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Sample records for antiviral signaling protein

  1. Cherry Valley Ducks Mitochondrial Antiviral-Signaling Protein-Mediated Signaling Pathway and Antiviral Activity Research.

    Science.gov (United States)

    Li, Ning; Hong, Tianqi; Li, Rong; Wang, Yao; Guo, Mengjiao; Cao, Zongxi; Cai, Yumei; Liu, Sidang; Chai, Tongjie; Wei, Liangmeng

    2016-01-01

    Mitochondrial antiviral-signaling protein (MAVS), an adaptor protein of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated signal pathway, is involved in innate immunity. In this study, Cherry Valley duck MAVS (duMAVS) was cloned from the spleen and analyzed. duMAVS was determined to have a caspase activation and recruitment domain at N-terminal, followed by a proline-rich domain and a transmembrane domain at C-terminal. Quantitative real-time PCR indicated that duMAVS was expressed in all tissues tested across a broad expression spectrum. The expression of duMAVS was significantly upregulated after infection with duck Tembusu virus (DTMUV). Overexpression of duMAVS could drive the activation of interferon (IFN)-β, nuclear factor-κB, interferon regulatory factor 7, and many downstream factors (such as Mx, PKR, OAS, and IL-8) in duck embryo fibroblast cells. What is more, RNA interference further confirmed that duMAVS was an important adaptor for IFN-β activation. The antiviral assay showed that duMAVS could suppress the various viral replications (DTMUV, novel reovirus, and duck plague virus) at early stages of infection. Overall, these results showed that the main signal pathway mediated by duMAVS and it had a broad-spectrum antiviral ability. This research will be helpful to better understanding the innate immune system of ducks.

  2. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    International Nuclear Information System (INIS)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

    2013-01-01

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways

  3. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo, E-mail: innks@khu.ac.kr

    2013-07-19

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

  4. Herpes simplex virus 1 infection dampens the immediate early antiviral innate immunity signaling from peroxisomes by tegument protein VP16.

    Science.gov (United States)

    Zheng, Chunfu; Su, Chenhe

    2017-02-21

    Herpes simplex virus 1 (HSV-1) is an archetypal member of the alphaherpesvirus subfamily with a large genome encoding over 80 proteins, many of which play a critical role in virus-host interactions and immune modulation. Upon viral infections, the host cells activate innate immune responses to restrict their replications. Peroxisomes, which have long been defined to regulate metabolic activities, are reported to be important signaling platforms for antiviral innate immunity. It has been verified that signaling from peroxisomal MAVS (MAVS-Pex) triggers a rapid interferon (IFN) independent IFN-stimulated genes (ISGs) production against invading pathogens. However, little is known about the interaction between DNA viruses such as HSV-1 and the MAVS-Pex mediated signaling. HSV-1 could activate the MAVS-Pex signaling pathway at a low multiplicity of infection (MOI), while infection at a high MOI dampens MAVS-Pex induced immediately early ISGs production. A high-throughput screen assay reveals that HSV-1 tegument protein VP16 inhibits the immediate early ISGs expression downstream of MAVS-Pex signaling. Moreover, the expression of ISGs was recovered when VP16 was knockdown with its specific short hairpin RNA. HSV-1 blocks MAVS-Pex mediated early ISGs production through VP16 to dampen the immediate early antiviral innate immunity signaling from peroxisomes.

  5. Over-expression of mitochondrial antiviral signaling protein inhibits coxsackievirus B3 infection by enhancing type-I interferons production

    Directory of Open Access Journals (Sweden)

    Zhang Qing-Meng

    2012-12-01

    Full Text Available Abstract Background Recent studies have revealed that Mitochondrial Antiviral Signaling (MAVS protein plays an essential role in the inhibition of viral infection through type I interferon (IFN pathway. It has been shown that 3C (pro cysteine protease of coxsackievirus B3 (CVB3 cleaves MAVS to inhibit type I IFNs induction. Other workers also found that MAVS knock-out mice suffered CVB3 susceptibility and severe histopathological change. Accordingly,our experiments were designed to explore the protection of over-expressing MAVS against CVB3 infection and the possible mechanism. Results In this study, HeLa cells (transfected with MAVS constructs pre- or post- exposure to CVB3 were used to analyze the function of exogenous MAVS on CVB3 infection. The results revealed that though CVB3 infection induced production of type I IFNs, viral replication and cell death were not effectively inhibited. Similarly, exogenous MAVS increased type I IFNs moderately. Morever, we observed robust production of type I IFNs in CVB3 post-infected HeLa cells thereby successfully inhibiting CVB3 infection, as well formation of cytopathic effect (CPE and cell death. Finally, introduction of exogenous MAVS into CVB3 pre-infected cells also restricted viral infection efficiently by greatly up-regulating IFNs. Conclusions In summary, exogenous MAVS effectively prevents and controls CVB3 infection by modulating and promoting the production of type I IFNs. The IFNs level in MAVS over-expressing cells is still tightly regulated by CVB3 infection. Thus, the factors that up-regulate MAVS might be an alternative prescription in CVB3-related syndromes by enhancing IFNs production.

  6. Lab-Attenuated Rabies Virus Causes Abortive Infection and Induces Cytokine Expression in Astrocytes by Activating Mitochondrial Antiviral-Signaling Protein Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Bin Tian

    2018-01-01

    Full Text Available Rabies is an ancient disease but remains endemic in most parts of the world and causes approximately 59,000 deaths annually. The mechanism through which the causative agent, rabies virus (RABV, evades the host immune response and infects the host central nervous system (CNS has not been completely elucidated thus far. Our previous studies have shown that lab-attenuated, but not wild-type (wt, RABV activates the innate immune response in the mouse and dog models. In this present study, we demonstrate that lab-attenuated RABV causes abortive infection in astrocytes, the most abundant glial cells in the CNS. Furthermore, we found that lab-attenuated RABV produces more double-stranded RNA (dsRNA than wt RABV, which is recognized by retinoic acid-inducible gene I (RIG-I or melanoma differentiation-associated protein 5 (MDA5. Activation of mitochondrial antiviral-signaling protein (MAVS, the common adaptor molecule for RIG-I and MDA5, results in the production of type I interferon (IFN and the expression of hundreds of IFN-stimulated genes, which suppress RABV replication and spread in astrocytes. Notably, lab-attenuated RABV replicates in a manner identical to that of wt RABV in MAVS−/− astrocytes. It was also found that lab-attenuated, but not wt, RABV induces the expression of inflammatory cytokines via the MAVS- p38/NF-κB signaling pathway. These inflammatory cytokines increase the blood–brain barrier permeability and thus enable immune cells and antibodies infiltrate the CNS parenchyma, resulting in RABV control and elimination. In contrast, wt RABV restricts dsRNA production and thus evades innate recognition by RIG-I/MDA5 in astrocytes, which could be one of the mechanisms by which wt RABV evades the host immune response in resident CNS cells. Our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated RABV in the CNS.

  7. Regulation of mitochondrial antiviral signaling (MAVS) expression and signaling by the mitochondria-associated endoplasmic reticulum membrane (MAM) protein Gp78.

    Science.gov (United States)

    Jacobs, Jana L; Zhu, Jianzhong; Sarkar, Saumendra N; Coyne, Carolyn B

    2014-01-17

    In a previous study, we identified the E3 ubiquitin ligase Gp78 by RNAi high-throughput screening as a gene whose depletion restricted enterovirus infection. In the current study, we show that Gp78, which localizes to the ER-mitochondria interface, is a regulator of RIG-I-like receptor (RLR) antiviral signaling. We show that depletion of Gp78 results in a robust decrease of vesicular stomatitis virus (VSV) infection and a corresponding enhancement of type I interferon (IFN) signaling. Mechanistically, we show that Gp78 modulates type I IFN induction by altering both the expression and signaling of the mitochondria-localized RLR adaptor mitochondrial antiviral signaling (MAVS). Expression of mutants of Gp78 that abolish its E3 ubiquitin ligase and its participation in ER-associated degradation (ERAD) lost their ability to degrade MAVS, but surprisingly maintained their ability to repress RLR signaling. In contrast, Gp78 lacking its entire C terminus lost both its ability to degrade MAVS and repress RLR signaling. We show that Gp78 interacts with both the N- and C-terminal domains of MAVS via its C-terminal RING domain, and that this interaction is required to abrogate Gp78-mediated attenuation of MAVS signaling. Our data thus implicate two parallel pathways by which Gp78 regulates MAVS signaling; one pathway requires its E3 ubiquitin ligase and ERAD activity to directly degrade MAVS, whereas the other pathway occurs independently of these activities, but requires the Gp78 RING domain and occurs via a direct association between this region and MAVS.

  8. Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses.

    Science.gov (United States)

    Biacchesi, Stéphane; LeBerre, Monique; Lamoureux, Annie; Louise, Yoann; Lauret, Emilie; Boudinot, Pierre; Brémont, Michel

    2009-08-01

    Viral infection triggers host innate immune responses through cellular sensor molecules which activate multiple signaling cascades that induce the production of interferons (IFN) and other cytokines. The recent identification of mammalian cytoplasmic viral RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and their mitochondrial adaptor, the mitochondrial antiviral signaling protein (MAVS), also called IPS-1, VISA, and Cardif, highlights the significance of these molecules in the induction of IFN. Teleost fish also possess a strong IFN system, but nothing is known concerning the RLRs and their downstream adaptor. In this study, we cloned MAVS cDNAs from several fish species (including salmon and zebrafish) and showed that they were orthologs of mammalian MAVS. We demonstrated that overexpression of these mitochondrial proteins in fish cells led to a constitutive induction of IFN and IFN-stimulated genes (ISGs). MAVS-overexpressing cells were almost fully protected against RNA virus infection, with a strong inhibition of both DNA and RNA virus replication (1,000- and 10,000-fold decreases, respectively). Analyses of MAVS deletion mutants showed that both the N-terminal CARD-like and C-terminal transmembrane domains, but not the central proline-rich region, were indispensable for MAVS signaling function. In addition, we cloned the cDNAs encoding a RIG-I-like molecule from salmonid and cyprinid cell lines. Like the case with MAVS, overexpression of RIG-I CARDs in fish cells led to a strong induction of both IFN and ISGs, conferring on fish cells full protection against RNA virus infection. This report provides the first demonstration that teleost fish possess a functional RLR pathway in which MAVS may play a central role in the induction of the innate immune response.

  9. Antiviral effects of the milk protein lactoferrin

    NARCIS (Netherlands)

    Berkhout, B.; Floris, R.; Recio, I.; Visser, S.

    2003-01-01

    Milk forms a rich source of biologically interesting components and the protein fraction is known to facilitate many different biological functions. In this manuscript, we focus on the antiviral properties of the milk protein lactoferrin (LF), in particular against the human immunodeficiency virus

  10. Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

    DEFF Research Database (Denmark)

    Rasmussen, Simon Brandtoft; Sørensen, Louise Nørgaard; Malmgaard, Lene

    2007-01-01

    Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes...... simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-alpha/beta) response is derived from several cell types and induced independently of TLR9...... and fibroblasts, where the virus was able to replicate, HSV-induced IFN-alpha/beta production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms...

  11. DMPD: Negative regulation of cytoplasmic RNA-mediated antiviral signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18703349 Negative regulation of cytoplasmic RNA-mediated antiviral signaling. Komur...Show Negative regulation of cytoplasmic RNA-mediated antiviral signaling. PubmedID 18703349 Title Negative r...egulation of cytoplasmic RNA-mediated antiviral signaling. Authors Komuro A, Bamm

  12. DMPD: Regulation of mitochondrial antiviral signaling pathways. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18549796 Regulation of mitochondrial antiviral signaling pathways. Moore CB, Ting J...P. Immunity. 2008 Jun;28(6):735-9. (.png) (.svg) (.html) (.csml) Show Regulation of mitochondrial antiviral signaling pathways.... PubmedID 18549796 Title Regulation of mitochondrial antiviral signaling pathways. Author

  13. Antiviral and Inflammatory Cellular Signaling Associated with Enterovirus 71 Infection

    Directory of Open Access Journals (Sweden)

    Yuefei Jin

    2018-03-01

    Full Text Available Enterovirus 71 (EV71 infection has become a major threat to global public health, especially in infants and young children. Epidemiological studies have indicated that EV71 infection is responsible for severe and even fatal cases of hand, foot, and mouth disease (HFMD. Accumulated evidence indicates that EV71 infection triggers a plethora of interactive signaling pathways, resulting in host immune evasion and inflammatory response. This review mainly covers the effects of EV71 infection on major antiviral and inflammatory cellular signal pathways. EV71 can activate cellular signaling networks including multiple cell surface and intracellular receptors, intracellular kinases, calcium flux, and transcription factors that regulate antiviral innate immunity and inflammatory response. Cellular signaling plays a critical role in the regulation of host innate immune and inflammatory pathogenesis. Elucidation of antiviral and inflammatory cellular signaling pathways initiated by EV71 will not only help uncover the potential mechanisms of EV71 infection-induced pathogenesis, but will also provide clues for the design of therapeutic strategies against EV71 infection.

  14. Cloning and expression of antiviral/ribosome-inactivating protein ...

    Indian Academy of Sciences (India)

    Madhu urs

    2007-12-16

    Dec 16, 2007 ... Many higher plant species belonging to various taxonomic families are known to produce endogenous, non-stress induced inhibitor proteins called antiviral proteins (AVPs). Many of these AVPs have ribosome-inhibiting rRNA N- glycosidase activity and are known as ribosome-inactivating proteins (RIPs).

  15. Antibacterial and antiviral effects of milk proteins and derivatives thereof

    NARCIS (Netherlands)

    Florisa, René; Recio, Isidra; Berkhout, Ben; Visser, Servaas

    2003-01-01

    Milk forms a rich source of biologically interesting components. In particular, its protein fraction is known to encompass many kinds of biological functions. In this review we focus on antibacterial and antiviral proper-ties of milk proteins and milk protein derivatives. The latter include

  16. Cloning and expression of antiviral/ribosome-inactivating protein ...

    Indian Academy of Sciences (India)

    Madhu urs

    2007-12-16

    Dec 16, 2007 ... The cleaved and purified recombinant. BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity, and imparted a high level of resistance against the tobacco mosaic virus (TMV). [Choudhary N, Kapoor H C and Lodha M L 2008 Cloning and expression of antiviral/ribosome-inactivating protein from ...

  17. Testing of disease-resistance of pokeweed antiviral protein gene ...

    African Journals Online (AJOL)

    Transformation of pokeweed antiviral protein gene (PAP) into plants was shown to improve plant resistance to several viruses or fungi pathogens with no much negative effect on plant growth. The non-virulent defective PAP inhibits only the virus but does not interfere with the host. A non-virulent defective PAP gene ...

  18. Identification of DreI as an antiviral factor regulated by RLR signaling pathway.

    Directory of Open Access Journals (Sweden)

    Shun Li

    Full Text Available BACKGROUND: Retinoic acid-inducible gene I (RIG-I-like receptors (RLRs had been demonstrated to prime interferon (IFN response against viral infection via the conserved RLR signaling in fish, and a novel fish-specific gene, the grass carp reovirus (GCRV-induced gene 2 (Gig2, had been suggested to play important role in host antiviral response. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we cloned and characterized zebrafish Gig2 homolog (named Danio rerio Gig2-I, DreI, and revealed its antiviral role and expressional regulation signaling pathway. RT-PCR, Western blot and promoter activity assay indicate that DreI can be induced by poly I:C, spring viremia of carp virus (SVCV and recombinant IFN (rIFN, showing that DreI is a typical ISG. Using the pivotal signaling molecules of RLR pathway, including RIG-I, MDA5 and IRF3 from crucian carp, it is found that DreI expression is regulated by RLR cascade and IRF3 plays an important role in this regulation. Furthermore, promoter mutation assay confirms that the IFN-stimulated regulatory elements (ISRE in the 5' flanking region of DreI is essential for its induction. Finally, overexpression of DreI leads to establish a strong antiviral state against SVCV and Rana grylio virus (RGV infection in EPC (Epithelioma papulosum cyprinid cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that DreI is an antiviral protein, which is regulated by RLR signaling pathway.

  19. Mx Proteins: Antiviral Gatekeepers That Restrain the Uninvited

    Science.gov (United States)

    Verhelst, Judith; Hulpiau, Paco

    2013-01-01

    SUMMARY Fifty years after the discovery of the mouse Mx1 gene, researchers are still trying to understand the molecular details of the antiviral mechanisms mediated by Mx proteins. Mx proteins are evolutionarily conserved dynamin-like large GTPases, and GTPase activity is required for their antiviral activity. The expression of Mx genes is controlled by type I and type III interferons. A phylogenetic analysis revealed that Mx genes are present in almost all vertebrates, usually in one to three copies. Mx proteins are best known for inhibiting negative-stranded RNA viruses, but they also inhibit other virus families. Recent structural analyses provide hints about the antiviral mechanisms of Mx proteins, but it is not known how they can suppress such a wide variety of viruses lacking an obvious common molecular pattern. Perhaps they interact with a (partially) symmetrical invading oligomeric structure, such as a viral ribonucleoprotein complex. Such an interaction may be of a fairly low affinity, in line with the broad target specificity of Mx proteins, yet it would be strong enough to instigate Mx oligomerization and ring assembly. Such a model is compatible with the broad “substrate” specificity of Mx proteins: depending on the size of the invading viral ribonucleoprotein complexes that need to be wrapped, the assembly process would consume the necessary amount of Mx precursor molecules. These Mx ring structures might then act as energy-consuming wrenches to disassemble the viral target structure. PMID:24296571

  20. Antiviral signaling protein MITA acts as a tumor suppressor in breast cancer by regulating NF-κB induced cell death.

    Science.gov (United States)

    Bhatelia, Khyati; Singh, Aru; Tomar, Dhanendra; Singh, Kritarth; Sripada, Lakshmi; Chagtoo, Megha; Prajapati, Paresh; Singh, Rochika; Godbole, Madan M; Singh, Rajesh

    2014-02-01

    Emerging evidences suggest that chronic inflammation is one of the major causes of tumorigenesis. The role of inflammation in regulation of breast cancer progression is not well established. Recently Mediator of IRF3 Activation (MITA) protein has been identified that regulates NF-κB and IFN pathways. Role of MITA in the context of inflammation and cancer progression has not been investigated. In the current report, we studied the role of MITA in the regulation of cross talk between cell death and inflammation in breast cancer cells. The expression of MITA was significantly lower on in estrogen receptor (ER) positive breast cancer cells than ER negative cells. Similarly, it was significantly down regulated in tumor tissue as compared to the normal tissue. The overexpression of MITA in MCF-7 and T47D decreases the cell proliferation and increases the cell death by activation of caspases. MITA positively regulates NF-κB transcription factor, which is essential for MITA induced cell death. The activation of NF-κB induces TNF-α production which further sensitizes MITA induced cell death by activation of death receptor pathway through capsase-8. MITA expression decreases the colony forming units and migration ability of MCF-7 cells. Thus, our finding suggests that MITA acts as a tumor suppressor which is down regulated during tumorigenesis providing survival advantage to tumor cell. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Innate antiviral immune signaling, viral evasion and modulation by HIV-1.

    Science.gov (United States)

    Rustagi, Arjun; Gale, Michael

    2014-03-20

    The intracellular innate antiviral response in human cells is an essential component of immunity against virus infection. As obligate intracellular parasites, all viruses must evade the actions of the host cell's innate immune response in order to replicate and persist. Innate immunity is induced when pathogen recognition receptors of the host cell sense viral products including nucleic acid as "non-self". This process induces downstream signaling through adaptor proteins to activate latent transcription factors that drive the expression of genes encoding antiviral and immune modulatory effector proteins that restrict virus replication and regulate adaptive immunity. The interferon regulatory factors (IRFs) are transcription factors that play major roles in innate immunity. In particular, IRF3 is activated in response to infection by a range of viruses including RNA viruses, DNA viruses and retroviruses. Among these viruses, human immunodeficiency virus type 1 (HIV-1) remains a major global health problem mediating chronic infection in millions of people wherein recent studies show that viral persistence is linked with the ability of the virus to dysregulate and evade the innate immune response. In this review, we discuss viral pathogen sensing, innate immune signaling pathways and effectors that respond to viral infection, the role of IRF3 in these processes and how it is regulated by pathogenic viruses. We present a contemporary overview of the interplay between HIV-1 and innate immunity, with a focus on understanding how innate immune control impacts infection outcome and disease. Copyright © 2013. Published by Elsevier Ltd.

  2. HIV Exploits Antiviral Host Innate GCN2-ATF4 Signaling for Establishing Viral Replication Early in Infection.

    Science.gov (United States)

    Jiang, Guochun; Santos Rocha, Clarissa; Hirao, Lauren A; Mendes, Erica A; Tang, Yuyang; Thompson, George R; Wong, Joseph K; Dandekar, Satya

    2017-05-02

    Antiviral innate host defenses against acute viral infections include suppression of host protein synthesis to restrict viral protein production. Less is known about mechanisms by which viral pathogens subvert host antiviral innate responses for establishing their replication and dissemination. We investigated early innate defense against human immunodeficiency virus (HIV) infection and viral evasion by utilizing human CD4 + T cell cultures in vitro and a simian immunodeficiency virus (SIV) model of AIDS in vivo Our data showed that early host innate defense against the viral infection involves GCN2-ATF4 signaling-mediated suppression of global protein synthesis, which is exploited by the virus for supporting its own replication during early viral infection and dissemination in the gut mucosa. Suppression of protein synthesis and induction of protein kinase GCN2-ATF4 signaling were detected in the gut during acute SIV infection. These changes diminished during chronic viral infection. HIV replication induced by serum deprivation in CD4 + T cells was linked to the induction of ATF4 that was recruited to the HIV long terminal repeat (LTR) to promote viral transcription. Experimental inhibition of GCN2-ATF4 signaling either by a specific inhibitor or by amino acid supplementation suppressed the induction of HIV expression. Enhancing ATF4 expression through selenium administration resulted in reactivation of latent HIV in vitro as well as ex vivo in the primary CD4 + T cells isolated from patients receiving suppressive antiretroviral therapy (ART). In summary, HIV/SIV exploits the early host antiviral response through GCN2-ATF4 signaling by utilizing ATF4 for activating the viral LTR transcription to establish initial viral replication and is a potential target for HIV prevention and therapy. IMPORTANCE Understanding how HIV overcomes host antiviral innate defense response in order to establish infection and dissemination is critical for developing prevention and

  3. DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.

    Science.gov (United States)

    Yoo, Ji-Seung; Takahasi, Kiyohiro; Ng, Chen Seng; Ouda, Ryota; Onomoto, Koji; Yoneyama, Mitsutoshi; Lai, Janice Ching; Lattmann, Simon; Nagamine, Yoshikuni; Matsui, Tadashi; Iwabuchi, Kuniyoshi; Kato, Hiroki; Fujita, Takashi

    2014-03-01

    RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).

  4. DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.

    Directory of Open Access Journals (Sweden)

    Ji-Seung Yoo

    2014-03-01

    Full Text Available RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA-dependent protein kinase (PKR activation, which has been shown to be essential for the formation of antiviral stress granule (avSG. We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR.

  5. Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses

    DEFF Research Database (Denmark)

    Hamming, Ole Jensen; Terczynska-Dyla, Ewa; Vieyres, Gabrielle

    2013-01-01

    to treatment with type I interferon. Here, we show that the IFNL4 gene encodes an active type III interferon, named IFNλ4, which signals through the IFNλR1 and IL-10R2 receptor chains. Recombinant IFNλ4 is antiviral against both HCV and coronaviruses at levels comparable to IFNλ3. However, the secretion....... Together, these findings result in the paradox that IFNλ4 is strongly antiviral but a disadvantage during HCV infection...

  6. Mitochondria and antiviral innate immunity

    OpenAIRE

    Koshiba, Takumi; Bashiruddin, Nasir; Kawabata, Shunichiro

    2011-01-01

    Mitochondria, dynamic organelles that undergo continuous cycles of fusion and fission, are the powerhouses of eukaryotic cells. Recent research indicates that mitochondria also act as platforms for antiviral immunity in vertebrates. Mitochondrial-mediated antiviral immunity depends on activation of the retinoic acid-inducible gene I (RIG-I)-like receptors signal transduction pathway and the participation of the mitochondrial outer membrane adaptor protein “mitochondrial antiviral signaling (M...

  7. The antiviral activity of the milk protein lactoferrin against the human immunodeficiency virus type 1

    NARCIS (Netherlands)

    Berkhout, Ben; Floris, René; Recio, Isidra; Visser, Servaas

    2004-01-01

    Milk forms a rich source of biologically interesting components and the protein fraction is known to facilitate many different biological functions. In this manuscript, we review the antiviral properties of the milk protein lactoferrin (LF). In particular, we will describe its antiviral activity

  8. HIV Exploits Antiviral Host Innate GCN2-ATF4 Signaling for Establishing Viral Replication Early in Infection

    Directory of Open Access Journals (Sweden)

    Guochun Jiang

    2017-05-01

    Full Text Available Antiviral innate host defenses against acute viral infections include suppression of host protein synthesis to restrict viral protein production. Less is known about mechanisms by which viral pathogens subvert host antiviral innate responses for establishing their replication and dissemination. We investigated early innate defense against human immunodeficiency virus (HIV infection and viral evasion by utilizing human CD4+ T cell cultures in vitro and a simian immunodeficiency virus (SIV model of AIDS in vivo. Our data showed that early host innate defense against the viral infection involves GCN2-ATF4 signaling-mediated suppression of global protein synthesis, which is exploited by the virus for supporting its own replication during early viral infection and dissemination in the gut mucosa. Suppression of protein synthesis and induction of protein kinase GCN2-ATF4 signaling were detected in the gut during acute SIV infection. These changes diminished during chronic viral infection. HIV replication induced by serum deprivation in CD4+ T cells was linked to the induction of ATF4 that was recruited to the HIV long terminal repeat (LTR to promote viral transcription. Experimental inhibition of GCN2-ATF4 signaling either by a specific inhibitor or by amino acid supplementation suppressed the induction of HIV expression. Enhancing ATF4 expression through selenium administration resulted in reactivation of latent HIV in vitro as well as ex vivo in the primary CD4+ T cells isolated from patients receiving suppressive antiretroviral therapy (ART. In summary, HIV/SIV exploits the early host antiviral response through GCN2-ATF4 signaling by utilizing ATF4 for activating the viral LTR transcription to establish initial viral replication and is a potential target for HIV prevention and therapy.

