GM-CSF treatment is not effective in congenital neutropenia patients due to its inability to activate NAMPT signaling.

PubMed

Koch, Corinna; Samareh, Bardia; Morishima, Tatsuya; Mir, Perihan; Kanz, Lothar; Zeidler, Cornelia; Skokowa, Julia; Welte, Karl

2017-03-01

Severe congenital neutropenia (CN) is a bone marrow failure syndrome characterized by an absolute neutrophil count (ANC) below 500 cells/μL and recurrent, life-threatening bacterial infections. Treatment with granulocyte colony-stimulating factor (G-CSF) increases the ANC in the majority of CN patients. In contrary, granulocyte-monocyte colony-stimulating factor (GM-CSF) fails to increase neutrophil numbers in CN patients in vitro and in vivo, suggesting specific defects in signaling pathways downstream of GM-CSF receptor. Recently, we detected that G-CSF induces granulopoiesis in CN patients by hyperactivation of nicotinamide phosphoribosyl transferase (NAMPT)/Sirtuin 1 signaling in myeloid cells. Here, we demonstrated that, in contrast to G-CSF, GM-CSF failed to induce NAMPT-dependent granulopoiesis in CN patients. We further identified NAMPT signaling as an essential downstream effector of the GM-CSF pathway in myelopoiesis.

The role of aquaporin-5 in cancer cell migration: A potential active participant.

PubMed

Jensen, Helene H; Login, Frédéric H; Koffman, Jennifer S; Kwon, Tae-Hwan; Nejsum, Lene N

2016-10-01

Emerging data identifies the water channel aquaporin-5 as a major player in multiple cancers. Over-expression of aquaporin-5 has been associated with increased metastasis and poor prognosis, suggesting that aquaporin-5 may enhance cancer cell migration. This review aims to highlight the current knowledge and hypothesis regarding downstream signaling partners of aquaporin-5 in relation to cancer cell migration. The molecular mechanisms that link aquaporin-5 to cell migration are not completely understood. Aquaporin-5 may promote cell movement by increasing water uptake into the front of the cell allowing local swelling. Aquaporin-5 may also activate extracellular-regulated kinases, increasing proliferation and potentially stimulating the migration machinery. Thus, further studies are warranted to identify the underlying mechanisms and signaling pathways. This will reveal whether aquaporin-5 and downstream effectors could be targets for developing new cancer therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pathogen effectors target Arabidopsis EDS1 and alter its interactions with immune regulators.

PubMed

Bhattacharjee, Saikat; Halane, Morgan K; Kim, Sang Hee; Gassmann, Walter

2011-12-09

Plant resistance proteins detect the presence of specific pathogen effectors and initiate effector-triggered immunity. Few immune regulators downstream of resistance proteins have been identified, none of which are known virulence targets of effectors. We show that Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of basal resistance and of effector-triggered immunity specifically mediated by Toll-interleukin-1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR) resistance proteins, forms protein complexes with the TIR-NB-LRR disease resistance proteins RPS4 and RPS6 and with the negative immune regulator SRFR1 at a cytoplasmic membrane. Further, the cognate bacterial effectors AvrRps4 and HopA1 disrupt these EDS1 complexes. Tight association of EDS1 with TIR-NB-LRR-mediated immunity may therefore derive mainly from being guarded by TIR-NB-LRR proteins, and activation of this branch of effector-triggered immunity may directly connect to the basal resistance signaling pathway via EDS1.

Prostate Cancer Epigenome

PubMed Central

Chinaranagari, Swathi; Sharma, Pankaj; Bowen, Nathan J.; Chaudhary, Jaideep

2018-01-01

Prostate cancer is a major health burden within the ever-increasingly aging US population. The molecular mechanisms involved in prostate cancer are diverse and heterogeneous. In this context, epigenetic changes, both global and gene specific, are now an emerging alternate mechanism in disease initiation and progression. The three major risk factors in prostate cancer: age, geographic ancestry, and environment are all influenced by epigenetics and additional significant insight is required to gain an understanding of the underlying mechanisms. The androgen receptor and its downstream effector pathways, central to prostate cancer initiation and progression, are subject to a multitude of epigenetic alterations. In this review we focus on the global perspective of epigenetics and the use of recent next-generation sequencing platforms to interrogate epigenetic changes in the prostate cancer genome. PMID:25421658

Prostate cancer epigenome.

PubMed

Chinaranagari, Swathi; Sharma, Pankaj; Bowen, Nathan J; Chaudhary, Jaideep

2015-01-01

Prostate cancer is a major health burden within the ever-increasingly aging US population. The molecular mechanisms involved in prostate cancer are diverse and heterogeneous. In this context, epigenetic changes, both global and gene specific, are now an emerging alternate mechanism in disease initiation and progression. The three major risk factors in prostate cancer: age, geographic ancestry, and environment are all influenced by epigenetics and additional significant insight is required to gain an understanding of the underlying mechanisms. The androgen receptor and its downstream effector pathways, central to prostate cancer initiation and progression, are subject to a multitude of epigenetic alterations. In this review we focus on the global perspective of epigenetics and the use of recent next-generation sequencing platforms to interrogate epigenetic changes in the prostate cancer genome.

Complement Factor B is the Downstream Effector of Toll-Like Receptors and Plays an Important Role in a Mouse Model of Severe Sepsis¶

PubMed Central

Zou, Lin; Feng, Yan; Li, Yan; Zhang, Ming; Chen, Chan; Cai, Jiayan; Gong, Yu; Wang, Larry; Thurman, Joshua M.; Wu, Xiaobo; Atkinson, John P.; Chao, Wei

2013-01-01

Severe sepsis involves massive activation of the innate immune system and leads to high mortality. Previous studies have demonstrated that various types of Toll-like receptors (TLRs) mediate a systemic inflammatory response and contribute to organ injury and mortality in animal models of severe sepsis. However, the downstream mechanisms responsible for TLR-mediated septic injury are poorly understood. Here, we show that activation of TLR2, TLR3 and TLR4 markedly enhanced complement factor B (cfB) synthesis and release by macrophages and cardiac cells. Polymicrobial sepsis, created by cecal ligation and puncture (CLP) in a mouse model, augmented cfB levels in the serum, peritoneal cavity and major organs including the kidney and heart. CLP also led to the alternative pathway (AP) activation, C3 fragment deposition in the kidney and heart, and cfB-dependent C3dg elevation. Bacteria isolated from septic mice activated the serum AP via a factor D-dependent manner. MyD88 deletion attenuated cfB/C3 up-regulation as well as cleavage induced by polymicrobial infection. Importantly, during sepsis, absence of cfB conferred a protective effect with improved survival and cardiac function, and markedly attenuated acute kidney injury. cfB deletion also led to increased neutrophil migratory function during the early phase of sepsis, decreased local and systemic bacterial load, attenuated cytokine production and reduced neutrophil reactive oxygen species production. Together, our data indicate that cfB acts as a downstream effector of TLR signaling and plays a critical role in the pathogenesis of severe bacterial sepsis. PMID:24154627

The ventromedial hypothalamus mediates predator fear memory

PubMed Central

Silva, Bianca A.; Mattucci, Camilla; Kryzwkowski, Piotr; Cuozzo, Rachel; Carbonari, Laura; Gross, Cornelius T.

2016-01-01

The amygdala has been shown to be essential for the processing of acute and learned fear across animal species. However, the downstream neural circuits that mediate these fear responses differ depending on the nature of the threat, with separate pathways identified for predator, conspecific, and physically harmful threats. In particular, the dorsomedial part of the ventromedial hypothalamus (VHMdm) is critical for the expression of defensive responses to predator. Here, we tested the hypothesis that this circuit also participates in predator fear memory by transient pharmacogenetic inhibition of VMHdm and its downstream effector, the dorsal periaqueductal grey, during predator fear learning in the mouse. Our data demonstrate that neural activity in VMHdm is required for both the acquisition and recall of predator fear memory, while that of its downstream effector, the dorsal periaqueductal grey, is required only for the acute expression of fear. These findings are consistent with a role for the medial hypothalamus in encoding an internal emotional state of fear. PMID:26991018

Msx genes are important apoptosis effectors downstream of the Shh/Gli3 pathway in the limb.

PubMed

Lallemand, Yvan; Bensoussan, Vardina; Cloment, Cécile Saint; Robert, Benoît

2009-07-15

In tetrapods, the anteroposterior (AP) patterning of the limb is under the control of the antagonistic activities of the secreted factor Sonic hedgehog (Shh) and Gli3R, the truncated repressor form of the transcription factor Gli3. In this report, we show that Msx1 and Msx2 are targets and downstream effectors of Gli3R. Consequently, in Shh null mutants, Msx genes are overexpressed and, furthermore, partially responsible for the limb phenotype. This is exemplified by the fact that reducing Msx activity in Shh mutants partially restores a normal limb development. Finally, we show that the main action of the Msx genes, in both normal and Shh(-/-) limb development, is to control cell death in the mesenchyme. We propose that, in the limb, Msx genes act downstream of the Shh/Gli3 pathway by transducing BMP signaling and that, in the absence of Shh signaling, their deregulation contributes to the extensive apoptosis that impairs limb development.

CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma.

PubMed

An, Rong; Wang, Yisong; Voeller, Donna; Gower, Arjan; Kim, In-Kyu; Zhang, Yu-Wen; Giaccone, Giuseppe

2016-05-17

Anaplastic lymphoma kinase (ALK) gene rearrangements are oncogenic drivers in a small subset of patients with non-small-cell lung cancer (NSCLC). The ALK inhibitors are highly effective in NSCLC patients harboring ALK rearrangements; however, most patients acquire resistance to the therapy following an initial response. Mechanisms of acquired resistance are complex. We used LC-MS/MS-based phosphotyrosine-peptide profiling in the EML4-ALK rearranged H3122 and H2228 cells treated with ALK inhibitors, to identify downstream effectors of ALK. We then used Western blot, siRNA experiments, cell proliferation, viability and migration assays to validate our findings. We identified CRKL as a novel downstream effector of ALK signaling. We demonstrated that CRKL tyrosine phosphorylation was repressed by pharmacological inhibition or small interfering RNA (siRNA) knockdown of ALK in the ALK-rearranged cells. More importantly, CRKL knockdown attenuated their cell proliferation, viability, and migration, but it had no effect on ALK phosphorylation and expression in these cells. Furthermore, CRKL tyrosine phosphorylation was inhibited by dasatinib (an inhibitor of ABL and SRC kinases), which in combination with the ALK inhibitor crizotinib displayed a synergistic inhibitory effect in vitro. In conclusion, our study suggests that CRKL is a key downstream effector of ALK, and combined inhibition of ALK and CRKL may represent an effective strategy for treating ALK-rearranged NSCLC patients.

MAPKAP kinase 2 (MK2)-dependent and independent models of blister formation in pemphigus vulgaris

PubMed Central

Mao, Xuming; Li, Hong; Sano, Yasuyo; Gaestel, Matthias; Park, Jin Mo; Payne, Aimee S.

2013-01-01

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by autoantibodies to the keratinocyte adhesion protein desmoglein (Dsg) 3. Previous studies suggest that PV pathogenesis involves p38 mitogen activated protein kinase-dependent and -independent pathways. However, p38 is a difficult protein to study and therapeutically target because it has four isoforms and multiple downstream effectors. In the current study, we identify MAPKAP kinase 2 (MK2) as a downstream effector of p38 signaling in PV and describe MK2-dependent and -independent mechanisms of blister formation using passive transfer of human anti-Dsg IgG4 mAbs to neonatal mice. In human keratinocytes, PV mAbs activate MK2 in a dose-dependent manner. MK2 is also activated in human pemphigus skin blisters, causing translocation of MK2 from the nucleus to the cytosol. Small molecule inhibition of MK2 and silencing of MK2 expression block PV mAb-induced Dsg3 endocytosis in human keratinocytes. Additionally, small molecule inhibition and genetic deletion of p38α and MK2 inhibit spontaneous, but not induced, suprabasal blisters by PV mAbs in mouse passive transfer models. Collectively, these data suggest that MK2 is a key downstream effector of p38 that can modulate PV autoantibody pathogenicity. MK2 inhibition may be a valuable adjunctive therapy for control of pemphigus blistering. PMID:23657501

Src-dependent EGFR transactivation regulates lung inflammation via downstream signaling involving ERK1/2, PI3Kδ/Akt and NFκB induction in a murine asthma model.

PubMed

El-Hashim, Ahmed Z; Khajah, Maitham A; Renno, Waleed M; Babyson, Rhema S; Uddin, Mohib; Benter, Ibrahim F; Ezeamuzie, Charles; Akhtar, Saghir

2017-08-30

The molecular mechanisms underlying asthma pathogenesis are poorly characterized. In this study, we investigated (1) whether Src mediates epidermal growth factor receptor (EGFR) transactivation; (2) if ERK1/2, PI3Kδ/Akt and NF-κB are signaling effectors downstream of Src/EGFR activation; and (3) if upstream inhibition of Src/EGFR is more effective in downregulating the allergic inflammation than selective inhibition of downstream signaling pathways. Allergic inflammation resulted in increased phosphorylation of EGFR, Akt, ERK1/2 and IκB in the lung tissues from ovalbumin (OVA)-challenged BALB/c mice. Treatment with inhibitors of Src (SU6656) or EGFR (AG1478) reduced EGFR phosphorylation and downstream signaling which resulted in the inhibition of the OVA-induced inflammatory cell influx in bronchoalveolar lavage fluid (BALF), perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyper-responsiveness. Treatment with pathway-selective inhibitors for ERK1/2 (PD89059) and PI3Kδ/Akt (IC-87114) respectively, or an inhibitor of NF-κB (BAY11-7085) also reduced the OVA-induced asthmatic phenotype but to a lesser extent compared to Src/EGFR inhibition. Thus, Src via EGFR transactivation and subsequent downstream activation of multiple pathways regulates the allergic airway inflammatory response. Furthermore, a broader upstream inhibition of Src/EGFR offers an attractive therapeutic alternative in the treatment of asthma relative to selectively targeting the individual downstream signaling effectors.

The Emerging Role of the Hippo Pathway in Lung Cancers: Clinical Implications.

PubMed

Teoh, Seong Lin; Das, Srijit

2017-11-30

The incidence of lung cancers has increased globally. Increased exposure to tobacco, passive smoking, less consumption of vegetables and fruits and occupational exposure to asbestos, arsenic and chromium are the main risk factors. The pathophysiology of lung cancer is complex and not well understood. Various microRNAs, genes and pathways are associated with lung cancers. The genes involved in lung cancers produce proteins involved in cell growth, differentiation, different cell cycles, apoptosis, immune modulation, tumor spread and progression. The Hippo pathway (also known as the Salvador-Warts-Hippo pathway) is the latest emerging concept in cancers. The Hippo pathway plays an important role in controlling the size of the tissue and organ by virtue of its action on cell proliferation and apoptosis. In the present review, we highlight the mammalian Hippo pathway, role of its core members, its upstream regulators, downstream effectors and the resistance cases in lung cancers. Specific interaction of Mer with cell surface hyaluronan receptor CD44 is vital in cell contact inhibition, thereby activating Hippo pathway. Both transcription co-activators YAP and TAZ (also known as WWTR1, being homologs of Drosophila Yki) are important regulators of proliferation and apoptosis, and serve as major downstream effectors of the Hippo pathway. Mutation of NF2, the upstream regulator of Hippo pathway is linked to the cancers. Targeting YAP and TAZ may be important for future drug delivery and treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The CLAVATA receptor FASCIATED EAR2 responds to distinct CLE peptides by signaling through two downstream effectors.

PubMed

Je, Byoung Il; Xu, Fang; Wu, Qingyu; Liu, Lei; Meeley, Robert; Gallagher, Joseph P; Corcilius, Leo; Payne, Richard J; Bartlett, Madelaine E; Jackson, David

2018-03-15

Meristems contain groups of indeterminate stem cells, which are maintained by a feedback loop between CLAVATA ( CLV ) and WUSCHEL ( WUS ) signaling. CLV signaling involves the secretion of the CLV3 peptide and its perception by a number of Leucine-Rich-Repeat (LRR) receptors, including the receptor-like kinase CLV1 and the receptor-like protein CLV2 coupled with the CORYNE (CRN) pseudokinase. CLV2, and its maize ortholog FASCIATED EAR2 (FEA2) appear to function in signaling by CLV3 and several related CLV3/EMBRYO-SURROUNDING REGION (CLE) peptide ligands. Nevertheless, how signaling specificity is achieved remains unknown. Here we show that FEA2 transmits signaling from two distinct CLE peptides, the maize CLV3 ortholog ZmCLE7 and ZmFON2-LIKE CLE PROTEIN1 (ZmFCP1) through two different candidate downstream effectors, the alpha subunit of the maize heterotrimeric G protein COMPACT PLANT2 (CT2), and ZmCRN. Our data provide a novel framework to understand how diverse signaling peptides can activate different downstream pathways through common receptor proteins. © 2018, Je et al.

Oncogenic NOTCH1 control of MYC and PI3K: challenges and opportunities for anti-NOTCH1 therapy in T-ALL

PubMed Central

Palomero, Teresa; Ferrando, Adolfo

2008-01-01

The identification of activating mutations in NOTCH1 in the majority of T-cell acute lymphoblastic leukemias and lymphomas (T-ALL) has brought much interest in inhibiting NOTCH1 signaling as therapeutic target in this disease. Small molecule inhibitors of the γ-secretase complex, which mediates a critical proteolytic cleavage required for NOTCH1 activation, hold the promise of becoming an effective molecular therapy against relapsed and refractory T-ALL. Recent progress in the elucidation of the transcriptional regulatory networks downstream of oncogenic NOTCH1 has uncovered a central role of NOTCH1 signaling in promoting leukemic cell growth and revealed an intricate circuitry that connects NOTCH1 signaling with MYC and the PI3K-AKT signaling pathway. The identification of the downstream effector pathways controlled by NOTCH1 should pave the way for the rational design of anti-NOTCH1 therapies for the treatment of T-ALL. PMID:18765521

Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors.

PubMed

Kast, David J; Yang, Changsong; Disanza, Andrea; Boczkowska, Malgorzata; Madasu, Yadaiah; Scita, Giorgio; Svitkina, Tatyana; Dominguez, Roberto

2014-04-01

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

AKT in cancer: new molecular insights and advances in drug development

PubMed Central

Mundi, Prabhjot S.; Sachdev, Jasgit; McCourt, Carolyn

2016-01-01

The phosphatidylinositol‐3 kinase (PI3K)–AKT pathway is one of the most commonly dysregulated pathways in all of cancer, with somatic mutations, copy number alterations, aberrant epigenetic regulation and increased expression in a number of cancers. The carefully maintained homeostatic balance of cell division and growth on one hand, and programmed cell death on the other, is universally disturbed in tumorigenesis, and downstream effectors of the PI3K–AKT pathway play an important role in this disturbance. With a wide array of downstream effectors involved in cell survival and proliferation, the well‐characterized direct interactions of AKT make it a highly attractive yet elusive target for cancer therapy. Here, we review the salient features of this pathway, evidence of its role in promoting tumorigenesis and recent progress in the development of therapeutic agents that target AKT. PMID:27232857

Upstream regulators and downstream effectors of NF-κB in Alzheimer's disease.

PubMed

Shi, Zhe-Min; Han, Ya-Wei; Han, Xiao-Hui; Zhang, Kun; Chang, Ya-Nan; Hu, Zhi-Mei; Qi, Hai-Xia; Ting, Chen; Zhen, Zhang; Hong, Wei

2016-07-15

Since Alzheimer's disease (AD) is becoming the prevalent dementia in the whole world, more underlying mechanisms are emerging. Long time has the transcription factor NF-κB been identified to participate in AD pathogenesis, various studies have focused on the causes and effects of AD that are linked to NF-κB. In this review we discuss diverse environmental stimuli including oxidative stress, neuroinflammation and metabolism, involved signaling pathways such as PI3K/AKT, MAPK and AGE/RAGE/GSK-3 and newly found ncRNAs that mediate neuron toxicity or neuron protection through NF-κB activation and the following response associated with the same factors in AD. These may provide future orientation of investigation at transcription level and support efficient treatment to AD by a better understanding of the upstream regulators and downstream effectors of NF-κB. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular pathways: regulation of metabolism by RB.

PubMed

Clem, Brian F; Chesney, Jason

2012-11-15

The discovery of the retinoblastoma (RB-1) gene as a tumor suppressor that is disrupted in a majority of human cancers either via direct or indirect genetic alterations has resulted in increased interest in its functions and downstream effectors. Although the canonical pathway that links this tumor suppressor to human cancers details its interaction with the E2F transcription factors and cell-cycle progression, recent studies have shown an essential role for RB-1 in the suppression of glycolytic and glutaminolytic metabolism. Characterization of the precise metabolic transporters and enzymes suppressed by the RB-E2F axis should enable the identification of small molecule antagonists that have selective and potent antitumor properties. ©2012 AACR.

Recent advances in the development of p21-activated kinase inhibitors.

PubMed

Coleman, Natalia; Kissil, Joseph

2012-04-01

The p21-activated kinases (PAKs) are downstream effectors of the small G-proteins of the Rac and cdc42 family and have been implicated as essential for cell proliferation and survival. Recent studies have also demonstrated the promise of PAKs as therapeutic targets in various types of cancers. The PAKs are divided into two major groups (group I and II) based on sequence similarities. Although the different roles the PAK groups might play are not well understood, recent efforts have focused on the identification of kinase inhibitors that can discriminate between the two groups. In this review these efforts and newly identified inhibitors will be described and future directions discussed.

Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Se-Hee; Schmitt, Christopher E.; Woolls, Melissa J.

Highlights: ► VEGF-A signaling regulates the segregation of axial vessels. ► VEGF-A signaling is mediated by PKC and ERK in this process. ► Ectopic activation of ERK is sufficient to rescue defects in vessel segregation. -- Abstract: Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that vascular endothelial growth factor-A (Vegf-A)more » signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and phosphatidylinositol 3-kinases (PI3 K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process.« less

Ras-Related Small GTPases RalA and RalB Regulate Cellular Survival After Ionizing Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kidd, Ambrose R.; Snider, Jared L.; Martin, Timothy D.

2010-09-01

Purpose: Oncogenic activation of Ras renders cancer cells resistant to ionizing radiation (IR), but the mechanisms have not been fully characterized. The Ras-like small GTPases RalA and RalB are downstream effectors of Ras function and are critical for both tumor growth and survival. The Ral effector RalBP1/RLIP76 mediates survival of mice after whole-body irradiation, but the role of the Ral GTPases themselves in response to IR is unknown. We have investigated the role of RalA and RalB in cellular responses to IR. Methods and Materials: RalA, RalB, and their major effectors RalBP1 and Sec5 were knocked down by stable expressionmore » of short hairpin RNAs in the K-Ras-dependent pancreatic cancer-derived cell line MIA PaCa-2. Radiation responses were measured by standard clonogenic survival assays for reproductive survival, {gamma}H2AX expression for double-strand DNA breaks (DSBs), and poly(ADP-ribose)polymerase (PARP) cleavage for apoptosis. Results: Knockdown of K-Ras, RalA, or RalB reduced colony-forming ability post-IR, and knockdown of either Ral isoform decreased the rate of DSB repair post-IR. However, knockdown of RalB, but not RalA, increased cell death. Surprisingly, neither RalBP1 nor Sec5 suppression affected colony formation post-IR. Conclusions: Both RalA and RalB contribute to K-Ras-dependent IR resistance of MIA PaCa-2 cells. Sensitization due to suppressed Ral expression is likely due in part to decreased efficiency of DNA repair (RalA and RalB) and increased susceptibility to apoptosis (RalB). Ral-mediated radioresistance does not depend on either the RalBP1 or the exocyst complex, the two best-characterized Ral effectors, and instead may utilize an atypical or novel effector.« less

Potent and Selective Peptide-based Inhibition of the G Protein Gαq*

PubMed Central

Charpentier, Thomas H.; Waldo, Gary L.; Lowery-Gionta, Emily G.; Krajewski, Krzysztof; Strahl, Brian D.; Kash, Thomas L.; Harden, T. Kendall; Sondek, John

2016-01-01

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq. A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2. In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells. PMID:27742837

Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

PubMed

Charpentier, Thomas H; Waldo, Gary L; Lowery-Gionta, Emily G; Krajewski, Krzysztof; Strahl, Brian D; Kash, Thomas L; Harden, T Kendall; Sondek, John

2016-12-02

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gα q binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gα q within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gα q in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gα q A representative peptide was specific for active Gα q because it did not bind inactive Gα q or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ 1 γ 2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gα q ; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gα q in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gα q -dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gα q in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gα q in cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

PubMed

Simon, Katharina; Hennen, Stephanie; Merten, Nicole; Blättermann, Stefanie; Gillard, Michel; Kostenis, Evi; Gomeza, Jesus

2016-01-08

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, In-Hyun; Erbay, Ebru; Nuzzi, Paul

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added atmore » a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector.« less

Pan-cancer transcriptomic analysis associates long non-coding RNAs with key mutational driver events

PubMed Central

Ashouri, Arghavan; Sayin, Volkan I.; Van den Eynden, Jimmy; Singh, Simranjit X.; Papagiannakopoulos, Thales; Larsson, Erik

2016-01-01

Thousands of long non-coding RNAs (lncRNAs) lie interspersed with coding genes across the genome, and a small subset has been implicated as downstream effectors in oncogenic pathways. Here we make use of transcriptome and exome sequencing data from thousands of tumours across 19 cancer types, to identify lncRNAs that are induced or repressed in relation to somatic mutations in key oncogenic driver genes. Our screen confirms known coding and non-coding effectors and also associates many new lncRNAs to relevant pathways. The associations are often highly reproducible across cancer types, and while many lncRNAs are co-expressed with their protein-coding hosts or neighbours, some are intergenic and independent. We highlight lncRNAs with possible functions downstream of the tumour suppressor TP53 and the master antioxidant transcription factor NFE2L2. Our study provides a comprehensive overview of lncRNA transcriptional alterations in relation to key driver mutational events in human cancers. PMID:28959951

Essential roles of Gab1 tyrosine phosphorylation in growth factor-mediated signaling and angiogenesis.

PubMed

Wang, Weiye; Xu, Suowen; Yin, Meimei; Jin, Zheng Gen

2015-02-15

Growth factors and their downstream receptor tyrosine kinases (RTKs) mediate a number of biological processes controlling cell function. Adaptor (docking) proteins, which consist exclusively of domains and motifs that mediate molecular interactions, link receptor activation to downstream effectors. Recent studies have revealed that Grb2-associated-binders (Gab) family members (including Gab1, Gab2, and Gab3), when phosphorylated on tyrosine residues, provide binding sites for multiple effector proteins, such as Src homology-2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) and phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, thereby playing important roles in transducing RTKs-mediated signals into pathways with diversified biological functions. Here, we provide an up-to-date overview on the domain structure and biological functions of Gab1, the most intensively studied Gab family protein, in growth factor signaling and biological functions, with a special focus on angiogenesis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

PubMed Central

Oran, Amanda R.; Adams, Clare M.; Zhang, Xiao-yong; Gennaro, Victoria J.; Pfeiffer, Harla K.; Mellert, Hestia S.; Seidel, Hans E.; Mascioli, Kirsten; Kaplan, Jordan; Gaballa, Mahmoud R.; Shen, Chen; Rigoutsos, Isidore; King, Michael P.; Cotney, Justin L.; Arnold, Jamie J.; Sharma, Suresh D.; Martinez, Ubaldo E.; Vakoc, Christopher R.; Chodosh, Lewis A.; Thompson, James E.; Bradner, James E.; Cameron, Craig E.; Shadel, Gerald S.; Eischen, Christine M.; McMahon, Steven B.

2016-01-01

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors. PMID:27590350

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer.

PubMed

Oran, Amanda R; Adams, Clare M; Zhang, Xiao-Yong; Gennaro, Victoria J; Pfeiffer, Harla K; Mellert, Hestia S; Seidel, Hans E; Mascioli, Kirsten; Kaplan, Jordan; Gaballa, Mahmoud R; Shen, Chen; Rigoutsos, Isidore; King, Michael P; Cotney, Justin L; Arnold, Jamie J; Sharma, Suresh D; Martinez-Outschoorn, Ubaldo E; Vakoc, Christopher R; Chodosh, Lewis A; Thompson, James E; Bradner, James E; Cameron, Craig E; Shadel, Gerald S; Eischen, Christine M; McMahon, Steven B

2016-11-08

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

Active photosynthetic inhibition mediated by MPK3/MPK6 is critical to effector-triggered immunity

PubMed Central

Su, Jianbin; Yang, Liuyi; Zhu, Qiankun; Wu, Hongjiao; He, Yi; Liu, Yidong; Xu, Juan; Jiang, Dean

2018-01-01

Extensive research revealed tremendous details about how plants sense pathogen effectors during effector-triggered immunity (ETI). However, less is known about downstream signaling events. In this report, we demonstrate that prolonged activation of MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MPKs), is essential to ETI mediated by both coiled coil-nucleotide binding site-leucine rich repeats (CNLs) and toll/interleukin-1 receptor nucleotide binding site-leucine rich repeats (TNLs) types of R proteins. MPK3/MPK6 activation rapidly alters the expression of photosynthesis-related genes and inhibits photosynthesis, which promotes the accumulation of superoxide (O2•−) and hydrogen peroxide (H2O2), two major reactive oxygen species (ROS), in chloroplasts under light. In the chemical-genetically rescued mpk3 mpk6 double mutants, ETI-induced photosynthetic inhibition and chloroplastic ROS accumulation are compromised, which correlates with delayed hypersensitive response (HR) cell death and compromised resistance. Furthermore, protection of chloroplasts by expressing a plastid-targeted cyanobacterial flavodoxin (pFLD) delays photosynthetic inhibition and compromises ETI. Collectively, this study highlights a critical role of MPK3/MPK6 in manipulating plant photosynthetic activities to promote ROS accumulation in chloroplasts and HR cell death, which contributes to the robustness of ETI. Furthermore, the dual functionality of MPK3/MPK6 cascade in promoting defense and inhibiting photosynthesis potentially allow it to orchestrate the trade-off between plant growth and defense in plant immunity. PMID:29723186

Advances in Therapeutic Fc Engineering – Modulation of IgG-Associated Effector Functions and Serum Half-life

PubMed Central

Saxena, Abhishek; Wu, Donghui

2016-01-01

Today, monoclonal immunoglobulin gamma (IgG) antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs) is achieved through both its antigen-binding fragment (Fab) and crystallizable fragment (Fc). Fab can specifically recognize tumor-associated antigen (TAA) and thus modulate TAA-linked downstream signaling pathways that may lead to the inhibition of tumor growth, induction of tumor apoptosis, and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis. Moreover, Fc is the region interacting with the neonatal Fc receptor in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anticancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce the cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering-based mAbs under clinical trials. PMID:28018347

Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses.

PubMed

Heidrich, Katharina; Wirthmueller, Lennart; Tasset, Céline; Pouzet, Cécile; Deslandes, Laurent; Parker, Jane E

2011-12-09

Pathogen effectors are intercepted by plant intracellular nucleotide binding-leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll-interleukin-1 receptor)-NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment-specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

Expanded functions for a family of plant intracellular immune receptors beyond specific recognition of pathogen effectors

PubMed Central

Bonardi, Vera; Tang, Saijun; Stallmann, Anna; Roberts, Melinda; Cherkis, Karen; Dangl, Jeffery L.

2011-01-01

Plants and animals deploy intracellular immune receptors that perceive specific pathogen effector proteins and microbial products delivered into the host cell. We demonstrate that the ADR1 family of Arabidopsis nucleotide-binding leucine-rich repeat (NB-LRR) receptors regulates accumulation of the defense hormone salicylic acid during three different types of immune response: (i) ADRs are required as “helper NB-LRRs” to transduce signals downstream of specific NB-LRR receptor activation during effector-triggered immunity; (ii) ADRs are required for basal defense against virulent pathogens; and (iii) ADRs regulate microbial-associated molecular pattern-dependent salicylic acid accumulation induced by infection with a disarmed pathogen. Remarkably, these functions do not require an intact P-loop motif for at least one ADR1 family member. Our results suggest that some NB-LRR proteins can serve additional functions beyond canonical, P-loop–dependent activation by specific virulence effectors, extending analogies between intracellular innate immune receptor function from plants and animals. PMID:21911370

The Genome Biology of Effector Gene Evolution in Filamentous Plant Pathogens.

PubMed

Sánchez-Vallet, Andrea; Fouché, Simone; Fudal, Isabelle; Hartmann, Fanny E; Soyer, Jessica L; Tellier, Aurélien; Croll, Daniel

2018-05-16

Filamentous pathogens, including fungi and oomycetes, pose major threats to global food security. Crop pathogens cause damage by secreting effectors that manipulate the host to the pathogen's advantage. Genes encoding such effectors are among the most rapidly evolving genes in pathogen genomes. Here, we review how the major characteristics of the emergence, function, and regulation of effector genes are tightly linked to the genomic compartments where these genes are located in pathogen genomes. The presence of repetitive elements in these compartments is associated with elevated rates of point mutations and sequence rearrangements with a major impact on effector diversification. The expression of many effectors converges on an epigenetic control mediated by the presence of repetitive elements. Population genomics analyses showed that rapidly evolving pathogens show high rates of turnover at effector loci and display a mosaic in effector presence-absence polymorphism among strains. We conclude that effective pathogen containment strategies require a thorough understanding of the effector genome biology and the pathogen's potential for rapid adaptation. Expected final online publication date for the Annual Review of Phytopathology Volume 56 is August 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The Hippo signaling functions through the Notch signaling to regulate intrahepatic bile duct development in mammals.

PubMed

Wu, Nan; Nguyen, Quy; Wan, Ying; Zhou, Tiaohao; Venter, Julie; Frampton, Gabriel A; DeMorrow, Sharon; Pan, Duojia; Meng, Fanyin; Glaser, Shannon; Alpini, Gianfranco; Bai, Haibo

2017-07-01

The Hippo signaling pathway and the Notch signaling pathway are evolutionary conserved signaling cascades that have important roles in embryonic development of many organs. In murine liver, disruption of either pathway impairs intrahepatic bile duct development. Recent studies suggested that the Notch signaling receptor Notch2 is a direct transcriptional target of the Hippo signaling pathway effector YAP, and the Notch signaling is a major mediator of the Hippo signaling in maintaining biliary cell characteristics in adult mice. However, it remains to be determined whether the Hippo signaling pathway functions through the Notch signaling in intrahepatic bile duct development. We found that loss of the Hippo signaling pathway tumor suppressor Nf2 resulted in increased expression levels of the Notch signaling pathway receptor Notch2 in cholangiocytes but not in hepatocytes. When knocking down Notch2 on the background of Nf2 deficiency in mouse livers, the excessive bile duct development induced by Nf2 deficiency was suppressed by heterozygous and homozygous deletion of Notch2 in a dose-dependent manner. These results implicated that Notch signaling is one of the downstream effectors of the Hippo signaling pathway in regulating intrahepatic bile duct development.

The Hippo signaling functions through the Notch signaling to regulate intrahepatic bile duct development in mammals

PubMed Central

Wu, Nan; Nguyen, Quy; Wan, Ying; Zhou, Tiaohao; Venter, Julie; Frampton, Gabriel A; DeMorrow, Sharon; Pan, Duojia; Meng, Fanyin; Glaser, Shannon; Alpini, Gianfranco; Bai, Haibo

2018-01-01

The Hippo signaling pathway and the Notch signaling pathway are evolutionary conserved signaling cascades that have important roles in embryonic development of many organs. In murine liver, disruption of either pathway impairs intrahepatic bile duct development. Recent studies suggested that the Notch signaling receptor Notch2 is a direct transcriptional target of the Hippo signaling pathway effector YAP, and the Notch signaling is a major mediator of the Hippo signaling in maintaining biliary cell characteristics in adult mice. However, it remains to be determined whether the Hippo signaling pathway functions through the Notch signaling in intrahepatic bile duct development. We found that loss of the Hippo signaling pathway tumor suppressor Nf2 resulted in increased expression levels of the Notch signaling pathway receptor Notch2 in cholangiocytes but not in hepatocytes. When knocking down Notch2 on the background of Nf2 deficiency in mouse livers, the excessive bile duct development induced by Nf2 deficiency was suppressed by heterozygous and homozygous deletion of Notch2 in a dose-dependent manner. These results implicated that Notch signaling is one of the downstream effectors of the Hippo signaling pathway in regulating intrahepatic bile duct development. PMID:28581486

mTOR signaling for biological control and cancer.

PubMed

Alayev, Anya; Holz, Marina K

2013-08-01

Mammalian target of rapamycin (mTOR) is a major intersection that connects signals from the extracellular milieu to corresponding changes in intracellular processes. When abnormally regulated, the mTOR signaling pathway is implicated in a wide spectrum of cancers, neurological diseases, and proliferative disorders. Therefore, pharmacological agents that restore the regulatory balance of the mTOR pathway could be beneficial for a great number of diseases. This review summarizes current understanding of mTOR signaling and some unanswered questions in the field. We describe the composition of the mTOR complexes, upstream signals that activate mTOR, and physiological processes that mTOR regulates. We also discuss the role of mTOR and its downstream effectors in cancer, obesity and diabetes, and autism. Copyright © 2013 Wiley Periodicals, Inc.

Dragging ras back in the ring.

PubMed

Stephen, Andrew G; Esposito, Dominic; Bagni, Rachel K; McCormick, Frank

2014-03-17

Ras proteins play a major role in human cancers but have not yielded to therapeutic attack. Ras-driven cancers are among the most difficult to treat and often excluded from therapies. The Ras proteins have been termed "undruggable," based on failures from an era in which understanding of signaling transduction, feedback loops, redundancy, tumor heterogeneity, and Ras' oncogenic role was poor. Structures of Ras oncoproteins bound to their effectors or regulators are unsolved, and it is unknown precisely how Ras proteins activate their downstream targets. These knowledge gaps have impaired development of therapeutic strategies. A better understanding of Ras biology and biochemistry, coupled with new ways of targeting undruggable proteins, is likely to lead to new ways of defeating Ras-driven cancers. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure-Guided Strategy for the Development of Potent Bivalent ERK Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lechtenberg, Bernhard C.; Mace, Peter D.; Sessions, E. Hampton

ERK is the effector kinase of the RAS-RAF-MEK-ERK signaling cascade, which promotes cell transformation and malignancy in many cancers and is thus a major drug target in oncology. Kinase inhibitors targeting RAF or MEK are already used for the treatment of certain cancers, such as melanoma. Although the initial response to these drugs can be dramatic, development of drug resistance is a major challenge, even with combination therapies targeting both RAF and MEK. Importantly, most resistance mechanisms still rely on activation of the downstream effector kinase ERK, making it a promising target for drug development efforts. Here, we report themore » design and structural/functional characterization of a set of bivalent ERK inhibitors that combine a small molecule inhibitor that binds to the ATP-binding pocket with a peptide that selectively binds to an ERK protein interaction surface, the D-site recruitment site (DRS). Our studies show that the lead bivalent inhibitor, SBP3, has markedly improved potency compared to the small molecule inhibitor alone. Unexpectedly, we found that SBP3 also binds to several ERK-related kinases that contain a DRS, highlighting the importance of experimentally verifying the predicted specificity of bivalent inhibitors. However, SBP3 does not target any other kinases belonging to the same CMGC branch of the kinome. Additionally, our modular click chemistry inhibitor design facilitates the generation of different combinations of small molecule inhibitors with ERK-targeting peptides.« less

Factors determining electrostatic fields in molecular dynamics simulations of the Ras/effector interface.

PubMed

Ensign, Daniel L; Webb, Lauren J

2011-12-01

Using molecular dynamics simulations, we explore geometric and physical factors contributing to calculated electrostatic fields at the binding surface of the GTPase Ras with a spectroscopically labeled variant of a downstream effector, the Ras-binding domain of Ral guanine nucleotide dissociation stimulator (RalGDS). A related system (differing by mutation of one amino acid) has been studied in our group using vibrational Stark effect spectroscopy, a technique sensitive to electrostatic fields. Electrostatic fields were computed using the AMBER 2003 force field and averaged over snapshots from molecular dynamics simulation. We investigate geometric factors by exploring how the orientation of the spectroscopic probe changes on Ras-effector binding. In addition, we explore the physical origin of electrostatic fields at our spectroscopic probe by comparing contributions to the field from discrete components of the system, such as explicit solvent, residues on the Ras surface, and residues on the RalGDS surface. These models support our experimental hypothesis that vibrational Stark shifts are caused by Ras binding to its effector and not the structural rearrangements of the effector surface or probe reorientation on Ras-effector binding, for at least some of our experimental probes. These calculations provide physical insight into the origin, magnitude, and importance of electrostatic fields in protein-protein interactions and suggest new experiments to probe the field's role in protein docking. Copyright © 2011 Wiley-Liss, Inc.

Altered Gene Regulation and Synaptic Morphology in "Drosophila" Learning and Memory Mutants

ERIC Educational Resources Information Center

Guan, Zhuo; Buhl, Lauren K.; Quinn, William G.; Littleton, J. Troy

2011-01-01

Genetic studies in "Drosophila" have revealed two separable long-term memory pathways defined as anesthesia-resistant memory (ARM) and long-lasting long-term memory (LLTM). ARM is disrupted in "radish" ("rsh") mutants, whereas LLTM requires CREB-dependent protein synthesis. Although the downstream effectors of ARM and LLTM are distinct, pathways…

Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

PubMed

Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

2015-07-15

Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes

PubMed Central

Marwaha, Rituraj; Arya, Subhash B.; Jagga, Divya; Kaur, Harmeet

2017-01-01

Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. PMID:28325809

Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Dustin C.; Ceresa, Brian P.

2008-11-01

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less

Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila

PubMed Central

Marshall, Lynne; Rideout, Elizabeth J; Grewal, Savraj S

2012-01-01

The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. Here, we show that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. We find that TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, we show that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, we find that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth. PMID:22367393

Calcium signaling in mammalian egg activation and embryo development: Influence of subcellular localization

PubMed Central

Miao, Yi-Liang; Williams, Carmen J.

2012-01-01

Calcium (Ca2+) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca2+ signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca2+ sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca2+ in many cell types and the impact of cellular localization on Ca2+ signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca2+ is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca2+ release and effectors of Ca2+ signals. We then summarize studies exploring how Ca2+ directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca2+ signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe areas for future research. PMID:22888043

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding.

PubMed

Manga, Prashiela; Bis, Sabina; Knoll, Kristen; Perez, Beremis; Orlow, Seth J

2010-10-01

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.

Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

USDA-ARS?s Scientific Manuscript database

Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diverse Xanthomonas strains in citrus.

PubMed

Shantharaj, Deepak; Römer, Patrick; Figueiredo, Jose F L; Minsavage, Gerald V; Krönauer, Christina; Stall, Robert E; Moore, Gloria A; Fisher, Latanya C; Hu, Yang; Horvath, Diana M; Lahaye, Thomas; Jones, Jeffrey B

2017-09-01

Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs3 1EBE ), and fused this engineered promoter with multiple EBEs (ProBs3 14EBE ) to either the β-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs3 1EBE and ProBs3 14EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs3 14EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs3 14EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Combover/CG10732, a Novel PCP Effector for Drosophila Wing Hair Formation

PubMed Central

Fagan, Jeremy K.; Dollar, Gretchen; Lu, Qiuheng; Barnett, Austen; Pechuan Jorge, Joaquin; Schlosser, Andreas; Pfleger, Cathie; Adler, Paul; Jenny, Andreas

2014-01-01

The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho Kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb)/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh. PMID:25207969

Phosphorylation at Ser-181 of oncogenic KRAS is required for tumor growth.

PubMed

Barceló, Carles; Paco, Noelia; Morell, Mireia; Alvarez-Moya, Blanca; Bota-Rabassedas, Neus; Jaumot, Montserrat; Vilardell, Felip; Capella, Gabriel; Agell, Neus

2014-02-15

KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors. ©2013 AACR.

Functionally Redundant RXLR Effectors from Phytophthora infestans Act at Different Steps to Suppress Early flg22-Triggered Immunity

PubMed Central

Fraiture, Malou; Liu, Xiaoyu; Boevink, Petra C.; Gilroy, Eleanor M.; Chen, Ying; Kandel, Kabindra; Sessa, Guido; Birch, Paul R. J.; Brunner, Frédéric

2014-01-01

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans. PMID:24763622

CB1 Cannabinoid Receptors Modulate Kinase and Phosphatase Activity during Extinction of Conditioned Fear in Mice

ERIC Educational Resources Information Center

Kamprath, Kornelia; Hermann, Heike; Lutz, Beat; Marsicano, Giovanni; Cannich, Astrid; Wotjak, Carsten T.

2004-01-01

Cannabinoid receptors type 1 (CB1) play a central role in both short-term and long-term extinction of auditory-cued fear memory. The molecular mechanisms underlying this function remain to be clarified. Several studies indicated extracellular signal-regulated kinases (ERKs), the phosphatidylinositol 3-kinase with its downstream effector AKT, and…

Activation of Exchange Protein Activated by Cyclic-AMP Enhances Long-Lasting Synaptic Potentiation in the Hippocampus

ERIC Educational Resources Information Center

Gelinas, Jennifer N.; Banko, Jessica L.; Peters, Melinda M.; Klann, Eric; Weeber, Edwin J.; Nguyen, Peter V.

2008-01-01

cAMP is a critical second messenger implicated in synaptic plasticity and memory in the mammalian brain. Substantial evidence links increases in intracellular cAMP to activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream effectors (transcription factors, receptors, protein kinases) necessary for long-term…

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

PubMed

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

Rho/Rho kinase and phosphoinositide 3-kinase are parallel pathways in the development of spontaneous arterial tone in deoxycorticosterone acetate-salt hypertension.

PubMed

Wehrwein, Erica A; Northcott, Carrie A; Loberg, Robert D; Watts, Stephanie W

2004-06-01

Hypertension is characterized by abnormal vascular contractility and function. Arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats develop spontaneous tone that is not observed in arteries from normotensive rats. Inhibition of phosphoinositide 3-kinase (PI3-kinase) by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) reduces spontaneous tone development. The Rho/Rho-kinase pathway has been suggested to play a role in hypertension and may be dependent on PI3-kinase activity. We hypothesized that Rhokinase is involved in spontaneous tone development and that Rho/Rho-kinase is a downstream effector of PI3-kinase. Using endothelium-denuded aortic strips in isolated tissue bath, we demonstrated that (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) (Y27632) (1 microM), a Rho-kinase inhibitor, significantly reduced spontaneous tone in the DOCA aorta but that it did not affect sham aorta basal tone (DOCA 63.5 +/- 15.9 versus sham 1.2 +/- 0.4 total change in percentage of phenylephrine contraction). We examined the interaction between the PI3-kinase and Rho pathways by observing the effects of LY294002 on a Rhokinase effector, myosin phosphatase (MYPT), and Y27632 on a PI3-kinase effector, Akt, using Western blot analysis. Inhibition of PI3-kinase reduced spontaneous tone, but it had no effect on the phosphorylation status of MYPT, indicating that PI3-kinase is not a downstream effector of Rho/Rho-kinase. These data indicate that there is little interaction between the Rho/Rhokinase and PI3-kinase pathways in the DOCA-salt aorta, and the two pathways seem to operate in parallel in supporting spontaneous arterial tone. These data reflect spontaneous tone only and do not rule out the possibility of interaction between these pathways in agonist-stimulated tone.

Solitary chemosensory cells and bitter taste receptor signaling in human sinonasal mucosa.

PubMed

Barham, Henry P; Cooper, Sarah E; Anderson, Catherine B; Tizzano, Marco; Kingdom, Todd T; Finger, Tom E; Kinnamon, Sue C; Ramakrishnan, Vijay R

2013-06-01

Solitary chemosensory cells (SCCs) are specialized cells in the respiratory epithelium that respond to noxious chemicals including bacterial signaling molecules. SCCs express components of bitter taste transduction including the taste receptor type 2 (TAS2R) bitter taste receptors and downstream signaling effectors: α-Gustducin, phospholipase Cβ2 (PLCβ2), and transient receptor potential cation channel subfamily M member 5 (TRPM5). When activated, SCCs evoke neurogenic reflexes, resulting in local inflammation. The purpose of this study was to test for the presence SCCs in human sinonasal epithelium, and to test for a correlation with inflammatory disease processes such as allergic rhinitis and chronic rhinosinusitis. Patient demographics and biopsies of human sinonasal mucosa were obtained from control patients (n = 7) and those with allergic rhinitis and/or chronic rhinosinusitis (n = 15). Reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), and immunohistochemistry were used to determine whether expression of signaling effectors was altered in diseased patients. RT-PCR demonstrated that bitter taste receptors TAS2R4, TAS2R14, and TAS2R46, and downstream signaling effectors α-Gustducin, PLCβ2, and TRPM5 are expressed in the inferior turbinate, middle turbinate, septum, and uncinate of both control and diseased patients. PLCβ2/TRPM5-immunoreactive SCCs were identified in the sinonasal mucosa of both control and diseased patients. qPCR showed similar expression of α-Gustducin and TRPM5 in the uncinate process of control and diseased groups, and there was no correlation between level of expression and 22-item Sino-Nasal Outcomes Test (SNOT-22) or pain scores. SCCs are present in human sinonasal mucosa in functionally relevant areas. Expression level of signaling effectors was similar in control and diseased patients and did not correlate with measures of pain and inflammation. Further study into these pathways may provide insight into nasal inflammatory diseases and may offer potential therapeutic targets. © 2013 ARS-AAOA, LLC.

The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes.

PubMed

Marwaha, Rituraj; Arya, Subhash B; Jagga, Divya; Kaur, Harmeet; Tuli, Amit; Sharma, Mahak

2017-04-03

Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. © 2017 Marwaha et al.

Bacterial virulence effectors and their activities.

PubMed

Hann, Dagmar R; Gimenez-Ibanez, Selena; Rathjen, John P

2010-08-01

The major virulence strategy for plant pathogenic bacteria is deployment of effector molecules within the host cytoplasm. Each bacterial strain possesses a set of 20-30 effectors which have overlapping activities, are functionally interchangeable, and diverge in composition between strains. Effectors target host molecules to suppress immunity. Two main strategies are apparent. Effectors that target host proteins seem to attack conserved structural domains but otherwise lack specificity. On the other hand, those that influence host gene transcription directly do so with extreme specificity. In both cases, examples are known where the host has exploited effector-target affinities to establish immune recognition of effectors. The molecular activity of each effector links virulence and immune outcomes. Copyright 2010 Elsevier Ltd. All rights reserved.

Kinetic and Structural Insights into the Mechanism of AMPylation by VopS Fic Domain*

PubMed Central

Luong, Phi; Kinch, Lisa N.; Brautigam, Chad A.; Grishin, Nick V.; Tomchick, Diana R.; Orth, Kim

2010-01-01

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP. PMID:20410310

Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model.

PubMed

Yang, Zhao; Yang, Fan; Zhang, Daolai; Liu, Zhixin; Lin, Amy; Liu, Chuan; Xiao, Peng; Yu, Xiao; Sun, Jin-Peng

2017-09-01

Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions not only by mediating receptor desensitization and internalization, but also by redirecting signaling to G protein-independent pathways via interactions with numerous downstream effector molecules. Accumulating evidence over the past decade has given rise to the phospho-barcode hypothesis, which states that ligand-specific phosphorylation patterns of a receptor direct its distinct functional outcomes. Our recent work using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance ( 19 F-NMR) spectroscopy led to the flute model, which provides preliminary insight into the receptor phospho-coding mechanism, by which receptor phosphorylation patterns are recognized by an array of phosphate-binding pockets on arrestin and are translated into distinct conformations. These selective conformations are recognized by various effector molecules downstream of arrestin. The phospho-barcoding mechanism enables arrestin to recognize a wide range of phosphorylation patterns of GPCRs, contributing to their diverse functions. Copyright © 2017 by The Author(s).

A Phytophthora sojae effector PsCRN63 forms homo-/hetero-dimers to suppress plant immunity via an inverted association manner.

PubMed

Li, Qi; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Chen, Yanyu; Zhou, Jian-Min; Dou, Daolong

2016-05-31

Oomycete pathogens produce a large number of effectors to promote infection. Their mode of action are largely unknown. Here we show that a Phytophthora sojae effector, PsCRN63, suppresses flg22-induced expression of FRK1 gene, a molecular marker in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). However, PsCRN63 does not suppress upstream signaling events including flg22-induced MAPK activation and BIK1 phosphorylation, indicating that it acts downstream of MAPK cascades. The PsCRN63-transgenic Arabidopsis plants showed increased susceptibility to bacterial pathogen Pseudomonas syringae pathovar tomato (Pst) DC3000 and oomycete pathogen Phytophthora capsici. The callose deposition were suppressed in PsCRN63-transgenic plants compared with the wild-type control plants. Genes involved in PTI were also down-regulated in PsCRN63-transgenic plants. Interestingly, we found that PsCRN63 forms an dimer that is mediated by inter-molecular interactions between N-terminal and C-terminal domains in an inverted association manner. Furthermore, the N-terminal and C-terminal domains required for the dimerization are widely conserved among CRN effectors, suggesting that homo-/hetero-dimerization of Phytophthora CRN effectors is required to exert biological functions. Indeed, the dimerization was required for PTI suppression and cell death-induction activities of PsCRN63.

Oomycetes, effectors, and all that jazz.

PubMed

Bozkurt, Tolga O; Schornack, Sebastian; Banfield, Mark J; Kamoun, Sophien

2012-08-01

Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inhibition of Ras for cancer treatment: the search continues

PubMed Central

Baines, Antonio T.; Xu, Dapeng; Der, Channing J.

2012-01-01

Background The RAS oncogenes (HRAS, NRAS and KRAS) comprise the most frequently mutated class of oncogenes in human cancers (33%), stimulating intensive effort in developing anti-Ras inhibitors for cancer treatment. Discussion Despite intensive effort, to date no effective anti-Ras strategies have successfully made it to the clinic. We present an overview of past and ongoing strategies to inhibit oncogenic Ras in cancer. Conclusions Since approaches to directly target mutant Ras have not been successful, most efforts have focused on indirect approaches to block Ras membrane association or downstream effector signaling. While inhibitors of effector signaling are currently under clinical evaluation, genome-wide unbiased genetic screens have identified novel directions for future anti-Ras drug discovery. PMID:22004085

Mechanotransduction in skeletal muscle

PubMed Central

Burkholder, Thomas J.

2007-01-01

Mechanical signals are critical to the development and maintenance of skeletal muscle, but the mechanisms that convert these shape changes to biochemical signals is not known. When a deformation is imposed on a muscle, changes in cellular and molecular conformations link the mechanical forces with biochemical signals, and the close integration of mechanical signals with electrical, metabolic, and hormonal signaling may disguise the aspect of the response that is specific to the mechanical forces. The mechanically induced conformational change may directly activate downstream signaling and may trigger messenger systems to activate signaling indirectly. Major effectors of mechanotransduction include the ubiquitous mitogen activated protein kinase (MAP) and phosphatidylinositol-3’ kinase (PI-3K), which have well described receptor dependent cascades, but the chain of events leading from mechanical stimulation to biochemical cascade is not clear. This review will discuss the mechanics of biological deformation, loading of cellular and molecular structures, and some of the principal signaling mechanisms associated with mechanotransduction. PMID:17127292

Mechanotransduction in skeletal muscle.

