Adaptation of a eukaryote-like ProRS to a prokaryote-like tRNAPro.

Prolyl-tRNA synthetases (ProRSs) are unique among aminoacyl-tRNA synthetases (aaRSs) in having two distinct structural architectures across different organisms: prokaryote-like (P-type) and eukaryote/archaeon-like (E-type). Interestingly, Bacillus thuringiensis harbors both types, with P-type (BtProRS1) and E-type ProRS (BtProRS2) coexisting. Despite their differences, both enzymes are constitutively expressed and functional in vivo. Similar to BtProRS1, BtProRS2 selectively charges the P-type tRNAPro and displays higher halofuginone tolerance than canonical E-type ProRS. However, these two isozymes recognize the primary identity elements of the P-type tRNAPro-G72 and A73 in the acceptor stem-through distinct mechanisms. Moreover, BtProRS2 exhibits significantly higher tolerance to stresses (such as heat, hydrogen peroxide, and dithiothreitol) than BtProRS1 does. This study underscores how an E-type ProRS adapts to a P-type tRNAPro and how it may contribute to the bacterium's survival under stress conditions.

Sequence-specific targeting of Caenorhabditis elegans C-Ala to the D-loop of tRNAAla.

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.

Gain of C-Ala enables AlaRS to target the L-shaped tRNAAla.

Unlike many other aminoacyl-tRNA synthetases, alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout biology. While Caenorhabditis elegans cytoplasmic AlaRS (CeAlaRSc) retains the prototype structure, its mitochondrial counterpart (CeAlaRSm) contains only a residual C-terminal domain (C-Ala). We demonstrated herein that the C-Ala domain from CeAlaRSc robustly binds both tRNA and DNA. It bound different tRNAs but preferred tRNAAla. Deletion of this domain from CeAlaRSc sharply reduced its aminoacylation activity, while fusion of this domain to CeAlaRSm selectively and distinctly enhanced its aminoacylation activity toward the elbow-containing (or L-shaped) tRNAAla. Phylogenetic analysis showed that CeAlaRSm once possessed the C-Ala domain but later lost most of it during evolution, perhaps in response to the deletion of the T-arm (part of the elbow) from its cognate tRNA. This study underscores the evolutionary gain of C-Ala for docking AlaRS to the L-shaped tRNAAla.

Human Thg1 displays tRNA-inducible GTPase activity.

tRNAHis guanylyltransferase (Thg1) catalyzes the 3'-5' incorporation of guanosine into position -1 (G-1) of tRNAHis. G-1 is unique to tRNAHis and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNAHis opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNAHis opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAsHis with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74-76), how HsThg1 prevents successive 3'-5' incorporation of G-1/G-2/G-3 into mitochondrial tRNAHis (tRNAmHis) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNAmHis indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNAmHis by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNAmHisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1. This study suggests that HsThg1 may downregulate the level of GTP through its GTPase activity to prevent multiple-GTP incorporation into tRNAmHis.

Thrombin-Induced Microglia Activation Modulated through Aryl Hydrocarbon Receptors.

Thrombin is a multifunctional serine protein which is closely related to neurodegenerative disorders. The Aryl hydrocarbon receptor (AhR) is well expressed in microglia cells involving inflammatory disorders of the brain. However, it remains unclear as to how modulation of AhR expression by thrombin is related to the development of neurodegeneration disorders. In this study, we investigated the role of AhR in the development of thrombin-induced neurodegenerative processes, especially those concerning microglia. The primary culture of either wild type or AhR deleted microglia, as well as BV-2 cell lines, was used for an in vitro study. Hippocampal slice culture and animals with either wild type or with AhR deleted were used for the ex vivo and in vivo studies. Simulations of ligand protein docking showed a strong integration between the thrombin and AhR. In thrombin-triggered microglia cells, deleting AhR escalated both the NO release and iNOS expression. Such effects were abolished by the administration of the AhR agonist. In thrombin-activated microglia cells, downregulating AhR increased the following: vascular permeability, pro-inflammatory genetic expression, MMP-9 activity, and the ratio of M1/M2 phenotype. In the in vivo study, thrombin induced the activation of microglia and their volume, thereby contributing to the deterioration of neurobehavior. Deleting AhR furthermore aggravated the response in terms of impaired neurobehavior, increasing brain edema, aggregating microglia, and increasing neuronal death. In conclusion, thrombin caused the activation of microglia through increased vessel permeability, expression of inflammatory response, and phenotype of M1 microglia, as well the MMP activity. Deleting AhR augmented the above detrimental effects. These findings indicate that the modulation of AhR is essential for the regulation of thrombin-induced brain damages and that the AhR agonist may harbor the potentially therapeutic effect in thrombin-induced neurodegenerative disorder.