  9. Zinc-finger antiviral protein inhibits XMRV infection.

    Directory of Open Access Journals (Sweden)

    Xinlu Wang

    Full Text Available BACKGROUND: The zinc-finger antiviral protein (ZAP is a host factor that specifically inhibits the replication of certain viruses, including Moloney murine leukemia virus (MoMLV, HIV-1, and certain alphaviruses and filoviruses. ZAP binds to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. The common features of ZAP-responsive RNA sequences remain elusive and thus whether a virus is susceptible to ZAP can only be determined experimentally. Xenotropic murine leukemia virus-related virus (XMRV is a recently identified γ-retrovirus that was originally thought to be involved in prostate cancer and chronic fatigue syndrome but recently proved to be a laboratory artefact. Nonetheless, XMRV as a new retrovirus has been extensively studied. Since XMRV and MoMLV share only 67.9% sequence identity in the 3'UTRs, which is the target sequence of ZAP in MoMLV, whether XMRV is susceptible to ZAP remains to be determined. FINDINGS: We constructed an XMRV-luc vector, in which the coding sequences of Gag-Pol and part of Env were replaced with luciferase-coding sequence. Overexpression of ZAP potently inhibited the expression of XMRV-luc in a ZAP expression-level-dependent manner, while downregulation of endogenous ZAP rendered cells more sensitive to infection. Furthermore, ZAP inhibited the spreading of replication-competent XMRV. Consistent with the previously reported mechanisms by which ZAP inhibits viral infection, ZAP significantly inhibited the accumulation of XMRV-luc mRNA in the cytoplasm. The ZAP-responsive element in XMRV mRNA was mapped to the 3'UTR. CONCLUSIONS: ZAP inhibits XMRV replication by preventing the accumulation of viral mRNA in the cytoplasm. Documentation of ZAP inhibiting XMRV helps to broaden the spectrum of ZAP's antiviral activity. Comparison of the target sequences of ZAP in XMRV and MoMLV helps to better understand the features of ZAP-responsive elements.

  10. Biological activities of the antiviral protein BE27 from sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Iglesias, Rosario; Citores, Lucía; Di Maro, Antimo; Ferreras, José M

    2015-02-01

    The ribosome inactivating protein BE27 displays several biological activities in vitro that could result in a broad action against several types of pathogens. Beetin 27 (BE27), a ribosome-inactivating protein (RIP) from sugar beet (Beta vulgaris L.) leaves, is an antiviral protein induced by virus and signaling compounds such as hydrogen peroxide and salicylic acid. Its role as a defense protein has been attributed to its RNA polynucleotide:adenosine glycosidase activity. Here we tested other putative activities of BE27 that could have a defensive role against pathogens finding that BE27 displays rRNA N-glycosidase activity against yeast and Agrobacterium tumefaciens ribosomes, DNA polynucleotide:adenosine glycosidase activity against herring sperm DNA, and magnesium-dependent endonuclease activity against the supercoiled plasmid PUC19 (nicking activity). The nicking activity could be a consequence of an unusual conformation of the BE27 active site, similar to that of PD-L1, a RIP from Phytolacca dioica L. leaves. Additionally, BE27 possesses superoxide dismutase activity, thus being able to produce the signal compound hydrogen peroxide. BE27 is also toxic to COLO 320 cells, inducing apoptosis in these cells by either activating the caspase pathways and/or inhibiting protein synthesis. The combined effect of these biological activities could result in a broad action against several types of pathogens such as virus, bacteria, fungi or insects.

  11. La protéine CG4572 de Drosophile et la propagation du signal ARNi immun antiviral

    OpenAIRE

    Karlikow, Margot

    2015-01-01

    During viral infection, cell survival will depend on adequately giving, receiving and processing information to establish an efficient antiviral immune response. Cellular communication is therefore essential to allow the propagation of immune signals that will confer protection to the entire organism.The major antiviral defense in insects is the RNA interference (RNAi) mechanism that is activated by detection of viral double-stranded RNA (dsRNA). The antiviral RNAi mechanism can be divided in...

  12. RIG-I antiviral signaling drives interleukin-23 production and psoriasis-like skin disease.

    Science.gov (United States)

    Zhu, Huiyuan; Lou, Fangzhou; Yin, Qianqian; Gao, Yuanyuan; Sun, Yang; Bai, Jing; Xu, Zhenyao; Liu, Zhaoyuan; Cai, Wei; Ke, Fang; Zhang, Lingyun; Zhou, Hong; Wang, Hong; Wang, Gang; Chen, Xiang; Zhang, Hongxin; Wang, Zhugang; Ginhoux, Florent; Lu, Chuanjian; Su, Bing; Wang, Honglin

    2017-05-01

    Retinoic acid inducible-gene I (RIG-I) functions as one of the major sensors of RNA viruses. DDX58 , which encodes the RIG-I protein, has been newly identified as a susceptibility gene in psoriasis. Here, we show that the activation of RIG-I by 5'ppp-dsRNA, its synthetic ligand, directly causes the production of IL-23 and triggers psoriasis-like skin disease in mice. Repeated injections of IL-23 to the ears failed to induce IL-23 production and a full psoriasis-like skin phenotype, in either germ-free or RIG-I-deficient mice. RIG-I is also critical for a full development of skin inflammation in imiquimod (IMQ)-induced psoriasis-like mouse model. Furthermore, RIG-I-mediated endogenous IL-23 production was mainly confined to the CD11c + dendritic cells (DCs) via nuclear factor-kappa B (NF-κB) signaling, and stimulated RIG-I expression in an auto-regulatory feedback loop. Thus, our data suggest that the dysregulation in the antiviral immune responses of hosts through the innate pattern recognition receptors may trigger the skin inflammatory conditions in the pathophysiology of psoriasis. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  13. Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response.

    Science.gov (United States)

    Conn, Kristen L; Wasson, Peter; McFarlane, Steven; Tong, Lily; Brown, James R; Grant, Kyle G; Domingues, Patricia; Boutell, Chris

    2016-05-01

    Small ubiquitin-like modifier (SUMO) is used by the intrinsic antiviral immune response to restrict viral pathogens, such as herpes simplex virus 1 (HSV-1). Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined. We show that unconjugated SUMO levels are largely maintained throughout infection regardless of the presence of ICP0, the HSV-1 SUMO-targeted ubiquitin ligase. Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate. These data highlight the continued importance for SUMO signaling throughout infection. We show that the SUMO ligase protein inhibitor of activated STAT 4 (PIAS4) is upregulated during HSV-1 infection and localizes to nuclear domains that contain viral DNA. PIAS4 is recruited to sites associated with HSV-1 genome entry through SUMO interaction motif (SIM)-dependent mechanisms that are destabilized by ICP0. In contrast, PIAS4 accumulates in replication compartments through SIM-independent mechanisms irrespective of ICP0 expression. Depletion of PIAS4 enhances the replication of ICP0-null mutant HSV-1, which is susceptible to restriction by the intrinsic antiviral immune response. The mechanisms of PIAS4-mediated restriction are synergistic with the restriction mechanisms of a characterized intrinsic antiviral factor, promyelocytic leukemia protein, and are antagonized by ICP0. We provide the first evidence that PIAS4 is an intrinsic antiviral factor. This novel role for PIAS4 in intrinsic antiviral immunity contrasts with the known roles of PIAS proteins as suppressors of innate immunity. Posttranslational modifications with small ubiquitin-like modifier (SUMO) proteins regulate multiple aspects of host immunity and viral replication. The protein inhibitor of activated STAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of

  14. Identification of alternatively translated Tetherin isoforms with differing antiviral and signaling activities.

    Directory of Open Access Journals (Sweden)

    Luis J Cocka

    2012-09-01

    Full Text Available Tetherin (BST-2/CD317/HM1.24 is an IFN induced transmembrane protein that restricts release of a broad range of enveloped viruses. Important features required for Tetherin activity and regulation reside within the cytoplasmic domain. Here we demonstrate that two isoforms, derived by alternative translation initiation from highly conserved methionine residues in the cytoplasmic domain, are produced in both cultured human cell lines and primary cells. These two isoforms have distinct biological properties. The short isoform (s-Tetherin, which lacks 12 residues present in the long isoform (l-Tetherin, is significantly more resistant to HIV-1 Vpu-mediated downregulation and consequently more effectively restricts HIV-1 viral budding in the presence of Vpu. s-Tetherin Vpu resistance can be accounted for by the loss of serine-threonine and tyrosine motifs present in the long isoform. By contrast, the l-Tetherin isoform was found to be an activator of nuclear factor-kappa B (NF-κB signaling whereas s-Tetherin does not activate NF-κB. Activation of NF-κB requires a tyrosine-based motif found within the cytoplasmic tail of the longer species and may entail formation of l-Tetherin homodimers since co-expression of s-Tetherin impairs the ability of the longer isoform to activate NF-κB. These results demonstrate a novel mechanism for control of Tetherin antiviral and signaling function and provide insight into Tetherin function both in the presence and absence of infection.

  15. Positive selection and increased antiviral activity associated with the PARP-containing isoform of human zinc-finger antiviral protein.

    Directory of Open Access Journals (Sweden)

    Julie A Kerns

    2008-01-01

    Full Text Available Intrinsic immunity relies on specific recognition of viral epitopes to mount a cell-autonomous defense against viral infections. Viral recognition determinants in intrinsic immunity genes are expected to evolve rapidly as host genes adapt to changing viruses, resulting in a signature of adaptive evolution. Zinc-finger antiviral protein (ZAP from rats was discovered to be an intrinsic immunity gene that can restrict murine leukemia virus, and certain alphaviruses and filoviruses. Here, we used an approach combining molecular evolution and cellular infectivity assays to address whether ZAP also acts as a restriction factor in primates, and to pinpoint which protein domains may directly interact with the virus. We find that ZAP has evolved under positive selection throughout primate evolution. Recurrent positive selection is only found in the poly(ADP-ribose polymerase (PARP-like domain present in a longer human ZAP isoform. This PARP-like domain was not present in the previously identified and tested rat ZAP gene. Using infectivity assays, we found that the longer isoform of ZAP that contains the PARP-like domain is a stronger suppressor of murine leukemia virus expression and Semliki forest virus infection. Our study thus finds that human ZAP encodes a potent antiviral activity against alphaviruses. The striking congruence between our evolutionary predictions and cellular infectivity assays strongly validates such a combined approach to study intrinsic immunity genes.

  16. Alpha-lipoic acid effects on brain glial functions accompanying double-stranded RNA antiviral and inflammatory signaling.

    Science.gov (United States)

    Scumpia, Philip O; Kelly-Scumpia, Kindra; Stevens, Bruce R

    2014-01-01

    Double-stranded RNAs (dsRNA) serve as viral ligands that trigger innate immunity in astrocytes and microglial, as mediated through Toll-like receptor 3 (TLR3) and dsRNA-dependent protein kinase (PKR). Beneficial transient TLR3 and PKR anti-viral signaling can become deleterious when events devolve into inflammation and cytotoxicity. Viral products in the brain cause glial cell dysfunction, and are a putative etiologic factor in neuropsychiatric disorders, notably schizophrenia, bipolar disorder, Parkinson's, and autism spectrum. Alpha-lipoic acid (LA) has been proposed as a possible therapeutic neuroprotectant. The objective of this study was to test our hypothesis that LA can control untoward antiviral mechanisms associated with neural dysfunction. Utilizing rat brain glial cultures (91% astrocytes:9% microglia) treated with PKR- and TLR3-ligand/viral mimetic dsRNA, polyinosinic-polycytidylic acid (polyI:C), we report in vitro glial antiviral signaling and LA reduction of the effects of this signaling. LA blunted the dsRNA-stimulated expression of IFNα/β-inducible genes Mx1, PKR, and TLR3. And in polyI:C treated cells, LA promoted gene expression of rate-limiting steps that benefit healthy neural redox status in glutamateric systems. To this end, LA decreased dsRNA-induced inflammatory signaling by downregulating IL-1β, IL-6, TNFα, iNOS, and CAT2 transcripts. In the presence of polyI:C, LA prevented cultured glial cytotoxicity which was correlated with increased expression of factors known to cooperatively control glutamate/cystine/glutathione redox cycling, namely glutamate uptake transporter GLAST/EAAT1, γ-glutamyl cysteine ligase catalytic and regulatory subunits, and IL-10. Glutamate exporting transporter subunits 4F2hc and xCT were downregulated by LA in dsRNA-stimulated glia. l-Glutamate net uptake was inhibited by dsRNA, and this was relieved by LA. Glutathione synthetase mRNA levels were unchanged by dsRNA or LA. This study demonstrates the protective

  17. Assessment of the antiviral properties of recombinant surfactant protein D against influenza B virus in vitro.

    Science.gov (United States)

    Hillaire, Marine L B; van Eijk, Martin; Vogelzang-van Trierum, Stella E; Nieuwkoop, Nella J; van Riel, Debby; Fouchier, Ron A M; Kuiken, Thijs; Osterhaus, Albert D M E; Haagsman, Henk P; Rimmelzwaan, Guus F

    2015-01-02

    The armamentarium of antiviral drugs against influenza viruses is limited. Furthermore, influenza viruses emerge that are resistant to existing antiviral drugs like the M2 and NA inhibitors. Therefore, there is an urgent need for the development of novel classes of antiviral drugs. Here we investigated the antiviral properties of recombinant porcine surfactant protein D (RpSP-D), an innate defense molecule with lectin properties, against influenza B viruses. We have previously shown that porcine SP-D has more potent neutralizing activity against influenza A viruses than human SP-D. Here we show that RpSP-D neutralizes influenza B viruses efficiently and inhibited the binding of these viruses to epithelial cells of the human trachea. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling.

    Science.gov (United States)

    Zhang, Qingzhan; Yoo, Dongwan

    2016-12-02

    Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are emerged and reemerging viruses in pigs, and together with transmissible gastroenteritis virus (TGEV), pose significant economic concerns to the swine industry. These viruses infect epithelial cells of the small intestine and cause watery diarrhea, dehydration, and a high mortality in neonatal piglets. Type I interferons (IFN-α/β) are major antiviral cytokines forming host innate immunity, and in turn, these enteric coronaviruses have evolved to modulate the host innate immune signaling during infection. Accumulating evidence however suggests that IFN induction and signaling in the intestinal epithelial cells differ from other epithelial cells, largely due to distinct features of the gut epithelial mucosal surface and commensal microflora, and it appears that type III interferon (IFN-λ) plays a key role to maintain the antiviral state in the gut. This review describes the recent understanding on the immune evasion strategies of porcine enteric coronaviruses and the role of different types of IFNs for intestinal antiviral innate immunity. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Viral Proteins That Bind Double-Stranded RNA: Countermeasures Against Host Antiviral Responses

    OpenAIRE

    Krug, Robert M.

    2014-01-01

    Several animal viruses encode proteins that bind double-stranded RNA (dsRNA) to counteract host dsRNA-dependent antiviral responses. This article discusses the structure and function of the dsRNA-binding proteins of influenza A virus and Ebola viruses (EBOVs).

  20. Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

    Directory of Open Access Journals (Sweden)

    Jaume Torres

    2015-06-01

    Full Text Available Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i the envelope protein in coronaviruses and (ii the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity.

  1. Inhibition of antiviral innate immunity by birnavirus VP3 protein via blockage of viral double-stranded RNA binding to the host cytoplasmic RNA detector MDA5.

    Science.gov (United States)

    Ye, Chengjin; Jia, Lu; Sun, Yanting; Hu, Boli; Wang, Lun; Lu, Xingmeng; Zhou, Jiyong

    2014-10-01

    Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity, but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I interferon (IFN) production but antagonized the antiviral activity of beta interferon (IFN-β) in DF-1 cells pretreated with IFN-α/β. Dual-luciferase assays and inducible expression systems demonstrated that IBDV protein VP3 significantly inhibited IFN-β expression stimulated by naked IBDV genomic double-stranded RNA (dsRNA). The VP3 protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-β expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5. MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-β did not function effectively during birnavirus infection, and the viral protein VP3 significantly inhibited IFN-β expression stimulated by naked viral genomic dsRNA. We also show that VP3 blocks MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral protein VP3 is an inhibitor of the antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway

  2. Charge modification of plasma and milk proteins results in antiviral active compounds

    NARCIS (Netherlands)

    Swart, P J; Harmsen, M C; Kuipers, M E; Van Dijk, A A; Van Der Strate, B W; Van Berkel, P H; Nuijens, J H; Smit, C; Witvrouw, M; De Clercq, E; de Béthune, M P; Pauwels, R; Meijer, D K

    1999-01-01

    Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti-HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various

  3. Filovirus proteins for antiviral drug discovery: Structure/function bases of the replication cycle.

    Science.gov (United States)

    Martin, Baptiste; Canard, Bruno; Decroly, Etienne

    2017-05-01

    Filoviruses are important pathogens that cause severe and often fatal hemorrhagic fever in humans, for which no approved vaccines and antiviral treatments are yet available. In an earlier article (Martin et al., Antiviral Research, 2016), we reviewed the role of the filovirus surface glycoprotein in replication and as a target for drugs and vaccines. In this review, we focus on recent findings on the filovirus replication machinery and how they could be used for the identification of new therapeutic targets and the development of new antiviral compounds. First, we summarize the recent structural and functional advances on the molecules involved in filovirus replication/transcription cycle, particularly the NP, VP30, VP35 proteins, and the "large" protein L, which harbors the RNA-dependent RNA polymerase (RdRp) and mRNA capping activities. These proteins are essential for viral mRNA synthesis and genome replication, and consequently they constitute attractive targets for drug design. We then describe how these insights into filovirus replication mechanisms and the structure/function characterization of the involved proteins have led to the development of new and innovative antiviral strategies that may help reduce the filovirus disease case fatality rate through post-exposure or prophylactic treatments. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Antiviral and antitumor activities of the protein fractions from the ...

    African Journals Online (AJOL)

    In this study, we present the extraction and purification of protein fractions from the larvae of the housefly, Musca domestica. The bioactivities of the protein fractions were indicated by pseudorabies virus (PRV) and human lung cancer cell line A 549. The crude protein fractions had no toxicity to chick embryo fibroblast-like ...

  5. Nuclear trafficking of proteins from RNA viruses: potential target for antivirals?

    Science.gov (United States)

    Caly, Leon; Wagstaff, Kylie M; Jans, David A

    2012-09-01

    A key aspect of the infectious cycle of many viruses is the transport of specific viral proteins into the host cell nucleus to perturb the antiviral response. Examples include a number of RNA viruses that are significant human pathogens, such as human immunodeficiency virus (HIV)-1, influenza A, dengue, respiratory syncytial virus and rabies, as well agents that predominantly infect livestock, such as Rift valley fever virus and Venezuelan equine encephalitis virus. Inhibiting the nuclear trafficking of viral proteins as a therapeutic strategy offers an attractive possibility, with important recent progress having been made with respect to HIV-1 and dengue. The results validate nuclear protein import as an antiviral target, and suggest the identification and development of nuclear transport inhibitors as a viable therapeutic approach for a range of human and zoonotic pathogenic viruses. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Antiviral and antitumor activities of the protein fractions from the ...

    African Journals Online (AJOL)

    AJL

    2012-05-15

    May 15, 2012 ... was bought from the China Center for Type Culture Collection of. Wuhan University (Wuhan, China). ... cultures were exposed to 100 µl of the crude protein fractions. (dilution rates were 1:1, 1:10 or 1:100) on .... The values represent the average of three replications. Asterisks denote significant differences ...

  7. Antiviral Protein of Momordica charantia L. Inhibits Different Subtypes of Influenza A

    Directory of Open Access Journals (Sweden)

    Viroj Pongthanapisith

    2013-01-01

    Full Text Available The new antiviral activity of the protein extracted from Momordica charantia was determined with different subtypes of influenza A. The protein was purified from the seed of M. charantia using an anion exchanger and a Fast Protein Liquid Chromatography (FPLC system. At the concentration of 1.401 mg/mL, the protein did not exhibit cytotoxicity in Madin-Darby canine kidney cells (MDCK but inhibited FFU influenza A/PR/8/34 H1N1 virus at 56.50%, 65.72%, and 100% inhibition by the protein treated before the virus (pretreated, the protein treated alongside with the virus (simultaneously treated, and the protein treated after the virus (posttreated during incubation, respectively. Using 5, 25, and 100 TCID50 of influenza A/New Caledonia/20/99 H1N1, A/Fujian/411/01 H3N2 and A/Thailand/1(KAN-1/2004 H5N1, the IC50 was calculated to be 100, 150, and 200; 75, 175, and 300; and 40, 75, and 200 μg/mL, respectively. Our present finding indicated that the plant protein inhibited not only H1N1 and H3N2 but also H5N1 subtype. As a result of the broad spectrum of its antiviral activity, this edible plant can be developed as an effective therapeutic agent against various and even new emerging subtypes of influenza A.

  8. Human cytomegaloviruses expressing yellow fluorescent fusion proteins--characterization and use in antiviral screening.

    Directory of Open Access Journals (Sweden)

    Sarah Straschewski

    Full Text Available Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity. We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP fused with the viral proteins IE-2, ppUL32 (pp150, and ppUL83 (pp65. In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI. The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus-infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.

  9. Biophysical Properties and Antiviral Activities of Measles Fusion Protein Derived Peptide Conjugated with 25-Hydroxycholesterol.

    Science.gov (United States)

    Gomes, Bárbara; Santos, Nuno C; Porotto, Matteo

    2017-10-31

    Measles virus (MV) infection is re-emerging, despite the availability of an effective vaccine. The mechanism of MV entry into a target cell relies on coordinated action between the MV hemagglutinin (H) receptor binding protein and the fusion envelope glycoprotein (F) which mediates fusion between the viral and cell membranes. Peptides derived from the C -terminal heptad repeat (HRC) of F can interfere with this process, blocking MV infection. As previously described, biophysical properties of HRC-derived peptides modulate their antiviral potency. In this work, we characterized a MV peptide fusion inhibitor conjugated to 25-hydroxycholesterol (25HC), a cholesterol derivative with intrinsic antiviral activity, and evaluated its interaction with membrane model systems and human blood cells. The peptide (MV.

  10. Autophagy Proteins in Viral Exocytosis and Anti-Viral Immune Responses

    Directory of Open Access Journals (Sweden)

    Christian Münz

    2017-10-01

    Full Text Available Abstract: Autophagy-related (Atg gene-encoded proteins were originally described for their crucial role in macroautophagy, a catabolic pathway for cytoplasmic constituent degradation in lysosomes. Recently it has become clear that modules of this machinery can also be used to influence endo- and exocytosis. This mini review discusses how these alternative Atg functions support virus replication and viral antigen presentation on major histocompatibility (MHC class I and II molecules. A better understanding of the modular use of the macroautophagy machinery might enable us to manipulate these alternative functions of Atg proteins during anti-viral therapies and to attenuate virus-induced immune pathologies.

  11. Regulators of G-protein-signaling proteins: negative modulators of G-protein-coupled receptor signaling.

    Science.gov (United States)

    Woodard, Geoffrey E; Jardín, Isaac; Berna-Erro, A; Salido, Gines M; Rosado, Juan A

    2015-01-01

    Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Both antiviral activity and intracellular localization of chicken Mx protein depend on a polymorphism at amino acid position 631.

    Science.gov (United States)

    Sasaki, Keisuke; Yoneda, Akihiro; Ninomiya, Akinori; Kawahara, Manabu; Watanabe, Tomomasa

    2013-01-04

    The Mx protein is known to inhibit the multiplication of several RNA viruses. In chickens, a polymorphism at amino acid position 631 (631 aa) of Mx protein has been suggested to be involved in the antiviral ability against vesicular stomatitis virus (VSV) and influenza virus, indicating that a Ser-to-Asn substitution at 631 aa is the source of this antiviral ability. However, how the substitution at 631 aa contributes to the antiviral activity remains to be clarified. In this study, we investigated differences in antiviral activity against VSV and intracellular localization between Ser and Asn types at 631 aa of the chicken Mx protein. The results showed that chicken Mx protein with an Asn at 631 aa inhibited VSV multiplication and Mx distribution in a granular-like pattern in the cytoplasm. However, Mx carrying the Ser type did not inhibit viral growth and homogenous spread throughout the cytoplasm. Furthermore, we found that replacing Ser with Asn at 631 aa provided Mx with antiviral activity against VSV, with Mx showing granular-like distribution in the cytoplasm. These results demonstrated that a single amino acid polymorphism at 631 aa of the chicken Mx protein altered both the antiviral activity and intracellular localization. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L.

    Directory of Open Access Journals (Sweden)

    Frédéric Sorgeloos

    Full Text Available Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.

  14. Inhibition of enterovirus 71 (EV-71 infections by a novel antiviral peptide derived from EV-71 capsid protein VP1.

    Directory of Open Access Journals (Sweden)

    Chee Wah Tan

    Full Text Available Enterovirus 71 (EV-71 is the main causative agent of hand, foot and mouth disease (HFMD. In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50 values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.

