Human lysyl-tRNA synthetase phosphorylation promotes HIV-1 proviral DNA transcription.

Human lysyl-tRNA synthetase (LysRS) was previously shown to be re-localized from its normal cytoplasmic location in a multi-aminoacyl-tRNA synthetase complex (MSC) to the nucleus of HIV-1 infected cells. Nuclear localization depends on S207 phosphorylation but the nuclear function of pS207-LysRS in the HIV-1 lifecycle is unknown. Here, we show that HIV-1 replication was severely reduced in a S207A-LysRS knock-in cell line generated by CRISPR/Cas9; this effect was rescued by S207D-LysRS. LysRS phosphorylation up-regulated HIV-1 transcription, as did direct transfection of Ap4A, an upstream transcription factor 2 (USF2) activator that is synthesized by pS207-LysRS. Overexpressing an MSC-derived peptide known to stabilize LysRS MSC binding inhibited HIV-1 replication. Transcription of HIV-1 proviral DNA and other USF2 target genes was reduced in peptide-expressing cells. We propose that nuclear pS207-LysRS generates Ap4A, leading to activation of HIV-1 transcription. Our results suggest a new role for nuclear LysRS in facilitating HIV-1 replication and new avenues for antiviral therapy.

Role of Nudt2 in Anchorage-Independent Growth and Cell Migration of Human Melanoma.

Nudt2 encodes a diadenosine tetraphosphate (Ap4A) hydrolase that catalyzes the hydrolysis of Ap4A and is involved in the lysyl tRNA synthetase-Ap4A-Nudt2 (LysRS-Ap4A-Nudt2) signaling pathway. We have previously demonstrated that this pathway is active in non-small cell lung cancer. Nudt2 was shown to be involved in cell proliferation in breast cancer, making it an important target in cancer therapy. Currently, the function of Nudt2 in malignant melanoma has not been demonstrated. Therefore, we investigated the role played by Nudt2 in the growth of human melanoma. Our study showed that Nudt2 knockdown suppressed anchorage-independent growth of human melanoma cells in vitro. The in vivo effect of Nudt2 was determined by investigating the role played by Nudt2 knockdown on the ability of the cells to form tumors in a mice xenograft model. Nudt2 knockdown significantly suppressed tumor growth in this model. Moreover, overexpression of Nudt2 resulted in an increase in anchorage-independent growth of these cells, whereas Nudt2 knockdown decreased their migration. In addition, Nudt2 knockdown reduced vimentin expression. Vimentin is one of the mesenchymal markers that are involved in the epithelial mesenchymal transition (EMT) process. Thus, Nudt2 plays an important role in promoting anchorage-independent growth and cell migration in melanoma.

The Distinct Effects of the Mitochondria-Targeted STAT3 Inhibitors Mitocur-1 and Mitocur-3 on Mast Cell and Mitochondrial Functions.

There is accumulating evidence that mitochondria and mitochondrial STAT3 are involved in the activation of mast cells. The mitochondria-targeted curcuminoids Mitocur-1 and Mitocur-3 have been suggested to reduce antigen-dependent mast cell activation by inhibiting mitochondrial STAT3. The aim of the current work was to investigate the mechanisms of action of these mitocurcuminoids on mast cells and mitochondrial functions. The pretreatment of rat basophilic leukemia cells RBL-2H3 with Mitocur-1 and Mitocur-3 decreased antigen-dependent degranulation but did not affect spontaneous degranulation. Both compounds caused mitochondrial fragmentation and increased mitochondrial ROS. Inhibition of Drp1 prevented mitochondrial fragmentation induced by Mitocur-3 but not by Mitocur-1. The antioxidant N-acetylcysteine inhibited mitochondrial fission induced by Mitocur-1 but not Mitocur-3. Mitochondrial fragmentation caused by Mitocur-3 but not Mitocur-1 was accompanied by activation of Drp1 and AMPK. These data suggest a distinct mechanism of action of mitocurcuminoids on the mitochondria of RBL-2H3 cells: Mitocur-3 stimulated AMPK and caused Drp1-dependent mitochondrial fragmentation, while Mitocur-1-induced mitochondrial fission was ROS-dependent. This difference may contribute to the higher toxicity of Mitocur-3 compared to Mitocur-1. The findings contribute to further drug development for inflammatory and allergic diseases.