PubMed

Burkholder, Thomas J

2007-01-01

Mechanical signals are critical to the development and maintenance of skeletal muscle, but the mechanisms that convert these shape changes to biochemical signals is not known. When a deformation is imposed on a muscle, changes in cellular and molecular conformations link the mechanical forces with biochemical signals, and the close integration of mechanical signals with electrical, metabolic, and hormonal signaling may disguise the aspect of the response that is specific to the mechanical forces. The mechanically induced conformational change may directly activate downstream signaling and may trigger messenger systems to activate signaling indirectly. Major effectors of mechanotransduction include the ubiquitous mitogen activated protein kinase (MAP) and phosphatidylinositol-3' kinase (PI-3K), which have well described receptor dependent cascades, but the chain of events leading from mechanical stimulation to biochemical cascade is not clear. This review will discuss the mechanics of biological deformation, loading of cellular and molecular structures, and some of the principal signaling mechanisms associated with mechanotransduction.

PAX3-FOXO1: Zooming in on an "undruggable" target.

PubMed

Wachtel, Marco; Schäfer, Beat W

2018-06-01

Driver oncogenes are prime targets for therapy in tumors many of which, including leukemias and sarcomas, express recurrent fusion transcription factors. One specific example for such a cancer type is alveolar rhabdomyosarcoma, which is associated in the majority of cases with the fusion protein PAX3-FOXO1. Since fusion transcription factors are challenging targets for development of small molecule inhibitors, indirect inhibitory strategies for this type of oncogenes represent a more promising approach. One can envision strategies at different molecular levels including upstream modifiers and activators, epigenetic and transcriptional co-regulators, and downstream effector targets. In this review, we will discuss the current knowledge regarding potential therapeutic targets that might contribute to indirect interference with PAX3-FOXO1 activity in alveolar rhabdomyosarcoma at the different molecular levels and extrapolate these findings to fusion transcription factors in general. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.

PubMed

Tartaglia, Marco; Gelb, Bruce D

2010-12-01

RAS GTPases control a major signaling network implicated in several cellular functions, including cell fate determination, proliferation, survival, differentiation, migration, and senescence. Within this network, signal flow through the RAF-MEK-ERK pathway-the first identified mitogen-associated protein kinase (MAPK) cascade-mediates early and late developmental processes controlling morphology determination, organogenesis, synaptic plasticity, and growth. Signaling through the RAS-MAPK cascade is tightly controlled; and its enhanced activation represents a well-known event in oncogenesis. Unexpectedly, in the past few years, inherited dysregulation of this pathway has been recognized as the cause underlying a group of clinically related disorders sharing facial dysmorphism, cardiac defects, reduced postnatal growth, ectodermal anomalies, variable cognitive deficits, and susceptibility to certain malignancies as major features. These disorders are caused by heterozygosity for mutations in genes encoding RAS proteins, regulators of RAS function, modulators of RAS interaction with effectors, or downstream signal transducers. Here, we provide an overview of the phenotypic spectrum associated with germline mutations perturbing RAS-MAPK signaling, the unpredicted molecular mechanisms converging toward the dysregulation of this signaling cascade, and major genotype-phenotype correlations. © 2010 New York Academy of Sciences.

The death effector domain-containing DEDD forms a complex with Akt and Hsp90, and supports their stability

PubMed Central

Kurabe, Nobuya; Mori, Mayumi; Kurokawa, Jun; Taniguchi, Kaori; Aoyama, Hisatoshi; Atsuda, Kazuhiro; Nishijima, Akemi; Odawara, Nariaki; Harada, Saori; Nakashima, Katsuhiko; Arai, Satoko; Miyazaki, Toru

2010-01-01

Insulin secretion and glucose transport are the major mechanisms to balance glucose homeostasis. Recently, we found that the death effector domain-containing DEDD inhibits cyclin-dependent kinase 1 (Cdk1) function, thereby preventing Cdk1-dependent inhibitory phosphorylation of S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase (PI3K), which overall results in maintenance of S6K1 activity. Here we newly show that DEDD forms a complex with Akt and heat-shock protein 90 (Hsp90), and supports the stability of both proteins. Hence, in DEDD−/− mice, Akt protein levels are diminished in skeletal muscles and adipose tissues, which interferes with the translocation of glucose transporter 4 (GLUT4) upon insulin stimulation, leading to inefficient incorporation of glucose in these organs. Interestingly, as for the activation of S6K1, suppression of Cdk1 is involved in the stabilization of Akt protein by DEDD, since diminishment of Cdk1 in DEDD−/− cells via siRNA expression or treatment with a Cdk1-inhibitor, increases both Akt and Hsp90 protein levels. Such multifaceted involvement of DEDD in glucose homeostasis by supporting both insulin secretion (via maintenance of S6K1 activity) and glucose uptake (via stabilizing Akt protein), may suggest an association of DEDD-deficiency with the pathogenesis of type 2 diabetes mellitus. PMID:20043882

Chemosensors in the Nose: Guardians of the Airways

PubMed Central

Tizzano, Marco

2013-01-01

The G-protein-coupled receptor molecules and downstream effectors that are used by taste buds to detect sweet, bitter, and savory tastes are also utilized by chemoresponsive cells of the airways to detect irritants. Here, we describe the different cell types in the airways that utilize taste-receptor signaling to trigger protective epithelial and neural responses to potentially dangerous toxins and bacterial infection. PMID:23280357

ErbB2 Trafficking and Signaling in Human Vestibular Schwannomas

DTIC Science & Technology

2009-10-01

Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012...10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 11. SPONSOR/MONITOR’S REPORT...6- In collaboration with Dr. Joseph Cioffi, Medical College of Wisconsin, we have demonstrated that microRNA 21, a downstream effector of ErbB2

PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways

PubMed Central

Fantauzzo, Katherine A.; Soriano, Philippe

2014-01-01

Previous studies have identified phosphatidylinositol 3-kinase (PI3K) as the main downstream effector of PDGFRα signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRα is unable to bind PI3K (PdgfraPI3K/PI3K) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt downstream from PI3K-mediated PDGFRα signaling, we immunoprecipitated Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates and analyzed the peptides by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). Our analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. We demonstrate that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that PdgfraPI3K/PI3K-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53. We show that Ybox1 binds both the Trp53 promoter and the p53 protein and that expression of Trp53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, we demonstrate that introduction of a Trp53-null allele attenuates the vertebral defects found in PdgfraPI3K/PI3K neonates. Our findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRα signaling that regulates survival and proliferation during skeletal development in vivo. PMID:24788519

Impact of end effector technology on telemanipulation performance

NASA Technical Reports Server (NTRS)

Bejczy, A. K.; Szakaly, Z.; Ohm, T.

1990-01-01

Generic requirements for end effector design are briefly summarized as derived from generic functional and operational requirements. Included is a brief summary of terms and definitions related to end effector technology. The second part contains a brief overview of end effector technology work as JPL during the past ten years, with emphasis on the evolution of new mechanical, sensing and control capabilities of end effectors. The third and major part is devoted to the description of current end effector technology. The ongoing work addresses mechanical, sensing and control details with emphasis on mechanical ruggedness, increased resolution in sensing, and close electronic and control integration with overall telemanipulator control system.

Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

PubMed Central

Lippé, Roger; Miaczynska, Marta; Rybin, Vladimir; Runge, Anja; Zerial, Marino

2001-01-01

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases. PMID:11452015

Unique catalytic activities and scaffolding of p21 activated kinase-1 in cardiovascular signaling.

PubMed

Ke, Yunbo; Lei, Ming; Wang, Xin; Solaro, R John

2013-09-27

P21 activated kinase-1 (Pak1) has diverse functions in mammalian cells. Although a large number of phosphoproteins have been designated as Pak1 substrates from in vitro studies, emerging evidence has indicated that Pak1 may function as a signaling molecule through a unique molecular mechanism - scaffolding. By scaffolding, Pak1 delivers signals through an auto-phosphorylation-induced conformational change without transfer of a phosphate group to its immediate downstream effector(s). Here we review evidence for this regulatory mechanism based on structural and functional studies of Pak1 in different cell types and research models as well as in vitro biochemical assays. We also discuss the implications of Pak1 scaffolding in disease-related signaling processes and the potential in cardiovascular drug development.

The NO-cGMP-PKG Signaling Pathway Regulates Synaptic Plasticity and Fear Memory Consolidation in the Lateral Amygdala via Activation of ERK/MAP Kinase

ERIC Educational Resources Information Center

Ota, Kristie T.; Pierre, Vicki J.; Ploski, Jonathan E.; Queen, Kaila; Schafe, Glenn E.

2008-01-01

Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and…

Rho GTPases, their post-translational modifications, disease-associated mutations and pharmacological inhibitors.

PubMed

Olson, Michael F

2018-05-04

The 20 members of the Rho GTPase family are key regulators of a wide-variety of biological activities. In response to activation, they signal via downstream effector proteins to induce dynamic alterations in the organization of the actomyosin cytoskeleton. In this review, post-translational modifications, mechanisms of dysregulation identified in human pathological conditions, and the ways that Rho GTPases might be targeted for chemotherapy will be discussed.

Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krauthammer, Michael; Kong, Yong; Ha, Byung Hak

We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequentmore » in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1{sup P29S}) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1{sup P29S} showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.« less

The Tandem CARDs of NOD2: Intramolecular Interactions and Recognition of RIP2

PubMed Central

Fridh, Veronica; Rittinger, Katrin

2012-01-01

Caspase recruitment domains (CARDs) are homotypic protein interaction modules that link the stimulus-dependent assembly of large signaling platforms such as inflammasomes to the activation of downstream effectors that often include caspases and kinases and thereby play an important role in the regulation of inflammatory and apoptotic signaling pathways. NOD2 belongs to the NOD-like (NLR) family of intracellular pattern recognition receptors (PRR) and induces activation of the NF-κB pathway in response to the recognition of bacterial components. This process requires the specific recognition of the CARD of the protein kinase RIP2 by the tandem CARDs of NOD2. Here we demonstrate that the tandem CARDs of NOD2 are engaged in an intramolecular interaction that is important for the structural stability of this region. Using a combination of ITC and pull-down experiments we identify distinct surface areas that are involved in the intramolecular tandem CARD interaction and the interaction with the downstream effector RIP2. Our findings indicate that while CARDa of NOD2 might be the primary binding partner of RIP2 the two CARDs of NOD2 do not act independently of one another but may cooperate to from a binding surface that is distinct from that of single CARDs. PMID:22470564

Mesodermal Tbx1 is required for patterning the proximal mandible in mice

PubMed Central

Aggarwal, Vimla S.; Carpenter, Courtney; Freyer, Laina; Liao, Jun; Petti, Marilena; Morrow, Bernice E.

2010-01-01

Defects in the lower jaw, or mandible, occur commonly either as isolated malformations or in association with genetic syndromes. Understanding its formation and genetic pathways required for shaping its structure in mammalian model organisms will shed light into the pathogenesis of malformations in humans. The lower jaw is derived from the mandibular process of the first pharyngeal arch (MdPA1) during embryogenesis. Integral to the development of the mandible, is the signaling interplay between Fgf8 and Bmp4 in the rostral ectoderm and their downstream effector genes in the underlying neural crest derived mesenchyme. The non-neural crest MdPA1 core mesoderm is needed to form muscles of mastication, but its role in patterning the mandible is unknown. Here, we show that mesoderm specific deletion of Tbx1, a T- box transcription factor and gene for velo-cardio-facial/DiGeorge syndrome, results in defects in formation of the proximal mandible by shifting expression of Fgf8, Bmp4 and their downstream effector genes in mouse embryos at E10.5. This occurs without significant changes in cell proliferation or apoptosis at the same stage. Our results elucidate a new function for the non-neural crest core mesoderm and specifically, mesodermal Tbx1, in shaping the lower jaw. PMID:20501333

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

MIX and match: mobile T6SS MIX-effectors enhance bacterial fitness

PubMed Central

Salomon, Dor

2016-01-01

ABSTRACT Protein secretion systems that mediate interbacterial competition secret a wide repertoire of antibacterial toxins. A major player in these competitions is the newly discovered bacterial type VI secretion system (T6SS). We recently found that a subset of polymorphic MIX-effectors, which are a widespread class of effectors secreted by T6SSs, are horizontally shared between marine bacteria and are used to diversify their T6SS effector repertoires, thus enhancing their environmental fitness. In this commentary, I expand on the ideas that were introduced in the previous report, and further speculate on the possible mobility of other MIX-effectors. In addition, I discuss the possible role of horizontal gene transfer in the dissemination of MIX-effectors through bacterial genomes, as well as its possible role in diversifying the T6SS effector repertoire. PMID:27066305

Activation of ATM by DNA Damaging Agents

DTIC Science & Technology

2004-09-01

risk for breast cancer . Since many anti-tumor chemotherapeutics used in breast cancer treatment have the capacity to induce DNA DSBs, I have...of a subset of downstream effectors of ATM in two human breast cancer cell lines. Studies are now underway to identify proteins that interact with ATM...implications for the treatment of breast cancer patients harboring mutations in ATM. 14. SUBJECT TERMS 15. NUMBER OF PAGES signal transduction, DNA damage and

Vav family exchange factors: an integrated regulatory and functional view

PubMed Central

Bustelo, Xosé R

2014-01-01

The Vav family is a group of tyrosine phosphorylation-regulated signal transduction molecules hierarchically located downstream of protein tyrosine kinases. The main function of these proteins is to work as guanosine nucleotide exchange factors (GEFs) for members of the Rho GTPase family. In addition, they can exhibit a variety of catalysis-independent roles in specific signaling contexts. Vav proteins play essential signaling roles for both the development and/or effector functions of a large variety of cell lineages, including those belonging to the immune, nervous, and cardiovascular systems. They also contribute to pathological states such as cancer, immune-related dysfunctions, and atherosclerosis. Here, I will provide an integrated view about the evolution, regulation, and effector properties of these signaling molecules. In addition, I will discuss the pros and cons for their potential consideration as therapeutic targets. PMID:25483299

NOD-like receptor cooperativity in effector-triggered immunity.

PubMed

Griebel, Thomas; Maekawa, Takaki; Parker, Jane E

2014-11-01

Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are basic elements of innate immunity in plants and animals. Whereas animal NLRs react to conserved microbe- or damage-associated molecular patterns, plant NLRs intercept the actions of diverse pathogen virulence factors (effectors). In this review, we discuss recent genetic and molecular evidence for functional NLR pairs, and discuss the significance of NLR self-association and heteromeric NLR assemblies in the triggering of downstream signaling pathways. We highlight the versatility and impact of cooperating NLR pairs that combine pathogen sensing with the initiation of defense signaling in both plant and animal immunity. We propose that different NLR receptor molecular configurations provide opportunities for fine-tuning resistance pathways and enhancing the host's pathogen recognition spectrum to keep pace with rapidly evolving microbial populations. Copyright © 2014. Published by Elsevier Ltd.

The effector gene xopAE of Xanthomonas euvesicatoria 85-10 is part of an operon and encodes an E3 ubiquitin ligase.

PubMed

Popov, Georgy; Majhi, Bharat Bhusan; Sessa, Guido

2018-05-21

The type III effector XopAE from the Xanthomonas euvesicatoria strain 85-10 ( Xe 85-10) was previously shown to inhibit plant immunity and enhance pathogen-induced disease symptoms. Evolutionary analysis of 60 xopAE alleles ( AEal ) revealed that the xopAE locus is conserved in multiple Xanthomonas species. The majority of xopAE alleles (55 out of 60) encodes a single ORF ( xopAE ), while in 5 alleles, including AEal 37 of the Xe 85-10 strain, a frame-shift splits the locus into two ORFs ( hpaF and a truncated xopAE ). To test whether the second ORF of AEal 37 ( xopAE 85-10 ) is translated, we examined expression of YFP fused downstream to truncated or mutant forms of the locus in Xanthomonas bacteria. YFP fluorescence was detected at maximal levels when the reporter was in proximity of an internal ribosome-binding site upstream to a rare ATT start codon in the xopAE 85-10 ORF, but severely reduced when these elements were abolished. In agreement with the notion that xopAE 85- 10 is a functional gene, its protein product was translocated into plant cells by the type III secretion system and translocation was dependent on its upstream ORF hpaF. Homology modeling predicted that XopAE 85-10 contains an E3 ligase XL-box domain at the C-terminus, and in vitro assays demonstrated that this domain displays mono-ubiquitination activity. Remarkably, the XL-box was essential for XopAE 85-10 to inhibit PAMP-induced gene expression in Arabidopsis protoplasts. Together, these results indicate that the xopAE 85-10 gene resides in a functional operon, which utilizes the alternative start codon ATT, and encodes a novel XL-box E3 ligase. Importance Xanthomonas bacteria utilize a type III secretion system to cause disease in many crops. This study provides insights into evolution, translocation and biochemical function of the XopAE type III secreted effector contributing to the understanding of Xanthomonas-host interactions. We establish XopAE as core effector of seven Xanthomonas species and elucidate evolution of the Xanthomonas euvesicatoria xopAE locus, which contains an operon encoding a truncated effector. Our findings indicate that this operon evolved from the split of a multi-domains gene into two ORFs that conserved the original domain function. Analysis of xopAE 85-10 translation provides the first evidence for translation initiation from an ATT codon in Xanthomonas Our data demonstrate that XopAE 85-10 is an XL-box E3 ubiquitin ligase and provide insights into structure and function of this effector family. Copyright © 2018 American Society for Microbiology.

A novel lymphoid enhancer-binding factor 1-cytoglobin axis promotes extravasation of osteosarcoma cells into the lungs.

PubMed

Pongsuchart, Mongkol; Kuchimaru, Takahiro; Yonezawa, Sakiko; Tran, Diem Thi Phuong; Kha, Nguyen The; Hoang, Ngoc Thi Hong; Kadonosono, Tetsuya; Kizaka-Kondoh, Shinae

2018-06-21

Lung metastasis is a major cause of mortality in patients with osteosarcoma (OS). A better understanding of the molecular mechanism of OS lung metastasis may facilitate development of new therapeutic strategies to prevent the metastasis. We have established high- and low-metastatic sublines (LM8-H and LM8-L respectively) from Dunn OS cell line LM8 by using in vivo image-guided screening. Among the genes whose expression was significantly increased in LM8-H compared to LM8-L, the transcription factor lymphoid enhancer-binding factor 1 (LEF1) was identified as a factor that promotes LM8-H cell extravasation into the lungs. To identify downstream effectors of LEF1 that are involved in OS lung metastasis, 13 genes were selected based on the LM8 microarray data and genome-wide meta-analysis of a public database for OS patients. Among them, the cytoglobin (Cygb) gene was identified as a key effector in promoting OS extravasation into the lungs. CYGB overexpression increased the extravasation ability of LM8-L cells, whereas knocking out the Cygb gene in LM8-H cells reduced this ability. Our results uncovered a novel LEF1-CYGB axis in OS lung metastasis and may open a new avenue for developing therapeutic strategies to prevent OS lung metastasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

MENA is a transcriptional target of the Wnt/beta-catenin pathway.

PubMed

Najafov, Ayaz; Seker, Tuncay; Even, Ipek; Hoxhaj, Gerta; Selvi, Osman; Ozel, Duygu Esen; Koman, Ahmet; Birgül-İyison, Necla

2012-01-01

Wnt/β-catenin signalling pathway plays important roles in embryonic development and carcinogenesis. Overactivation of the pathway is one of the most common driving forces in major cancers such as colorectal and breast cancers. The downstream effectors of the pathway and its regulation of carcinogenesis and metastasis are still not very well understood. In this study, which was based on two genome-wide transcriptomics screens, we identify MENA (ENAH, Mammalian enabled homologue) as a novel transcriptional target of the Wnt/β-catenin signalling pathway. We show that the expression of MENA is upregulated upon overexpression of degradation-resistant β-catenin. Promoters of all mammalian MENA homologues contain putative binding sites for Tcf4 transcription factor--the primary effector of the Wnt/β-catenin pathway and we demonstrate functionality of these Tcf4-binding sites using luciferase reporter assays and overexpression of β-catenin, Tcf4 and dominant-negative Tcf4. In addition, lithium chloride-mediated inhibition of GSK3β also resulted in increase in MENA mRNA levels. Chromatin immunoprecipitation showed direct interaction between β-catenin and MENA promoter in Huh7 and HEK293 cells and also in mouse brain and liver tissues. Moreover, overexpression of Wnt1 and Wnt3a ligands increased MENA mRNA levels. Additionally, knock-down of MENA ortholog in D. melanogaster eyeful and sensitized eye cancer fly models resulted in increased tumor and metastasis formations. In summary, our study identifies MENA as novel nexus for the Wnt/β-catenin and the Notch signalling cascades.

Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins.

PubMed

Hewezi, Tarek

2015-10-01

Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. © 2015 American Society of Plant Biologists. All Rights Reserved.

Therapeutic Potential of Targeting PAK Signaling.

PubMed

Senapedis, William; Crochiere, Marsha; Baloglu, Erkan; Landesman, Yosef

2016-01-01

The therapeutic potential of targeting p21-Activated Kinases (PAK1 - 6) for the treatment of cancer has recently gained traction in the biotech industry. Many pharmaceutically-viable ATP competitive inhibitors have been through different stages of pre-clinical development with only a single compound evaluated in human trails (PF-3758309). The best studied functional roles of PAK proteins are control of cell adhesion and migration. PAK proteins are known downstream effectors of Ras signaling with PAK expression elevated in cancer (pancreatic, colon, breast, lung and other solid tumors). In addition altered PAK expression is a confirmed driver of this disease, especially in tumors harboring oncogenic Ras. However, there are very few examples of gain-of-function PAK mutations, as a majority of the cancer types have elevated PAK expression due to gene amplification or transcriptional modifications. There is a substantial number of known substrates affected by this aberrant PAK activity. One particular substrate, β-catenin, has garnered interest given its importance in both normal and cancer cell development. These data place PAK proteins between two major signaling pathways in cancer (Ras and β -catenin), making therapeutic targeting of PAKs an intriguing approach for the treatment of a broad array of oncological malignancies.

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome

PubMed Central

van Bueren, Kelly Lammerts; Papangeli, Irinna; Rochais, Francesca; Pearce, Kerra; Roberts, Catherine; Calmont, Amelie; Szumska, Dorota; Kelly, Robert G.; Bhattacharya, Shoumo; Scambler, Peter J.

2010-01-01

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of β-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1lacZ (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1−/− mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures. PMID:20122914

Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

PubMed

Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

2017-01-01

Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

In vivo insertion pool sequencing identifies virulence factors in a complex fungal–host interaction

PubMed Central

Uhse, Simon; Pflug, Florian G.; Stirnberg, Alexandra; Ehrlinger, Klaus; von Haeseler, Arndt

2018-01-01

Large-scale insertional mutagenesis screens can be powerful genome-wide tools if they are streamlined with efficient downstream analysis, which is a serious bottleneck in complex biological systems. A major impediment to the success of next-generation sequencing (NGS)-based screens for virulence factors is that the genetic material of pathogens is often underrepresented within the eukaryotic host, making detection extremely challenging. We therefore established insertion Pool-Sequencing (iPool-Seq) on maize infected with the biotrophic fungus U. maydis. iPool-Seq features tagmentation, unique molecular barcodes, and affinity purification of pathogen insertion mutant DNA from in vivo-infected tissues. In a proof of concept using iPool-Seq, we identified 28 virulence factors, including 23 that were previously uncharacterized, from an initial pool of 195 candidate effector mutants. Because of its sensitivity and quantitative nature, iPool-Seq can be applied to any insertional mutagenesis library and is especially suitable for genetically complex setups like pooled infections of eukaryotic hosts. PMID:29684023

Role of TRP ion channels in cancer and tumorigenesis.

PubMed

Shapovalov, George; Ritaine, Abigael; Skryma, Roman; Prevarskaya, Natalia

2016-05-01

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

Protein kinase A activates the Hippo pathway to modulate cell proliferation and differentiation

PubMed Central

Yu, Fa-Xing; Zhang, Yifan; Park, Hyun Woo; Jewell, Jenna L.; Chen, Qian; Deng, Yaoting; Pan, Duojia; Taylor, Susan S.; Lai, Zhi-Chun; Guan, Kun-Liang

2013-01-01

The Hippo tumor suppressor pathway plays an important role in tissue homeostasis that ensures development of functional organs at proper size. The YAP transcription coactivator is a major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinases Lats1/2. It has recently been shown that YAP activity is regulated by G-protein-coupled receptor signaling. Here we demonstrate that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate Lats kinases and YAP phosphorylation. We also show that inactivation of YAP is crucial for PKA-induced adipogenesis. In addition, PKA activation in Drosophila inhibits the expression of Yorki (Yki, a YAP ortholog) target genes involved in cell proliferation and death. Taken together, our study demonstrates that Hippo–YAP is a key signaling branch of cAMP and PKA and reveals new insight into mechanisms of PKA in regulating a broad range of cellular functions. PMID:23752589

An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer.

PubMed

Vallejo, Adrian; Perurena, Naiara; Guruceaga, Elisabet; Mazur, Pawel K; Martinez-Canarias, Susana; Zandueta, Carolina; Valencia, Karmele; Arricibita, Andrea; Gwinn, Dana; Sayles, Leanne C; Chuang, Chen-Hua; Guembe, Laura; Bailey, Peter; Chang, David K; Biankin, Andrew; Ponz-Sarvise, Mariano; Andersen, Jesper B; Khatri, Purvesh; Bozec, Aline; Sweet-Cordero, E Alejandro; Sage, Julien; Lecanda, Fernando; Vicent, Silve

2017-02-21

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers.

A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

PubMed

Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

2017-09-19

Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuang, Xianghong; Shen, Jianjun; Wong, Paul K.Y.

Abnormal thymocyte development with thymic lymphomagenesis inevitably occurs in Atm-/- mice, indicating that ATM plays a pivotal role in regulating postnatal thymocyte development and preventing thymic lymphomagenesis. The mechanism for ATM controls these processes is unclear. We have shown previously that c-Myc, an oncoprotein regulated by the mammalian target of rapamycin (mTOR), is overexpressed in Atm-/- thymocytes. Here, we show that inhibition of mTOR signaling with its specific inhibitor, rapamycin, suppresses normal thymocyte DNA synthesis by downregulating 4EBP1, but not S6K, and that 4EBP1 phosphorylation and cyclin D1 expression are coordinately increased in Atm-/- thymocytes. Administration of rapamycin to Atm-/-more » mice attenuates elevated phospho-4EBP1, c-Myc and cyclin D1 in their thymocytes, and delays thymic lymphoma development. These results indicate that mTOR downstream effector 4EBP1 is essential for normal thymocyte proliferation, but deregulation of 4EBP1 in Atm deficiency is a major factor driving thymic lymphomagenesis in the animals.« less

SPIN1, negatively regulated by miR-148/152, enhances Adriamycin resistance via upregulating drug metabolizing enzymes and transporter in breast cancer.

PubMed

Chen, Xu; Wang, Ya-Wen; Gao, Peng

2018-05-09

Spindlin1 (SPIN1), a protein highly expressed in several human cancers, has been correlated with tumorigenesis and development. Alterations of drug metabolizing enzymes and drug transporters are major determinants of chemoresistance in tumor cells. However, whether the metabolizing enzymes and transporters are under the control of SPIN1 in breast cancer chemoresistance has not yet been defined. SPIN1 expression in breast cancer cells and tissues was detected by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Chemosensitivity assays in vitro and in vivo were performed to determine the effect of SPIN1 on Adriamycin resistance. Downstream effectors of SPIN1 were screened by microarray and confirmed by qRT-PCR and Western blot. Luciferase assay and Western blot were used to identify miRNAs regulating SPIN1. We showed that SPIN1 was significantly elevated in drug-resistant breast cancer cell lines and tissues, compared with the chemosensitive ones. SPIN1 enhanced Adriamycin resistance of breast cancer cells in vitro, and downregulation of SPIN1 by miRNA could decrease Adriamycin resistance in vivo. Mechanistically, drug metabolizing enzymes and transporter CYP2C8, UGT2B4, UGT2B17 and ABCB4 were proven to be downstream effectors of SPIN1. Notably, SPIN1 was identified as a direct target of the miR-148/152 family (miR-148a-3p, miR-148b-3p and miR-152-3p). As expected, miR-148a-3p, miR-148b-3p or miR-152-3p could increase Adriamycin sensitivity in breast cancer cells in vitro. Moreover, high expression of SPIN1 or low expression of the miR-148/152 family predicted poorer survival in breast cancer patients. Our results establish that SPIN1, negatively regulated by the miR-148/152 family, enhances Adriamycin resistance in breast cancer via upregulating the expression of drug metabolizing enzymes and drug transporter.

Regulation of Transient Receptor Potential channels by the phospholipase C pathway

PubMed Central

Rohacs, Tibor

2013-01-01

Transient Receptor Potential (TRP) channels were discovered while analyzing visual mutants in drosophila. The protein encoded by the transient receptor potential (trp) gene is a Ca2+ permeable cation channel activated downstream of the phospholipase C (PLC) pathway. While searching for homologues in other organisms, a surprisingly large number of mammalian TRP channels were cloned. The regulation of TRP channels is quite diverse, but many of them are either activated downstream of the PLC pathway, or modulated by it. This review will summarize the current knowledge on regulation of TRP channels by the PLC pathway, with special focus on TRPC-s, which can be considered as effectors of the PLC pathway, and the heat and capsaicin sensitive TRPV1, which is modulated by the PLC pathway in a complex manner. PMID:23916247

Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing

NASA Astrophysics Data System (ADS)

Wülfing, Christoph; Purtic, Bozidar; Klem, Jennifer; Schatzle, John D.

2003-06-01

Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.

Elucidating the Tumor-Suppressive Role of SLITs in Maintaining the Basal Cell Niche

DTIC Science & Technology

2011-10-01

2011) • Discovered Rac as a downstream effector of SLIT/ROBO1 signaling and potential link to Abl signaling in the gland (Macias et al., in preparation...into mechanisms by which normal stem/progenitor cells are regulated, leading to potential insights into how they may be deregulated upon cancerous...of AGMs in breast cancer tumorigenesis and progression and consider their potential in development of new diagnostic markers and therapeutics. AGMs in

Murine natural killer immunoreceptors use distinct proximal signaling complexes to direct cell function

PubMed Central

May, Rebecca M.; Okumura, Mariko; Hsu, Chin-Jung; Bassiri, Hamid; Yang, Enjun; Rak, Gregory; Mace, Emily M.; Philip, Naomi H.; Zhang, Weiguo; Baumgart, Tobias; Orange, Jordan S.; Nichols, Kim E.

2013-01-01

Signaling pathways leading to natural killer (NK)–cell effector function are complex and incompletely understood. Here, we investigated the proximal signaling pathways downstream of the immunotyrosine-based activation motif (ITAM) bearing activating receptors. We found that the adaptor molecule SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is recruited to microclusters at the plasma membrane in activated NK cells and that this is required for initiation of downstream signaling and multiple NK-cell effector functions in vitro and in vivo. Surprisingly, we found that 2 types of proximal signaling complexes involving SLP-76 were formed. In addition to the canonical membrane complex formed between SLP-76 and linker for activation of T cells (LAT) family members, a novel LAT family–independent SLP-76–dependent signaling pathway was identified. The LAT family–independent pathway involved the SH2 domain of SLP-76 and adhesion and degranulation-promoting adaptor protein (ADAP). Both the LAT family–dependent and ADAP-dependent pathway contributed to interferon-gamma production and cytotoxicity; however, they were not essential for other SLP-76–dependent events, including phosphorylation of AKT and extracellular signal–related kinase and cellular proliferation. These results demonstrate that NK cells possess an unexpected bifurcation of proximal ITAM-mediated signaling, each involving SLP-76 and contributing to optimal NK-cell function. PMID:23407547

Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

PubMed Central

Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

2015-01-01

Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

PubMed

Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

2017-04-01

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064. © 2016 AlphaMed Press.

Hydrogen sulfide as an oxygen sensor.

PubMed

Olson, Kenneth R

2015-02-10

Although oxygen (O2)-sensing cells and tissues have been known for decades, the identity of the O2-sensing mechanism has remained elusive. Evidence is accumulating that O2-dependent metabolism of hydrogen sulfide (H2S) is this enigmatic O2 sensor. The elucidation of biochemical pathways involved in H2S synthesis and metabolism have shown that reciprocal H2S/O2 interactions have been inexorably linked throughout eukaryotic evolution; there are multiple foci by which O2 controls H2S inactivation, and the effects of H2S on downstream signaling events are consistent with those activated by hypoxia. H2S-mediated O2 sensing has been demonstrated in a variety of O2-sensing tissues in vertebrate cardiovascular and respiratory systems, including smooth muscle in systemic and respiratory blood vessels and airways, carotid body, adrenal medulla, and other peripheral as well as central chemoreceptors. Information is now needed on the intracellular location and stoichometry of these signaling processes and how and which downstream effectors are activated by H2S and its metabolites. Development of specific inhibitors of H2S metabolism and effector activation as well as cellular organelle-targeted compounds that release H2S in a time- or environmentally controlled way will not only enhance our understanding of this signaling process but also provide direction for future therapeutic applications.

Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition.

PubMed

Mammoto, Akiko; Huang, Sui; Moore, Kimberly; Oh, Philmo; Ingber, Donald E

2004-06-18

Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana.

PubMed

Choi, Sera; Jayaraman, Jay; Segonzac, Cécile; Park, Hye-Jee; Park, Hanbi; Han, Sang-Wook; Sohn, Kee Hoon

2017-01-01

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium -mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa , Psa -NZ V13 and Psa -NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana . Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium -mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1 , NDR1 , or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana . In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta .

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana

PubMed Central

Choi, Sera; Jayaraman, Jay; Segonzac, Cécile; Park, Hye-Jee; Park, Hanbi; Han, Sang-Wook; Sohn, Kee Hoon

2017-01-01

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium-mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa, Psa-NZ V13 and Psa-NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana. Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium-mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1, NDR1, or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana. In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta. PMID:29326748

Serial killers: ordering caspase activation events in apoptosis.

PubMed

Slee, E A; Adrain, C; Martin, S J

1999-11-01

Caspases participate in the molecular control of apoptosis in several guises; as triggers of the death machinery, as regulatory elements within it, and ultimately as a subset of the effector elements of the machinery itself. The mammalian caspase family is steadily growing and currently contains 14 members. At present, it is unclear whether all of these proteases participate in apoptosis. Thus, current research in this area is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of cell death. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation. As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling (upstream) and effector (downstream) roles based upon their prodomain lengths. In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme. Here, we discuss the apoptosis-associated caspase cascade and the hierarchy of caspase activation events within it.

TCR Signal Strength Alters T–DC Activation and Interaction Times and Directs the Outcome of Differentiation

PubMed Central

van Panhuys, Nicholas

2016-01-01

The ability of CD4+ T cells to differentiate into effector subsets underpins their ability to shape the immune response and mediate host protection. During T cell receptor-induced activation of CD4+ T cells, both the quality and quantity of specific activatory peptide/MHC ligands have been shown to control the polarization of naive CD4+ T cells in addition to co-stimulatory and cytokine-based signals. Recently, advances in two-­photon microscopy and tetramer-based cell tracking methods have allowed investigators to greatly extend the study of the role of TCR signaling in effector differentiation under in vivo conditions. In this review, we consider data from recent in vivo studies analyzing the role of TCR signal strength in controlling the outcome of CD4+ T cell differentiation and discuss the role of TCR in controlling the critical nature of CD4+ T cell interactions with dendritic cells during activation. We further propose a model whereby TCR signal strength controls the temporal aspects of T–DC interactions and the implications for this in mediating the downstream signaling events, which influence the transcriptional and epigenetic regulation of effector differentiation. PMID:26834747

Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

PubMed Central

Gao, Xiquan; Cox, Kevin L.; He, Ping

2014-01-01

An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP), which is called PAMP-triggered immunity (PTI). The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI). Calcium (Ca2+) signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs) have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF)-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response. PMID:27135498

Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

PubMed Central

Chen, Changlong; Chen, Yongpan; Jian, Heng; Yang, Dan; Dai, Yiran; Pan, Lingling; Shi, Fengwei; Yang, Shanshan; Liu, Qian

2018-01-01

Heterodera avenae is one of the most important plant pathogens and causes vast losses in cereal crops. As a sedentary endoparasitic nematode, H. avenae secretes effectors that modify plant defenses and promote its biotrophic infection of its hosts. However, the number of effectors involved in the interaction between H. avenae and host defenses remains unclear. Here, we report the identification of putative effectors in H. avenae that regulate plant defenses on a large scale. Our results showed that 78 of the 95 putative effectors suppressed programmed cell death (PCD) triggered by BAX and that 7 of the putative effectors themselves caused cell death in Nicotiana benthamiana. Among the cell-death-inducing effectors, three were found to be dependent on their specific domains to trigger cell death and to be expressed in esophageal gland cells by in situ hybridization. Ten candidate effectors that suppressed BAX-triggered PCD also suppressed PCD triggered by the elicitor PsojNIP and at least one R-protein/cognate effector pair, suggesting that they are active in suppressing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Notably, with the exception of isotig16060, these putative effectors could also suppress PCD triggered by cell-death-inducing effectors from H. avenae, indicating that those effectors may cooperate to promote nematode parasitism. Collectively, our results indicate that the majority of the tested effectors of H. avenae may play important roles in suppressing cell death induced by different elicitors in N. benthamiana. PMID:29379510

Plant-bacterial pathogen interactions mediated by type III effectors.

PubMed

Feng, Feng; Zhou, Jian-Min

2012-08-01

Effectors secreted by the bacterial type III system play a central role in the interaction between Gram-negative bacterial pathogens and their host plants. Recent advances in the effector studies have helped cementing several key concepts concerning bacterial pathogenesis, plant immunity, and plant-pathogen co-evolution. Type III effectors use a variety of biochemical mechanisms to target specific host proteins or DNA for pathogenesis. The identifications of their host targets led to the identification of novel components of plant innate immune system. Key modules of plant immune signaling pathways such as immune receptor complexes and MAPK cascades have emerged as a major battle ground for host-pathogen adaptation. These modules are attacked by multiple type III effectors, and some components of these modules have evolved to actively sense the effectors and trigger immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Antipsychotics activate the TGFβ pathway effector SMAD3

PubMed Central

Cohen, T.; Sundaresh, S.; Levine, F.

2014-01-01

Although effective in treating an array of neurological disorders, antipsychotics are associated with deleterious metabolic side effects. Through high-throughput screening, we previously identified phenothiazine antipsychotics as modulators of the human insulin promoter. Here, we extended our initial finding to structurally diverse typical and atypical antipsychotics. We then identified the TGFβ pathway as being involved in the effect of antipsychotics on the insulin promoter, finding that antipsychotics activated SMAD3, a downstream effector of the TGFβ pathway, through a receptor distinct from the TGFβ receptor family and known neurotransmitter receptor targets of antipsychotics. Of note, antipsychotics that do not cause metabolic side effects did not activate SMAD3. In vivo relevance was demonstrated by reanalysis of gene expression data from human brains treated with antipsychotics, which showed altered expression of SMAD3 responsive genes. This work raises the possibility that antipsychotics could be designed that retain beneficial CNS activity while lacking deleterious metabolic side effects. PMID:22290122

PI3K: A Crucial Piece in the RAS Signaling Puzzle.

PubMed

Krygowska, Agata Adelajda; Castellano, Esther

2018-06-01

RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Caspase 3 promotes genetic instability and carcinogenesis

PubMed Central

Liu, Xinjian; He, Yujun; Li, Fang; Huang, Qian; Kato, Takamitsu A.; Hall, Russell P; Li, Chuan-Yuan

2015-01-01

Summary Apoptosis is typically considered an anti-oncogenic process since caspase activation can promote the elimination of genetically unstable or damaged cells. We report that a central effector of apoptosis, caspase 3, facilitates, rather than suppresses, chemical and radiation-induced genetic instability and carcinogenesis. We found that a significant fraction of mammalian cells treated with ionizing radiation can survive, despite caspase 3 activation. Moreover, this sublethal activation of caspase 3 promoted persistent DNA damage and oncogenic transformation. In addition, chemically-induced skin carcinogenesis was significantly reduced in mice genetically deficient in caspase 3. Furthermore, attenuation of Endo G activity significantly reduced radiation-induced DNA damage and oncogenic transformation, identifying Endo G as a downstream effector of caspase 3 in this pathway. Our findings suggest that rather than acting as a broad inhibitor of carcinogenesis, caspase 3 activation may contribute to genome instability and play a pivotal role in tumor formation following damage. PMID:25866249

Ral signaling pathway in health and cancer.

PubMed

Moghadam, Adel Rezaei; Patrad, Elham; Tafsiri, Elham; Peng, Warner; Fangman, Benjamin; Pluard, Timothy J; Accurso, Anthony; Salacz, Michael; Shah, Kushal; Ricke, Brandon; Bi, Danse; Kimura, Kyle; Graves, Leland; Najad, Marzieh Khajoie; Dolatkhah, Roya; Sanaat, Zohreh; Yazdi, Mina; Tavakolinia, Naeimeh; Mazani, Mohammad; Amani, Mojtaba; Ghavami, Saeid; Gartell, Robyn; Reilly, Colleen; Naima, Zaid; Esfandyari, Tuba; Farassati, Faris

2017-12-01

The Ral (Ras-Like) signaling pathway plays an important role in the biology of cells. A plethora of effects is regulated by this signaling pathway and its prooncogenic effectors. Our team has demonstrated the overactivation of the RalA signaling pathway in a number of human malignancies including cancers of the liver, ovary, lung, brain, and malignant peripheral nerve sheath tumors. Additionally, we have shown that the activation of RalA in cancer stem cells is higher in comparison with differentiated cancer cells. In this article, we review the role of Ral signaling in health and disease with a focus on the role of this multifunctional protein in the generation of therapies for cancer. An improved understanding of this pathway can lead to development of a novel class of anticancer therapies that functions on the basis of intervention with RalA or its downstream effectors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

PubMed Central

Liu, Yunxiao; Lan, Xia; Song, Shiren; Yin, Ling; Dry, Ian B.; Qu, Junjie; Xiang, Jiang; Lu, Jiang

2018-01-01

Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria. PMID:29706971

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene

PubMed Central

Lilley, Catherine J.; Maqbool, Abbas; Wu, Duqing; Yusup, Hazijah B.; Jones, Laura M.; Birch, Paul R. J.; Urwin, Peter E.

2018-01-01

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function. PMID:29641602

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

PubMed

Ali, Ramadan A; Camick, Christina; Wiles, Katherine; Walseth, Timothy F; Slama, James T; Bhattacharya, Sumit; Giovannucci, David R; Wall, Katherine A

2016-02-26

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells*

PubMed Central

Ali, Ramadan A.; Camick, Christina; Wiles, Katherine; Walseth, Timothy F.; Slama, James T.; Bhattacharya, Sumit; Giovannucci, David R.; Wall, Katherine A.

2016-01-01

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca2+ mobilizing second messenger discovered to date, has been implicated in Ca2+ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca2+ signaling or the identity of the Ca2+ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca2+ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca2+ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca2+ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca2+ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca2+ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. PMID:26728458

Cereal powdery mildew effectors: a complex toolbox for an obligate pathogen.

PubMed

Bourras, Salim; Praz, Coraline R; Spanu, Pietro D; Keller, Beat

2018-02-15

Cereal powdery mildews are major pathogens of cultivated monocot crops, and all are obligate biotrophic fungi that can only grow and reproduce on living hosts. This lifestyle is combined with extreme host specialization where every mildew subspecies (referred to as forma specialis) can only infect one plant species. Recently there has been much progress in our understanding of the possible roles effectors play in this complex host-pathogen interaction. Here, we review current knowledge on the origin, evolution, and mode of action of cereal mildew effectors, with a particular focus on recent advances in the identification of bona fide effectors and avirulence effector proteins from wheat and barley powdery mildews. Copyright © 2018 Elsevier Ltd. All rights reserved.

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida.

PubMed

Thorpe, Peter; Mantelin, Sophie; Cock, Peter Ja; Blok, Vivian C; Coke, Mirela C; Eves-van den Akker, Sebastian; Guzeeva, Elena; Lilley, Catherine J; Smant, Geert; Reid, Adam J; Wright, Kathryn M; Urwin, Peter E; Jones, John T

2014-10-23

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Causes and Consequences of Replication Stress

PubMed Central

Zeman, Michelle K.; Cimprich, Karlene A.

2015-01-01

Replication stress is a complex phenomenon which has serious implications for genome stability, cell survival, and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATM- and Rad3-related (ATR). ATR and its downstream effectors stabilize and help to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding these pathways may be key to diagnosis and treatment of human diseases caused by defective responses to replication stress. PMID:24366029

Functional Validation of H2 Relaxin and Its Downstream Effectors as Mediators, Therapeutic Targets, and Potential Biomarkers of Prostate Cancer Progression

DTIC Science & Technology

2009-06-01

increased translocation of NFkappaB to the nucleus, LNCaP stably transfected with H2 relaxin are more resistant to treatment with chemotherapeutic...the PKA and NFkappaB signaling pathways promotes CR growth and chemo-resistance respectively. 15. SUBJECT TERMS H2 relaxin, castrate resistant...increased translocation of NFkappaB to the nucleus. Although not specified in the statement of work, through interaction with Dr. Allen Gao’s lab we

Functional Validation of H2 Relaxin, and its Downstream Effectors, as Mediators, Therapeutic Targets and Potential Biomarkers of Prostate Cancer Progression

DTIC Science & Technology

2011-06-01

contribute to CR growth. Inhibition of PKA attenuated RLN2-mediated AR activity, inhibited proliferation and caused a small but significant increase in...detection of p53 abnormalities and increases their predictive value. BJU Int 94: 996–1002. Silvertown JD, Geddes BJ, Summerlee AJ. (2003). Adenovirus ...in facilitating H2 relaxin-mediated CR growth of CaP cells. Inhibition of PKA attenuated RLN2-mediated AR activity, inhibited proliferation and

Emerging role of the Jun N-terminal kinase interactome in human health.

PubMed

Guo, Xiao-Xi; An, Su; Yang, Yang; Liu, Ying; Hao, Qian; Tang, Tao; Xu, Tian-Rui

2018-02-08

The c-Jun N-terminal kinases (JNKs) are located downstream of Ras-mitogen activated protein kinase signaling cascades. More than 20 years of study has shown that JNKs control cell fate and many cellular functions. JNKs and their interacting proteins form a complicated network with diverse biological functions and physiological effects. Members of the JNK interactome include Jun, amyloid precursor protein, and insulin receptor substrate. Recent studies have shown that the JNK interactome is involved in tumorigenesis, neuron development, and insulin resistance. In this review, we summarize the features of the JNK interactome and classify its members into three groups: upstream regulators, downstream effectors, and scaffold partners. We also highlight the unique cellular signaling mechanisms of JNKs and provide more insights into the roles of the JNK interactome in human diseases. © 2018 International Federation for Cell Biology.

Wnt some lose some: transcriptional governance of stem cells by Wnt/β-catenin signaling

PubMed Central

Lien, Wen-Hui; Fuchs, Elaine

2014-01-01

In mammals, Wnt/β-catenin signaling features prominently in stem cells and cancers, but how and for what purposes have been matters of much debate. In this review, we summarize our current knowledge of Wnt/β-catenin signaling and its downstream transcriptional regulators in normal and malignant stem cells. We centered this review largely on three types of stem cells—embryonic stem cells, hair follicle stem cells, and intestinal epithelial stem cells—in which the roles of Wnt/β-catenin have been extensively studied. Using these models, we unravel how many controversial issues surrounding Wnt signaling have been resolved by dissecting the diversity of its downstream circuitry and effectors, often leading to opposite outcomes of Wnt/β-catenin-mediated regulation and differences rooted in stage- and context-dependent effects. PMID:25030692

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000[OPEN

PubMed Central

Lewis, Laura A.; Polanski, Krzysztof; de Torres-Zabala, Marta; Bowden, Laura; Jenkins, Dafyd J.; Hill, Claire; Baxter, Laura; Truman, William; Prusinska, Justyna; Hickman, Richard; Wild, David L.; Ott, Sascha; Buchanan-Wollaston, Vicky; Beynon, Jim

2015-01-01

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. PMID:26566919

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

PubMed

Lewis, Laura A; Polanski, Krzysztof; de Torres-Zabala, Marta; Jayaraman, Siddharth; Bowden, Laura; Moore, Jonathan; Penfold, Christopher A; Jenkins, Dafyd J; Hill, Claire; Baxter, Laura; Kulasekaran, Satish; Truman, William; Littlejohn, George; Prusinska, Justyna; Mead, Andrew; Steinbrenner, Jens; Hickman, Richard; Rand, David; Wild, David L; Ott, Sascha; Buchanan-Wollaston, Vicky; Smirnoff, Nick; Beynon, Jim; Denby, Katherine; Grant, Murray

2015-11-01

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. © 2015 American Society of Plant Biologists. All rights reserved.

NIK1-mediated translation suppression functions as a plant antiviral immunity mechanism.

PubMed

Zorzatto, Cristiane; Machado, João Paulo B; Lopes, Kênia V G; Nascimento, Kelly J T; Pereira, Welison A; Brustolini, Otávio J B; Reis, Pedro A B; Calil, Iara P; Deguchi, Michihito; Sachetto-Martins, Gilberto; Gouveia, Bianca C; Loriato, Virgílio A P; Silva, Marcos A C; Silva, Fabyano F; Santos, Anésia A; Chory, Joanne; Fontes, Elizabeth P B

2015-04-30

Plants and plant pathogens are subject to continuous co-evolutionary pressure for dominance, and the outcomes of these interactions can substantially impact agriculture and food security. In virus-plant interactions, one of the major mechanisms for plant antiviral immunity relies on RNA silencing, which is often suppressed by co-evolving virus suppressors, thus enhancing viral pathogenicity in susceptible hosts. In addition, plants use the nucleotide-binding and leucine-rich repeat (NB-LRR) domain-containing resistance proteins, which recognize viral effectors to activate effector-triggered immunity in a defence mechanism similar to that employed in non-viral infections. Unlike most eukaryotic organisms, plants are not known to activate mechanisms of host global translation suppression to fight viruses. Here we demonstrate in Arabidopsis that the constitutive activation of NIK1, a leucine-rich repeat receptor-like kinase (LRR-RLK) identified as a virulence target of the begomovirus nuclear shuttle protein (NSP), leads to global translation suppression and translocation of the downstream component RPL10 to the nucleus, where it interacts with a newly identified MYB-like protein, L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB), to downregulate translational machinery genes fully. LIMYB overexpression represses ribosomal protein genes at the transcriptional level, resulting in protein synthesis inhibition, decreased viral messenger RNA association with polysome fractions and enhanced tolerance to begomovirus. By contrast, the loss of LIMYB function releases the repression of translation-related genes and increases susceptibility to virus infection. Therefore, LIMYB links immune receptor LRR-RLK activation to global translation suppression as an antiviral immunity strategy in plants.

Dendritic Cytoskeletal Architecture Is Modulated by Combinatorial Transcriptional Regulation in Drosophila melanogaster.

PubMed

Das, Ravi; Bhattacharjee, Shatabdi; Patel, Atit A; Harris, Jenna M; Bhattacharya, Surajit; Letcher, Jamin M; Clark, Sarah G; Nanda, Sumit; Iyer, Eswar Prasad R; Ascoli, Giorgio A; Cox, Daniel N

2017-12-01

Transcription factors (TFs) have emerged as essential cell autonomous mediators of subtype specific dendritogenesis; however, the downstream effectors of these TFs remain largely unknown, as are the cellular events that TFs control to direct morphological change. As dendritic morphology is largely dictated by the organization of the actin and microtubule (MT) cytoskeletons, elucidating TF-mediated cytoskeletal regulatory programs is key to understanding molecular control of diverse dendritic morphologies. Previous studies in Drosophila melanogaster have demonstrated that the conserved TFs Cut and Knot exert combinatorial control over aspects of dendritic cytoskeleton development, promoting actin and MT-based arbor morphology, respectively. To investigate transcriptional targets of Cut and/or Knot regulation, we conducted systematic neurogenomic studies, coupled with in vivo genetic screens utilizing multi-fluor cytoskeletal and membrane marker reporters. These analyses identified a host of putative Cut and/or Knot effector molecules, and a subset of these putative TF targets converge on modulating dendritic cytoskeletal architecture, which are grouped into three major phenotypic categories, based upon neuromorphometric analyses: complexity enhancer, complexity shifter, and complexity suppressor. Complexity enhancer genes normally function to promote higher order dendritic growth and branching with variable effects on MT stabilization and F-actin organization, whereas complexity shifter and complexity suppressor genes normally function in regulating proximal-distal branching distribution or in restricting higher order branching complexity, respectively, with spatially restricted impacts on the dendritic cytoskeleton. Collectively, we implicate novel genes and cellular programs by which TFs distinctly and combinatorially govern dendritogenesis via cytoskeletal modulation. Copyright © 2017 by the Genetics Society of America.

RSK3 is required for concentric myocyte hypertrophy in an activated Raf1 model for Noonan syndrome.

PubMed

Passariello, Catherine L; Martinez, Eliana C; Thakur, Hrishikesh; Cesareo, Maria; Li, Jinliang; Kapiloff, Michael S

2016-04-01

Noonan syndrome (NS) is a congenital disorder resulting from mutations of the Ras-Raf signaling pathway. Hypertrophic cardiomyopathy associated with RAF1 "RASopathy" mutations is a major risk factor for heart failure and death in NS and has been attributed to activation of MEK1/2-ERK1/2 mitogen-activated protein kinases. We recently discovered that type 3 p90 ribosomal S6 kinase (RSK3) is an ERK effector that is required, like ERK1/2, for concentric myocyte hypertrophy in response to pathological stress such as pressure overload. In order to test whether RSK3 also contributes to NS-associated hypertrophic cardiomyopathy, RSK3 knock-out mice were crossed with mice bearing the Raf1(L613V) human NS mutation. We confirmed that Raf1(L613V) knock-in confers a NS-like phenotype, including cardiac hypertrophy. Active RSK3 was increased in Raf1(L613V) mice. Constitutive RSK3 gene deletion prevented the Raf1(L613V)-dependent concentric growth in width of the cardiac myocyte and attenuated cardiac hypertrophy in female mice. These results are consistent with RSK3 being an important mediator of ERK1/2-dependent growth in RASopathy. In conjunction with previously published data showing that RSK3 is important for pathological remodeling of the heart, these data suggest that targeting of this downstream MAP-kinase pathway effector should be considered in the treatment of RASopathy-associated hypertrophic cardiomyopathy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oxidative stress, caspase-3 activation and cleavage of ROCK-1 play an essential role in MeHg-induced cell death in primary astroglial cells.

PubMed

Dos Santos, Alessandra Antunes; López-Granero, Caridad; Farina, Marcelo; Rocha, João B T; Bowman, Aaron B; Aschner, Michael

2018-03-01

Methylmercury is a toxic environmental contaminant that elicits significant toxicity in humans. The central nervous system is the primary target of toxicity, and is particularly vulnerable during development. Rho-associated protein kinase 1 (ROCK-1) is a major downstream effector of the small GTPase RhoA and a direct substrate of caspase-3. The activation of ROCK-1 is necessary for membrane blebbing during apoptosis. In this work, we examined whether MeHg could affect the RhoA/ROCK-1 signaling pathway in primary cultures of mouse astrocytes. Exposure of cells with 10 μM MeHg decreased cellular viability after 24 h of incubation. This reduction in viability was preceded by a significant increase in intracellular and mitochondrial reactive oxygen species levels, as well as a reduced NAD + /NADH ratio. MeHg also induced an increase in mitochondrial-dependent caspase-9 and caspase-3, while the levels of RhoA protein expression were reduced or unchanged. We further found that MeHg induced ROCK-1 cleavage/activation and promoted LIMK1 and MYPT1 phosphorylation, both of which are the best characterized ROCK-1 downstream targets. Inhibiting ROCK-1 and caspases activation attenuated the MeHg-induced cell death. Collectively, these findings are the first to show that astrocytes exposed to MeHg showed increased cleavage/activation of ROCK-1, which was independent of the small GTPase RhoA. Copyright © 2018. Published by Elsevier Ltd.

Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells

PubMed Central

Smith, Stephen P.; Scarpini, Cinzia G.; Groves, Ian J.; Odle, Richard I.; Coleman, Nicholas

2016-01-01

Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of ‘master regulators’ for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins. PMID:27457222

A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib

PubMed Central

Yang, Lida; Kaur, Harmandeep; Pestel, Jenny; Looso, Mario; Nolte, Hendrik; Krishnan, Ramesh K.; Bünemann, Moritz; Offermanns, Stefan; Swiercz, Jakub M.

2017-01-01

Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor βPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration. PMID:28007914

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

PubMed

Suda, Yasuyuki; Tachikawa, Hiroyuki; Inoue, Ichiro; Kurita, Tomokazu; Saito, Chieko; Kurokawa, Kazuo; Nakano, Akihiko; Irie, Kenji

2018-02-01

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Parthanatos, a messenger of death

PubMed Central

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2015-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s multiple roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include, but are not limited to DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its active role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 over activation underlies cell death in experimental models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into designing PARP-1 inhibitors and understanding mechanisms downstream of PARP-1 over activation. PARP-1 overactivation may kill by depleting cellular energy through nicotinamide adenine dinucleotide (NAD+) consumption, and by releasing the cell death effector apoptosis-inducing factor (AIF). Unexpectedly, recent evidence shows that poly-ADP ribose (PAR) polymer itself, and not the consumption of NAD+ is the source of cytotoxicity. Thus, PAR polymer acts as a cell death effector downstream of PARP-1-mediated cell death signaling. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will summarize the proposed mechanisms by which PARP-1 overactivation kills. We will present evidence for parthanatos, and the questions raised by these recent findings. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 over activation. PMID:19273119

HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

PubMed Central

Choi, Yiseul; Ko, Young San; Park, Jinju; Choi, Youngsun; Kim, Younghoon; Pyo, Jung-Soo; Jang, Bo Gun; Hwang, Douk Ho; Kim, Woo Ho; Lee, Byung Lan

2016-01-01

AIM To investigated the relationships between HER2, c-Jun N-terminal kinase (JNK) and protein kinase B (AKT) with respect to metastatic potential of HER2-positive gastric cancer (GC) cells. METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility. CONCLUSION HER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC. PMID:27895401

The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.

PubMed

Gibson, Monica Prasad; Zhu, Qinglin; Wang, Suzhen; Liu, Qilin; Liu, Ying; Wang, Xiaofang; Yuan, Baozhi; Ruest, L Bruno; Feng, Jian Q; D'Souza, Rena N; Qin, Chunlin; Lu, Yongbo

2013-03-08

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

Phytophthora suppressor of RNA silencing 2 is a conserved RxLR effector that promotes infection in soybean and Arabidopsis thaliana.

PubMed

Xiong, Qin; Ye, Wenwu; Choi, Duseok; Wong, James; Qiao, Yongli; Tao, Kai; Wang, Yuanchao; Ma, Wenbo

2014-12-01

The genus Phytophthora consists of notorious and emerging pathogens of economically important crops. Each Phytophthora genome encodes several hundreds of cytoplasmic effectors, which are believed to manipulate plant immune response inside the host cells. However, the majority of Phytophthora effectors remain functionally uncharacterized. We recently discovered two effectors from the soybean stem and root rot pathogen Phytophthora sojae with the activity to suppress RNA silencing in plants. These effectors are designated Phytophthora suppressor of RNA silencing (PSRs). Here, we report that the P. sojae PSR2 (PsPSR2) belongs to a conserved and widespread effector family in Phytophthora. A PsPSR2-like effector produced by P. infestans (PiPSR2) can also suppress RNA silencing in plants and promote Phytophthora infection, suggesting that the PSR2 family effectors have conserved functions in plant hosts. Using Agrobacterium rhizogenes-mediated hairy roots induction, we demonstrated that the expression of PsPSR2 rendered hypersusceptibility of soybean to P. sojae. Enhanced susceptibility was also observed in PsPSR2-expressing Arabidopsis thaliana plants during Phytophthora but not bacterial infection. These experiments provide strong evidence that PSR2 is a conserved Phytophthora effector family that performs important virulence functions specifically during Phytophthora infection of various plant hosts.

A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta.

PubMed

Zhang, Lei; Davies, Laura J; Elling, Axel A

2015-01-01

Root-knot nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands of nematodes and secreted into plant tissue through a needle-like stylet. Effectors characterized to date have been shown to mediate processes essential for nematode pathogenesis. To gain an insight into their site of action and putative function, the subcellular localization of 13 previously isolated Meloidogyne incognita effectors was determined. Translational fusions were created between effectors and EGFP-GUS (enhanced green fluorescent protein-β-glucuronidase) reporter genes, which were transiently expressed in tobacco leaf cells. The majority of effectors localized to the cytoplasm, with one effector, 7H08, imported into the nuclei of plant cells. Deletion analysis revealed that the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. As a result of the nuclear localization of the effector, 7H08 was tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Effector biology of plant-associated organisms: concepts and perspectives.

PubMed

Win, J; Chaparro-Garcia, A; Belhaj, K; Saunders, D G O; Yoshida, K; Dong, S; Schornack, S; Zipfel, C; Robatzek, S; Hogenhout, S A; Kamoun, S

2012-01-01

Every plant is closely associated with a variety of living organisms. Therefore, deciphering how plants interact with mutualistic and parasitic organisms is essential for a comprehensive understanding of the biology of plants. The field of plant-biotic interactions has recently coalesced around an integrated model. Major classes of molecular players both from plants and their associated organisms have been revealed. These include cell surface and intracellular immune receptors of plants as well as apoplastic and host-cell-translocated (cytoplasmic) effectors of the invading organism. This article focuses on effectors, molecules secreted by plant-associated organisms that alter plant processes. Effectors have emerged as a central class of molecules in our integrated view of plant-microbe interactions. Their study has significantly contributed to advancing our knowledge of plant hormones, plant development, plant receptors, and epigenetics. Many pathogen effectors are extraordinary examples of biological innovation; they include some of the most remarkable proteins known to function inside plant cells. Here, we review some of the key concepts that have emerged from the study of the effectors of plant-associated organisms. In particular, we focus on how effectors function in plant tissues and discuss future perspectives in the field of effector biology.

Acetyl L-carnitine targets adenosine triphosphate synthase in protecting zebrafish embryos from toxicities induced by verapamil and ketamine: An in vivo assessment.

PubMed

Guo, Xiaoqing; Dumas, Melanie; Robinson, Bonnie L; Ali, Syed F; Paule, Merle G; Gu, Qiang; Kanungo, Jyotshna

2017-02-01

Verapamil is a Ca 2 + channel blocker and is highly prescribed as an anti-anginal, antiarrhythmic and antihypertensive drug. Ketamine, an antagonist of the Ca 2 + -permeable N-methyl-d-aspartate-type glutamate receptors, is a pediatric anesthetic. Previously we have shown that acetyl l-carnitine (ALCAR) reverses ketamine-induced attenuation of heart rate and neurotoxicity in zebrafish embryos. Here, we used 48 h post-fertilization zebrafish embryos that were exposed to relevant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo. In 48 h post-fertilization embryos, 2 mm ketamine reduced heart rate in a 2 or 4 h exposure and 0.5 mm ALCAR neutralized this effect. ALCAR could reverse ketamine's effect, possibly through a compensatory mechanism involving extracellular Ca 2 + entry through L-type Ca 2 + channels that ALCAR is known to activate. Hence, we used verapamil to block the L-type Ca 2 + channels. Verapamil was more potent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific times of exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamil plus ketamine, even in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an inhibitor of intracellular Ca 2 + release suggesting that ALCAR acts via effectors downstream of Ca 2 + . In fact, ALCAR's protective effect was blunted by oligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca 2 + during adenosine triphosphate generation. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in abrogating ketamine- and verapamil-induced developmental toxicities. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae.

PubMed

Liao, Hsien-Ching; Chen, Mei-Yu

2012-02-24

The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts.

PubMed

Petre, Benjamin; Lorrain, Cécile; Saunders, Diane G O; Win, Joe; Sklenar, Jan; Duplessis, Sébastien; Kamoun, Sophien

2016-04-01

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast-targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N-terminal signal-dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells. © 2015 John Wiley & Sons Ltd.

A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths

USGS Publications Warehouse

Boudinot, Pierre; Zou, Jun; Ota, Tatsuya; Buonocore, Francesco; Scapigliati, Giuseppe; Canapa, Adriana; Cannon, John; Litman, Gary; Hansen, John D.

2014-01-01

The recent availability of both robust transcriptome and genome resources for coelacanth (Latimeria chalumnae) has led to unique discoveries for coelacanth immunity such as the lack of IgM, a central component of adaptive immunity. This study was designed to more precisely address the origins and evolution of gene families involved in the initial recognition and response to microbial pathogens, which effect innate immunity. Several multigene families involved in innate immunity are addressed, including: Toll-like receptors (TLRs), retinoic acid inducible gene 1 (RIG1)-like receptors (RLRs), the nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), diverse immunoglobulin domain-containing proteins (DICP) and modular domain immune-type receptors (MDIRs). Our analyses also include the tripartite motif-containing proteins (TRIM), which are involved in pathogen recognition as well as the positive regulation of antiviral immunity. Finally, this study addressed some of the downstream effectors of the antimicrobial response including IL-1 family members, type I and II interferons (IFN) and IFN-stimulated effectors (ISGs). Collectively, the genes and gene families in coelacanth that effect innate immune functions share characteristics both in content, structure and arrangement with those found in tetrapods but not in teleosts. The findings support the sister group relationship of coelacanth fish with tetrapods.

G protein signaling in the parasite Entamoeba histolytica

PubMed Central

Bosch, Dustin E; Siderovski, David P

2013-01-01

The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling–Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surface receptor capping. A remarkably large family of 91 Rab GTPases has multiple roles in a complex amebic vesicular trafficking system required for phagocytosis and pinocytosis and secretion of known virulence factors, such as amebapores and cysteine proteases. Although much remains to be discovered, recent studies of G protein signaling in E. histolytica have enhanced our understanding of parasitic pathogenesis and have also highlighted possible targets for pharmacological manipulation. PMID:23519208

Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations

PubMed Central

Verboon, Jeffrey M.; Rahe, Travis K.; Rodriguez-Mesa, Evelyn; Parkhurst, Susan M.

2015-01-01

Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non–immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott–Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex–independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations. PMID:25739458

Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila.

PubMed

Hing, H; Xiao, J; Harden, N; Lim, L; Zipursky, S L

1999-06-25

The SH2/SH3 adaptor protein Dock has been proposed to transduce signals from guidance receptors to the actin cytoskeleton in Drosophila photoreceptor (R cell) growth cones. Here, we demonstrate that Drosophila p21-activated kinase (Pak) is required in a Dock pathway regulating R cell axon guidance and targeting. Dock and Pak colocalize to R cell axons and growth cones, physically interact, and their loss-of-function phenotypes are indistinguishable. Normal patterns of R cell connectivity require Pak's kinase activity and binding sites for both Dock and Cdc42/Rac. A membrane-tethered form of Pak (Pak(myr) acts as a dominant gain-of-function protein. Retinal expression of Pak(myr) rescues the R cell connectivity phenotype in dock mutants. These data establish Pak as a critical regulator of axon guidance and a downstream effector of Dock in vivo.

Wave2 activates serum response element via its VCA region and functions downstream of Rac.

PubMed

Ishiguro, Kazuhiro; Cao, Zhifang; Ilasca, Marco Lopez; Ando, Takafumi; Xavier, Ramnik

2004-12-10

WAVE2 is a member of the WASP/WAVE family of protein effectors of actin reorganization and cell movement. In this report, we demonstrate that WAVE2 overexpression induces serum response element (SRE) activation through serum response factor. A WAVE2 mutant lacking the VCA region did not induce SRE activation and actin polymerization. WAVE2-induced SRE activation was blocked by exposure of cells to Latrunculin A, or overexpression of actin mutant R62D. The DeltaVCA mutant inhibited Rac V12-induced SRE activation, suggesting that WAVE2 lies downstream of Rac. Similar deletion of the VCA domain of WASP attenuated Cdc42 V12-mediated SRE activation, suggesting that WAVE2 acts in relation to Rac as WASP acts in relation to Cdc42. WAVE2 overexpression did not activate NF-kappaB.

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

PubMed

Naor, Adit; Panas, Michael W; Marino, Nicole; Coffey, Michael J; Tonkin, Christopher J; Boothroyd, John C

2018-04-03

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma , we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔ myr1 , and RHΔ asp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔ myr1 - but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions. IMPORTANCE Toxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins. Copyright © 2018 Naor et al.

Xenon Preconditioning Protects against Renal Ischemic-Reperfusion Injury via HIF-1α Activation

PubMed Central

Ma, Daqing; Lim, Ta; Xu, Jing; Tang, Haidy; Wan, Yanjie; Zhao, Hailin; Hossain, Mahmuda; Maxwell, Patrick H.; Maze, Mervyn

2009-01-01

The mortality rate from acute kidney injury after major cardiovascular operations can be as high as 60%, and no therapies have been proved to prevent acute kidney injury in this setting. Here, we show that preconditioning with the anesthetic gas xenon activates hypoxia-inducible factor 1α (HIF-1α) and its downstream effectors erythropoietin and vascular endothelial growth factor in a time-dependent manner in the kidneys of adult mice. Xenon increased the efficiency of HIF-1α translation via modulation of the mammalian target of rapamycin pathway. In a model of renal ischemia-reperfusion injury, xenon provided morphologic and functional renoprotection; hydrodynamic injection of HIF-1α small interfering RNA demonstrated that this protection is HIF-1α dependent. These results suggest that xenon preconditioning is a natural inducer of HIF-1α and that administration of xenon before renal ischemia can prevent acute renal failure. If these data are confirmed in the clinical setting, then preconditioning with xenon may be beneficial before procedures that temporarily interrupt renal perfusion. PMID:19144758

The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity

PubMed Central

Heimbucher, Thomas; Liu, Zheng; Bossard, Carine; McCloskey, Richard; Carrano, Andrea C.; Riedel, Christian G.; Tanasa, Bogdan; Klammt, Christian; Fonslow, Bryan R.; Riera, Celine E.; Lillemeier, Bjorn F.; Kemphues, Kenneth; Yates, John R.; O'Shea, Clodagh; Hunter, Tony; Dillin, Andrew

2015-01-01

SUMMARY One of the major determinants of aging in organisms ranging from worms to man are FOXO family transcription factors, which are downstream effectors of Insulin/IGF-1 signaling (IIS). The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity. PMID:26154057

Essential role of protein kinase C delta in platelet signaling, alpha IIb beta 3 activation, and thromboxane A2 release.

PubMed

Yacoub, Daniel; Théorêt, Jean-François; Villeneuve, Louis; Abou-Saleh, Haissam; Mourad, Walid; Allen, Bruce G; Merhi, Yahye

2006-10-06

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCdelta, but not PKCalpha or PKCbeta, is required for collagen-induced phospholipase C-dependent signaling, activation of alpha(IIb)beta(3), and platelet aggregation. Analysis of PKCdelta phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by alpha(IIb)beta(3) outside-in signaling. Moreover, PKCdelta triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A(2), which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCdelta signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A(2).

S6K1 in the central nervous system regulates energy expenditure via MC4R/CRH pathways in response to deprivation of an essential amino acid.

PubMed

Xia, Tingting; Cheng, Ying; Zhang, Qian; Xiao, Fei; Liu, Bin; Chen, Shanghai; Guo, Feifan

2012-10-01

It is well established that the central nervous system (CNS), especially the hypothalamus, plays an important role in regulating energy homeostasis and lipid metabolism. We have previously shown that hypothalamic corticotropin-releasing hormone (CRH) is critical for stimulating fat loss in response to dietary leucine deprivation. The molecular mechanisms underlying the CNS regulation of leucine deprivation-stimulated fat loss are, however, still largely unknown. Here, we used intracerebroventricular injection of adenoviral vectors to identify a novel role for hypothalamic p70 S6 kinase 1 (S6K1), a major downstream effector of the kinase mammalian target of rapamycin, in leucine deprivation stimulation of energy expenditure. Furthermore, we show that the effect of hypothalamic S6K1 is mediated by modulation of Crh expression in a melanocortin-4 receptor-dependent manner. Taken together, our studies provide a new perspective for understanding the regulation of energy expenditure by the CNS and the importance of cross-talk between nutritional control and regulation of endocrine signals.

DISC1 regulates new neuron development in the adult brain via modulation of AKT-mTOR signaling through KIAA1212.

PubMed

Kim, Ju Young; Duan, Xin; Liu, Cindy Y; Jang, Mi-Hyeon; Guo, Junjie U; Pow-anpongkul, Nattapol; Kang, Eunchai; Song, Hongjun; Ming, Guo-li

2009-09-24

Disrupted-in-schizophrenia 1 (DISC1), a susceptibility gene for major mental illnesses, regulates multiple aspects of embryonic and adult neurogenesis. Here, we show that DISC1 suppression in newborn neurons of the adult hippocampus leads to overactivated signaling of AKT, another schizophrenia susceptibility gene. Mechanistically, DISC1 directly interacts with KIAA1212, an AKT binding partner that enhances AKT signaling in the absence of DISC1, and DISC1 binding to KIAA1212 prevents AKT activation in vitro. Functionally, multiple genetic manipulations to enhance AKT signaling in adult-born neurons in vivo exhibit similar defects as DISC1 suppression in neuronal development that can be rescued by pharmacological inhibition of mammalian target of rapamycin (mTOR), an AKT downstream effector. Our study identifies the AKT-mTOR signaling pathway as a critical DISC1 target in regulating neuronal development and provides a framework for understanding how multiple susceptibility genes may functionally converge onto a common pathway in contributing to the etiology of certain psychiatric disorders.

Functional characterization of bovine TIRAP and MyD88 in mediating bacterial lipopolysaccharide-induced endothelial NF-kappaB activation and apoptosis.

PubMed

Cates, Elizabeth A; Connor, Erin E; Mosser, David M; Bannerman, Douglas D

2009-11-01

Mastitis is a prevalent disease in dairy cows. Gram-negative bacteria, which express the pro-inflammatory molecule lipopolysaccharide (LPS), are responsible for the majority of acute clinical cases of mastitis. Previous studies have identified differential susceptibility of human and bovine endothelial cells (EC) to the pro-inflammatory and injury-inducing effects of LPS. The Toll-like receptor (TLR)-4 signaling pathway, which is activated by LPS, has been well studied in humans, but not in ruminants. Human myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NF-kappaB and apoptotic signaling pathways. To assess the role of the bovine orthologs of these proteins in bovine TLR-4 signaling, dominant-negative constructs were expressed in bovine EC, and LPS-induced NF-kappaB activation and apoptosis evaluated. The results from this study indicate that bovine MyD88 and TIRAP play functional roles in transducing LPS signaling from TLR-4 to downstream effector molecules involved in NF-kappaB activation, and that TIRAP promotes apoptotic signaling.

p130Cas scaffolds the signalosome to direct adaptor-effector cross talk during Kaposi's sarcoma-associated herpesvirus trafficking in human microvascular dermal endothelial cells.

PubMed

Bandyopadhyay, Chirosree; Veettil, Mohanan Valiya; Dutta, Sujoy; Chandran, Bala

2014-12-01

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3β1, αVβ3, and αVβ5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. Eukaryotic cell adaptor molecules, without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies human microvascular dermal endothelial (HMVEC-d) cellular scaffold protein p130Cas (Crk-associated substrate) as a platform to promote Kaposi's sarcoma-associated herpesvirus (KSHV) trafficking. Early during KSHV de novo infection, p130Cas associates with lipid rafts and scaffolds EphrinA2 (EphA2)-associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal cross talk of the KSHV entry-associated upstream signal complex from the immediate downstream trafficking-associated signalosome, consequently routing KSHV toward lysosomal degradation and eventually blocking KSHV infection and associated malignancies. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Quantification of disease expression conferred by three host gene-necrotrophic effector interactions in the wheat-Parastagonospora nodorum pathosystem

USDA-ARS?s Scientific Manuscript database

Septoria nodorum blotch (SNB), which is a major foliar disease on wheat is caused by the necrotrophic fungus Parastagonospora nodorum. The wheat-P. nodorum pathosystem involves the recognition of necrotrophic effectors (NEs) secreted by P. nodorum by corresponding wheat NE sensitivity genes. Recogni...

MLKL activation triggers NLRP3-mediated processing and release of IL-1β independent of gasdermin-D

PubMed Central

Gutierrez, Kimberley D.; Davis, Michael A.; Daniels, Brian P.; Olsen, Tayla M.; Ralli-Jain, Pooja; Tait, Stephen W.G.; Gale, Michael; Oberst, Andrew

2017-01-01

Necroptosis is a form of programmed cell death defined by activation of the kinase RIPK3 and its downstream effector, the pseudokinase MLKL. Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. Here, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1β. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1β released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1β independent of the recently described pyroptotic effector gasdermin-D. Together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1β independent of gasdermin-D. PMID:28130493

Intestinal Effector T Cells in Health and Disease

PubMed Central

Maynard, Craig L.; Weaver, Casey T.

2011-01-01

Summary Crohn’s disease and ulcerative colitis are the two major forms of chronic relapsing inflammatory disorders of the human intestines collectively referred to as inflammatory bowel disease (IBD). Though a complex set of autoinflammatory disorders that can be precipitated by diverse genetic and environmental factors, a feature that appears common to IBD pathogenesis is a dysregulated effector T cell response to the commensal microbiota. Due to the heightened effector T cell activity in IBD, developmental and functional pathways that give rise to these cells are potential targets for therapeutic intervention. In this review, we highlight recent advances in our understanding of effector T cell biology in the context of intestinal immune regulation and speculate on their potential clinical significance. PMID:19766082

Kinome Analysis of Receptor-Induced Phosphorylation in Human Natural Killer Cells

PubMed Central

König, Sebastian; Nimtz, Manfred; Scheiter, Maxi; Ljunggren, Hans-Gustaf; Bryceson, Yenan T.; Jänsch, Lothar

2012-01-01

Background Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation. Results A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. Conclusions The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses. PMID:22238634

Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol 3-Kinase Signal Transduction by Altering Plasma Membrane Packing*

PubMed Central

Van Aelst, Britt; Devloo, Rosalie; Zachée, Pierre; t'Kindt, Ruben; Sandra, Koen; Vandekerckhove, Philippe; Compernolle, Veerle; Feys, Hendrik B.

2016-01-01

Psoralen and ultraviolet A light (PUVA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft-versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC50), 8.4 ± 1.1 versus 4.3 ± 1.1 μm) and glycoprotein VI (EC50, 1.61 ± 0.85 versus 0.26 ± 0.21 μg/ml) but not PAR4 (EC50, 50 ± 1 versus 58 ± 1 μm) signal transduction. Our findings were confirmed in T-cells from graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases. PMID:27687726

A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib.

PubMed

Sun, Tianliang; Yang, Lida; Kaur, Harmandeep; Pestel, Jenny; Looso, Mario; Nolte, Hendrik; Krasel, Cornelius; Heil, Daniel; Krishnan, Ramesh K; Santoni, Marie-Josée; Borg, Jean-Paul; Bünemann, Moritz; Offermanns, Stefan; Swiercz, Jakub M; Worzfeld, Thomas

2017-01-02

Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor βPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration. © 2017 Sun et al.

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

PubMed Central

Venkatareddy, Madhusudan; Verma, Rakesh; Kalinowski, Anne; Patel, Sanjeevkumar R.; Shisheva, Assia

2016-01-01

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34–deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux. PMID:26825532

The alpha subunit of Go modulates cell proliferation and differentiation through interactions with Necdin.

PubMed

Ju, Hyunhee; Lee, Sujin; Kang, Sunghak; Kim, Sung-Soo; Ghil, Sungho

2014-07-10

Heterotrimeric GTP-binding proteins (G-proteins) play an important role in mediating signal transduction generated by neurotransmitters or hormones. Go, a member of the Gi/Go subfamily, is the most abundant G-protein found in the brain. Recently, the alpha subunit of Go (Gαo) was characterized as an inducer of neuronal differentiation. However, its underlying molecular mechanisms have remained unclear to date, since the downstream effectors of Gαo are ambiguous. A neurally differentiated embryonal carcinoma-derived protein (Necdin) was isolated as an interacting partner for Gαo from a mouse brain cDNA library using yeast two-hybrid screening. Interactions between the proteins were confirmed with several affinity binding assays, both in vitro and in vivo. Necdin interacted directly and preferentially with activated Gαo, compared to wild-type protein. Interestingly, Gαo did not interact with Gαi, despite high sequence homology between the two proteins. We subsequently analyzed whether Gαo modulates the cellular activities of Necdin. Notably, expression of Gαo significantly augmented Necdin-mediated cellular responses, such as proliferation and differentiation. Moreover, activation of type 1 cannabinoid receptor (CB1R), a Gi/oα-coupled receptor, augmented cell growth suppression, which was mediated by Gαo and Necdin in U87MG cells containing CB1R, Gαo, and Necdin as normal components. These results collectively suggest that Necdin is a candidate downstream effector for Gαo. Our findings provide novel insights into the cellular roles of Gαo and its coupled receptor.

Signaling through G protein coupled receptors.

PubMed

Tuteja, Narendra

2009-10-01

Heterotrimeric G proteins (Galpha, Gbeta/Ggamma subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane alpha-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Galpha subunit. This leads to the dissociation of Gbeta/Ggamma dimer from Galpha. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Galpha-GTP is hydrolyzed to GDP and Galpha becomes inactive (Galpha-GDP), which leads to its re-association with the Gbeta/Ggamma dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.

Taste Receptor Signaling-- From Tongues to Lungs

PubMed Central

Kinnamon, Sue C.

2013-01-01

Taste buds are the transducing endorgans of gustation. Each taste bud comprises 50–100 elongated cells, which extend from the basal lamina to the surface of the tongue, where their apical microvilli encounter taste stimuli in the oral cavity. Salts and acids utilize apically located ion channels for transduction, while bitter, sweet and umami (glutamate) stimuli utilize G protein coupled receptors (GPCRs) and second messenger signaling mechanisms. This review will focus on GPCR signaling mechanisms. Two classes of taste GPCRs have been identified, the T1Rs for sweet and umami (glutamate) stimuli, and the T2Rs for bitter stimuli. These low affinity GPCRs all couple to the same downstream signaling effectors that include Gβγ activation of PLCβ2, IP3-mediated release of Ca2+ from intracellular stores, and Ca2+-dependent activation of the monovalent selective cation channel, TrpM5. These events lead to membrane depolarization, action potentials, and release of ATP as a transmitter to activate gustatory afferents. The Gα subunit, α-gustducin, activates a phosphodiesterase to decrease intracellular cAMP levels, although the precise targets of cAMP have not been identified. With the molecular identification of the taste GPCRs, it has become clear that taste signaling is not limited to taste buds, but occurs in many cell types of the airways. These include solitary chemosensory cells, ciliated epithelial cells, and smooth muscle cells. Bitter receptors are most abundantly expressed in the airways, where they respond to irritating chemicals and promote protective airway reflexes, utilizing the same downstream signaling effectors as taste cells. PMID:21481196

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon

2011-11-11

Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation.more » Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.« less

Hydrogen Sulfide as an Oxygen Sensor

PubMed Central

2015-01-01

Abstract Significance Although oxygen (O2)-sensing cells and tissues have been known for decades, the identity of the O2-sensing mechanism has remained elusive. Evidence is accumulating that O2-dependent metabolism of hydrogen sulfide (H2S) is this enigmatic O2 sensor. Recent Advances The elucidation of biochemical pathways involved in H2S synthesis and metabolism have shown that reciprocal H2S/O2 interactions have been inexorably linked throughout eukaryotic evolution; there are multiple foci by which O2 controls H2S inactivation, and the effects of H2S on downstream signaling events are consistent with those activated by hypoxia. H2S-mediated O2 sensing has been demonstrated in a variety of O2-sensing tissues in vertebrate cardiovascular and respiratory systems, including smooth muscle in systemic and respiratory blood vessels and airways, carotid body, adrenal medulla, and other peripheral as well as central chemoreceptors. Critical Issues Information is now needed on the intracellular location and stoichometry of these signaling processes and how and which downstream effectors are activated by H2S and its metabolites. Future Directions Development of specific inhibitors of H2S metabolism and effector activation as well as cellular organelle-targeted compounds that release H2S in a time- or environmentally controlled way will not only enhance our understanding of this signaling process but also provide direction for future therapeutic applications. Antioxid. Redox Signal. 22, 377–397. “Nothing in Biology Makes Sense Except in the Light of Evolution” —Theodosius Dobzhansky (29) PMID:24801248

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

PubMed Central

2017-01-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility.

PubMed

Caillaud, Marie-Cécile; Piquerez, Sophie J M; Fabro, Georgina; Steinbrenner, Jens; Ishaque, Naveed; Beynon, Jim; Jones, Jonathan D G

2012-01-01

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria in addition to their better-characterized role in suppressing plant defence. However, the specific mechanisms by which these effectors promote virulence remain unclear. To address this question, we examined changes in subcellular architecture using live-cell imaging during the compatible interaction between the oomycete Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. We monitored host-cell restructuring of subcellular compartments within plant mesophyll cells during haustoria ontogenesis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection, in particular to the tonoplast, which was located close to the extra-haustorial membrane surrounding the haustorium. We also investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. We identified two major classes of HaRxL effector based on localization: nuclear-localized effectors and membrane-localized effectors. Further, we identified a single effector, HaRxL17, that associated with the tonoplast in uninfected cells and with membranes around haustoria, probably the extra-haustorial membrane, in infected cells. Functional analysis of selected effector candidates in planta revealed that HaRxL17 enhances plant susceptibility. The roles of subcellular changes and effector localization, with specific reference to the potential role of HaRxL17 in plant cell membrane trafficking, are discussed with respect to Hpa virulence. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6.

PubMed

Häcker, Hans; Redecke, Vanessa; Blagoev, Blagoy; Kratchmarova, Irina; Hsu, Li-Chung; Wang, Gang G; Kamps, Mark P; Raz, Eyal; Wagner, Hermann; Häcker, Georg; Mann, Matthias; Karin, Michael

2006-01-12

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.

Integration of T Cell Receptor, Notch and Cytokine Signals Programs in Mouse γδ T Cell Effector Differentiation.

PubMed

Zarin, Payam; In, Tracy S H; Chen, Edward L Y; Singh, Jastaranpreet; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Anderson, Michele K; Zúñiga-Pflücker, Juan Carlos

2018-05-13

γδ T-cells perform a wide range of tissue and disease specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon-γ (IFNγ) and interleukin-17 (IL-17) producing γδ T-cells remain unknown. Here, we define the cues involved in the functional programming of γδ T-cells, by examining the roles of T-cell receptor (TCR), Notch, and cytokine-receptor signaling. KN6 γδTCR-transduced Rag2 -/- T-cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL-17 producing γδ T cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL-1β, IL-21 and IL-23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL-17 producing γδ T-cell subsets. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Dynamic and thermodynamic response of the Ras protein Cdc42Hs upon association with the effector domain of PAK3

PubMed Central

Moorman, Veronica R.; Valentine, Kathleen G.; Bédard, Sabrina; Kasinath, Vignesh; Dogan, Jakob; Love, Fiona M.; Wand, A. Joshua

2014-01-01

Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type GTPase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21 activated kinase 3 (PAK3) is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation were measured to investigate the dynamical changes in activated GMPPCP•Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with a sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately −10 kcal mol−1 at 298 K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs become more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring become more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding. PMID:25109462

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants.

PubMed

MacLean, Allyson M; Sugio, Akiko; Makarova, Olga V; Findlay, Kim C; Grieve, Victoria M; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A

2011-10-01

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.

Two distinct secretion systems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzae

PubMed Central

Giraldo, Martha C.; Dagdas, Yasin F.; Gupta, Yogesh K.; Mentlak, Thomas A.; Yi, Mihwa; Martinez-Rocha, Ana Lilia; Saitoh, Hiromasa; Terauchi, Ryohei; Talbot, Nicholas J.; Valent, Barbara

2013-01-01

To cause plant diseases, pathogenic micro-organisms secrete effector proteins into host tissue to suppress immunity and support pathogen growth. Bacterial pathogens have evolved several distinct secretion systems to target effector proteins, but whether fungi, which cause the major diseases of most crop species, also require different secretory mechanisms is not known. Here we report that the rice blast fungus Magnaporthe oryzae possesses two distinct secretion systems to target effectors during plant infection. Cytoplasmic effectors, which are delivered into host cells, preferentially accumulate in the biotrophic interfacial complex, a novel plant membrane-rich structure associated with invasive hyphae. We show that the biotrophic interfacial complex is associated with a novel form of secretion involving exocyst components and the Sso1 t-SNARE. By contrast, effectors that are secreted from invasive hyphae into the extracellular compartment follow the conventional secretory pathway. We conclude that the blast fungus has evolved distinct secretion systems to facilitate tissue invasion. PMID:23774898

The role of cGMP signalling in regulating life cycle progression of Plasmodium.

PubMed

Hopp, Christine S; Bowyer, Paul W; Baker, David A

2012-08-01

The 3'-5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is the main mediator of cGMP signalling in the malaria parasite. This article reviews the role of PKG in Plasmodium falciparum during gametogenesis and blood stage schizont rupture, as well as the role of the Plasmodium berghei orthologue in ookinete differentiation and motility, and liver stage schizont development. The current views on potential effector proteins downstream of PKG and the mechanisms that may regulate cyclic nucleotide levels are presented. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

PubMed

Rodriguez, Patricia A; Escudero-Martinez, Carmen; Bos, Jorunn I B

2017-03-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M persicae -host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence.

PubMed

Eves-van den Akker, Sebastian; Laetsch, Dominik R; Thorpe, Peter; Lilley, Catherine J; Danchin, Etienne G J; Da Rocha, Martine; Rancurel, Corinne; Holroyd, Nancy E; Cotton, James A; Szitenberg, Amir; Grenier, Eric; Montarry, Josselin; Mimee, Benjamin; Duceppe, Marc-Olivier; Boyes, Ian; Marvin, Jessica M C; Jones, Laura M; Yusup, Hazijah B; Lafond-Lapalme, Joël; Esquibet, Magali; Sabeh, Michael; Rott, Michael; Overmars, Hein; Finkers-Tomczak, Anna; Smant, Geert; Koutsovoulos, Georgios; Blok, Vivian; Mantelin, Sophie; Cock, Peter J A; Phillips, Wendy; Henrissat, Bernard; Urwin, Peter E; Blaxter, Mark; Jones, John T

2016-06-10

The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.

A Novel Positive Feedback Loop Mediated by the Docking Protein Gab1 and Phosphatidylinositol 3-Kinase in Epidermal Growth Factor Receptor Signaling

PubMed Central

Rodrigues, Gerard A.; Falasca, Marco; Zhang, Zhongtao; Ong, Siew Hwa; Schlessinger, Joseph

2000-01-01

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4,5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR. PMID:10648629

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

PubMed

Tyler, Brett M; Kale, Shiv D; Wang, Qunqing; Tao, Kai; Clark, Helen R; Drews, Kelly; Antignani, Vincenzo; Rumore, Amanda; Hayes, Tristan; Plett, Jonathan M; Fudal, Isabelle; Gu, Biao; Chen, Qinghe; Affeldt, Katharyn J; Berthier, Erwin; Fischer, Gregory J; Dou, Daolong; Shan, Weixing; Keller, Nancy P; Martin, Francis; Rouxel, Thierry; Lawrence, Christopher B

2013-06-01

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense.

PubMed

Zhang, Mengmeng; Su, Jianbin; Zhang, Yan; Xu, Juan; Zhang, Shuqun

2018-05-09

Mitogen-activated protein kinase (MAPK) cascades are key signaling modules downstream of receptors/sensors that perceive endogenous and exogenous stimuli such as hormones, peptide ligands, and pathogen-derived patterns/effectors. In this review, we summarize recent advances in the establishment of MAPK cascades as unified signaling modules downstream of receptor-like kinases (RLKs) and receptor-like proteins (RLPs) in plant growth and defense, the identification of components connecting the RLK/RLP receptor complexes to the MAPK cascades, and the interactions between MAPK and hormone signaling pathways. We also propose a set of criteria for defining the physiological substrates of plant MAPKs. With only a limited number of MAPK components, multiple functional pathways often share the same MAPK cascade. As a result, understanding the signaling specificity, which requires detailed information about the spatiotemporal expression of the components involved, their complex formation, and the consequence of substrate phosphorylation, is central to our study of MAPK functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Focal Adhesion Kinase Is Required for Intestinal Regeneration and Tumorigenesis Downstream of Wnt/c-Myc Signaling

PubMed Central

Ashton, Gabrielle H.; Morton, Jennifer P.; Myant, Kevin; Phesse, Toby J.; Ridgway, Rachel A.; Marsh, Victoria; Wilkins, Julie A.; Athineos, Dimitris; Muncan, Vanesa; Kemp, Richard; Neufeld, Kristi; Clevers, Hans; Brunton, Valerie; Winton, Douglas J.; Wang, Xiaoyan; Sears, Rosalie C.; Clarke, Alan R.; Frame, Margaret C.; Sansom, Owen J.

2012-01-01

SUMMARY The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis. Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer. PMID:20708588

Effector prediction in host-pathogen interaction based on a Markov model of a ubiquitous EPIYA motif

PubMed Central

2010-01-01

Background Effector secretion is a common strategy of pathogen in mediating host-pathogen interaction. Eight EPIYA-motif containing effectors have recently been discovered in six pathogens. Once these effectors enter host cells through type III/IV secretion systems (T3SS/T4SS), tyrosine in the EPIYA motif is phosphorylated, which triggers effectors binding other proteins to manipulate host-cell functions. The objectives of this study are to evaluate the distribution pattern of EPIYA motif in broad biological species, to predict potential effectors with EPIYA motif, and to suggest roles and biological functions of potential effectors in host-pathogen interactions. Results A hidden Markov model (HMM) of five amino acids was built for the EPIYA-motif based on the eight known effectors. Using this HMM to search the non-redundant protein database containing 9,216,047 sequences, we obtained 107,231 sequences with at least one EPIYA motif occurrence and 3115 sequences with multiple repeats of the EPIYA motif. Although the EPIYA motif exists among broad species, it is significantly over-represented in some particular groups of species. For those proteins containing at least four copies of EPIYA motif, most of them are from intracellular bacteria, extracellular bacteria with T3SS or T4SS or intracellular protozoan parasites. By combining the EPIYA motif and the adjacent SH2 binding motifs (KK, R4, Tarp and Tir), we built HMMs of nine amino acids and predicted many potential effectors in bacteria and protista by the HMMs. Some potential effectors for pathogens (such as Lawsonia intracellularis, Plasmodium falciparum and Leishmania major) are suggested. Conclusions Our study indicates that the EPIYA motif may be a ubiquitous functional site for effectors that play an important pathogenicity role in mediating host-pathogen interactions. We suggest that some intracellular protozoan parasites could secrete EPIYA-motif containing effectors through secretion systems similar to the T3SS/T4SS in bacteria. Our predicted effectors provide useful hypotheses for further studies. PMID:21143776

Partial Diversity Generates Effector Immunity Specificity of the Bac41-Like Bacteriocins of Enterococcus faecalis Clinical Strains.

PubMed

Kurushima, Jun; Ike, Yasuyoshi; Tomita, Haruyoshi

2016-09-01

Bacteriocin 41 (Bac41) is the plasmid-encoded bacteriocin produced by the opportunistic pathogen Enterococcus faecalis Its genetic determinant consists of bacL1 (effector), bacL2 (regulator), bacA (effector), and bacI (immunity). The secreted effectors BacL1 and BacA coordinate to induce the lytic cell death of E. faecalis Meanwhile, the immunity factor BacI provides self-resistance to the Bac41 producer, E. faecalis, against the action of BacL1 and BacA. In this study, we demonstrated that more than half of the 327 clinical strains of E. faecalis screened had functional Bac41 genes. Analysis of the genetic structure of the Bac41 genes in the DNA sequences of the E. faecalis strains revealed that the Bac41-like genes consist of a relatively conserved region and a variable region located downstream from bacA Based on similarities in the variable region, the Bac41-like genes could be classified into type I, type IIa, and type IIb. Interestingly, the distinct Bac41 types had specific immunity factors for self-resistance, BacI1 or BacI2, and did not show cross-immunity to the other type of effector. We also demonstrated experimentally that the specificity of the immunity was determined by the combination of the C-terminal region of BacA and the presence of the unique BacI1 or BacI2 factor. These observations suggested that Bac41-like bacteriocin genes are extensively disseminated among E. faecalis strains in the clinical environment and can be grouped into at least three types. It was also indicated that the partial diversity results in specificity of self-resistance which may offer these strains a competitive advantage. Bacteriocins are antibacterial effectors produced by bacteria. In general, a bacteriocin-coding gene is accompanied by a cognate immunity gene that confers self-resistance on the bacteriocin-producing bacterium itself. We demonstrated that one of the bacteriocins, Bac41, is disseminated among E. faecalis clinical strains and the Bac41 subtypes with partial diversity. The Bac41-like bacteriocins were found to be classified into type I, type IIa, and type IIb by variation of the cognate immunity factors. The antibacterial activity of the respective effectors was specifically inhibited by the immunity factor from the same type of Bac41 but not the other types. This specificity of effector-immunity pairs suggests that bacteriocin genes might have evolved to change the immunity specificity to acquire an advantage in interbacterial competition. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The cell fate determinant Scribble is required for maintenance of hematopoietic stem cell function.

PubMed

Mohr, Juliane; Dash, Banaja P; Schnoeder, Tina M; Wolleschak, Denise; Herzog, Carolin; Tubio Santamaria, Nuria; Weinert, Sönke; Godavarthy, Sonika; Zanetti, Costanza; Naumann, Michael; Hartleben, Björn; Huber, Tobias B; Krause, Daniela S; Kähne, Thilo; Bullinger, Lars; Heidel, Florian H

2018-05-01

Cell fate determinants influence self-renewal potential of hematopoietic stem cells. Scribble and Llgl1 belong to the Scribble polarity complex and reveal tumor-suppressor function in drosophila. In hematopoietic cells, genetic inactivation of Llgl1 leads to expansion of the stem cell pool and increases self-renewal capacity without conferring malignant transformation. Here we show that genetic inactivation of its putative complex partner Scribble results in functional impairment of hematopoietic stem cells (HSC) over serial transplantation and during stress. Although loss of Scribble deregulates transcriptional downstream effectors involved in stem cell proliferation, cell signaling, and cell motility, these effectors do not overlap with transcriptional targets of Llgl1. Binding partner analysis of Scribble in hematopoietic cells using affinity purification followed by mass spectometry confirms its role in cell signaling and motility but not for binding to polarity modules described in drosophila. Finally, requirement of Scribble for self-renewal capacity also affects leukemia stem cell function. Thus, Scribble is a regulator of adult HSCs, essential for maintenance of HSCs during phases of cell stress.

Interleukin-4-dependent innate collaboration between iNKT cells and B-1 B cells controls adaptative contact sensitivity

PubMed Central

Campos, Regis A; Szczepanik, Marian; Itakura, Atsuko; Lisbonne, Mariette; Dey, Neelendu; Leite-de-Moraes, Maria C; Askenase, Philip W

2006-01-01

We showed that hepatic Vα14+ invariant natural killer T (iNKT) cells, via their rapid interleukin (IL)-4 production, activate B-1 cells to initiate contact sensitivity (CS). This innate collaboration was absent in IL-4–/– and signal transducer and activator of transcription (STAT)-6–/– mice and was inhibited by anti-IL-4 treatment. These mice have defective CS because they fail to locally recruit the sensitized effector T cells of acquired immunity. Their CS is reconstituted by transfer of downstream-acting 1-day immune B-1 cells from wild-type mice. Responses were not reconstituted with B-1 cells from IL-4 receptor-α–/– or STAT-6–/– mice, nor by IL-4 treatment of B cell-deficient mice at immunization. Finally, IL-4 was preferentially and transiently produced by hepatic iNKT cells within 7 min after sensitization to mediate collaboration between innate-like iNKT cells and the B-1 B cells that participate in the recruitment of effector T cells in vivo. PMID:16556268

Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression.

PubMed

Pinto, Rita; Hansen, Lars; Hintze, John; Almeida, Raquel; Larsen, Sylvester; Coskun, Mehmet; Davidsen, Johanne; Mitchelmore, Cathy; David, Leonor; Troelsen, Jesper Thorvald; Bennett, Eric Paul

2017-07-27

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The enhancement of stress-related memory by glucocorticoids depends on synapsin-Ia/Ib

PubMed Central

Revest, J-M; Kaouane, N; Mondin, M; Le Roux, A; Rougé-Pont, F; Vallée, M; Barik, J; Tronche, F; Desmedt, A; Piazza, P V

2010-01-01

The activation of glucocorticoid receptors (GR) by glucocorticoids increases stress-related memory through the activation of the MAPK signaling pathway and the downstream transcription factor Egr-1. Here, using converging in vitro and in vivo approaches, respectively, GR-expressing cell lines, culture of hippocampal neurons, and GR genetically modified mice (GRNesCre), we identified synapsin-Ia/Ib as one of the effectors of the glucocorticoid signaling cascade. Stress and glucocorticoid-induced activation of the GR modulate synapsin-Ia/Ib through two complementary mechanisms. First, glucocorticoids driving Egr-1 expression increase the expression of synapsin-Ia/Ib, and second, glucocorticoids driving MAPK activation increase its phosphorylation. Finally, we showed that blocking fucosylation of synapsin-Ia/Ib in the hippocampus inhibits its expression and prevents the glucocorticoid-mediated increase in stress-related memory. In conclusion, our data provide a complete molecular pathway (GR/Egr-1/MAPK/Syn-Ia/Ib) through which stress and glucocorticoids enhance the memory of stress-related events and highlight the function of synapsin-Ia/Ib as molecular effector of the behavioral effects of stress. PMID:20368707

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans

PubMed Central

Frische, Ester W; Pellis-van Berkel, Wendy; van Haaften, Gijs; Cuppen, Edwin; Plasterk, Ronald H A; Tijsterman, Marcel; Bos, Johannes L; Zwartkruis, Fried J T

2007-01-01

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell–cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the α-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration. PMID:17989692

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans.

PubMed

Frische, Ester W; Pellis-van Berkel, Wendy; van Haaften, Gijs; Cuppen, Edwin; Plasterk, Ronald H A; Tijsterman, Marcel; Bos, Johannes L; Zwartkruis, Fried J T

2007-12-12

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell-cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the alpha-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration.

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

PubMed Central

Pathan, Akbar Ali Khan; Panthi, Bhavana; Khan, Zahid; Koppula, Purushotham Reddy; Alanazi, Mohammed Saud; Sachchidanand; Parine, Narasimha Reddy; Chourasia, Mukesh

2016-01-01

Objective Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results Interestingly, the designed compounds exhibit a binding preference for the “off” state over “on” state conformation of K-Ras protein. Moreover, the designed compounds’ interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski’s rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. Conclusion Thus, through the current study, we propose targeting only “off” state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein. PMID:27217775

Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

PubMed

Li, Ting; Liu, Nannan

2017-12-01

This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat Model.

PubMed

Ubba, Vaibhave; Soni, Upendra Kumar; Chadchan, Sangappa; Maurya, Vineet Kumar; Kumar, Vijay; Maurya, Ruchika; Chaturvedi, Himanshu; Singh, Rajender; Dwivedi, Anila; Jha, Rajesh Kumar

2017-05-01

The function of RHOG, a RAC1 activator, was explored in the ovary during ovarian follicular development and pathological conditions. With the help of immunoblotting and immunolocalization, we determined the expression and localization of RHOG in normal (estrous cycle) and polycystic ovaries using Sprague Dawley (SD) rat model. Employing polymerase chain reaction and flow cytometry, we analyzed the transcript and expression levels of downstream molecules of RHOG, DOCK1, and RAC1 in the polycystic ovarian syndrome (PCOS) ovary along with normal antral follicular theca and granulosa cells after dehydroepiandrosterone (DHEA) supplementation. The effect of RHOG knockdown on DOCK1, VAV, and RAC1 expression was evaluated in the human ovarian cells (SKOV3), theca cells, and granulosa cells from SD rats with the help of flow cytometry. Oocyte at secondary follicles along with stromal cells showed optimal expression of RHOG. Immunoblotting of RHOG revealed its maximum expression at diestrus and proestrus, which was downregulated at estrus stage. Mild immunostaining of RHOG was also present in the theca and granulosa cells of the secondary and antral follicles. Polycystic ovary exhibited weak immunostaining for RHOG and that was corroborated by immunoblotting-based investigations. RHOG effectors DOCK1 and ELMO1 were found reduced in the ovary in PCOS condition/DHEA. RHOG silencing reduced the expression of DOCK1 and RAC1 in the theca and granulosa cells from SD rat antral follicles and that was mirrored in the human ovarian cells. Collectively, RHOG can mediate signaling through downstream effectors DOCK1 and RAC1 during ovarian follicular development (theca and granulosa cells and oocyte), but DHEA downregulated them in the PCOS ovary.

Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gebhard, Catherine; Staehli, Barbara E.; Zurich Center for Integrative Human Physiology

2011-11-04

Highlights: Black-Right-Pointing-Pointer The nuclear enzyme PARP-1 is a downstream effector of oxidative stress. Black-Right-Pointing-Pointer PARP-1 protects from oxidative stress induced endothelial dysfunction. Black-Right-Pointing-Pointer This effect is mediated through inhibition of vasoconstrictor prostanoid production. Black-Right-Pointing-Pointer Thus, PARP-1 may play a protective role as antioxidant defense mechanism. -- Abstract: Background: Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress. Methods: PARP-1(-/-) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings weremore » suspended in organ chambers for isometric tension recording to analyze vascular function. Results: PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(-/-), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(-/-) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, L-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(-/-) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(-/-) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(-/-) mice (p < 0.05 vs. PQ treated PARP-1(+/+). Conclusion: PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.« less

Effects of mutant human Ki-ras{sup G12C} gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.

2008-08-15

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras{sup G12C} allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 {mu}g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras{sup G12C} allele in the lung, and resulted in themore » development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 {mu}g/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 {mu}g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 {mu}g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras{sup G12C} expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.« less

MEK1 inhibits cardiac PPARα activity by direct interaction and prevents its nuclear localization.

PubMed

el Azzouzi, Hamid; Leptidis, Stefanos; Bourajjaj, Meriem; van Bilsen, Marc; da Costa Martins, Paula A; De Windt, Leon J

2012-01-01

The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK1/2) pathways. In response to increased workload, the mitogen-activated protein kinase kinase (MAPKK) MEK1 has been shown to be active. Studies embarking on mitogen-activated protein kinase (MAPK) signaling cascades in the heart have indicated peroxisome-proliferators activated-receptors (PPARs) as downstream effectors that can be regulated by this signaling cascade. Despite the importance of PPARα in controlling cardiac metabolism, little is known about the relationship between MAPK signaling and cardiac PPARα signaling. Using co-immunoprecipitation and immunofluorescence approaches we show a complex formation of PPARα with MEK1 and not with ERK1/2. Binding of PPARα to MEK1 is mediated via a LXXLL motif and results in translocation from the nucleus towards the cytoplasm, hereby disabling the transcriptional activity of PPARα. Mice subjected to voluntary running-wheel exercise showed increased cardiac MEK1 activation and complex formation with PPARα, subsequently resulting in reduced PPARα activity. Inhibition of MEK1, using U0126, blunted this effect. Here we show that activation of the MEK1-ERK1/2 pathway leads to specific inhibition of PPARα transcriptional activity. Furthermore we show that this inhibitory effect is mediated by MEK1, and not by its downstream effector kinase ERK1/2, through a mechanism involving direct binding to PPARα and subsequent stimulation of PPARα export from the nucleus.

Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells.

PubMed

Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

2018-02-01

The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf . Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s.

Chemotherapeutic Potential of 2-[Piperidinoethoxyphenyl]-3-Phenyl-2H-Benzo(b)pyran in Estrogen Receptor- Negative Breast Cancer Cells: Action via Prevention of EGFR Activation and Combined Inhibition of PI-3-K/Akt/FOXO and MEK/Erk/AP-1 Pathways

PubMed Central

Saxena, Ruchi; Chandra, Vishal; Manohar, Murli; Hajela, Kanchan; Debnath, Utsab; Prabhakar, Yenamandra S.; Saini, Karan Singh; Konwar, Rituraj; Kumar, Sandeep; Megu, Kaling; Roy, Bal Gangadhar; Dwivedi, Anila

2013-01-01

Inhibition of epidermal growth factor receptor (EGFR) signaling is considered to be a promising treatment strategy for estrogen receptor (ER)-negative breast tumors. We have investigated here the anti-breast cancer properties of a novel anti-proliferative benzopyran compound namely, 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b)pyran (CDRI-85/287) in ER- negative and EGFR- overexpressing breast cancer cells. The benzopyran compound selectively inhibited the EGF-induced growth of MDA-MB 231 cells and ER-negative primary breast cancer cell culture. The compound significantly reduced tumor growth in xenograft of MDA-MB 231 cells in nude mice. The compound displayed better binding affinity for EGFR than inhibitor AG1478 as demonstrated by molecular docking studies. CDRI-85/287 significantly inhibited the activation of EGFR and downstream effectors MEK/Erk and PI-3-K/Akt. Subsequent inhibition of AP-1 promoter activity resulted in decreased transcription activation and expression of c-fos and c-jun. Dephosphorylation of downstream effectors FOXO-3a and NF-κB led to increased expression of p27 and decreased expression of cyclin D1 which was responsible for decreased phosphorylation of Rb and prevented the transcription of E2F- dependent genes involved in cell cycle progression from G1/S phase. The compound induced apoptosis via mitochondrial pathway and it also inhibited EGF-induced invasion of MDA-MB 231 cells as evidenced by decreased activity of MMP-9 and expression of CTGF. These results indicate that benzopyran compound CDRI-85/287 could constitute a powerful new chemotherapeutic agent against ER-negative and EGFR over-expressing breast tumors. PMID:23840429

Activation of the Hedgehog Signaling Pathway in the Developing Lens Stimulates Ectopic FoxE3 Expression and Disruption in Fiber Cell Differentiation

PubMed Central

Kerr, Christine L.; Huang, Jian; Williams, Trevor; West-Mays, Judith A.

2012-01-01

Purpose. The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. Methods. We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. Results. Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. Conclusions. Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3. PMID:22491411

Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity in vitro and in vivo

PubMed Central

Morelli, E; Leone, E; Cantafio, M E Gallo; Di Martino, M T; Amodio, N; Biamonte, L; Gullà, A; Foresta, U; Pitari, M R; Botta, C; Rossi, M; Neri, A; Munshi, N C; Anderson, K C; Tagliaferri, P; Tassone, P

2015-01-01

Interferon regulatory factor 4 (IRF4) is an attractive therapeutic target in multiple myeloma (MM). We here report that expression of IRF4 mRNA inversely correlates with microRNA (miR)-125b in MM patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive expression of miR-125b-5p by lentiviral vectors or transfection with synthetic mimics impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells in vitro. Apoptotic and autophagy-associated cell death were triggered in MM cells on miR-125b-5p ectopic expression. Importantly, we found that the anti-MM activity of miR-125b-5p was mediated via direct downregulation of IRF4 and its downstream effector BLIMP-1. Moreover, inhibition of IRF4 translated into downregulation of c-Myc, caspase-10 and cFlip, relevant IRF4-downstream effectors. Finally, in vivo intra-tumor or systemic delivery of formulated miR-125b-5p mimics against human MM xenografts in severe combined immunodeficient/non-obese diabetic mice induced significant anti-tumor activity and prolonged survival. Taken together, our findings provide evidence that miR-125b, differently from other hematologic malignancies, has tumor-suppressor activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials. PMID:25987254

4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

PubMed Central

Shives, Katherine D.; Massey, Aaron R.; May, Nicholas A.; Morrison, Thomas E.; Beckham, J. David

2016-01-01

West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome. PMID:27763553

Caspase-3 Is Transiently Activated without Cell Death during Early Antigen Driven Expansion of CD8+ T Cells In Vivo

PubMed Central

McComb, Scott; Mulligan, Rebecca; Sad, Subash

2010-01-01

Background CD8+ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8+ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8+ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8+ T cell responses has yet to be examined. Methods and Findings We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8+ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8+ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3hi and caspase-3low CD8+ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62Llow) CD8+ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8+ cells remained active caspase-3low throughout the contraction phase. Conclusions Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8+ T cells. Furthermore, the contraction of CD8+ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3. PMID:21203525

Evolution of the vertebrate insulin receptor substrate (Irs) gene family.