  15. Cigarette Smoke Dampens Anti-viral Signaling in Small Airway Epithelial Cells by Disrupting TLR3 Cleavage.

    Science.gov (United States)

    Duffney, Parker F; McCarthy, Claire E; Nogales, Aitor; Thatcher, Thomas H; Martinez-Sobrido, Luis; Phipps, Richard P; Sime, Patricia J

    2017-12-14

    Cigarette smokers and people exposed to secondhand smoke are at an increased risk for pulmonary viral infections, yet the mechanism responsible for this heightened susceptibility is not understood. To understand the effect of cigarette smoke on susceptibility to viral infection we used an air-liquid interface culture system, and exposed primary human small airway epithelial cells (SAEC) to whole cigarette smoke followed by treatment with the viral mimetic polyinosinic polycytidylic acid (poly I:C) or influenza A virus (IAV). We found that prior smoke exposure strongly inhibited production of pro-inflammatory (interleukin 6 and interleukin 8) and anti-viral (interferon gamma induced protein 10, IP-10 and interferons) mediators in SAECs in response to poly I:C and IAV infection. Impaired antiviral responses corresponded to increased infection with IAV. This was associated with a decrease in phosphorylation of the key antiviral transcription factor interferon response factor (IRF3). Here we found that cigarette smoke exposure inhibited activation of toll-like receptor (TLR) 3 by impairing TLR3 cleavage, which was required for downstream phosphorylation of IRF3 and production of IP-10. These results identify a novel mechanism by which cigarette smoke exposure impairs antiviral responses in lung epithelial cells, which may contribute to increased susceptibility to respiratory infections.

  16. The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA.

    Science.gov (United States)

    Zhong, Bo; Zhang, Lu; Lei, Caoqi; Li, Ying; Mao, Ai-Ping; Yang, Yan; Wang, Yan-Yi; Zhang, Xiao-Lian; Shu, Hong-Bing

    2009-03-20

    Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

  17. Assessment of the antiviral properties of recombinant surfactant protein D against influenza B virus in vitro

    NARCIS (Netherlands)

    Hillaire, Marine L.B.; van Eijk, Martin; Vogelzang-van Trierum, Stella E; Nieuwkoop, Nella J; van Riel, Debby; Fouchier, Ron A M; Kuiken, Thijs; Osterhaus, Albert D.M.E.; Haagsman, Henk P.; Rimmelzwaan, Guus F

    2015-01-01

    The armamentarium of antiviral drugs against influenza viruses is limited. Furthermore, influenza viruses emerge that are resistant to existing antiviral drugs like the M2 and NA inhibitors. Therefore, there is an urgent need for the development of novel classes of antiviral drugs. Here we

  18. High Expression of Antiviral Proteins in Mucosa from Individuals Exhibiting Resistance to Human Immunodeficiency Virus.

    Science.gov (United States)

    Gonzalez, Sandra Milena; Taborda, Natalia Andrea; Feria, Manuel Gerónimo; Arcia, David; Aguilar-Jiménez, Wbeimar; Zapata, Wildeman; Rugeles, María Teresa

    2015-01-01

    Several soluble factors have been reported to have the capacity of inhibiting HIV replication at different steps of the virus life cycle, without eliminating infected cells and through enhancement of specific cellular mechanisms. Yet, it is unclear if these antiviral factors play a role in the protection from HIV infection or in the control of viral replication. Here we evaluated two cohorts: i) one of 58 HIV-exposed seronegative individuals (HESNs) who were compared with 59 healthy controls (HCs), and ii) another of 13 HIV-controllers who were compared with 20 HIV-progressors. Peripheral blood, oral and genital mucosa and gut-associated lymphoid tissue (GALT) samples were obtained to analyze the mRNA expression of ELAFIN, APOBEC3G, SAMHD1, TRIM5α, RNase 7 and SerpinA1 using real-time PCR. HESNs exhibited higher expression of all antiviral factors in peripheral blood mononuclear cells (PBMCs), oral or genital mucosa when compared with HCs. Furthermore, HIV-controllers exhibited higher levels of SerpinA1 in GALT. These findings suggest that the activity of these factors is compartmentalized and that these proteins have a predominant role depending on the tissue to avoid the infection, reduce the viral load and modulate the susceptibility to HIV infection.

  19. High Expression of Antiviral Proteins in Mucosa from Individuals Exhibiting Resistance to Human Immunodeficiency Virus.

    Directory of Open Access Journals (Sweden)

    Sandra Milena Gonzalez

    Full Text Available Several soluble factors have been reported to have the capacity of inhibiting HIV replication at different steps of the virus life cycle, without eliminating infected cells and through enhancement of specific cellular mechanisms. Yet, it is unclear if these antiviral factors play a role in the protection from HIV infection or in the control of viral replication. Here we evaluated two cohorts: i one of 58 HIV-exposed seronegative individuals (HESNs who were compared with 59 healthy controls (HCs, and ii another of 13 HIV-controllers who were compared with 20 HIV-progressors. Peripheral blood, oral and genital mucosa and gut-associated lymphoid tissue (GALT samples were obtained to analyze the mRNA expression of ELAFIN, APOBEC3G, SAMHD1, TRIM5α, RNase 7 and SerpinA1 using real-time PCR.HESNs exhibited higher expression of all antiviral factors in peripheral blood mononuclear cells (PBMCs, oral or genital mucosa when compared with HCs. Furthermore, HIV-controllers exhibited higher levels of SerpinA1 in GALT.These findings suggest that the activity of these factors is compartmentalized and that these proteins have a predominant role depending on the tissue to avoid the infection, reduce the viral load and modulate the susceptibility to HIV infection.

  20. Guanylate-binding protein 1 participates in cellular antiviral response to dengue virus

    Directory of Open Access Journals (Sweden)

    Pan Wen

    2012-11-01

    Full Text Available Abstract Background Dengue virus (DENV, the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus found in tropical and sub-tropical regions around the world. Vaccines against DENV are currently unavailable. Guanylate-binding protein 1 (GBP1 is one of the Interferon (IFN stimulated genes (ISGs and has been shown important for host immune defense against various pathogens. However, the role of GBP1 during DENV infection remains unclarified. In this study, we evaluated the relevance of GBP1 to DENV infection in in vitro model. Findings Quantitative RT-PCR (qRT-PCR and Western blot showed that the expression of mouse Gbp1 was dramatically upregulated in DENV-infected RAW264.7 cells. The intracellular DENV loads were significantly higher in Gbp1 silenced cells compared with controls. The expression levels of selective anti-viral cytokines were decreased in Gbp1 siRNA treated cells, while the transcription factor activity of NF-κB was impaired upon GBP1 silencing during infection. Conclusions Our data suggested that GBP1 plays an antiviral role during DENV infection.

  1. Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins.

    Science.gov (United States)

    Bulanova, Daria; Ianevski, Aleksandr; Bugai, Andrii; Akimov, Yevhen; Kuivanen, Suvi; Paavilainen, Henrik; Kakkola, Laura; Nandania, Jatin; Turunen, Laura; Ohman, Tiina; Ala-Hongisto, Hanna; Pesonen, Hanna M; Kuisma, Marika S; Honkimaa, Anni; Walton, Emma L; Oksenych, Valentyn; Lorey, Martina B; Guschin, Dmitry; Shim, Jungmin; Kim, Jinhee; Than, Thoa T; Chang, So Young; Hukkanen, Veijo; Kulesskiy, Evgeny; Marjomaki, Varpu S; Julkunen, Ilkka; Nyman, Tuula A; Matikainen, Sampsa; Saarela, Jani S; Sane, Famara; Hober, Didier; Gabriel, Gülsah; De Brabander, Jef K; Martikainen, Miika; Windisch, Marc P; Min, Ji-Young; Bruzzone, Roberto; Aittokallio, Tero; Vähä-Koskela, Markus; Vapalahti, Olli; Pulk, Arto; Velagapudi, Vidya; Kainov, Denis E

    2017-09-25

    Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.

  2. Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Perry

    Full Text Available Ubiquitin (Ub is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs. However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1, a critical mediator of the unfolded protein response (UPR. WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1 through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.

  3. Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses

    Science.gov (United States)

    Goździcka-Józefiak, Anna

    2017-01-01

    Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine. PMID:29104238

  4. Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses.

    Science.gov (United States)

    Musidlak, Oskar; Nawrot, Robert; Goździcka-Józefiak, Anna

    2017-11-01

    Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine.

  5. Predicting Secretory Proteins with SignalP

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2017-01-01

    SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the secretory pathway, but are not necessarily secreted. After a brief introduction to the biology of signal peptides and the history...

  6. Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling

    Directory of Open Access Journals (Sweden)

    Yi-Hsuan Wu

    2015-12-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α and GTPase myxovirus resistance 1 (MX1—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E and enterovirus 71 (EV71 infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.

  7. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2015-09-28

    searched using PUBMED and. Google Scholar). The protein-protein interaction data obtain- ed were knitted together to reconstruct the entire signalling cascade. The diagram of the reconstructed pathway was drawn using ...

  8. Peptide Signals Encode Protein Localization▿

    OpenAIRE

    Russell, Jay H.; Keiler, Kenneth C.

    2007-01-01

    Many bacterial proteins are localized to precise intracellular locations, but in most cases the mechanism for encoding localization information is not known. Screening libraries of peptides fused to green fluorescent protein identified sequences that directed the protein to helical structures or to midcell. These peptides indicate that protein localization can be encoded in 20-amino-acid peptides instead of complex protein-protein interactions and raise the possibility that the location of a ...

  9. Transmissible gastroenteritis virus: identification of M protein-binding peptide ligands with antiviral and diagnostic potential.

    Science.gov (United States)

    Zou, Hao; Zarlenga, Dante S; Sestak, Karol; Suo, Siqingaowa; Ren, Xiaofeng

    2013-09-01

    The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p<0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response

    Directory of Open Access Journals (Sweden)

    Andrea Martins-da-Silva

    2018-01-01

    Full Text Available Hematophagous insects transmit infectious diseases. Sand flies are vectors of leishmaniasis, but can also transmit viruses. We have been studying immune responses of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. We identified a non-specific antiviral response in L. longipalpis LL5 embryonic cells when treated with non-specific double-stranded RNAs (dsRNAs. This response is reminiscent of interferon response in mammals. We are investigating putative effectors for this antiviral response. Secreted molecules have been implicated in immune responses, including interferon-related responses. We conducted a mass spectrometry analysis of conditioned medium from LL5 cells 24 and 48 h after dsRNA or mock treatment. We identified 304 proteins. At 24 h, 19 proteins had an abundance equal or greater than 2-fold change, while the levels of 17 proteins were reduced when compared to control cells. At the 48 h time point, these numbers were 33 and 71, respectively. The two most abundant secreted peptides at 24 h in the dsRNA-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (FKBP, a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. The transcription profile of most candidates did not follow the pattern of secreted protein abundance.

  11. The presence of the iron-sulfur motif is important for the conformational stability of the antiviral protein, Viperin.

    Directory of Open Access Journals (Sweden)

    Shubhasis Haldar

    Full Text Available Viperin, an antiviral protein, has been shown to contain a CX(3CX(2C motif, which is conserved in the radical S-adenosyl-methionine (SAM enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a low-resolution predicted structure. The results show that Viperin is an α-β protein containing iron-sulfur cluster at the center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. Conformational analyses using circular dichroism and steady state fluorescence spectroscopy indicate that the mutant proteins are partially unfolded, conformationally unstable and aggregation prone. The lack of conformational stability of the mutant proteins may have direct relevance to the absence of their antiviral activity.

  12. Roles for Regulator of G Protein Signaling Proteins in Synaptic Signaling and Plasticity.

    Science.gov (United States)

    Gerber, Kyle J; Squires, Katherine E; Hepler, John R

    2016-02-01

    The regulator of G protein signaling (RGS) family of proteins serves critical roles in G protein-coupled receptor (GPCR) and heterotrimeric G protein signal transduction. RGS proteins are best understood as negative regulators of GPCR/G protein signaling. They achieve this by acting as GTPase activating proteins (GAPs) for Gα subunits and accelerating the turnoff of G protein signaling. Many RGS proteins also bind additional signaling partners that either regulate their functions or enable them to regulate other important signaling events. At neuronal synapses, GPCRs, G proteins, and RGS proteins work in coordination to regulate key aspects of neurotransmitter release, synaptic transmission, and synaptic plasticity, which are necessary for central nervous system physiology and behavior. Accumulating evidence has revealed key roles for specific RGS proteins in multiple signaling pathways at neuronal synapses, regulating both pre- and postsynaptic signaling events and synaptic plasticity. Here, we review and highlight the current knowledge of specific RGS proteins (RGS2, RGS4, RGS7, RGS9-2, and RGS14) that have been clearly demonstrated to serve critical roles in modulating synaptic signaling and plasticity throughout the brain, and we consider their potential as future therapeutic targets. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  13. CNS activity of Pokeweed Anti-viral Protein (PAP in mice infected with Lymphocytic Choriomeningitis Virus (LCMV

    Directory of Open Access Journals (Sweden)

    Tibbles Heather E

    2005-02-01

    Full Text Available Abstract Background Others and we have previously described the potent in vivo and in vitro activity of the broad-spectrum antiviral agent PAP (Pokeweed antiviral protein against a wide range of viruses. The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on the survival of mice challenged with lymphocytic choriomeningitis virus (LCMV. Methods We examined the therapeutic effect of PAP in CBA mice inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation. Results PAP exhibits significant in vivo anti- LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV. At non-toxic dose levels, PAP significantly prolonged survival in the absence of the majority of disease-associated symptoms. The median survival time of PAP-treated mice was >21 days as opposed to 7 days median survival for the control (p = 0.0069. Conclusion Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice.

  14. Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR

    Directory of Open Access Journals (Sweden)

    Chinchar V Gregory

    2011-03-01

    Full Text Available Abstract Background Ranaviruses (family Iridoviridae are important pathogens of lower vertebrates. However, little is known about how they circumvent the immune response of their hosts. Many ranaviruses contain a predicted protein, designated vIF2α, which shows homology with the eukaryotic translation initiation factor 2α. In analogy to distantly related proteins found in poxviruses vIF2α might act as an inhibitor of the antiviral protein kinase PKR. Results We have characterized the function of vIF2α from Rana catesbeiana virus Z (RCV-Z. Multiple sequence alignments and secondary structure prediction revealed homology of vIF2α with eIF2α throughout the S1-, helical- and C-terminal domains. Genetic and biochemical analyses showed that vIF2α blocked the toxic effects of human and zebrafish PKR in a heterologous yeast system. Rather than complementing eIF2α function, vIF2α acted in a manner comparable to the vaccinia virus (VACV K3L protein (K3, a pseudosubstrate inhibitor of PKR. Both vIF2α and K3 inhibited human PKR-mediated eIF2α phosphorylation, but not PKR autophosphorylation on Thr446. In contrast the E3L protein (E3, another poxvirus inhibitor of PKR, inhibited both Thr446 and eIF2α Ser51 phosphorylation. Interestingly, phosphorylation of eIF2α by zebrafish PKR was inhibited by vIF2α and E3, but not by K3. Effective inhibition of PKR activity coincided with increased PKR expression levels, indicative of relieved autoinhibition of PKR expression. Experiments with vIF2α deletion constructs, showed that both the N-terminal and helical domains were sufficient for inhibition of PKR, whereas the C-terminal domain was dispensable. Conclusions Our results show that RCV-Z vIF2α is a functional inhibitor of human and zebrafish PKR, and probably functions in similar fashion as VACV K3. This constitutes an important step in understanding the interaction of ranaviruses and the host innate immune system.

  15. Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses.

    Science.gov (United States)

    Fuchs, Jonas; Hölzer, Martin; Schilling, Mirjam; Patzina, Corinna; Schoen, Andreas; Hoenen, Thomas; Zimmer, Gert; Marz, Manja; Weber, Friedemann; Müller, Marcel A; Kochs, Georg

    2017-08-01

    Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts. IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity

  16. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... [Ganguli P, Chowdhury S, Bhowmick R and Sarkar RR 2015 Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: A ... cells and tissues by studying different signalling pathways, such as Hedgehog ...... Murray JD 2003 On the mechanochemical theory of biological.

  17. Cissampelos sympodialis has anti-viral effect inhibiting dengue non-structural viral protein-1 and pro-inflammatory mediators

    Directory of Open Access Journals (Sweden)

    Fagner Carvalho Leite

    Full Text Available ABSTRACT Dengue is the most important viral infection transmitted among humans by arthropod-borne. There are currently no vaccines or specific therapeutical treatment. Therefore, immunomodulatory compounds from plants have been widely examined for their antiviral effects. Cissampelos sympodialis Eichler, Menispermaceae, has scientifically proven to present immunomodulatory activities. Here we assessed the antiviral activity of leaf hydroalcoholic extract, warifteine or methylwarifteine from C. sympodialis in an in vitro dengue virus infection model. The results demonstrated that leaf hydroalcoholic extract or warifteine/methylwarifteine treatment did not reduce dengue virus-Ag+ hepatocyte (Huh-7 cell rates in present experimental conditions. However, we assessed the potential antiviral effect of leaf hydroalcoholic extract or warifteine/methylwarifteine on dengue virus-infection by the production of inflammatory molecules, TNF-α, MIF, IL-8 and PGE2. Dengue virus infection enhanced TNF-α, MIF, IL-8 and PGE2 production in infected Huh-7 cells and leaf hydroalcoholic extract but not warifteine/methylwarifteine treatments, significantly reduced these molecules in infected cells. In dengue virus-infected Huh-7 cells, non-structural protein-1 is produced and leaf hydroalcoholic extract significantly inhibited it independently of alkaloids. Our findings imply that leaf hydroalcoholic extract may attenuate dengue virus infection in Huh-7 cells by inhibiting the enhanced of pro-inflammatory mediators and non-structural protein-1 production induce by dengue virus independently of warifteine/methywarifteine its major compound.

  18. Protein cysteine oxidation in redox signaling

    DEFF Research Database (Denmark)

    Forman, Henry Jay; Davies, Michael J; Krämer, Anna C

    2017-01-01

    Oxidation of critical signaling protein cysteines regulated by H2O2 has been considered to involve sulfenic acid (RSOH) formation. RSOH may subsequently form either a sulfenyl amide (RSNHR') with a neighboring amide, or a mixed disulfide (RSSR') with another protein cysteine or glutathione...... of the species previously identified as the "sulfenome" - the cellular complement of reversibly-oxidized thiol proteins generated via sulfenic acids....

  19. The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response.

    Directory of Open Access Journals (Sweden)

    Preeti Bharaj

    2016-09-01

    Full Text Available For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I system. Nipah virus (NiV, a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus, is known to encode for four P gene-derived viral proteins (P/C/W/V with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M, which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε. We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258 in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new

  20. Inhibition of CRM1-mediated nuclear export of influenza A nucleoprotein and nuclear export protein as a novel target for antiviral drug development.

    Science.gov (United States)

    Chutiwitoonchai, Nopporn; Mano, Takafumi; Kakisaka, Michinori; Sato, Hirotaka; Kondoh, Yasumitsu; Osada, Hiroyuki; Kotani, Osamu; Yokoyama, Masaru; Sato, Hironori; Aida, Yoko

    2017-07-01

    An anti-influenza compound, DP2392-E10 based on inhibition of the nuclear export function of the viral nucleoprotein-nuclear export signal 3 (NP-NES3) domain was successfully identified by our previous high-throughput screening system. Here, we demonstrated that DP2392-E10 exerts its antiviral effect by inhibiting replication of a broad range of influenza A subtypes. In regard to the molecular mechanism, we revealed that DP2392-E10 inhibits nuclear export of both viral NP and nuclear export protein (NEP). More specifically, in vitro pull-down assays revealed that DP2392-E10 directly binds cellular CRM1, which mediates nuclear export of NP and NEP. In silico docking suggested that DP2392-E10 binds at a region close to the HEAT9 and HEAT10 domains of CRM1. Together, these results indicate that the CRM1-mediated nuclear export function of influenza virus represents a new potential target for antiviral drug development, and also provide a core structure for a novel class of inhibitors that target this function. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Elevated sodium chloride drives type I interferon signaling in macrophages and increases antiviral resistance.

    Science.gov (United States)

    Zhang, Wu-Chang; Du, Lin-Juan; Zheng, Xiao-Jun; Chen, Xiao-Qing; Shi, Chaoji; Chen, Bo-Yan; Sun, Xue-Nan; Li, Chao; Zhang, Yu-Yao; Liu, Yan; Xiao, Hui; Leng, Qibin; Jiang, Xinquan; Zhang, Zhiyuan; Sun, Shuyang; Duan, Sheng-Zhong

    2018-01-19

    Type I IFN production and signaling in macrophages play critical roles in innate immune responses. High salt ( i.e. high concentrations of NaCl) has been proposed to be an important environmental factor that influences immune responses in multiple ways. However, it remains unknown whether high salt regulates type I IFN production and signaling in macrophages. Here, we demonstrated that high salt promoted IFNβ production and its signaling in both human and mouse macrophages, and consequentially primed macrophages for strengthened immune sensing and signaling when challenged with viruses or viral nucleic acid analogues. Using both pharmacological inhibitors and RNA interference we showed that these effects of high salt on IFNβ signaling were mediated by the p38 MAPK/ATF2/AP1 signaling pathway. Consistently, high salt increased resistance to vesicle stomatitis virus (VSV) infection in vitro. In vivo data indicated that a high-salt diet protected mice from lethal VSV infection. Taken together, these results identify high salt as a crucial regulator of type I IFN production and signaling, shedding important new light on the regulation of innate immune responses. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. MCPIP1 is a positive regulator of type I interferons antiviral activity.

    Science.gov (United States)

    Qian, Liping; Zuo, Yibo; Deng, Wenjun; Miao, Ying; Liu, Jin; Yuan, Yukang; Guo, Tingting; Zhang, Liting; Jin, Jun; Wang, Jun; Zheng, Hui

    2018-04-15

    Type-I interferons (IFN-I) are widely used for antiviral immunotherapy in clinic. Therefore, identification of the regulators of IFN-I antiviral activity is important for developing novel targets for IFN-based antiviral therapy. Monocyte chemoattractant protein 1-induced protein 1 (MCPIP1) is critical for cellular inflammatory responses. However, the roles of MCPIP1 in interferons (IFNs)-mediated antiviral immunity are unexplored. In this study, we demonstrate for the first time that MCPIP1 is an important positive regulator of IFNs antiviral activity. We found that MCPIP1 can promote innate antiviral immunity independently of both its RNase and deubiquitinase activity. Furthermore, we reveal that MCPIP1 is an IFN-induced positive feedback signal molecule which promotes IFN-I-mediated antiviral efficacy. Mechanistically, MCPIP1 does not affect the activation of JAK/STAT upstream of IFN-I signaling, but significantly promotes IFN-I signaling by enhancing ISRE promoter activity and expression of interferon-stimulated genes (ISGs). And MCPIP1-mediated activation of IFN-I signaling is independently of its RNase and deubiquitinase activity. These findings uncover a novel innate antiviral mechanism mediated by the IFN-MCPIP1 axis, and may provide potential targets for enhancing IFNs antiviral therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Cucumber mosaic virus coat protein modulates the accumulation of 2b protein and antiviral silencing that causes symptom recovery in planta.

    Directory of Open Access Journals (Sweden)

    Xiao-Peng Zhang

    2017-07-01

    Full Text Available Shoot apical meristems (SAM are resistant to most plant viruses due to RNA silencing, which is restrained by viral suppressors of RNA silencing (VSRs to facilitate transient viral invasion of the SAM. In many cases chronic symptoms and long-term virus recovery occur, but the underlying mechanisms are poorly understood. Here, we found that wild-type Cucumber mosaic virus (CMVWT invaded the SAM transiently, but was subsequently eliminated from the meristems. Unexpectedly, a CMV mutant, designated CMVRA that harbors an alanine substitution in the N-terminal arginine-rich region of the coat protein (CP persistently invaded the SAM and resulted in visible reductions in apical dominance. Notably, the CMVWT virus elicited more potent antiviral silencing than CMVRA in newly emerging leaves of infected plants. However, both viruses caused severe symptoms with minimal antiviral silencing effects in the Arabidopsis mutants lacking host RNA-DEPENDENT RNA POLYMERASE 6 (RDR6 or SUPPRESSOR OF GENE SILENCING 3 (SGS3, indicating that CMVWT induced host RDR6/SGS3-dependent antiviral silencing. We also showed that reduced accumulation of the 2b protein is elicited in the CMVWT infection and consequently rescues potent antiviral RNA silencing. Indeed, co-infiltration assays showed that the suppression of posttranscriptional gene silencing mediated by 2b is more severely compromised by co-expression of CPWT than by CPRA. We further demonstrated that CPWT had high RNA binding activity leading to translation inhibition in wheat germ systems, and CPWT was associated with SGS3 into punctate granules in vivo. Thus, we propose that the RNAs bound and protected by CPWT possibly serve as templates of RDR6/SGS3 complexes for siRNA amplification. Together, these findings suggest that the CMV CP acts as a central hub that modulates antiviral silencing and VSRs activity, and mediates viral self-attenuation and long-term symptom recovery.

  4. NEOGLYCOPROTEINS AS CARRIERS FOR ANTIVIRAL DRUGS - SYNTHESIS AND ANALYSIS OF PROTEIN DRUG CONJUGATES

    NARCIS (Netherlands)

    Molema, Grietje; Jansen, Robert W.; Visser, Jan; Herdewijn, Piet; Moolenaar, Frits; Meijer, Dirk K.F.