Neuronal Death Caused by HMGB1-Evoked via Inflammasomes from Thrombin-Activated Microglia Cells.

Microglial cells are a macrophage-like cell type residing within the CNS. These cells evoke pro-inflammatory responses following thrombin-induced brain damage. Inflammasomes, which are large caspase-1-activating protein complexes, play a critical role in mediating the extracellular release of HMGB1 in activated immune cells. The exact role of inflammasomes in microglia activated by thrombin remains unclear, particularly as it relates to the downstream functions of HMGB1. After receiving microinjections of thrombin, Sprague Dawley rats of 200 to 250 gm were studied in terms of behaviors and immunohistochemical staining. Primary culture of microglia cells and BV-2 cells were used for the assessment of signal pathways. In a water maze test and novel object recognition analysis, microinjections of thrombin impaired rats' short-term and long-term memory, and such detrimental effects were alleviated by injecting anti-HMGB-1 antibodies. After thrombin microinjections, the increased oxidative stress of neurons was aggravated by HMGB1 injections but attenuated by anti-HMGB-1 antibodies. Such responses occurred in parallel with the volume of activated microglia cells, as well as their expressions of HMGB-1, IL-1ß, IL-18, and caspase-I. In primary microglia cells and BV-2 cell lines, thrombin also induced NO release and mRNA expressions of iNOS, IL-1ß, IL-18, and activated caspase-I. HMGB-1 aggravated these responses, which were abolished by anti-HMGB-1 antibodies. In conclusion, thrombin induced microglia activation through triggering inflammasomes to release HMGB1, contributing to neuronal death. Such an action was counteracted by the anti-HMGB-1 antibodies. The refinement of HMGB-1 modulated the neuro-inflammatory response, which was attenuated in thrombin-associated neurodegenerative disorder.

Naturally occurring dual recognition of tRNAHis substrates with and without a universal identity element.

The extra 5' guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis (CetRNAnHis) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNAHis (CetRNAmHis) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNAmHis strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNAnHis without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNAHis identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNAHis effectively converted a non-cognate tRNA to a tRNAHis-like substrate. Our study suggests that a new balance between identity elements of tRNAHis relieves HisRS from the absolute requirement for G-1.

Evolutionary gain of highly divergent tRNA specificities by two isoforms of human histidyl-tRNA synthetase.

The discriminator base N73 is a key identity element of tRNAHis. In eukaryotes, N73 is an "A" in cytoplasmic tRNAHis and a "C" in mitochondrial tRNAHis. We present evidence herein that yeast histidyl-tRNA synthetase (HisRS) recognizes both A73 and C73, but somewhat prefers A73 even within the context of mitochondrial tRNAHis. In contrast, humans possess two distinct yet closely related HisRS homologues, with one encoding the cytoplasmic form (with an extra N-terminal WHEP domain) and the other encoding its mitochondrial counterpart (with an extra N-terminal mitochondrial targeting signal). Despite these two isoforms sharing high sequence similarities (81% identity), they strongly preferred different discriminator bases (A73 or C73). Moreover, only the mitochondrial form recognized the anticodon as a strong identity element. Most intriguingly, swapping the discriminator base between the cytoplasmic and mitochondrial tRNAHis isoacceptors conveniently switched their enzyme preferences. Similarly, swapping seven residues in the active site between the two isoforms readily switched their N73 preferences. This study suggests that the human HisRS genes, while descending from a common ancestor with dual function for both types of tRNAHis, have acquired highly specialized tRNA recognition properties through evolution.