PubMed

Al-Salam, Ahmad; Irwin, David M

2017-06-23

Insulin receptor substrate (Irs) proteins are essential for insulin signaling as they allow downstream effectors to dock with, and be activated by, the insulin receptor. A family of four Irs proteins have been identified in mice, however the gene for one of these, IRS3, has been pseudogenized in humans. While it is known that the Irs gene family originated in vertebrates, it is not known when it originated and which members are most closely related to each other. A better understanding of the evolution of Irs genes and proteins should provide insight into the regulation of metabolism by insulin. Multiple genes for Irs proteins were identified in a wide variety of vertebrate species. Phylogenetic and genomic neighborhood analyses indicate that this gene family originated very early in vertebrae evolution. Most Irs genes were duplicated and retained in fish after the fish-specific genome duplication. Irs genes have been lost of various lineages, including Irs3 in primates and birds and Irs1 in most fish. Irs3 and Irs4 experienced an episode of more rapid protein sequence evolution on the ancestral mammalian lineage. Comparisons of the conservation of the proteins sequences among Irs paralogs show that domains involved in binding to the plasma membrane and insulin receptors are most strongly conserved, while divergence has occurred in sequences involved in interacting with downstream effector proteins. The Irs gene family originated very early in vertebrate evolution, likely through genome duplications, and in parallel with duplications of other components of the insulin signaling pathway, including insulin and the insulin receptor. While the N-terminal sequences of these proteins are conserved among the paralogs, changes in the C-terminal sequences likely allowed changes in biological function.

Structure of the Dominant Negative S17N Mutant of Ras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nassar, N.; Singh, K; Garcia-Diaz, M

2010-01-01

The use of the dominant negative mutant of Ras has been crucial in elucidating the cellular signaling of Ras in response to the activation of various membrane-bound receptors. Although several point mutants of Ras exhibit a dominant negative effect, the asparagine to serine mutation at position 17 (S17N) remains the most popular and the most effective at inhibiting the activation of endogenous Ras. It is now widely accepted that the dominant negative effect is due to the ability of the mutant to sequester upstream activators and its inability to activate downstream effectors. Here, we present the crystal structure of RasS17Nmore » in the GDP-bound form. In the three molecules that populate the asymmetric unit, the Mg{sup 2+} ion that normally coordinates the {beta}-phosphate is absent because of steric hindrance from the Asn17 side chain. Instead, a Ca{sup 2+} ion is coordinating the {alpha}-phosphate. Also absent from one molecule is electron density for Phe28, a conserved residue that normally stabilizes the nucleotide's guanine base. Except for Phe28, the nucleotide makes conserved interactions with Ras. Combined, the inability of Phe28 to stabilize the guanine base and the absence of a Mg{sup 2+} ion to neutralize the negative charges on the phosphates explain the weaker affinity of GDP for Ras. Our data suggest that the absence of the Mg{sup 2+} should also dramatically affect GTP binding to Ras and the proper positioning of Thr35 necessary for the activation of switch 1 and the binding to downstream effectors, a prerequisite for the triggering of signaling pathways.« less

Phytoplasma Effector SAP54 Induces Indeterminate Leaf-Like Flower Development in Arabidopsis Plants1[C][W][OA

PubMed Central

MacLean, Allyson M.; Sugio, Akiko; Makarova, Olga V.; Findlay, Kim C.; Grieve, Victoria M.; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A.

2011-01-01

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches’ broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host. PMID:21849514

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

PubMed

Demirci, Selami; Doğan, Ayşegül; Apdik, Hüseyin; Tuysuz, Emre Can; Gulluoglu, Sukru; Bayrak, Omer Faruk; Şahin, Fikrettin

2018-01-01

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fıbroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

Interfamily Transfer of Dual NB-LRR Genes Confers Resistance to Multiple Pathogens

PubMed Central

Narusaka, Mari; Kubo, Yasuyuki; Hatakeyama, Katsunori; Imamura, Jun; Ezura, Hiroshi; Nanasato, Yoshihiko; Tabei, Yutaka; Takano, Yoshitaka; Shirasu, Ken; Narusaka, Yoshihiro

2013-01-01

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens. PMID:23437080

TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

PubMed Central

Ku, Amy T; Shaver, Timothy M; Rao, Ajay S; Howard, Jeffrey M; Rodriguez, Christine N; Miao, Qi; Garcia, Gloria; Le, Diep; Yang, Diane; Borowiak, Malgorzata; Cohen, Daniel N; Chitsazzadeh, Vida; Diwan, Abdul H; Tsai, Kenneth Y; Nguyen, Hoang

2017-01-01

The transcription factor TCF7L1 is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in skin tumorigenesis has not yet been defined. Here we document TCF7L1 upregulation in skin squamous cell carcinoma (SCC) and demonstrate that TCF7L1 overexpression increases tumor incidence, tumor multiplicity, and malignant progression in the chemically induced mouse model of skin SCC. Additionally, we show that downregulation of TCF7L1 and its paralogue TCF7L2 reduces tumor growth in a xenograft model of human skin SCC. Using separation-of-function mutants, we show that TCF7L1 promotes tumor growth, enhances cell migration, and overrides oncogenic RAS-induced senescence independently of its interaction with β-catenin. Through transcriptome profiling and combined gain- and loss-of-function studies, we identified LCN2 as a major downstream effector of TCF7L1 that drives tumor growth. Our findings establish a tumor-promoting role for TCF7L1 in skin and elucidate the mechanisms underlying its tumorigenic capacity. DOI: http://dx.doi.org/10.7554/eLife.23242.001 PMID:28467300

Yorkie Facilitates Organ Growth and Metamorphosis in Bombyx.

PubMed

Liu, Shumin; Zhang, Panli; Song, Hong-Sheng; Qi, Hai-Sheng; Wei, Zhao-Jun; Zhang, Guozheng; Zhan, Shuai; Liu, Zhihong; Li, Sheng

2016-01-01

The Hippo pathway, which was identified from genetic screens in the fruit fly, Drosophila melanogaster, has a major size-control function in animals. All key components of the Hippo pathway, including the transcriptional coactivator Yorkie that is the most critical substrate and downstream effector of the Hippo kinase cassette, are found in the silkworm, Bombyx mori. As revealed by microarray and quantitative real-time PCR, expression of Hippo pathway genes is particularly enriched in several mitotic tissues, including the ovary, testis, and wing disc. Developmental profiles of Hippo pathway genes are generally similar (with the exception of Yorkie) within each organ, but vary greatly in different tissues showing nearly opposing expression patterns in the wing disc and the posterior silk gland (PSG) on day 2 of the prepupal stage. Importantly, the reduction of Yorkie expression by RNAi downregulated Yorkie target genes in the ovary, decreased egg number, and delayed larval-pupal-adult metamorphosis. In contrast, baculovirus-mediated Yorkie(CA) overexpression upregulated Yorkie target genes in the PSG, increased PSG size, and accelerated larval-pupal metamorphosis. Together the results show that Yorkie potentially facilitates organ growth and metamorphosis, and suggest that the evolutionarily conserved Hippo pathway is critical for size control, particularly for PSG growth, in the silkworm.

A key role for mitochondria in endothelial signaling by plasma cysteine/cystine redox potential

PubMed Central

Go, Young-Mi; Park, Heonyong; Koval, Michael; Orr, Michael; Reed, Matthew; Liang, Yongliang; Smith, Debra; Pohl, Jan; Jones, Dean P.

2011-01-01

The redox potential of the plasma cysteine/cystine couple (EhCySS) is oxidized in association with risk factors for cardiovascular disease (CVD), including age, smoking, type 2 diabetes, obesity, and alcohol abuse. Previous in vitro findings support a cause–effect relationship for extracellular EhCySS in cell signaling pathways associated with CVD, including those controlling monocyte adhesion to endothelial cells. In this study, we provide evidence that mitochondria are a major source of reactive oxygen species (ROS) in the signaling response to a more oxidized extracellular EhCySS. This increase in ROS was blocked by overexpression of mitochondrial thioredoxin-2 (Trx2) in endothelial cells from Trx2-transgenic mice, suggesting that mitochondrial thiol antioxidant status plays a key role in this redox signaling mechanism. Mass spectrometry-based redox proteomics showed that several classes of plasma membrane and cytoskeletal proteins involved in inflammation responded to this redox switch, including vascular cell adhesion molecule, integrins, actin, and several Ras family GTPases. Together, the data show that the proinflammatory effects of oxidized plasma EhCySS are due to a mitochondrial signaling pathway that is mediated through redox control of downstream effector proteins. PMID:19879942

Protein S Negatively Regulates Neural Stem Cell Self-Renewal through Bmi-1 Signaling

PubMed Central

Zelentsova-Levytskyi, Katya; Talmi, Ziv; Abboud-Jarrous, Ghada; Capucha, Tal; Sapir, Tamar; Burstyn-Cohen, Tal

2017-01-01

Revealing the molecular mechanisms underlying neural stem cell self-renewal is a major goal toward understanding adult brain homeostasis. The self-renewing potential of neural stem and progenitor cells (NSPCs) must be tightly regulated to maintain brain homeostasis. We recently reported the expression of Protein S (PROS1) in adult hippocampal NSPCs, and revealed its role in regulation of NSPC quiescence and neuronal differentiation. Here, we investigate the effect of PROS1 on NSPC self-renewal and show that genetic ablation of Pros1 in neural progenitors increased NSPC self-renewal by 50%. Mechanistically, we identified the upregulation of the polycomb complex protein Bmi-1 and repression of its downstream effectors p16Ink4a and p19Arf to promote NSPC self-renewal in Pros1-ablated cells. Rescuing Pros1 expression restores normal levels of Bmi-1 signaling, and reverts the proliferation and enhanced self-renewal phenotypes observed in Pros1-deleted cells. Our study identifies PROS1 as a novel negative regulator of NSPC self-renewal. We conclude PROS1 is instructive for NSPC differentiation by negatively regulating Bmi-1 signaling in adult and embryonic neural stem cells. PMID:28512399

p38 MAPK inhibitors: a patent review (2012 - 2013).

PubMed

Bühler, Stefanie; Laufer, Stefan A

2014-05-01

The p38 MAPK is a ubiquitous target in the research-based pharmaceutical industry. It plays a decisive role in the regulation of the production of proinflammatory cytokines. Since novel biological therapies have revolutionized the treatment of chronic inflammatory diseases, an intensive global search is underway for small molecules for the same application. Herein, the patents and the corresponding publications of international companies, which focus on the development and identification of a new generation of small-molecule p38 inhibitors, are summarized. The most promising approach is the development of linear binders, which induce a glycine flip at Gly110 of the kinase hinge region by a carbonyl oxygen atom of the respective ligand. The major focus of the patent works was the application of molecules in new indications. Previous applications were in the treatment of rheumatoid arthritis; currently, there are several new applications, including pulmonary diseases, cancer and Alzheimer's disease. Targeting p38 upstream kinases and downstream effectors has also proved to be a very promising step in the development of more effective inhibitors. A further trend is drug combination, applied to a wide range of indications, such as chronic obstructive pulmonary disease and cancer.

The Hippo pathway in heart development, regeneration, and diseases

PubMed Central

Zhou, Qi; Li, Li; Zhao, Bin; Guan, Kun-Liang

2015-01-01

The heart is the first organ formed during mammalian development. A properly sized and functional heart is vital throughout the entire lifespan. Loss of cardiomyocytes due to injury or diseases leads to heart failure, which is a major cause of human morbidity and mortality. Unfortunately, regenerative potential of the adult heart is very limited. The Hippo pathway is a recently identified signaling cascade that plays an evolutionarily conserved role in organ size control by inhibiting cell proliferation, promoting apoptosis, regulating fates of stem/ progenitor cells, and in some circumstances, limiting cell size. Interestingly, research indicates a key role of this pathway in regulation of cardiomyocyte proliferation and heart size. Inactivation of the Hippo pathway or activation of its downstream effector, the Yes-associated protein (YAP) transcription co-activator, improves cardiac regeneration. Several known upstream signals of the Hippo pathway such as mechanical stress, G-protein-coupled receptor (GPCR) signaling, and oxidative stress, are known to play critical roles in cardiac physiology. In addition, YAP has been shown to regulate cardiomyocyte fate through multiple transcriptional mechanisms. In this review, we summarize and discuss current findings regarding the roles and mechanisms of the Hippo pathway in heart development, injury, and regeneration. PMID:25858067

Scavenging nucleic acid debris to combat autoimmunity and infectious disease

NASA Astrophysics Data System (ADS)

Holl, Eda K.; Shumansky, Kara L.; Borst, Luke B.; Burnette, Angela D.; Sample, Christopher J.; Ramsburg, Elizabeth A.; Sullenger, Bruce A.

2016-08-01

Nucleic acid-containing debris released from dead and dying cells can be recognized as damage-associated molecular patterns (DAMPs) or pattern-associated molecular patterns (PAMPs) by the innate immune system. Inappropriate activation of the innate immune response can engender pathological inflammation and autoimmune disease. To combat such diseases, major efforts have been made to therapeutically target the pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) that recognize such DAMPs and PAMPs, or the downstream effector molecules they engender, to limit inflammation. Unfortunately, such strategies can limit the ability of the immune system to combat infection. Previously, we demonstrated that nucleic acid-binding polymers can act as molecular scavengers and limit the ability of artificial nucleic acid ligands to activate PRRs. Herein, we demonstrate that nucleic acid scavengers (NASs) can limit pathological inflammation and nucleic acid-associated autoimmunity in lupus-prone mice. Moreover, we observe that such NASs do not limit an animal’s ability to combat viral infection, but rather their administration improves survival when animals are challenged with lethal doses of influenza. These results indicate that molecules that scavenge extracellular nucleic acid debris represent potentially safer agents to control pathological inflammation associated with a wide range of autoimmune and infectious diseases.

Proteomic Characterization of the World Trade Center dust-activated mdig and c-myc signaling circuit linked to multiple myeloma

PubMed Central

Wu, Kai; Li, Lingzhi; Thakur, Chitra; Lu, Yongju; Zhang, Xiangmin; Yi, Zhengping; Chen, Fei

2016-01-01

Several epidemiological studies suggested an increased incidence rate of multiple myeloma (MM) among first responders and other individuals who exposed to World Trade Center (WTC) dust. In this report, we provided evidence showing that WTC dust is potent in inducing mdig protein and/or mRNA in bronchial epithelial cells, B cells and MM cell lines. An increased mdig expression in MM bone marrow was observed, which is associated with the disease progression and prognosis of the MM patients. Through integrative genomics and proteomics approaches, we further demonstrated that mdig directly interacts with c-myc and JAK1 in MM cell lines, which contributes to hyperactivation of the IL-6-JAK-STAT3 signaling important for the pathogenesis of MM. Genetic silencing of mdig reduced activity of the major downstream effectors in the IL-6-JAK-STAT3 pathway. Taken together, these data suggest that WTC dust may be one of the key etiological factors for those who had been exposed for the development of MM by activating mdig and c-myc signaling circuit linked to the IL-6-JAK-STAT3 pathway essential for the tumorigenesis of the malignant plasma cells. PMID:27833099

The hippo pathway in heart development, regeneration, and diseases.

PubMed

Zhou, Qi; Li, Li; Zhao, Bin; Guan, Kun-Liang

2015-04-10

The heart is the first organ formed during mammalian development. A properly sized and functional heart is vital throughout the entire lifespan. Loss of cardiomyocytes because of injury or diseases leads to heart failure, which is a major cause of human morbidity and mortality. Unfortunately, regenerative potential of the adult heart is limited. The Hippo pathway is a recently identified signaling cascade that plays an evolutionarily conserved role in organ size control by inhibiting cell proliferation, promoting apoptosis, regulating fates of stem/progenitor cells, and in some circumstances, limiting cell size. Interestingly, research indicates a key role of this pathway in regulation of cardiomyocyte proliferation and heart size. Inactivation of the Hippo pathway or activation of its downstream effector, the Yes-associated protein transcription coactivator, improves cardiac regeneration. Several known upstream signals of the Hippo pathway such as mechanical stress, G-protein-coupled receptor signaling, and oxidative stress are known to play critical roles in cardiac physiology. In addition, Yes-associated protein has been shown to regulate cardiomyocyte fate through multiple transcriptional mechanisms. In this review, we summarize and discuss current findings on the roles and mechanisms of the Hippo pathway in heart development, injury, and regeneration. © 2015 American Heart Association, Inc.

Plant-pathogen interactions: toward development of next-generation disease-resistant plants.

PubMed

Nejat, Naghmeh; Rookes, James; Mantri, Nitin L; Cahill, David M

2017-03-01

Briskly evolving phytopathogens are dire threats to our food supplies and threaten global food security. From the recent advances made toward high-throughput sequencing technologies, understanding of pathogenesis and effector biology, and plant innate immunity, translation of these means into new control tools is being introduced to develop durable disease resistance. Effectoromics as a powerful genetic tool for uncovering effector-target genes, both susceptibility genes and executor resistance genes in effector-assisted breeding, open up new avenues to improve resistance. TALENs (Transcription Activator-Like Effector Nucleases), engineered nucleases and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are breakthrough and powerful techniques for genome editing, providing efficient mechanisms for targeted crop protection strategies in disease resistance programs. In this review, major advances in plant disease management to confer durable disease resistance and novel strategies for boosting plant innate immunity are highlighted.

Bacterial effectors target the plant cell nucleus to subvert host transcription.

PubMed

Canonne, Joanne; Rivas, Susana

2012-02-01

In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells.

PAC exhibits potent anti-colon cancer properties through targeting cyclin D1 and suppressing epithelial-to-mesenchymal transition.

PubMed

Al-Qasem, Abeer; Al-Howail, Huda A; Al-Swailem, Mashael; Al-Mazrou, Amer; Al-Otaibi, Basem; Al-Jammaz, Ibrahim; Al-Khalaf, Huda H; Aboussekhra, Abdelilah

2016-03-01

Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Although response rates and overall survival have been improved in recent years, resistance to multiple drug combinations is inevitable. Therefore, the development of more efficient drugs, with fewer side effects is urgently needed. To this end, we have investigated in the present report the effect of PAC, a novel cucumin analogue, on CRC cells both in vitro and in vivo. We have shown that PAC induces apoptosis, mainly via the internal mitochondrial route, and inhibits cell proliferation through delaying the cell cycle at G2/M phase. Interestingly, the pro-apoptotic effect was mediated through STAT3-dependent down-regulation of cyclin D1 and its downstream target survivin. Indeed, change in the expression level of cyclin D1 modulated the expression of survivin and the response of CRC cells to PAC. Furthermore, using the ChIP assay, we have shown PAC-dependent reduction in the binding of STAT3 to the cyclin D1 promoter in vivo. Additionally, PAC suppressed the epithelial-to-mesenchymal process through down-regulating the mesenchymal markers (N-cadherin, vimentin and Twist1) and inhibiting the invasion/migration abilities of the CRC cells via repressing the pro-migration/invasion protein kinases AKT and ERK1/2. In addition, PAC inhibited tumor growth and repressed the JAK2/STAT3, AKT/mTOR and MEK/ERK pathways as well as their common downstream effectors cyclin D1 and survivin in humanized CRC xenografts. Collectively, these results indicate that PAC has potent anti-CRC effects, and therefore could constitute an effective alternative chemotherapeutic agent, which may consolidate the adjuvant treatment of colon cancer. © 2015 Wiley Periodicals, Inc.

The Arabidopsis cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC4 work in the same signaling pathway to regulate pathogen defense and floral transition.

PubMed

Chin, Kimberley; DeFalco, Thomas A; Moeder, Wolfgang; Yoshioka, Keiko

2013-10-01

Arabidopsis (Arabidopsis thaliana) cyclic nucleotide-gated ion channels (CNGCs) form a large family consisting of 20 members and have been implicated in Ca(2+) signaling related to various physiological processes, such as pathogen defense, development, and thermotolerance. The null mutant of AtCNGC2, defense, no death (dnd1), exhibits autoimmune phenotypes, while it is impaired in mounting the hypersensitive response, which is a hallmark of effector-triggered (i.e. RESISTANCE-gene mediated) resistance. It has been suggested that AtCNGC2 is involved in defense responses and likely other aspects of physiology through its role as a Ca(2+)-conducting channel. However, the downstream signaling components and its relation with AtCNGC4, which is the closest paralog of AtCNGC2, remain elusive. Despite the fact that cngc4 mutants display almost identical phenotypes to those seen in cngc2 mutants, not much is known about their relationship. Here, we report the identification and characterization of the Arabidopsis mutant repressor of defense no death1 (rdd1), obtained from a suppressor screen of a transfer DNA insertion knockout mutant of AtCNGC2 in order to identify downstream components of dnd1-mediated signal transduction. rdd1 suppressed the majority of dnd1-mediated phenotypes except Ca(2+) hypersensitivity. In addition, rdd1 also suppressed the dnd1-mediated late-flowering phenotype that was discovered in this study. Our genetic analysis conducted to elucidate the relationship between AtCNGC2 and AtCNGC4 indicates that RDD1 is also involved in AtCNGC4-mediated signal transduction. Lastly, bimolecular fluorescence complementation analysis suggests that AtCNGC2 and AtCNGC4 are likely part of the same channel complex.

A New Strategy to Control and Eradicate "Undruggable" Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology.

PubMed

Van Sciver, Robert E; Lee, Michael P; Lee, Caroline Dasom; Lafever, Alex C; Svyatova, Elizaveta; Kanda, Kevin; Colliver, Amber L; Siewertsz van Reesema, Lauren L; Tang-Tan, Angela M; Zheleva, Vasilena; Bwayi, Monicah N; Bian, Minglei; Schmidt, Rebecca L; Matrisian, Lynn M; Petersen, Gloria M; Tang, Amy H

2018-05-14

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely "undruggable". Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.

CFTR modulates RPS27 gene expression using chloride anion as signaling effector.

PubMed

Valdivieso, Ángel G; Mori, Consuelo; Clauzure, Mariángeles; Massip-Copiz, Macarena; Santa-Coloma, Tomás A

2017-11-01

In Cystic Fibrosis (CF), the impairment of the CFTR channel activity leads to a variety of alterations, including differential gene expression. However, the CFTR signaling mechanisms remain unclear. Recently, culturing IB3-1 CF cells under different intracellular Cl - concentrations ([Cl - ] i ), we observed several Cl - -dependent genes and further characterized one of them as RPS27. Thus, we hypothesized that Cl - might act as a signaling effector for CFTR signaling. Here, to test this idea, we study RPS27 expression in T84 cells modulating the CFTR activity by using CFTR inhibitors. First, we observed that incubation of T84 cells with increasing concentrations of the CFTR inhibitors CFTR(inh)-172 or GlyH-101 determined a progressive increase in the relative [Cl - ] i (using the Cl - fluorescent probe SPQ). The [Cl - ] i rise was concomitant with a dose-dependent down-regulation of RPS27. These results imply that CFTR inhibition produce Cl - accumulation and that RPS27 expression can be modulated by CFTR inhibition. Therefore, Cl - behaves as a signaling effector for CFTR in the modulation of RPS27 expression. In addition, the IL-1β receptor antagonist IL1RN or the JNK inhibitor SP600125, both restored the down-regulation of RPS27 induced by CFTRinh-172, implying a role of autocrine IL-1β and JNK signaling downstream of Cl - in RPS27 modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Three-dimensional structure of the NLRP7 pyrin domain: insight into pyrin-pyrin-mediated effector domain signaling in innate immunity.

PubMed

Pinheiro, Anderson S; Proell, Martina; Eibl, Clarissa; Page, Rebecca; Schwarzenbacher, Robert; Peti, Wolfgang

2010-08-27

The innate immune system provides an initial line of defense against infection. Nucleotide-binding domain- and leucine-rich repeat-containing protein (NLR or (NOD-like)) receptors play a critical role in the innate immune response by surveying the cytoplasm for traces of intracellular invaders and endogenous stress signals. NLRs themselves are multi-domain proteins. Their N-terminal effector domains (typically a pyrin or caspase activation and recruitment domain) are responsible for driving downstream signaling and initiating the formation of inflammasomes, multi-component complexes necessary for cytokine activation. However, the currently available structures of NLR effector domains have not yet revealed the mechanism of their differential modes of interaction. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP7 (NLRP7 PYD) obtained by NMR spectroscopy. The NLRP7 PYD adopts a six-alpha-helix bundle death domain fold. A comparison of conformational and dynamics features of the NLRP7 PYD with other PYDs showed distinct differences for helix alpha3 and loop alpha2-alpha3, which, in NLRP7, is stabilized by a strong hydrophobic cluster. Moreover, the NLRP7 and NLRP1 PYDs have different electrostatic surfaces. This is significant, because death domain signaling is driven by electrostatic contacts and stabilized by hydrophobic interactions. Thus, these results provide new insights into NLRP signaling and provide a first molecular understanding of inflammasome formation.

SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda

PubMed Central

Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J.; Clem, Rollie J.

2013-01-01

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. PMID:23474489

Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs.

PubMed

Ham, Jong Hyun; Majerczak, Doris R; Nomura, Kinya; Mecey, Christy; Uribe, Francisco; He, Sheng-Yang; Mackey, David; Coplin, David L

2009-06-01

The broadly conserved AvrE-family of type III effectors from gram-negative plant-pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato or Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and subcellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well.

The effector candidate repertoire of the arbuscular mycorrhizal fungus Rhizophagus clarus.

PubMed

Sędzielewska Toro, Kinga; Brachmann, Andreas

2016-02-09

Arbuscular mycorrhizal fungi (AMF) form an ecologically important symbiosis with more than two thirds of studied land plants. Recent studies of plant-pathogen interactions showed that effector proteins play a key role in host colonization by controlling the plant immune system. We hypothesise that also for symbiotic-plant interactions the secreted effectome of the fungus is a major component of communication and the conservation level of effector proteins between AMF species may be indicative whether they play a fundamental role. In this study, we used a bioinformatics pipeline to predict and compare the effector candidate repertoire of the two AMF species, Rhizophagus irregularis and Rhizophagus clarus. Our in silico pipeline revealed a list of 220 R. irregularis candidate effector genes that create a valuable information source to elucidate the mechanism of plant infection and colonization by fungi during AMF symbiotic interaction. While most of the candidate effectors show no homologies to known domains or proteins, the candidates with homologies point to potential roles in signal transduction, cell wall modification or transcription regulation. A remarkable aspect of our work is presence of a large portion of the effector proteins involved in symbiosis, which are not unique to each fungi or plant species, but shared along the Glomeromycota phylum. For 95% of R. irregularis candidates we found homologs in a R. clarus genome draft generated by Illumina high-throughput sequencing. Interestingly, 9% of the predicted effectors are at least as conserved between the two Rhizophagus species as proteins with housekeeping functions (similarity > 90%). Therefore, we state that this group of highly conserved effector proteins between AMF species may play a fundamental role during fungus-plant interaction. We hypothesise that in symbiotic interactions the secreted effectome of the fungus might be an important component of communication. Identification and functional characterization of the primary AMF effectors that regulate symbiotic development will help in understanding the mechanisms of fungus-plant interaction.

Increased expression of TLR9 associated with pro-inflammatory S100A8 and IL-8 in diabetic wounds could lead to unresolved inflammation in type 2 diabetes mellitus (T2DM) cases with impaired wound healing.

PubMed

Singh, Kanhaiya; Agrawal, Neeraj K; Gupta, Sanjeev K; Sinha, Pratima; Singh, Kiran

2016-01-01

Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia which causes a chain of abrupt biochemical and physiological changes. Immune dys-regulation is the hallmark of T2DM that could contribute to prolonged inflammation causing transformation of wounds into non-healing chronic ulcers. Toll like receptor -9 (TLR9) is a major receptor involved in innate immune regulation. TLR9 activation induces release of pro-inflammatory molecules like S100A8 and interleukin-8 (IL-8) by myeloid cells causing migration of myeloid cells to the site of inflammation. We hypothesized that pro-inflammatory S100A8 and IL-8 proteins could cause persistent inflammation in chronic wounds like diabetic foot ulcer (DFU) and may contribute to impaired wound healing in T2DM patients. Expression of TLR9 and its downstream effector molecules S100A8, and IL-8 were analyzed in chronic diabetic wound and non-diabetic control wound tissue samples by semiquantitative reverse transcriptase - polymerase chain reaction (RT-PCR), quantitative RT-PCR, western blot and immunofluorescence. CD11b(+)CD33(+) myeloid cells were analyzed by flow cytometry. TLR9 message and protein were higher in diabetic wounds compared to control wounds (p=0.03, t=2.21 for TLR9 mRNA; p=<0.001, t=4.21 for TLR9 protein). TLR9 down-stream effector molecules S100A8 and IL-8 were also increased in diabetic wounds (p=0.003, t=3.1 for S100A8 mRNA; p=0.04, t=2.04 for IL-8). CD11b(+) CD33(+) myeloid cells were decreased in T2DM as compared to non-diabetic controls (p=0.001, t=3.6). DFU subjects had higher levels of CD11b(+) CD33(+) myeloid cells as compared to non-DFU T2DM control (p=0.003, t=2.8). Infection in the wound microenvironment could be the cause of increase in CD11b(+)CD33(+) myeloid cells in DFU (p=0.03, t=2.5). The up-regulation of myeloid cell-derived pro-inflammatory molecules S100A8 and IL-8 in combination with lower levels of CD11b(+) CD33(+) myeloid cells may cause the impairment of wound healing in T2DM subjects leading to chronic ulcers. Copyright © 2016 Elsevier Inc. All rights reserved.

Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling

PubMed Central

Gami, Minaxi S; Iser, Wendy B; Hanselman, Keaton B; Wolkow, Catherine A

2006-01-01

Background In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals. Results This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes. Conclusion A screen for suppressors of PI3K mutant phenotypes identified activating mutations in two known pathway components, providing insights into their regulation. In particular, the interdependence of akt-1 and pdk-1, even in activated forms, supports the existence of AGE-1-independent pathways for these phospholipid-dependent kinases. Phenotypic analysis of these alleles shows that the larval and adult outputs of AGE-1/PI3K are fully separable in these mutants. PMID:17020605

The role of p21-activated kinases in hepatocellular carcinoma metastasis.

PubMed

Tse, Edith Yuk Ting; Ching, Yick Pang

2014-01-01

The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed.

Shigella

PubMed Central

Marteyn, Benoit; Gazi, Anastasia; Sansonetti, Philippe

2012-01-01

Much is known about the molecular effectors of pathogenicity of gram-negative enteric pathogens, among which Shigella can be considered a model. This is due to its capacity to recapitulate the multiple steps required for a pathogenic microbe to survive close to its mucosal target, colonize and then invade its epithelial surface, cause its inflammatory destruction and simultaneously regulate the extent of the elicited innate response to likely survive the encounter and achieve successful subsequent transmission. These various steps of the infectious process represent an array of successive environmental conditions to which the bacteria need to successfully adapt. These conditions represent the selective pressure that triggered the “arms race” in which Shigella acquired the genetic and molecular effectors of its pathogenic armory, including the regulatory hierarchies that regulate the expression and function of these effectors. They also represent cues through which Shigella achieves the temporo-spatial expression and regulation of its virulence effectors. The role of such environmental cues has recently become obvious in the case of the major virulence effector of Shigella, the type three secretion system (T3SS) and its dedicated secreted virulence effectors. It needs to be better defined for other major virulence components such as the LPS and peptidoglycan which are used as examples here, in addition to the T3SS as models of regulation as it relates to the assembly and functional regulation of complex macromolecular systems of the bacterial surface. This review also stresses the need to better define what the true and relevant environmental conditions can be at the various steps of the progression of infection. The “identity” of the pathogen differs depending whether it is cultivated under in vitro or in vivo conditions. Moreover, this “identity” may quickly change during its progression into the infected tissue. Novel concepts and relevant tools are needed to address this challenge in microbial pathogenesis. PMID:22356862

ISG15 Functions as an Interferon-Mediated Antiviral Effector Early in the Murine Norovirus Life Cycle

PubMed Central

Rodriguez, Marisela R.; Monte, Kristen; Thackray, Larissa B.

2014-01-01

ABSTRACT Human noroviruses (HuNoV) are the leading cause of nonbacterial gastroenteritis worldwide. Similar to HuNoV, murine noroviruses (MNV) are enteric pathogens spread via the fecal-oral route and have been isolated from numerous mouse facilities worldwide. Type I and type II interferons (IFN) restrict MNV-1 replication; however, the antiviral effectors impacting MNV-1 downstream of IFN signaling are largely unknown. Studies using dendritic cells, macrophages, and mice deficient in free and conjugated forms of interferon-stimulated gene 15 (ISG15) revealed that ISG15 conjugation contributes to protection against MNV-1 both in vitro and in vivo. ISG15 inhibited a step early in the viral life cycle upstream of viral genome transcription. Directly transfecting MNV-1 RNA into IFN-stimulated mouse embryonic fibroblasts (MEFs) and bone marrow-derived dendritic cells (BMDC) lacking ISG15 conjugates bypassed the antiviral activity of ISG15, further suggesting that ISG15 conjugates restrict the MNV-1 life cycle at the viral entry/uncoating step. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of early stages of MNV-1 replication. IMPORTANCE Type I IFNs are important in controlling murine norovirus 1 (MNV-1) infections; however, the proteins induced by IFNs that restrict viral growth are largely unknown. This report reveals that interferon-stimulated gene 15 (ISG15) mitigates MNV-1 replication both in vitro and in vivo. In addition, it shows that ISG15 inhibits MNV-1 replication by targeting an early step in the viral life cycle, MNV-1 entry and/or uncoating. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of viral entry/uncoating. PMID:24899198

Activation of ERK and JNK signaling pathways by mycotoxin citrinin in human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, C.-H.; Yu, F.-Y.; Wang, L.-T.

2009-06-15

Mycotoxin citrinin (CTN) is commonly found in foods and feeds that are contaminated/inoculated with Penicillium, Aspergillus and Monascus species. The exposure of human embryonic kidney (HEK293) and HeLa cells to CTN resulted in a dose-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), ERK1/2 and JNK. In HEK293 cultures, the administering of CTN increased both the mRNA and protein levels of egr-1, c-fos and c-jun genes; additionally, the ERK1/2 pathway contributed to the upregulation of Egr-1 and c-Fos protein expression. CTN treatment also induced the transcription activity of Egr-1 and AP-1 proteins, as evidenced by luciferase reportermore » assays. Bioinformatic analyses indicated two genes Gadd45{beta} and MMP3 have Egr-1 and AP-1 response elements in their promoters, respectively. Furthermore, co-exposure of HEK293 cells to CTN and MAPK pathway inhibitors demonstrated that CTN increased the levels of Gadd45{beta} mRNA through ERK1/2 signaling pathway and up-regulated the MMP3 transcripts majorly via JNK pathway. Finally, CTN-triggered caspase 3 activity was significantly reduced in the presence of MAPK inhibitors. Our results suggest that CTN positively regulates ERK1/2 and JNK pathways as well as their downstream effectors in human cells; activated MAPK pathways are also involved in CTN-induced apoptosis.« less

β-Adrenergic modulation of cancer cell proliferation: available evidence and clinical perspectives.

PubMed

Coelho, Marisa; Soares-Silva, Cátia; Brandão, Daniela; Marino, Franca; Cosentino, Marco; Ribeiro, Laura

2017-02-01

In this review, we aimed to present and discuss the available preclinical and epidemiological evidences regarding the modulation of cancer cell proliferation by β-adrenoceptors (β-AR), with a specific focus on the putative effects of β-blockers according to their pharmacological properties. A comprehensive review of the published literature was conducted, and the evidences concerning the involvement of β-AR in cancer as well as the possible role of β-blockers were selected and discussed. The majority of reviewed studies show that: (1) All the cancer types express both β1- and β2-AR, with the exception of neuroblastoma only seeming to express β2-AR; (2) adrenergic agonists are able to increase proliferation of several types of cancers; (3) the proliferative effect seems to be mediated by both β1- and β2-AR; (4) binding to β-AR results in a cAMP transient flux which activates two major downstream effector systems: protein kinase A and EPAC and (5) β-blockers might be putative adjuvants for cancer treatment. Overall, the reviewed studies show strong evidences that β-AR activation, through several intracellular mechanisms, modulate tumor cell proliferation suggesting β-blockers can be a feasible therapeutic approach to antagonize β-adrenergic response or have a protective effect per se. This review highlight the need for intensifying the research not only on the molecular mechanisms underlying the β-adrenergic influence in cancer, but also on the implications of biased agonism of β-blockers as potential antitumor agents.

The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity.

PubMed

Medina, Cesar Augusto; Reyes, Paola Andrea; Trujillo, Cesar Augusto; Gonzalez, Juan Luis; Bejarano, David Alejandro; Montenegro, Nathaly Andrea; Jacobs, Jonathan M; Joe, Anna; Restrepo, Silvia; Alfano, James R; Bernal, Adriana

2018-03-01

Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 possesses 17 predicted T3Es belonging to the Xanthomonas outer protein (Xop) class. This work aimed to characterize nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1 and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) using heterologous systems. AvrBs2, XopR and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs2 and XopAO1 in Xam during cassava infection. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Specific Eph receptor-cytoplasmic effector signaling mediated by SAM-SAM domain interactions.

PubMed

Wang, Yue; Shang, Yuan; Li, Jianchao; Chen, Weidi; Li, Gang; Wan, Jun; Liu, Wei; Zhang, Mingjie

2018-05-11

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain many Eph SAM mutations identified in patients suffering from cancers and other diseases. © 2018, Wang et al.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

PubMed

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Nanorobotic end-effectors: Design, fabrication, and in situ characterization

NASA Astrophysics Data System (ADS)

Fan, Zheng

Nano-robotic end-effectors have promising applications for nano-fabrication, nano-manufacturing, nano-optics, nano-medical, and nano-sensing; however, low performances of the conventional end-effectors have prevented the widespread utilization of them in various fields. There are two major difficulties in developing the end-effectors: their nano-fabrication and their advanced characterization in the nanoscale. Here we introduce six types of end-effectors: the nanotube fountain pen (NFP), the super-fine nanoprobe, the metal-filled carbon nanotube (m CNT)-based sphere-on-pillar (SOP) nanoantennas, the tunneling nanosensor, and the nanowire-based memristor. The investigations on the NFP are focused on nano-fluidics and nano-fabrications. The NFP could direct write metallic "inks" and fabricating complex metal nanostructures from 0D to 3D with a position servo control, which is critically important to future large-scale, high-throughput nanodevice production. With the help of NFP, we could fabricate the end-effectors such as super-fine nanoprobe and m CNT-based SOP nanoantennas. Those end-effectors are able to detect local flaws or characterize the electrical/mechanical properties of the nanostructure. Moreover, using electron-energy-loss-spectroscopy (EELS) technique during the operation of the SOP optical antenna opens a new basis for the application of nano-robotic end-effectors. The technique allows advanced characterization of the physical changes, such as carrier diffusion, that are directly responsible for the device's properties. As the device was coupled with characterization techniques of scanning-trasmission-electron-microscopy (STEM), the development of tunneling nanosensor advances this field of science into quantum world. Furthermore, the combined STEM-EELS technique plays an important role in our understanding of the memristive switching performance in the nanowire-based memristor. The developments of those nano-robotic end-effectors expend the study abilities in investigating the in situ nanotechnology, providing efficient ways in in situ nanostructure fabrication and the advanced characterization of the nanomaterials.

COX-derived prostanoid pathways in gastrointestinal cancer development and progression: novel targets for prevention and intervention.

PubMed

Cathcart, Mary-Clare; O'Byrne, Kenneth J; Reynolds, John V; O'Sullivan, Jacintha; Pidgeon, Graham P

2012-01-01

Arachidonic acid metabolism through cyclooxygenase (COX) pathways leads to the generation of biologically active eicosanoids. Eicosanoid expression levels vary during development and progression of gastrointestinal (GI) malignancies. COX-2 is the major COX-isoform responsible for G.I. cancer development/progression. COX-2 expression increases during progression from a normal to cancerous state. Evidence from observational studies has demonstrated that chronic NSAID use reduces the risk of cancer development, while both incidence and risk of death due to G.I. cancers were significantly reduced by daily aspirin intake. A number of randomized controlled trials (APC trial, Prevention of Sporadic Adenomatous Polyps trial, APPROVe trial) have also shown a significant protective effect in patients receiving selective COX-2 inhibitors. However, chronic use of selective COX-2 inhibitors at high doses was associated with increased cardiovascular risk, while NSAIDs have also been associated with increased risk. More recently, downstream effectors of COX-signaling have been investigated in cancer development/progression. PGE(2), which binds to both EP and PPAR receptors, is the major prostanoid implicated in the carcinogenesis of G.I. cancers. The role of TXA(2) in G.I. cancers has also been examined, although further studies are required to uncover its role in carcinogenesis. Other prostanoids investigated include PGD(2) and its metabolite 15d-PGJ2, PGF(1α) and PGI(2). Targeting these prostanoids in G.I. cancers has the promise of avoiding cardiovascular toxicity associated with chronic selective COX-2 inhibition, while maintaining anti-tumor reactivity. A progressive sequence from normal to pre-malignant to a malignant state has been identified in G.I. cancers. In this review, we will discuss the role of the COX-derived prostanoids in G.I. cancer development and progression. Targeting these downstream prostanoids for chemoprevention and/or treatment of G.I. cancers will also be discussed. Finally, we will highlight the latest pre-clinical technologies as well as avenues for future investigation in this highly topical research field. Copyright © 2011 Elsevier B.V. All rights reserved.

Structural basis of death domain signaling in the p75 neurotrophin receptor

PubMed Central

Lin, Zhi; Tann, Jason Y; Goh, Eddy TH; Kelly, Claire; Lim, Kim Buay; Gao, Jian Fang; Ibanez, Carlos F

2015-01-01

Death domains (DDs) mediate assembly of oligomeric complexes for activation of downstream signaling pathways through incompletely understood mechanisms. Here we report structures of complexes formed by the DD of p75 neurotrophin receptor (p75NTR) with RhoGDI, for activation of the RhoA pathway, with caspase recruitment domain (CARD) of RIP2 kinase, for activation of the NF-kB pathway, and with itself, revealing how DD dimerization controls access of intracellular effectors to the receptor. RIP2 CARD and RhoGDI bind to p75NTR DD at partially overlapping epitopes with over 100-fold difference in affinity, revealing the mechanism by which RIP2 recruitment displaces RhoGDI upon ligand binding. The p75NTR DD forms non-covalent, low-affinity symmetric dimers in solution. The dimer interface overlaps with RIP2 CARD but not RhoGDI binding sites, supporting a model of receptor activation triggered by separation of DDs. These structures reveal how competitive protein-protein interactions orchestrate the hierarchical activation of downstream pathways in non-catalytic receptors. DOI: http://dx.doi.org/10.7554/eLife.11692.001 PMID:26646181

Pax8 and Pax2a function synergistically in otic specification, downstream of the Foxi1 and Dlx3b transcription factors.

PubMed

Hans, Stefan; Liu, Dong; Westerfield, Monte

2004-10-01

The vertebrate inner ear arises from an ectodermal thickening, the otic placode, that forms adjacent to the presumptive hindbrain. Previous studies have suggested that competent ectodermal cells respond to Fgf signals from adjacent tissues and express two highly related paired box transcription factors Pax2a and Pax8 in the developing placode. We show that compromising the functions of both Pax2a and Pax8 together blocks zebrafish ear development, leaving only a few residual otic cells. This suggests that Pax2a and Pax8 are the main effectors downstream of Fgf signals. Our results further provide evidence that pax8 expression and pax2a expression are regulated by two independent factors, Foxi1 and Dlx3b, respectively. Combined loss of both factors eliminates all indications of otic specification. We suggest that the Foxi1-Pax8 pathway provides an early 'jumpstart' of otic specification that is maintained by the Dlx3b-Pax2a pathway.

Parthanatos, a messenger of death.

PubMed

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2009-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 overactivation underlies cell death in models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into understanding mechanisms downstream of PARP-1 overactivation. Recent evidence shows that poly-ADP ribose (PAR) polymer itself can act as a cell death effector downstream of PARP-1. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will present evidence and questions raised by these recent findings, and summarize the proposed mechanisms by which PARP-1 overactivation kills. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 overactivation.

GSK3β regulates AKT-induced central nervous system axon regeneration via an eIF2Bε-dependent, mTORC1-independent pathway.

PubMed

Guo, Xinzheng; Snider, William D; Chen, Bo

2016-03-14

Axons fail to regenerate after central nervous system (CNS) injury. Modulation of the PTEN/mTORC1 pathway in retinal ganglion cells (RGCs) promotes axon regeneration after optic nerve injury. Here, we report that AKT activation, downstream of Pten deletion, promotes axon regeneration and RGC survival. We further demonstrate that GSK3β plays an indispensable role in mediating AKT-induced axon regeneration. Deletion or inactivation of GSK3β promotes axon regeneration independently of the mTORC1 pathway, whereas constitutive activation of GSK3β reduces AKT-induced axon regeneration. Importantly, we have identified eIF2Bε as a novel downstream effector of GSK3β in regulating axon regeneration. Inactivation of eIF2Bε reduces both GSK3β and AKT-mediated effects on axon regeneration. Constitutive activation of eIF2Bε is sufficient to promote axon regeneration. Our results reveal a key role of the AKT-GSK3β-eIF2Bε signaling module in regulating axon regeneration in the adult mammalian CNS.

Systematic functional characterization of resistance to PI3K inhibition in breast cancer

PubMed Central

Treacy, Daniel J.; Luo, Flora; Ghandi, Mahmoud; Castel, Pau; Scaltriti, Maurizio; Baselga, Jose; Garraway, Levi A.

2016-01-01

PIK3CA (which encodes the phosphoinositide-3 kinase (PI3K) alpha isoform) is the most frequently mutated oncogene in breast cancer. Small-molecule PI3K inhibitors have shown promise in clinical trials; however, intrinsic and acquired resistance limits their utility. We used a systematic gain-of-function approach to identify genes whose upregulation confers resistance to the PI3K inhibitor BYL719 in breast cancer cells. Among the validated resistance genes, PIM kinases conferred resistance by maintaining downstream PI3K effector activation in an AKT-independent manner. Concurrent pharmacological inhibition of PIM and PI3K overcame this resistance mechanism. We also observed upregulated PIM expression and activity in a subset of breast cancer biopsies with clinical resistance to PI3K inhibitors. PIM1 overexpression is mutually exclusive with PIK3CA mutation in treatment-naïve breast cancers, suggesting downstream functional redundancy. Together, these results offer new insights into resistance to PI3K inhibitors and support clinical studies of combined PIM/PI3K inhibition in a subset of PIK3CA-mutant cancers. PMID:27604488

Inhibition of mammalian target of rapamycin activation in the rostral anterior cingulate cortex attenuates pain-related aversion in rats.

PubMed

Lu, Bo; Jiang, Jingyan; Sun, Jianliang; Xiao, Chun; Meng, Bo; Zheng, Jinwei; Li, Xiaoyu; Wang, Ruichun; Wu, Guorong; Chen, Junping

2016-09-01

Pain is a complex experience that comprises both sensory and affective dimensions. Mammalian target of rapamycin (mTOR) plays an important role in the modulation of neuronal plasticity associated with the pathogenesis of pain sensation. However, the role of mTOR in pain affect is unclear. Using a formalin-induced conditioned place avoidance (F-CPA) test, the current study investigated the effects of the mTOR specific inhibitor rapamycin on noxious stimulation induced aversion in the rostral anterior cingulate cortex (rACC). Intraplantar injection of 5% formalin was associated with significant activation of mTOR, as well as p70 ribosomal S6 protein (p70S6K), its downstream effector, in the rACC. The inhibition of mTOR activation with rapamycin disrupted pain-related aversion; however, this inhibition did not affect formalin-induced spontaneous nociceptive behaviors in rats. These findings demonstrated for the first time that mTOR and its downstream pathway in the rACC contribute to the induction of pain-related negative emotion. Copyright © 2016 Elsevier B.V. All rights reserved.

A rho-binding protein kinase C-like activity is required for the function of protein kinase N in Drosophila development.

PubMed

Betson, Martha; Settleman, Jeffrey

2007-08-01

The Rho GTPases interact with multiple downstream effectors to exert their biological functions, which include important roles in tissue morphogenesis during the development of multicellular organisms. Among the Rho effectors are the protein kinase N (PKN) proteins, which are protein kinase C (PKC)-like kinases that bind activated Rho GTPases. The PKN proteins are well conserved evolutionarily, but their biological role in any organism is poorly understood. We previously determined that the single Drosophila ortholog of mammalian PKN proteins, Pkn, is a Rho/Rac-binding kinase essential for Drosophila development. By performing "rescue" studies with various Pkn mutant constructs, we have defined the domains of Pkn required for its role during Drosophila development. These studies suggested that Rho, but not Rac binding is important for Pkn function in development. In addition, we determined that the kinase domain of PKC53E, a PKC family kinase, can functionally substitute for the kinase domain of Pkn during development, thereby exemplifying the evolutionary strategy of "combining" functional domains to produce proteins with distinct biological activities. Interestingly, we also identified a requirement for Pkn in wing morphogenesis, thereby revealing the first postembryonic function for Pkn.

Structural basis for viral 5′-PPP-RNA recognition by human IFIT proteins

PubMed Central

Abbas, Yazan M.; Pichlmair, Andreas; Górna, Maria W.; Superti-Furga, Giulio; Nagar, Bhushan

2016-01-01

IFIT proteins are interferon-inducible, innate immune effector molecules that are thought to confer antiviral defence through disruption of protein-protein interactions in the host translation initiation machinery. However, recently it was discovered that IFITs could directly recognize viral RNA bearing a 5′-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here, we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an N-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double stranded viral PPP-RNA, RIG-I. Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence specific manner and requires approximately a 3-nucleotide 5′-overhang. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 were found to cause a defect in the anti-viral response by HEK cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA and lend insight into their downstream effector function. PMID:23334420

Unravelling Immunoglobulin G Fc N-Glycosylation: A Dynamic Marker Potentiating Predictive, Preventive and Personalised Medicine.

PubMed

Russell, Alyce; Adua, Eric; Ugrina, Ivo; Laws, Simon; Wang, Wei

2018-01-29

Multiple factors influence immunoglobulin G glycosylation, which in turn affect the glycoproteins' function on eliciting an anti-inflammatory or pro-inflammatory response. It is prudent to underscore these processes when considering the use of immunoglobulin G N -glycan moieties as an indication of disease presence, progress, or response to therapeutics. It has been demonstrated that the altered expression of genes that encode enzymes involved in the biosynthesis of immunoglobulin G N -glycans, receptors, or complement factors may significantly modify immunoglobulin G effector response, which is important for regulating the immune system. The immunoglobulin G N -glycome is highly heterogenous; however, it is considered an interphenotype of disease (a link between genetic predisposition and environmental exposure) and so has the potential to be used as a dynamic biomarker from the perspective of predictive, preventive, and personalised medicine. Undoubtedly, a deeper understanding of how the multiple factors interact with each other to alter immunoglobulin G glycosylation is crucial. Herein we review the current literature on immunoglobulin G glycoprotein structure, immunoglobulin G Fc glycosylation, associated receptors, and complement factors, the downstream effector functions, and the factors associated with the heterogeneity of immunoglobulin G glycosylation.

RhoJ Regulates Melanoma Chemoresistance by Suppressing Pathways that Sense DNA Damage

PubMed Central

Ho, Hsiang; Aruri, Jayavani; Kapadia, Rubina; Mehr, Hootan; White, Michael A.; Ganesan, Anand K.

2012-01-01

Melanomas resist conventional chemotherapeutics in part through intrinsic disrespect of apoptotic checkpoint activation. In this study, using an unbiased genome-wide RNAi screen we identified RhoJ and its effector Pak1, as key modulators of melanoma cell sensitivity to DNA damage. We find that RhoJ activates Pak1 in response to drug-induced DNA damage, which then uncouples ATR from its downstream effectors, ultimately resulting in a blunted DNA damage response (DDR). In addition, ATR suppression leads to the decreased phosphorylation of ATF2, and consequent increased expression of the melanocyte survival gene Sox10 resulting in a higher DDR threshold required to engage melanoma cell death. In the setting of normal melanocyte behavior, this regulatory relationship may facilitate appropriate epidermal melanization in response to UV-induced DNA damage. However, pathological pathway activation during oncogenic transformation produces a tumor that is intrinsically resistant to chemotherapy and has the propensity to accumulate additional mutations. These findings identify DNA damage agents and pharmacological inhibitors of RhoJ/PAK1 as novel synergistic agents that can be used to treat melanomas that are resistant to conventional chemotherapies. PMID:22971344

Interplay between cardiac transcription factors and non-coding RNAs in predisposing to atrial fibrillation.

PubMed

Mikhailov, Alexander T; Torrado, Mario

2018-05-12

There is growing evidence that putative gene regulatory networks including cardio-enriched transcription factors, such as PITX2, TBX5, ZFHX3, and SHOX2, and their effector/target genes along with downstream non-coding RNAs can play a potentially important role in the process of adaptive and maladaptive atrial rhythm remodeling. In turn, expression of atrial fibrillation-associated transcription factors is under the control of upstream regulatory non-coding RNAs. This review broadly explores gene regulatory mechanisms associated with susceptibility to atrial fibrillation-with key examples from both animal models and patients-within the context of both cardiac transcription factors and non-coding RNAs. These two systems appear to have multiple levels of cross-regulation and act coordinately to achieve effective control of atrial rhythm effector gene expression. Perturbations of a dynamic expression balance between transcription factors and corresponding non-coding RNAs can provoke the development or promote the progression of atrial fibrillation. We also outline deficiencies in current models and discuss ongoing studies to clarify remaining mechanistic questions. An understanding of the function of transcription factors and non-coding RNAs in gene regulatory networks associated with atrial fibrillation risk will enable the development of innovative therapeutic strategies.

Reverse engineering GTPase programming languages with reconstituted signaling networks.

PubMed

Coyle, Scott M

2016-07-02

The Ras superfamily GTPases represent one of the most prolific signaling currencies used in Eukaryotes. With these remarkable molecules, evolution has built GTPase networks that control diverse cellular processes such as growth, morphology, motility and trafficking. (1-4) Our knowledge of the individual players that underlie the function of these networks is deep; decades of biochemical and structural data has provided a mechanistic understanding of the molecules that turn GTPases ON and OFF, as well as how those GTPase states signal by controlling the assembly of downstream effectors. However, we know less about how these different activities work together as a system to specify complex dynamic signaling outcomes. Decoding this molecular "programming language" would help us understand how different species and cell types have used the same GTPase machinery in different ways to accomplish different tasks, and would also provide new insights as to how mutations to these networks can cause disease. We recently developed a bead-based microscopy assay to watch reconstituted H-Ras signaling systems at work under arbitrary configurations of regulators and effectors. (5) Here we highlight key observations and insights from this study and propose extensions to our method to further study this and other GTPase signaling systems.

MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1β Independently of Gasdermin-D.

PubMed

Gutierrez, Kimberley D; Davis, Michael A; Daniels, Brian P; Olsen, Tayla M; Ralli-Jain, Pooja; Tait, Stephen W G; Gale, Michael; Oberst, Andrew

2017-03-01

Necroptosis is a form of programmed cell death defined by activation of the kinase receptor interacting protein kinase 3 and its downstream effector, the pseudokinase mixed lineage kinase domain-like (MLKL). Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. In this study, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1β. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1β released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1β independently of the recently described pyroptotic effector gasdermin-D. Taken together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1β independently of gasdermin-D. Copyright © 2017 by The American Association of Immunologists, Inc.

Epac2 Mediates cAMP-Dependent Potentiation of Neurotransmission in the Hippocampus

PubMed Central

Fernandes, Herman B.; Riordan, Sean; Nomura, Toshihiro; Remmers, Christine L.; Kraniotis, Stephen; Marshall, John J.; Kukreja, Lokesh; Vassar, Robert

2015-01-01

Presynaptic terminal cAMP elevation plays a central role in plasticity at the mossy fiber-CA3 synapse of the hippocampus. Prior studies have identified protein kinase A as a downstream effector of cAMP that contributes to mossy fiber LTP (MF-LTP), but the potential contribution of Epac2, another cAMP effector expressed in the MF synapse, has not been considered. We investigated the role of Epac2 in MF-CA3 neurotransmission using Epac2−/− mice. The deletion of Epac2 did not cause gross alterations in hippocampal neuroanatomy or basal synaptic transmission. Synaptic facilitation during short trains was not affected by loss of Epac2 activity; however, both long-term plasticity and forskolin-mediated potentiation of MFs were impaired, demonstrating that Epac2 contributes to cAMP-dependent potentiation of transmitter release. Examination of synaptic transmission during long sustained trains of activity suggested that the readily releasable pool of vesicles is reduced in Epac2−/− mice. These data suggest that cAMP elevation uses an Epac2-dependent pathway to promote transmitter release, and that Epac2 is required to maintain the readily releasable pool at MF synapses in the hippocampus. PMID:25904804

Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype.

PubMed

Lamers, Ideke J C; Reijnders, Margot R F; Venselaar, Hanka; Kraus, Alison; Jansen, Sandra; de Vries, Bert B A; Houge, Gunnar; Gradek, Gyri Aasland; Seo, Jieun; Choi, Murim; Chae, Jong-Hee; van der Burgt, Ineke; Pfundt, Rolph; Letteboer, Stef J F; van Beersum, Sylvia E C; Dusseljee, Simone; Brunner, Han G; Doherty, Dan; Kleefstra, Tjitske; Roepman, Ronald

2017-11-02

The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization. Copyright © 2017 American Society of Human Genetics. All rights reserved.

Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

PubMed Central

Kuzmanov, Uros; Guo, Hongbo; Buchsbaum, Diana; Cosme, Jake; Abbasi, Cynthia; Isserlin, Ruth; Sharma, Parveen; Gramolini, Anthony O.; Emili, Andrew

2016-01-01

Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function. PMID:27742792

Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.

PubMed

Sharma, Rahul; Xia, Xiaojuan; Cano, Liliana M; Evangelisti, Edouard; Kemen, Eric; Judelson, Howard; Oome, Stan; Sambles, Christine; van den Hoogen, D Johan; Kitner, Miloslav; Klein, Joël; Meijer, Harold J G; Spring, Otmar; Win, Joe; Zipper, Reinhard; Bode, Helge B; Govers, Francine; Kamoun, Sophien; Schornack, Sebastian; Studholme, David J; Van den Ackerveken, Guido; Thines, Marco

2015-10-05

Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

PubMed Central

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-01

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

PubMed

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-09

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhadurihauck, Anjuli; Li, Lei; Li, Qianqian

Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer, using tumor-infiltrating lymphocytes (TILs) expanded in vitro. Tumor-infiltrating cytotoxic T lymphocytes (TICTLs) play a prominent role in cancer control. TILs terminally differentiate in response to immunosuppressive environments within tumors, and thus are slow to expand and challenging to maintain both in vitro and in patients. To reverse this exhaustion, we utilize a nuclear protein delivery system that exposes TICTLs to the SOX2, Oct-4, and NANOG (SON) proteins. Unlike activated naïve CTLs (effector CTLs), TICTLs respond favorably to SON treatment, exhibiting steady proliferation and extended survivability independent of cytokinemore » and antigen stimulation. Though TICTLs treated with SON (STICTLs) still express T cell receptors as well as other critical downstream components, they are unresponsive to antigen challenge, suggesting that SON treatment regresses TICTLs into a state similar to that of an early double negative T cell. Our findings indicate the TICTL response to SON proteins is unique when compared to effector CTLs, suggesting TICTLs may be sensitive to regulation by other lineage-specific transcription factors and opening a promising new avenue into cancer immunotherapy. To our knowledge, this is the first report on lineage reprogramming of TILs using protein stem cell transcription factors delivered directly to the nucleus. -- Highlights: •TICTLs are sensitive to reprogramming by proteins of stem cell transcription factors, but effector CTLs were not. •TICTLs are regressed back to an early double negative T cell stage. •TCR signaling is deregulated by these transcription factors.« less

SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda.

PubMed

Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J; Clem, Rollie J

2013-05-01

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India

PubMed Central

Chandele, Anmol; Sewatanon, Jaturong; Gunisetty, Sivaram; Singla, Mohit; Onlamoon, Nattawat; Akondy, Rama S.; Kissick, Haydn Thomas; Nayak, Kaustuv; Reddy, Elluri Seetharami; Kalam, Haroon; Kumar, Dhiraj; Verma, Anil; Panda, HareKrushna; Wang, Siyu; Angkasekwinai, Nasikarn; Pattanapanyasat, Kovit; Chokephaibulkit, Kulkanya; Lodha, Rakesh; Kabra, Sushil; Ahmed, Rafi

2016-01-01

ABSTRACT Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR− CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue. IMPORTANCE Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro. Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects. PMID:27707928

Leishmania donovani Utilize Sialic Acids for Binding and Phagocytosis in the Macrophages through Selective Utilization of Siglecs and Impair the Innate Immune Arm.

PubMed

Roy, Saptarshi; Mandal, Chitra

2016-08-01

Leishmania donovani, belonging to a unicellular protozoan parasite, display the differential level of linkage-specific sialic acids on their surface. Sialic acids binding immunoglobulin-like lectins (siglecs) are a class of membrane-bound receptors present in the haematopoetic cell lineages interact with the linkage-specific sialic acids. Here we aimed to explore the utilization of sialic acids by Leishmania donovani for siglec-mediated binding, phagocytosis, modulation of innate immune response and signaling pathways for establishment of successful infection in the host. We have found enhanced binding of high sialic acids containing virulent strains (AG83+Sias) with siglec-1 and siglec-5 present on macrophages compared to sialidase treated AG83+Sias (AG83-Sias) and low sialic acids-containing avirulent strain (UR6) by flow cytometry. This specific receptor-ligand interaction between sialic acids and siglecs were further confirmed by confocal microscopy. Sialic acids-siglec-1-mediated interaction of AG83+Sias with macrophages induced enhanced phagocytosis. Additionally, sialic acids-siglec-5 interaction demonstrated reduced ROS, NO generation and Th2 dominant cytokine response upon infection with AG83+Sias in contrast to AG83-Sias and UR6. Sialic acids-siglecs binding also facilitated multiplication of intracellular amastigotes. Moreover, AG83+Sias induced sialic acids-siglec-5-mediated upregulation of host phosphatase SHP-1. Such sialic acids-siglec interaction was responsible for further downregulation of MAPKs (p38, ERK and JNK) and PI3K/Akt pathways followed by the reduced translocation of p65 subunit of NF-κβ to the nucleus from cytosol in the downstream signaling pathways. This sequence of events was reversed in AG83-Sias and UR6-infected macrophages. Besides, siglec-knockdown macrophages also showed the reversal of AG83+Sias infection-induced effector functions and downstream signaling events. Taken together, this study demonstrated that virulent parasite (AG83+Sias) establish a unique sialic acids-mediated binding and subsequent phagocytosis in the host cell through the selective exploitation of siglec-1. Additionally, sialic acids-siglec-5 interaction altered the downstream signaling pathways which contributed impairment of immune effector functions of macrophages. To the best of our knowledge, this is a comprehensive report describing sialic acids-siglec interactions and their role in facilitating uptake of the virulent parasite within the host.

Interaction of fenoterol stereoisomers with β2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

PubMed

Reinartz, Michael T; Kälble, Solveig; Wainer, Irving W; Seifert, Roland

2015-05-01

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the β2-adrenoceptor (β2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using β2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro.

PubMed

Dorjgotov, Dulguun; Jurca, Manuela E; Fodor-Dunai, Csilla; Szucs, Attila; Otvös, Krisztina; Klement, Eva; Bíró, Judit; Fehér, Attila

2009-04-02

Plants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.

Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation

PubMed Central

Šolman, Maja; Ligabue, Alessio; Blaževitš, Olga; Jaiswal, Alok; Zhou, Yong; Liang, Hong; Lectez, Benoit; Kopra, Kari; Guzmán, Camilo; Härmä, Harri; Hancock, John F; Aittokallio, Tero; Abankwa, Daniel

2015-01-01

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer. DOI: http://dx.doi.org/10.7554/eLife.08905.001 PMID:26274561

Disease implications of the Hippo/YAP pathway

PubMed Central

Plouffe, Steven W; Hong, Audrey W; Guan, Kun-Liang

2015-01-01

The Hippo signaling pathway is important for controlling organ size and tissue homeostasis. Originally identified in Drosophila melanogaster, the core components of the Hippo pathway are highly conserved in mammals. The Hippo pathway can be modulated by a wide range of stimuli, including G protein coupled receptor (GPCR) signaling, changes in the actin cytoskeleton, cell-cell contact, and cell polarity. When activated, the Hippo pathway functions as a tumor suppressor to limit cell growth. However, dysregulation by genetic inactivation of core pathway components, or amplification or gene fusion of its downstream effectors, results in increased cell proliferation and decreased apoptosis and differentiation. Not surprisingly, this can lead to tissue overgrowth, tumorigenesis, and many other diseases. PMID:25702974

Integrating non-coding RNAs in JAK-STAT regulatory networks

PubMed Central

Witte, Steven; Muljo, Stefan A

2014-01-01

Being a well-characterized pathway, JAK-STAT signaling serves as a valuable paradigm for studying the architecture of gene regulatory networks. The discovery of untranslated or non-coding RNAs, namely microRNAs and long non-coding RNAs, provides an opportunity to elucidate their roles in such networks. In principle, these regulatory RNAs can act as downstream effectors of the JAK-STAT pathway and/or affect signaling by regulating the expression of JAK-STAT components. Examples of interactions between signaling pathways and non-coding RNAs have already emerged in basic cell biology and human diseases such as cancer, and can potentially guide the identification of novel biomarkers or drug targets for medicine. PMID:24778925

Domain requirements for the Dock adapter protein in growth- cone signaling.

PubMed

Rao, Y; Zipursky, S L

1998-03-03

Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons.

Cotinine: A Therapy for Memory Extinction in Post-traumatic Stress Disorder.

PubMed

Mendoza, Cristhian; Barreto, George E; Iarkov, Alexandre; Tarasov, Vadim V; Aliev, Gjumrakch; Echeverria, Valentina

2018-01-15

Post-traumatic stress disorder (PTSD) is a mental disorder that may develop after exposure to exceptionally threatening or unescapable horrifying events. Actual therapies fail to alleviate the emotional suffering and cognitive impairment associated with this disorder, mostly because they are ineffective in treating the failure to extinguish trauma memories in a great percentage of those affected. In this review, current behavioral, cellular, and molecular evidence supporting the use of cotinine for treating PTSD are reviewed. The role of the positive modulation by cotinine of the nicotinic acetylcholine receptors (nAChRs) and their downstream effectors, the protection of astroglia, and the inhibition of microglia in the PTSD brain are also discussed.

Development of transplantable human chordoma xenograft for preclinical assessment of novel therapeutic strategies.

PubMed

Bozzi, Fabio; Manenti, Giacomo; Conca, Elena; Stacchiotti, Silvia; Messina, Antonella; Dagrada, GianPaolo; Gronchi, Alessandro; Panizza, Pietro; Pierotti, Marco A; Tamborini, Elena; Pilotti, Silvana

2014-01-01

Chordomas are rare and indolent bone tumors that arise in the skull base and mobile spine. Distant metastases occur in >20% of cases, but morbidity and mortality are mainly related to local relapses that affect the majority of patients. Standard chemotherapy has modest activity, whereas new targeted therapies (alone or in combination) have some activity in controlling disease progression. However, the scarcity of preclinical models capable of testing in vivo responses to these therapies hampers the development of new medical strategies. In this study, 8 chordoma samples taken from 8 patients were implanted in nude mice. Four engrafted successfully and gave rise to tumor masses that were analyzed histologically, by means of fluorescence in situ hybridization and biochemical techniques. The data relating to each of the mouse tumors were compared with those obtained from the corresponding human tumor. All 4 engraftments retained the histological, genetic and biochemical features of the human tumors they came from. In one epidermal growth factor receptor(EGFR)-positive xenograft, responsiveness to lapatinib was evaluated by comparing the pre- and post treatment findings. The treatment induced a low-level, heterogeneous switching off of EGFR and its downstream signaling effectors. Overall, this model is very close to human chordoma and represents a new means of undertaking preclinical investigations and developing tailored therapies.

The therapeutic effects of Rho-ROCK inhibitors on CNS disorders

PubMed Central

Kubo, Takekazu; Yamaguchi, Atsushi; Iwata, Nobuyoshi; Yamashita, Toshihide

2008-01-01

Rho-kinase (ROCK) is a serine/threonine kinase and one of the major downstream effectors of the small GTPase Rho. The Rho-ROCK pathway is involved in many aspects of neuronal functions including neurite outgrowth and retraction. The Rho-ROCK pathway becomes an attractive target for the development of drugs for treating central nervous system (CNS) disorders, since it has been recently revealed that this pathway is closely related to the pathogenesis of several CNS disorders such as spinal cord injuries, stroke, and Alzheimer’s disease (AD). In the adult CNS, injured axons regenerate poorly due to the presence of myelin-associated axonal growth inhibitors such as myelin-associated glycoprotein (MAG), Nogo, oligodendrocyte-myelin glycoprotein (OMgp), and the recently identified repulsive guidance molecule (RGM). The effects of these inhibitors are reversed by blockade of the Rho-ROCK pathway in vitro, and the inhibition of this pathway promotes axonal regeneration and functional recovery in the injured CNS in vivo. In addition, the therapeutic effects of the Rho-ROCK inhibitors have been demonstrated in animal models of stroke. In this review, we summarize the involvement of the Rho-ROCK pathway in CNS disorders such as spinal cord injuries, stroke, and AD and also discuss the potential of Rho-ROCK inhibitors in the treatment of human CNS disorders. PMID:18827856

Regulation of the metastasis suppressor Nm23-H1 by tumor viruses

PubMed Central

Banerjee, Shuvomoy; Jha, Hem Chandra

2018-01-01

Metastasis is the most common cause of cancer mortality. To increase the survival of patients, it is necessary to develop more effective methods for treating as well as preventing metastatic diseases. Recent advancement of knowledge in cancer metastasis provides the basis for development of targeted molecular therapeutics aimed at the tumor cell or its interaction with the host microenvironment. Metastasis suppressor genes (MSGs) are promising targets for inhibition of the metastasis process. During the past decade, functional significance of these genes, their regulatory pathways, and related downstream effector molecules have become a major focus of cancer research. Nm23-H1, first in the family of Nm23 human homologues, is a well-characterized, anti-metastatic factor linked with a large number of human malignancies. Mounting evidence to date suggests an important role for Nm23-H1 in reducing virus-induced tumor cell motility and migration. A detailed understanding of the molecular association between oncogenic viral antigens with Nm23-H1 may reveal the underlying mechanisms for tumor virus-associated malignancies. In this review, we will focus on the recent advances to our understanding of the molecular basis of oncogenic virus-induced progression of tumor metastasis by deregulation of Nm23-H1. PMID:25199839

Yorkie Facilitates Organ Growth and Metamorphosis in Bombyx

PubMed Central

Liu, Shumin; Zhang, Panli; Song, Hong-Sheng; Qi, Hai-Sheng; Wei, Zhao-Jun; Zhang, Guozheng; Zhan, Shuai; Liu, Zhihong; Li, Sheng

2016-01-01

The Hippo pathway, which was identified from genetic screens in the fruit fly, Drosophila melanogaster, has a major size-control function in animals. All key components of the Hippo pathway, including the transcriptional coactivator Yorkie that is the most critical substrate and downstream effector of the Hippo kinase cassette, are found in the silkworm, Bombyx mori. As revealed by microarray and quantitative real-time PCR, expression of Hippo pathway genes is particularly enriched in several mitotic tissues, including the ovary, testis, and wing disc. Developmental profiles of Hippo pathway genes are generally similar (with the exception of Yorkie) within each organ, but vary greatly in different tissues showing nearly opposing expression patterns in the wing disc and the posterior silk gland (PSG) on day 2 of the prepupal stage. Importantly, the reduction of Yorkie expression by RNAi downregulated Yorkie target genes in the ovary, decreased egg number, and delayed larval-pupal-adult metamorphosis. In contrast, baculovirus-mediated YorkieCA overexpression upregulated Yorkie target genes in the PSG, increased PSG size, and accelerated larval-pupal metamorphosis. Together the results show that Yorkie potentially facilitates organ growth and metamorphosis, and suggest that the evolutionarily conserved Hippo pathway is critical for size control, particularly for PSG growth, in the silkworm. PMID:27489496

The mitogen-activated protein kinase (MAPK) cascade controls phosphatase and tensin homolog (PTEN) expression through multiple mechanisms.

PubMed

Ciuffreda, Ludovica; Di Sanza, Cristina; Cesta Incani, Ursula; Eramo, Adriana; Desideri, Marianna; Biagioni, Francesca; Passeri, Daniela; Falcone, Italia; Sette, Giovanni; Bergamo, Paola; Anichini, Andrea; Sabapathy, Kanaga; McCubrey, James A; Ricciardi, Maria Rosaria; Tafuri, Agostino; Blandino, Giovanni; Orlandi, Augusto; De Maria, Ruggero; Cognetti, Francesco; Del Bufalo, Donatella; Milella, Michele

2012-06-01

The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent "escape" mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade.

Targeting oncogenic KRAS in non-small cell lung cancer cells by phenformin inhibits growth and angiogenesis.

PubMed

Wang, Zhi Dong; Wei, Sheng Quan; Wang, Qin Yi

2015-01-01

Tumors require a vascular supply to grow and can achieve this via the expression of pro-angiogenic growth factors. Many potential oncogenic mutations have been identified in tumor angiogenesis. Somatic mutations in the small GTPase KRAS are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated anti-tumor activity both in vitro and in vivo. The extracellular regulated protein kinases (ERK) signaling is known to be a major cellular target of biguanides. Based on KRAS activates several down-stream effectors leading to the stimulation of the RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAF/MEK/ERK) and phosphatidylinositol-3-kinase (PI3K) pathways, we investigated the anti-tumor effects of biguanides on the proliferation of KRAS-mutated tumor cells in vitro and on KRAS-driven tumor growth in vivo. In cancer cells harboring oncogenic KRAS, phenformin switches off the ERK pathway and inhibit the expression of pro-angiogenic molecules. In tumor xenografts harboring the KRAS mutation, phenformin extensively modifies the tumor growth causing abrogation of angiogenesis. These results strongly suggest that significant therapeutic advantage may be achieved by phenformin anti-angiogenesis for the treatment of tumor.

Comparative transcriptional profiling identifies takeout as a gene that regulates life span

PubMed Central

Bauer, Johannes; Antosh, Michael; Chang, Chengyi; Schorl, Christoph; Kolli, Santharam; Neretti, Nicola; Helfand, Stephen L.

2010-01-01

A major challenge in translating the positive effects of dietary restriction (DR) for the improvement of human health is the development of therapeutic mimics. One approach to finding DR mimics is based upon identification of the proximal effectors of DR life span extension. Whole genome profiling of DR in Drosophila shows a large number of changes in gene expression, making it difficult to establish which changes are involved in life span determination as opposed to other unrelated physiological changes. We used comparative whole genome expression profiling to discover genes whose change in expression is shared between DR and two molecular genetic life span extending interventions related to DR, increased dSir2 and decreased Dmp53 activity. We find twenty-one genes shared among the three related life span extending interventions. One of these genes, takeout, thought to be involved in circadian rhythms, feeding behavior and juvenile hormone binding is also increased in four other life span extending conditions: Rpd3, Indy, chico and methuselah. We demonstrate takeout is involved in longevity determination by specifically increasing adult takeout expression and extending life span. These studies demonstrate the power of comparative whole genome transcriptional profiling for identifying specific downstream elements of the DR life span extending pathway. PMID:20519778

Human melanoma cells resistant to MAPK inhibitors can be effectively targeted by inhibition of the p90 ribosomal S6 kinase

PubMed Central

Kosnopfel, Corinna; Sinnberg, Tobias; Sauer, Birgit; Niessner, Heike; Schmitt, Anja; Makino, Elena; Forschner, Andrea; Hailfinger, Stephan; Garbe, Claus; Schittek, Birgit

2017-01-01

The clinical availability of small molecule inhibitors specifically targeting mutated BRAF marked a significant breakthrough in melanoma therapy. Despite a dramatic anti-tumour activity and improved patient survival, rapidly emerging resistance, however, greatly limits the clinical benefit. The majority of the already described resistance mechanisms involve a reactivation of the MAPK signalling pathway. The p90 ribosomal S6 kinase (RSK), a downstream effector of the MAPK signalling cascade, has been reported to enhance survival of melanoma cells in response to chemotherapy. Here, we can show that RSK activity is significantly increased in human melanoma cells with acquired resistance to the BRAFV600E/K inhibitor vemurafenib. Interestingly, inhibition of RSK signalling markedly impairs the viability of vemurafenib resistant melanoma cells and is effective both in two-dimensional and in three-dimensional culture systems, especially in a chronic, long-term application. The effect of RSK inhibition can be partly replicated by downregulation of the well-known RSK target, Y-box binding protein 1 (YB-1). Intriguingly, RSK inhibition also retains its efficacy in melanoma cells with combined resistance to vemurafenib and the MEK inhibitor trametinib. These data suggest that active RSK signalling might be an attractive novel therapeutic target in melanoma with acquired resistance to MAPK pathway inhibitors. PMID:28415756

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways*

PubMed Central

Kovacevic, Zaklina; Menezes, Sharleen V.; Sahni, Sumit; Kalinowski, Danuta S.; Bae, Dong-Hun; Lane, Darius J. R.; Richardson, Des R.

2016-01-01

N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-β pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors. PMID:26534963

Anionic lipids and the cytoskeletal proteins MreB and RodZ define the spatio-temporal distribution and function of membrane stress controller PspA in Escherichia coli.

PubMed

Jovanovic, Goran; Mehta, Parul; Ying, Liming; Buck, Martin

2014-11-01

All cell types must maintain the integrity of their membranes. The conserved bacterial membrane-associated protein PspA is a major effector acting upon extracytoplasmic stress and is implicated in protection of the inner membrane of pathogens, formation of biofilms and multi-drug-resistant persister cells. PspA and its homologues in Gram-positive bacteria and archaea protect the cell envelope whilst also supporting thylakoid biogenesis in cyanobacteria and higher plants. In enterobacteria, PspA is a dual function protein negatively regulating the Psp system in the absence of stress and acting as an effector of membrane integrity upon stress. We show that in Escherichia coli the low-order oligomeric PspA regulatory complex associates with cardiolipin-rich, curved polar inner membrane regions. There, cardiolipin and the flotillin 1 homologue YqiK support the PspBC sensors in transducing a membrane stress signal to the PspA-PspF inhibitory complex. After stress perception, PspA high-order oligomeric effector complexes initially assemble in polar membrane regions. Subsequently, the discrete spatial distribution and dynamics of PspA effector(s) in lateral membrane regions depend on the actin homologue MreB and the peptidoglycan machinery protein RodZ. The consequences of loss of cytoplasmic membrane anionic lipids, MreB, RodZ and/or YqiK suggest that the mode of action of the PspA effector is closely associated with cell envelope organization. © 2014 The Authors.

Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation.

PubMed

Cressman, William J; Beckett, Dorothy

2016-01-19

Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5'-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein's dimerization free energy by -4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5'-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5'-AMP binding at the distant effector binding site.

Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells

PubMed Central

Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

2018-01-01

Background The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf. Methods Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Results Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Conclusions Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s. PMID:29581812

The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation

PubMed Central

Warnhoff, Kurt; Murphy, John T.; Kumar, Sandeep; Schneider, Daniel L.; Peterson, Michelle; Hsu, Simon; Guthrie, James; Robertson, J. David; Kornfeld, Kerry

2014-01-01

The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance. PMID:25330323

METCAM/MUC18 promoted tumorigenesis of human breast cancer SK-BR-3 cells in a dosage-specific manner.

PubMed

Huang, Chang-Yu; Wu, Guang-Jer

2016-04-01

Overexpression of METCAM/MUC18, an immunoglobulin-like cell-adhesion molecule, promotes tumorigenesis and progression of human breast cancer cells. We also observed an intriguing phenomenon that a high-expressing SK-BR-3 clone manifested a transient tumor suppression effect in vivo. The purpose of this study was to understand if this was caused by clonal variation, METCAM/MUC18-dosage effect, or the number of cells injected. Several G418-resistant clones of SK-BR-3, expressing different levels of METCAM/MUC18, were obtained for testing effects of human METCAM/MUC18 on in vitro motility, invasiveness, and anchorage-independent colony formation (in vitro tumorigenicity) and in vivo tumorigenesis in female Balb/C athymic nude mice. Tumor sections were made for histology and immunohistochemistry analyses, and tumor lysates for Western blot analysis to determine the effects of human METCAM/MUC18 expression on levels of various downstream effectors. METCAM/MUC18 promoted in vitro motility, invasiveness, and in vitro tumorigenicity of SK-BR-3 cells in a dosage-specific manner. Overexpression of METCAM/MUC18 could promote in vivo tumorigenesis of SK-BR-3 cells even when one tenth of the previously used cell number (5 × 10(5)) was injected and in vivo tumorigenesis of SK-BR-3 cells was directly proportional to the dosage of the protein. The previously observed transient tumor suppression effect from the same clone was no longer observed. The downstream effector, such as phospho-AKT/AKT ratio, was elevated in the tumors. Transient suppression observed previously in the clone was caused by injection of a high cell number (2 × 10(6)-5 × 10(6)). METCAM/MUC18 positively promotes tumorigenesis of SK-BR-3 cells by increasing the survival and proliferation pathway. Copyright © 2016. Published by Elsevier B.V.

Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.

PubMed

Rhee, Ho Sung; Closser, Michael; Guo, Yuchun; Bashkirova, Elizaveta V; Tan, G Christopher; Gifford, David K; Wichterle, Hynek

2016-12-21

Generic spinal motor neuron identity is established by cooperative binding of programming transcription factors (TFs), Isl1 and Lhx3, to motor-neuron-specific enhancers. How expression of effector genes is maintained following downregulation of programming TFs in maturing neurons remains unknown. High-resolution exonuclease (ChIP-exo) mapping revealed that the majority of enhancers established by programming TFs are rapidly deactivated following Lhx3 downregulation in stem-cell-derived hypaxial motor neurons. Isl1 is released from nascent motor neuron enhancers and recruited to new enhancers bound by clusters of Onecut1 in maturing neurons. Synthetic enhancer reporter assays revealed that Isl1 operates as an integrator factor, translating the density of Lhx3 or Onecut1 binding sites into transient enhancer activity. Importantly, independent Isl1/Lhx3- and Isl1/Onecut1-bound enhancers contribute to sustained expression of motor neuron effector genes, demonstrating that outwardly stable expression of terminal effector genes in postmitotic neurons is controlled by a dynamic relay of stage-specific enhancers. Copyright © 2016 Elsevier Inc. All rights reserved.

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

PubMed

Hatsugai, Noriyuki; Igarashi, Daisuke; Mase, Keisuke; Lu, You; Tsuda, Yayoi; Chakravarthy, Suma; Wei, Hai-Lei; Foley, Joseph W; Collmer, Alan; Glazebrook, Jane; Katagiri, Fumiaki

2017-09-15

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses. © 2017 The Authors.

Philippines' downstream sector poised for growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1992-05-11

This paper reports that the Philippines' downstream sector is poised for sharp growth. Despite a slip in refined products demand in recent years, Philippines products demand will rebound sharply by 2000, East-West Center (EWC), Honolulu, predicts. Philippines planned refinery expansions are expected to meet that added demand, EWC Director Fereidun Fesharaki says. Like the rest of the Asia-Pacific region, product specifications are changing, but major refiners in the area expect to meet the changes without major case outlays. At the same time, Fesharaki says, push toward deregulation will further bolster the outlook for the Philippines downstream sector.

Mast cells in airway diseases and interstitial lung disease.

PubMed

Cruse, Glenn; Bradding, Peter

2016-05-05

Mast cells are major effector cells of inflammation and there is strong evidence that mast cells play a significant role in asthma pathophysiology. There is also a growing body of evidence that mast cells contribute to other inflammatory and fibrotic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. This review discusses the role that mast cells play in airway diseases and highlights how mast cell microlocalisation within specific lung compartments and their cellular interactions are likely to be critical for their effector function in disease. Published by Elsevier B.V.

Farnesyl transferase inhibitors: a major breakthrough in anticancer therapy? Naples, 12 April 2002.

PubMed

Caponigro, Francesco

2002-09-01

An international meeting focused on farnesyl transferase inhibitors (FTIs) was held in Naples on 12 April 2002 and represented an excellent occasion to gather most of the clinicians who are involved in clinical trials with this class of new compounds. Oncogene mutations of the gene occur in approximately 30% of all human cancers and may have prognostic significance. Ras protein is normally synthesized as pro-Ras, which undergoes a number of post-translational modifications, among which farnesylation. Processed Ras proteins localize to the inner surface of the plasma membrane, and function as a molecular switch that cycles between an inactive and an active form. When in its active form, either because of the binding of an external ligand or because of its constitutive activation, Ras activates several downstream effectors, such as Raf-1, Rac, Rho and phospahtidylinositol-3 kinase, which mediate important cellular functions, such as proliferation, cytoskeletal organization and others. Interruption of the Ras signaling pathway can be basically achieved in three ways, i.e. inhibition of Ras protein expression through antisense oligonucleotides, prevention of Ras membrane localization and inhibition of Ras downstream effectors. SCH 66336 (lonafarnib; Sarasar), a tricyclic orally active FTI, has been the first of these compounds to undergo clinical development. The toxicity profile observed in all completed phase I/II trials has been fairly similar, since gastrointestinal tract toxicity (nausea, vomiting and diarrhea) and fatigue have generally qualified as dose-limiting toxicity (DLT). One objective response in a patient with pretreated non-small cell lung cancer (NSCLC) was observed. Based on preclinical evidence of synergism between lonafarnib and other anticancer agents, combination studies have been started. In particular, lonafarnib has been combined both with gemcitabine and with paclitaxel in phase I studies. Nausea, vomiting, diarrhea and myelosuppression represented DLTs in these studies, in which an encouraging clinical activity was observed, in particular in pancreatic carcinoma (lonafarnib plus gemcitabine) and in NSCLC (lonafarnib plus paclitaxel). R115777 (Zarnestra) is another novel orally active FT competitive inhibitor in clinical development. Single-agent phase I/II studies have shown that myelotoxicity and neurotoxicity are DLTs, intermittent schedule is probably better tolerated and antitumor activity is observed particularly in breast cancer. A number of combination studies with R115777 have been carried out; taken as a whole, they show that the drug can be easily combined with several anticancer agents and phase III trials exploring the potential benefit from incorporation of R115777 into active chemotherapy regimens are indicated. Two other FTIs are in an earlier stage of clinical development. BMS-214662 has the main advantage of being cytotoxic in nature, rather than cytostatic; in particular, potent antitumor activity in human tumor xenografts of different histologies has been reported. A major drawback for BMS-214662 is its severe gastrointestinal and liver toxicities, which prevent the achievement of adequate systemic exposures following the oral route. L-778,123 has been stopped in its clinical development due to its severe and unexpected toxicity, i.e. grade 4 thrombocytopenia and significant Q-T prolongation.

Conserved electrostatic fields at the Ras-effector interface measured through vibrational Stark effect spectroscopy explain the difference in tilt angle in the Ras binding domains of Raf and RalGDS.

PubMed

Walker, David M; Wang, Ruifei; Webb, Lauren J

2014-10-07

Vibrational Stark effect (VSE) spectroscopy was used to measure the electrostatic fields present at the interface of the human guanosine triphosphatase (GTPase) Ras docked with the Ras binding domain (RBD) of the protein kinase Raf. Nine amino acids located on the surface of Raf were selected for labeling with a nitrile vibrational probe. Eight of the probe locations were situated along the interface of Ras and Raf, and one probe was 2 nm away on the opposite side of Raf. Vibrational frequencies of the nine Raf nitrile probes were compared both in the monomeric, solvated protein and when docked with wild-type (WT) Ras to construct a comprehensive VSE map of the Ras-Raf interface. Molecular dynamics (MD) simulations employing an umbrella sampling strategy were used to generate a Boltzmann-weighted ensemble of nitrile positions in both the monomeric and docked complexes to determine the effect that docking has on probe location and orientation and to aid in the interpretation of VSE results. These results were compared to an identical study that was previously conducted on nine nitrile probes on the RBD of Ral guanidine dissociation stimulator (RalGDS) to make comparisons between the docked complexes formed when either of the two effectors bind to WT Ras. This comparison finds that there are three regions of conserved electrostatic fields that are formed upon docking of WT Ras with both downstream effectors. Conservation of this pattern in the docked complex then results in different binding orientations observed in otherwise structurally similar proteins. This work supports an electrostatic cause of the known binding tilt angle between the Ras-Raf and Ras-RalGDS complexes.

Ras and relatives--job sharing and networking keep an old family together.

PubMed

Ehrhardt, Annette; Ehrhardt, Götz R A; Guo, Xuecui; Schrader, John W

2002-10-01

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

Perturbation of Maize Phenylpropanoid Metabolism by an AvrE Family Type III Effector from Pantoea stewartii1[OPEN

PubMed Central

Asselin, Jo Ann E.; Lin, Jinshan; Perez-Quintero, Alvaro L.; Gentzel, Irene; Majerczak, Doris; Opiyo, Stephen O.; Zhao, Wanying; Paek, Seung-Mann; Kim, Min Gab; Coplin, David L.; Blakeslee, Joshua J.; Mackey, David

2015-01-01

AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of Pantoea stewartii ssp. stewartii (Pnss) to proliferate efficiently and cause wilt and leaf blight symptoms in maize (Zea mays) plants. Notably, when WtsE is delivered by a heterologous system into the leaf cells of susceptible maize seedlings, it alone produces water-soaked disease symptoms reminiscent of those produced by Pnss. Thus, WtsE is a pathogenicity and virulence factor in maize, and an Escherichia coli heterologous delivery system can be used to study the activity of WtsE in isolation from other factors produced by Pnss. Transcriptional profiling of maize revealed the effects of WtsE, including induction of genes involved in secondary metabolism and suppression of genes involved in photosynthesis. Targeted metabolite quantification revealed that WtsE perturbs maize metabolism, including the induction of coumaroyl tyramine. The ability of mutant WtsE derivatives to elicit transcriptional and metabolic changes in susceptible maize seedlings correlated with their ability to promote disease. Furthermore, chemical inhibitors that block metabolic flux into the phenylpropanoid pathways targeted by WtsE also disrupted the pathogenicity and virulence activity of WtsE. While numerous metabolites produced downstream of the shikimate pathway are known to promote plant defense, our results indicate that misregulated induction of phenylpropanoid metabolism also can be used to promote pathogen virulence. PMID:25635112

Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

PubMed Central

Rothmeier, Eva; Pfaffinger, Gudrun; Hoffmann, Christine; Harrison, Christopher F.; Grabmayr, Heinrich; Repnik, Urska; Hannemann, Mandy; Wölke, Stefan; Bausch, Andreas; Griffiths, Gareth; Müller-Taubenberger, Annette; Itzen, Aymelt; Hilbi, Hubert

2013-01-01

The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila. PMID:24068924

RhoA orchestrates glycolysis for Th2 cell differentiation and allergic airway inflammation

PubMed Central

Yang, Jun-Qi; Kalim, Khalid W.; Li, Yuan; Zhang, Shuangmin; Hinge, Ashwini; Filippi, Marie-Dominique; Zheng, Yi; Guo, Fukun

2015-01-01

Background Mitochondrial metabolism is known to be important for T cell activation. However, its involvement in effector T cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T cell activation and effector cell differentiation and function remains largely unknown. Objective We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for Th2 cell differentiation and Th2-mediated allergic airway inflammation. Methods Conditional RhoA-deficient mice were generated by crossing RhoAflox/flox mice with CD2-Cre transgenic mice. Effects of RhoA on Th2 differentiation were evaluated by in vitro Th2-polarized culture conditions, and in vivo in ovalbumin (OVA)-induced allergic airway inflammation. Cytokines were measured by intracellular staining and ELISA. T cell metabolism was measured by Seahorse XF24 Analyzer and flow cytometry. Results Disruption of RhoA inhibited T cell activation and Th2 differentiation in vitro and prevented the development of allergic airway inflammation in vivo, with no effect on Th1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to Th2 cell differentiation and allergic airway inflammation via regulating IL-4 receptor mRNA expression and Th2-specific signaling events. Finally, inhibition of Rho-associated protein kinase (ROCK), an immediate downstream effector of RhoA, blocked Th2 differentiation and allergic airway inflammation. Conclusion RhoA is a key component of the signaling cascades leading to Th2-differentiation and allergic airway inflammation, at least in part, through the control of T cell metabolism and via ROCK pathway. PMID:26100081

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression

PubMed Central

Tavares, Catarina; Eloy, Catarina; Melo, Miguel; Gaspar da Rocha, Adriana; Pestana, Ana; Batista, Rui; Rios, Elisabete; Sobrinho Simões, Manuel

2018-01-01

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression. PMID:29757257

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression.

PubMed

Tavares, Catarina; Eloy, Catarina; Melo, Miguel; Gaspar da Rocha, Adriana; Pestana, Ana; Batista, Rui; Bueno Ferreira, Luciana; Rios, Elisabete; Sobrinho Simões, Manuel; Soares, Paula

2018-05-13

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression.

JIL-1 and Su(var)3-7 Interact Genetically and Counteract Each Other's Effect on Position-Effect Variegation in Drosophila

PubMed Central

Deng, Huai; Cai, Weili; Wang, Chao; Lerach, Stephanie; Delattre, Marion; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

2010-01-01

The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. We show that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, we show that in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms was unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity. PMID:20457875

Plasticity of gene-regulatory networks controlling sex determination: of masters, slaves, usual suspects, newcomers, and usurpators.

PubMed

Herpin, Amaury; Schartl, Manfred

2015-10-01

Sexual dimorphism is one of the most pervasive and diverse features of animal morphology, physiology, and behavior. Despite the generality of the phenomenon itself, the mechanisms controlling how sex is determined differ considerably among various organismic groups, have evolved repeatedly and independently, and the underlying molecular pathways can change quickly during evolution. Even within closely related groups of organisms for which the development of gonads on the morphological, histological, and cell biological level is undistinguishable, the molecular control and the regulation of the factors involved in sex determination and gonad differentiation can be substantially different. The biological meaning of the high molecular plasticity of an otherwise common developmental program is unknown. While comparative studies suggest that the downstream effectors of sex-determining pathways tend to be more stable than the triggering mechanisms at the top, it is still unclear how conserved the downstream networks are and how all components work together. After many years of stasis, when the molecular basis of sex determination was amenable only in the few classical model organisms (fly, worm, mouse), recently, sex-determining genes from several animal species have been identified and new studies have elucidated some novel regulatory interactions and biological functions of the downstream network, particularly in vertebrates. These data have considerably changed our classical perception of a simple linear developmental cascade that makes the decision for the embryo to develop as male or female, and how it evolves. © 2015 The Authors.

Functional Characterization of the Mannitol Promoter of Pseudomonas fluorescens DSM 50106 and Its Application for a Mannitol-Inducible Expression System for Pseudomonas putida KT2440

PubMed Central

Hoffmann, Jana; Altenbuchner, Josef

2015-01-01

A new pBBR1MCS-2-derived vector containing the Pseudomonas fluorescens DSM10506 mannitol promoter PmtlE and mtlR encoding its AraC/XylS type transcriptional activator was constructed and optimized for low basal expression. Mannitol, arabitol, and glucitol-inducible gene expression was demonstrated with Pseudomonas putida and eGFP as reporter gene. The new vector was applied for functional characterization of PmtlE. Identification of the DNA binding site of MtlR was achieved by in vivo eGFP measurement with PmtlE wild type and mutants thereof. Moreover, purified MtlR was applied for detailed in vitro investigations using electrophoretic mobility shift assays and DNaseI footprinting experiments. The obtained data suggest that MtlR binds to PmtlE as a dimer. The proposed DNA binding site of MtlR is AGTGC-N5-AGTAT-N7-AGTGC-N5-AGGAT. The transcription activation mechanism includes two binding sites with different binding affinities, a strong upstream binding site and a weaker downstream binding site. The presence of the weak downstream binding site was shown to be necessary to sustain mannitol-inducibility of PmtlE. Two possible functions of mannitol are discussed; the effector might stabilize binding of the second monomer to the downstream half site or promote transcription activation by inducing a conformational change of the regulator that influences the contact to the RNA polymerase. PMID:26207762

Immunohistochemical evalulation of activated Ras and Rac1 as potential downstream effectors of aquaporin-5 in breast cancer in vivo.

PubMed

Jensen, Helene H; Login, Frédéric H; Park, Ji-Young; Kwon, Tae-Hwan; Nejsum, Lene N

2017-11-25

Aberrant levels of aquaporin-5 (AQP5) expression have been observed in several types of cancer, including breast cancer, where AQP5 overexpression is associated with metastasis and poor prognosis. In cultured cancer cells, AQP5 facilitates cell migration and activates Ras signaling. Both increased cell migration and Ras activation are associated with cancer metastasis, but so far it is unknown if AQP5 also affects these processes in vivo. Therefore, we investigated if high AQP5 expression in breast cancer tissue correlated with increased activation of Ras and of Rac1, which is a GTPase also involved in cell migration. This was accomplished by immunohistochemical analysis of invasive ductal carcinoma of breast tissue sections from human patients, followed by qualitative and quantitative correlation analysis between AQP5 and activated Ras and Rac1. Immunohistochemistry revealed that activation of Ras and Rac1 was positively correlated. There was, however, no correlation between high AQP5 expression and activation of Ras, whereas a nonsignificant, but positive, tendency between the levels of AQP5 and activated Rac1 levels was observed. In summary, this is the first report that correlates AQP5 expression levels to downstream signaling partners in breast cancer tissue sections. The results suggest Rac1 as a potential downstream signaling partner of AQP5 in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Fragment-based drug discovery of potent and selective MKK3/6 inhibitors.

PubMed

Adams, Mark; Kobayashi, Toshitake; Lawson, J David; Saitoh, Morihisa; Shimokawa, Kenichiro; Bigi, Simone V; Hixon, Mark S; Smith, Christopher R; Tatamiya, Takayuki; Goto, Masayuki; Russo, Joseph; Grimshaw, Charles E; Swann, Steven

2016-02-01

The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sonic Hedgehog functions through dynamic changes in temporal competence in the developing ventral telencephalon

PubMed Central

Sousa, Vitor H.; Fishell, Gord

2010-01-01

Morphogens act during development to provide graded spatial information that controls patterning and cell lineage specification in the nervous system. The role of morphogen signaling in instructing the expression of downstream effector transcription factors has been well established. However, a key requirement for morphogen signaling is the existence of functional intracellular machinery able to mediate the appropriate response in target cells. Here we suggest that dynamic changes in the temporal responses to Shh in the developing ventral telencephalon occur through alterations in progenitor competence. We suggest these developmental changes in competence are mediated by a transcriptional mechanism that intrinsically integrates information from the distinct signaling pathways that act to pattern the telencephalic neuroepithelium. PMID:20466536

Immunoglobulin class switch DNA recombination: induction, targeting and beyond

PubMed Central

Xu, Zhenming; Zan, Hong; Pone, Egest J.; Mai, Thach; Casali, Paolo

2012-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus is central to the maturation of the antibody response and critically requires the AID cytidine deaminase. CSR entails changes of the chromatin state and transcriptional activation of the IgH locus upstream and downstream switch (S) regions that are to undergo S-S DNA recombination, induction of AID, and targeting of CSR factors to S regions by 14-3-3 adaptors and as enabled by the transcription machinery and histone modifications. In this Review, we focus on recent advances in CSR induction and targeting. We also outline an integrated model of the assembly of macromolecular complexes that transduce critical epigenetic information to enzymatic effectors of the CSR machinery. PMID:22728528

Matrix-Dependent Perturbation of TGFβ Signaling and Disease

PubMed Central

Doyle, Jefferson J.; Gerber, Elizabeth E.; Dietz, Harry C.

2012-01-01

Transforming growth factor beta (TGFβ) is a multipotent cytokine that is sequestered in the extracellular matrix (ECM) through interactions with a number of ECM proteins. The ECM serves to concentrate latent TGFβ at sites of intended function, to influence the bioavailability and/or function of TGFβ activators, and perhaps to regulate the intrinsic performance of cell surface effectors of TGFβ signal propagation. The downstream consequences of TGFβ signaling cascades in turn provide feedback modulation of the ECM. This review covers recent examples of how genetic mutations in constituents of the ECM or TGFβ signaling cascade result in altered ECM homeostasis, cellular performance and ultimately disease, with an emphasis on emerging therapeutic strategies that seek to capitalize on this refined mechanistic understanding. PMID:22641039

Domain requirements for the Dock adapter protein in growth- cone signaling

PubMed Central

Rao, Yong; Zipursky, S. Lawrence

1998-01-01

Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons. PMID:9482841

Rho-associated coiled-coil containing kinases (ROCK)

PubMed Central

Julian, Linda; Olson, Michael F

2014-01-01

Rho-associated coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase.1–5 They belong to the AGC family of serine/threonine kinases6 and play vital roles in facilitating actomyosin cytoskeleton contractility downstream of RhoA and RhoC activation. Since their discovery, ROCK kinases have been extensively studied, unveiling their manifold functions in processes including cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs have been identified, ROCK1 (also called ROCK I, ROKβ, Rho-kinase β, or p160ROCK) and ROCK2 (also known as ROCK II, ROKα, or Rho kinase), hereafter collectively referred to as ROCK. In this review, we will focus on the structure, regulation, and functions of ROCK. PMID:25010901

Angiotensin Mediated Oxidative Stress and Neuroprotective Potential of Antioxidants and AT1 Receptor Blockers.

PubMed

Prusty, Shakti Ketan; Sahu, Pratap Kumar; Subudhi, Bharat Bhusan

2017-01-01

Oxidative stress in brain underlies the major neurological disorders including Alzheimer's disease (AD) and Parkinson's disease (PD). Peripherally, Angiotensin-II is a major effector of inflammation. Identification of its capacity to access brain during hypertension, as well as location of central renin angiotensin system have led to its recognition as the major effector of oxidative stress in brain. Clinical uses of antioxidants to antagonize this oxidative stress have mostly failed. In this scenario, AT1 blockers have been investigated to prevent neurodegeneration. Although it has shown promise, clinical efficacy is limited to few drugs including telmisartan mainly due to the poor brain availability of others. In this review we aim to analyze the potential of antioxidants to reduce oxidative stress in brain. We have given critical analysis of the approaches for re-purposing of AT1 blockers against oxidative stress induced neurodegeneration. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Effector mechanism of magnolol-induced apoptosis in human lung squamous carcinoma CH27 cells

PubMed Central

Yang, Shu-Er; Hsieh, Ming-Tsuen; Tsai, Tung-Hu; Hsu, Shih-Lan

2003-01-01

Magnolol, an active component isolated from the root and stem bark of Magnolia officinalis, has been reported to exhibit antitumour effects, but little is known about its molecular mechanisms of action. Magnolol inhibited proliferation of human lung squamous carcinoma CH27 cells at low concentrations (10–40 μM), and induced apoptosis at high concentrations (80–100 μM). Treatment with 80 μM magnolol significantly increased the expression of Bad and Bcl-XS proteins, whereas it decreased the expression of Bcl-XL. Overexpression of Bcl-2 protected CH27 cells against magnolol-triggered apoptosis. Magnolol treatment resulted in accumulation of cytosolic cytochrome c and activation of caspase-9 and downstream caspases (caspase-3 and -6). Pretreatment with z-VAD-fmk markedly inhibited magnolol-induced cell death, but did not prevent cytosolic cytochrome c accumulation. Magnolol induced a modest and persistent JNK activation and ERK inactivation in CH27 cells without evident changes in the protein levels. The responsiveness of JNK and ERK to magnolol suggests the involvement of these kinases in the initiation of the apoptosis process. These results indicate that regulation of the Bcl-2 family, accumulation of cytosolic cytochrome c, and activation of caspase-9 and caspase-3 may be the effector mechanisms of magnolol-induced apoptosis. PMID:12522090

Visual–Motor Transformations Within Frontal Eye Fields During Head-Unrestrained Gaze Shifts in the Monkey

PubMed Central

Sajad, Amirsaman; Sadeh, Morteza; Keith, Gerald P.; Yan, Xiaogang; Wang, Hongying; Crawford, John Douglas

2015-01-01

A fundamental question in sensorimotor control concerns the transformation of spatial signals from the retina into eye and head motor commands required for accurate gaze shifts. Here, we investigated these transformations by identifying the spatial codes embedded in visually evoked and movement-related responses in the frontal eye fields (FEFs) during head-unrestrained gaze shifts. Monkeys made delayed gaze shifts to the remembered location of briefly presented visual stimuli, with delay serving to dissociate visual and movement responses. A statistical analysis of nonparametric model fits to response field data from 57 neurons (38 with visual and 49 with movement activities) eliminated most effector-specific, head-fixed, and space-fixed models, but confirmed the dominance of eye-centered codes observed in head-restrained studies. More importantly, the visual response encoded target location, whereas the movement response mainly encoded the final position of the imminent gaze shift (including gaze errors). This spatiotemporal distinction between target and gaze coding was present not only at the population level, but even at the single-cell level. We propose that an imperfect visual–motor transformation occurs during the brief memory interval between perception and action, and further transformations from the FEF's eye-centered gaze motor code to effector-specific codes in motor frames occur downstream in the subcortical areas. PMID:25491118

SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase*

PubMed Central

Takeshita, Yuji; Hashimoto, Yuichi; Nawa, Mikiro; Uchino, Hiroyuki; Matsuoka, Masaaki

2013-01-01

Humanin is a secreted bioactive peptide that suppresses cell toxicity caused by a variety of insults. The neuroprotective effect of Humanin against Alzheimer disease (AD)-related death is mediated by the binding of Humanin to its heterotrimeric Humanin receptor composed of ciliary neurotrophic receptor α, WSX-1, and gp130, as well as the activation of intracellular signaling pathways including a JAK2 and STAT3 signaling axis. Despite the elucidation of the signaling pathways by which Humanin mediates its neuroprotection, the transcriptional targets of Humanin that behaves as effectors of Humanin remains undefined. In the present study, Humanin increased the mRNA and protein expression of SH3 domain-binding protein 5 (SH3BP5), which has been known to be a JNK interactor, in neuronal cells. Similar to Humanin treatment, overexpression of SH3BP5 inhibited AD-related neuronal death, while siRNA-mediated knockdown of endogenous SH3BP5 expression attenuated the neuroprotective effect of Humanin. These results indicate that SH3BP5 is a downstream effector of Humanin. Furthermore, biochemical analysis has revealed that SH3BP5 binds to JNK and directly inhibits JNK through its two putative mitogen-activated protein kinase interaction motifs (KIMs). PMID:23861391

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector, phyllogen, induces phyllody

PubMed Central

Maejima, Kensaku; Iwai, Ryo; Himeno, Misako; Komatsu, Ken; Kitazawa, Yugo; Fujita, Naoko; Ishikawa, Kazuya; Fukuoka, Misato; Minato, Nami; Yamaji, Yasuyuki; Oshima, Kenro; Namba, Shigetou

2014-01-01

Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant-pathogenic bacteria that transform plant floral organs into leaf-like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma-secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma-infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two-hybrid and in planta transient co-expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin–proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody-inducing gene family of ‘phyllogens’. PMID:24597566

T cell Bim levels reflect responses to anti–PD-1 cancer therapy

PubMed Central

Dronca, Roxana S.; Liu, Xin; Harrington, Susan M.; Chen, Lingling; Cao, Siyu; Kottschade, Lisa A.; McWilliams, Robert R.; Block, Matthew S.; Nevala, Wendy K.; Thompson, Michael A.; Mansfield, Aaron S.; Park, Sean S.; Markovic, Svetomir N.

2016-01-01

Immune checkpoint therapy with PD-1 blockade has emerged as an effective therapy for many advanced cancers; however, only a small fraction of patients achieve durable responses. To date, there is no validated blood-based means of predicting the response to PD-1 blockade. We report that Bim is a downstream signaling molecule of the PD-1 pathway, and its detection in T cells is significantly associated with expression of PD-1 and effector T cell markers. High levels of Bim in circulating tumor-reactive (PD-1+CD11ahiCD8+) T cells were prognostic of poor survival in patients with metastatic melanoma who did not receive anti–PD-1 therapy and were also predictive of clinical benefit in patients with metastatic melanoma who were treated with anti–PD-1 therapy. Moreover, this circulating tumor-reactive T cell population significantly decreased after successful anti–PD-1 therapy. Our study supports a crucial role of Bim in both T cell activation and apoptosis as regulated by PD-1 and PD-L1 interactions in effector CD8+ T cells. Measurement of Bim levels in circulating T cells of patients with cancer may provide a less invasive strategy to predict and monitor responses to anti–PD-1 therapy, although future prospective analyses are needed to validate its utility. PMID:27182556

Nuclear accumulation of the Arabidopsis immune receptor RPS4 is necessary for triggering EDS1-dependent defense.

PubMed

Wirthmueller, Lennart; Zhang, Yan; Jones, Jonathan D G; Parker, Jane E

2007-12-04

Recognition of specific pathogen molecules inside the cell by nucleotide-binding domain and leucine-rich repeat (NB-LRR) receptors constitutes an important layer of innate immunity in plants. Receptor activation triggers host cellular reprogramming involving transcriptional potentiation of basal defenses and localized programmed cell death. The sites and modes of action of NB-LRR receptors are, however, poorly understood. Arabidopsis Toll/Interleukin-1 (TIR) type NB-LRR receptor RPS4 recognizes the bacterial type III effector AvrRps4. We show that epitope-tagged RPS4 expressed under its native regulatory sequences distributes between endomembranes and nuclei in healthy and AvrRps4-triggered tissues. RPS4 accumulation in the nucleus, mediated by a bipartite nuclear localization sequence (NLS) at its C terminus, is necessary for triggering immunity through authentic activation by AvrRps4 in Arabidopsis or as an effector-independent "deregulated" receptor in tobacco. A strikingly conserved feature of TIR-NB-LRR receptors is their recruitment of the nucleocytoplasmic basal-defense regulator EDS1 in resistance to diverse pathogens. We find that EDS1 is an indispensable component of RPS4 signaling and that it functions downstream of RPS4 activation but upstream of RPS4-mediated transcriptional reprogramming in the nucleus.