    In order to investigate whether neoglycoproteins can potentially act as carriers for targeting of antiviral drugs to certain cell types in the body, various neoglycoproteins were synthesized using thiophosgene-activated p-aminophenyl sugar derivatives. These neoglycoproteins were conjugated with the

  5. Melittin-loaded immunoliposomes against viral surface proteins, a new approach to antiviral therapy

    NARCIS (Netherlands)

    Falco Gracia, J.A.; Barrajon-Catalan, E.; Menendez-Gutierrez, M.P.; Coll, J.; Micol, V.; Estepa, A.

    2013-01-01

    In this study, melittin, a well-characterized pore-forming lytic amphiphilic peptide susceptible to be vehiculized in lipid membranes, has been utilized to study their antiviral properties. For this purpose, an assay based on melittin loaded-immunoliposomes previously described by our group was

  6. Interaction research on an antiviral molecule that targets the coat protein of southern rice black-streaked dwarf virus.

    Science.gov (United States)

    Ran, Longlu; Ding, Yan; Luo, Liangzhi; Gan, Xiuhai; Li, Xiangyang; Chen, Yongzhong; Hu, Deyu; Song, Baoan

    2017-10-01

    Southern rice black-streaked dwarf virus (SRBSDV) coat protein (P10) is the key protein required for viral transmission and host plant infection and is thus a promising target for anti-SRBSDV agent screening. In this study, P10 was obtained from Escherichia coli through cloning, expression, and purification. The antiviral agent Ningnanmycin was selected as control, and a series of antiviral compounds based on the structural scaffold of ferulic acid were analyzed. Size-exclusion chromatography analysis results showed that compound F27 can alter the aggregation of P10 proteins. Furthermore, fluorescence titration and microscale thermophoresis assay results indicated that F27 binds to P10 with K A of 5.75×10 5 M -1 and K D of 7.81μM. The ligand- and receptor-based three-dimensional quantitative structure-activity analyses were performed to determine the requirements for the interaction between the carboxyl structures and P10s. On the basis of the obtained models and information, we provided insights regarding the design and optimization of novel molecules as anti-SRBSDV agents. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Ubiquitin in signaling and protein quality control

    DEFF Research Database (Denmark)

    Al-Saoudi, Sofie Vincents

    Protein ubiquitylation is an important post-translational modification that holds a variety of cellular functions. This Ph.D. thesis is comprised of two studies, of which one focused on ubiquitylation related to inflammatory signaling, and the other on the role of the ubiquitin-proteasome system...

  8. Glycosphingolipid–Protein Interaction in Signal Transduction

    Directory of Open Access Journals (Sweden)

    Domenico Russo

    2016-10-01

    Full Text Available Glycosphingolipids (GSLs are a class of ceramide-based glycolipids essential for embryo development in mammals. The synthesis of specific GSLs depends on the expression of distinctive sets of GSL synthesizing enzymes that is tightly regulated during development. Several reports have described how cell surface receptors can be kept in a resting state or activate alternative signalling events as a consequence of their interaction with GSLs. Specific GSLs, indeed, interface with specific protein domains that are found in signalling molecules and which act as GSL sensors to modify signalling responses. The regulation exerted by GSLs on signal transduction is orthogonal to the ligand–receptor axis, as it usually does not directly interfere with the ligand binding to receptors. Due to their properties of adjustable production and orthogonal action on receptors, GSLs add a new dimension to the control of the signalling in development. GSLs can, indeed, dynamically influence progenitor cell response to morphogenetic stimuli, resulting in alternative differentiation fates. Here, we review the available literature on GSL–protein interactions and their effects on cell signalling and development.

  9. Antiviral immunity in marine molluscs.

    Science.gov (United States)

    Green, Timothy J; Raftos, David; Speck, Peter; Montagnani, Caroline

    2015-09-01

    Marine molluscs, like all living organisms, are constantly exposed to viruses and have evolved efficient antiviral defences. We review here recent developments in molluscan antiviral immunity against viruses belonging to the order Herpesvirales. Emerging results suggest an interferon-like response and autophagy are involved in the antiviral defence of bivalves to viral infection. Multi-functional plasma proteins from gastropods and bivalves have been identified to have broad-spectrum antiviral activity against mammalian viruses. The antiviral defences present in molluscs can be enhanced by genetic selection, as shown by the presence of oyster strains specifically resistant to ostreid herpesvirus type 1. Whether varying amounts or different isoforms of these antiviral plasma proteins contributes to genetic resistance is worthy of further research. Other evolutionarily conserved antiviral mechanisms, such as RNA interference and apoptosis, still need further characterization.

  10. Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

    Science.gov (United States)

    Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

    2013-12-01

    The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Frizzled receptors signal through G proteins.

    Science.gov (United States)

    Nichols, Andrea S; Floyd, Desiree H; Bruinsma, Stephen P; Narzinski, Kirk; Baranski, Thomas J

    2013-06-01

    Frizzled receptors have long been thought to couple to G proteins but biochemical evidence supporting such an interaction has been lacking. Here we expressed mammalian Wnt-Frizzled fusion proteins in Saccharomyces cerevisiae and tested the receptors' ability to activate the yeast mitogen-activated protein kinase (MAPK) pathway via heterotrimeric G proteins. Our results show that Frizzled receptors can interact with Gαi, Gαq, and Gαs proteins, thus confirming that Frizzled functions as a G protein coupled receptor (GPCR). However, the activity level of Frizzled-mediated G protein signaling was much lower than that of a typical GPCR and, surprisingly, was highest when coupled to Gαs. The Frizzled/Gαs interaction was further established in vivo as Drosophila expressing a loss-of-function Gαs allele rescued the photoreceptor differentiation phenotype of Frizzled mutant flies. Together, these data point to an important role for Frizzled as a nontraditional GPCR that preferentially couples to Gαs heterotrimeric G proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

    Science.gov (United States)

    Gaya, Mauro; Barral, Patricia; Burbage, Marianne; Aggarwal, Shweta; Montaner, Beatriz; Warren Navia, Andrew; Aid, Malika; Tsui, Carlson; Maldonado, Paula; Nair, Usha; Ghneim, Khader; Fallon, Padraic G; Sekaly, Rafick-Pierre; Barouch, Dan H; Shalek, Alex K; Bruckbauer, Andreas; Strid, Jessica; Batista, Facundo D

    2018-01-25

    B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Structure and Function of the Non-Structural Protein of Dengue Virus and its Applications in Antiviral Therapy.

    Science.gov (United States)

    Xie, Qian; Zhang, Bao; Yu, JianHai; Wu, Qinghua; Yang, Fangji; Cao, Hong; Zhao, Wei

    2017-01-01

    Dengue fever, a type of global and tropical infectious disease, and its prevention has become a challenging issue worldwide. Antibody-dependent enhancement effects and the virus pathogenic mechanism have not yet been fully elucidated, hindering the development of dengue fever prevention and suitable drug treatment. There is currently no specific prevention and therapy in clinical trials, however, in recent years, studies have focused on the pathogenesis and treatment of dengue. Research focusing on dengue virus nonstructural protein in special drugs for the prevention and control of dengue fever is a new progress leading to improved understanding regarding the prevention and control of dengue fever and suitable drugs for the treatment. The main challenges regarding the structure of dengue virus nonstructural protein and the drugs for antiviral therapy are summarized in this paper.

  14. Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform

    Science.gov (United States)

    Nguyen, Nhung T.H.; Now, Hesung; Kim, Woo-Jong; Kim, Nari; Yoo, Joo-Yeon

    2016-01-01

    RIG-I is a key cytosolic RNA sensor that mediates innate immune defense against RNA virus. Aberrant RIG-I activity leads to severe pathological states such as autosomal dominant multi-system disorder, inflammatory myophathies and dermatomyositis. Therefore, identification of regulators that ensure efficient defense without harmful immune-pathology is particularly critical to deal with RIG-I-associated diseases. Here, we presented the inflammatory inducible FAT10 as a novel negative regulator of RIG-I-mediated inflammatory response. In various cell lines, FAT10 protein is undetectable unless it is induced by pro-inflammatory cytokines. FAT10 non-covalently associated with the 2CARD domain of RIG-I, and inhibited viral RNA-induced IRF3 and NF-kB activation through modulating the RIG-I protein solubility. We further demonstrated that FAT10 was recruited to RIG-I-TRIM25 to form an inhibitory complex where FAT10 was stabilized by E3 ligase TRIM25. As the result, FAT10 inhibited the antiviral stress granules formation contains RIG-I and sequestered the active RIG-I away from the mitochondria. Our study presented a novel mechanism to dampen RIG-I activity. Highly accumulated FAT10 is observed in various cancers with pro-inflammatory environment, therefore, our finding which uncovered the suppressive effect of the accumulated FAT10 during virus-mediated inflammatory response may also provide molecular clue to understand the carcinogenesis related with infection and inflammation. PMID:26996158

  15. A recombinant, infectious human parainfluenza virus type 3 expressing the enhanced green fluorescent protein for use in high-throughput antiviral assays

    Science.gov (United States)

    Roth, Jason P.; Li, Joseph K.-K.; Smee, Donald F.; Morrey, John D.; Barnard, Dale L.

    2009-01-01

    The ability to rescue an infectious, recombinant, negative-stranded, RNA virus from a cDNA clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted into the human parainfluenza virus type 3 (HPIV-3) antigenome and a recombinant, infectious virus was rescued. Maximum EGFP expression levels, measured by fluorescence, were seen at day 3. Comparison of a three-day, viral expressed EGFP fluorescence assay to a seven-day, neutral red assay, based on complete cell destruction in virus infected MA-104 cells, yielded Z′-factor values of 0.83 and 0.70, respectively. A three-day, endpoint EGFP-based antiviral assay and a seven-day, endpoint neutral red based antiviral assay were run in parallel to establish antiviral sensitivity profiles of 23 compounds based on selective index (SI) values. Using an SI threshold of 10, the EGFP-based antiviral assay had a sensitivity of 100% and a specificity of 54%. Thus, the use of an EGFP-based antiviral assay for testing potential antiviral compounds against HPIV-3 in a high-throughput format may be justified. PMID:19189850

  16. Organizing signal transduction through A-kinase anchoring proteins (AKAPs).

    Science.gov (United States)

    Logue, Jeremy S; Scott, John D

    2010-11-01

    A fundamental role for protein-protein interactions in the organization of signal transduction pathways is evident. Anchoring, scaffolding and adapter proteins function to enhance the precision and directionality of these signaling events by bringing enzymes together. The cAMP signaling pathway is organized by A-kinase anchoring proteins. This family of proteins assembles enzyme complexes containing the cAMP-dependent protein kinase, phosphoprotein phosphatases, phosphodiesterases and other signaling effectors to optimize cellular responses to cAMP and other second messengers. Selected A-kinase anchoring protein signaling complexes are highlighted in this minireview. © 2010 The Authors Journal compilation © 2010 FEBS.

  17. Cutaneous RANK-RANKL Signaling Upregulates CD8-Mediated Antiviral Immunity during Herpes simplex Virus Infection by Preventing Virus-Induced Langerhans Cell Apoptosis.

    Science.gov (United States)

    Klenner, Lars; Hafezi, Wali; Clausen, Björn E; Lorentzen, Eva U; Luger, Thomas A; Beissert, Stefan; Kühn, Joachim E; Loser, Karin

    2015-11-01

    Herpes simplex virus-type 1 (HSV-1) causes the majority of cutaneous viral infections. Viral infections are controlled by the immune system, and CD8(+) cytotoxic T-lymphocytes (CTLs) have been shown to be crucial during the clearance of HSV-1 infections. Although epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to come into contact with the virus, it has been shown that the processing of viral antigens and the differentiation of antiviral CTLs are mediated by migratory CD103(+) dermal DCs and CD8α(+) lymph node-resident DCs. In vivo regulatory T-cells (Tregs) are implicated in the regulation of antiviral immunity and we have shown that signaling via the receptor activator of NF-κB (RANK) and its ligand RANKL mediates the peripheral expansion of Tregs. However, in addition to expanding Tregs, RANK-RANKL interactions are involved in the control of antimicrobial immunity by upregulating the priming of CD4(+) effector T cells in LCMV infection or by the generation of parasite-specific CD8(+) T cells in Trypanosoma cruzi infection. Here, we demonstrate that cutaneous RANK-RANKL signaling is critical for the induction of CD8-mediated antiviral immune responses during HSV-1 infection of the skin by preventing virus-induced LC apoptosis, improving antigen transport to regional lymph nodes, and increasing the CTL priming capacity of lymph node DCs.

  18. Silencing herpes simplex virus type 1 capsid protein encoding genes by siRNA: a promising antiviral therapeutic approach.

    Directory of Open Access Journals (Sweden)

    Fujun Jin

    Full Text Available Herpes simplex virus type 1 (HSV-1, a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18 and VP5 (UL19 individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

  19. Silencing herpes simplex virus type 1 capsid protein encoding genes by siRNA: a promising antiviral therapeutic approach.

    Science.gov (United States)

    Jin, Fujun; Li, Shen; Zheng, Kai; Zhuo, Cuiqin; Ma, Kaiqi; Chen, Maoyun; Wang, Qiaoli; Zhang, Peizhuo; Fan, Jianglin; Ren, Zhe; Wang, Yifei

    2014-01-01

    Herpes simplex virus type 1 (HSV-1), a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs) against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18) and VP5 (UL19) individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

  20. Understanding the neurospecificity of Prion protein signaling.

    Science.gov (United States)

    Schneider, Benoit; Pietri, Mathea; Pradines, Elodie; Loubet, Damien; Launay, Jean-Marie; Kellermann, Odile; Mouillet-Richard, Sophie

    2011-01-01

    The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. The presence of PrP(C) at the surface of neurons is an absolute requirement for the development of prion diseases and corruption of PrP(C) function(s) within an infectious context emerges as a proximal cause for PrP(Sc)-induced neurodegeneration. Experimental evidence gained over the past decade indicates that PrP(C) has the capacity to mobilize promiscuous signal transduction cascades that, notably, contribute to cell homeostasis. Beyond ubiquitous effectors, much data converge onto a neurospecificity of PrP(C) signaling, which may be the clue to neuronal cell demise in prion disorders. In this article, we highlight the requirement of PrP(C) for TSEs-associated neurodegeneration and review the current knowledge of PrP(C)-dependent signal transduction in neuronal cells and its implications for PrP(Sc)-mediated neurotoxicity.

  1. Myxoma Virus dsRNA Binding Protein M029  Inhibits the Type I IFN‐Induced Antiviral State in a  Highly Species‐Specific Fashion

    Directory of Open Access Journals (Sweden)

    Masmudur M. Rahman

    2017-02-01

    Full Text Available Myxoma virus (MYXV is Leporipoxvirus that possesses a specific rabbit‐restricted host tropism but exhibits a much broader  cellular host range in cultured cells. MYXV is able to efficiently  block all aspects of the type I interferon (IFN‐induced  antiviral  state  in rabbit cells, partially in  human  cells  and  very  poorly  in  mouse  cells.  The mechanism(s of this species‐specific inhibition of  type I IFN‐induced antiviral state is not well understood. Here we demonstrate that MYXV encoded  protein  M029, a truncated relative of the vaccinia virus (VACV E3 double‐stranded RNA (dsRNA  binding  protein  that  inhibits  protein  kinase  R (PKR,  can  also  antagonize the type I IFN‐induced  antiviral state in a highly species‐specific manner. In cells pre‐treated with type I IFN prior to  infection,  MYXV  exploits  M029  to  overcome  the  induced  antiviral  state completely in rabbit cells,  partially  in  human  cells,  but  not at all in mouse cells. However, in cells pre‐infected with MYXV,  IFN‐induced  signaling  is fully  inhibited  even  in the  absence  of M029 in cells from all three species,  suggesting  that  other  MYXV  protein(s  apart  from  M029  block  IFN  signaling  in  a  speciesindependent  manner.  We  also  show  that  the  antiviral  state  induced in rabbit, human or mouse cells  by  type  I IFN  can  inhibit M029‐knockout MYXV even when PKR is genetically knocked‐out, suggesting  that  M029  targets  other  host  proteins  for  this  antiviral state inhibition. Thus, the MYXV  dsRNA  binding  protein  M029  not  only  antagonizes  PKR  from  multiple  species  but  also blocks the  type I IFN antiviral state independently of PKR in a highly species‐specific fashion.

  2. Myxoma Virus dsRNA Binding Protein M029  Inhibits the Type I IFN-Induced Antiviral State in a  Highly Species-Specific Fashion.

    Science.gov (United States)

    Rahman, Masmudur M; McFadden, Grant

    2017-02-02

    Myxoma virus (MYXV) is Leporipoxvirus that possesses a specific rabbit-restricted host tropism but exhibits a much broader  cellular host range in cultured cells. MYXV is able to efficiently  block all aspects of the type I interferon (IFN)-induced  antiviral  state  in rabbit cells, partially in  human  cells  and  very  poorly  in  mouse  cells.  The mechanism(s) of this species-specific inhibition of  type I IFN-induced antiviral state is not well understood. Here we demonstrate that MYXV encoded  protein  M029, a truncated relative of the vaccinia virus (VACV) E3 double-stranded RNA (dsRNA)  binding  protein  that  inhibits  protein  kinase  R (PKR),  can  also  antagonize the type I IFN-induced  antiviral state in a highly species-specific manner. In cells pre-treated with type I IFN prior to  infection,  MYXV  exploits  M029  to  overcome  the  induced  antiviral  state completely in rabbit cells,  partially  in  human  cells,  but  not at all in mouse cells. However, in cells pre-infected with MYXV,  IFN-induced  signaling  is fully  inhibited  even  in the  absence  of M029 in cells from all three species,  suggesting  that  other  MYXV  protein(s)  apart  from  M029  block  IFN  signaling  in  a  speciesindependent  manner.  We  also  show  that  the  antiviral  state  induced in rabbit, human or mouse cells  by  type  I IFN  can  inhibit M029-knockout MYXV even when PKR is genetically knocked-out, suggesting  that  M029  targets  other  host  proteins  for  this  antiviral state inhibition. Thus, the MYXV  dsRNA  binding  protein  M029  not  only  antagonizes  PKR  from  multiple  species  but  also blocks the  type I IFN antiviral state independently of PKR in a highly species-specific fashion.

  3. Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

    Directory of Open Access Journals (Sweden)

    Balart Luis A

    2010-10-01

    Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

  4. TRIM32 protein modulates type I interferon induction and cellular antiviral response by targeting MITA/STING protein for K63-linked ubiquitination.

    Science.gov (United States)

    Zhang, Jing; Hu, Ming-Ming; Wang, Yan-Yi; Shu, Hong-Bing

    2012-08-17

    Viral infection activates several transcription factors including NF-κB and IRF3, which collaborate to induce type I interferons (IFNs) and innate antiviral response. MITA (also called STING) is a critical adaptor protein that links virus-sensing receptors to IRF3 activation upon infection by both RNA and DNA pathogens. Here we show that the E3 ubiquitin ligase tripartite motif protein 32 (TRIM32) ubiquitinated MITA and dramatically enhanced MITA-mediated induction of IFN-β. Overexpression of TRIM32 potentiated virus-triggered IFNB1 expression and cellular antiviral response. Consistently, knockdown of TRIM32 had opposite effects. TRIM32 interacted with MITA, and was located at the mitochondria and endoplasmic reticulum. TRIM32 targeted MITA for K63-linked ubiquitination at K20/150/224/236 through its E3 ubiquitin ligase activity, which promoted the interaction of MITA with TBK1. These findings suggest that TRIM32 is an important regulatory protein for innate immunity against both RNA and DNA viruses by targeting MITA for K63-linked ubiquitination and downstream activation.

  5. Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Holger B Kramer

    2010-05-01

    Full Text Available The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT, termed virus inhibitory peptide (VIRIP, was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

  6. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells

    Science.gov (United States)

    Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negat...

  7. Detection of the antiviral activity of epicatechin isolated from Salacia crassifolia (Celastraceae) against Mayaro virus based on protein C homology modelling and virtual screening.

    Science.gov (United States)

    Ferreira, P G; Ferraz, A C; Figueiredo, J E; Lima, C F; Rodrigues, V G; Taranto, A G; Ferreira, J M S; Brandão, G C; Vieira-Filho, S A; Duarte, L P; de Brito Magalhães, C L; de Magalhães, J C

    2018-02-24

    Mayaro fever, caused by Mayaro virus (MAYV) is a sub-lethal disease with symptoms that are easily confused with those of dengue fever, except for polyarthralgia, which may culminate in physical incapacitation. Recently, outbreaks of MAYV have been documented in metropolitan areas, and to date, there is no therapy or vaccine available. Moreover, there is no information regarding the three-dimensional structure of the viral proteins of MAYV, which is important in the search for antivirals. In this work, we constructed a three-dimensional model of protein C of MAYV by homology modelling, and this was employed in a manner similar to that of receptors in virtual screening studies to evaluate 590 molecules as prospective antiviral agents. In vitro bioassays were utilized to confirm the potential antiviral activity of the flavonoid epicatechin isolated from Salacia crassifolia (Celastraceae). The virtual screening showed that six flavonoids were promising ligands for protein C. The bioassays showed potent antiviral action of epicatechin, which protected the cells from almost all of the effects of viral infection. An effective concentration (EC 50 ) of 0.247 μmol/mL was observed with a selectivity index (SI) of 7. The cytotoxicity assay showed that epicatechin has low toxicity, with a 50% cytotoxic concentration (CC 50 ) greater than 1.723 µmol/mL. Epicatechin was found to be twice as potent as the reference antiviral ribavirin. Furthermore, a replication kinetics assay showed a strong inhibitory effect of epicatechin on MAYV growth, with a reduction of at least four logs in virus production. Our results indicate that epicatechin is a promising candidate for further testing as an antiviral agent against Mayaro virus and other alphaviruses.

  8. Influenza A Virus Protein PA-X Contributes to Viral Growth and Suppression of the Host Antiviral and Immune Responses.

    Science.gov (United States)

    Hayashi, Tsuyoshi; MacDonald, Leslie A; Takimoto, Toru

    2015-06-01

    Influenza virus infection causes global inhibition of host protein synthesis in infected cells. This host shutoff is thought to allow viruses to escape from the host antiviral response, which restricts virus replication and spread. Although the mechanism of host shutoff is unclear, a novel viral protein expressed by ribosomal frameshifting, PA-X, was found to play a major role in influenza virus-induced host shutoff. However, little is known about the impact of PA-X expression on currently circulating influenza A virus pathogenicity and the host antiviral response. In this study, we rescued a recombinant influenza A virus, A/California/04/09 (H1N1, Cal), containing mutations at the frameshift motif in the polymerase PA gene (Cal PA-XFS). Cal PA-XFS expressed significantly less PA-X than Cal wild type (WT). Cal WT, but not Cal PA-XFS, induced degradation of host β-actin mRNA and suppressed host protein synthesis, supporting the idea that PA-X induces host shutoff via mRNA decay. Moreover, Cal WT inhibited beta interferon (IFN-β) expression and replicated more rapidly than Cal PA-XFS in human respiratory cells. Mice infected with Cal PA-XFS had significantly lower levels of viral growth and greater expression of IFN-β mRNA in their lungs than mice infected with Cal WT. Importantly, more antihemagglutinin and neutralizing antibodies were produced in Cal PA-XFS-infected mice than in Cal WT-infected mice, despite the lower level of virus replication in the lungs. Our data indicate that PA-X of the pandemic H1N1 virus has a strong impact on viral growth and the host innate and acquired immune responses to influenza virus. Virus-induced host protein shutoff is considered to be a major factor allowing viruses to evade innate and acquired immune recognition. We provide evidence that the 2009 H1N1 influenza A virus protein PA-X plays a role in virus replication and inhibition of host antiviral response by means of its host protein synthesis shutoff activity both in vitro

  9. Positive selection drives rapid evolution of certain amino acid residues in an evolutionarily highly conserved interferon-inducible antiviral protein of fishes.

    Science.gov (United States)

    Padhi, Abinash

    2013-01-01

    Viperin, an evolutionarily highly conserved interferon-inducible multifunctional protein, has previously been reported to exhibit antiviral activity against a wide range of DNA and RNA viruses. Utilizing the complete nucleotide coding sequence data of fish viperin antiviral genes, and employing the maximum likelihood-based codon substitution models, the present study reports the pervasive role of positive selection in the evolution of viperin antiviral protein in fishes. The overall rate of nonsynonymous (dN) to synonymous (dS) substitutions (dN/dS) for the three functional domains of viperin (N-terminal, central domain and C-terminal) were 1.1, 0.12, and 0.24, respectively. Codon-by-codon substitution analyses have revealed that while most of the positively selected sites were located at the N-terminal amphipathic α-helix domain, few amino acid residues at the C-terminal domain were under positive selection. However, none of the sites in the central domain were under positive selection. These results indicate that, although viperin is evolutionarily highly conserved, the three functional domains experienced differential selection pressures. Taken together with the results of previous studies, the present study suggests that the persistent antagonistic nature of surrounding infectious viral pathogens might be the likely cause for such adaptive evolutionary changes of certain amino acids in fish viperin antiviral protein.

