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.

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.

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.

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.

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.

A tryptophan-rich peptide acts as a transcription activation domain.

BACKGROUND: Eukaryotic transcription activators normally consist of a sequence-specific DNA-binding domain (DBD) and a transcription activation domain (AD). While many sequence patterns and motifs have been defined for DBDs, ADs do not share easily recognizable motifs or structures. RESULTS: We report herein that the N-terminal domain of yeast valyl-tRNA synthetase can function as an AD when fused to a DNA-binding protein, LexA, and turn on reporter genes with distinct LexA-responsive promoters. The transcriptional activity was mainly attributed to a five-residue peptide, WYDWW, near the C-terminus of the N domain. Remarkably, the pentapeptide per se retained much of the transcriptional activity. Mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide (resulting in W5) significantly enhanced its activity (~1.8-fold), while mutations which substituted aromatic residues with alanine residues severely impaired its activity. Accordingly, a much more active peptide, pentatryptophan (W7), was produced, which elicited ~3-fold higher activity than that of the native pentapeptide and the N domain. Further study indicated that W7 mediates transcription activation through interacting with the general transcription factor, TFIIB. CONCLUSIONS: Since W7 shares no sequence homology or features with any known transcription activators, it may represent a novel class of AD.

Promoting the formation of an active synthetase/tRNA complex by a nonspecific tRNA-binding domain.

Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K(d) approximately 0.5 microm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.

Translation initiation from a naturally occurring non-AUG codon in Saccharomyces cerevisiae.

Although previous studies have already shown that both cytoplasmic and mitochondrial activities of glycyl-tRNA synthetase are provided by a single gene, GRS1,in the yeast Saccharomyces cerevisiae, the mechanism by which this occurs remains unclear. Evidence presented here indicates that this bifunctional property is actually a result of two distinct translational products alternatively generated from a single transcript of this gene. Except for an amino-terminal 23-amino acid extension, these two isoforms have the same polypeptide sequence and function exclusively in their respective compartments under normal conditions. Reporter gene assays further suggest that this leader peptide can function independently as a mitochondrial targeting signal and plays the major role in the subcellular localization of the isoforms. Additionally, whereas the short protein is translationally initiated from a traditional AUG triplet, the longer isoform is generated from an upstream inframe UUG codon. To our knowledge, GRS1 appears to be the first example in the yeast wherein a functional protein isoform is initiated from a naturally occurring non-AUG codon. The results suggest that non-AUG initiation might be a mechanism existing throughout all kingdoms.

Mitochondrial form of a tRNA synthetase can be made bifunctional by manipulating its leader peptide.

Previous studies showed that yeast VAS1 encodes both the cytoplasmic and mitochondrial forms of valyl-tRNA synthetase (ValRS), using alternative transcription and translation. The ValRS isoforms have identical polypeptide sequences, except for a 46-amino acid leader peptide that functions as a mitochondrial targeting signal. Although the two forms of the enzyme exhibit indistinguishable tRNA specificities in vitro, they cannot substitute for each other in vivo because of their different localizations. Here we show that the 46-residue leader sequence can be divided into two nonoverlapping peptides, each of which retains the ability to target the enzyme into mitochondria. The engineered proteins (with truncated leader sequences) are dual-targeted, rescuing both the cytoplasmic and mitochondrial defects of a vas1 knockout strain. Thus, in addition to alternative splicing and alternative translation initiation as mechanisms by which a single gene can encode cytoplasmic and mitochondrial activities, the inherent characteristics of a single polypeptide may enable it to be distributed simultaneously between two cellular compartments. This mechanism may explain how certain other single genes in Saccharomyces cerevisiae provide dual functions.