A WHEP Domain Regulates the Dynamic Structure and Activity of Caenorhabditis elegans Glycyl-tRNA Synthetase.

WHEP domains exist in certain eukaryotic aminoacyl-tRNA synthetases and play roles in tRNA or protein binding. We present evidence herein that cytoplasmic and mitochondrial forms of Caenorhabditis elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1) through alternative initiation of translation. The cytoplasmic form possessed an N-terminal WHEP domain, whereas its mitochondrial isoform possessed an extra N-terminal sequence consisting of an mitochondrial targeting signal and an appended domain. Cross-species complementation assays showed that CeGRS1 effectively rescued the cytoplasmic and mitochondrial defects of a yeast GRS1 knock-out strain. Although both forms of CeGlyRS efficiently charged the cytoplasmic tRNAs(Gly) of C. elegans, the mitochondrial form was much more efficient than its cytoplasmic counterpart in charging the mitochondrial tRNA(Gly) isoacceptor, which carries a defective TψC hairpin. Despite the WHEP domain per se lacking tRNA binding activity, deletion of this domain reduced the catalytic efficiency of the enzyme. Most interestingly, the deletion mutant possessed a higher thermal stability and a somewhat lower structural flexibility. Our study suggests a role for the WHEP domain as a regulator of the dynamic structure and activity of the enzyme.

Modulating the Structure and Function of an Aminoacyl-tRNA Synthetase Cofactor by Biotinylation.

Arc1p is a yeast-specific tRNA-binding protein that forms a ternary complex with glutamyl-tRNA synthetase (GluRSc) and methionyl-tRNA synthetase (MetRS) in the cytoplasm to regulate their catalytic activities and subcellular distributions. Despite Arc1p not being involved in any known biotin-dependent reaction, it is a natural target of biotin modification. Results presented herein show that biotin modification had no obvious effect on the growth-supporting activity, subcellular distribution, tRNA binding, or interactions of Arc1p with GluRSc and MetRS. Nevertheless, biotinylation of Arc1p was temperature dependent; raising the growth temperature from 30 to 37 °C drastically reduced its biotinylation level. As a result, Arc1p purified from a yeast culture that had been grown overnight at 37 °C was essentially biotin free. Non-biotinylated Arc1p was more heat stable, more flexible in structure, and more effective than its biotinylated counterpart in promoting glutamylation activity of the otherwise inactive GluRSc at 37 °C in vitro Our study suggests that the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes.

The regulation of transcriptome responses in zebrafish embryo exposure to triadimefon.

The residue of triadimefon (TDF) (a pesticide) has become the pollutant in water due to its intensive use in agriculture and medicine, and its stability in water leaching from soil and vegetation. In this study, RNA-seq, a high-throughput method was performed, to analyze the global expression of differential expressed genes (DEGs) in zebrafish embryos treated with TDF (10 µg/mL) from fertilization to 72 h post-fertilization (hpf) as compared with that in the control group (without TDF treatment). Two cDNA libraries were generated from treated and non-treated embryos, respectively. With the 79.4% and 78.8% of reads mapped to the reference, it was observed that many differential genes were expressed between the two libraries. The most 20 differentially expressed up-regulated or down-regulated genes were involving in the signaling transduction, the activation of many genes related to cytochrome P450 enzymes, and molecular metabolism. Validation of seven genes expression confirmed RNA-seq results. The transcriptome sequences were further subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and showed diverse biological functions and metabolic pathways. The data from this study contributed to a better understanding of the potential consequences of fish exposed to TDF, and to evaluate the potential threat of TDF to fish population in the aquatic environment. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 217-226, 2017.