  10. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan

    2016-01-26

    In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

  11. Complementary roles of Fas-associated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity.

    Science.gov (United States)

    Lu, Jennifer V; Weist, Brian M; van Raam, Bram J; Marro, Brett S; Nguyen, Long V; Srinivas, Prathna; Bell, Bryan D; Luhrs, Keith A; Lane, Thomas E; Salvesen, Guy S; Walsh, Craig M

    2011-09-13

    Caspase-8 (casp8) is required for extrinsic apoptosis, and mice deficient in casp8 fail to develop and die in utero while ultimately failing to maintain the proliferation of T cells, B cells, and a host of other cell types. Paradoxically, these failures are not caused by a defect in apoptosis, but by a presumed proliferative function of this protease. Indeed, following mitogenic stimulation, T cells lacking casp8 or its adaptor protein FADD (Fas-associated death domain protein) develop a hyperautophagic morphology, and die a programmed necrosis-like death process termed necroptosis. Recent studies have demonstrated that receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3 together facilitate TNF-induced necroptosis, but the precise role of RIPKs in the demise of T cells lacking FADD or casp8 activity is unknown. Here we demonstrate that RIPK3 and FADD have opposing and complementary roles in promoting T-cell clonal expansion and homeostasis. We show that the defective proliferation of T cells bearing an interfering form of FADD (FADDdd) is rescued by crossing with RIPK3(-/-) mice, although such rescue ultimately leads to lymphadenopathy. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that FADD, casp8, and RIPK3 are all essential for clonal expansion, contraction, and antiviral responses. Finally, we demonstrate that caspase-mediated cleavage of RIPK1-containing necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis in the face of death receptor signaling. These studies highlight the "two-faced" nature of casp8 activity, promoting clonal expansion in some situations and apoptotic demise in others.

  12. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

    Science.gov (United States)

    Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

    2016-01-01

    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. The interferon-inducible DNA-sensor protein IFI16: a key player in the antiviral response.

    Science.gov (United States)

    Dell'Oste, Valentina; Gatti, Deborah; Giorgio, Alessandro G; Gariglio, Marisa; Landolfo, Santo; De Andrea, Marco

    2015-01-01

    IFI16, a member of the IFN-inducible PYHIN-200 gene family, displays multifaceted activity due to its ability to bind to various target proteins and, in turn, modulate a variety cell functions including proliferation, differentiation, apoptosis/pyroptosis, senescence, and in? ammation. The last few year have seen major advances in our knowledge of IFI16 antiviral activity and its role in the immune response. Indeed, a wealth of evidence now supports a key role of IFI16 in the activation of innate immunity and viral restriction against Herpesviruses and Lentiviruses, such that the definition of IFI16 as a "restriction factor" is now widely accepted. However, most viruses have developed their own unique strategy to antagonize IFI16, leading to a modification or disruption of its function. This review summarizes our current understanding of how viral replication is sensed and then inhibited by IFI16 protein and the viral strategies employed to defeat this host defense mechanism. We will focus mainly on Herpesviruses, although recent discoveries on the role of IFI16 in lentiviral infection will also be considered.

  14. Rational design of highly potent HIV-1 fusion inhibitory proteins: Implication for developing antiviral therapeutics

    International Nuclear Information System (INIS)

    Ni Ling; Gao, George F.; Tien Po

    2005-01-01

    Recombinant protein containing one heptad-repeat 1 (HR1) segment and one HR2 segment of the HIV-1 gp41 (HR1-HR2) has been shown to fold into thermally stable six-helix bundle, representing the fusogenic core of gp41. In this study, we have used the fusogenic core as a scaffold to design HIV-1 fusion inhibitory proteins by linking another HR1 to the C terminus of HR1-HR2 (HR121) or additional HR2 to the N terminus of HR1-HR2 (HR212). Both recombinant proteins could be abundantly and solubly expressed and easily purified, exhibiting high stability and potent inhibitory activity on HIV-1 fusion with IC 50 values of 16.2 ± 2.8 and 2.8 ± 0.63 nM, respectively. These suggest that these rationally designed proteins can be further developed as novel anti-HIV-1 therapeutics

  15. Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

    Science.gov (United States)

    Chan, Chi-Ping; Yuen, Chun-Kit; Cheung, Pak-Hin Hinson; Fung, Sin-Yee; Lui, Pak-Yin; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

    2018-03-07

    PACT is a double-stranded RNA-binding protein that has been implicated in host-influenza A virus (IAV) interaction. PACT facilitates the action of RIG-I in the activation of the type I IFN response, which is suppressed by the viral nonstructural protein NS1. PACT is also known to interact with the IAV RNA polymerase subunit PA. Exactly how PACT exerts its antiviral activity during IAV infection remains to be elucidated. In the current study, we demonstrated the interplay between PACT and IAV polymerase. Induction of IFN-β by the IAV RNP complex was most robust when both RIG-I and PACT were expressed. PACT-dependent activation of IFN-β production was suppressed by the IAV polymerase subunits, polymerase acidic protein, polymerase basic protein 1 (PB1), and PB2. PACT associated with PA, PB1, and PB2. Compromising PACT in IAV-infected A549 cells resulted in the augmentation of viral RNA (vRNA) transcription and replication and IFN-β production. Furthermore, vRNA replication was boosted by knockdown of PACT in both A549 cells and IFN-deficient Vero cells. Thus, the antiviral activity of PACT is mediated primarily via its interaction with and inhibition of IAV polymerase. Taken together, our findings reveal a new facet of the host-IAV interaction in which the interplay between PACT and IAV polymerase affects the outcome of viral infection and antiviral response.-Chan, C.-P., Yuen, C.-K., Cheung, P.-H. H., Fung, S.-Y., Lui, P.-Y., Chen, H., Kok, K.-H., Jin, D.-Y. Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

  16. Interaction Research on the Antiviral Molecule Dufulin Targeting on Southern Rice Black Streaked Dwarf Virus P9-1 Nonstructural Protein

    Directory of Open Access Journals (Sweden)

    Zhenchao Wang

    2015-03-01

    Full Text Available ern rice black streaked dwarf virus (SRBSDV causes severe harm to rice production. Unfortunately, studies on effective antiviral drugs against SRBSDV and interaction mechanism of antiviral molecule targeting on SRBSDV have not been reported. This study found dufulin (DFL, an ideal anti-SRBSDV molecule, and investigated the interactions of DFL targeting on the nonstructural protein P9-1. The biological sequence information and bonding characterization of DFL to four kinds of P9-1 protein were described with fluorescence titration (FT and microscale thermophoresis (MST assays. The sequence analysis indicated that P9-1 had highly-conserved C- and N-terminal amino acid residues and a hypervariable region that differed from 131 aa to 160 aa. Consequently, wild-type (WT-His-P9-1, 23 C-terminal residues truncated (TR-ΔC23-His-P9-1, 6 N-terminal residues truncated (TR-ΔN6-His-P9-1, and Ser138 site-directed (MU-138-His-P9-1 mutant proteins were expressed. The FT and MST assay results indicated that DFL bounded to WT-His-P9-1 with micromole affinity and the 23 C-terminal amino acids were the potential targeting site. This system, which combines a complete sequence analysis, mutant protein expression, and binding action evaluating system, could further advance the understanding of the interaction abilities between antiviral drugs and their targets.

  17. Lopinavir up-regulates expression of the antiviral protein ribonuclease L in human papillomavirus-positive cervical carcinoma cells.

    Science.gov (United States)

    Batman, Gavin; Oliver, Anthony W; Zehbe, Ingeborg; Richard, Christina; Hampson, Lynne; Hampson, Ian N

    2011-01-01

    We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism. SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used. Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression. These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.

  18. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA1 TLR signaling molecules MAVS IPS1, KIAA1271, VISA VISA_(gene) Mitochondrial antiviral-signaling pr...otein CARD adapter inducing interferon beta, Interferon beta promoter stimulator protein... 1, Putative NF-kappa-B-activating protein 031N, Virus-induced-signaling adapter 9606 Homo sapiens Q7Z434 57506 2VGQ 57506 ...

  19. Multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity.

    Directory of Open Access Journals (Sweden)

    Sophiya Karki

    Full Text Available The zinc finger antiviral protein (ZAP is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV, the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.

  20. Acyclic nucleoside phosphonate antivirals activate gene expression of monocyte chemotactic protein 1 and 3.

    Czech Academy of Sciences Publication Activity Database

    Potměšil, Petr; Holý, Antonín; Kmoníčková, Eva; Křížková, Jana; Zídek, Zdeněk

    2007-01-01

    Roč. 14, č. 1 (2007), s. 59-66 ISSN 1021-7770 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z40550506 Keywords : Acyclic nucleoside phosponate * HIV * Monocyte chemotactic protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.024, year: 2007

  1. dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

    Directory of Open Access Journals (Sweden)

    Eliane F. Meurs

    2012-10-01

    Full Text Available The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2a. As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2a kinase, its participation in the regulation of the NF-kB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV. PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2a-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages.

  2. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation of subs...... the Arabidopsis thaliana MAPKs MPK3, 4, and 6 and MAP2Ks MKK1, 2, 4, and 5. Future work needs to focus on identifying substrates of MAPKs, and on understanding how specificity is achieved among MAPK signaling pathways.......Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

  3. Protein phosphatase 2A: the Trojan Horse of cellular signaling.

    Science.gov (United States)

    Sontag, E

    2001-01-01

    Dynamic phosphorylation and dephosphorylation of proteins are fundamental mechanisms utilized by cells to transduce signals. Whereas transduction by protein kinases has been a major focus of studies in the last decade, protein phosphatase 2A (PP2A) enzymes emerge in this millenium as the most fashionable players in cellular signaling. Viral proteins target specific PP2A enzymes in order to deregulate chosen cellular pathways in the host and promote viral progeny. The observation that a variety of viruses utilize PP2A to alienate cellular behavior emphasizes the fundamental importance of PP2A in signal transduction. This review will primarily focus on discussing the uniqueness of PP2A regulation and uncovering the critical role played by protein-protein interactions in the modulation of PP2A signaling. Moreover, the place of PP2A in signaling pathways and its functional significance for human diseases will be discussed.

  4. The ubiquitin-like protein PLIC-1 or ubiquilin 1 inhibits TLR3-Trif signaling.

    Directory of Open Access Journals (Sweden)

    Nabanita Biswas

    Full Text Available The innate immune responses to virus infection are initiated by either Toll-like receptors (TLR3/7/8/9 or cytoplasmic double-stranded RNA (dsRNA-recognizing RNA helicases RIG-I and MDA5. To avoid causing injury to the host, these signaling pathways must be switched off in time by negative regulators.Through yeast-two hybrid screening, we found that an ubiquitin-like protein named protein linking integrin-associated protein to cytoskeleton 1(PLIC-1 or Ubiquilin 1 interacted with the Toll/interleukin-1 receptor (TIR domain of TLR4. Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway. Concomitantly, reduction of endogenous PLIC-1 by short-hairpin interfering RNA (shRNA enhanced TLR3 activation both in luciferase reporter assays as well as in new castle disease virus (NDV infected cells. An interaction between PLIC-1 and Trif was confirmed in co-immunoprecipitation (Co-IP and GST-pull-down assays. Subsequent confocal microscopic analysis revealed that PLIC-1 and Trif colocalized with the autophagosome marker LC3 in punctate subcellular structures. Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

  5. Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; Peters, Stephan L. M.; Michel, Martin C.; Alewijnse, Astrid E.

    2008-01-01

    G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization

  6. Inducible protein-10 as a predictive marker of antiviral hepatitis C treatment

    DEFF Research Database (Denmark)

    Neesgaard, Bastian; Ruhwald, Morten; Weis, Nina

    2017-01-01

    AIM: To investigate interferon-γ-inducible protein-10's (IP-10) potential to anticipate rapid (RVR)- and sustained virological responses (SVR) to chronic hepatitis C (CHC) treatment. METHODS: We included case series examining RVR or SVR in relation to 24 or 48 wk treatment for CHC, in patients...... treatment free for at least six months, with genotype 1 or 4, and in relation to 24 wk treatment for genotype 2 and 3, with pegylated interferon in combination with ribavirin. Patients had to have both a baseline IP-10 level as well as a hepatitis C virus (HCV)-RNA determination 4 wk after treatment...... initiation or 24 wk after end of treatment. Studies including patients with liver diseases other than CHC, human immunodeficiency virus-infection, treatment with immunosuppresents or cytostatica, alcohol dependency or active intravenous drug-use were excluded. We found 81 articles by searching the MEDLINE...

  7. Biased and g protein-independent signaling of chemokine receptors

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie

    2014-01-01

    ), different receptors (with the same ligand), or different tissues or cells (for the same ligand-receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may...

  8. Regulator of G-protein signaling - 5 (RGS5 is a novel repressor of hedgehog signaling.

    Directory of Open Access Journals (Sweden)

    William M Mahoney

    Full Text Available Hedgehog (Hh signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc and smoothened (Smo. Recent studies identify Smo as a G-protein coupled receptor (GPCR-like protein that signals through large G-protein complexes which contain the Gαi subunit. We hypothesize Regulator of G-Protein Signaling (RGS proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs for GTP-bound Gαi, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP, we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.

  9. Mx1 and Mx2 key antiviral proteins are surprisingly lost in toothed whales.

    Science.gov (United States)

    Braun, Benjamin A; Marcovitz, Amir; Camp, J Gray; Jia, Robin; Bejerano, Gill

    2015-06-30

    Viral outbreaks in dolphins and other Delphinoidea family members warrant investigation into the integrity of the cetacean immune system. The dynamin-like GTPase genes Myxovirus 1 (Mx1) and Mx2 defend mammals against a broad range of viral infections. Loss of Mx1 function in human and mice enhances infectivity by multiple RNA and DNA viruses, including orthomyxoviruses (influenza A), paramyxoviruses (measles), and hepadnaviruses (hepatitis B), whereas loss of Mx2 function leads to decreased resistance to HIV-1 and other viruses. Here we show that both Mx1 and Mx2 have been rendered nonfunctional in Odontoceti cetaceans (toothed whales, including dolphins and orcas). We discovered multiple exon deletions, frameshift mutations, premature stop codons, and transcriptional evidence of decay in the coding sequence of both Mx1 and Mx2 in four species of Odontocetes. We trace the likely loss event for both proteins to soon after the divergence of Odontocetes and Mystocetes (baleen whales) ∼33-37 Mya. Our data raise intriguing questions as to what drove the loss of both Mx1 and Mx2 genes in the Odontoceti lineage, a double loss seen in none of 56 other mammalian genomes, and suggests a hitherto unappreciated fundamental genetic difference in the way these magnificent mammals respond to viral infections.

  10. Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity

    Science.gov (United States)

    Lee, Eun-Young; Lee, Hyun-Cheol; Kim, Hyun-Kwan; Jang, Song Yee; Park, Seong-Jun; Kim, Yong-Hoon; Kim, Jong Hwan; Hwang, Jungwon; Kim, Jae-Hoon; Kim, Tae-Hwan; Arif, Abul; Kim, Seon-Young; Choi, Young-Ki; Lee, Cheolju; Lee, Chul-Ho; Jung, Jae U; Fox, Paul L; Kim, Sunghoon; Lee, Jong-Soo; Kim, Myung Hee

    2016-01-01

    The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/−) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection. PMID:27595231

  11. A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

    Science.gov (United States)

    King, Benjamin R; Hershkowitz, Dylan; Eisenhauer, Philip L; Weir, Marion E; Ziegler, Christopher M; Russo, Joanne; Bruce, Emily A; Ballif, Bryan A; Botten, Jason

    2017-08-01

    Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV. IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend

  12. Morbillivirus v proteins exhibit multiple mechanisms to block type 1 and type 2 interferon signalling pathways.

    Directory of Open Access Journals (Sweden)

    Senthil K Chinnakannan

    Full Text Available Morbilliviruses form a closely related group of pathogenic viruses which encode three non-structural proteins V, W and C in their P gene. Previous studies with rinderpest virus (RPV and measles virus (MeV have demonstrated that these non-structural proteins play a crucial role in blocking type I (IFNα/β and type II (IFNγ interferon action, and various mechanisms have been proposed for these effects. We have directly compared four important morbilliviruses, rinderpest (RPV, measles virus (MeV, peste des petits ruminants virus (PPRV and canine distemper virus (CDV. These viruses and their V proteins could all block type I IFN action. However, the viruses and their V proteins had varying abilities to block type II IFN action. The ability to block type II IFN-induced gene transcription correlated with co-precipitation of STAT1 with the respective V protein, but there was no correlation between co-precipitation of either STAT1 or STAT2 and the abilities of the V proteins to block type I IFN-induced gene transcription or the creation of the antiviral state. Further study revealed that the V proteins of RPV, MeV, PPRV and CDV could all interfere with phosphorylation of the interferon-receptor-associated kinase Tyk2, and the V protein of highly virulent RPV could also block the phosphorylation of another such kinase, Jak1. Co-precipitation studies showed that morbillivirus V proteins all form a complex containing Tyk2 and Jak1. This study highlights the ability of morbillivirus V proteins to target multiple components of the IFN signalling pathways to control both type I and type II IFN action.

  13. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins

    DEFF Research Database (Denmark)

    Hansen, J A; Lindberg, K; Hilton, D J

    1999-01-01

    In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS pro...

  14. Dystrophin complex functions as a scaffold for signalling proteins.

    Science.gov (United States)

    Constantin, Bruno

    2014-02-01

    Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part

  15. Inhibition of cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal.

    Directory of Open Access Journals (Sweden)

    Tyler M Sharp

    2010-10-01

    Full Text Available Protein trafficking between the endoplasmic reticulum (ER and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development.

  16. LocSigDB: a database of protein localization signals

    Science.gov (United States)

    Negi, Simarjeet; Pandey, Sanjit; Srinivasan, Satish M.; Mohammed, Akram; Guda, Chittibabu

    2015-01-01

    LocSigDB (http://genome.unmc.edu/LocSigDB/) is a manually curated database of experimental protein localization signals for eight distinct subcellular locations; primarily in a eukaryotic cell with brief coverage of bacterial proteins. Proteins must be localized at their appropriate subcellular compartment to perform their desired function. Mislocalization of proteins to unintended locations is a causative factor for many human diseases; therefore, collection of known sorting signals will help support many important areas of biomedical research. By performing an extensive literature study, we compiled a collection of 533 experimentally determined localization signals, along with the proteins that harbor such signals. Each signal in the LocSigDB is annotated with its localization, source, PubMed references and is linked to the proteins in UniProt database along with the organism information that contain the same amino acid pattern as the given signal. From LocSigDB webserver, users can download the whole database or browse/search for data using an intuitive query interface. To date, LocSigDB is the most comprehensive compendium of protein localization signals for eight distinct subcellular locations. Database URL: http://genome.unmc.edu/LocSigDB/ PMID:25725059

  17. BAR domain proteins regulate Rho GTPase signaling.

    Science.gov (United States)

    Aspenström, Pontus

    2014-01-01

    BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

  18. Regulation, Signaling, and Physiological Functions of G-Proteins.

    Science.gov (United States)

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Signaling within Allosteric Machines: Signal Transmission Pathways Inside G Protein-Coupled Receptors.

    Science.gov (United States)

    Bartuzi, Damian; Kaczor, Agnieszka A; Matosiuk, Dariusz

    2017-07-15

    In recent years, our understanding of function of G protein-coupled receptors (GPCRs) has changed from a picture of simple signal relays, transmitting only a particular signal to a particular G protein heterotrimer, to versatile machines, capable of various responses to different stimuli and being modulated by various factors. Some recent reports provide not only the data on ligands/modulators and resultant signals induced by them, but also deeper insights into exact pathways of signal migration and mechanisms of signal transmission through receptor structure. Combination of these computational and experimental data sheds more light on underlying mechanisms of signal transmission and signaling bias in GPCRs. In this review we focus on available clues on allosteric pathways responsible for complex signal processing within GPCRs structures, with particular emphasis on linking compatible in silico- and in vitro-derived data on the most probable allosteric connections.

  20. The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein

    Energy Technology Data Exchange (ETDEWEB)

    Hoenen, Antje [School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane (Australia); Gillespie, Leah [Department of Microbiology, La Trobe University, Melbourne (Australia); Department of Microbiology and Immunology, University of Melbourne, Melbourne (Australia); Morgan, Garry; Heide, Peter van der [Institute for Molecular Bioscience, University of Queensland, Brisbane (Australia); Khromykh, Alexander [School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane (Australia); Australian Infectious Diseases Research Centre, University of Queensland, Brisbane (Australia); Mackenzie, Jason, E-mail: jason.mackenzie@unimelb.edu.au [Department of Microbiology, La Trobe University, Melbourne (Australia); Department of Microbiology and Immunology, University of Melbourne, Melbourne (Australia)

    2014-01-05

    Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNV{sub KUN}) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNV{sub KUN} particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNV{sub KUN} under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNV{sub KUN} assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNV{sub KUN} assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system. - Highlights: • We show that the ISG MxA can recognize the West Nile virus capsid protein. • Interaction between WNV C protein and MxA induces cytoplasmic fibrils. • MxA can be retargeted to the ER to restrict WNV particle release. • WNV assembly process is a strategy to avoid MxA recognition.

  1. The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein

    International Nuclear Information System (INIS)

    Hoenen, Antje; Gillespie, Leah; Morgan, Garry; Heide, Peter van der; Khromykh, Alexander; Mackenzie, Jason

    2014-01-01

    Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNV KUN ) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNV KUN particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNV KUN under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNV KUN assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNV KUN assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system. - Highlights: • We show that the ISG MxA can recognize the West Nile virus capsid protein. • Interaction between WNV C protein and MxA induces cytoplasmic fibrils. • MxA can be retargeted to the ER to restrict WNV particle release. • WNV assembly process is a strategy to avoid MxA recognition

  2. Involvement of a putative intercellular signal-recognizing G protein ...

    African Journals Online (AJOL)

    Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena. PN Agbedanu, MT Brewer, TA Day, MJ Kimber, KL Anderson, SK Rasmussen, MA Rasmussen, SA Carlson ...

  3. Protein phosphorylation and its role in archaeal signal transduction

    Science.gov (United States)

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

    2016-01-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  4. Influenza A virus encoding secreted Gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins.

    Directory of Open Access Journals (Sweden)

    Nadine Eckert

    Full Text Available Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI zanamivir and the host cell interferon-inducible transmembrane (IFITM proteins 1-3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.

  5. Bone morphogenetic protein signalling in colorectal cancer

    NARCIS (Netherlands)

    Hardwick, James C.; Kodach, Liudmila L.; Offerhaus, G. Johan; van den Brink, Gijs R.

    2008-01-01

    Much of the current understanding of colorectal cancer stems from the study of rare, inherited colorectal cancer syndromes. Mutations in the bone morphogenetic protein (BMP) pathway have been found in juvenile polyposis, an inherited polyposis syndrome that predisposes to colorectal cancer. The

  6. Interferon induced IFIT family genes in host antiviral defense.

    Science.gov (United States)

    Zhou, Xiang; Michal, Jennifer J; Zhang, Lifan; Ding, Bo; Lunney, Joan K; Liu, Bang; Jiang, Zhihua

    2013-01-01

    Secretion of interferons (IFNs) from virus-infected cells is a hallmark of host antiviral immunity and in fact, IFNs exert their antiviral activities through the induction of antiviral proteins. The IFN-induced protein with tetratricopeptide repeats (IFITs) family is among hundreds of IFN-stimulated genes. This family contains a cluster of duplicated loci. Most mammals have IFIT1, IFIT2, IFIT3 and IFIT5; however, bird, marsupial, frog and fish have only IFIT5. Regardless of species, IFIT5 is always adjacent to SLC16A12. IFIT family genes are predominantly induced by type I and type III interferons and are regulated by the pattern recognition and the JAK-STAT signaling pathway. IFIT family proteins are involved in many processes in response to viral infection. However, some viruses can escape the antiviral functions of the IFIT family by suppressing IFIT family genes expression or methylation of 5' cap of viral molecules. In addition, the variants of IFIT family genes could significantly influence the outcome of hepatitis C virus (HCV) therapy. We believe that our current review provides a comprehensive picture for the community to understand the structure and function of IFIT family genes in response to pathogens in human, as well as in animals.

  7. How fast does a signal propagate through proteins?

    Directory of Open Access Journals (Sweden)

    Hui T Young

    Full Text Available As the molecular basis of signal propagation in the cell, proteins are regulated by perturbations, such as mechanical forces or ligand binding. The question arises how fast such a signal propagates through the protein molecular scaffold. As a first step, we have investigated numerically the dynamics of force propagation through a single (Ala[Formula: see text] protein following a sudden increase in the stretching forces applied to its end termini. The force propagates along the backbone into the center of the chain on the picosecond scale. Both conformational and tension dynamics are found in good agreement with a coarse-grained theory of force propagation through semiflexible polymers. The speed of force propagation of [Formula: see text]50Å ps(-1 derived from these simulations is likely to determine an upper speed limit of mechanical signal transfer in allosteric proteins or molecular machines.

  8. Rap G protein signal in normal and disordered lymphohematopoiesis.

    Science.gov (United States)

    Minato, Nagahiro

    2013-09-10

    Rap proteins (Rap1, Rap2a, b, c) are small molecular weight GTPases of the Ras family. Rap G proteins mediate diverse cellular events such as cell adhesion, proliferation, and gene activation through various signaling pathways. Activation of Rap signal is regulated tightly by several specific regulatory proteins including guanine nucleotide exchange factors and GTPase-activating proteins. Beyond cell biological studies, increasing attempts have been made in the past decade to define the roles of Rap signal in specific functions of normal tissue systems as well as in cancer. In the immune and hematopoietic systems, Rap signal plays crucial roles in the development and function of essentially all lineages of lymphocytes and hematopoietic cells, and importantly, deregulated Rap signal may lead to unique pathological conditions depending on the affected cell types, including various types of leukemia and autoimmunity. The phenotypical studies have unveiled novel, even unexpected functional aspects of Rap signal in cells from a variety of tissues, providing potentially important clues for controlling human diseases, including malignancy. © 2013 Elsevier Inc. All rights reserved.