Structural basis for viral 5'-PPP-RNA recognition by human IFIT proteins.

PubMed

Abbas, Yazan M; Pichlmair, Andreas; Górna, Maria W; Superti-Furga, Giulio; Nagar, Bhushan

2013-02-07

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defence through disruption of protein-protein interactions in the host translation-initiation machinery. However, it was recently discovered that IFITs can directly recognize viral RNA bearing a 5'-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an amino-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single-stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double-stranded viral PPP-RNA, retinoic acid-inducible gene I (RIG-I, also known as DDX58). Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence-specific manner and requires a 5'-overhang of approximately three nucleotides. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 was found to cause a defect in the antiviral response by human embryonic kidney cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA, and lend insight into their downstream effector function.

Epac2 Mediates cAMP-Dependent Potentiation of Neurotransmission in the Hippocampus.

PubMed

Fernandes, Herman B; Riordan, Sean; Nomura, Toshihiro; Remmers, Christine L; Kraniotis, Stephen; Marshall, John J; Kukreja, Lokesh; Vassar, Robert; Contractor, Anis

2015-04-22

Presynaptic terminal cAMP elevation plays a central role in plasticity at the mossy fiber-CA3 synapse of the hippocampus. Prior studies have identified protein kinase A as a downstream effector of cAMP that contributes to mossy fiber LTP (MF-LTP), but the potential contribution of Epac2, another cAMP effector expressed in the MF synapse, has not been considered. We investigated the role of Epac2 in MF-CA3 neurotransmission using Epac2(-/-) mice. The deletion of Epac2 did not cause gross alterations in hippocampal neuroanatomy or basal synaptic transmission. Synaptic facilitation during short trains was not affected by loss of Epac2 activity; however, both long-term plasticity and forskolin-mediated potentiation of MFs were impaired, demonstrating that Epac2 contributes to cAMP-dependent potentiation of transmitter release. Examination of synaptic transmission during long sustained trains of activity suggested that the readily releasable pool of vesicles is reduced in Epac2(-/-) mice. These data suggest that cAMP elevation uses an Epac2-dependent pathway to promote transmitter release, and that Epac2 is required to maintain the readily releasable pool at MF synapses in the hippocampus. Copyright © 2015 the authors 0270-6474/15/356544-10$15.00/0.

Citron kinase controls abscission through RhoA and anillin

PubMed Central

Gai, Marta; Camera, Paola; Dema, Alessandro; Bianchi, Federico; Berto, Gaia; Scarpa, Elena; Germena, Giulia; Di Cunto, Ferdinando

2011-01-01

The small GTPase RhoA plays a crucial role in the different stages of cytokinesis, including contractile ring formation, cleavage furrow ingression, and midbody abscission. Citron kinase (CIT-K), a protein required for cytokinesis and conserved from insects to mammals, is currently considered a cytokinesis-specific effector of active RhoA. In agreement with previous observations, we show here that, as in Drosophila cells, CIT-K is specifically required for abscission in mammalian cells. However, in contrast with the current view, we provide evidence that CIT-K is an upstream regulator rather than a downstream effector of RhoA during late cytokinesis. In addition, we show that CIT-K is capable of physically and functionally interacting with the actin-binding protein anillin. Active RhoA and anillin are displaced from the midbody in CIT-K-depleted cells, while only anillin, but not CIT-K, is affected if RhoA is inactivated in late cytokinesis. The overexpression of CIT-K and of anillin leads to abscission delay. However, the delay produced by CIT-K overexpression can be reversed by RhoA inactivation, while the delay produced by anillin overexpression is RhoA-independent. Altogether, these results indicate that CIT-K is a crucial abscission regulator that may promote midbody stability through active RhoA and anillin. PMID:21849473

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P.

PubMed

Zhang, Xiaoxiao; Farah, Nadya; Rolston, Laura; Ericsson, Daniel J; Catanzariti, Ann-Maree; Bernoux, Maud; Ve, Thomas; Bendak, Katerina; Chen, Chunhong; Mackay, Joel P; Lawrence, Gregory J; Hardham, Adrienne; Ellis, Jeffrey G; Williams, Simon J; Dodds, Peter N; Jones, David A; Kobe, Bostjan

2018-05-01

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Nicotine/cigarette smoke promotes metastasis of pancreatic cancer through α7nAChR-mediated MUC4 upregulation.

PubMed

Momi, N; Ponnusamy, M P; Kaur, S; Rachagani, S; Kunigal, S S; Chellappan, S; Ouellette, M M; Batra, S K

2013-03-14

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared with the normal pancreas, and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette smoke extract and nicotine, which is the major component of CS, significantly upregulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via the α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. In addition, we demonstrated that nicotine-mediated MUC4 upregulation promotes the PC cell migration through the activation of the downstream effectors, such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in vivo studies showed a marked increase in the mean pancreatic tumor weight (low dose (100 mg/m(3) total suspended particulate (TSP)), P=0.014; high dose (247 mg/m(3) TSP), P=0.02) and significant tumor metastasis to various distant organs in the CS-exposed mice, orthotopically implanted with luciferase-transfected PC cells, as compared with the sham controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine (low dose, 155.88±35.96 ng/ml; high dose, 216.25±29.95 ng/ml) and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings revealed for the first time that CS upregulates the MUC4 mucin in PC via the α7nAChR/JAK2/STAT3 downstream signaling cascade, thereby promoting metastasis of PC.

Nicotine/Cigarette-smoke Promotes Metastasis of Pancreatic Cancer Through α7nAChR-mediated MUC4 Up-regulation

PubMed Central

Momi, Navneet; Ponnusamy, Moorthy P.; Kaur, Sukhwinder; Rachagani, Satyanarayana; Kunigal, Sateesh S; Chellappan, Srikumar; Ouellette, Michel M; Batra, Surinder K

2012-01-01

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well-established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared to the normal pancreas and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette-smoke-extract (CSE) and nicotine, which is the major component of CS, significantly up-regulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. Additionally, we demonstrated that nicotine-mediated MUC4 up-regulation promotes the PC cell migration through the activation of the downstream effectors such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in-vivo studies demonstrated a dramatic increase in the mean pancreatic tumor weight [low-dose (100 mg/m3 TSP), p=0.014; high-dose (247 mg/m3 TSP), p=0.02] and significant tumor metastasis to various distant organs in the CS-exposed-mice, orthotopically implanted with luciferase-transfected PC cells, as compared to the sham-controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine [low-dose, 155.88±35.96 ng/ml; high-dose, 216.25±29.95 ng/ml] and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings revealed for the first time that CS up-regulates the MUC4 mucin in PC via α7nAChR/JAK2/STAT3 downstream signaling cascade, thereby promoting metastasis of pancreatic cancer. PMID:22614008

Zebrafish hhex, nk2.1a, and pax2.1 regulate thyroid growth and differentiation downstream of Nodal-dependent transcription factors.

PubMed

Elsalini, Osama A; von Gartzen, Julia; Cramer, Matthias; Rohr, Klaus B

2003-11-01

During zebrafish development, the thyroid primordium initiates expression of molecular markers such as hhex and nk2.1a in the endoderm prior to pharynx formation. As expected for an endodermally derived organ, initiation of thyroid development depends on Nodal signalling. We find that it also depends on three downstream effectors of Nodal activity, casanova (cas), bonnie and clyde (bon), and faust (fau)/gata5. Despite their early Nodal-dependent expression in the endoderm, both hhex and nk2.1a are only required relatively late during thyroid development. In hhex and nk2.1a loss-of-function phenotypes, thyroid development is initiated and arrests only after the primordium has evaginated from the pharyngeal epithelium. Thus, like pax2.1, both hhex and nk2.1a have similarly late roles in differentiation or growth of thyroid follicular cells, and here, we show that all three genes act in parallel rather than in a single pathway. Our functional analysis suggests that these genes have similar roles as in mammalian thyroid development, albeit in a different temporal mode of organogenesis.

Transmembrane adaptor protein PAG1 is a novel tumor suppressor in neuroblastoma

PubMed Central

Agarwal, Saurabh; Ghosh, Rajib; Chen, Zaowen; Lakoma, Anna; Gunaratne, Preethi H.; Kim, Eugene S.; Shohet, Jason M.

2016-01-01

(NB) is the most common extracranial pediatric solid tumor with high mortality rates. The tyrosine kinase c-Src has been known to play an important role in differentiation of NB cells, but the mechanism of c-Src regulation has not been defined. Here, we characterize PAG1 (Cbp, Csk binding protein), a central inhibitor of c-Src and other Src family kinases, as a novel tumor suppressor in NB. Clinical cohort analysis demonstrate that low expression of PAG1 is a significant prognostic factor for high stage disease, increased relapse, and worse overall survival for children with NB. PAG1 knockdown in NB cells promotes proliferation and anchorage-independent colony formation with increased activation of AKT and ERK downstream of c-Src, while PAG1 overexpression significantly rescues these effects. In vivo, PAG1 overexpression significantly inhibits NB tumorigenicity in an orthotopic xenograft model. Our results establish PAG1 as a potent tumor suppressor in NB by inhibiting c-Src and downstream effector pathways. Thus, reactivation of PAG1 and inhibition of c-Src kinase activity represents an important novel therapeutic approach for high-risk NB. PMID:26993602

Gab1 Mediates Hepatocyte Growth Factor-Stimulated Mitogenicity and Morphogenesis in Multipotent Myeloid Cells

PubMed Central

Felici, Angelina; Giubellino, Alessio; Bottaro, Donald P.

2012-01-01

Hepatocyte growth factor (HGF)-stimulated mitogenesis, motogenesis and morphogenesis in various cell types begins with activation of the Met receptor tyrosine kinase and the recruitment of intracellular adaptors and kinase substrates. The adapter protein Gab1 is a critical effector and substrate of activated Met, mediating morphogenesis, among other activities, in epithelial cells. To define its role downstream of Met in hematopoietic cells, Gab1 was expressed in the HGF-responsive, Gab1-negative murine myeloid cell line 32D. Interestingly, the adhesion and motility of Gab1-expressing cells were significantly greater than parental cells, independent of growth factor treatment. Downstream of activated Met, Gab1 expression was specifically associated with rapid Shp-2 recruitment and activation, increased mitogenic potency, suppression of GATA-1 expression and concomitant upregulation of GATA-2 transcription. In addition to enhanced proliferation, continuous culture of Gab1-expressing 32D cells in HGF resulted in cell attachment, filopodia extension and phenotypic changes suggestive of monocytic differentiation. Our results suggest that in myeloid cells, Gab1 is likely to enhance HGF mitogenicity by coupling Met to Shp-2 and GATA-2 expression, thereby potentially contributing to normal myeloid differentiation as well as oncogenic transformation. PMID:20506405

Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila.

PubMed

Tsarouhas, Vasilios; Yao, Liqun; Samakovlis, Christos

2014-04-15

Metazoans have evolved efficient mechanisms for epidermal repair and survival following injury. Several cellular responses and key signaling molecules that are involved in wound healing have been identified in Drosophila, but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair are poorly understood. The Ret-like receptor tyrosine kinase (RTK) Stitcher (Stit, also known as Cad96Ca) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce restoration of the extracellular barrier. Here, we describe the immediate downstream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src family tyrosine kinases bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src family kinases (SFKs) control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier-repair genes. Our analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode for Src kinases and their central functions during epidermal wound healing in vivo.

Reduction in ins-7 gene expression in non-neuronal cells of high glucose exposed Caenorhabditis elegans protects from reactive metabolites, preserves neuronal structure and head motility, and prolongs lifespan.

PubMed

Mendler, Michael; Riedinger, Christin; Schlotterer, Andrea; Volk, Nadine; Fleming, Thomas; Herzig, Stephan; Nawroth, Peter P; Morcos, Michael

2017-02-01

Glucose derived metabolism generates reactive metabolites affecting the neuronal system and lifespan in C. elegans. Here, the role of the insulin homologue ins-7 and its downstream effectors in the generation of high glucose induced neuronal damage and shortening of lifespan was studied. In C. elegans high glucose conditions induced the expression of the insulin homologue ins-7. Abrogating ins-7 under high glucose conditions in non-neuronal cells decreased reactive oxygen species (ROS)-formation and accumulation of methylglyoxal derived advanced glycation endproducts (AGEs), prevented structural neuronal damage and normalised head motility and lifespan. The restoration of lifespan by decreased ins-7 expression was dependent on the concerted action of sod-3 and glod-4 coding for the homologues of iron-manganese superoxide dismutase and glyoxalase 1, respectively. Under high glucose conditions mitochondria-mediated oxidative stress and glycation are downstream targets of ins-7. This impairs the neuronal system and longevity via a non-neuronal/neuronal crosstalk by affecting sod-3 and glod-4, thus giving further insight into the pathophysiology of diabetic complications. Copyright © 2017 Elsevier Inc. All rights reserved.

Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

PubMed Central

Satpathy, Shankha; Wagner, Sebastian A; Beli, Petra; Gupta, Rajat; Kristiansen, Trine A; Malinova, Dessislava; Francavilla, Chiara; Tolar, Pavel; Bishop, Gail A; Hostager, Bruce S; Choudhary, Chunaram

2015-01-01

B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association with effector proteins and with endo-lysosomal compartments. In addition, we show that BCL10 is modified by LUBAC-mediated linear ubiquitylation, and demonstrate an important function of LUBAC in BCR-induced NF-κB signaling. Our results offer a global and integrated view of BCR signaling, and the provided datasets can serve as a valuable resource for further understanding BCR signaling networks. PMID:26038114

cAMP signaling in skeletal muscle adaptation: hypertrophy, metabolism, and regeneration

PubMed Central

Stewart, Randi

2012-01-01

Among organ systems, skeletal muscle is perhaps the most structurally specialized. The remarkable subcellular architecture of this tissue allows it to empower movement with instructions from motor neurons. Despite this high degree of specialization, skeletal muscle also has intrinsic signaling mechanisms that allow adaptation to long-term changes in demand and regeneration after acute damage. The second messenger adenosine 3′,5′-monophosphate (cAMP) not only elicits acute changes within myofibers during exercise but also contributes to myofiber size and metabolic phenotype in the long term. Strikingly, sustained activation of cAMP signaling leads to pronounced hypertrophic responses in skeletal myofibers through largely elusive molecular mechanisms. These pathways can promote hypertrophy and combat atrophy in animal models of disorders including muscular dystrophy, age-related atrophy, denervation injury, disuse atrophy, cancer cachexia, and sepsis. cAMP also participates in muscle development and regeneration mediated by muscle precursor cells; thus, downstream signaling pathways may potentially be harnessed to promote muscle regeneration in patients with acute damage or muscular dystrophy. In this review, we summarize studies implicating cAMP signaling in skeletal muscle adaptation. We also highlight ligands that induce cAMP signaling and downstream effectors that are promising pharmacological targets. PMID:22354781

Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

PubMed Central

Lee-Sherick, Alisa B.; Zhang, Weihe; Menachof, Kelly K.; Hill, Amanda A.; Rinella, Sean; Kirkpatrick, Gregory; Page, Lauren S.; Stashko, Michael A.; Jordan, Craig T.; Wei, Qi; Liu, Jing; Zhang, Dehui; DeRyckere, Deborah; Wang, Xiaodong; Frye, Stephen; Earp, H. Shelton; Graham, Douglas K.

2015-01-01

Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML. PMID:25762638

Fgfr1 regulates development through the combinatorial use of signaling proteins.

PubMed

Brewer, J Richard; Molotkov, Andrei; Mazot, Pierre; Hoch, Renée V; Soriano, Philippe

2015-09-01

Fibroblast growth factor (Fgf) signaling governs multiple processes important in development and disease. Many lines of evidence have implicated Erk1/2 signaling induced through Frs2 as the predominant effector pathway downstream from Fgf receptors (Fgfrs), but these receptors can also signal through other mechanisms. To explore the functional significance of the full range of signaling downstream from Fgfrs in mice, we engineered an allelic series of knock-in point mutations designed to disrupt Fgfr1 signaling functions individually and in combination. Analysis of each mutant indicates that Frs2 binding to Fgfr1 has the most pleiotropic functions in development but also that the receptor uses multiple proteins additively in vivo. In addition to Frs2, Crk proteins and Plcγ also contribute to Erk1/2 activation, affecting axis elongation and craniofacial and limb development and providing a biochemical mechanism for additive signaling requirements. Disruption of all known signaling functions diminished Erk1/2 and Plcγ activation but did not recapitulate the peri-implantation Fgfr1-null phenotype. This suggests that Erk1/2-independent signaling pathways are functionally important for Fgf signaling in vivo. © 2015 Brewer et al.; Published by Cold Spring Harbor Laboratory Press.

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome.

PubMed

Kuo, Caroline Y; Long, Joseph D; Campo-Fernandez, Beatriz; de Oliveira, Satiro; Cooper, Aaron R; Romero, Zulema; Hoban, Megan D; Joglekar, Alok V; Lill, Georgia R; Kaufman, Michael L; Fitz-Gibbon, Sorel; Wang, Xiaoyan; Hollis, Roger P; Kohn, Donald B

2018-05-29

X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Curcumin and Emodin Down-Regulate TGF-β Signaling Pathway in Human Cervical Cancer Cells

PubMed Central

Thacker, Pooja Chandrakant; Karunagaran, Devarajan

2015-01-01

Cervical cancer is the major cause of cancer related deaths in women, especially in developing countries and Human Papilloma Virus infection in conjunction with multiple deregulated signaling pathways leads to cervical carcinogenesis. TGF-β signaling in later stages of cancer is known to induce epithelial to mesenchymal transition promoting tumor growth. Phytochemicals, curcumin and emodin, are effective as chemopreventive and chemotherapeutic compounds against several cancers including cervical cancer. The main objective of this work was to study the effect of curcumin and emodin on TGF-β signaling pathway and its functional relevance to growth, migration and invasion in two cervical cancer cell lines, SiHa and HeLa. Since TGF-β and Wnt/β-catenin signaling pathways are known to cross talk having common downstream targets, we analyzed the effect of TGF-β on β-catenin (an important player in Wnt/β-catenin signaling) and also studied whether curcumin and emodin modulate them. We observed that curcumin and emodin effectively down regulate TGF-β signaling pathway by decreasing the expression of TGF-β Receptor II, P-Smad3 and Smad4, and also counterbalance the tumorigenic effects of TGF-β by inhibiting the TGF-β-induced migration and invasion. Expression of downstream effectors of TGF-β signaling pathway, cyclinD1, p21 and Pin1, was inhibited along with the down regulation of key mesenchymal markers (Snail and Slug) upon curcumin and emodin treatment. Curcumin and emodin were also found to synergistically inhibit cell population and migration in SiHa and HeLa cells. Moreover, we found that TGF-β activates Wnt/β-catenin signaling pathway in HeLa cells, and curcumin and emodin down regulate the pathway by inhibiting β-catenin. Taken together our data provide a mechanistic basis for the use of curcumin and emodin in the treatment of cervical cancer. PMID:25786122

MicroRNA cluster miR-17-92 Cluster in Exosomes Enhance Neuroplasticity and Functional Recovery After Stroke in Rats.

PubMed

Xin, Hongqi; Katakowski, Mark; Wang, Fengjie; Qian, Jian-Yong; Liu, Xian Shuang; Ali, Meser M; Buller, Benjamin; Zhang, Zheng Gang; Chopp, Michael

2017-03-01

Multipotent mesenchymal stromal cell (MSC) harvested exosomes are hypothesized as the major paracrine effectors of MSCs. In vitro, the miR-17-92 cluster promotes oligodendrogenesis, neurogenesis, and axonal outgrowth. We, therefore, investigated whether the miR-17-92 cluster-enriched exosomes harvested from MSCs transfected with an miR-17-92 cluster plasmid enhance neurological recovery compared with control MSC-derived exosomes. Rats subjected to 2 hours of transient middle cerebral artery occlusion were intravenously administered miR-17-92 cluster-enriched exosomes, control MSC exosomes, or liposomes and were euthanized 28 days post-middle cerebral artery occlusion. Histochemistry, immunohistochemistry, and Golgi-Cox staining were used to assess dendritic, axonal, synaptic, and myelin remodeling. Expression of phosphatase and tensin homolog and activation of its downstream proteins, protein kinase B, mechanistic target of rapamycin, and glycogen synthase kinase 3β in the peri-infarct region were measured by means of Western blots. Compared with the liposome treatment, both exosome treatment groups exhibited significant improvement of functional recovery, but miR-17-92 cluster-enriched exosome treatment had significantly more robust effects on improvement of neurological function and enhancements of oligodendrogenesis, neurogenesis, and neurite remodeling/neuronal dendrite plasticity in the ischemic boundary zone (IBZ) than the control MSC exosome treatment. Moreover, miR-17-92 cluster-enriched exosome treatment substantially inhibited phosphatase and tensin homolog, a validated miR-17-92 cluster target gene, and subsequently increased the phosphorylation of phosphatase and tensin homolog downstream proteins, protein kinase B, mechanistic target of rapamycin, and glycogen synthase kinase 3β compared with control MSC exosome treatment. Our data suggest that treatment of stroke with tailored exosomes enriched with the miR-17-92 cluster increases neural plasticity and functional recovery after stroke, possibly via targeting phosphatase and tensin homolog to activate the PI3K/protein kinase B/mechanistic target of rapamycin/glycogen synthase kinase 3β signaling pathway. © 2017 American Heart Association, Inc.

Curcumin and emodin down-regulate TGF-β signaling pathway in human cervical cancer cells.

PubMed

Thacker, Pooja Chandrakant; Karunagaran, Devarajan

2015-01-01

Cervical cancer is the major cause of cancer related deaths in women, especially in developing countries and Human Papilloma Virus infection in conjunction with multiple deregulated signaling pathways leads to cervical carcinogenesis. TGF-β signaling in later stages of cancer is known to induce epithelial to mesenchymal transition promoting tumor growth. Phytochemicals, curcumin and emodin, are effective as chemopreventive and chemotherapeutic compounds against several cancers including cervical cancer. The main objective of this work was to study the effect of curcumin and emodin on TGF-β signaling pathway and its functional relevance to growth, migration and invasion in two cervical cancer cell lines, SiHa and HeLa. Since TGF-β and Wnt/β-catenin signaling pathways are known to cross talk having common downstream targets, we analyzed the effect of TGF-β on β-catenin (an important player in Wnt/β-catenin signaling) and also studied whether curcumin and emodin modulate them. We observed that curcumin and emodin effectively down regulate TGF-β signaling pathway by decreasing the expression of TGF-β Receptor II, P-Smad3 and Smad4, and also counterbalance the tumorigenic effects of TGF-β by inhibiting the TGF-β-induced migration and invasion. Expression of downstream effectors of TGF-β signaling pathway, cyclinD1, p21 and Pin1, was inhibited along with the down regulation of key mesenchymal markers (Snail and Slug) upon curcumin and emodin treatment. Curcumin and emodin were also found to synergistically inhibit cell population and migration in SiHa and HeLa cells. Moreover, we found that TGF-β activates Wnt/β-catenin signaling pathway in HeLa cells, and curcumin and emodin down regulate the pathway by inhibiting β-catenin. Taken together our data provide a mechanistic basis for the use of curcumin and emodin in the treatment of cervical cancer.

A New Strategy to Control and Eradicate “Undruggable” Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology

PubMed Central

Lee, Michael P.; Lee, Caroline Dasom; Lafever, Alex C.; Svyatova, Elizaveta; Kanda, Kevin; Collier, Amber L.; Siewertsz van Reesema, Lauren L.; Tang-Tan, Angela M.; Zheleva, Vasilena; Bwayi, Monicah N.; Bian, Minglei; Schmidt, Rebecca L.; Petersen, Gloria M.

2018-01-01

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely “undruggable”. Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future. PMID:29757973

Sprouty is a cytoplasmic target of adenoviral E1A oncoproteins to regulate the receptor tyrosine kinase signalling pathway

PubMed Central

2011-01-01

Background Oncoproteins encoded by the early region of adenoviruses have been shown to be powerful tools to study gene regulatory mechanisms, which affect major cellular events such as proliferation, differentiation, apoptosis and oncogenic transformation. They are possesing a key role to favor viral replication via their interaction with multiple cellular proteins. In a yeast two-hybrid screen we have identified Sprouty1 (Spry1) as a target of adenoviral E1A Oncoproteins. Spry proteins are central and complex regulators of the receptor tyrosine kinase (RTK) signalling pathway. The deregulation of Spry family members is often associated with alterations of the RTK signalling and its downstream effectors, leading to the ERK pathway. Results Here, we confirm our yeast two-hybrid data, showing the interaction between Spry1 and E1A in GST pull-down and immunoprecipitation assays. We also demonstrated the interaction of E1A with two further Spry isoforms. Using deletion mutants we identified the N-terminus and the CR conserved region (CR) 3 of E1A- and the C-terminal half of Spry1, which contains the highly conserved Spry domain, as the essential sites for direct interaction between Spry and E1A. Immunofluorescent microscopy data revealed a co-localization of E1A13S with Spry1 in the cytoplasm. SRE and TRE reporter assays demonstrated that co-expression of Spry1 with E1A13S abolishes the inhibitory function of Spry1 in RTK signalling, which is consequently accompanied with a decrease of E1A13S-induced gene expression. Conclusions These results establish Spry1 as a cytoplasmic localized cellular target for E1A oncoproteins to regulate the RTK signalling pathway, and consequently cellular events downstream of RTK that are essential for viral replication and transformation. PMID:21518456

The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mak, Amanda Nga-Sze; Bradley, Philip; Cernadas, Raul A.

2012-02-10

DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand.more » Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector-DNA recognition.« less

Mobile and stationary laser weapon demonstrators of Rheinmetall Waffe Munition

NASA Astrophysics Data System (ADS)

Ludewigt, K.; Riesbeck, Th.; Baumgärtel, Th.; Schmitz, J.; Graf, A.; Jung, M.

2014-10-01

For some years Rheinmetall Waffe Munition has successfully developed, realised and tested a variety of versatile high energy laser (HEL) weapon systems for air- and ground-defence scenarios like C-RAM, UXO clearing. By employing beam superimposition technology and a modular laser weapon concept, the total optical power has been successively increased. Stationary weapon platforms and now military mobile vehicles were equipped with high energy laser effectors. Our contribution summarises the most recent development stages of Rheinmetalls high energy laser weapon program. We present three different vehicle based HEL demonstrators: the 5 kW class Mobile HEL Effector Track V integrated in an M113 tank, the 20 kW class Mobile HEL Effector Wheel XX integrated in a multirole armoured vehicle GTK Boxer 8x8 and the 50 kW class Mobile HEL Effector Container L integrated in a reinforced container carried by an 8x8 truck. As a highlight, a stationary 30 kW Laser Weapon Demonstrator shows the capability to defeat saturated attacks of RAM targets and unmanned aerial vehicles. 2013 all HEL demonstrators were tested in a firing campaign at the Rheinmetall testing centre in Switzerland. Major results of these tests are presented.

Divergent and convergent modes of interaction between wheat and Puccinia graminis f. sp. tritici isolates revealed by the comparative gene co-expression network and genome analyses.

PubMed

Rutter, William B; Salcedo, Andres; Akhunova, Alina; He, Fei; Wang, Shichen; Liang, Hanquan; Bowden, Robert L; Akhunov, Eduard

2017-04-12

Two opposing evolutionary constraints exert pressure on plant pathogens: one to diversify virulence factors in order to evade plant defenses, and the other to retain virulence factors critical for maintaining a compatible interaction with the plant host. To better understand how the diversified arsenals of fungal genes promote interaction with the same compatible wheat line, we performed a comparative genomic analysis of two North American isolates of Puccinia graminis f. sp. tritici (Pgt). The patterns of inter-isolate divergence in the secreted candidate effector genes were compared with the levels of conservation and divergence of plant-pathogen gene co-expression networks (GCN) developed for each isolate. Comprative genomic analyses revealed substantial level of interisolate divergence in effector gene complement and sequence divergence. Gene Ontology (GO) analyses of the conserved and unique parts of the isolate-specific GCNs identified a number of conserved host pathways targeted by both isolates. Interestingly, the degree of inter-isolate sub-network conservation varied widely for the different host pathways and was positively associated with the proportion of conserved effector candidates associated with each sub-network. While different Pgt isolates tended to exploit similar wheat pathways for infection, the mode of plant-pathogen interaction varied for different pathways with some pathways being associated with the conserved set of effectors and others being linked with the diverged or isolate-specific effectors. Our data suggest that at the intra-species level pathogen populations likely maintain divergent sets of effectors capable of targeting the same plant host pathways. This functional redundancy may play an important role in the dynamic of the "arms-race" between host and pathogen serving as the basis for diverse virulence strategies and creating conditions where mutations in certain effector groups will not have a major effect on the pathogen's ability to infect the host.

Transposons to toxins: the provenance, architecture and diversification of a widespread class of eukaryotic effectors

PubMed Central

Zhang, Dapeng; Burroughs, A. Maxwell; Vidal, Newton D.; Iyer, Lakshminarayan M.; Aravind, L.

2016-01-01

Enzymatic effectors targeting nucleic acids, proteins and other cellular components are the mainstay of conflicts across life forms. Using comparative genomics we identify a large class of eukaryotic proteins, which include effectors from oomycetes, fungi and other parasites. The majority of these proteins have a characteristic domain architecture with one of several N-terminal ‘Header’ domains, which are predicted to play a role in trafficking of these effectors, including a novel version of the Ubiquitin fold. The Headers are followed by one or more diverse C-terminal domains, such as restriction endonuclease (REase), protein kinase, HNH endonuclease, LK-nuclease (a RNase) and multiple distinct peptidase domains, which are predicted to carry their toxicity determinants. The most common types of these proteins appear to have originated from prokaryotic transposases (e.g. TN7 and Mu) and combine a CDC6/ORC1-STAND clade NTPase domain with a C-terminal REase domain. Other than the so-called Crinkler effectors of oomycetes and fungi, these effectors are encoded by other eukaryotic parasites such as trypanosomatids (the RHS proteins) and the rhizarian Plasmodiophora, and symbionts like Capsaspora. Remarkably, we also find these proteins in free-living eukaryotes, including several viridiplantae, fungi, amoebozoans and animals. These versions might either still be transposons or function in other poorly understood eukaryote-specific inter-organismal and inter-genomic conflicts. These include the Medea1 selfish element of Tribolium that spreads via post-zygotic killing. We present a unified mechanism for the recombination-dependent diversification and action of this widespread class of molecular weaponry deployed across diverse conflicts ranging from parasitic to free-living forms. PMID:27060143

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

PubMed Central

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8–13% and reliably measures NK cell- or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. PMID:17617419

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

PubMed

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

CD134/CD137 Dual Costimulation-Elicited IFN-γ Maximizes Effector T Cell Function but Limits Treg Expansion

PubMed Central

Rose, Marie-Clare St.; Taylor, Roslyn A.; Bandyopadhyay, Suman; Qui, Harry Z.; Hagymasi, Adam T.; Vella, Anthony T.; Adler, Adam J.

2012-01-01

T cell tolerance to tumor antigens represents a major hurdle in generating tumor immunity. Combined administration of agonistic monoclonal antibodies to the costimulatory receptors CD134 plus CD137 can program T cells responding to tolerogenic antigen to undergo expansion and effector T cell differentiation, and also elicits tumor immunity. Nevertheless, CD134 and CD137 agonists can also engage inhibitory immune components. To understand how immune stimulatory versus inhibitory components are regulated during CD134 plus CD137 dual costimulation, the current study utilized a model where dual costimulation programs T cells encountering a highly tolerogenic self-antigen to undergo effector differentiation. IFN-γ was found to play a pivotal role in maximizing the function of effector T cells while simultaneously limiting the expansion of CD4+CD25+Foxp3+ Tregs. In antigen-responding effector T cells, IFN-γ operates via a direct cell-intrinsic mechanism to cooperate with IL-2 to program maximal expression of granzyme B. Simultaneously, IFN-γ limits expression of the IL-2 receptor alpha chain (CD25) and IL-2 signaling through a mechanism that does not involve T-bet-mediated repression of IL-2. IFN-γ also limited CD25 and Foxp3 expression on bystanding CD4+Foxp3+ Tregs, and limited the potential of these Tregs to expand. These effects could not be explained by the ability of IFN-γ to limit IL-2 availability. Taken together, during dual costimulation IFN-γ interacts with IL-2 through distinct mechanisms to program maximal expression of effector molecules in antigen-responding T cells while simultaneously limiting Treg expansion. PMID:23295363

Vegetative compatibility groups partition variation in the virulence of Verticillium dahliae on strawberry

PubMed Central

Fan, Rong; Cockerton, Helen M.; Armitage, Andrew D.; Bates, Helen; Cascant-Lopez, Emma; Antanaviciute, Laima; Xu, Xiangming; Hu, Xiaoping

2018-01-01

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector. PMID:29451893

Vegetative compatibility groups partition variation in the virulence of Verticillium dahliae on strawberry.

PubMed

Fan, Rong; Cockerton, Helen M; Armitage, Andrew D; Bates, Helen; Cascant-Lopez, Emma; Antanaviciute, Laima; Xu, Xiangming; Hu, Xiaoping; Harrison, Richard J

2018-01-01

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector.

Computational Model of the Insect Pheromone Transduction Cascade

PubMed Central

Gu, Yuqiao; Lucas, Philippe; Rospars, Jean-Pierre

2009-01-01

A biophysical model of receptor potential generation in the male moth olfactory receptor neuron is presented. It takes into account all pre-effector processes—the translocation of pheromone molecules from air to sensillum lymph, their deactivation and interaction with the receptors, and the G-protein and effector enzyme activation—and focuses on the main post-effector processes. These processes involve the production and degradation of second messengers (IP3 and DAG), the opening and closing of a series of ionic channels (IP3-gated Ca2+ channel, DAG-gated cationic channel, Ca2+-gated Cl− channel, and Ca2+- and voltage-gated K+ channel), and Ca2+ extrusion mechanisms. The whole network is regulated by modulators (protein kinase C and Ca2+-calmodulin) that exert feedback inhibition on the effector and channels. The evolution in time of these linked chemical species and currents and the resulting membrane potentials in response to single pulse stimulation of various intensities were simulated. The unknown parameter values were fitted by comparison to the amplitude and temporal characteristics (rising and falling times) of the experimentally measured receptor potential at various pheromone doses. The model obtained captures the main features of the dose–response curves: the wide dynamic range of six decades with the same amplitudes as the experimental data, the short rising time, and the long falling time. It also reproduces the second messenger kinetics. It suggests that the two main types of depolarizing ionic channels play different roles at low and high pheromone concentrations; the DAG-gated cationic channel plays the major role for depolarization at low concentrations, and the Ca2+-gated Cl− channel plays the major role for depolarization at middle and high concentrations. Several testable predictions are proposed, and future developments are discussed. PMID:19300479

Surface Water Response Modeling

EPA Science Inventory

During response to spills, or for facility planning, the vulnerability of downstream water resources is a major concern. How long and at what concentration do spilled contaminants reach downstream receptors? Models have the potential to answer these questions, but only if they ...

Host response to Candida albicans bloodstream infection and sepsis

PubMed Central

Duggan, Seána; Leonhardt, Ines; Hünniger, Kerstin; Kurzai, Oliver

2015-01-01

Candida albicans is a major cause of bloodstream infection which may present as sepsis and septic shock - major causes of morbidity and mortality world-wide. After invasion of the pathogen, innate mechanisms govern the early response. Here, we outline the models used to study these mechanisms and summarize our current understanding of innate immune responses during Candida bloodstream infection. This includes protective immunity as well as harmful responses resulting in Candida induced sepsis. Neutrophilic granulocytes are considered principal effector cells conferring protection and recognize C. albicans mainly via complement receptor 3. They possess a range of effector mechanisms, contributing to elimination of the pathogen. Neutrophil activation is closely linked to complement and modulated by activated mononuclear cells. A thorough understanding of these mechanisms will help in creating an individualized approach to patients suffering from systemic candidiasis and aid in optimizing clinical management. PMID:25785541

CD200 is induced by ERK and is a potential therapeutic target in melanoma

PubMed Central

Petermann, Kimberly B.; Rozenberg, Gabriela I.; Zedek, Daniel; Groben, Pamela; McKinnon, Karen; Buehler, Christin; Kim, William Y.; Shields, Janiel M.; Penland, Shannon; Bear, James E.; Thomas, Nancy E.; Serody, Jonathan S.; Sharpless, Norman E.

2007-01-01

Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM. PMID:18008004

A Homologue of an Operon Required for DNA Transfer in Agrobacterium Is Required in Brucella abortus for Virulence and Intracellular Multiplication

PubMed Central

Sieira, Rodrigo; Comerci, Diego J.; Sánchez, Daniel O.; Ugalde, Rodolfo A.

2000-01-01

As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment. PMID:10940027

Podocyte-Specific GLUT4-Deficient Mice Have Fewer and Larger Podocytes and Are Protected From Diabetic Nephropathy

PubMed Central

Guzman, Johanna; Jauregui, Alexandra N.; Merscher-Gomez, Sandra; Maiguel, Dony; Muresan, Cristina; Mitrofanova, Alla; Diez-Sampedro, Ana; Szust, Joel; Yoo, Tae-Hyun; Villarreal, Rodrigo; Pedigo, Christopher; Molano, R. Damaris; Johnson, Kevin; Kahn, Barbara; Hartleben, Bjoern; Huber, Tobias B.; Saha, Jharna; Burke, George W.; Abel, E. Dale; Brosius, Frank C.; Fornoni, Alessia

2014-01-01

Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria. PMID:24101677

Tamoxifen reduces P-gp-mediated multidrug resistance via inhibiting the PI3K/Akt signaling pathway in ER-negative human gastric cancer cells.

PubMed

Mao, Zonglei; Zhou, Jin; Luan, Junwei; Sheng, Weihua; Shen, Xiaochun; Dong, Xiaoqiang

2014-03-01

Multidrug resistance (MDR), mediated by overexpression of drug efflux transporters such as P-glycoprotein (P-gp), is a major problem limiting successful chemotherapy of gastric cancer. Tamoxifen (TAM), a triphenylethylene nonsteroidal antiestrogen agent, shows broad-spectrum antitumor properties. Emerging studies demonstrated that TAM could significantly reduce the MDR in a variety of human cancers. Here we investigated the effects and possible underlying mechanisms of action of TAM on the reversion of MDR in ER-negative human gastric cancer cells. Our results demonstrated that in MDR phenotype SGC7901/CDDP gastric cancer cells TAM dramatically lowered the IC50 of CDDP, 5-FU and ADM, increased the intracellular Rhodamine123 accumulation and induced G0/G1 phase arrest, while G2/M phase decreased accordingly. Furthermore, at the molecular level, TAM substantially decreased the expression of P-gp, p-Akt and the Akt-regulated downstream effectors such as p-GSK-3β, p-BAD, Bcl-XL and cyclinD1 proteins without affecting the expression of t-Akt, t-GSK-3β, t-BAD proteins in SGC7901/CDDP cells. Thus, our findings demonstrate that TAM reverses P-gp-mediated gastric cancer cell MDR via inhibiting the PI3K/Akt signaling pathway. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

The non-receptor tyrosine kinase Lyn controls neutrophil adhesion by recruiting the CrkL–C3G complex and activating Rap1 at the leading edge

PubMed Central

He, Yuan; Kapoor, Ashish; Cook, Sara; Liu, Shubai; Xiang, Yang; Rao, Christopher V.; Kenis, Paul J. A.; Wang, Fei

2011-01-01

Establishing new adhesions at the extended leading edges of motile cells is essential for stable polarity and persistent motility. Despite recent identification of signaling pathways that mediate polarity and chemotaxis in neutrophils, little is known about molecular mechanisms governing cell–extracellular-matrix (ECM) adhesion in these highly polarized and rapidly migrating cells. Here, we describe a signaling pathway in neutrophils that is essential for localized integrin activation, leading edge attachment and persistent migration during chemotaxis. This pathway depends upon Gi-protein-mediated activation and leading edge recruitment of Lyn, a non-receptor tyrosine kinase belonging to the Src kinase family. We identified the small GTPase Rap1 as a major downstream effector of Lyn to regulate neutrophil adhesion during chemotaxis. Depletion of Lyn in neutrophil-like HL-60 cells prevented chemoattractant-induced Rap1 activation at the leading edge of the cell, whereas ectopic expression of Rap1 largely rescued the defects induced by Lyn depletion. Furthermore, Lyn controls spatial activation of Rap1 by recruiting the CrkL–C3G protein complex to the leading edge. Together, these results provide novel mechanistic insights into the poorly understood signaling network that controls leading edge adhesion during chemotaxis of neutrophils, and possibly other amoeboid cells. PMID:21628423

Calcium Nutrition and Extracellular Calcium Sensing: Relevance for the Pathogenesis of Osteoporosis, Cancer and Cardiovascular Diseases

PubMed Central

Peterlik, Meinrad; Kállay, Enikoe; Cross, Heide S.

2013-01-01

Through a systematic search in Pubmed for literature, on links between calcium malnutrition and risk of chronic diseases, we found the highest degree of evidence for osteoporosis, colorectal and breast cancer, as well as for hypertension, as the only major cardiovascular risk factor. Low calcium intake apparently has some impact also on cardiovascular events and disease outcome. Calcium malnutrition can causally be related to low activity of the extracellular calcium-sensing receptor (CaSR). This member of the family of 7-TM G-protein coupled receptors allows extracellular Ca2+ to function as a “first messenger” for various intracellular signaling cascades. Evidence demonstrates that Ca2+/CaSR signaling in functional linkage with vitamin D receptor (VDR)-activated pathways (i) promotes osteoblast differentiation and formation of mineralized bone; (ii) targets downstream effectors of the canonical and non-canonical Wnt pathway to inhibit proliferation and induce differentiation of colorectal cancer cells; (iii) evokes Ca2+ influx into breast cancer cells, thereby activating pro-apoptotic intracellular signaling. Furthermore, Ca2+/CaSR signaling opens Ca2+-sensitive K+ conductance channels in vascular endothelial cells, and also participates in IP3-dependent regulation of cytoplasmic Ca2+, the key intermediate of cardiomyocyte functions. Consequently, impairment of Ca2+/CaSR signaling may contribute to inadequate bone formation, tumor progression, hypertension, vascular calcification and, probably, cardiovascular disease. PMID:23340319

Development of transplantable human chordoma xenograft for preclinical assessment of novel therapeutic strategies

PubMed Central

Bozzi, Fabio; Manenti, Giacomo; Conca, Elena; Stacchiotti, Silvia; Messina, Antonella; Dagrada, GianPaolo; Gronchi, Alessandro; Panizza, Pietro; Pierotti, Marco A.; Tamborini, Elena; Pilotti, Silvana

2014-01-01

Background Chordomas are rare and indolent bone tumors that arise in the skull base and mobile spine. Distant metastases occur in >20% of cases, but morbidity and mortality are mainly related to local relapses that affect the majority of patients. Standard chemotherapy has modest activity, whereas new targeted therapies (alone or in combination) have some activity in controlling disease progression. However, the scarcity of preclinical models capable of testing in vivo responses to these therapies hampers the development of new medical strategies. Methods In this study, 8 chordoma samples taken from 8 patients were implanted in nude mice. Four engrafted successfully and gave rise to tumor masses that were analyzed histologically, by means of fluorescence in situ hybridization and biochemical techniques. The data relating to each of the mouse tumors were compared with those obtained from the corresponding human tumor. Results All 4 engraftments retained the histological, genetic and biochemical features of the human tumors they came from. In one epidermal growth factor receptor(EGFR)-positive xenograft, responsiveness to lapatinib was evaluated by comparing the pre- and posttreatment findings. The treatment induced a low-level, heterogeneous switching off of EGFR and its downstream signaling effectors. Conclusions Overall, this model is very close to human chordoma and represents a new means of undertaking preclinical investigations and developing tailored therapies. PMID:24366975

Silibinin Induced Human Glioblastoma Cell Apoptosis Concomitant with Autophagy through Simultaneous Inhibition of mTOR and YAP.

PubMed

Bai, Zhuan-Li; Tay, Vincent; Guo, Shu-Zhong; Ren, Juan; Shu, Mao-Guo

2018-01-01

Silibinin, also known as silybin, is the major flavonolignan isolated from Silybum marianum . Although previous reports demonstrated that silibinin exhibits significant tumor suppressor activities in various cancers by promoting cell apoptosis, it was also shown to trigger autophagy to counteract apoptosis induced by exogenous stresses in several types of cells. However, there is no report to address the role of silibinin induced autophagy in human A172 and SR glioblastoma cells. Our study showed that silibinin treatment not only inhibited the metabolic activities of glioblastoma cells but also promoted their apoptosis through the regulation of caspase 3 and PARP-1 in concentration- and time-dependent manners. Meanwhile, silibinin induced autophagy through upregulation of microtubule-associated protein a light chain 3- (LC3-) II. And autophagy inhibition with chloroquine, a lysosomotropic agent, significantly enhanced silibinin induced glioblastoma cell apoptosis. Moreover, silibinin dose-dependently downregulated the phosphorylation levels of mTOR at Ser-2448, p70S6K at Thr-389, and 4E-BP1 at Thr-37/46. Furthermore, the expression of YAP, the downstream effector of Hippo signal pathway, was also suppressed by silibinin. These results suggested that silibinin induced glioblastoma cell apoptosis concomitant with autophagy which might be due to simultaneous inhibition of mTOR and YAP and silibinin induced autophagy exerted a protective role against cell apoptosis in both A172 and SR cells.

Quantitative proteomics-based analysis supports a significant role of GTG proteins in regulation of ABA response in Arabidopsis roots.

PubMed

Alvarez, Sophie; Roy Choudhury, Swarup; Hicks, Leslie M; Pandey, Sona

2013-03-01

Abscisic acid (ABA) is proposed to be perceived by multiple receptors in plants. We have previously reported on the role of two GPCR-type G-proteins (GTG proteins) as plasma membrane-localized ABA receptors in Arabidopsis thaliana. However, due to the presence of multiple transmembrane domains, detailed structural and biochemical characterization of GTG proteins remains limited. Since ABA induces substantial changes in the proteome of plants, a labeling LC-based quantitative proteomics approach was applied to elucidate the global effects and possible downstream targets of GTG1/GTG2 proteins. Quantitative differences in protein abundance between wild-type and gtg1gtg2 were analyzed for evaluation of the effect of ABA on the root proteome and its dependence on the presence of functional GTG1/GTG2 proteins. The results presented in this study reveal the most comprehensive ABA-responsive root proteome reported to date in Arabidopsis. Notably, the majority of ABA-responsive proteins required the presence of GTG proteins, supporting their key role in ABA signaling. These observations were further confirmed by additional experiments. Overall, comparison of the ABA-dependent protein abundance changes in wild-type versus gtg1gtg2 provides clues to their possible links with some of the well-established effectors of the ABA signaling pathways and their role in mediating phytohormone cross-talk.

NopP, a phosphorylated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii.

PubMed

Skorpil, Peter; Saad, Maged M; Boukli, Nawal M; Kobayashi, Hajime; Ares-Orpel, Florencia; Broughton, William J; Deakin, William J

2005-09-01

Rhizobium sp. NGR234 nodulates many plants, some of which react to proteins secreted via a type three secretion system (T3SS) in a positive- (Flemingia congesta, Tephrosia vogelii) or negative- (Crotalaria juncea, Pachyrhizus tuberosus) manner. T3SSs are devices that Gram-negative bacteria use to inject effector proteins into the cytoplasm of eukaryotic cells. The only two rhizobial T3SS effector proteins characterized to date are NopL and NopP of NGR234. NopL can be phosphorylated by plant kinases and we show this to be true for NopP as well. Mutation of nopP leads to a dramatic reduction in nodule numbers on F. congesta and T. vogelii. Concomitant mutation of nopL and nopP further diminishes nodulation capacity to levels that, on T. vogelii, are lower than those produced by the T3SS null mutant NGR(Omega)rhcN. We also show that the T3SS of NGR234 secretes at least one additional effector, which remains to be identified. In other words, NGR234 secretes a cocktail of effectors, some of which have positive effects on nodulation of certain plants while others are perceived negatively and block nodulation. NopL and NopP are two components of this mix that extend the ability of NGR234 to nodulate certain legumes.

pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion

PubMed Central

Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario

2016-01-01

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity–primed T cells acquired cytotoxic activity earlier than high affinity–primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity–stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. PMID:27799622

Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

PubMed

de Lange, Orlando; Schreiber, Tom; Schandry, Niklas; Radeck, Jara; Braun, Karl Heinz; Koszinowski, Julia; Heuer, Holger; Strauß, Annett; Lahaye, Thomas

2013-08-01

Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Integral Characterization of Defective BDNF/TrkB Signalling in Neurological and Psychiatric Disorders Leads the Way to New Therapies

PubMed Central

Tejeda, Gonzalo S.; Díaz-Guerra, Margarita

2017-01-01

Enhancement of brain-derived neurotrophic factor (BDNF) signalling has great potential in therapy for neurological and psychiatric disorders. This neurotrophin not only attenuates cell death but also promotes neuronal plasticity and function. However, an important challenge to this approach is the persistence of aberrant neurotrophic signalling due to a defective function of the BDNF high-affinity receptor, tropomyosin-related kinase B (TrkB), or downstream effectors. Such changes have been already described in several disorders, but their importance as pathological mechanisms has been frequently underestimated. This review highlights the relevance of an integrative characterization of aberrant BDNF/TrkB pathways for the rational design of therapies that by combining BDNF and TrkB targets could efficiently promote neurotrophic signalling. PMID:28134845

Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein.

PubMed

Tao, Shi-Cong; Gao, You-Shui; Zhu, Hong-Yi; Yin, Jun-Hui; Chen, Yi-Xuan; Zhang, Yue-Lei; Guo, Shang-Chun; Zhang, Chang-Qing

2016-06-03

The pH of extracellular fluids is a basic property of the tissue microenvironment and is normally maintained at 7.40 ± 0.05 in humans. Many pathological circumstances, such as ischemia, inflammation, and tumorigenesis, result in the reduction of extracellular pH in the affected tissues. In this study, we reported that the osteogenic differentiation of BMSCs was significantly inhibited by decreases in the extracellular pH. Moreover, we demonstrated that proton-sensing GPR4 signaling mediated the proton-induced inhibitory effects on the osteogenesis of BMSCs. Additionally, we found that YAP was the downstream effector of GPR4 signaling. Our findings revealed that the extracellular pH modulates the osteogenic responses of BMSCs by regulating the proton-sensing GPR4-YAP pathway.

Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

PubMed Central

Cattenoz, Pierre B.; Popkova, Anna; Southall, Tony D.; Aiello, Giuseppe; Brand, Andrea H.; Giangrande, Angela

2016-01-01

High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

Anti-fibrotic effects of a novel small compound on the regulation of cytokine production in a mouse model of colorectal fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imai, Jin; Department of Gastroenterology, Tokai University School of Medicine, Kanagawa; Hozumi, Katsuto, E-mail: hozumi@is.icc.u-tokai.ac.jp

Intestinal fibrotic stricture is a major complication of inflammatory bowel disease. Despite its clinical importance, anti-fibrotic therapy has not been implemented. Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to tissue fibrosis. We have previously shown that the administration of a small compound, HSc025, which promotes the nuclear translocation of YB-1 as a downstream effector of IFN-γ and antagonizes TGF-β/Smad signaling, improves fibrosis in several murine tissues. In this study, we evaluated the anti-fibrotic effect of HSc025 on colorectal fibrosis in TNBS-induced murine chronic colitis. Daily oral administration of HSc025 (3, 15 and 75 mg/kg) suppressed collagenmore » production and decreased the severity of colorectal fibrosis in a dose-dependent manner. In addition, the local production of TGF-β was decreased after HSc025 treatment, whereas that of IL-13 and TNF-α was not affected. HSc025 administration maintained the level of IFN-γ production, even at a late stage when IFN-γ production was lost without the drug treatment. These results demonstrate that HSc025 could be a therapeutic candidate for intestinal fibrosis in inflammatory bowel disease that acts by altering the local production of cytokines, as well as by directly suppressing collagen production. - Highlights: • Colorectal fibrosis of TNBS-induced colitis was attenuated by HSc025 administration. • Local production of TGF-b was suppressed by the modulation of TGF-b/IFN-g signaling. • Derepression of IFN-g production was induced by the drug treatment.« less

Noncanonical control of C. elegans germline apoptosis by the insulin/IGF-1 and Ras/MAPK signaling pathways.

PubMed

Perrin, A J; Gunda, M; Yu, B; Yen, K; Ito, S; Forster, S; Tissenbaum, H A; Derry, W B

2013-01-01

The insulin/IGF-1 pathway controls a number of physiological processes in the nematode worm Caenorhabditis elegans, including development, aging and stress response. We previously found that the Akt/PKB ortholog AKT-1 dampens the apoptotic response to genotoxic stress in the germline by negatively regulating the p53-like transcription factor CEP-1. Here, we report unexpected rearrangements to the insulin/IGF-1 pathway, whereby the insulin-like receptor DAF-2 and 3-phosphoinositide-dependent protein kinase PDK-1 oppose AKT-1 to promote DNA damage-induced apoptosis. While DNA damage does not affect phosphorylation at the PDK-1 site Thr350/Thr308 of AKT-1, it increased phosphorylation at Ser517/Ser473. Although ablation of daf-2 or pdk-1 completely suppressed akt-1-dependent apoptosis, the transcriptional activation of CEP-1 was unaffected, suggesting that daf-2 and pdk-1 act independently or downstream of cep-1 and akt-1. Ablation of the akt-1 paralog akt-2 or the downstream target of the insulin/IGF-1 pathway daf-16 (a FOXO transcription factor) restored sensitivity to damage-induced apoptosis in daf-2 and pdk-1 mutants. In addition, daf-2 and pdk-1 mutants have reduced levels of phospho-MPK-1/ERK in their germ cells, indicating that the insulin/IGF-1 pathway promotes Ras signaling in the germline. Ablation of the Ras effector gla-3, a negative regulator of mpk-1, restored sensitivity to apoptosis in daf-2 mutants, suggesting that gla-3 acts downstream of daf-2. In addition, the hypersensitivity of let-60/Ras gain-of-function mutants to damage-induced apoptosis was suppressed to wild-type levels by ablation of daf-2. Thus, insulin/IGF-1 signaling selectively engages AKT-2/DAF-16 to promote DNA damage-induced germ cell apoptosis downstream of CEP-1 through the Ras pathway.

Structure of a bacterial type III secretion system in contact with a host membrane in situ

NASA Astrophysics Data System (ADS)

Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

2015-12-01

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking `pump-action' conformational changes that underpin effector injection.

Feasibility study for clinical application of caspase-3 inhibitors in Pemphigus vulgaris.

PubMed

Hariton, William V J; Galichet, Arnaud; Vanden Berghe, Tom; Overmiller, Andrew M; Mahoney, My G; Declercq, Wim; Müller, Eliane J

2017-12-01

The potentially severe side effects of systemic corticosteroids and immunosuppressants used in Pemphigus vulgaris (PV) call for novel therapeutic approaches. In this context, pharmacological inhibition of major pathogenic signalling effectors represents a promising alternative. However, we have also shown that overinhibition of effectors required for epidermal homeostasis can exacerbate PV pathophysiology implicating transepidermal keratinocyte fragility. A feedforward target validation therefore preferentially includes studies on knockout mouse models. We previously reported on successful amelioration of PV blisters following inhibition of non-apoptotic, low-level caspase-3. Here, we use conditional, keratinocyte-specific caspase-3-deficient mice (casp3 EKO ) to demonstrate (i) absence of keratinocyte fragility upon injection of the potent Dsg3-specific antibody AK23 and (ii) amelioration of blistering on the background of known signalling effectors. Our results provide the experimental proof of concept justifying translation of the caspase-3 inhibitor approach into PV clinical trials. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Structure of a bacterial type III secretion system in contact with a host membrane in situ.