Structural switch of lysyl-tRNA synthetase between translation and transcription.

Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207 phosphorylation provokes a new conformer of LysRS that inactivates its translational function but activates its transcriptional function. The crystal structure of an MSC subcomplex established that LysRS is held in the MSC by binding to the N terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap(4)A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.

The pLysRS-Ap4A Pathway in Mast Cells Regulates the Switch from Host Defense to a Pathological State.

The innate and adaptive immune systems play an essential role in host defense against pathogens. Various signal transduction pathways monitor and balance the immune system since an imbalance may promote pathological states such as allergy, inflammation, and cancer. Mast cells have a central role in the regulation of the innate/adaptive immune system and are involved in the pathogenesis of many inflammatory and allergic diseases by releasing inflammatory mediators such as histamines, proteases, chemotactic factors, and cytokines. Although various signaling pathways are associated with mast cell activation, our discovery and characterization of the pLysRS-Ap4A signaling pathway in these cells provided an additional important step towards a full understanding of the intracellular mechanisms involved in mast cell activation. In the present review, we will discuss in depth this signaling pathway's contribution to host defense and the pathological state.

The Role Played by Transcription Factor E3 in Modulating Cardiac Hypertrophy.

Transcription factor E3 (TFE3), which is a key regulator of cellular adaptation, is expressed in most tissues, including the heart, and is reportedly overexpressed during cardiac hypertrophy. In this study, TFE3's role in cardiac hypertrophy was investigated. To understand TFE3's physiological importance in cardiac hypertrophy, pressure-overload cardiac hypertrophy was induced through transverse aortic constriction (TAC) in both wild-type (WT) and TFE3 knockout mice (TFE3-/-). Eleven weeks after TAC induction, cardiac hypertrophy was observed in both WT and TFE3-/- mice. However, significant reductions in ejection fraction and fractional shortening were observed in WT mice compared to TFE3-/- mice. To understand the mechanism, we found that myosin heavy chain (Myh7), which increases during hemodynamic overload, was lower in TFE3-/- TAC mice than in WT TAC mice, whereas extracellular signal-regulated protein kinases (ERK) phosphorylation, which confers cardioprotection, was lower in the left ventricles of WT mice than in TFE3-/- mice. We also found high expressions of TFE3, histone, and MYH7 and low expression of pERK in the normal human heart compared to the hypertensive heart. In the H9c2 cell line, we found that ERK inhibition caused TFE3 nuclear localization. In addition, we found that MYH7 was associated with TFE3, and during TFE3 knockdown, MYH7 and histone were downregulated. Therefore, we showed that TFE3 expression was increased in the mouse model of cardiac hypertrophy and tissues from human hypertensive hearts, whereas pERK was decreased reversibly, which suggested that TFE3 is involved in cardiac hypertrophy through TFE3-histone-MYH7-pERK signaling.

Pyruvate dehydrogenase has a major role in mast cell function, and its activity is regulated by mitochondrial microphthalmia transcription factor.

BACKGROUND: We have recently observed that oxidative phosphorylation-mediated ATP production is essential for mast cell function. Pyruvate dehydrogenase (PDH) is the main regulator of the Krebs cycle and is located upstream of the electron transport chain. However, the role of PDH in mast cell function has not been described. Microphthalmia transcription factor (MITF) regulates the development, number, and function of mast cells. Localization of MITF to the mitochondria and its interaction with mitochondrial proteins has not been explored. OBJECTIVE: We sought to explore the role played by PDH in mast cell exocytosis and to determine whether MITF is localized in the mitochondria and involved in regulation of PDH activity. METHODS: Experiments were performed in vitro by using human and mouse mast cells, as well as rat basophil leukemia cells, and in vivo in mice. The effect of PDH inhibition on mast cell function was examined. PDH interaction with MITF was measured before and after immunologic activation. Furthermore, mitochondrial localization of MITF and its effect on PDH activity were determined. RESULTS: PDH is essential for immunologically mediated degranulation of mast cells. After activation, PDH is serine dephosphorylated. In addition, for the first time, we show that MITF is partially located in the mitochondria and interacts with PDH. This interaction is dependent on the phosphorylation state of PDH. Furthermore, mitochondrial MITF regulates PDH activity. CONCLUSION: The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases.