  9. Linking proteins to signaling pathways for experiment design and evaluation.

    Directory of Open Access Journals (Sweden)

    Illés J Farkas

    Full Text Available Biomedical experimental work often focuses on altering the functions of selected proteins. These changes can hit signaling pathways, and can therefore unexpectedly and non-specifically affect cellular processes. We propose PathwayLinker, an online tool that can provide a first estimate of the possible signaling effects of such changes, e.g., drug or microRNA treatments. PathwayLinker minimizes the users' efforts by integrating protein-protein interaction and signaling pathway data from several sources with statistical significance tests and clear visualization. We demonstrate through three case studies that the developed tool can point out unexpected signaling bias in normal laboratory experiments and identify likely novel signaling proteins among the interactors of known drug targets. In our first case study we show that knockdown of the Caenorhabditis elegans gene cdc-25.1 (meant to avoid progeny may globally affect the signaling system and unexpectedly bias experiments. In the second case study we evaluate the loss-of-function phenotypes of a less known C. elegans gene to predict its function. In the third case study we analyze GJA1, an anti-cancer drug target protein in human, and predict for this protein novel signaling pathway memberships, which may be sources of side effects. Compared to similar services, a major advantage of PathwayLinker is that it drastically reduces the necessary amount of manual literature searches and can be used without a computational background. PathwayLinker is available at http://PathwayLinker.org. Detailed documentation and source code are available at the website.

  10. Isolation of Rhp-PSP, a member of YER057c/YjgF/UK114 protein family with antiviral properties, from the photosynthetic bacterium Rhodopseudomonas palustris strain JSC-3b.

    Science.gov (United States)

    Su, Pin; Feng, Tuizi; Zhou, Xuguo; Zhang, Songbai; Zhang, Yu; Cheng, Ju'e; Luo, Yuanhua; Peng, Jing; Zhang, Zhuo; Lu, Xiangyang; Zhang, Deyong; Liu, Yong

    2015-11-04

    Rhodopseudomonas palustris strain JSC-3b isolated from a water canal adjacent to a vegetable field produces a protein that was purified by bioactivity-guided fractionation based on ammonium sulfate precipitation, ion-exchange absorption and size exclusion. The protein was further identified as an endoribonuclease L-PSP (Liver-Perchloric acid-soluble protein) by shotgun mass spectrometry analysis and gene identification, and it is member of YER057c/YjgF/UK114 protein family. Herein, this protein is designated Rhp-PSP. Rhp-PSP exhibited significant inhibitory activities against tobacco mosaic virus (TMV) in vivo and in vitro. To our knowledge, this represents the first report on the antiviral activity of a protein of the YER057c/YjgF/UK114 family and also the first antiviral protein isolated from R. palustris. Our research provides insight into the potential of photosynthetic bacterial resources in biological control of plant virus diseases and sustainable agriculture.

  11. Signal regulatory proteins (SIRPS) are secreted presynaptic organizing molecules.

    Science.gov (United States)

    Umemori, Hisashi; Sanes, Joshua R

    2008-12-05

    Formation of chemical synapses requires exchange of organizing signals between the synaptic partners. Using synaptic vesicle aggregation in cultured neurons as a marker of presynaptic differentiation, we purified candidate presynaptic organizers from mouse brain. A major bioactive species was the extracellular domain of signal regulatory protein alpha (SIRP-alpha), a transmembrane immunoglobulin superfamily member concentrated at synapses. The extracellular domain of SIRP-alpha is cleaved and shed in a developmentally regulated manner. The presynaptic organizing activity of SIRP-alpha is mediated in part by CD47. SIRP-alpha homologues, SIRP-beta and -gamma also have synaptic vesicle clustering activity. The effects of SIRP-alpha are distinct from those of another presynaptic organizer, FGF22: the two proteins induced vesicle clusters of different sizes, differed in their ability to promote neurite branching, and acted through different receptors and signaling pathways. SIRP family proteins may act together with other organizing molecules to pattern synapses.

  12. Intramolecular Conformational Changes Optimize Protein Kinase C Signaling#

    Science.gov (United States)

    Antal, Corina E.; Violin, Jonathan D.; Kunkel, Maya T.; Skovsø, Søs

    2014-01-01

    Summary Optimal tuning of enzyme signaling is critical for cellular homeostasis. We use fluorescence resonance energy transfer reporters in live cells to follow conformational transitions that tune the affinity of a multi-domain signal transducer, protein kinase C, for optimal response to second messengers. This enzyme comprises two diacylglycerol sensors, the C1A and C1B domains, whose intrinsic affinity for ligand is sufficiently high that the enzyme would be in a ligand-engaged, active state if not for mechanisms that mask its domains. We show that both diacylglycerol sensors are exposed in newly-synthesized protein kinase C and that conformational transitions following priming phosphorylations mask the domains such that the lower affinity sensor, the C1B domain, is the primary diacylglycerol binder. Protein kinase C's conformational rearrangements serve as a paradigm for how multi-module transducers optimize their dynamic range of signaling. PMID:24631122

  13. Protein and signaling networks in vertebrate photoreceptor cells

    Directory of Open Access Journals (Sweden)

    Karl-Wilhelm eKoch

    2015-11-01

    Full Text Available Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cGMP and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase GRK1 under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases and is regulated by specific neuronal Ca2+-sensor proteins called GCAPs. At least one guanylate cyclase (ROS-GC1 was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments.

  14. G protein signaling in the parasite Entamoeba histolytica

    Science.gov (United States)

    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

  15. Role of Regulators of G Protein Signaling Proteins in Bone Physiology and Pathophysiology.

    Science.gov (United States)

    Jules, Joel; Yang, Shuying; Chen, Wei; Li, Yi-Ping

    2015-01-01

    Regulators of G protein signaling (RGS) proteins enhance the intrinsic GTPase activity of α subunits of the heterotrimeric G protein complex of G protein-coupled receptors (GPCRs) and thereby inactivate signal transduction initiated by GPCRs. The RGS family consists of nearly 37 members with a conserved RGS homology domain which is critical for their GTPase accelerating activity. RGS proteins are expressed in most tissues, including heart, lung, brain, kidney, and bone and play essential roles in many physiological and pathological processes. In skeletal development and bone homeostasis as well as in many bone disorders, RGS proteins control the functions of various GPCRs, including the parathyroid hormone receptor type 1 and calcium-sensing receptor and also regulate various critical signaling pathways, such as Wnt and calcium oscillations. This chapter will discuss the current findings on the roles of RGS proteins in regulating signaling of key GPCRs in skeletal development and bone homeostasis. We also will examine the current updates of RGS proteins' regulation of calcium oscillations in bone physiology and highlight the roles of RGS proteins in selected bone pathological disorders. Despite the recent advances in bone and mineral research, RGS proteins remain understudied in the skeletal system. Further understanding of the roles of RGS proteins in bone should not only provide great insights into the molecular basis of various bone diseases but also generate great therapeutic drug targets for many bone diseases. © 2015 Elsevier Inc. All rights reserved.

  16. Structural Reconstruction of Protein-Protein Complexes Involved in Intracellular Signaling.

    Science.gov (United States)

    Kirsch, Klára; Sok, Péter; Reményi, Attila

    2016-01-01

    Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity.

  17. G protein-coupled receptor accessory proteins and signaling: pharmacogenomic insights.

    Science.gov (United States)

    Thompson, Miles D; Cole, David E C; Jose, Pedro A; Chidiac, Peter

    2014-01-01

    The identification and characterization of the genes encoding G protein-coupled receptors (GPCRs) and the proteins necessary for the processes of ligand binding, GPCR activation, inactivation, and receptor trafficking to the membrane are discussed in the context of human genetic disease. In addition to functional GPCR variants, the identification of genetic disruptions affecting proteins necessary to GPCR functions have provided insights into the function of these pathways. Gsα and Gβ subunit polymorphisms have been found to result in complex phenotypes. Disruptions in accessory proteins that normally modify or organize heterotrimeric G-protein coupling may also result in disease states. These include the contribution of variants of the regulator of G protein signaling (RGS) protein to hypertension; the role variants of the activator of G protein signaling (AGS) proteins to phenotypes (such as the type III AGS8 variant to hypoxia); the contribution of G protein-coupled receptor kinase (GRK) proteins, such as GRK4, in disorders such as hypertension. The role of accessory proteins in GPCR structure and function is discussed in the context of genetic disorders associated with disruption of the genes that encode them. An understanding of the pharmacogenomics of GPCR and accessory protein signaling provides the basis for examining both GPCR pharmacogenetics and the genetics of monogenic disorders that result from disruption of given receptor systems.

  18. Protein import into plant mitochondria: signals, machinery, processing, and regulation.

    Science.gov (United States)

    Murcha, Monika W; Kmiec, Beata; Kubiszewski-Jakubiak, Szymon; Teixeira, Pedro F; Glaser, Elzbieta; Whelan, James

    2014-12-01

    The majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. The interferon response circuit in antiviral host defense.

    Science.gov (United States)

    Haller, O; Weber, F

    2009-01-01

    Viruses have learned to multiply in the face of a powerful innate and adaptive immune response of the host. They have evolved multiple strategies to evade the interferon (IFN) system which would otherwise limit virus growth at an early stage of infection. IFNs induce the synthesis of a range of antiviral proteins which serve as cell-autonomous intrinsic restriction factors. For example, the dynamin-like MxA GTPase inhibits the multiplication of influenza and bunyaviruses (such as La Crosse virus, Hantaan virus, Rift Valley Fever virus, and Crimean-Congo hemorrhagic fever virus) by binding and sequestering the nucleocapsid protein into large perinuclear complexes. To overcome such intracellular restrictions, virulent viruses either inhibit IFN synthesis, bind and inactivate secreted IFN molecules, block IFN-activated signaling, or disturb the action of IFN-induced antiviral proteins. Many viruses produce specialized proteins to disarm the danger signal or express virulence genes that target members of the IFN regulatory factor family (IRFs) or components of the JAK-STAT signaling pathway. An alternative evasion strategy is based on extreme viral replication speed which out-competes the IFN response. The identification of viral proteins with IFN antagonistic functions has great implications for disease prevention and therapy. Virus mutants lacking IFN antagonistic properties represent safe yet highly immunogenic candidate vaccines. Furthermore, novel drugs intercepting viral IFN-antagonists could be used to disarm the viral intruders.

  20. 14-3-3 proteins in guard cell signaling

    Directory of Open Access Journals (Sweden)

    Valérie eCotelle

    2016-01-01

    Full Text Available Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses.

  1. The signaling helix: a common functional theme in diverse signaling proteins

    Directory of Open Access Journals (Sweden)

    Aravind L

    2006-09-01

    Full Text Available Abstract Background The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood. Results Using sensitive sequence analysis methods we identify a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins, such as the Dimerization-Histidine phosphotransfer module of histidine kinases, the intra-cellular domains of the chemotaxis receptors, inter-GAF domain helical linkers and the α-helical HAMP module. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains such as histidine kinases, cNMP cyclase, PP2C phosphatases, NtrC-like AAA+ ATPases and diguanylate cyclases. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. The sequence conservation pattern of the S-helix revealed the presence of a unique constellation of polar residues in the dimer-interface positions within the central heptad of the coiled-coil formed by the S-helix. Conclusion Combining these observations with previously reported mutagenesis studies on different S-helix-containing proteins we suggest that it functions as a switch that prevents constitutive activation of linked downstream

  2. Redox Signaling Regulated by Cysteine Persulfide and Protein Polysulfidation.

    Science.gov (United States)

    Kasamatsu, Shingo; Nishimura, Akira; Morita, Masanobu; Matsunaga, Tetsuro; Abdul Hamid, Hisyam; Akaike, Takaaki

    2016-12-15

    For decades, reactive persulfide species including cysteine persulfide (CysSSH) have been known to exist endogenously in organisms. However, the physiological significance of endogenous persulfides remains poorly understood. That cystathionine β-synthase and cystathionine γ-lyase produced CysSSH from cystine was recently demonstrated. An endogenous sulfur transfer system involving CysSSH evidently generates glutathione persulfide (GSSH) that exists at concentrations greater than 100 μM in vivo. Because reactive persulfide species such as CysSSH and GSSH have higher nucleophilicity than parental cysteine (Cys) and glutathione do, these reactive species exhibit strong scavenging activities against oxidants, e.g., hydrogen peroxide, and electrophiles, which contributes to redox signaling regulation. Also, several papers indicated that various proteins and enzymes have Cys polysulfides including CysSSH at their specific Cys residues, which is called protein polysulfidation. Apart from the redox signaling regulatory mechanism, another plausible function of protein polysulfidation is providing protection for protein thiol residues against irreversible chemical modification caused by oxidants and electrophiles. Elucidation of the redox signaling regulatory mechanism of reactive persulfide species including small thiol molecules and thiol-containing proteins should lead to the development of new therapeutic strategies and drug discoveries for oxidative and electrophilic stress-related diseases.

  3. Dynamic protein complexes regulate NF-kappaB signaling.

    Science.gov (United States)

    Wegener, E; Krappmann, D

    2008-01-01

    NF-kappaB is a major regulator of the first-line defense against invading pathogens, antigen-specific adaptive immune responses or chemical stress. Stimulation either by extracellular ligands (e.g., inflammatory cytokines, microbial pathogens, peptide antigens) or by intracellular Stressors (e.g., genotoxic drugs) initiates signal-specific pathways that all converge at the IkappaB kinase (IKK) complex, the gatekeeper for NF-kappaB activation. During recent years, considerable progress has been made in understanding the function of NF-kappaB in the regulation of cell growth, survival and apoptosis. In this review, we will focus on the regulation of large signaling complexes on the route to NF-kappaB. Recently published data demonstrate that the assembly, maintenance and activity of the IKK complex determine downstream activation of NF-kappaB. In addition, dynamic complexes upstream of IKK are formed in response to tumor necrosis factor (TNF), antigenic peptides or DNA-damaging agents. Clustering of signaling adaptors promotes the association and activation of ubiquitin ligases that trigger the conjugation of regulatory ubiquitin to target proteins. Ubiquitination serves as a platform to recruit the IKK complex and potentially other protein kinases to trigger IKK activation. These findings support a concept whereby protein complex assembly induces regulatory ubiquitination, which in turn recruits and activates protein kinases. Notably, the great interest in a detailed description of the mechanisms that regulate NF-kappaB activity stems from many observations that link dysregulated NF-kappaB signaling with the onset or progression of various diseases, including cancer, chronic inflammation, cardiovascular disorders and neurodegenerative diseases. Thus, the formation of large signaling clusters and regulatory ubiquitin chains represents promising targets for pharmacological intervention to modulate NF-kappaB signal transduction in disease.

  4. Viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways.

    Science.gov (United States)

    Rajsbaum, Ricardo; García-Sastre, Adolfo

    2013-08-01

    Early innate and cell-intrinsic responses are essential to protect host cells against pathogens. In turn, viruses have developed sophisticated mechanisms to establish productive infections by counteracting host innate immune responses. Increasing evidence indicates that these antiviral factors may have a dual role by directly inhibiting viral replication as well as by sensing and transmitting signals to induce antiviral cytokines. Recent studies have pointed at new, unappreciated mechanisms of viral evasion of host innate protective responses including manipulating the host ubiquitin (Ub) system. Virus-mediated inhibition of antiviral factors by Ub-dependent degradation is emerging as a crucial mechanism for evading the antiviral response. In addition, recent studies have uncovered new mechanisms by which virus-encoded proteins inhibit Ub and Ub-like (Ubl) modification of host proteins involved in innate immune signaling pathways. Here we discuss recent findings and novel strategies that viruses have developed to counteract these early innate antiviral defenses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Biomolecular Simulation of Base Excision Repair and Protein Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Straatsma, TP; McCammon, J A; Miller, John H; Smith, Paul E; Vorpagel, Erich R; Wong, Chung F; Zacharias, Martin W

    2006-03-03

    The goal of the Biomolecular Simulation of Base Excision Repair and Protein Signaling project is to enhance our understanding of the mechanism of human polymerase-β, one of the key enzymes in base excision repair (BER) and the cell-signaling enzymes cyclic-AMP-dependent protein kinase. This work used molecular modeling and simulation studies to specifically focus on the • dynamics of DNA and damaged DNA • dynamics and energetics of base flipping in DNA • mechanism and fidelity of nucleotide insertion by BER enzyme human polymerase-β • mechanism and inhibitor design for cyclic-AMP-dependent protein kinase. Molecular dynamics simulations and electronic structure calculations have been performed using the computer resources at the Molecular Science Computing Facility at the Environmental Molecular Sciences Laboratory.

  6. Emerging Roles of Regulators of G Protein Signaling (RGS) Proteins in the Immune System.

    Science.gov (United States)

    Druey, Kirk M

    2017-01-01

    The regulators of G protein signaling (RGS) proteins are a large, evolutionarily conserved group of intracellular proteins expressed in every cell type and tissue throughout the body including the immune system. Through their signature GTPase-activating protein (GAP) activity on heterotrimeric G proteins and interactions with signaling complexes and membrane constituents (e.g., lipids), RGS proteins determine the intensity and duration of G protein-coupled receptor-induced responses. They may also have a function in generating intracellular signaling gradients necessary for the directional migration of leukocytes to inflamed tissues containing local accumulations of chemoattractants. Although physiological functions of most RGS proteins in leukocytes and lymphoid organs are largely unknown, it appears thus far that deficiency of individual RGS proteins in mice does not affect homeostatic immune responses in the absence of immunogenic challenge and/or microbial infection. Although aberrant expression of some RGS proteins has been linked to dysregulated immunity and/or neoplasia in humans, there are no human diseases attributed to specific RGS dysfunction. Here, we highlight mostly published work describing expression and functions of the core group of RGS proteins that were among the first discovered, in both innate and adaptive immune processes, with particular emphasis on cell trafficking. © 2017 Elsevier Inc. All rights reserved.

  7. Single-cell analysis of G-protein signal transduction.

    Science.gov (United States)

    Clister, Terri; Mehta, Sohum; Zhang, Jin

    2015-03-13

    The growing use of fluorescent biosensors to directly probe the spatiotemporal dynamics of biochemical processes in living cells has revolutionized the study of intracellular signaling. In this review, we summarize recent developments in the use of biosensors to illuminate the molecular details of G-protein-coupled receptor (GPCR) signaling pathways, which have long served as the model for our understanding of signal transduction, while also offering our perspectives on the future of this exciting field. Specifically, we highlight several ways in which biosensor-based single-cell analyses are being used to unravel many of the enduring mysteries that surround these diverse signaling pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

    Science.gov (United States)

    Abu-Taha, Issam H; Heijman, Jordi; Feng, Yuxi; Vettel, Christiane; Dobrev, Dobromir; Wieland, Thomas

    2018-02-01

    Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

  9. Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling.

    Directory of Open Access Journals (Sweden)

    Michiel van Gent

    2014-02-01

    Full Text Available Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR. TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpesviruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs. The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

  10. LSm14A Plays a Critical Role in Antiviral Immune Responses by Regulating MITA Level in a Cell-Specific Manner.

    Science.gov (United States)

    Liu, Tian-Tian; Yang, Qing; Li, Mi; Zhong, Bo; Ran, Yong; Liu, Li-Li; Yang, Yan; Wang, Yan-Yi; Shu, Hong-Bing

    2016-06-15

    Viral infection triggers induction of antiviral cytokines and effectors, which are critical mediators of innate antiviral immune response. It has been shown that the processing body-associated protein LSm14A is involved in the induction of antiviral cytokines in cell lines but in vivo evidence is lacking. By generating LSm14A-deficient mice, in this study, we show that LSm14A plays a critical and specific role in the induction of antiviral cytokines in dendritic cells (DCs) but not in macrophages and fibroblasts. Induction of antiviral cytokines triggered by the DNA viruses HSV-1 and murid herpesvirus 68 and the RNA virus vesicular stomatitis virus but not Sendai virus was impaired in Lsm14a(-/-) DCs, which is correlated to the functions of the adaptor protein MITA/STING in the antiviral signaling pathways. LSm14A deficiency specifically downregulated MITA/STING level in DCs by impairing its nuclear mRNA precursor processing and subsequently impaired antiviral innate and adaptive immune responses. Our findings reveal a nuclear mRNA precursor processing and cell-specific regulatory mechanism of antiviral immune responses. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Middle east respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response.

    Science.gov (United States)

    Siu, Kam-Leung; Yeung, Man Lung; Kok, Kin-Hang; Yuen, Kit-San; Kew, Chun; Lui, Pak-Yin; Chan, Chi-Ping; Tse, Herman; Woo, Patrick C Y; Yuen, Kwok-Yung; Jin, Dong-Yan

    2014-05-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes severe disease in human. MERS-CoV is closely related to bat coronaviruses HKU4 and HKU5. Evasion of the innate antiviral response might contribute significantly to MERS-CoV pathogenesis, but the mechanism is poorly understood. In this study, we characterized MERS-CoV 4a protein as a novel immunosuppressive factor that antagonizes type I interferon production. MERS-CoV 4a protein contains a double-stranded RNA-binding domain capable of interacting with poly(I · C). Expression of MERS-CoV 4a protein suppressed the interferon production induced by poly(I · C) or Sendai virus. RNA binding of MERS-CoV 4a protein was required for IFN antagonism, a property shared by 4a protein of bat coronavirus HKU5 but not by the counterpart in bat coronavirus HKU4. MERS-CoV 4a protein interacted with PACT in an RNA-dependent manner but not with RIG-I or MDA5. It inhibited PACT-induced activation of RIG-I and MDA5 but did not affect the activity of downstream effectors such as RIG-I, MDA5, MAVS, TBK1, and IRF3. Taken together, our findings suggest a new mechanism through which MERS-CoV employs a viral double-stranded RNA-binding protein to circumvent the innate antiviral response by perturbing the function of cellular double-stranded RNA-binding protein PACT. PACT targeting might be a common strategy used by different viruses, including Ebola virus and herpes simplex virus 1, to counteract innate immunity. Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging and highly lethal human pathogen. Why MERS-CoV causes severe disease in human is unclear, and one possibility is that MERS-CoV is particularly efficient in counteracting host immunity, including the sensing of virus invasion. It will therefore be critical to clarify how MERS-CoV cripples the host proteins that sense viruses and to compare MERS-CoV with its ancestral viruses in bats in the counteraction of virus sensing

  12. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.

    2013-09-03

    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  13. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling

    DEFF Research Database (Denmark)

    Blagoev, B.; Kratchmarova, I.; Ong, S.E.

    2003-01-01

    employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR...... and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did...

  14. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

  15. Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein.

    Science.gov (United States)

    Leyme, Anthony; Marivin, Arthur; Maziarz, Marcin; DiGiacomo, Vincent; Papakonstantinou, Maria P; Patel, Prachi P; Blanco-Canosa, Juan B; Walawalkar, Isha A; Rodriguez-Davila, Gonzalo; Dominguez, Isabel; Garcia-Marcos, Mikel

    2017-11-28

    Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gβγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

  16. Duplicated RGS (Regulator of G-protein signaling) proteins exhibit conserved biochemical but differential transcriptional regulation of heterotrimeric G-protein signaling in Brassica species.

    Science.gov (United States)

    Kumar, Roshan; Bisht, Naveen C

    2018-02-01

    G-alpha (Gα) and 'Regulator of G-protein Signaling (RGS)' proteins are the two key components primarily involved in regulation of heterotrimeric G-proteins signaling across phyla. Unlike Arabidopsis thaliana, our knowledge about G-protein regulation in polyploid Brassica species is sparse. In this study, we identified one Gα and two RGS genes each from three species of Brassica 'U' triangle and assessed the effects of whole genome triplication on the divergence of gene sequence and structure, protein-protein interaction, biochemical activities, and gene expression. Sequence and phylogenetic analysis revealed that the deduced Gα and RGS proteins are evolutionarily conserved across Brassica species. The duplicated RGS proteins of each Brassica species interacted with their cognate Gα but displayed varying levels of interaction strength. The Gα and the duplicated RGS proteins of Brassica species exhibited highly conserved G-protein activities when tested under in-vitro conditions. Expression analysis of the B. rapa RGS genes revealed a high degree of transcriptional differentiation across the tested tissue types and in response to various elicitors, particularly under D-glucose, salt and phytohormone treatments. Taken together, our results suggest that the RGS-mediated regulation of G-protein signaling in Brassica species is predominantly governed by stage and condition-specific expression differentiation of the duplicated RGS genes.

  17. Role of protein dynamics in transmembrane receptor signalling

    DEFF Research Database (Denmark)

    Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

    2018-01-01

    Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... and computation have been used to study receptor dynamics. Recent studies on three distinct classes of receptors (G-protein coupled receptors, ligand-gated ion-channels and single-pass receptors) are highlighted to show that conformational changes across a range of time-scales and length-scales are central...

  18. Antiviral lectins: Selective inhibitors of viral entry.

    Science.gov (United States)

    Mitchell, Carter A; Ramessar, Koreen; O'Keefe, Barry R

    2017-06-01

    Many natural lectins have been reported to have antiviral activity. As some of these have been put forward as potential development candidates for preventing or treating viral infections, we have set out in this review to survey the literature on antiviral lectins. The review groups lectins by structural class and class of source organism we also detail their carbohydrate specificity and their reported antiviral activities. The review concludes with a brief discussion of several of the pertinent hurdles that heterologous proteins must clear to be useful clinical candidates and cites examples where such studies have been reported for antiviral lectins. Though the clearest path currently being followed is the use of antiviral lectins as anti-HIV microbicides via topical mucosal administration, some investigators have also found systemic efficacy against acute infections following subcutaneous administration. Published by Elsevier B.V.

  19. Viral RNase3 Co-Localizes and Interacts with the Antiviral Defense Protein SGS3 in Plant Cells

    Science.gov (United States)

    Weinheimer, Isabel; Haikonen, Tuuli; Ala-Poikela, Marjo; Moser, Mirko; Streng, Janne; Rajamäki, Minna-Liisa; Valkonen, Jari P. T.

