containing proteins in yeast. We predict that methylarginine efflux is a crucial coping mechanism to avoid methylarginine‐mediated cell toxicity due to inhibition of nitric oxide synthesis. This work examines yeast methylarginines under stress for the first time and reveals new effects of methylarginine. This will contribute to the knowledge of how cells respond to stress and impacts what we know about autophagy and amino acid metabolism.Support or Funding InformationWe thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for support (RGPIN‐2015–04450 Discovery Grant to A.F., USRA to E.Z., and Alexander Graham Bell CGS‐D to J.I.B.).

Proton Nuclear Magnetic Resonance Metabolomics Corroborates Serine Hydroxymethyltransferase as the Primary Target of 2-Aminoacrylate in a ridA Mutant of Salmonella enterica.

The reactive intermediate deaminase RidA (EC 3.5.99.10) is conserved across all domains of life and deaminates reactive enamine species. When Salmonella enterica ridA mutants are grown in minimal medium, 2-aminoacrylate (2AA) accumulates, damages several pyridoxal 5'-phosphate (PLP)-dependent enzymes, and elicits an observable growth defect. Genetic studies suggested that damage to serine hydroxymethyltransferase (GlyA), and the resultant depletion of 5,10-methelenetetrahydrofolate (5,10-mTHF), was responsible for the observed growth defect. However, the downstream metabolic consequence from GlyA damage by 2AA remains relatively unexplored. This study sought to use untargeted proton nuclear magnetic resonance (1H NMR) metabolomics to determine whether the metabolic state of an S. enterica ridA mutant was accurately reflected by characterizing growth phenotypes. The data supported the conclusion that metabolic changes in a ridA mutant were due to the IlvA-dependent generation of 2AA, and that the majority of these changes were a consequence of damage to GlyA. While many of the metabolic differences for a ridA mutant could be explained, changes in some metabolites were not easily modeled, suggesting that additional levels of metabolic complexity remain to be unraveled.IMPORTANCE The accumulation of the reactive enamine intermediate 2-aminoacrylate (2AA) elicits global metabolic stress in many prokaryotes and eukaryotes by simultaneously damaging multiple pyridoxal 5'-phosphate (PLP)-dependent enzymes. This work employed 1H NMR to expand our understanding of the consequence(s) of 2AA stress on metabolite pools and effectively identify the metabolic changes stemming from one damaged target: GlyA. This study shows that nutrient supplementation during 1H NMR metabolomics experiments can disentangle complex metabolic outcomes stemming from a general metabolic stress. Metabolomics shows great potential to complement classical reductionist approaches to cost-effectively accelerate the rate of progress in expanding our global understanding of metabolic network structure and physiology. To that end, this work demonstrates the utility in implementing nutrient supplementation and genetic perturbation into metabolomics workflows as a means to connect metabolic outputs to physiological phenomena and establish causal relationships.

Dissecting the Function of MADS-Box Transcription Factors in Orchid Reproductive Development.

The orchid family (Orchidaceae) represents the second largest angiosperm family, having over 900 genera and 27,000 species in almost all over the world. Orchids have evolved a myriad of intriguing ways in order to survive extreme weather conditions, acquire nutrients, and attract pollinators for reproduction. The family of MADS-box transcriptional factors have been shown to be involved in the control of many developmental processes and responses to environmental stresses in eukaryotes. Several findings in different orchid species have elucidated that MADS-box genes play critical roles in the orchid growth and development. An in-depth understanding of their ecological adaptation will help to generate more interest among breeders and produce novel varieties for the floriculture industry. In this review, we summarize recent findings of MADS-box transcription factors in regulating various growth and developmental processes in orchids, in particular, the floral transition and floral patterning. We further discuss the prospects for the future directions in light of new genome resources and gene editing technologies that could be applied in orchid research and breeding.

Checkpoint inhibition of origin firing prevents DNA topological stress.

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

A consensus set of genetic vulnerabilities to ATR inhibition.

The response to DNA replication stress in eukaryotes is under the control of the ataxia–telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase ε accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity and the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.