LysRS serves as a key signaling molecule in the immune response by regulating gene expression.

Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.

Erbin is a negative modulator of cardiac hypertrophy.

ErbB2 interacting protein (Erbin) is a widely expressed protein and participates in inhibition of several intracellular signaling pathways. Its mRNA has been found to be present in relatively high levels in the heart. However, its physiological role in the heart has not been explored. In the present work, we elucidated the role of Erbin in cardiac hypertrophy. Cardiac hypertrophy was induced in mice either by isoproterenol administration or by aortic constriction. The level of Erbin was significantly decreased in both models. Erbin(-/-) mice rapidly develop decompensated cardiac hypertrophy, and following severe pressure overload all Erbin(-/-) mice died from heart failure. Down-regulation of Erbin expression was also observed in biopsies derived from human failing hearts. It is known that Erbin inhibits Ras-mediated activation of the extracellular signal-regulated kinase (ERK) by binding to Soc-2 suppressor of clear homolog (Shoc2). Our data clearly show that ERK phosphorylation is enhanced in the heart tissues of Erbin(-/-) mice. Furthermore, we clearly demonstrate here that Erbin associates with Shoc2 in both whole hearts and in cardiomyocytes, and that in the absence of Erbin, Raf is phosphorylated and binds Shoc2, resulting in ERK phosphorylation. In conclusion, Erbin is an inhibitor of pathological cardiac hypertrophy, and this inhibition is mediated, at least in part, by modulating ERK signaling.

Mitochondrial STAT3 plays a major role in IgE-antigen-mediated mast cell exocytosis.

BACKGROUND: The involvement of mitochondrial oxidative phosphorylation (OXPHOS) in mast cell exocytosis was recently suggested by the finding that mitochondria translocate to exocytosis sites upon mast cell activation. In parallel, mitochondrial signal transducer and activator of transcription 3 (STAT3) was found to be involved in ATP production. However, the regulation of mitochondrial STAT3 function and its connection to mast cell exocytosis is unknown. OBJECTIVE: We sought to explore the role played by mitochondrial STAT3 in mast cell exocytosis. METHODS: Experiments were performed in vitro with human and mouse mast cells and rat basophilic leukemia (RBL) cells and in vivo in mice. OXPHOS activity was measured after immunologic activation. The expression of STAT3, extracellular signal-regulated kinase 1/2, and protein inhibitor of activated STAT3 in the mitochondria during mast cell activation was determined, as was the effect of STAT3 inhibition on OXPHOS activity and mast cell function. RESULTS: Here we show that mitochondrial STAT3 is essential for immunologically mediated degranulation of human and mouse mast cells and RBL cells. Additionally, in IgE-antigen-activated RBL cells, mitochondrial STAT3 was phosphorylated on serine 727 in an extracellular signal-regulated kinase 1/2-dependent manner, which was followed by induction of OXPHOS activity. Furthermore, the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3, was found to inhibit OXPHOS activity in the mitochondria, resulting in inhibition of mast cell degranulation. Moreover, mice injected with Stattic, a STAT3 inhibitor, had a significant decrease in histamine secretion. CONCLUSION: These results provide the first evidence of a regulatory role for mitochondrial STAT3 in mast cell functions, and therefore mitochondrial STAT3 could serve as a new target for the manipulation of allergic diseases.

Amino-acyl tRNA synthetases generate dinucleotide polyphosphates as second messengers: functional implications.

In this chapter we describe aminoacyl-tRNA synthetase (aaRS) production of dinucleotide polyphosphate in response to stimuli, their interaction with various signaling pathways, and the role of diadenosine tetraphosphate and diadenosine triphosphate as second messengers. The primary role of aaRS is to mediate aminoacylation of cognate tRNAs, thereby providing a central role for the decoding of genetic code during protein translation. However, recent studies suggest that during evolution, "moonlighting" or non-canonical roles were acquired through incorporation of additional domains, leading to regulation by aaRSs of a spectrum of important biological processes, including cell cycle control, tissue differentiation, cellular chemotaxis, and inflammation. In addition to aminoacylation of tRNA, most aaRSs can also produce dinucleotide polyphosphates in a variety of physiological conditions. The dinucleotide polyphosphates produced by aaRS are biologically active both extra- and intra-cellularly, and seem to function as important signaling molecules. Recent findings established the role of dinucleotide polyphosphates as second messengers.