    2016-01-01

    Sweet potato chlorotic stunt virus (SPCSV; family Closteroviridae) encodes a Class 1 RNase III endoribonuclease (RNase3) that suppresses post-transcriptional RNA interference (RNAi) and eliminates antiviral defense in sweetpotato plants (Ipomoea batatas). For RNAi suppression, RNase3 cleaves double-stranded small interfering RNAs (ds-siRNA) and long dsRNA to fragments that are too short to be utilized in RNAi. However, RNase3 can suppress only RNAi induced by sense RNA. Sense-mediated RNAi involves host suppressor of gene silencing 3 (SGS3) and RNA–dependent RNA polymerase 6 (RDR6). In this study, subcellular localization and host interactions of RNase3 were studied in plant cells. RNase3 was found to interact with SGS3 of sweetpotato and Arabidopsis thaliana when expressed in leaves, and it localized to SGS3/RDR6 bodies in the cytoplasm of leaf cells and protoplasts. RNase3 was also detected in the nucleus. Co-expression of RNase3 and SGS3 in leaf tissue enhanced the suppression of RNAi, as compared with expression of RNase3 alone. These results suggest additional mechanisms needed for efficient RNase3-mediated suppression of RNAi and provide new information about the subcellular context and phase of the RNAi pathway in which RNase3 realizes RNAi suppression. PMID:27391019

  20. La respuesta inmune antiviral

    Directory of Open Access Journals (Sweden)

    Rainel Sánchez de la Rosa

    1998-02-01

    Full Text Available Se expone que los virus son parásitos intracelulares obligados, puesto que no tienen metabolismo propio; esto obliga al sistema inmune a poner en marcha sus mecanismos más especializados para reconocer y eliminar, tanto a los virus libres, como a las células infectadas. Se señala que las células presentadoras de antígenos, los linfocitos B y los T unidos al complejo mayor de histocompatibilidad, forman parte de la organización de la respuesta inmune antiviral; la inducción de esta respuesta con proteínas, péptidos y ADN desnudo, son alternativas actuales tanto en la prevención como en el tratamiento de las infecciones viralesIt is explained that viruses are compulsory intracellular parasites, since they don't have their own metabolism, which makes the immune system to start its mest specialized mechanisms to recognize and eliminate the free viruses and the infected cells. It is stated that the cells presenting antigens, and the B and T lymphocytes together with the major histocompatibility complex, are part of the organization of the immune antiviral response. The induction of this response with proteins, peptides and naked DNA are the present alternatives for the prevention and treatment of viral infections

  1. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  2. Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling.

    Science.gov (United States)

    Chakravorty, David; Gookin, Timothy E; Milner, Matthew J; Yu, Yunqing; Assmann, Sarah M

    2015-09-01

    Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling. © 2015 American Society of Plant Biologists. All Rights Reserved.

  3. The Tudor domain protein Spindlin1 is involved in intrinsic antiviral defense against incoming hepatitis B Virus and herpes simplex virus type 1.

    Directory of Open Access Journals (Sweden)

    Aurélie Ducroux

    2014-09-01

    Full Text Available Hepatitis B virus infection (HBV is a major risk factor for the development of hepatocellular carcinoma. HBV replicates from a covalently closed circular DNA (cccDNA that remains as an episome within the nucleus of infected cells and serves as a template for the transcription of HBV RNAs. The regulatory protein HBx has been shown to be essential for cccDNA transcription in the context of infection. Here we identified Spindlin1, a cellular Tudor-domain protein, as an HBx interacting partner. We further demonstrated that Spindlin1 is recruited to the cccDNA and inhibits its transcription in the context of infection. Spindlin1 knockdown induced an increase in HBV transcription and in histone H4K4 trimethylation at the cccDNA, suggesting that Spindlin1 impacts on epigenetic regulation. Spindlin1-induced transcriptional inhibition was greater for the HBV virus deficient for the expression of HBx than for the HBV WT virus, suggesting that HBx counteracts Spindlin1 repression. Importantly, we showed that the repressive role of Spindlin1 is not limited to HBV transcription but also extends to other DNA virus that replicate within the nucleus such as Herpes Simplex Virus type 1 (HSV-1. Taken together our results identify Spindlin1 as a critical component of the intrinsic antiviral defense and shed new light on the function of HBx in HBV infection.

  4. The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense.

    Science.gov (United States)

    Rossi, Marika; Vallino, Marta; Abbà, Simona; Ciuffo, Marina; Balestrini, Raffaella; Genre, Andrea; Turina, Massimo

    2015-01-01

    The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV6710, with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CPwt, in situ immunolocalization experiments indicated that CP6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.

  5. Promotion of bone morphogenetic protein signaling by tetraspanins and glycosphingolipids.

    Directory of Open Access Journals (Sweden)

    Zhiyu Liu

    2015-05-01

    Full Text Available Bone morphogenetic proteins (BMPs belong to the transforming growth factor β (TGFβ superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like "Sma/Mab" signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.

  6. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction

    Science.gov (United States)

    Hu, Jianfei; Neiswinger, Johnathan; Zhang, Jin; Zhu, Heng; Qian, Jiang

    2015-01-01

    Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process. PMID:26393507

  7. Identification of G protein-coupled receptor signaling pathway proteins in marine diatoms using comparative genomics.

    Science.gov (United States)

    Port, Jesse A; Parker, Micaela S; Kodner, Robin B; Wallace, James C; Armbrust, E Virginia; Faustman, Elaine M

    2013-07-24

    The G protein-coupled receptor (GPCR) signaling pathway plays an essential role in signal transmission and response to external stimuli in mammalian cells. Protein components of this pathway have been characterized in plants and simpler eukaryotes such as yeast, but their presence and role in unicellular photosynthetic eukaryotes have not been determined. We use a comparative genomics approach using whole genome sequences and gene expression libraries of four diatoms (Pseudo-nitzschia multiseries, Thalassiosira pseudonana, Phaeodactylum tricornutum and Fragilariopsis cylindrus) to search for evidence of GPCR signaling pathway proteins that share sequence conservation to known GPCR pathway proteins. The majority of the core components of GPCR signaling were well conserved in all four diatoms, with protein sequence similarity to GPCRs, human G protein α- and β-subunits and downstream effectors. There was evidence for the Gγ-subunit and thus a full heterotrimeric G protein only in T. pseudonana. Phylogenetic analysis of putative diatom GPCRs indicated similarity but deep divergence to the class C GPCRs, with branches basal to the GABAB receptor subfamily. The extracellular and intracellular regions of these putative diatom GPCR sequences exhibited large variation in sequence length, and seven of these sequences contained the necessary ligand binding domain for class C GPCR activation. Transcriptional data indicated that a number of the putative GPCR sequences are expressed in diatoms under various stress conditions in culture, and that many of the GPCR-activated signaling proteins, including the G protein, are also expressed. The presence of sequences in all four diatoms that code for the proteins required for a functional mammalian GPCR pathway highlights the highly conserved nature of this pathway and suggests a complex signaling machinery related to environmental perception and response in these unicellular organisms. The lack of evidence for some GPCR pathway

  8. Signal peptide of eosinophil cationic protein is toxic to cells lacking signal peptide peptidase

    International Nuclear Information System (INIS)

    Wu, C.-M.; Chang, Margaret Dah-Tsyr

    2004-01-01

    Eosinophil cationic protein (ECP) is a toxin secreted by activated human eosinophils. The properties of mature ECP have been well studied but those of the signal peptide of ECP (ECPsp) are not clear. In this study, several chimeric proteins containing N-terminal fusion of ECPsp were generated, and introduced into Escherichia coli, Pichia pastoris, and human epidermoid carcinoma cell line A431 to study the function of ECPsp. We found that expression of ECPsp chimeric proteins inhibited the growth of E. coli and P. pastoris but not A431 cells. Primary sequence analysis and in vitro transcription/translation of ECPsp have revealed that it is a potential substrate for human signal peptide peptidase (hSPP), an intramembrane protease located in endoplasmic reticulum. In addition, knockdown of the hSPP mRNA expression in ECPsp-eGFP/A431 cells caused the growth inhibitory effect, whereas complementally expression of hSPP in P. pastoris system rescued the cell growth. Taken together, we have demonstrated that ECPsp is a toxic signal peptide, and expression of hSPP protects the cells from growth inhibition

  9. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients.

    Directory of Open Access Journals (Sweden)

    Jakob G Jespersen

    Full Text Available BACKGROUND: Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR, glycogen synthase kinase 3β (GSK3β and forkhead box O (FoxO pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU patients compared with healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, and muscle ring finger protein 1 (MuRF1; and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1, FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6, tumor necrosis factor α (TNF-α and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2=0.36, p<0.05 between insulin infusion dose and phosphorylated Akt was demonstrated. CONCLUSIONS/SIGNIFICANCE: We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.

  10. Prolactin receptor and signal transduction to milk protein genes

    Energy Technology Data Exchange (ETDEWEB)

    Djiane, J.; Daniel, N.; Bignon, C. [Unite d`Endocrinologie Moleculaire, Jouy en Josas (France)] [and others

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

  11. Dose and Size-Dependent Antiviral Effects of Silver Nanoparticles on Feline Calicivirus, a Human Norovirus Surrogate.

    Science.gov (United States)

    Bekele, Aschalew Z; Gokulan, Kuppan; Williams, Katherine M; Khare, Sangeeta

    2016-05-01

    Silver nanoparticles (AgNPs) as antibacterial agents are incorporated in many consumer products, while the use as antiviral agents is an ongoing area of research. We evaluated the antiviral properties of AgNPs of variable sizes (10, 75, and 110 nm) and doses (25, 50, and 100 μg/mL) at different contact time points against feline calicivirus (FCV), a surrogate for norovirus. Antiviral effects of the AgNPs were determined by comparing the infectivity of FCV, the appearance of cytopathic effects (CPEs), and the integrity of the viral capsid protein in viral suspension treated with AgNPs with the untreated controls. The 10 nm AgNPs at 50 and 100 μg/mL concentrations inactivated the FCV beyond the limit of detection, resulting in a decrease of up to 6.5 log10 viral titer, prevented development of CPEs, and reduction in the western blot band signal of the viral capsid protein. No significant antiviral effect was observed for the 75 and 110 nm AgNPs. Conclusions and Applications: These results demonstrate that the antiviral effects of AgNPs are both size and dose dependent, thus potential applications of AgNPs as antiviral agents to prevent contamination of foodborne viruses need to consider size and dose effects.

  12. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    Energy Technology Data Exchange (ETDEWEB)

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  13. Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling.

    Directory of Open Access Journals (Sweden)

    Christopher M Tracy

    Full Text Available G protein β subunits (Gβ play essential roles in phototransduction as part of G protein βγ (Gβγ and regulator of G protein signaling 9 (RGS9-Gβ5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2 and RGS9-Gβ5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling.

  14. Uridine affects liver protein glycosylation, insulin signaling, and heme biosynthesis.

    Directory of Open Access Journals (Sweden)

    Yasuyo Urasaki

    Full Text Available Purines and pyrimidines are complementary bases of the genetic code. The roles of purines and their derivatives in cellular signal transduction and energy metabolism are well-known. In contrast, the roles of pyrimidines and their derivatives in cellular function remain poorly understood. In this study, the roles of uridine, a pyrimidine nucleoside, in liver metabolism are examined in mice. We report that short-term uridine administration in C57BL/6J mice increases liver protein glycosylation profiles, reduces phosphorylation level of insulin signaling proteins, and activates the HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. Short-term uridine administration is also associated with reduced liver hemin level and reduced ability for insulin-stimulated blood glucose removal during an insulin tolerance test. Some of the short-term effects of exogenous uridine in C57BL/6J mice are conserved in transgenic UPase1-/- mice with long-term elevation of endogenous uridine level. UPase1-/- mice exhibit activation of the liver HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. UPase1-/- mice also exhibit impaired ability for insulin-stimulated blood glucose removal. However, other short-term effects of exogenous uridine in C57BL/6J mice are not conserved in UPase1-/- mice. UPase1-/- mice exhibit normal phosphorylation level of liver insulin signaling proteins and increased liver hemin concentration compared to untreated control C57BL/6J mice. Contrasting short-term and long-term consequences of uridine on liver metabolism suggest that uridine exerts transient effects and elicits adaptive responses. Taken together, our data support potential roles of pyrimidines and their derivatives in the regulation of liver metabolism.

  15. Heat Shock Proteins as Danger Signals for Cancer Detection

    International Nuclear Information System (INIS)

    Seigneuric, Renaud; Mjahed, Hajare; Gobbo, Jessica; Joly, Anne-Laure; Berthenet, Kevin; Shirley, Sarah; Garrido, Carmen

    2011-01-01

    First discovered in 1962, heat shock proteins (HSPs) are highly studied with about 35,500 publications on the subject to date. HSPs are highly conserved, function as molecular chaperones for a large panel of “client” proteins and have strong cytoprotective properties. Induced by many different stress signals, they promote cell survival in adverse conditions. Therefore, their roles have been investigated in several conditions and pathologies where HSPs accumulate, such as in cancer. Among the diverse mammalian HSPs, some members share several features that may qualify them as cancer biomarkers. This review focuses mainly on three inducible HSPs: HSP27, HPS70, and HSP90. Our survey of recent literature highlights some recurring weaknesses in studies of the HSPs, but also identifies findings that indicate that some HSPs have potential as cancer biomarkers for successful clinical applications.

  16. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    Directory of Open Access Journals (Sweden)

    Koul Sweaty

    2004-06-01

    Full Text Available Abstract The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.

  17. Two mechanistically distinct immune evasion proteins of cowpox virus combine to avoid antiviral CD8 T cells

    OpenAIRE

    Byun, Minji; Verweij, Marieke C.; Pickup, David J.; Wiertz, Emmanuel J. H. J.; Hansen, Ted H.; Yokoyama, Wayne M.

    2009-01-01

    Downregulation of MHC class I on the cell surface is an immune evasion mechanism shared by many DNA viruses including cowpox virus. Previously, a cowpox virus protein, CPXV203, was shown to downregulate MHC class I. Here, we report that CPXV12 is the only other MHC class I regulating protein of cowpox virus and it uses a mechanism distinct from that of CPXV203. Whereas CPXV203 retains fully assembled MHC class I by exploiting the KDEL-mediated endoplasmic reticulum retention pathway, CPXV12 b...

  18. Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

    Directory of Open Access Journals (Sweden)

    Juliana L. Dreyfuss

    2009-09-01

    Full Text Available Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam

  19. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  20. Interaction of antivirals with a heptameric bundle model of the p7 protein of hepatitis C virus.

    Science.gov (United States)

    Dahl, Sophie L; Kalita, Monoj Mon; Fischer, Wolfgang B

    2018-04-01

    A series of ligands are known experimentally to affect the infectivity cycle of the hepatitis C virus. The target protein for the ligands is proposed to be p7, a 63 amino acid polytopic channel-forming protein, with possibly two transmembrane domains. Protein p7 is found to assemble into functional oligomers of various sizes, depending on the genotype (GT). Nine ligands are docked to various sites of a computationally derived heptameric bundle of p7 of GT1a. The energy of interaction, here binding energy, is calculated using three different docking programs (Autodock, MOE, LeadIT). Three protein regions are defined to which the ligands are placed, the loop region and the site with the termini as well as the mid-region which is supposed to track poses inside the putative pore. A common feature is that the loop sites and poses either within the pore or at the intermonomer space of the bundle are preferred for all ligands with proposed binding energies smaller than -10 kJ/mol. BIT225, benzamine, amantadine, and NN-DNJ show good overall scoring. © 2017 John Wiley & Sons A/S.

  1. The antiviral protein human lactoferrin is distributed in the body to cytomegalovirus (CMV) infection-prone cells and tissues

    NARCIS (Netherlands)

    Beljaars, Leonie; Bakker, Hester I; van der Strate, Barry W A; Smit, Catharina; Duijvestijn, Adrian M; Meijer, Dirk K F; Molema, Grietje

    Purpose. Lactoferrin has anti-Cytomegalovirus (CMV) and -HIV properties in vitro. However, the pharmacokinetic behavior of the 80-kD protein has not been well defined. We, therefore, assessed the plasma decay and body distribution of lactoferrin after intravenous administration to freely moving

  2. R4 Regulator of G Protein Signaling (RGS) Proteins in Inflammation and Immunity.

    Science.gov (United States)

    Xie, Zhihui; Chan, Eunice C; Druey, Kirk M

    2016-03-01

    G protein-coupled receptors (GPCRs) have important functions in both innate and adaptive immunity, with the capacity to bridge interactions between the two arms of the host responses to pathogens through direct recognition of secreted microbial products or the by-products of host cells damaged by pathogen exposure. In the mid-1990s, a large group of intracellular proteins was discovered, the regulator of G protein signaling (RGS) family, whose main, but not exclusive, function appears to be to constrain the intensity and duration of GPCR signaling. The R4/B subfamily--the focus of this review--includes RGS1-5, 8, 13, 16, 18, and 21, which are the smallest RGS proteins in size, with the exception of RGS3. Prominent roles in the trafficking of B and T lymphocytes and macrophages have been described for RGS1, RGS13, and RGS16, while RGS18 appears to control platelet and osteoclast functions. Additional G protein independent functions of RGS13 have been uncovered in gene expression in B lymphocytes and mast cell-mediated allergic reactions. In this review, we discuss potential physiological roles of this RGS protein subfamily, primarily in leukocytes having central roles in immune and inflammatory responses. We also discuss approaches to target RGS proteins therapeutically, which represents a virtually untapped strategy to combat exaggerated immune responses leading to inflammation.

  3. The Human Cytomegalovirus Major Immediate-Early Proteins as Antagonists of Intrinsic and Innate Antiviral Host Responses

    Directory of Open Access Journals (Sweden)

    Michael Nevels

    2009-11-01

    Full Text Available The major immediate-early (IE gene of human cytomegalovirus (CMV is believed to have a decisive role in acute infection and its activity is an important indicator of viral reactivation from latency. Although a variety of gene products are expressed from this region, the 72-kDa IE1 and the 86-kDa IE2 nuclear phosphoproteins are the most abundant and important. Both proteins have long been recognized as promiscuous transcriptional regulators. More recently, a critical role of the IE1 and IE2 proteins in counteracting nonadaptive host cell defense mechanisms has been revealed. In this review we will briefly summarize the available literature on IE1- and IE2-dependent mechanisms contributing to CMV evasion from intrinsic and innate immune responses.

  4. The V protein of Tioman virus is incapable of blocking type I interferon signaling in human cells.

    Directory of Open Access Journals (Sweden)

    Grégory Caignard

    Full Text Available The capacity of a virus to cross species barriers is determined by the development of bona fide interactions with cellular components of new hosts, and in particular its ability to block IFN-α/β antiviral signaling. Tioman virus (TioV, a close relative of mumps virus (MuV, has been isolated in giant fruit bats in Southeast Asia. Nipah and Hendra viruses, which are present in the same bat colonies, are highly pathogenic in human. Despite serological evidences of close contacts between TioV and human populations, whether TioV is associated to some human pathology remains undetermined. Here we show that in contrast to the V protein of MuV, the V protein of TioV (TioV-V hardly interacts with human STAT2, does not degrade STAT1, and cannot block IFN-α/β signaling in human cells. In contrast, TioV-V properly binds to human STAT3 and MDA5, and thus interferes with IL-6 signaling and IFN-β promoter induction in human cells. Because STAT2 binding was previously identified as a host restriction factor for some Paramyxoviridae, we established STAT2 sequence from giant fruit bats, and binding to TioV-V was tested. Surprisingly, TioV-V interaction with STAT2 from giant fruit bats is also extremely weak and barely detectable. Altogether, our observations question the capacity of TioV to appropriately control IFN-α/β signaling in both human and giant fruit bats that are considered as its natural host.

  5. Innate and intrinsic antiviral immunity in skin.

    Science.gov (United States)

    Kawamura, Tatsuyoshi; Ogawa, Youichi; Aoki, Rui; Shimada, Shinji

    2014-09-01

    As the body's most exposed interface with the environment, the skin is constantly challenged by potentially pathogenic microbes, including viruses. To sense the invading viruses, various types of cells resident in the skin express many different pattern-recognition receptors (PRRs) such as C-type lectin receptors (CLRs), Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and cytosolic DNA sensors, that can detect the pathogen-associated molecular patterns (PAMPs) of the viruses. The detection of viral PAMPs initiates two major innate immune signaling cascades: the first involves the activation of the downstream transcription factors, such as interferon regulatory factors (IRFs), nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1), which cooperate to induce the transcription of type I interferons and pro-inflammatory cytokines. The second signaling pathway involves the caspase-1-mediated processing of IL-1β and IL-18 through the formation of an inflammasome complex. Cutaneous innate immunity including the production of the innate cytokines constitutes the first line of host defence that limits the virus dissemination from the skin, and also plays an important role in the activation of adaptive immune response, which represents the second line of defence. More recently, the third immunity "intrinsic immunity" has emerged, that provides an immediate and direct antiviral defense mediated by host intrinsic restriction factors. This review focuses on the recent advances regarding the antiviral immune systems, highlighting the innate and intrinsic immunity against the viral infections in the skin, and describes how viral components are recognized by cutaneous immune systems. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  7. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.......Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors...

  8. DEVELOPMENT OF ANTIVIRAL AGENTS

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. DEVELOPMENT OF ANTIVIRAL AGENTS. Chandipura virus can be regarded as a model system to design and develop antiviral agents. These agents could be small molecules or RNA/PNA aptamers or Antisense RNA to speicific gene sequence in the viral genome.

  9. Protein Phosphatase, Mg2+/Mn2+-dependent 1A controls the innate antiviral and antibacterial response of macrophages during HIV-1 and Mycobacterium tuberculosis infection

    Science.gov (United States)

    Sun, Jim; Schaaf, Kaitlyn; Duverger, Alexandra; Wolschendorf, Frank; Speer, Alexander; Wagner, Frederic; Niederweis, Michael; Kutsch, Olaf

    2016-01-01

    Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. While some research has described how each pathogen accelerates the course of infection of the other pathogen by compromising the immune system, very little is known about the molecular biology of HIV-1/Mtb co-infection at the host cell level. This is somewhat surprising, as both pathogens are known to replicate and persist in macrophages. We here identify Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) as a molecular link between Mtb infection and increased HIV-1 susceptibility of macrophages. We demonstrate that both Mtb and HIV-1 infection induce the expression of PPM1A in primary human monocyte/macrophages and THP-1 cells. Genetic manipulation studies revealed that increased PPMA1 expression rendered THP-1 cells highly susceptible to HIV-1 infection, while depletion of PPM1A rendered them relatively resistant to HIV-1 infection. At the same time, increased PPM1A expression abrogated the ability of THP-1 cells to respond to relevant bacterial stimuli with a proper cytokine/chemokine secretion response, blocked their chemotactic response and impaired their ability to phagocytose bacteria. These data suggest that PPM1A, which had previously been shown to play a role in the antiviral response to Herpes Simplex virus infection, also governs the antibacterial response of macrophages to bacteria, or at least to Mtb infection. PPM1A thus seems to play a central role in the innate immune response of macrophages, implying that host directed therapies targeting PPM1A could be highly beneficial, in particular for HIV/Mtb co-infected patients. PMID:27004401

  10. Replication-competent recombinant porcine reproductive and respiratory syndrome (PRRS) viruses expressing indicator proteins and antiviral cytokines.

    Science.gov (United States)

    Sang, Yongming; Shi, Jishu; Sang, Wenjing; Rowland, Raymond R R; Blecha, Frank

    2012-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN) genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129). Coding regions of exogenous genes, which included Renilla luciferase (Rluc), green and red fluorescent proteins (GFP and DsRed, respectively) and several interferons (IFNs), were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 ° IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101) in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV) vaccines, which are capable of reversing subverted innate immune responses and may induce more

  11. Ubiquitin-fusion as a strategy to modulate protein half-life: A3G antiviral activity revisited

    International Nuclear Information System (INIS)

    Cadima-Couto, Iris; Freitas-Vieira, Acilino; Nowarski, Roni; Britan-Rosich, Elena; Kotler, Moshe; Goncalves, Joao

    2009-01-01

    The human APOBEC3G (A3G) is a potent inhibitor of HIV-1 replication and its activity is suppressed by HIV-1 virion infectivity factor (Vif). Vif neutralizes A3G mainly by inducing its degradation in the proteasome and blocking its incorporation into HIV-1 virions. Assessing the time needed for A3G incorporation into virions is, therefore, important to determine how quickly Vif must act to induce its degradation. We show that modelling the intracellular half-life of A3G can induce its Vif-independent targeting to the ubiquitin-proteasome system. By using various amino acids (X) in a cleavable ubiquitin-X-A3G fusion, we demonstrate that the half-life (t1/2) of X-A3G can be manipulated. We show that A3G molecules with a half-life of 13 min are incorporated into virions, whereas those with a half-life shorter than 5 min were not. The amount of X-A3G incorporated into virions increases from 13 min (Phe-A3G) to 85 min (Asn-A3G) and remains constant after this time period. Interestingly, despite the presence of similar levels of Arg-A3G (t1/2 = 28 min) and Asp-A3G (t1/2 = 65 min) into HIV-1 Δvif virions, inhibition of viral infectivity was only evident in the presence of A3G proteins with a longer half-life (t1/2 ≥ 65 min).

  12. Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling.

    Directory of Open Access Journals (Sweden)

    Tina L Gumienny

    2010-05-01

    Full Text Available Bone morphogenetic protein (BMP pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-10 as a positive extracellular regulator of BMP-like receptor signaling. SMA-10 acts genetically in a BMP-like (Sma/Mab pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-10 and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane proteins. SMA-10 is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-10(lf animals with the Drosophila ortholog lambik, showing that SMA-10 physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.