Vanderwaltozyma polyspora possesses two glycyl-tRNA synthetase genes: one constitutive and one inducible.

Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein synthesis. We herein present evidence that the yeast Vanderwaltozyma polyspora possesses two paralogous glycyl-tRNA synthetase (GlyRS) genes-GRS1 and GRS2. Paradoxically, GRS1 provided functions in both the cytoplasm and mitochondria, while GRS2 was essentially silent under normal growth conditions. Expression of GRS2 could be activated by stresses such as high pH or ethanol and most effectively by high temperature. The expressed GlyRS2 protein was exclusively found in the cytoplasm and more stable under heat-shock conditions (37°C) than under normal growth conditions (30°C) in vivo. In addition, GRS2 effectively rescued the cytoplasmic defect of a Saccharomyces cerevisiae GRS1 knockout strain when expressed from a constitutive promoter. Moreover, the purified GlyRS2 enzyme was fairly active at both 30°C and 37°C in glycylation of yeast tRNA in vitro. However, unexpectedly, the purified GlyRS2 enzyme was practically inactive at temperature above 40°C in vitro. Our study suggests that GRS2 is an inducible gene that acts under stress conditions where GlyRS1 may be insufficient, unavailable, or rendered inactive.

Alanyl-tRNA synthetase genes of Vanderwaltozyma polyspora arose from duplication of a dual-functional predecessor of mitochondrial origin.

In eukaryotes, the cytoplasmic and mitochondrial forms of a given aminoacyl-tRNA synthetase (aaRS) are typically encoded by two orthologous nuclear genes, one of eukaryotic origin and the other of mitochondrial origin. We herein report a novel scenario of aaRS evolution in yeast. While all other yeast species studied possess a single nuclear gene encoding both forms of alanyl-tRNA synthetase (AlaRS), Vanderwaltozyma polyspora, a yeast species descended from the same whole-genome duplication event as Saccharomyces cerevisiae, contains two distinct nuclear AlaRS genes, one specifying the cytoplasmic form and the other its mitochondrial counterpart. The protein sequences of these two isoforms are very similar to each other. The isoforms are actively expressed in vivo and are exclusively localized in their respective cellular compartments. Despite the presence of a promising AUG initiator candidate, the gene encoding the mitochondrial form is actually initiated from upstream non-AUG codons. A phylogenetic analysis further revealed that all yeast AlaRS genes, including those in V. polyspora, are of mitochondrial origin. These findings underscore the possibility that contemporary AlaRS genes in V. polyspora arose relatively recently from duplication of a dual-functional predecessor of mitochondrial origin.

Trans-kingdom rescue of Gln-tRNAGln synthesis in yeast cytoplasm and mitochondria.

Aminoacylation of transfer RNA(Gln) (tRNA(Gln)) is performed by distinct mechanisms in different kingdoms and represents the most diverged route of aminoacyl-tRNA synthesis found in nature. In Saccharomyces cerevisiae, cytosolic Gln-tRNA(Gln) is generated by direct glutaminylation of tRNA(Gln) by glutaminyl-tRNA synthetase (GlnRS), whereas mitochondrial Gln-tRNA(Gln) is formed by an indirect pathway involving charging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNA(Gln) amidotransferase. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for its yeast homologue in vivo. We report herein that the same fusion enzyme, upon being imported into mitochondria, substituted the indirect pathway for Gln-tRNA(Gln) synthesis as well, despite significant differences in the identity determinants of E. coli and yeast cytosolic and mitochondrial tRNA(Gln) isoacceptors. Fusion of Arc1p to the bacterial enzyme significantly enhanced its aminoacylation activity towards yeast tRNA(Gln) isoacceptors in vitro. Our study provides a mechanism by which trans-kingdom rescue of distinct pathways of Gln-tRNA(Gln) synthesis can be conferred by a single enzyme.

Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate.

Prolyl-tRNA synthetase (ProRS), belonging to the family of aminoacyl-tRNA synthetases responsible for pairing specific amino acids with their respective tRNAs, is categorized into two distinct types: the eukaryote/archaeon-like type (E-type) and the prokaryote-like type (P-type). Notably, these types are specific to their corresponding cognate tRNAs. In an intriguing paradox, Thermus thermophilus ProRS (TtProRS) aligns with the E-type ProRS but selectively charges the P-type tRNAPro, featuring the bacterium-specific acceptor-stem elements G72 and A73. This investigation reveals TtProRS's notable resilience to the inhibitor halofuginone, a synthetic derivative of febrifugine emulating Pro-A76, resembling the characteristics of the P-type ProRS. Furthermore, akin to the P-type ProRS, TtProRS identifies its cognate tRNA through recognition of the acceptor-stem elements G72/A73, along with the anticodon elements G35/G36. However, in contrast to the P-type ProRS, which relies on a strictly conserved R residue within the bacterium-like motif 2 loop for recognizing G72/A73, TtProRS achieves this through a non-conserved sequence, RTR, within the otherwise non-interacting eukaryote-like motif 2 loop. This investigation sheds light on the adaptive capacity of a typically conserved housekeeping enzyme to accommodate a novel substrate.

Recognition of tRNAHis in an RNase P-Free Nanoarchaeum.

The 5' extra guanosine with 5'-monophosphate at position -1 (G-1) of tRNAHis (p-tRNAHis) is a nearly universal feature that establishes tRNAHis identity. G-1 is either genome encoded and retained after processing by RNase P (RNase P) or posttranscriptionally incorporated by tRNAHis guanylyltransferase (Thg1) after RNase P cleavage. However, RNase P is not found in the hyperthermophilic archaeum Nanoarchaeum equitans; instead, all of its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 5'-triphosphate (ppp-tRNAs). How N. equitans histidyl-tRNA synthetase (NeHisRS) recognizes its cognate tRNA (NetRNAHis) is of particular interest. In this paper, we show that G-1 serves as the major identity element of NetRNAHis, with its anticodon performing a similar role, though to a lesser extent. Moreover, NeHisRS distinctly preferred p-tRNAHis over ppp-tRNAHis (~5-fold difference). Unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme could charge tRNAsHis with A73 and C73 with nearly equal efficiency. As a result, mutation at the C73-recognition amino acid residue Q112 had only a minor effect (<2-fold reduction). This study suggests that NeHisRS has evolved to disregard C73, but it still maintains its evolutionarily preserved preference toward tRNAHis with 5'-monophosphate. IMPORTANCE Mature tRNAHis has, at its 5'-terminus, an extra guanosine with 5'-monophosphate, designated G-1. G-1 is the major recognition element for histidyl-tRNA synthetase (HisRS), regardless of whether it is of eukaryotic or prokaryotic origin. However, in the hyperthermophilic archaeum Nanoarchaeum equitans, all its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 5'-triphosphate. This piqued our curiosity about whether N. equitans histidyl-tRNA synthetase (NeHisRS) prefers tRNAHis with 5'-triphosphate. We show herein that G-1 is still the major recognition element for NeHisRS. However, unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme shows almost the same preference for C73 and A73. Most intriguingly, NeHisRS still prefers 5'-monophosphate over 5'-triphosphate. It thus appears that the preference of HisRS for tRNAHis with 5'-monophosphate emerged very early in evolution.

A naturally occurring mini-alanyl-tRNA synthetase.