PubMed

Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R; Hayward, Richard D

2015-12-11

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking 'pump-action' conformational changes that underpin effector injection.

Assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure.

PubMed

Katen, Sarah P; Tan, Zhenning; Chirapu, Srinivas Reddy; Finn, M G; Zlotnick, Adam

2013-08-06

Hepatitis B virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized heteroaryldihydropyrimidine compounds but favors a unique quasiequivalent location on the capsid surface. Thus, this pocket is a promiscuous drug-binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biochemical analysis of force-sensitive responses using a large-scale cell stretch device.

PubMed

Renner, Derrick J; Ewald, Makena L; Kim, Timothy; Yamada, Soichiro

2017-09-03

Physical force has emerged as a key regulator of tissue homeostasis, and plays an important role in embryogenesis, tissue regeneration, and disease progression. Currently, the details of protein interactions under elevated physical stress are largely missing, therefore, preventing the fundamental, molecular understanding of mechano-transduction. This is in part due to the difficulty isolating large quantities of cell lysates exposed to force-bearing conditions for biochemical analysis. We designed a simple, easy-to-fabricate, large-scale cell stretch device for the analysis of force-sensitive cell responses. Using proximal biotinylation (BioID) analysis or phospho-specific antibodies, we detected force-sensitive biochemical changes in cells exposed to prolonged cyclic substrate stretch. For example, using promiscuous biotin ligase BirA* tagged α-catenin, the biotinylation of myosin IIA increased with stretch, suggesting the close proximity of myosin IIA to α-catenin under a force bearing condition. Furthermore, using phospho-specific antibodies, Akt phosphorylation was reduced upon stretch while Src phosphorylation was unchanged. Interestingly, phosphorylation of GSK3β, a downstream effector of Akt pathway, was also reduced with stretch, while the phosphorylation of other Akt effectors was unchanged. These data suggest that the Akt-GSK3β pathway is force-sensitive. This simple cell stretch device enables biochemical analysis of force-sensitive responses and has potential to uncover molecules underlying mechano-transduction.

Visual-Motor Transformations Within Frontal Eye Fields During Head-Unrestrained Gaze Shifts in the Monkey.

PubMed

Sajad, Amirsaman; Sadeh, Morteza; Keith, Gerald P; Yan, Xiaogang; Wang, Hongying; Crawford, John Douglas

2015-10-01

A fundamental question in sensorimotor control concerns the transformation of spatial signals from the retina into eye and head motor commands required for accurate gaze shifts. Here, we investigated these transformations by identifying the spatial codes embedded in visually evoked and movement-related responses in the frontal eye fields (FEFs) during head-unrestrained gaze shifts. Monkeys made delayed gaze shifts to the remembered location of briefly presented visual stimuli, with delay serving to dissociate visual and movement responses. A statistical analysis of nonparametric model fits to response field data from 57 neurons (38 with visual and 49 with movement activities) eliminated most effector-specific, head-fixed, and space-fixed models, but confirmed the dominance of eye-centered codes observed in head-restrained studies. More importantly, the visual response encoded target location, whereas the movement response mainly encoded the final position of the imminent gaze shift (including gaze errors). This spatiotemporal distinction between target and gaze coding was present not only at the population level, but even at the single-cell level. We propose that an imperfect visual-motor transformation occurs during the brief memory interval between perception and action, and further transformations from the FEF's eye-centered gaze motor code to effector-specific codes in motor frames occur downstream in the subcortical areas. © The Author 2014. Published by Oxford University Press.

Cation dyshomeostasis and cardiomyocyte necrosis: the Fleckenstein hypothesis revisited.

PubMed

Borkowski, Brian J; Cheema, Yaser; Shahbaz, Atta U; Bhattacharya, Syamal K; Weber, Karl T

2011-08-01

An ongoing loss of cardiomyocytes to apoptotic and necrotic cell death pathways contributes to the progressive nature of heart failure. The pathophysiological origins of necrotic cell loss relate to the neurohormonal activation that accompanies acute and chronic stressor states and which includes effector hormones of the adrenergic nervous system. Fifty years ago, Albrecht Fleckenstein and coworkers hypothesized the hyperadrenergic state, which accompanies such stressors, causes cardiomyocyte necrosis based on catecholamine-initiated excessive intracellular Ca(2+) accumulation (EICA), and mitochondrial Ca(2+) overloading in particular, in which the ensuing dysfunction and structural degeneration of these organelles leads to necrosis. In recent years, two downstream factors have been identified which, together with EICA, constitute a signal-transducer-effector pathway: (i) mitochondria-based induction of oxidative stress, in which the rate of reactive oxygen metabolite generation exceeds their rate of detoxification by endogenous antioxidant defences; and (ii) the opening of the mitochondrial inner membrane permeability transition pore (mPTP) followed by organellar swelling and degeneration. The pathogenesis of stress-related cardiomyopathy syndromes is likely related to this pathway. Other factors which can account for cytotoxicity in stressor states include: hypokalaemia; ionized hypocalcaemia and hypomagnesaemia with resultant elevations in parathyroid hormone serving as a potent mediator of EICA; and hypozincaemia with hyposelenaemia, which compromise antioxidant defences. Herein, we revisit the Fleckenstein hypothesis of EICA in leading to cardiomyocyte necrosis and the central role played by mitochondria.

MERTK signaling in the retinal pigment epithelium regulates the tyrosine phosphorylation of GDP dissociation inhibitor alpha from the GDI/CHM family of RAB GTPase effectors.

PubMed

Shelby, Shameka J; Feathers, Kecia L; Ganios, Anna M; Jia, Lin; Miller, Jason M; Thompson, Debra A

2015-11-01

Photoreceptor outer segments (OS) in the vertebrate retina undergo a process of continual renewal involving shedding of disc membranes that are cleared by phagocytic uptake into the retinal pigment epithelium (RPE). In dystrophic Royal College of Surgeons (RCS) rats, OS phagocytosis is blocked by a mutation in the gene encoding the receptor tyrosine kinase MERTK. To identify proteins tyrosine-phosphorylated downstream of MERTK in the RPE, MALDI-mass spectrometry with peptide-mass fingerprinting was used in comparative studies of RCS congenic and dystrophic rats. At times corresponding to peak phagocytic activity, the RAB GTPase effector GDP dissociation inhibitor alpha (GDI1) was found to undergo tyrosine phosphorylation only in congenic rats. In cryosections of native RPE/choroid, GDI1 colocalized with MERTK and the intracellular tyrosine-kinase SRC. In cultured RPE-J cells, and in transfected heterologous cells, MERTK stimulated SRC-mediated tyrosine phosphorylation of GDI1. In OS-fed RPE-J cells, GDI1 colocalized with MERTK and SRC on apparent phagosomes located near the apical membrane. In addition, both GDI1 and RAB5, a regulator of vesicular transport, colocalized with ingested OS. Taken together, these findings identify a novel role of MERTK signaling in membrane trafficking in the RPE that is likely to subserve mechanisms of phagosome formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Endoderm-specific deletion of Tbx1 reveals an FGF-independent role for Tbx1 in pharyngeal apparatus morphogenesis

PubMed Central

Jackson, Abigail; Kasah, Sahrunizam; Mansour, Suzanne L.; Morrow, Bernice; Basson, M. Albert

2015-01-01

Background The T-box transcription factor Tbx1, is essential for the normal development of multiple organ systems in the embryo. One of the most striking phenotypes in Tbx1−/− embryos is the failure of the caudal pharyngeal pouches to evaginate from the foregut endoderm. Despite considerable interest in the role of Tbx1 in development, the mechanisms whereby Tbx1 controls caudal pouch formation have remained elusive. In particular, the question as to how Tbx1 expression in the pharyngeal endoderm regulates pharyngeal pouch morphogenesis in the mouse embryo is not known. Results To address this question, we produced mouse embryos in which Tbx1 was specifically deleted from the pharyngeal endoderm and as expected, embryos failed to form caudal pharyngeal pouches. To determine the molecular mechanism, we examined expression of Fgf3 and Fgf8 ligands and downstream effectors. Although Fgf8 expression is greatly reduced in Tbx1-deficient endoderm, FGF signaling levels are unaffected. Furthermore, pouch morphogenesis is only partially perturbed by the loss of both Fgf3 and Fgf8 from the endoderm, indicating that neither are required for pouch formation. Conclusions Tbx1 deletion from the pharyngeal endoderm is sufficient to cause caudal pharyngeal arch segmentation defects by FGF-independent effectors that remain to be identified. PMID:24812002

Autochthonous tumors driven by Rb1 loss have an ongoing requirement for the RBP2 histone demethylase.

PubMed

McBrayer, Samuel K; Olenchock, Benjamin A; DiNatale, Gabriel J; Shi, Diana D; Khanal, Januka; Jennings, Rebecca B; Novak, Jesse S; Oser, Matthew G; Robbins, Alissa K; Modiste, Rebecca; Bonal, Dennis; Moslehi, Javid; Bronson, Roderick T; Neuberg, Donna; Nguyen, Quang-De; Signoretti, Sabina; Losman, Julie-Aurore; Kaelin, William G

2018-04-17

Inactivation of the retinoblastoma gene ( RB1 ) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1 +/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1 +/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1 -null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

PEDF attenuates insulin-dependent molecular pathways of glucose homeostasis in skeletal myocytes.

PubMed

Carnagarin, Revathy; Dharmarajan, Arun M; Dass, Crispin R

2016-02-15

Pigment epithelium-derived factor (PEDF) is an anti-angiogenic serpin associated with insulin resistance in metabolic disorders such as diabetes, metabolic syndrome, obesity and polycystic ovarian syndrome. While the mechanism of PEDF induced-insulin resistance of metabolic disorders has been attributed to its inflammatory and lipolytic effects, little evidence exists to support a direct role of PEDF in mediating insulin resistance. Here, we seminally provide evidence that PEDF can inhibit insulin signal transduction governing glucose homeostasis from the receptor to the effector phosphorylation through Akt/PKB-dependent and -independent pathways in mouse and human skeletal muscle cell lines. PEDF attenuates the insulin-dependent molecular axes of glucose metabolism. Exposure of skeletal myocytes to PEDF attenuates insulin-dependent insulin receptor autophosphorylation, tyrosine phosphorylation of insulin receptor substrate 1, and dual loop phosphorylation-activation of Akt. PEDF significantly inhibits the downstream effector - glycogen synthase kinase (and thereby the glycogenic axis of insulin signalling). PEDF turned off both the molecular switches of GLUT4 translocation: IRS-Akt/PKB-AS160 mediated and IR-pCbl-dependent GLUT4 translocation (the molecular axis of glucose uptake). These findings implicate a direct effect of PEDF on multiple insulin-dependent molecular mechanisms of glucose homeostasis in skeletal muscle cells, thereby enabling it to contribute to peripheral insulin resistance at the cellular level. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Cation dyshomeostasis and cardiomyocyte necrosis: the Fleckenstein hypothesis revisited

PubMed Central

Borkowski, Brian J.; Cheema, Yaser; Shahbaz, Atta U.; Bhattacharya, Syamal K.; Weber, Karl T.

2011-01-01

An ongoing loss of cardiomyocytes to apoptotic and necrotic cell death pathways contributes to the progressive nature of heart failure. The pathophysiological origins of necrotic cell loss relate to the neurohormonal activation that accompanies acute and chronic stressor states and which includes effector hormones of the adrenergic nervous system. Fifty years ago, Albrecht Fleckenstein and coworkers hypothesized the hyperadrenergic state, which accompanies such stressors, causes cardiomyocyte necrosis based on catecholamine-initiated excessive intracellular Ca2+ accumulation (EICA), and mitochondrial Ca2+ overloading in particular, in which the ensuing dysfunction and structural degeneration of these organelles leads to necrosis. In recent years, two downstream factors have been identified which, together with EICA, constitute a signal–transducer–effector pathway: (i) mitochondria-based induction of oxidative stress, in which the rate of reactive oxygen metabolite generation exceeds their rate of detoxification by endogenous antioxidant defences; and (ii) the opening of the mitochondrial inner membrane permeability transition pore (mPTP) followed by organellar swelling and degeneration. The pathogenesis of stress-related cardiomyopathy syndromes is likely related to this pathway. Other factors which can account for cytotoxicity in stressor states include: hypokalaemia; ionized hypocalcaemia and hypomagnesaemia with resultant elevations in parathyroid hormone serving as a potent mediator of EICA; and hypozincaemia with hyposelenaemia, which compromise antioxidant defences. Herein, we revisit the Fleckenstein hypothesis of EICA in leading to cardiomyocyte necrosis and the central role played by mitochondria. PMID:21398641

Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration

PubMed Central

Zhang, Lijuan; Du, Jianhai; Justus, Sally; Hsu, Chun-Wei; Bonet-Ponce, Luis; Wu, Wen-Hsuan; Tsai, Yi-Ting; Wu, Wei-Pu; Jia, Yading; Duong, Jimmy K.; Mahajan, Vinit B.; Lin, Chyuan-Sheng; Wang, Shuang; Hurley, James B.

2016-01-01

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy–mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders. PMID:27841758

Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of immunoglobulins with skewed affinity for FcγR2a and FcγR2b.

PubMed

Ackerman, Margaret E; Dugast, Anne-Sophie; McAndrew, Elizabeth G; Tsoukas, Stephen; Licht, Anna F; Irvine, Darrell J; Alter, Galit

2013-05-01

While development of an HIV vaccine that can induce neutralizing antibodies remains a priority, decades of research have proven that this is a daunting task. However, accumulating evidence suggests that antibodies with the capacity to harness innate immunity may provide some protection. While significant research has focused on the cytolytic properties of antibodies in acquisition and control, less is known about the role of additional effector functions. In this study, we investigated antibody-dependent phagocytosis of HIV immune complexes, and we observed significant differences in the ability of antibodies from infected subjects to mediate this critical effector function. We observed both quantitative differences in the capacity of antibodies to drive phagocytosis and qualitative differences in their FcγR usage profile. We demonstrate that antibodies from controllers and untreated progressors exhibit increased phagocytic activity, altered Fc domain glycosylation, and skewed interactions with FcγR2a and FcγR2b in both bulk plasma and HIV-specific IgG. While increased phagocytic activity may directly influence immune activation via clearance of inflammatory immune complexes, it is also plausible that Fc receptor usage patterns may regulate the immune response by modulating downstream signals following phagocytosis--driving passive degradation of internalized virus, release of immune modulating cytokines and chemokines, or priming of a more effective adaptive immune response.

Trends in Regenerative Medicine: Repigmentation in Vitiligo Through Melanocyte Stem Cell Mobilization.

PubMed

Birlea, Stanca A; Costin, Gertrude-E; Roop, Dennis R; Norris, David A

2017-07-01

Vitiligo is the most frequent human pigmentary disorder, characterized by progressive autoimmune destruction of mature epidermal melanocytes. Of the current treatments offering partial and temporary relief, ultraviolet (UV) light is the most effective, coordinating an intricate network of keratinocyte and melanocyte factors that control numerous cellular and molecular signaling pathways. This UV-activated process is a classic example of regenerative medicine, inducing functional melanocyte stem cell populations in the hair follicle to divide, migrate, and differentiate into mature melanocytes that regenerate the epidermis through a complex process involving melanocytes and other cell lineages in the skin. Using an in-depth correlative analysis of multiple experimental and clinical data sets, we generated a modern molecular research platform that can be used as a working model for further research of vitiligo repigmentation. Our analysis emphasizes the active participation of defined molecular pathways that regulate the balance between stemness and differentiation states of melanocytes and keratinocytes: p53 and its downstream effectors controlling melanogenesis; Wnt/β-catenin with proliferative, migratory, and differentiation roles in different pigmentation systems; integrins, cadherins, tetraspanins, and metalloproteinases, with promigratory effects on melanocytes; TGF-β and its effector PAX3, which control differentiation. Our long-term goal is to design pharmacological compounds that can specifically activate melanocyte precursors in the hair follicle in order to obtain faster, better, and durable repigmentation. © 2016 Wiley Periodicals, Inc.

Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

PubMed

Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

2014-02-01

During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

PubMed

Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

2015-11-01

Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. © 2015 Wiley Periodicals, Inc.

Trends in Regenerative Medicine: Repigmentation in Vitiligo Through Melanocyte Stem Cell Mobilization

PubMed Central

Birlea, Stanca A.; Costin, Gertrude-E.; Roop, Dennis R.; Norris, David A.

2017-01-01

Vitiligo is the most frequent human pigmentary disorder, characterized by progressive autoimmune destruction of mature epidermal melanocytes. Of the current treatments offering partial and temporary relief, ultraviolet (UV) light is the most effective, coordinating an intricate network of keratinocyte and melanocyte factors that control numerous cellular and molecular signaling pathways. This UV-activated process is a classic example of regenerative medicine, inducing functional melanocyte stem cell populations in the hair follicle to divide, migrate, and differentiate into mature melanocytes that regenerate the epidermis through a complex process involving melanocytes and other cell lineages in the skin. Using an in-depth correlative analysis of multiple experimental and clinical data sets, we generated a modern molecular research platform that can be used as a working model for further research of vitiligo repigmentation. Our analysis emphasizes the active participation of defined molecular pathways that regulate the balance between stemness and differentiation states of melanocytes and keratinocytes: p53 and its downstream effectors controlling melanogenesis; Wnt/β-catenin with proliferative, migratory, and differentiation roles in different pigmentation systems; integrins, cadherins, tetraspanins, and metalloproteinases, with promigratory effects on melanocytes; TGF-β and its effector PAX3, which control differentiation. Our long-term goal is to design pharmacological compounds that can specifically activate melanocyte precursors in the hair follicle in order to obtain faster, better, and durable repigmentation. PMID:28029168

Controlled branched-chain amino acids auxotrophy in Listeria monocytogenes allows isoleucine to serve as a host signal and virulence effector

PubMed Central

Borovok, Ilya; Sigal, Nadejda

2018-01-01

Listeria monocytogenes (Lm) is a saprophyte and intracellular pathogen. Transition to the pathogenic state relies on sensing of host-derived metabolites, yet it remains unclear how these are recognized and how they mediate virulence gene regulation. We previously found that low availability of isoleucine signals Lm to activate the virulent state. This response is dependent on CodY, a global regulator and isoleucine sensor. Isoleucine-bound CodY represses metabolic pathways including branched-chain amino acids (BCAA) biosynthesis, however under BCAA depletion, as occurs during infection, BCAA biosynthesis is upregulated and isoleucine-unbound CodY activates virulence genes. While isoleucine was revealed as an important input signal, it was not identified how internal levels are controlled during infection. Here we show that Lm regulates BCAA biosynthesis via CodY and via a riboregulator located upstream to the BCAA biosynthesis genes, named Rli60. rli60 is transcribed when BCAA levels drop, forming a ribosome-mediated attenuator that cis-regulates the downstream genes according to BCAA supply. Notably, we found that Rli60 restricts BCAA production, essentially starving Lm, a mechanism that is directly linked to virulence, as it controls the internal isoleucine pool and thereby CodY activity. This controlled BCAA auxotrophy likely evolved to enable isoleucine to serve as a host signal and virulence effector. PMID:29529043

Controlled branched-chain amino acids auxotrophy in Listeria monocytogenes allows isoleucine to serve as a host signal and virulence effector.

PubMed

Brenner, Moran; Lobel, Lior; Borovok, Ilya; Sigal, Nadejda; Herskovits, Anat A

2018-03-01

Listeria monocytogenes (Lm) is a saprophyte and intracellular pathogen. Transition to the pathogenic state relies on sensing of host-derived metabolites, yet it remains unclear how these are recognized and how they mediate virulence gene regulation. We previously found that low availability of isoleucine signals Lm to activate the virulent state. This response is dependent on CodY, a global regulator and isoleucine sensor. Isoleucine-bound CodY represses metabolic pathways including branched-chain amino acids (BCAA) biosynthesis, however under BCAA depletion, as occurs during infection, BCAA biosynthesis is upregulated and isoleucine-unbound CodY activates virulence genes. While isoleucine was revealed as an important input signal, it was not identified how internal levels are controlled during infection. Here we show that Lm regulates BCAA biosynthesis via CodY and via a riboregulator located upstream to the BCAA biosynthesis genes, named Rli60. rli60 is transcribed when BCAA levels drop, forming a ribosome-mediated attenuator that cis-regulates the downstream genes according to BCAA supply. Notably, we found that Rli60 restricts BCAA production, essentially starving Lm, a mechanism that is directly linked to virulence, as it controls the internal isoleucine pool and thereby CodY activity. This controlled BCAA auxotrophy likely evolved to enable isoleucine to serve as a host signal and virulence effector.

CD4 cells can be more efficient at tumor rejection than CD8 cells.

PubMed

Perez-Diez, Ainhoa; Joncker, Nathalie T; Choi, Kyungho; Chan, William F N; Anderson, Colin C; Lantz, Olivier; Matzinger, Polly

2007-06-15

Researchers designing antitumor treatments have long focused on eliciting tumor-specific CD8 cytotoxic T lymphocytes (CTL) because of their potent killing activity and their ability to reject transplanted organs. The resulting treatments, however, have generally been surprisingly poor at inducing complete tumor rejection, both in experimental models and in the clinic. Although a few scattered studies suggested that CD4 T "helper" cells might also serve as antitumor effectors, they have generally been studied mostly for their ability to enhance the activity of CTL. In this mouse study, we compared monoclonal populations of tumor-specific CD4 and CD8 T cells as effectors against several different tumors, and found that CD4 T cells eliminated tumors that were resistant to CD8-mediated rejection, even in cases where the tumors expressed major histocompatibility complex (MHC) class I molecules but not MHC class II. MHC class II expression on host tissues was critical, suggesting that the CD4 T cells act indirectly. Indeed, the CD4 T cells partnered with NK cells to obtain the maximal antitumor effect. These findings suggest that CD4 T cells can be powerful antitumor effector cells that can, in some cases, outperform CD8 T cells, which are the current "gold standard" effector cell in tumor immunotherapy.

Inferring the effective TOR-dependent network: a computational study in yeast

PubMed Central

2013-01-01

Background Calorie restriction (CR) is one of the most conserved non-genetic interventions that extends healthspan in evolutionarily distant species, ranging from yeast to mammals. The target of rapamycin (TOR) has been shown to play a key role in mediating healthspan extension in response to CR by integrating different signals that monitor nutrient-availability and orchestrating various components of cellular machinery in response. Both genetic and pharmacological interventions that inhibit the TOR pathway exhibit a similar phenotype, which is not further amplified by CR. Results In this paper, we present the first comprehensive, computationally derived map of TOR downstream effectors, with the objective of discovering key lifespan mediators, their crosstalk, and high-level organization. We adopt a systematic approach for tracing information flow from the TOR complex and use it to identify relevant signaling elements. By constructing a high-level functional map of TOR downstream effectors, we show that our approach is not only capable of recapturing previously known pathways, but also suggests potential targets for future studies. Information flow scores provide an aggregate ranking of relevance of proteins with respect to the TOR signaling pathway. These rankings must be normalized for degree bias, appropriately interpreted, and mapped to associated roles in pathways. We propose a novel statistical framework for integrating information flow scores, the set of differentially expressed genes in response to rapamycin treatment, and the transcriptional regulatory network. We use this framework to identify the most relevant transcription factors in mediating the observed transcriptional response, and to construct the effective response network of the TOR pathway. This network is hypothesized to mediate life-span extension in response to TOR inhibition. Conclusions Our approach, unlike experimental methods, is not limited to specific aspects of cellular response. Rather, it predicts transcriptional changes and post-translational modifications in response to TOR inhibition. The constructed effective response network greatly enhances understanding of the mechanisms underlying the aging process and helps in identifying new targets for further investigation of anti-aging regimes. It also allows us to identify potential network biomarkers for diagnosis and prognosis of age-related pathologies. PMID:24005029

Radioresistance of chordoma cells is associated with the ATM/ATR pathway, in which RAD51 serves as an important downstream effector.

PubMed

Zhang, Chao; Wang, Bing; Li, Lei; Li, Yawei; Li, Pengzhi; Lv, Guohua

2017-09-01

Surgery followed by radiotherapy is the standard treatment for chordomas, which are a rare but low-grade type of bone cancer arising from remnants of the embryonic notochord. However, disease recurrence following radiotherapy is common, most likely due to endogenous DNA repair mechanisms that promote cell survival upon radiation strikes. The ataxia telangiectasia mutated/ataxia telangiectasia mutated and Rad3 related (ATM/ATR)-mediated pathway has a critical role in DNA repair mechanisms; however, it has rarely been investigated in chordomas. In the present study, the expression of signal molecules related to the ATM/ATR pathway in chordoma tissues and adjacent normal tissues were initially examined using immunohistochemistry and western blot analysis. Chordoma U-CH1 and U-CH2 cells were subsequently used to investigate cell responses to ionizing radiation and the potential protective actions mediated by the ATM/ATR pathway. Phosphorylated (p)-ATM, p-ATR, γ-H2A histone family, member X (H2AX) and RAD51 were significantly upregulated in chordoma tissues relative to adjacent normal tissues (P<0.05). No significant reductions were observed in the viability of U-CH1 and U-CH2 cells following exposure to low-dose (1 and 2 Gy) radiation. Radiation (1 and 2 Gy) triggered a significant upregulation in p-ATM, γ-H2AX and RAD51 expression in U-CH1 cells (P<0.05), as well as a significant upregulation in p-ATM, p-ATR and RAD51 levels in U-CH2 cells (P<0.05). RAD51 knockdown increased the responses of both U-CH1 and U-CH2 cells to 1 Gy radiation, as evidenced by the significantly decreased cell viability and increased apoptosis rate (P<0.05). Collectively, the results of the present study indicated that radioresistance of chordoma cells is associated with the ATM/ATR pathway, in which RAD51 serves as an important downstream effector. Thus, RAD51 presents a promising therapeutic target for improving the outcome of radiotherapy treatment in chordomas.

Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles.

PubMed

Chen, Hsin-Yi; Wu, Chien-Ting; Tang, Chieh-Ju C; Lin, Yi-Nan; Wang, Won-Jing; Tang, Tang K

2017-08-15

Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly.Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles.

Profiling EGFR activity in head and neck squamous cell carcinoma by using a novel layered membrane Western blot technology.

PubMed

Patel, Vyomesh; Ramesh, Arun; Traicoff, June L; Baibakov, Galina; Emmert-Buck, Michael R; Gutkind, J Silvio; Knezevic, Vladimir

2005-05-01

Given the role of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), several rational approaches have now been utilized to abrogate tyrosine kinase activity and its disengagement from downstream signal transducers. Monitoring the activity of these molecules could potentially be useful to determine not only drug efficacy but also to identify HNSCC patients most likely to benefit from this type of therapy. In this study we have used a novel high throughput multi-layered Western blotting (MLWestern) method that allows the detection of multiple proteins from a single experiment in order to characterize key components in the EGFR signaling pathway in HNSCC cells. Total and activated forms of EGFR and the downstream effectors, Erk and Akt were readily detected in HNSCC cells, where in the control cells (HaCaT) these proteins could only be detected in EGF stimulated cells. Results from conventional Western blot and MLWestern were comparable. Clustering analysis of protein expression revealed similarities in cellular response between some of the cell lines indicative of similarities in their biological response. The data indicate that MLWestern can be potentially applied to identify molecular targets that could be used for rational therapeutic intervention strategies.

Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes.

PubMed

Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee; Geng, Fuqiang; Rafii, Shahin; Altorki, Nasser K; McGraw, Timothy E

2016-12-20

Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin's effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Growth Inhibition by Bupivacaine Is Associated with Inactivation of Ribosomal Protein S6 Kinase 1

PubMed Central

Beigh, Mushtaq Ahmad; Showkat, Mehvish; Bashir, Basharat; Bashir, Asma; Hussain, Mahboob ul; Andrabi, Khurshid Iqbal

2014-01-01

Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation. PMID:24605337

Role of redox signaling in the autonomous proliferative response of endothelial cells to hypoxia.

PubMed

Schäfer, M; Schäfer, C; Ewald, N; Piper, H M; Noll, Th

2003-05-16

Endothelial cells exhibit an autonomous proliferative response to hypoxia, independent of paracrine effectors. In cultured endothelial cells of porcine aorta, we analyzed the signaling of this response, with a focus on the roles of redox signaling and the MEK/ERK pathway. Transient hypoxia (1 hour) stimulated proliferation by 61+/-4% (n=16; P<0.05 versus control), quantified after 24 hours normoxic postincubation. Hypoxia induced an activation of ERK2 and of NAD(P)H oxidase and a burst of reactive oxygen species (ROS), determined by DCF fluorescence. To inhibit the MEK/ERK pathway, we used PD 98059 (PD, 20 micromol/L); to downregulate NAD(P)H oxidase, we applied p22phox antisense oligonucleotides; and to inhibit mitochondrial ROS generation, we used the ubiquinone derivate mitoQ (MQ, 10 micromol/L). All three inhibitions suppressed the proliferative response: PD inhibited NAD(P)H oxidase activation; p22phox antisense transfection did not inhibit ERK2 activation, but suppressed ROS production; and MQ inhibited ERK2 activation and ROS production. The autonomous proliferative response depends on the MEK/ERK pathway and redox signaling steps upstream and downstream of ERK. Located upstream is ROS generation by mitochondria, downstream is NAD(P)H oxidase.

Mitochondrial apoptotic pathway activation in the atria of heart failure patients due to mitral and tricuspid regurgitation.

PubMed

Chang, Jen-Ping; Chen, Mien-Cheng; Liu, Wen-Hao; Lin, Yu-Sheng; Huang, Yao-Kuang; Pan, Kuo-Li; Ho, Wan-Chun; Fang, Chih-Yuan; Chen, Chien-Jen; Chen, Huang-Chung

2015-08-01

Apoptosis occurs in atrial cardiomyocytes in mitral and tricuspid valve disease. The purpose of this study was to examine the respective roles of the mitochondrial and tumor necrosis factor-α receptor associated death domain (TRADD)-mediated death receptor pathways for apoptosis in the atrial cardiomyocytes of heart failure patients due to severe mitral and moderate-to-severe tricuspid regurgitation. This study comprised eighteen patients (7 patients with persistent atrial fibrillation and 11 in sinus rhythm). Atrial appendage tissues were obtained during surgery. Three purchased normal human left atrial tissues served as normal controls. Moderately-to-severely myolytic cardiomyocytes comprised 59.7±22.1% of the cardiomyocytes in the right atria and 52.4±12.9% of the cardiomyocytes in the left atria of mitral and tricuspid regurgitation patients with atrial fibrillation group and comprised 58.4±24.8% of the cardiomyocytes in the right atria of mitral and tricuspid regurgitation patients with sinus rhythm. In contrast, no myolysis was observed in the normal human adult left atrial tissue samples. Immunohistochemical analysis showed expression of cleaved caspase-9, an effector of the mitochondrial pathways, in the majority of right atrial cardiomyocytes (87.3±10.0%) of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial cardiomyocytes (90.6±31.4%) and left atrial cardiomyocytes (70.7±22.0%) of mitral and tricuspid regurgitation patients with atrial fibrillation. In contrast, only 5.7% of cardiomyocytes of the normal left atrial tissues showed strongly positive expression of cleaved caspase-9. Of note, none of the atrial cardiomyocytes in right atrial tissue in sinus rhythm and in the fibrillating right and left atria of mitral and tricuspid regurgitation patients, and in the normal human adult left atrial tissue samples showed cleaved caspase-8 expression, which is a downstream effector of TRADD of the death receptor pathway. Immunoblotting of atrial extracts showed that there was enhanced expression of cytosolic cytochrome c, an effector of the mitochondrial pathways, but no expression of membrane TRADD and cytosolic caspase-8 in the right atrial tissue of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial and left atrial tissues of mitral and tricuspid regurgitation patients with atrial fibrillation. Taken together, this study showed that mitochondrial pathway for apoptosis was activated in the right atria in sinus rhythm and in the left and right atria in atrial fibrillation of heart failure patients due to mitral and tricuspid regurgitation, and this mitochondrial pathway activation may contribute to atrial contractile dysfunction and enlargement in this clinical setting. Copyright © 2015. Published by Elsevier Inc.

Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease

PubMed Central

McLane, Laura M.; Steblyanko, Maria; Anikeeva, Nadia; Ablanedo-Terrazas, Yuria; Demers, Korey; Eller, Michael A.; Streeck, Hendrik; Jansson, Marianne; Sönnerborg, Anders; Canaday, David H.; Naji, Ali; Wherry, E. John; Robb, Merlin L.; Reyes-Teran, Gustavo; Sykulev, Yuri; Betts, Michael R.

2018-01-01

CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine β-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and β-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of β-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and β-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues. PMID:29652923

The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice.

PubMed

Park, Chan-Ho; Chen, Songbiao; Shirsekar, Gautam; Zhou, Bo; Khang, Chang Hyun; Songkumarn, Pattavipha; Afzal, Ahmed J; Ning, Yuese; Wang, Ruyi; Bellizzi, Maria; Valent, Barbara; Wang, Guo-Liang

2012-11-01

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.

Time-resolved dual transcriptomics reveal early induced Nicotiana benthamiana root genes and conserved infection-promoting Phytophthora palmivora effectors.

PubMed

Evangelisti, Edouard; Gogleva, Anna; Hainaux, Thomas; Doumane, Mehdi; Tulin, Frej; Quan, Clément; Yunusov, Temur; Floch, Kévin; Schornack, Sebastian

2017-05-11

Plant-pathogenic oomycetes are responsible for economically important losses in crops worldwide. Phytophthora palmivora, a tropical relative of the potato late blight pathogen, causes rotting diseases in many tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus. Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped in understanding how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce. We employed the model plant Nicotiana benthamiana to study the P. palmivora root infections at the cellular and molecular levels. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features. These results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection-relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.

Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells.

PubMed

Cremers, Niels A J; Lundvig, Ditte M S; van Dalen, Stephanie C M; Schelbergen, Rik F; van Lent, Peter L E M; Szarek, Walter A; Regan, Raymond F; Carels, Carine E; Wagener, Frank A D T G

2014-10-08

Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.

Nonmarket resource valuation in the postfire environment

Treesearch

David Calkin; Greg Jones; Kevin Hyde

2008-01-01

After the containment of large wildland fires, major onsite and downstream effects including lost soil productivity, watershed response, increased vulnerability to invasive weeds, and downstream sedimentation can cause threats to human life and property. Burned Area Emergency Response (BAER) teams are responsible for developing treatment plans to mitigate negative...

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

PubMed

Sperschneider, Jana; Gardiner, Donald M; Dodds, Peter N; Tini, Francesco; Covarelli, Lorenzo; Singh, Karam B; Manners, John M; Taylor, Jennifer M

2016-04-01

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au. © 2015 CSIRO New Phytologist © 2015 New Phytologist Trust.

Blocking Glycolytic Metabolism Increases Memory T Cells and Antitumor Function | Center for Cancer Research

Cancer.gov

CD8+ T cells are a major component of the cellular immune response, which is necessary to control a variety of bacterial and viral infections. CD8+ T cells also play a major role in the cell-mediated antitumor immune response. After encountering antigen, naïve CD8+ T cells undergo an extensive period of proliferation and expansion, and differentiate into effector cells and

Phosphorylated ribosomal S6 (p-rpS6) as a post-treatment indicator of HER2 signalling targeted drug resistance.

PubMed

Yang-Kolodji, Gloria; Mumenthaler, Shannon M; Mehta, Arjun; Ji, Lingyun; Tripathy, Debu

2015-01-01

To identify clinically relevant predictive biomarkers of trastuzumab resistance. MTT, FACS assays, immunoblotting and immunocytochemistry were used to phenotypically characterize drug responses of two cell models BT474R and SKBR3R. Student's t-test and Spearman's correlation were applied for statistic analysis. The activity of a downstream effector of the HER2 pathway phosphorylated ribosomal protein S6 (p-rpS6), was suppressed by trastuzumab in the parental cell lines yet remained unchanged in the resistant cells following treatment. The level of p-rpS6 was inversely correlated to the drug induced growth inhibition of trastuzumab-resistant cells when they are treated with selected HER2 targeting drugs. p-rpS6 is a robust post-treatment indicator of HER2 pathway-targeted therapy resistance.

p21-activated kinases and gastrointestinal cancer.

PubMed

He, Hong; Baldwin, Graham S

2013-01-01

p21-activated kinases (PAKs) were initially identified as effector proteins downstream from GTPases of the Rho family. To date, six members of the PAK family have been discovered in mammalian cells. PAKs play important roles in growth factor signalling, cytoskeletal remodelling, gene transcription, cell proliferation and oncogenic transformation. A large body of research has demonstrated that PAKs are up-regulated in several human cancers, and that their overexpression is linked to tumour progression and resistance to therapy. Structural and biochemical studies have revealed the mechanisms involved in PAK signalling, and opened the way to the development of PAK-targeted therapies for cancer treatment. Here we summarise recent findings from biological and clinical research on the role of PAKs in gastrointestinal cancer, and discuss the current status of PAK-targeted anticancer therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

mAKAP – A Master Scaffold for Cardiac Remodeling

PubMed Central

Passariello, Catherine L.; Li, Jinliang; Dodge-Kafka, Kimberly; Kapiloff, Michael S.

2014-01-01

Cardiac remodeling is regulated by an extensive intracellular signal transduction network. Each of the many signaling pathways in this network contributes uniquely to the control of cellular adaptation. In the last few years, it has become apparent that multimolecular signaling complexes or ‘signalosomes’ are important for fidelity in intracellular signaling and for mediating crosstalk between the different signaling pathways. These complexes integrate upstream signals and control downstream effectors. In the cardiac myocyte, the protein mAKAPβ serves as a scaffold for a large signalosome that is responsive to cAMP, calcium, hypoxia, and mitogen-activated protein kinase signaling. The main function of mAKAPβ signalosomes is to modulate stress-related gene expression regulated by the transcription factors NFATc, MEF2 and HIF-1α and type II histone deacetylases that control pathological cardiac hypertrophy. PMID:25551320

Effect of aging on microRNAs and regulation of pathogen recognition receptors

PubMed Central

Olivieri, Fabiola; Procopio, Antonio Dormenico

2014-01-01

Immunosenescence is the multifactorial age-associated immune deteriorization that leads to increased susceptibility to infections and decreased responses to vaccines. Recent studies have shown a fundamental role for microRNAs (miRNAs) in regulating immune responses, and nearly all the miRNAs involved in immune regulation show modulation during aging. Aging-associated miRNAs are largely negative regulators of the immune innate response and target central nodes of aging-associated networks, in particular, NF-κB, the downstream effector of TLR signals that leads to induction of proinflammatory responses. Multiple miRNAs have been reported to share similar regulatory activity. Here we review miRNA regulation of human innate immune recognition in aging, including both activation and resolution of inflammation, critical issues in detection, and areas of active investigation into our understanding of immunosenescence. PMID:24769423

HDAC9 promotes glioblastoma growth via TAZ-mediated EGFR pathway activation.

PubMed

Yang, Rui; Wu, Yanan; Wang, Mei; Sun, Zhongfeng; Zou, Jiahua; Zhang, Yundong; Cui, Hongjuan

2015-04-10

Histone deacetylase 9 (HDAC9), a member of class II HDACs, regulates a wide variety of normal and abnormal physiological functions. We found that HDAC9 is over-expressed in prognostically poor glioblastoma patients. Knockdown HDAC9 decreased proliferation in vitro and tumor formation in vivo. HDAC9 accelerated cell cycle in part by potentiating the EGFR signaling pathway. Also, HDAC9 interacted with TAZ, a key downstream effector of Hippo pathway. Knockdown of HDAC9 decreased the expression of TAZ. We found that overexpressed TAZ in HDAC9-knockdown cells abrogated the effects induced by HDAC9 silencing both in vitro and in vivo. We demonstrated that HDAC9 promotes tumor formation of glioblastoma via TAZ-mediated EGFR pathway activation, and provide the evidence for promising target for the treatment of glioblastoma.

The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment

PubMed Central

Johnson, Randy; Halder, Georg

2014-01-01

The Hippo signaling pathway is an emerging growth control and tumor suppressor pathway that regulates cell proliferation and stem cell functions. Defects in Hippo signaling and hyperactivation of its downstream effectors YAP and TAZ contribute to the development of cancer, suggesting that pharmacological inhibition of YAP and TAZ activity may be an effective anticancer strategy. Conversely, YAP and TAZ can also play beneficial roles in stimulating tissue repair and regeneration following injury, therefore activation of YAP and TAZ may be useful in these contexts. Recently, a complex network of intracellular and extracellular signaling pathways that modulate YAP and TAZ activities have been identified. Here we review the regulation of the Hippo signaling pathway, its functions in normal homeostasis and disease, and recent progress in the identification of small molecule pathway modulators. PMID:24336504

Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB.

PubMed

Zeng, Lei; Kuti, Miklos; Mujtaba, Shiraz; Zhou, Ming-Ming

2014-07-01

The fibroblast growth factor receptor (FGFR) substrate 2 (FRS2) family proteins function as scaffolding adapters for receptor tyrosine kinases (RTKs). The FRS2α proteins interact with RTKs through the phosphotyrosine-binding (PTB) domain and transfer signals from the activated receptors to downstream effector proteins. Here, we report the nuclear magnetic resonance structure of the FRS2α PTB domain bound to phosphorylated TrkB. The structure reveals that the FRS2α-PTB domain is comprised of two distinct but adjacent pockets for its mutually exclusive interaction with either nonphosphorylated juxtamembrane region of the FGFR, or tyrosine phosphorylated peptides TrkA and TrkB. The new structural insights suggest rational design of selective small molecules through targeting of the two conjunct pockets in the FRS2α PTB domain. © 2014 Wiley Periodicals, Inc.

The central amygdala circuits in fear regulation

NASA Astrophysics Data System (ADS)

Li, Bo

The amygdala is essential for fear learning and expression. The central amygdala (CeA), once viewed as a passive relay between the amygdala complex and downstream fear effectors, has emerged as an active participant in fear conditioning. However, how the CeA contributes to the learning and expression of fear remains unclear. Our recent studies in mice indicate that fear conditioning induces robust plasticity of excitatory synapses onto inhibitory neurons in the lateral subdivision of CeA (CeL). In particular, this plasticity is cell-type specific and is required for the formation of fear memory. In addition, sensory cues that predict threat can cause activation of the somatostatin-positive CeL neurons, which is sufficient to drive freezing behavior. Here I will report our recent findings regarding the circuit and cellular mechanisms underlying CeL function in fear processing.

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

PubMed

Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-07-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses

PubMed Central

Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2017-01-01

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation. PMID:28223697

Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

PubMed

Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2017-02-22

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

Dual-Color Luciferase Complementation for Chemokine Receptor Signaling.

PubMed

Luker, Kathryn E; Luker, Gary D

2016-01-01

Chemokine receptors may share common ligands, setting up potential competition for ligand binding, and association of activated receptors with downstream signaling molecules such as β-arrestin. Determining the "winner" of competition for shared effector molecules is essential for understanding integrated functions of chemokine receptor signaling in normal physiology, disease, and response to therapy. We describe a dual-color click beetle luciferase complementation assay for cell-based analysis of interactions of two different chemokine receptors, CXCR4 and ACKR3, with the intracellular scaffolding protein β-arrestin 2. This assay provides real-time quantification of receptor activation and signaling in response to chemokine CXCL12. More broadly, this general imaging strategy can be applied to quantify interactions of any set of two proteins that interact with a common binding partner. © 2016 Elsevier Inc. All rights reserved.

Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

PubMed Central

Servienė, Elena; Lukša, Juliana; Orentaitė, Irma

2012-01-01

Background Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. Principal Findings We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Significance Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action. PMID:23227207

Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

PubMed

Servienė, Elena; Lukša, Juliana; Orentaitė, Irma; Lafontaine, Denis L J; Urbonavičius, Jaunius

2012-01-01

Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frank, Evan A.; Birch, M. Eileen; Yadav, Jagjit S., E-mail: Jagjit.Yadav@uc.edu

Carbon nanotubes (CNTs) are rapidly emerging as high-priority occupational toxicants. CNT powders contain fibrous particles that aerosolize readily in places of manufacture and handling, posing an inhalation risk for workers. Studies using animal models indicate that lung exposure to CNTs causes prolonged inflammatory responses and diffuse alveolar injury. The mechanisms governing CNT-induced lung inflammation are not fully understood but have been suggested to involve alveolar macrophages (AMs). In the current study, we sought to systematically assess the effector role of AMs in vivo in the induction of lung inflammatory responses to CNT exposures and investigate their cell type-specific mechanisms. Multi-wallmore » CNTs characterized for various physicochemical attributes were used as the CNT type. Using an AM-specific depletion and repopulation approach in a mouse model, we unambiguously demonstrated that AMs are major effector cells necessary for the in vivo elaboration of CNT-induced lung inflammation. We further investigated in vitro AM responses and identified molecular targets which proved critical to pro-inflammatory responses in this model, namely MyD88 as well as MAPKs and Ca{sup 2} {sup +}/CamKII. We further demonstrated that MyD88 inhibition in donor AMs abrogated their capacity to reconstitute CNT-induced inflammation when adoptively transferred into AM-depleted mice. Taken together, this is the first in vivo demonstration that AMs act as critical effector cell types in CNT-induced lung inflammation and that MyD88 is required for this in vivo effector function. AMs and their cell type-specific mechanisms may therefore represent potential targets for future therapeutic intervention of CNT-related lung injury. - Highlights: • Demonstrated in vivo effector role of alveolar macrophages (AMs) in CNT toxicity • MyD88, MAPKs, and Ca{sup 2} {sup +}/CamKII are required for AM inflammatory responses in vitro. • MyD88 signaling is required for in vivo effector function of AMs. • MyD88 may be a potential target for intervention in CNT lung exposures.« less

Network biology discovers pathogen contact points in host protein-protein interactomes.

PubMed

Ahmed, Hadia; Howton, T C; Sun, Yali; Weinberger, Natascha; Belkhadir, Youssef; Mukhtar, M Shahid

2018-06-13

In all organisms, major biological processes are controlled by complex protein-protein interactions networks (interactomes), yet their structural complexity presents major analytical challenges. Here, we integrate a compendium of over 4300 phenotypes with Arabidopsis interactome (AI-1 MAIN ). We show that nodes with high connectivity and betweenness are enriched and depleted in conditional and essential phenotypes, respectively. Such nodes are located in the innermost layers of AI-1 MAIN and are preferential targets of pathogen effectors. We extend these network-centric analyses to Cell Surface Interactome (CSI LRR ) and predict its 35 most influential nodes. To determine their biological relevance, we show that these proteins physically interact with pathogen effectors and modulate plant immunity. Overall, our findings contrast with centrality-lethality rule, discover fast information spreading nodes, and highlight the structural properties of pathogen targets in two different interactomes. Finally, this theoretical framework could possibly be applicable to other inter-species interactomes to reveal pathogen contact points.

Crossed-uncrossed difference (CUD) in a new light: anatomy of the negative CUD in Poffenberger's paradigm.

PubMed

Derakhshan, I

2006-03-01

Crossed Uncrossed Differentials (CUDs) have long been used as surrogate for the interhemispheric transfer time (IHTT). Evidence is presented that macular vision is the province of the major hemisphere, wherein all commands are initiated regardless of the laterality of the effectors of such commands. Using clinical and time-resolved data it is shown also that the above arrangement (i.e. neural handedness) corresponds to the subject's behavioral avowed (avowed, self-declared) handedness only in a statistical sense; with a substantial minority of humanity displaying a disparity of neural and behavioral handedness. Evidence is provided that the negative CUD in previously reported studies was a reflection of such incongruity in those subjects studied. Thus, to lateralize the command center it is sufficient to determine the reaction time of two symmetrically located effectors on the body. The side with longer reaction time is ipsilateral to the major hemisphere, with the difference of the two sides commensurate to transcallosal IHTT.

Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

PubMed

D'Orsi, Beatrice; Bonner, Helena; Tuffy, Liam P; Düssmann, Heiko; Woods, Ina; Courtney, Michael J; Ward, Manus W; Prehn, Jochen H M

2012-02-01

Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

PubMed

Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

2016-02-01

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. Copyright © 2016 by the Genetics Society of America.

Growth of chronic myeloid leukemia cells is inhibited by infection with Ad-SH2-HA adenovirus that disrupts Grb2-Bcr-Abl complexes.

PubMed

Peng, Zhi; Luo, Hong-Wei; Yuan, Ying; Shi, Jing; Huang, Shi-Feng; Li, Chun-Li; Cao, Wei-Xi; Huang, Zong-Gan; Feng, Wen-Li

2011-05-01

The persistence of Bcr-Abl-positive cells in patients on imatinib therapy indicates that inhibition of the Bcr-Abl kinase activity alone might not be sufficient to eradicate the leukemia cells. Many downstream effectors of Bcr-Abl have been described, including activation of both the Grb2-SoS-Ras-MAPK and Grb2-Gab2-PI3K-Akt pathways. The Bcr-Abl-Grb2 interaction, which is mediated by the direct interaction of the Grb2 SH2 domain with the phospho-Bcr-Abl Y177, is required for activation of these signaling pathways. Therefore, disrupting their interaction represents a potential therapeutic strategy for inhibiting the oncogenic downstream signals of Bcr-Abl. Adenovirus Ad-SH2-HA expressing the Grb2 SH2 domain was constructed and applied in this study. As expected, Ad-SH2-HA efficiently infected CML cells and functioned by binding to the phospho-Bcr-Abl Y177 site, competitively disrupting the Grb2 SH2-phospho-Bcr-Abl Y177 complex. They induced potent anti-proliferation and apoptosis-inducing effects in CML cell lines. Moreover, the Ras, MAPK and Akt activities were significantly reduced in the Ad-SH2-HA treated cells. These were not observed with the point-mutated control adenovirus Ad-Sm-HA with abolished phospho-Bcr-Abl Y177 binding sites. These data indicate that, in addition to the direct targeting of Bcr-Abl, selective inhibition of its downstream signaling pathways may be a therapeutic option for CML, and the Ad-SH2-HA-mediated killing strategy could be explored as a promising anti-leukemia agent in CML.

T cells' immunological synapses induce polarization of brain astrocytes in vivo and in vitro: a novel astrocyte response mechanism to cellular injury.

PubMed

Barcia, Carlos; Sanderson, Nicholas S R; Barrett, Robert J; Wawrowsky, Kolja; Kroeger, Kurt M; Puntel, Mariana; Liu, Chunyan; Castro, Maria G; Lowenstein, Pedro R

2008-08-20

Astrocytes usually respond to trauma, stroke, or neurodegeneration by undergoing cellular hypertrophy, yet, their response to a specific immune attack by T cells is poorly understood. Effector T cells establish specific contacts with target cells, known as immunological synapses, during clearance of virally infected cells from the brain. Immunological synapses mediate intercellular communication between T cells and target cells, both in vitro and in vivo. How target virally infected astrocytes respond to the formation of immunological synapses established by effector T cells is unknown. Herein we demonstrate that, as a consequence of T cell attack, infected astrocytes undergo dramatic morphological changes. From normally multipolar cells, they become unipolar, extending a major protrusion towards the immunological synapse formed by the effector T cells, and withdrawing most of their finer processes. Thus, target astrocytes become polarized towards the contacting T cells. The MTOC, the organizer of cell polarity, is localized to the base of the protrusion, and Golgi stacks are distributed throughout the protrusion, reaching distally towards the immunological synapse. Thus, rather than causing astrocyte hypertrophy, antiviral T cells cause a major structural reorganization of target virally infected astrocytes. Astrocyte polarization, as opposed to hypertrophy, in response to T cell attack may be due to T cells providing a very focused attack, and thus, astrocytes responding in a polarized manner. A similar polarization of Golgi stacks towards contacting T cells was also detected using an in vitro allogeneic model. Thus, different T cells are able to induce polarization of target astrocytes. Polarization of target astrocytes in response to immunological synapses may play an important role in regulating the outcome of the response of astrocytes to attacking effector T cells, whether during antiviral (e.g. infected during HIV, HTLV-1, HSV-1 or LCMV infection), anti-transplant, autoimmune, or anti-tumor immune responses in vivo and in vitro.

Trio’s Rho-specific GEF domain is the missing Gαq effector in C. elegans

PubMed Central

Williams, Stacey L.; Lutz, Susanne; Charlie, Nicole K.; Vettel, Christiane; Ailion, Michael; Coco, Cassandra; Tesmer, John J.G.; Jorgensen, Erik M.; Wieland, Thomas; Miller, Kenneth G.

2007-01-01

The Gαq pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cβ [PLCβ]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Gαq pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCβ). Strains containing null mutations in both EGL-8 (PLCβ) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Gαq-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Gαq synergizes with Trio RhoGEF to activate RhoA. Activated Gαq and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio’s Rho-specific GEF domain is a major Gαq effector that, together with PLCβ, mediates the Gαq signaling that drives the locomotion, egg laying, and growth of the animal. PMID:17942708

Human symbionts inject and neutralize antibacterial toxins to persist in the gut.

PubMed

Wexler, Aaron G; Bao, Yiqiao; Whitney, John C; Bobay, Louis-Marie; Xavier, Joao B; Schofield, Whitman B; Barry, Natasha A; Russell, Alistair B; Tran, Bao Q; Goo, Young Ah; Goodlett, David R; Ochman, Howard; Mougous, Joseph D; Goodman, Andrew L

2016-03-29

The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes--one of two major phyla in the gut--also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.

T cell source of type 1 cytokines determines illness patterns in respiratory syncytial virus-infected mice.

PubMed Central

Tang, Y. W.; Graham, B. S.

1997-01-01

Manipulation of the cytokine microenvironment at the time of vaccination can influence immune responses to remote challenge, providing a strategy to study the molecular pathogenesis of respiratory syncytial virus (RSV) vaccine-enhanced disease in the mouse model. Although treatment with antibody against IL-4 or recombinant IL-12 (rIL-12) at the time of formalin-inactivated RSV vaccination induced a similar shift in the pattern of cytokine mRNA expression upon live virus challenge, anti-IL-4 treated mice had increased CD8+ cytotoxic T lymphocyte activity and reduced illness compared with rIL-12-treated mice. To define effector mechanisms responsible for these patterns, CD4+ and/or CD8+ T lymphocytes were selectively depleted in vivo at the time of RSV challenge. In rIL-12-treated mice, CD4+ lymphocytes made the largest contribution to IFN-gamma mRNA, RSV clearance, and illness, while in anti-IL-4 treated mice, CD8+ lymphocytes were the major effector. The effector responsible for virus clearance also mediated illness, suggesting that efficiency of virus clearance determined disease expression. These results demonstrate that the phenotype of effector cells involved in the immune response to virus challenge may be a more important determinant of disease than patterns of cytokine expression classically assigned to Th1 and Th2 lymphocytes. PMID:9151790

The host immune response to Clostridium difficile infection

PubMed Central

2013-01-01

Clostridium difficile infection (CDI) is the most common infectious cause of healthcare-acquired diarrhoea. Outcomes of C. difficile colonization are varied, from asymptomatic carriage to fulminant colitis and death, due in part to the interplay between the pathogenic virulence factors of the bacterium and the counteractive immune responses of the host. Secreted toxins A and B are the major virulence factors of C. difficile and induce a profound inflammatory response by intoxicating intestinal epithelial cells causing proinflammatory cytokine release. Host cell necrosis, vascular permeability and neutrophil infiltration lead to an elevated white cell count, profuse diarrhoea and in severe cases, dehydration, hypoalbuminaemia and toxic megacolon. Other bacterial virulence factors, including surface layer proteins and flagella proteins, are detected by host cell surface signal molecules that trigger downstream cell-mediated immune pathways. Human studies have identified a role for serum and faecal immunoglobulin levels in protection from disease, but the recent development of a mouse model of CDI has enabled studies into the precise molecular interactions that trigger the immune response during infection. Key effector molecules have been identified that can drive towards a protective anti-inflammatory response or a damaging proinflammatory response. The limitations of current antimicrobial therapies for CDI have led to the development of both active and passive immunotherapies, none of which have, as yet been formally approved for CDI. However, recent advances in our understanding of the molecular basis of host immune protection against CDI may provide an exciting opportunity for novel therapeutic developments in the future. PMID:25165542

Gα73B is a downstream effector of JAK/STAT signalling and a regulator of Rho1 in Drosophila haematopoiesis.