The functional role of Nudt2 in human triple negative breast cancer.

The main known function of Nudix hydrolase 2 (Nudt2) is to hydrolyze the secondary messenger diadenosine 5', 5'''-p1, p4-tetraphosphate (Ap4A). In this study we examined the role of Nudt2 in breast carcinoma through its expression in human invasive ductal carcinoma tissues, and its functions in human triple negative breast cancer (TNBC) cell lines. A significantly higher expression of Nudt2 was observed in human invasive ductal carcinoma tissues compared to that in normal breast tissue. Knockdown of Nudt2 in TNBC cell lines resulted in a significant reduction in cellular proliferation via the Ki67 marker, accompanied by G0/G1 phase cell cycle arrest, in the migration and invasion of these cells and in tumorigenicity and anchorage-independent growth. It can therefore be concluded that Nudt2 plays a significant role in promoting TNBC growth.

The enigma of the role of protein inhibitor of activated STAT3 (PIAS3) in the immune response.

Protein inhibitor of activated STAT3 (PIAS3), the main cellular inhibitor of signal transducers and activator of transcription 3 (STAT3), has been described as a modulator of DNA binding transcription factors. The exploration of the emerging roles of PIAS3 in immune regulation is a growing and fascinating field. Recent discoveries have shed new light on the key role of PIAS3 in the regulation of transcriptional activity, and on the molecular mechanism involved. These findings suggest that the known functions of this signalling molecule are merely the "tip of the iceberg". This article reviews the challenging questions regarding the link between PIAS3 and the intracellular signalling in immune cells. Some of the known functions of PIAS3 that potentially modulate key proteins in the immune system will also be discussed.

Interaction of two translational components, lysyl-tRNA synthetase and p40/37LRP, in plasma membrane promotes laminin-dependent cell migration.

Although human lysyl-tRNA synthetase (KRS), an enzyme for protein synthesis, is often highly expressed in various cancer cells, its pathophysiological implications have not been understood. Here we found that KRS induces cancer cell migration through interaction with the 67-kDa laminin receptor (67LR) that is converted from ribosomal subunit p40. On laminin signal, KRS was phosphorylated at the T52 residue by p38MAPK and dissociated from the cytosolic multi-tRNA synthetase complex for membrane translocation. The importance of T52 phosphorylation for membrane translocation of KRS was confirmed by site-directed mutagenesis. In the membrane, turnover of 67LR was controlled by Nedd4-mediated ubiquitination, and KRS inhibited ubiquitin-dependent degradation of 67LR, thereby enhancing laminin-induced cell migration. This work thus unveiled a unique function of KRS in the control of cell migration and its pathological implication in metastasis.

Transcription factor E3, a major regulator of mast cell-mediated allergic response.

BACKGROUND: Microphthalmia transcription factor, an MiT transcription family member closely related to transcription factor E3 (TFE3), is essential for mast cell development and survival. TFE3 was previously reported to play a role in the functions of B and T cells; however, its role in mast cells has not yet been explored. OBJECTIVE: We sought to explore the role played by TFE3 in mast cell function. METHODS: Mast cell numbers were evaluated by using toluidine blue staining. FACS analysis was used to determine percentages of Kit and FcεRI double-positive cells in the peritoneum of wild-type (WT) and TFE3 knockout (TFE3(-/-)) mice. Cytokine and inflammatory mediator secretion were measured in immunologically activated cultured mast cells derived from either knockout or WT mice. In vivo plasma histamine levels were measured after immunologic triggering of these mice. RESULTS: No significant differences in mast cell numbers between WT and TFE3(-/-) mice were observed in the peritoneum, lung, and skin. However, TFE3(-/-) mice showed a marked decrease in the number of Kit(+) and FcεRI(+) peritoneal and cultured mast cells. Surface expression levels of FcεRI in TFE3(-/-) peritoneal mast cells was significantly lower than in control cells. Cultured mast cells derived from TFE3(-/-) mice showed a marked decrease in degranulation and mediator secretion. In vivo experiments showed that the level of plasma histamine in TFE3(-/-) mice after an allergic trigger was substantially less than that seen in WT mice. CONCLUSION: TFE3 is a novel regulator of mast cell functions and as such could emerge as a new target for the manipulation of allergic diseases.