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology, consisting of catalytic, tRNA-recognition, editing, and C-Ala domains. The catalytic and tRNA-recognition domains catalyze aminoacylation, the editing domain hydrolyzes mischarged tRNAAla, and C-Ala-the major tRNA-binding module-targets the elbow of the L-shaped tRNAAla. Interestingly, a mini-AlaRS lacking the editing and C-Ala domains is recovered from the Tupanvirus of the amoeba Acanthamoeba castellanii. Here we show that Tupanvirus AlaRS (TuAlaRS) is phylogenetically related to its host's AlaRS. Despite lacking the conserved amino acid residues responsible for recognition of the identity element of tRNAAla (G3:U70), TuAlaRS still specifically recognized G3:U70-containing tRNAAla. In addition, despite lacking C-Ala, TuAlaRS robustly binds and charges microAla (an RNA substrate corresponding to the acceptor stem of tRNAAla) as well as tRNAAla, indicating that TuAlaRS exclusively targets the acceptor stem. Moreover, this mini-AlaRS could functionally substitute for yeast AlaRS in vivo. This study suggests that TuAlaRS has developed a new tRNA-binding mode to compensate for the loss of C-Ala.

Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug.

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs.

Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress.

Triple-negative breast cancer (TNBC) has been shown with high mitochondrial oxidative phosphorylation and production of reactive oxygen species (ROS). MnSOD (SOD2) is a mitochondrial antioxidant defense that has been implicated in inhibition of human malignancies. However, the impact of MnSOD on immunosuppressive macrophage functions and TNBC aggressiveness has never been explored. We found here that SOD2high is primarily observed in the aggressive subtypes of HER2(+) breast cancers and TNBCs patients. Further analyses demonstrated that the oncoprotein multiple copies in T-cell malignancy-1 (MCT-1 or MCTS1) induces mitochondrial superoxide dismutase (MnSOD) in TNBC cells by stabilizing the transcription factor Nrf2. SOD2high/MCTS1high expression correlates with a poor prognosis in breast cancer patients. MnSOD in TNBC cells functions as a prooxidant peroxidase that increases mitochondrial ROS (mROS) and adaptation to oxidative stress under the oncogenic effect. Interleukin-6 (IL-6) in the MCT-1 pathway elevates Nrf2/MnSOD and mROS levels. Knockdown of MnSOD inhibits TNBC cell invasion, breast cancer stem cells (BCSCs), mROS, and IL-6 excretion promoted by MCT-1. TNBC cells deficient in MnSOD prevent the polarization and chemotaxis of M2 macrophages but improve the ability of M1 macrophages to engulf cancer cells. Quenching mROS with MitoQ, a mitochondria-targeted non-metal-based antioxidant MnSOD mimics, effectively suppresses BCSCs and M2 macrophage invasion exacerbated by MnSOD and MCT-1. Consistently, silencing MnSOD impedes TNBC progression and intratumoral M2 macrophage infiltration. We revealed a novel stratagem for TNBC management involving targeting the MCT-1 oncogene-induced mitochondrial prooxidant MnSOD pathway, which prevents the development of an immunosuppressive tumor microenvironment.

Schizosaccharomyces pombe possesses two paralogous valyl-tRNA synthetase genes of mitochondrial origin.

Previous studies showed that VAS1 of Saccharomyces cerevisiae encodes both cytosolic and mitochondrial forms of valyl-tRNA synthetase (ValRS) through alternative initiation of translation. We show herein that except for Schizosaccharomyces pombe, all yeast species studied contained a single ValRS gene encoding both forms, and all of the mature protein forms deduced from those genes possessed an N-terminal appended domain (Ad) that was absent from their bacterial relatives. In contrast, S. pombe contained two distinct nuclear ValRS genes, one encoding the mitochondrial form and the other its cytosolic counterpart. Although the cytosolic form closely resembles other yeast ValRS sequences (approximately 60% identity), the mitochondrial form exhibits significant divergence from others (approximately 35% identity). Both genes are active and essential for the survival of the yeast. Most conspicuously, the mitochondrial form lacks the characteristic Ad. A phylogenetic analysis further suggested that both forms of S. pombe ValRS are of mitochondrial origin, and the mitochondrial form is ancestral to the cytoplasmic form.