PubMed

Bausek, Nina; Zeidler, Martin P

2014-01-01

JAK/STAT signalling regulates many essential developmental processes including cell proliferation and haematopoiesis, whereas its inappropriate activation is associated with the majority of myeloproliferative neoplasias and numerous cancers. Furthermore, high levels of JAK/STAT pathway signalling have also been associated with enhanced metastatic invasion by cancerous cells. Strikingly, gain-of-function mutations in the single Drosophila JAK homologue, Hopscotch, result in haemocyte neoplasia, inappropriate differentiation and the formation of melanised haemocyte-derived 'tumour' masses; phenotypes that are partly orthologous to human gain-of-function JAK2-associated pathologies. Here we show that Gα73B, a novel JAK/STAT pathway target gene, is necessary for JAK/STAT-mediated tumour formation in flies. In addition, although Gα73B does not affect haemocyte differentiation, it does regulate haemocyte morphology and motility under non-pathological conditions. We show that Gα73B is required for constitutive, but not injury-induced, activation of Rho1 and for the localisation of Rho1 into filopodia upon haemocyte activation. Consistent with these results, we also show that Rho1 interacts genetically with JAK/STAT signalling, and that wild-type levels of Rho1 are necessary for tumour formation. Our findings link JAK/STAT transcriptional outputs, Gα73B activity and Rho1-dependent cytoskeletal rearrangements and cell motility, therefore connecting a pathway associated with cancer with a marker indicative of invasiveness. As such, we suggest a mechanism by which JAK/STAT pathway signalling may promote metastasis.

Type I interferon (IFN-alpha/beta) rescues B-lymphocytes from apoptosis via PI3Kdelta/Akt, Rho-A, NFkappaB and Bcl-2/Bcl(XL).

PubMed

Badr, Gamal; Saad, Heba; Waly, Hanan; Hassan, Khadega; Abdel-Tawab, Hanem; Alhazza, Ibrahim M; Ahmed, Emad A

2010-01-01

Although IFN-alpha was reported to promote the survival of peripheral B-lymphocytes via the PI3-kinase-Akt pathway, the triggered signalling pathways involved in the protection of B cell from apoptosis need to be clarified. Using flow cytometry and western blot analysis, we have found that type 1 IFNs (IFN-alpha/beta) protect human B cells in culture from spontaneous apoptosis and from apoptosis mediated by anti-CD95 agonist, in a dose- and time-dependant manner. IFN-alpha/beta-mediated anti-apoptotic effect on human B cells was totally abrogated by blockade of IFNR1 chain. Our data indicate that PI3Kdelta, Rho-A, NFkappaB and Bcl-2/Bcl(XL) are active downstream of IFN receptors and are the major effectors of IFN-alpha/beta-rescued B cells from apoptosis. Furthermore, immunohistochemical results show marked reduction in numbers of CD20 positive B cell in both spleen and Peyer's patches from mice treated with anti-IFNR1 blocking antibody compared with control group. Moreover, ultrastructural observations of these organs show an obvious increase in apoptotic cells from mice treated with anti-IFNR1 blocking antibody. Our results provide more details about the triggered signalling pathways and the phosphorylation cascade which are involved in the protection of B cell from apoptosis after treatment with IFN-alpha/beta. Copyright 2010 Elsevier Inc. All rights reserved.

Implantation failure in mice with a disruption in Phospholipase C beta 1 gene: lack of embryonic attachment, aberrant steroid hormone signalling and defective endocannabinoid metabolism

PubMed Central

Filis, Panayiotis; Kind, Peter C.; Spears, Norah

2013-01-01

Phospholipase C beta 1 (PLCβ1) is a downstream effector of G-protein-coupled receptor signalling and holds central roles in reproductive physiology. Mice with a disruption in the Plcβ1 gene are infertile with pleiotropic reproductive defects, the major reproductive block in females being implantation failure. Here, PLCβ1 was demonstrated at the luminal and glandular epithelia throughout the pre- and peri-implantation period, with transient stromal expression during 0.5–1.5 days post coitum (dpc). Examination of implantation sites at 4.5 dpc showed that in females lacking functional PLCβ1 (knock-out (KO) females), embryos failed to establish proper contact with the uterine epithelium. Proliferating luminal epithelial cells were evident in KO implantation sites, indicating failure to establish a receptive uterus. Real-time PCR demonstrated that KO implantation sites had aberrant ovarian steroid signalling, with high levels of estrogen receptor α, lactoferrin and amphiregulin mRNA, while immunohistochemistry revealed very low levels of estrogen receptor α protein, possibly due to rapid receptor turnover. KO implantation sites expressed markedly less fatty acid amide hydrolase and monoacylglycerol lipase, indicating that endocannabinoid metabolism was also affected. Collectively, our results show that PLCβ1 is essential for uterine preparation for implantation, and that defective PLCβ1-mediated signalling during implantation is associated with aberrant ovarian steroid signalling and endocannabinoid metabolism. PMID:23295235

A combinatorial strategy for treating KRAS mutant lung cancer

PubMed Central

Manchado, Eusebio; Weissmueller, Susann; Morris, John P.; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T.; Gainor, Justin F.; Corcoran, Ryan B.; Engelman, Jeffrey A.; Rudin, Charles M.; Rosen, Neal; Lowe, Scott W.

2016-01-01

Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proven difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, an FDA-approved MEK inhibitor that acts downstream of KRAS to suppress signaling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA (shRNA) screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signaling rebound and adaptive drug resistance. As a consequence, genetic or pharmacologic inhibition of FGFR1 in combination with trametinib enhances tumor cell death in vitro and in vivo. This compensatory response shows distinct specificities – it is dominated by FGFR1 in KRAS mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patient tumors treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer. PMID:27338794

STAT5 Is Crucial to Maintain Leukemic Stem Cells in Acute Myelogenous Leukemias Induced by MOZ-TIF2

PubMed Central

Tam, Winnie F.; Hähnel, Patricia S.; Schüler, Andrea; Lee, Benjamin H.; Okabe, Rachel; Zhu, Nan; Pante, Saskia V.; Raffel, Glen; Mercher, Thomas; Wernig, Gerlinde; Bockamp, Ernesto; Sasca, Daniel; Kreft, Andreas; Robinson, Gertraud W.; Hennighausen, Lothar; Gilliland, D. Gary; Kindler, Thomas

2014-01-01

MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2–induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2–transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5−/− MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5+/+ MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option. PMID:23149921

Trypanosoma cruzi trans-sialidase: A potent and specific survival factor for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling

PubMed Central

Chuenkova, Marina V.; Furnari, Frank B.; Cavenee, Webster K.; Pereira, Miercio A.

2001-01-01

Patients infected with Trypanosoma cruzi may remain asymptomatic for decades and show signs of neuroregeneration in the peripheral nervous system (PNS). In the absence of such neuroregeneration, patients may die in part by extensive neuronal destruction in the gastrointestinal tract. Thus, T. cruzi may, despite their invasion of the PNS, directly prevent cell death to keep nerve destruction in check. Indeed, T. cruzi invasion of Schwann cells, their prime target in PNS, suppressed host-cell apoptosis caused by growth-factor deprivation. The trans-sialidase (TS) of T. cruzi and the Cys-rich domain of TS reproduced the antiapoptotic activity of the parasites at doses (≥3.0 nM) comparable or lower than those of bona fide mammalian growth factors. This effect was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). TS also activated Akt, a downstream effector of PI3K. Ectopic expression of TS in an unrelated parasite, Leishmania major, turned those parasites into activators of Akt in Schwann cells. In contrast, the Cys-rich domain of TS did not block apoptosis in Schwann cells overexpressing dominant-negative Akt or constitutively active PTEN, a negative regulator of PI3K/Akt signaling. The results demonstrate that T. cruzi, through its TS, triggers the survival of host Schwann cells via the PI3K/Akt pathway, suggesting a role for PI3K/Akt in the pathogenesis of Chagas' disease. PMID:11481434

Role of Rac1/WAVE2 Signaling in Mediating the Inhibitory Effects of γ-Tocotrienol on Mammary Cancer Cell Migration and Invasion.

PubMed

Algayadh, Ibrahim Gayadh; Dronamraju, Venkateshwararao; Sylvester, Paul William

2016-01-01

The majority of breast cancer deaths result from the progression of this disease to a metastatic phenotype. Rac1 and Cdc42 are Rho family members that together with their downstream effectors, Wiskott-Aldrich Syndrome protein-family verprolin-homologous protein 2 (WAVE2) and Arp2/3, play an important role in cytoskeletal reorganization and the formation of membrane protrusions that promote cancer cell migration and invasion. γ-Tocotrienol, is a natural isoform within the vitamin E family of compounds that inhibits breast cancer cell growth and progression by suppressing various signaling pathways involved in mitogenic signaling and metastatic progression. Studies were conducted to examine the effects of γ-tocotrienol on Rac1/WAVE2 signaling dependent migration and invasion in highly metastatic mouse +SA and human MDA-MB-231 mammary cancer cells. Exposure to γ-tocotrienol resulted in a dose-responsive decrease in Rac1/WAVE2 signaling as characterized by a suppression in the levels of Rac1/Cdc42, phospho-Rac1/Cdc42, WAVE2, Arp2, and Arp3 expression. Additional studies also demonstrated that similar treatment with γ-tocotrienol resulted in a significant reduction in tumor cell migration and invasion. Taken together, these findings indicate that γ-tocotrienol treatment effectively inhibits Rac1/WAVE2 signaling and reduces metastatic phenotypic expression in mammary cancer cells, suggesting that γ-tocotrienol may provide some benefit as a novel therapeutic approach in the treatment of metastatic breast cancer.

Noncanonical transforming growth factor β signaling in scleroderma fibrosis

PubMed Central

Trojanowska, Maria

2014-01-01

Purpose of review Persistent transforming growth factor β (TGF-β) signaling is the major factor contributing to scleroderma (SSc) fibrosis. This review will summarize recent progress on the noncanonical TGF-β signaling pathways and their role in SSc fibrosis. Recent findings Canonical TGF-β signaling involves activation of the TGF-β receptors and downstream signal transducers Smad2/3. The term noncanonical TGF-β signaling includes a variety of intracellular signaling pathways activated by TGF-β independently of Smad2/3 activation. There is evidence that these pathways play important role in SSc fibrosis. In a subset of SSc fibroblasts, a multiligand receptor complex consisting of TGF-β and CCN2 receptors drives constitutive activation of the Smad1 pathway. CCN2 is also a primary effector of this pathway, thus establishing an autocrine loop that amplifies TGF-β signaling. SSc fibroblasts also demonstrate reduced expression of endogenous antagonists of TGF-β signaling including transcriptional repressors, Friend leukemia integration-1 and perixosome proliferator-activated receptor-γ, as well as inhibitor of Smad3 phosphorylation, PTEN. PTEN is a key mediator of the cross-talk between the sphingosine kinase and the TGF-β pathways. Summary Discovery of the role of noncanonical TGF-β signaling in fibrosis offers new molecular targets for the antifibrotic therapies. Due to the heterogeneous nature of SSc, knowledge of these pathways could help to tailor the therapy to the individual patient depending on the activation status of a specific profibrotic pathway. PMID:19713852

Distinct Roles for CXCR6(+) and CXCR6(-) CD4(+) T Cells in the Pathogenesis of Chronic Colitis.

PubMed

Mandai, Yasushi; Takahashi, Daisuke; Hase, Koji; Obata, Yuuki; Furusawa, Yukihiro; Ebisawa, Masashi; Nakagawa, Tomoo; Sato, Toru; Katsuno, Tatsuro; Saito, Yasushi; Shimaoka, Takeshi; Yokosuka, Osamu; Yokote, Kotaro; Ohno, Hiroshi

2013-01-01

CD4(+) T cells play a central role in the development of inflammatory bowel disease (IBD) via high-level production of effector cytokines such as IFN-γ and TNF-α. To better characterize the colitogenic CD4(+) T cells, we examined their expression of CXCR6, a chemokine receptor that is expressed by T cells upon activation and is upregulated in several inflammatory diseases. We found that 80% of colonic lamina propria CD4(+) T cells expressed CXCR6 in the CD45RB(high) T cell-transferred colitis model. CXCR6 expression was similarly upregulated in inflamed mucosa of patients with Crohn's disease. Although surface marker analysis demonstrated that both CXCR6(+) and CXCR6(-) CD4(+) T-cell subsets consist of the cells with effector and effector-memory cells, the more cells in the CXCR6(+) subset produced IFN-γ and TNF-α compared to CXCR6(-) subset, and only the CXCR6(+) subset produced IL-17A. Nevertheless, adoptive retransfer of lamina propria CXCR6(+) T cells into Rag1 (-/-) recipients failed to induce the disease due to limited expansion of the transferred cells. By contrast, retransfer of CXCR6(-) cells evoked colitis similar to that observed in CD4(+)CD45RB(high) T cell-transferred mice, and resulted in their conversion into CXCR6(+) cells. Collectively, these observations suggest that the CXCR6(+)CD4(+) T-cell subset consists of terminally differentiated effector cells that serve as the major source of effector cytokines in the inflamed tissue, whereas CXCR6(-)CD4(+) T-cell subset serves as a colitogenic memory compartment that retains the ability to proliferate and differentiate into CXCR6(+)CD4(+) T cells.

Distinct Roles for CXCR6+ and CXCR6− CD4+ T Cells in the Pathogenesis of Chronic Colitis

PubMed Central

Hase, Koji; Obata, Yuuki; Furusawa, Yukihiro; Ebisawa, Masashi; Nakagawa, Tomoo; Sato, Toru; Katsuno, Tatsuro; Saito, Yasushi; Shimaoka, Takeshi; Yokosuka, Osamu; Yokote, Kotaro; Ohno, Hiroshi

2013-01-01

CD4+ T cells play a central role in the development of inflammatory bowel disease (IBD) via high-level production of effector cytokines such as IFN-γ and TNF-α. To better characterize the colitogenic CD4+ T cells, we examined their expression of CXCR6, a chemokine receptor that is expressed by T cells upon activation and is upregulated in several inflammatory diseases. We found that 80% of colonic lamina propria CD4+ T cells expressed CXCR6 in the CD45RBhigh T cell-transferred colitis model. CXCR6 expression was similarly upregulated in inflamed mucosa of patients with Crohn’s disease. Although surface marker analysis demonstrated that both CXCR6+ and CXCR6− CD4+ T-cell subsets consist of the cells with effector and effector-memory cells, the more cells in the CXCR6+ subset produced IFN-γ and TNF-α compared to CXCR6− subset, and only the CXCR6+ subset produced IL-17A. Nevertheless, adoptive retransfer of lamina propria CXCR6+ T cells into Rag1 −/− recipients failed to induce the disease due to limited expansion of the transferred cells. By contrast, retransfer of CXCR6− cells evoked colitis similar to that observed in CD4+CD45RBhigh T cell-transferred mice, and resulted in their conversion into CXCR6+ cells. Collectively, these observations suggest that the CXCR6+CD4+ T-cell subset consists of terminally differentiated effector cells that serve as the major source of effector cytokines in the inflamed tissue, whereas CXCR6−CD4+ T-cell subset serves as a colitogenic memory compartment that retains the ability to proliferate and differentiate into CXCR6+CD4+ T cells. PMID:23840334

Single-Cell Tracking Reveals a Role for Pre-Existing CCR5+ Memory Th1 Cells in the Control of Rhinovirus-A39 After Experimental Challenge in Humans.

PubMed

Muehling, Lyndsey M; Turner, Ronald B; Brown, Kenneth B; Wright, Paul W; Patrie, James T; Lahtinen, Sampo J; Lehtinen, Markus J; Kwok, William W; Woodfolk, Judith A

2018-01-17

Little is known about T cells that respond to human rhinovirus in vivo, due to timing of infection, viral diversity, and complex T-cell specificities. We tracked circulating CD4+ T cells with identical epitope specificities that responded to intranasal challenge with rhinovirus (RV)-A39, and we assessed T-cell signatures in the nose. Cells were monitored using a mixture of 2 capsid-specific major histocompatibility complex II tetramers over a 7-week period, before and after RV-A39 challenge, in 16 human leukocyte antigen-DR4+ subjects who participated in a trial of Bifidobacterium lactis (Bl-04) supplementation. Pre-existing tetramer+ T cells were linked to delayed viral shedding, enriched for activated CCR5+ Th1 effectors, and included a minor interleukin-21+ T follicular helper cell subset. After RV challenge, expansion and activation of virus-specific CCR5+ Th1 effectors was restricted to subjects who had a rise in neutralizing antibodies, and tetramer-negative CCR5+ effector memory types were comodulated. In the nose, CXCR3-CCR5+ T cells present during acute infection were activated effector memory type, whereas CXCR3+ cells were central memory type, and cognate chemokine ligands were elevated over baseline. Probiotic had no T-cell effects. We conclude that virus-specific CCR5+ effector memory CD4+ T cells primed by previous exposure to related viruses contribute to the control of rhinovirus. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Uncovering the Legionella genus effector repertoire - strength in diversity and numbers

PubMed Central

Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

2016-01-01

Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution. PMID:26752266

Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

PubMed

Tanaka, Shigeyuki; Djamei, Armin; Presti, Libera Lo; Schipper, Kerstin; Winterberg, Sarah; Amati, Simone; Becker, Dirk; Büchner, Heike; Kumlehn, Jochen; Reissmann, Stefanie; Kahmann, Regine

2015-01-01

The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery. Copyright © 2015 Elsevier GmbH. All rights reserved.

Piperine from black pepper inhibits activation-induced proliferation and effector function of T lymphocytes.

PubMed

Doucette, Carolyn D; Rodgers, Gemma; Liwski, Robert S; Hoskin, David W

2015-11-01

Piperine is a major alkaloid component of black pepper (Piper nigrum Linn), which is a widely consumed spice. Here, we investigated the effect of piperine on mouse T lymphocyte activation. Piperine inhibited polyclonal and antigen-specific T lymphocyte proliferation without affecting cell viability. Piperine also suppressed T lymphocyte entry into the S and G2 /M phases of the cell cycle, and decreased expression of G1 -associated cyclin D3, CDK4, and CDK6. In addition, piperine inhibited CD25 expression, synthesis of interferon-γ, interleukin (IL)-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. The inhibitory effect of piperine on T lymphocytes was associated with hypophosphorylation of Akt, extracellular signal-regulated kinase, and inhibitor of κBα, but not ZAP-70. The ability of piperine to inhibit several key signaling pathways involved in T lymphocyte activation and the acquisition of effector function suggests that piperine might be useful in the management of T lymphocyte-mediated autoimmune and chronic inflammatory disorders. © 2015 Wiley Periodicals, Inc.

Antigen-dependent proliferation and cytokine induction in respiratory syncytial virus-infected cotton rats reflect the presence of effector-memory T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richter, Bettina W.M.; Onuska, Jaya M.; Niewiesk, Stefan

2005-06-20

Respiratory syncytial virus (RSV) is a major cause of lower airway disease in infants and children. Immunity to RSV is not long lasting, resulting in re-occurring infections throughout life. Effective long-lived immunity results when central-memory T cells that proliferate vigorously and secrete IL-2 are present. In contrast, effector-memory T cells that mainly produce IFN-{gamma}, facilitate virus clearance but are not long lived. To identify the type of memory response induced after RSV-A (Long) infection, we characterized the kinetics of the antigen-specific immune response and identified the types of cytokines induced. RSV-specific lymphocytic proliferation following primary and secondary infection was similar,more » and in both cases responses waned within a short period of time. In addition, mRNA for IFN-{gamma} but not IL-2 was induced in RSV-specific CD4{sup +} T cells. This supports the idea that the presence of effector-memory rather than central-memory T cells contributes to the ineffectiveness of the immune response to RSV.« less

[Effector proteins of Clamidia].

PubMed

Kariagina, A S; Alekseevskiĭ, A V; Spirin, S A; Zigangirova, N A; Gintsburg, A L

2009-01-01

The review summarizes the recent published data on molecular mechanisms of Chlamidiae - host cell interaction, first of all on chlamydial effector proteins. Such proteins as well as III transport system proteins that transfer many effector proteins into host cytoplasm are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of the infection. Patogen proteins participating in the next stage, that is the intracellular inclusion traffic to the centrosome, are CT229 of C. trachomatis and Cpn0585 of C. pneumoniae, which interact with cellular Rab GTPases. In C. trachomatis, IncA protein plays a key role in chlamydial inclusions fusion, CT847 modulates life cycle of the host cell, LDA3 is essential in acquisition of nutrients. CPAF protease and inclusion membrane proteins IncG and CADD participate in suppression of apoptosis of infected cells. The proteases CPAF and CT441, as well as deubiquitinating ChlaDub1 protein, contribute to avoiding the immune response.

MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

PubMed Central

2010-01-01

Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer. PMID:20409325

Downstream Petroleum Mergers and Acquisitions by U.S. Major Oil Companies

EIA Publications

2009-01-01

A summary presentation of mergers and acquisitions by U.S. major oil companies (including the U.S. affiliates of foreign major oil companies). The presentation focuses on petroleum refining over the last several years through late 2009.

Suppression or activation of immune responses by predicted secreted proteins of the soybean rust pathogen Phakopsora pachyrhizi

USDA-ARS?s Scientific Manuscript database

Rust fungi, such as Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are intimately associated with plant cells. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characte...

Genome-scale identification of Legionella pneumophila effectors using a machine learning approach.

PubMed

Burstein, David; Zusman, Tal; Degtyar, Elena; Viner, Ram; Segal, Gil; Pupko, Tal

2009-07-01

A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF) was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine learning algorithms for the identification and characterization of bacterial pathogenesis determinants.

Analysis of the cytochrome c-dependent apoptosis apparatus in cells from human pancreatic carcinoma

PubMed Central

Gerhard, M C; Schmid, R M; Häcker, G

2002-01-01

Defects in the apoptotic system are likely to play a role in tumorigenesis. Pancreatic carcinoma cells are extremely resistant to apoptosis induction by chemotherapy suggesting that the apoptosis machinery is faulty. We investigated the integrity of the cytochrome c-dependent apoptotic apparatus in 10 human pancreatic carcinoma cell lines. Expression of Apaf-1, caspase-3, -6, -7, -8 and -9, Hsp-70 and XIAP was detected in all cell lines. The expression levels of Apaf-1 and caspase-8 were homogenous in all cell lines whereas differences in expression of other caspases were seen. In cytosolic fractions, all investigated caspases were processed in response to cytochrome c but the extent of processing varied between the cell lines. No stringent correlation between the amount of processing of caspase-9 and effector caspases was seen. Cytochrome c-induced effector caspase activity was quantitated by enzyme assay. Especially at low concentrations of added cytochrome c, this response varied greatly between the cell lines. These data demonstrate that the apoptotic system downstream of the mitochondria is qualitatively intact in pancreatic carcinoma. They further show that the response to cytochrome c can be quantitated in a cell-free system and that determinants other than mere expression of apoptotic molecules can regulate cytochrome c-induced apoptosis. British Journal of Cancer (2002) 86, 893–898. DOI: 10.1038/sj/bjc/6600171 www.bjcancer.com © 2002 Cancer Research UK PMID:11953820

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development.

PubMed

Lu, Cecilia S; Zhai, Bo; Mauss, Alex; Landgraf, Matthias; Gygi, Stephen; Van Vactor, David

2014-09-26

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

PubMed Central

Lu, Cecilia S.; Zhai, Bo; Mauss, Alex; Landgraf, Matthias; Gygi, Stephen; Van Vactor, David

2014-01-01

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins. PMID:25135978

Cell Intrinsic Galectin-3 Attenuates Neutrophil ROS-Dependent Killing of Candida by Modulating CR3 Downstream Syk Activation

PubMed Central

Wu, Sheng-Yang; Huang, Juin-Hua; Chen, Wen-Yu; Chan, Yi-Chen; Lin, Chun-Hung; Chen, Yee-Chun; Liu, Fu-Tong; Wu-Hsieh, Betty A.

2017-01-01

Invasive candidiasis is a leading cause of nosocomial bloodstream infection. Neutrophils are the important effector cells in host resistance to candidiasis. To investigate the modulation of neutrophil fungicidal function will advance our knowledge on the control of candidiasis. While recombinant galectin-3 enhances neutrophil phagocytosis of Candida, we found that intracellular galectin-3 downregulates neutrophil fungicidal functions. Co-immunoprecipitation and immunofluorescence staining reveal that cytosolic gal3 physically interacts with Syk in neutrophils after Candida stimulation. Gal3−/− neutrophils have higher level of Syk activation as well as greater abilities to generate reactive oxygen species (ROS) and kill Candida than gal3+/+ cells. While galectin-3 deficiency modulates neutrophil and macrophage activation and the recruitment of monocytes and dendritic cells, the deficiency does not affect the numbers of infiltrating neutrophils or macrophages. Galectin-3 deficiency ameliorates systemic candidiasis by reducing fungal burden, renal pathology, and mortality. Adoptive transfer experiments demonstrate that cell intrinsic galectin-3 negatively regulates neutrophil effector functions against candidiasis. Reducing galectin-3 expression or activity by siRNA or gal3 inhibitor TD139 enhances human neutrophil ROS production. Mice treated with TD139 have enhanced ability to clear the fungus. Our work unravels the mechanism by which galectin-3 regulates Syk-dependent neutrophil fungicidal functions and raises the possibility that blocking gal3 in neutrophils may be a promising therapeutic strategy for treating systemic candidiasis. PMID:28217127

The Influence of Macronutrients on Splanchnic and Hepatic Lymphocytes in Aging Mice.

PubMed

Le Couteur, David G; Tay, Szun S; Solon-Biet, Samantha; Bertolino, Patrick; McMahon, Aisling C; Cogger, Victoria C; Colakoglu, Feyza; Warren, Alessandra; Holmes, Andrew J; Pichaud, Nicolas; Horan, Martin; Correa, Carolina; Melvin, Richard G; Turner, Nigel; Ballard, J William O; Ruohonen, Kari; Raubenheimer, David; Simpson, Stephen J

2015-12-01

There is a strong association between aging, diet, and immunity. The effects of macronutrients and energy intake on splanchnic and hepatic lymphocytes were studied in 15 month old mice. The mice were ad-libitum fed 1 of 25 diets varying in the ratios and amounts of protein, carbohydrate, and fat over their lifetime. Lymphocytes in liver, spleen, Peyers patches, mesenteric lymph nodes, and inguinal lymph nodes were evaluated using flow cytometry. Low protein intake reversed aging changes in splenic CD4 and CD8 T cells, CD4:CD8 T cell ratio, memory/effector CD4 T cells and naïve CD4 T cells. A similar influence of total caloric intake in these ad-libitum fed mice was not apparent. Protein intake also influenced hepatic NK cells and B cells, while protein to carbohydrate ratio influenced hepatic NKT cells. Hepatosteatosis was associated with increased energy and fat intake and changes in hepatic Tregs, effector/memory T, and NK cells. Hepatic NK cells were also associated with body fat, glucose tolerance, and leptin levels while hepatic Tregs were associated with hydrogen peroxide production by hepatic mitochondria. Dietary macronutrients, particularly protein, influence splanchnic lymphocytes in old age, with downstream associations with mitochondrial function, liver pathology, and obesity-related phenotype. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Intracellular signaling by phospholipase D as a therapeutic target.

PubMed

Steed, P M; Chow, A H

2001-09-01

The pharmaceutical industry has recently focused on intracellular signaling as a means to integrate the multiple facets of complex disease states, such as inflammation, because these pathways respond to numerous extracellular signals and coordinate a collection of cell responses contributing to pathology. One critical aspect of intracellular signaling is regulation of key cell functions by lipid mediators, in particular the generation of a key mediator, phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine by phospholipase D (PLD). Research in this field has intensified, due in part to the recent cloning and partial characterization of the two PLD isoforms in mammalian cells, and this work has contributed significantly to our understanding of events downstream of PA generation. It is these effector functions of PLD activity that make this pathway attractive as a therapeutic target while the biochemical properties of the PLD isozymes make them amenable to small molecule intervention. Recent studies indicate that PA, and its immediate metabolites diacylglycerol and lyso-PA, affect numerous cellular pathways including ligand-mediated secretion, cytoskeletal reorganisations, respiratory burst, prostaglandin release, cell migration, cytokine release, and mitogenesis. This review summarises the data implicating signaling via PLD in these cell functions, obtained from: (i) molecular analyses of PLD/effector interactions, (ii) correlation between PA production and cell responses, (iii) experimental manipulation of PA levels, (iv) inhibition of PLD regulators, and (v) direct inhibition of PA production. The utility of targeting PLD signaling for the treatment of acute/chronic inflammation and other indications is discussed in light of these data.

G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

PubMed Central

El Far, Oussama; Betz, Heinrich

2002-01-01

G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

Multilayered Regulation of Ethylene Induction Plays a Positive Role in Arabidopsis Resistance against Pseudomonas syringae1[OPEN

PubMed Central

Guan, Rongxia; Su, Jianbin; Meng, Xiangzong; Li, Sen; Liu, Yidong; Xu, Juan; Zhang, Shuqun

2015-01-01

Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens. However, its function in plant bacterial resistance remains unclear. Here, we report a detailed analysis of ethylene induction in Arabidopsis (Arabidopsis thaliana) in response to Pseudomonas syringae pv tomato DC3000 (Pst). Ethylene biosynthesis is highly induced in both pathogen/microbe-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI), and the induction is potentiated by salicylic acid (SA) pretreatment. In addition, Pst actively suppresses PAMP-triggered ethylene induction in a type III secretion system-dependent manner. SA potentiation of ethylene induction is dependent mostly on MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and MPK3 and their downstream ACS2 and ACS6, two type I isoforms of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs). ACS7, a type III ACS whose expression is enhanced by SA pretreatment, is also involved. Pst expressing the avrRpt2 effector gene (Pst-avrRpt2), which is capable of triggering ETI, induces a higher level of ethylene production, and the elevated portion is dependent on SALICYLIC ACID INDUCTION DEFICIENT2 and NONEXPRESSER OF PATHOGENESIS-RELATED GENE1, two key players in SA biosynthesis and signaling. High-order ACS mutants with reduced ethylene induction are more susceptible to both Pst and Pst-avrRpt2, demonstrating a positive role of ethylene in plant bacterial resistance mediated by both PAMP-triggered immunity and ETI. PMID:26265775

Microenvironment mesenchymal cells protect ovarian cancer cell lines from apoptosis by inhibiting XIAP inactivation

PubMed Central

Castells, M; Milhas, D; Gandy, C; Thibault, B; Rafii, A; Delord, J-P; Couderc, B

2013-01-01

Epithelial ovarian carcinoma is characterized by high frequency of recurrence (70% of patients) and carboplatin resistance acquisition. Carcinoma-associated mesenchymal stem cells (CA-MSC) have been shown to induce ovarian cancer chemoresistance through trogocytosis. Here we examined CA-MSC properties to protect ovarian cancer cells from carboplatin-induced apoptosis. Apoptosis was determined by Propidium Iodide and Annexin-V-FITC labelling and poly-ADP-ribose polymerase cleavage analysis. We showed a significant increase of inhibitory concentration 50 and a 30% decrease of carboplatin-induced apoptosis in ovarian cancer cells incubated in the presence of CA-MSC-conditioned medium (CM). A molecular analysis of apoptosis signalling pathway in response to carboplatin revealed that the presence of CA-MSC CM induced a 30% decrease of effector caspases-3 and -7 activation and proteolysis activity. CA-MSC secretions promoted Akt and X-linked inhibitor of apoptosis protein (XIAP; caspase inhibitor from inhibitor of apoptosis protein (IAP) family) phosphorylation. XIAP depletion by siRNA strategy permitted to restore apoptosis in ovarian cancer cells stimulated by CA-MSC CM. The factors secreted by CA-MSC are able to confer chemoresistance to carboplatin in ovarian cancer cells through the inhibition of effector caspases activation and apoptosis blockade. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway and the phosphorylation of its downstream target XIAP underlined the implication of this signalling pathway in ovarian cancer chemoresistance. This study reveals the potentialities of targeting XIAP in ovarian cancer therapy. PMID:24176845

Serum-Nutrient Starvation Induces Cell Death Mediated by Bax and Puma That Is Counteracted by p21 and Unmasked by Bcl-xL Inhibition

PubMed Central

Braun, Frédérique; Bertin-Ciftci, Joséphine; Gallouet, Anne-Sophie; Millour, Julie; Juin, Philippe

2011-01-01

The cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) is a multifunctional protein known to promote cell cycle arrest and survival in response to p53-dependent and p53 independent stimuli. We herein investigated whether and how it might contribute to the survival of cancer cells that are in low-nutrient conditions during tumour growth, by culturing isogenic human colorectal cancer cell lines (HCT116) and breast cancer cell lines in a medium deprived in amino acids and serum. We show that such starvation enhances, independently from p53, the expression of p21 and that of the pro-apoptotic BH3-only protein Puma. Under these conditions, p21 prevents Puma and its downstream effector Bax from triggering the mitochondrial apoptotic pathway. This anti-apoptotic effect is exerted from the cytosol but it is unrelated to the ability of p21 to interfere with the effector caspase 3. The survival function of p21 is, however, overcome by RNA interference mediated Bcl-xL depletion, or by the pharmacological inhibitor ABT-737. Thus, an insufficient supply in nutrients may not have an overt effect on cancer cell viability due to p21 induction, but it primes these cells to die, and sensitizes them to the deleterious effects of Bcl-xL inhibitors regardless of their p53 status. PMID:21887277

Transfer of Motor Learning Is More Pronounced in Proximal Compared to Distal Effectors in Upper Extremities

PubMed Central

Aune, Tore K.; Aune, Morten A.; Ingvaldsen, Rolf P.; Vereijken, Beatrix

2017-01-01

The current experiment investigated generalizability of motor learning in proximal versus distal effectors in upper extremities. Twenty-eight participants were divided into three groups: training proximal effectors, training distal effectors, and no training control group (CG). Performance was tested pre- and post-training for specific learning and three learning transfer conditions: (1) bilateral learning transfer between homologous effectors, (2) lateral learning transfer between non-homologous effectors, and (3) bilateral learning transfer between non-homologous effectors. With respect to specific learning, both training groups showed significant, similar improvement for the trained proximal and distal effectors, respectively. In addition, there was significant learning transfer to all three transfer conditions, except for bilateral learning transfer between non-homologous effectors for the distal training group. Interestingly, the proximal training group showed significantly larger learning transfer to other effectors compared to the distal training group. The CG did not show significant improvements from pre- to post-test. These results show that learning is partly effector independent and generalizable to different effectors, even though transfer is suboptimal compared to specific learning. Furthermore, there is a proximal-distal gradient in generalizability, in that learning transfer from trained proximal effectors is larger than from trained distal effectors, which is consistent with neuroanatomical differences in activation of proximal and distal muscles. PMID:28943857

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

PubMed

Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

2012-05-01

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

Phorbol ester phorbol-12-myristate-13-acetate promotes anchorage-independent growth and survival of melanomas through MEK-independent activation of ERK1/2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jorgensen, Kjersti; Skrede, Martina; Cruciani, Veronique

2005-04-01

The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotidesmore » against PKC-{alpha}, or the PKC inhibitor Goe6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.« less

DISCO interacting protein 2 determines direction of axon projection under the regulation of c-Jun N-terminal kinase in the Drosophila mushroom body

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nitta, Yohei; Brain Research Institute, Niigata University; Sugie, Atsushi

Precisely controlled axon guidance for complex neuronal wiring is essential for appropriate neuronal function. c-Jun N-terminal kinase (JNK) was found to play a role in axon guidance recently as well as in cell proliferation, protection and apoptosis. In spite of many genetic and molecular studies on these biological processes regulated by JNK, how JNK regulates axon guidance accurately has not been fully explained thus far. To address this question, we use the Drosophila mushroom body (MB) as a model since the α/β axons project in two distinct directions. Here we show that DISCO interacting protein 2 (DIP2) is required formore » the accurate direction of axonal guidance. DIP2 expression is under the regulation of Basket (Bsk), the Drosophila homologue of JNK. We additionally found that the Bsk/DIP2 pathway is independent from the AP-1 transcriptional factor complex pathway, which is directly activated by Bsk. In conclusion, our findings revealed DIP2 as a novel effector downstream of Bsk modulating the direction of axon projection. - Highlights: • DIP2 is required for accurate direction of axon guidance in Drosophila mushroom body. • DIP2 is a downstream of JNK in the axon guidance of Drosophila mushroom body neuron. • JNK/DIP2 pathway is independent from JNK/AP-1 transcriptional factor complex pathway.« less

PI(3,4)P2 plays critical roles in the regulation of focal adhesion dynamics of MDA-MB-231 breast cancer cells.

PubMed

Fukumoto, Miki; Ijuin, Takeshi; Takenawa, Tadaomi

2017-05-01

Phosphoinositides play pivotal roles in the regulation of cancer cell phenotypes. Among them, phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ) localizes to the invadopodia, and positively regulates tumor cell invasion. In this study, we examined the effect of PI(3,4)P 2 on focal adhesion dynamics in MDA-MB-231 basal breast cancer cells. Knockdown of SHIP2, a phosphatidylinositol 3,4,5-trisphosphatase (PIP 3 ) 5-phosphatase that generates PI(3,4)P 2 , in MDA-MB-231 breast cancer cells, induced the development of focal adhesions and cell spreading, leading to the suppression of invasion. In contrast, knockdown of PTEN, a 3-phosphatase that de-phosphorylates PIP 3 and PI(3,4)P 2 , induced cell shrinkage and increased cell invasion. Interestingly, additional knockdown of SHIP2 rescued these phenotypes. Overexpression of the TAPP1 PH domain, which binds to PI(3,4)P 2 , and knockdown of Lpd, a downstream effector of PI(3,4)P 2 , resulted in similar phenotypes to those induced by SHIP2 knockdown. Taken together, our results suggest that inhibition of PI(3,4)P 2 generation and/or downstream signaling could be useful for inhibiting breast cancer metastasis. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

TALE and Shape: How to Make a Leaf Different.

PubMed

Di Giacomo, Elisabetta; Iannelli, Maria Adelaide; Frugis, Giovanna

2013-05-06

The Three Amino acid Loop Extension (TALE) proteins constitute an ancestral superclass of homeodomain transcription factors conserved in animals, plants and fungi. In plants they comprise two classes, KNOTTED1-LIKE homeobox (KNOX) and BEL1-like homeobox (BLH or BELL, hereafter referred to as BLH), which are involved in shoot apical meristem (SAM) function, as well as in the determination and morphological development of leaves, stems and inflorescences. Selective protein-protein interactions between KNOXs and BLHs affect heterodimer subcellular localization and target affinity. KNOXs exert their roles by maintaining a proper balance between undifferentiated and differentiated cell state through the modulation of multiple hormonal pathways. A pivotal function of KNOX in evolutionary diversification of leaf morphology has been assessed. In the SAM of both simple- and compound-leafed seed species, downregulation of most class 1 KNOX (KNOX1) genes marks the sites of leaf primordia initiation. However, KNOX1 expression is re-established during leaf primordia development of compound-leafed species to maintain transient indeterminacy and morphogenetic activity at the leaf margins. Despite the increasing knowledge available about KNOX1 protein function in plant development, a comprehensive view on their downstream effectors remains elusive. This review highlights the role of TALE proteins in leaf initiation and morphological plasticity with a focus on recent advances in the identification of downstream target genes and pathways.

Small Heat Shock Proteins Are Novel Common Determinants of Alcohol and Nicotine Sensitivity in Caenorhabditis elegans

PubMed Central

Johnson, James R.; Rajamanoharan, Dayani; McCue, Hannah V.; Rankin, Kim

2016-01-01

Addiction to drugs is strongly determined by multiple genetic factors. Alcohol and nicotine produce distinct pharmacological effects within the nervous system through discrete molecular targets; yet, data from family and twin analyses support the existence of common genetic factors for addiction in general. The mechanisms underlying addiction, however, are poorly described and common genetic factors for alcohol and nicotine remain unidentified. We investigated the role that the heat shock transcription factor, HSF-1, and its downstream effectors played as common genetic modulators of sensitivity to addictive substances. Using Caenorhabditis elegans, an exemplary model organism with substance dose-dependent responses similar to mammals, we demonstrate that HSF-1 altered sensitivity to both alcohol and nicotine. Using a combination of a targeted RNAi screen of downstream factors and transgenic approaches we identified that these effects were contingent upon the constitutive neuronal expression of HSP-16.48, a small heat shock protein (HSP) homolog of human α-crystallin. Furthermore we demonstrated that the function of HSP-16.48 in drug sensitivity surprisingly was independent of chaperone activity during the heat shock stress response. Instead we identified a distinct domain within the N-terminal region of the HSP-16.48 protein that specified its function in comparison to related small HSPs. Our findings establish and characterize a novel genetic determinant underlying sensitivity to diverse addictive substances. PMID:26773049

Global Phosphoproteomic Analysis of Insulin/Akt/mTORC1/S6K Signaling in Rat Hepatocytes.

PubMed

Zhang, Yuanyuan; Zhang, Yajie; Yu, Yonghao

2017-08-04

Insulin resistance is a hallmark of type 2 diabetes. Although multiple genetic and physiological factors interact to cause insulin resistance, deregulated signaling by phosphorylation is a common underlying mechanism. In particular, the specific phosphorylation-dependent regulatory mechanisms and signaling outputs of insulin are poorly understood in hepatocytes, which represents one of the most important insulin-responsive cell types. Using primary rat hepatocytes as a model system, we performed reductive dimethylation (ReDi)-based quantitative mass spectrometric analysis and characterized the phosphoproteome that is regulated by insulin as well as its key downstream kinases including Akt, mTORC1, and S6K. We identified a total of 12 294 unique, confidently localized phosphorylation sites and 3805 phosphorylated proteins in this single cell type. Detailed bioinformatic analysis on each individual data set identified both known and previously unrecognized targets of this key insulin downstream effector pathway. Furthermore, integrated analysis of the hepatic Akt/mTORC1/S6K signaling axis allowed the delineation of the substrate specificity of several close-related kinases within the insulin signaling pathway. We expect that the data sets will serve as an invaluable resource, providing the foundation for future hypothesis-driven research that helps delineate the molecular mechanisms that underlie the pathogenesis of type 2 diabetes and related metabolic syndrome.

Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins, is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo.

PubMed

Ettensohn, Charles A; Illies, Michele R; Oliveri, Paola; De Jong, Deborah L

2003-07-01

In the sea urchin embryo, the large micromeres and their progeny function as a critical signaling center and execute a complex morphogenetic program. We have identified a new and essential component of the gene network that controls large micromere specification, the homeodomain protein Alx1. Alx1 is expressed exclusively by cells of the large micromere lineage beginning in the first interphase after the large micromeres are born. Morpholino studies demonstrate that Alx1 is essential at an early stage of specification and controls downstream genes required for epithelial-mesenchymal transition and biomineralization. Expression of Alx1 is cell autonomous and regulated maternally through beta-catenin and its downstream effector, Pmar1. Alx1 expression can be activated in other cell lineages at much later stages of development, however, through a regulative pathway of skeletogenesis that is responsive to cell signaling. The Alx1 protein is highly conserved among euechinoid sea urchins and is closely related to the Cart1/Alx3/Alx4 family of vertebrate homeodomain proteins. In vertebrates, these proteins regulate the formation of skeletal elements of the limbs, face and neck. Our findings suggest that the ancestral deuterostome had a population of biomineral-forming mesenchyme cells that expressed an Alx1-like protein.

The Role of the Insulin/IGF System in Cancer: Lessons Learned from Clinical Trials and the Energy Balance-Cancer Link

PubMed Central

Bowers, Laura W.; Rossi, Emily L.; O’Flanagan, Ciara H.; deGraffenried, Linda A.; Hursting, Stephen D.

2015-01-01

Numerous epidemiological and pre-clinical studies have demonstrated that the insulin/insulin-like growth factor (IGF) system plays a key role in the development and progression of several types of cancer. Insulin/IGF signaling, in cooperation with chronic low-grade inflammation, is also an important contributor to the cancer-promoting effects of obesity. However, clinical trials for drugs targeting different components of this system have produced largely disappointing results, possibly due to the lack of predictive biomarker use and problems with the design of combination therapy regimens. With careful attention to the identification of likely patient responders and optimal drug combinations, the outcome of future trials may be improved. Given that insulin/IGF signaling is known to contribute to obesity-associated cancer, further investigation regarding the efficacy of drugs targeting this system and its downstream effectors in the obese patient population is warranted. PMID:26029167

Discovery of a potent, covalent BTK inhibitor for B-cell lymphoma.

PubMed

Wu, Hong; Wang, Wenchao; Liu, Feiyang; Weisberg, Ellen L; Tian, Bei; Chen, Yongfei; Li, Binhua; Wang, Aoli; Wang, Beilei; Zhao, Zheng; McMillin, Douglas W; Hu, Chen; Li, Hong; Wang, Jinhua; Liang, Yanke; Buhrlage, Sara J; Liang, Junting; Liu, Jing; Yang, Guang; Brown, Jennifer R; Treon, Steven P; Mitsiades, Constantine S; Griffin, James D; Liu, Qingsong; Gray, Nathanael S

2014-05-16

BTK is a member of the TEC family of non-receptor tyrosine kinases whose deregulation has been implicated in a variety of B-cell-related diseases. We have used structure-based drug design in conjunction with kinome profiling and cellular assays to develop a potent, selective, and irreversible BTK kinase inhibitor, QL47, which covalently modifies Cys481. QL47 inhibits BTK kinase activity with an IC50 of 7 nM, inhibits autophosphorylation of BTK on Tyr223 in cells with an EC50 of 475 nM, and inhibits phosphorylation of a downstream effector PLCγ2 (Tyr759) with an EC50 of 318 nM. In Ramos cells QL47 induces a G1 cell cycle arrest that is associated with pronounced degradation of BTK protein. QL47 inhibits the proliferation of B-cell lymphoma cancer cell lines at submicromolar concentrations.

Regulation of neuronal communication by G protein-coupled receptors.

PubMed

Huang, Yunhong; Thathiah, Amantha

2015-06-22

Neuronal communication plays an essential role in the propagation of information in the brain and requires a precisely orchestrated connectivity between neurons. Synaptic transmission is the mechanism through which neurons communicate with each other. It is a strictly regulated process which involves membrane depolarization, the cellular exocytosis machinery, neurotransmitter release from synaptic vesicles into the synaptic cleft, and the interaction between ion channels, G protein-coupled receptors (GPCRs), and downstream effector molecules. The focus of this review is to explore the role of GPCRs and G protein-signaling in neurotransmission, to highlight the function of GPCRs, which are localized in both presynaptic and postsynaptic membrane terminals, in regulation of intrasynaptic and intersynaptic communication, and to discuss the involvement of astrocytic GPCRs in the regulation of neuronal communication. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Flexible timing by temporal scaling of cortical responses

PubMed Central

Wang, Jing; Narain, Devika; Hosseini, Eghbal A.; Jazayeri, Mehrdad

2017-01-01

Musicians can perform at different tempos, speakers can control the cadence of their speech, and children can flexibly vary their temporal expectations of events. To understand the neural basis of such flexibility, we recorded from the medial frontal cortex of nonhuman primates trained to produce different time intervals with different effectors. Neural responses were heterogeneous, nonlinear and complex, and exhibited a remarkable form of temporal invariance: firing rate profiles were temporally scaled to match the produced intervals. Recording from downstream neurons in the caudate and thalamic neurons projecting to the medial frontal cortex indicated that this phenomenon originates within cortical networks. Recurrent neural network models trained to perform the task revealed that temporal scaling emerges from nonlinearities in the network and degree of scaling is controlled by the strength of external input. These findings demonstrate a simple and general mechanism for conferring temporal flexibility upon sensorimotor and cognitive functions. PMID:29203897

Drugging the undruggable Ras: mission possible?

PubMed Central

Cox, Adrienne D.; Fesik, Stephen W.; Kimmelman, Alec C.; Luo, Ji; Der, Channing J.

2015-01-01

Despite more than three decades of intensive effort, no effective pharmacologic inhibitors of the Ras oncoproteins have reached the clinic, prompting the widely held perception that Ras proteins are “undruggable”. However, there is renewed hope that this is not the case. In this review, we summarize the progress and promise of five key directions. First, we focus on the prospects of direct inhibitors of Ras. Second, we revisit the issue of whether blocking Ras membrane association is a viable approach. Third, we assess the status of targeting Ras downstream effector signalling, arguably the most favourable current direction. Fourth, we address whether the search for synthetic lethal interactors of mutant RAS still holds promise. Finally, Ras-mediated changes in cell metabolism have recently been described. Can these changes be exploited for new therapeutic directions? We conclude with perspectives on how additional complexities, not yet fully understood, may impact each of these approaches. PMID:25323927

How a mycoparasite employs g-protein signaling: using the example of trichoderma.

PubMed

Omann, Markus; Zeilinger, Susanne

2010-01-01

Mycoparasitic Trichoderma spp. act as potent biocontrol agents against a number of plant pathogenic fungi, whereupon the mycoparasitic attack includes host recognition followed by infection structure formation and secretion of lytic enzymes and antifungal metabolites leading to the host's death. Host-derived signals are suggested to be recognized by receptors located on the mycoparasite's cell surface eliciting an internal signal transduction cascade which results in the transcription of mycoparasitism-relevant genes. Heterotrimeric G proteins of fungi transmit signals originating from G-protein-coupled receptors mainly to the cAMP and the MAP kinase pathways resulting in regulation of downstream effectors. Components of the G-protein signaling machinery such as Gα subunits and G-protein-coupled receptors were recently shown to play crucial roles in Trichoderma mycoparasitism as they govern processes such as the production of extracellular cell wall lytic enzymes, the secretion of antifungal metabolites, and the formation of infection structures.

Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA

PubMed Central

Walther, Rhian F.; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J.; Pichaud, Franck

2016-01-01

Summary The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. PMID:27052178

Of mice and men: molecular genetics of congenital heart disease.

PubMed

Andersen, Troels Askhøj; Troelsen, Karin de Linde Lind; Larsen, Lars Allan

2014-04-01

Congenital heart disease (CHD) affects nearly 1 % of the population. It is a complex disease, which may be caused by multiple genetic and environmental factors. Studies in human genetics have led to the identification of more than 50 human genes, involved in isolated CHD or genetic syndromes, where CHD is part of the phenotype. Furthermore, mapping of genomic copy number variants and exome sequencing of CHD patients have led to the identification of a large number of candidate disease genes. Experiments in animal models, particularly in mice, have been used to verify human disease genes and to gain further insight into the molecular pathology behind CHD. The picture emerging from these studies suggest that genetic lesions associated with CHD affect a broad range of cellular signaling components, from ligands and receptors, across down-stream effector molecules to transcription factors and co-factors, including chromatin modifiers.

TALEN- and CRISPR/Cas9-Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid-Based Transfection.

PubMed

Hendriks, William T; Jiang, Xin; Daheron, Laurence; Cowan, Chad A

2015-08-03

Using custom-engineered nuclease-mediated genome editing, such as Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can be generated and differentiated into various cell types. This strategy of genome engineering in hPSCs will prove invaluable for studying human biology and disease. Here, we provide a detailed protocol for design and construction of TALEN and CRISPR vectors, testing of their nuclease activity, and delivery of TALEN or CRISPR vectors into hPSCs. In addition, we describe the use of single-stranded oligodeoxynucleotides (ssODNs) to introduce or repair point mutations. Next, we describe the identification of edited hPSC clones without antibiotic selection, including their clonal selection, genotyping, and expansion for downstream applications. Copyright © 2015 John Wiley & Sons, Inc.

How a Mycoparasite Employs G-Protein Signaling: Using the Example of Trichoderma

PubMed Central

Omann, Markus; Zeilinger, Susanne

2010-01-01

Mycoparasitic Trichoderma spp. act as potent biocontrol agents against a number of plant pathogenic fungi, whereupon the mycoparasitic attack includes host recognition followed by infection structure formation and secretion of lytic enzymes and antifungal metabolites leading to the host's death. Host-derived signals are suggested to be recognized by receptors located on the mycoparasite's cell surface eliciting an internal signal transduction cascade which results in the transcription of mycoparasitism-relevant genes. Heterotrimeric G proteins of fungi transmit signals originating from G-protein-coupled receptors mainly to the cAMP and the MAP kinase pathways resulting in regulation of downstream effectors. Components of the G-protein signaling machinery such as Gα subunits and G-protein-coupled receptors were recently shown to play crucial roles in Trichoderma mycoparasitism as they govern processes such as the production of extracellular cell wall lytic enzymes, the secretion of antifungal metabolites, and the formation of infection structures. PMID:21637351

Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

PubMed Central

Ashida, Hiroshi; Nakano, Hiroyasu; Sasakawa, Chihiro

2013-01-01

NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation. PMID:23754945

Fluid shear stress activates YAP1 to promote cancer cell motility

NASA Astrophysics Data System (ADS)

Lee, Hyun Jung; Diaz, Miguel F.; Price, Katherine M.; Ozuna, Joyce A.; Zhang, Songlin; Sevick-Muraca, Eva M.; Hagan, John P.; Wenzel, Pamela L.

2017-01-01

Mechanical stress is pervasive in egress routes of malignancy, yet the intrinsic effects of force on tumour cells remain poorly understood. Here, we demonstrate that frictional force characteristic of flow in the lymphatics stimulates YAP1 to drive cancer cell migration; whereas intensities of fluid wall shear stress (WSS) typical of venous or arterial flow inhibit taxis. YAP1, but not TAZ, is strictly required for WSS-enhanced cell movement, as blockade of YAP1, TEAD1-4 or the YAP1-TEAD interaction reduces cellular velocity to levels observed without flow. Silencing of TEAD phenocopies loss of YAP1, implicating transcriptional transactivation function in mediating force-enhanced cell migration. WSS dictates expression of a network of YAP1 effectors with executive roles in invasion, chemotaxis and adhesion downstream of the ROCK-LIMK-cofilin signalling axis. Altogether, these data implicate YAP1 as a fluid mechanosensor that functions to regulate genes that promote metastasis.

RhoA/Rho-kinase signaling: a therapeutic target in pulmonary hypertension.

PubMed

Barman, Scott A; Zhu, Shu; White, Richard E

2009-01-01

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling as well as inflammation. Rho-kinases (ROCKs) are one of the best-described effectors of the small G-protein RhoA, and ROCKs are involved in a variety of cellular functions including muscle cell contraction, proliferation and vascular inflammation through inhibition of myosin light chain phosphatase and activation of downstream mediators. A plethora of evidence in animal models suggests that heightened RhoA/ROCK signaling is important in the pathogenesis of pulmonary hypertension by causing enhanced constriction and remodeling of the pulmonary vasculature. Both animal and clinical studies suggest that ROCK inhibitors are effective for treatment of severe PAH with minimal risk, which supports the premise that ROCKs are important therapeutic targets in pulmonary hypertension and that ROCK inhibitors are a promising new class of drugs for this devastating disease.

Role of IκB kinase β in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex.

PubMed

Karim, Zubair A; Hensch, Nicole R; Qasim, Hanan; Alshbool, Fatima Z; Khasawneh, Fadi T

2018-06-02

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKβ is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKβ regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKβ regulates membrane fusion and the remodeling of the CBM complex formation. Copyright © 2018 Elsevier Inc. All rights reserved.

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix

PubMed Central

Williams, B. Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-01-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953