The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells.

In recent years, growing evidence has indicated the pivotal role of mitochondria in mast cell immunological activation. We have previously reported a decrease in degranulation and cytokine secretion following the inhibition of pyruvate dehydrogenase (PDH) either by CPI-613 (PDH inhibitor/anti-cancer drug) or through its interaction with mitochondrial microphthalmia-associated transcription factor (MITF). In the present study, we further explored the role played by mitochondrial MITF in mast cell exocytosis using rat basophil leukemia cells [RBL], as well as mouse bone marrow-derived mast cells (BMMCs). Here, we report that mast cell degranulation, cytokine secretion and oxidative phosphorylation (OXPHOS) activities were associated with phosphorylation of Serine 73 of mitochondrial MITF, controlled by extracellular signals regulated by protein kinase (ERK1/2) activity. Also, we report here that decreased OXPHOS activity following ERK1/2 inhibition (U0126 treatment) during IgE-Ag activation was mediated by the dephosphorylation of Serine 73 mitochondrial MITF, which inhibited its association with PDH. This led to a reduction in mast cell reactivity. In addition, a phosphorylation-mimicking mitochondrial MITF-S73D positively regulated the mitochondrial activity, thereby supporting mast cell degranulation. Thus, the present research findings highlight the prominence of mitochondrial MITF Serine 73 phosphorylation in immunologically activated mast cells.

A specific epitope of protein inhibitor of activated STAT3 is responsible for the induction of apoptosis in rat transformed mast cells.

Protein inhibitor of activated STAT3 (PIAS3) functions in vivo as a key molecule in suppressing the transcriptional activity of both microphthalmia transcription factor (MITF) and STAT3, two transcription factors that play a major role in the development, phenotypic expression, and survival of mast cells and melanocytes. In the present study we have investigated the role played by PIAS3 in the regulation of cell cycle in mast cells and melanocytes. We have characterized the biological role of a 23-aa domain derived from PIAS3 that induces apoptosis in these cells by inhibiting the transcriptional activity of both MITF and STAT3. This PIAS3 inhibitor peptide could serve as the beginning of an in depth study for the development of peptide inhibitors for MITF and STAT3.

Microphthalmia transcription factor isoforms in mast cells and the heart.

The microphthalmia transcription factor (Mitf) is critical for the survival and differentiation of a variety of cell types. While on the transcript level it has been noted that melanocytes and cardiomyocytes express specific Mitf isoforms, mast cells express several isoforms, mainly Mitf-H and Mitf-MC, whose function has not been thoroughly investigated. We found that in mast cells the expression of the specific Mitf isoforms is dependent on physiological stimuli that cause a major shifting of promoter usage and internal splicing. For example, activation of the c-kit signaling pathway almost totally abolished one of the main splice isoforms. Since cardiomyocytes express only the Mitf-H isoform, they were an ideal system to determine this isoform's physiological role. We identified that the expression of myosin light-chain 1a (MLC-1a) is regulated by Mitf-H. Interestingly, the transactivation of MLC-1a by Mitf-H in cardiomyocytes is decreased by overexpression of the splice form with exon 6a. In conclusion, we found that there is physiological switching of Mitf isoforms and that the promoter context and the cell context have a combined influence on gene expression programs.

[Military tract of medicine--the need and the vision].

Establishing the Military Tract in the Hebrew University School of Medicine is an opportunity to enrich the medical curriculum with contents relevant to better training of military physicians. Subjects such as hyperbaric medicine, hypobaric physiology, terror medicine, mass casualty event (MCE) and management of non-conventional injuries will be added to the regular M.D. curriculum. Medical education based on the elements of excellence: Judaism, Zionism, humanism and leadership, will be the foundations for the future generation of medical leaders in Israel for both the military and civilian systems. This leadership will be articulated in the clinical, research and administrative fields.

Corrigendum: The Role Played by Mitochondria in FcϵRI-Dependent Mast Cell Activation.

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