  13. Protein kinase C signaling and cell cycle regulation

    Directory of Open Access Journals (Sweden)

    Adrian R Black

    2013-01-01

    Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

  14. Replication-Competent Recombinant Porcine Reproductive and Respiratory Syndrome (PRRS Viruses Expressing Indicator Proteins and Antiviral Cytokines

    Directory of Open Access Journals (Sweden)

    Frank Blecha

    2012-01-01

    Full Text Available Porcine reproductive and respiratory syndrome virus (PRRSV can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129. Coding regions of exogenous genes, which included Renilla luciferase (Rluc, green and red fluorescent proteins (GFP and DsRed, respectively and several interferons (IFNs, were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 » IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101 in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV vaccines, which are capable of reversing subverted innate immune responses and

  15. Enterovirus 71 protease 2Apro targets MAVS to inhibit anti-viral type I interferon responses.

    Directory of Open Access Journals (Sweden)

    Bei Wang

    2013-03-01

    Full Text Available Enterovirus 71 (EV71 is the major causative pathogen of hand, foot, and mouth disease (HFMD. Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN responses by targeting the mitochondrial anti-viral signaling (MAVS protein--a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I and melanoma differentiation associated gene (MDA-5 viral recognition receptor signaling--upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2A(pro, but not the mutant 2A(pro (2A(pro-110 containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A(pro could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.

  16. Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

    Directory of Open Access Journals (Sweden)

    Walderik W. Zomerman

    2018-03-01

    Full Text Available Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog, group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteome

  17. Direct-acting Antivirals in Kidney Transplant Patients: Successful Hepatitis C Treatment and Short Term Reduction in Urinary Protein/Creatinine Ratios

    Directory of Open Access Journals (Sweden)

    Michael R. Goetsch

    2017-09-01

    Full Text Available Introduction: The role of Hepatitis C Virus (HCV clearance in long-term kidney graft survival is unknown. In this study, we examined short-term trends of urinary protein/creatinine (P/C ratios in a cohort of HCV-infected kidney transplant recipients with stable graft function and treated with direct-acting antivirals (DAAs. Methods: We conducted a retrospective study of 19 kidney transplant patients with chronic HCV infection treated with DAAs at the University of Alabama at Birmingham 1917 Viral Hepatitis Clinic between January 2013 and June 2016. Markers of glomerular damage were assessed using average protein/creatinine (P/C ratios measured pre- and post-treatment. We also described treatment efficacy using sustained virologic response at 12 weeks post-HCV treatment (SVR12. Results: The median age of the 19 patients included was 59 years (Q1=58, Q3=64 at completion of treatment. Of these patients, 68% were African American, 32% were White and 63% were male. The median time between kidney transplant and initiation of DAA therapy was 2.25 years (Q1=0.79, Q3=3.79. Post-treatment P/C ratios (median=0.127, Q1=0.090, Q3=0.220 were significantly lower (p=0.01 than pre-treatment ratios (median=0.168, Q1=0.118, Q3=0.385. P/C ratios decreased in 14 of 19 patients (74% with median change of -0.072 (median percent change= -40%. Post-treatment eGFRs (median=58.9, Q1=48.9, Q3=72.3 were not significantly different (p=0.82 than the pre-treatment values (median=57.0, Q1=48.8, Q3=67.8. Conclusions: In this preliminary study, there was a statistically significant decrease in P/C ratios associated with HCV clearance, suggesting a potential role for DAAs in improving kidney graft survival. Larger cohort studies will be needed to assess the clinical and long-term benefits of DAAs in this special population of HCV infected patients.

  18. Mitogen activated protein kinase signaling in the kidney: Target for intervention?

    NARCIS (Netherlands)

    de Borst, M.H.; Wassef, L.; Kelly, D.J.; van Goor, H.; Navis, Ger Jan

    2006-01-01

    Mitogen activated protein kinases (MAPKs) are intracellular signal transduction molecules, which connect cell-surface receptor signals to intracellular processes. MAPKs regulate a range of cellular activities including cell proliferation, gene expression, apoptosis, cell differentiation and cytokine

  19. Unraveling the cellular context of cyclic nucleotide signaling proteins by chemical proteomics

    NARCIS (Netherlands)

    Corradini, E.

    2015-01-01

    Understanding the molecular mechanisms which regulate signal transduction is fundamental to the development of therapeutic molecules for the treatment of several diseases. In particular, signaling proteins, such as cyclic nucleotide dependent enzymes are the orchestrators of many tissue functions.

  20. Hepatitis C virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (STAT) 1 protein expression.

    Science.gov (United States)

    Stone, Amy E L; Mitchell, Angela; Brownell, Jessica; Miklin, Daniel J; Golden-Mason, Lucy; Polyak, Stephen J; Gale, Michael J; Rosen, Hugo R

    2014-01-01

    Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.

  1. The evolution of regulators of G protein signalling proteins as drug targets - 20 years in the making: IUPHAR Review 21.

    Science.gov (United States)

    Sjögren, B

    2017-03-01

    Regulators of G protein signalling (RGS) proteins are celebrating the 20th anniversary of their discovery. The unveiling of this new family of negative regulators of G protein signalling in the mid-1990s solved a persistent conundrum in the G protein signalling field, in which the rate of deactivation of signalling cascades in vivo could not be replicated in exogenous systems. Since then, there has been tremendous advancement in the knowledge of RGS protein structure, function, regulation and their role as novel drug targets. RGS proteins play an important modulatory role through their GTPase-activating protein (GAP) activity at active, GTP-bound Gα subunits of heterotrimeric G proteins. They also possess many non-canonical functions not related to G protein signalling. Here, an update on the status of RGS proteins as drug targets is provided, highlighting advances that have led to the inclusion of RGS proteins in the IUPHAR/BPS Guide to PHARMACOLOGY database of drug targets. © 2017 The British Pharmacological Society.

  2. G protein coupled receptors signaling pathways implicate in inflammatory and immune response of rheumatoid arthritis.

    Science.gov (United States)

    Shu, Jinling; Zhang, Feng; Zhang, Lingling; Wei, Wei

    2017-05-01

    G protein-coupled receptors (GPCRs) are transmembrane receptor proteins, which allow the transfer of signals across the membrane. Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis and accompanied with inflammatory and abnormal immune response. GPCRs signaling pathways play a significant role in inflammatory and immune response processes including RA. In this review, we have focused on the advances in GPCRs signaling pathway implicating the inflammatory and immune response of RA. The signaling pathways of GPCRs-adenylyl cyclase (AC)-cyclic adenosine 3', 5'-monophosphate (cAMP) include β 2 adrenergic receptors (β 2 -ARs)-AC-cAMP signaling pathways, E-prostanoid2/4 (EP2/4)-AC-cAMP signaling pathways and so on. Regulatory proteins, such as G protein-coupled receptor kinases (GRKs) and β-arrestins, play important modulatory roles in GPCRs signaling pathway. GPCRs signaling pathway and regulatory proteins implicate the pathogenesis process of inflammatory and immune response. GPCRs-AC-cAMP signal pathways involve in the inflammatory and immune response of RA. Different signaling pathways are mediated by different receptors, such as β 2 -AR, PGE2 receptor, chemokines receptor, and adenosine receptor. GRKs and β-arrestins are crucial proteins in the regulation of GPCRs signaling pathways. The potential therapeutic targets as well as strategies to modulate GPCRs signaling pathway are new development trends.

  3. G-protein-coupled receptors mediate 14-3-3 signal transduction.

    Science.gov (United States)

    Li, Hua; Eishingdrelo, Alex; Kongsamut, Sathapana; Eishingdrelo, Haifeng

    2016-01-01

    G-protein-coupled receptor (GPCR)-interacting proteins likely participate in regulating GPCR signaling by eliciting specific signal transduction cascades, inducing cross-talk with other pathways, and fine tuning the signal. However, except for G-proteins and β-arrestins, other GPCR-interacting proteins are poorly characterized. 14-3-3 proteins are signal adaptors, and their participation in GPCR signaling is not well understood or recognized. Here we demonstrate that GPCR-mediated 14-3-3 signaling is ligand-regulated and is likely to be a more general phenomenon than suggested by the previous reports of 14-3-3 involvement with a few GPCRs. For the first time, we can pharmacologically characterize GPCR/14-3-3 signaling. We have shown that GPCR-mediated 14-3-3 signaling is phosphorylation-dependent, and that the GPCR/14-3-3 interaction likely occurs later than receptor desensitization and internalization. GPCR-mediated 14-3-3 signaling can be β-arrestin-independent, and individual agonists can have different potencies on 14-3-3 and β-arrestin signaling. GPCRs can also mediate the interaction between 14-3-3 and Raf-1. Our work opens up a new broad realm of previously unappreciated GPCR signal transduction. Linking GPCRs to 14-3-3 signal transduction creates the potential for the development of new research directions and provides a new signaling pathway for drug discovery.

  4. Critical role for cross-linking of trimeric lectin domains of surfactant protein D in antiviral activity against influenza A virus

    DEFF Research Database (Denmark)

    Tecle, Tesfaldet; White, Mitchell R; Sørensen, Grith Lykke

    2008-01-01

    and antiviral activity of NCRDs as assessed by haemagglutination and neuraminidase inhibition and by viral neutralization. mAb-mediated cross-linking also enabled NCRDs to induce viral aggregation and to increase viral uptake by neutrophils and virus-induced respiratory burst responses by these cells...

  5. Innate immunity to dengue virus infection and subversion of antiviral responses

    Science.gov (United States)

    Green, Angela M.; Beatty, P. Robert; Hadjilaou, Alexandros; Harris, Eva

    2014-01-01

    Dengue is a major public health issue in tropical and subtropical regions worldwide. The four serotypes of dengue virus (DENV1-4) are spread primarily by Aedes aegypti and Ae. albopictus mosquitoes, whose geographic range continues to expand. Humans are the only host for epidemic strains of DENV, and the virus has developed sophisticated mechanisms to evade human innate immune responses. The host cell's first line of defense begins with an intracellular signaling cascade resulting in production of interferon (IFN)-α/β, which promotes intracellular antiviral responses and helps initiates the adaptive response during the course of DENV infection. In response, DENV has developed numerous ways to subvert these intracellular antiviral responses and directly inhibit cellular signaling cascades. Specifically, DENV manipulates the unfolded protein response and autophagy to counter cellular stress and delay apoptosis. The DENV non-structural protein NS4B and subgenomic sfRNA interfere with the RNAi pathway by inhibiting the RNAse Dicer. During heterotypic secondary DENV infection, subneutralizing antibodies can enable viral uptake through Fcγ receptors and down-regulate signaling cascades initiated via the pattern recognition receptors TLR3 and MDA5/RIG-I, thus reducing the antiviral state of the cell. The DENV NS2B/3 protein cleaves human STING/MITA, interfering with induction of IFN-α/β. Finally, DENV NS2A, NS4A, and NS4B complex together to block STAT1 phosphorylation, while NS5 binds and promotes degradation of human STAT2, thus preventing formation of the STAT1/STAT2 heterodimer and its transcriptional induction of ISGs. Here we discuss the host innate immune response to DENV and the mechanisms of immune evasion DENV has developed to manipulate cellular antiviral responses. PMID:24316047

  6. Innate immunity to dengue virus infection and subversion of antiviral responses.

    Science.gov (United States)

    Green, Angela M; Beatty, P Robert; Hadjilaou, Alexandros; Harris, Eva

    2014-03-20

    Dengue is a major public health issue in tropical and subtropical regions worldwide. The four serotypes of dengue virus (DENV1-DENV4) are spread primarily by Aedes aegypti and Aedes albopictus mosquitoes, whose geographic range continues to expand. Humans are the only host for epidemic strains of DENV, and the virus has developed sophisticated mechanisms to evade human innate immune responses. The host cell's first line of defense begins with an intracellular signaling cascade resulting in production of interferon α/β (IFN-α/β), which promotes intracellular antiviral responses and helps initiates the adaptive response during the course of DENV infection. In response, DENV has developed numerous ways to subvert these intracellular antiviral responses and directly inhibit cellular signaling cascades. Specifically, DENV manipulates the unfolded protein response and autophagy to counter cellular stress and delay apoptosis. The DENV non-structural protein NS4B and subgenomic flavivirus RNA interfere with the RNA interference pathway by inhibiting the RNase Dicer. During heterotypic secondary DENV infection, subneutralizing antibodies can enable viral uptake through Fcγ receptors and down-regulate signaling cascades initiated via the pattern recognition receptors TLR-3 and MDA5/RIG-I, thus reducing the antiviral state of the cell. The DENV NS2B/3 protein cleaves human STING/MITA, interfering with induction of IFN-α/β. Finally, DENV NS2A, NS4A, and NS4B complex together to block STAT1 phosphorylation, while NS5 binds and promotes degradation of human STAT2, thus preventing formation of the STAT1/STAT2 heterodimer and its transcriptional induction of interferon stimulating genes. Here, we discuss the host innate immune response to DENV and the mechanisms of immune evasion that DENV has developed to manipulate cellular antiviral responses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Information theory in systems biology. Part II: protein-protein interaction and signaling networks.

    Science.gov (United States)

    Mousavian, Zaynab; Díaz, José; Masoudi-Nejad, Ali

    2016-03-01

    By the development of information theory in 1948 by Claude Shannon to address the problems in the field of data storage and data communication over (noisy) communication channel, it has been successfully applied in many other research areas such as bioinformatics and systems biology. In this manuscript, we attempt to review some of the existing literatures in systems biology, which are using the information theory measures in their calculations. As we have reviewed most of the existing information-theoretic methods in gene regulatory and metabolic networks in the first part of the review, so in the second part of our study, the application of information theory in other types of biological networks including protein-protein interaction and signaling networks will be surveyed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Antiviral Defense Mechanisms in Honey Bees.

    Science.gov (United States)

    Brutscher, Laura M; Daughenbaugh, Katie F; Flenniken, Michelle L

    2015-08-01

    Honey bees are significant pollinators of agricultural crops and other important plant species. High annual losses of honey bee colonies in North America and in some parts of Europe have profound ecological and economic implications. Colony losses have been attributed to multiple factors including RNA viruses, thus understanding bee antiviral defense mechanisms may result in the development of strategies that mitigate colony losses. Honey bee antiviral defense mechanisms include RNA-interference, pathogen-associated molecular pattern (PAMP) triggered signal transduction cascades, and reactive oxygen species generation. However, the relative importance of these and other pathways is largely uncharacterized. Herein we review the current understanding of honey bee antiviral defense mechanisms and suggest important avenues for future investigation.

  9. Distinct determinants in HIV-1 Vif and human APOBEC3 proteins are required for the suppression of diverse host anti-viral proteins.

    Directory of Open Access Journals (Sweden)

    Wenyan Zhang

    Full Text Available APOBEC3G (A3G and related cytidine deaminases of the APOBEC3 family of proteins are potent inhibitors of many retroviruses, including HIV-1. Formation of infectious HIV-1 requires the suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through the common mechanism of recruiting the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. The domains in Vif and various APOBEC3 proteins required for APOBEC3 recognition and degradation have not been fully characterized.In the present study, we have demonstrated that the regions of APOBEC3F (A3F that are required for its HIV-1-mediated binding and degradation are distinct from those reported for A3G. We found that the C-terminal cytidine deaminase domain (C-CDD of A3F alone is sufficient for its interaction with HIV-1 Vif and its Vif-mediated degradation. We also observed that the domains of HIV-1 Vif that are uniquely required for its functional interaction with full-length A3F are also required for the degradation of the C-CDD of A3F; in contrast, those Vif domains that are uniquely required for functional interaction with A3G are not required for the degradation of the C-CDD of A3F. Interestingly, the HIV-1 Vif domains required for the degradation of A3F are also required for the degradation of A3C and A3DE. On the other hand, the Vif domains uniquely required for the degradation of A3G are dispensable for the degradation of cytidine deaminases A3C and A3DE.Our data suggest that distinct regions of A3F and A3G are targeted by HIV-1 Vif molecules. However, HIV-1 Vif suppresses A3F, A3C, and A3DE through similar recognition determinants, which are conserved among Vif molecules from diverse HIV-1 strains. Mapping these determinants may be useful for the design of novel anti-HIV inhibitors.

  10. Identification of novel components in microProtein signalling

    DEFF Research Database (Denmark)

    Rodrigues, Vandasue Lily

    findings suggest that these interacting components are part of a larger repressor complex preventing premature floral transition. Till date, all the miPs described in plants target transcription factors. The lack of diversity of protein target classes can be attributed to the lack of functional...... characterization of smaller proteins. Using a computational approach, we identified putative microProteins that could target a diverse variety of protein classes. Using a synthetic microProtein approach, we demonstrate that miPs can target a diverse variety of target proteins, which makes them of interest...

  11. The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

    Directory of Open Access Journals (Sweden)

    M. A. Prado

    2001-01-01

    Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

  12. Molecular Mechanisms of Foot-and-Mouth Disease Virus Targeting the Host Antiviral Response

    Directory of Open Access Journals (Sweden)

    Miguel Rodríguez Pulido

    2017-06-01

    Full Text Available Foot-and-mouth disease virus (FMDV is the causative agent of an acute vesicular disease affecting pigs, cattle and other domestic, and wild animals worldwide. The aim of the host interferon (IFN response is to limit viral replication and spread. Detection of the viral genome and products by specialized cellular sensors initiates a signaling cascade that leads to a rapid antiviral response involving the secretion of type I- and type III-IFNs and other antiviral cytokines with antiproliferative and immunomodulatory functions. During co-evolution with their hosts, viruses have acquired strategies to actively counteract host antiviral responses and the balance between innate response and viral antagonism may determine the outcome of disease and pathogenesis. FMDV proteases Lpro and 3C have been found to antagonize the host IFN response by a repertoire of mechanisms. Moreover, the putative role of other viral proteins in IFN antagonism is being recently unveiled, uncovering sophisticated immune evasion strategies different to those reported to date for other members of the Picornaviridae family. Here, we review the interplay between antiviral responses induced by FMDV infection and viral countermeasures to block them. Research on strategies used by viruses to modulate immunity will provide insights into the function of host pathways involved in defense against pathogens and will also lead to development of new therapeutic strategies to fight virus infections.

  13. Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

    Directory of Open Access Journals (Sweden)

    El-Mahdy Sherif

    2010-05-01

    Full Text Available Abstract Background Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on lung defense are incompletely understood. Because airway epithelial cell responses to type II interferon (IFN are critical in regulation of defense against many respiratory viral infections, we hypothesized that cigarette smoke has inhibitory effects on IFN-γ-dependent antiviral mechanisms in epithelial cells in the airway. Methods Primary human tracheobronchial epithelial cells were first treated with cigarette smoke extract (CSE followed by exposure to both CSE and IFN-γ. Epithelial cell cytotoxicity and IFN-γ-induced signaling, gene expression, and antiviral effects against respiratory syncytial virus (RSV were tested without and with CSE exposure. Results CSE inhibited IFN-γ-dependent gene expression in airway epithelial cells, and these effects were not due to cell loss or cytotoxicity. CSE markedly inhibited IFN-γ-induced Stat1 phosphorylation, indicating that CSE altered type II interferon signal transduction and providing a mechanism for CSE effects. A period of CSE exposure combined with an interval of epithelial cell exposure to both CSE and IFN-γ was required to inhibit IFN-γ-induced cell signaling. CSE also decreased the inhibitory effect of IFN-γ on RSV mRNA and protein expression, confirming effects on viral infection. CSE effects on IFN-γ-induced Stat1 activation, antiviral protein expression, and inhibition of RSV infection were decreased by glutathione augmentation of epithelial cells using N-acetylcysteine or glutathione monoethyl ester, providing one strategy to alter cigarette smoke effects. Conclusions The results indicate that CSE inhibits the antiviral effects of IFN-γ, thereby presenting one explanation for increased susceptibility to respiratory viral infection in individuals exposed to cigarette smoke.

  14. Antiviral Polymer Therapeutics

    DEFF Research Database (Denmark)

    Smith, Anton Allen Abbotsford

    2014-01-01

    The field of drug delivery is in essence an exercise in engineered pharmacokinetics. Methods of doing so have been developed through the introduction of a vehicle carrying the drug, either by encapsulation or covalent attachment. The emergence of polymer therapeutics in anticancer therapy has...... garnered a great deal of interest due to the substantial room for improvement inherent to conventional chemotherapeutic agents. Chemotherapeutic agents and antiviral agents have a lot of features in common due to both of them typically targeting endogenous targets, unlike antibacterial compounds, though...... the examples of polymer therapeutics being applied as an antiviral treatment are few and far in-between. This work aims to explore antiviral therapeutics, specifically in context of hepatitis virus C (HCV) and HIV. The current treatment of hepatitis C consists of a combination of drugs, of which ribavirin...

  15. Protein conservation and variation suggest mechanisms of cell type-specific modulation of signaling pathways.

    Directory of Open Access Journals (Sweden)

    Martin H Schaefer

    2014-06-01

    Full Text Available Many proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors and the output (transcription factors layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types.

  16. A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

    Science.gov (United States)

    Jia, Lixia; Chisari, Mariangela; Maktabi, Mohammad H; Sobieski, Courtney; Zhou, Hao; Konopko, Aaron M; Martin, Brent R; Mennerick, Steven J; Blumer, Kendall J

    2014-02-28

    Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

  17. An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8(+) T cells and antibody when expressed from modified vaccinia Ankara.

    Science.gov (United States)

    Quinan, Bárbara R; Flesch, Inge E A; Pinho, Tânia M G; Coelho, Fabiana M; Tscharke, David C; da Fonseca, Flávio G

    2014-05-23

    Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8(+) T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8(+) T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8(+) T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8(+) T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8(+) T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Ophthalmic antiviral chemotherapy : An overview

    Directory of Open Access Journals (Sweden)

    Athmanathan Sreedharan

    1997-01-01

    Full Text Available Antiviral drug development has been slow due to many factors. One such factor is the difficulty to block the viral replication in the cell without adversely affecting the host cell metabolic activity. Most of the antiviral compounds are analogs of purines and pyramidines. Currently available antiviral drugs mainly inhibit viral nucleic acid synthesis, hence act only on actively replicating viruses. This article presents an overview of some of the commonly used antiviral agents in clinical ophthalmology.

  19. Signaling via G proteins mediates tumorigenic effects of GPR87

    DEFF Research Database (Denmark)

    Arfelt, Kristine Niss; Fares, Suzan; Sparre-Ulrich, Alexander H.

    2017-01-01

    G protein-coupled receptors (GPCRs) constitute a large protein family of seven transmembrane (7TM) spanning proteins that regulate multiple physiological functions. GPR87 is overexpressed in several cancers and plays a role in tumor cell survival. Here, the basal activity of GPR87 was investigated...... in transiently transfected HEK293 cells, revealing ligand-independent coupling to Gαi, Gαq and Gα12/13. Furthermore, GPR87 showed a ligand-independent G protein-dependent activation of the downstream transcription factors CREB, NFκB, NFAT and SRE. In tetracycline-induced Flp-In T-Rex-293 cells, GPR87 induced...

  20. Making Sense of G Proteins: Genetic analysis of sensory G protein signaling in the nematode C. elegans

    NARCIS (Netherlands)

    H. Lans (Hannes)

    2005-01-01

    textabstractAmong the key molecules involved in sensory perception are G proteins, which act in every cell to activate a cascade of signaling molecules in response to certain environmental cues. In this thesis, several studies on the role of G proteins in the sensory system of C. elegans are

  1. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  2. Conformational analysis of g protein-coupled receptor signaling by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Li, Sheng; Lee, Su Youn; Chung, Ka Young

    2015-01-01

    Conformational change and protein-protein interactions are two major mechanisms of membrane protein signal transduction, including G protein-coupled receptors (GPCRs). Upon agonist binding, GPCRs change conformation, resulting in interaction with downstream signaling molecules such as G proteins. To understand the precise signaling mechanism, studies have investigated the structural mechanism of GPCR signaling using X-ray crystallography, nuclear magnetic resonance (NMR), or electron paramagnetic resonance. In addition to these techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has recently been used in GPCR studies. HDX-MS measures the rate at which peptide amide hydrogens exchange with deuterium in the solvent. Exposed or flexible regions have higher exchange rates and excluded or ordered regions have lower exchange rates. Therefore, HDX-MS is a useful tool for studying protein-protein interfaces and conformational changes after protein activation or protein-protein interactions. Although HDX-MS does not give high-resolution structures, it analyzes protein conformations that are difficult to study with X-ray crystallography or NMR. Furthermore, conformational information from HDX-MS can help in the crystallization of X-ray crystallography by suggesting highly flexible regions. Interactions between GPCRs and downstream signaling molecules are not easily analyzed by X-ray crystallography or NMR because of the large size of the GPCR-signaling molecule complexes, hydrophobicity, and flexibility of GPCRs. HDX-MS could be useful for analyzing the conformational mechanism of GPCR signaling. In this chapter, we discuss details of HDX-MS for analyzing GPCRs using the β2AR-G protein complex as a model system. © 2015 Elsevier Inc. All rights reserved.

  3. Landscape mapping of functional proteins in insulin signal transduction and insulin resistance: a network-based protein-protein interaction analysis.

    Directory of Open Access Journals (Sweden)

    Chiranjib Chakraborty

    Full Text Available The type 2 diabetes has increased rapidly in recent years throughout the world. The insulin signal transduction mechanism gets disrupted sometimes and it's known as insulin-resistance. It is one of the primary causes associated with type-2 diabetes. The signaling mechanisms involved several proteins that include 7 major functional proteins such as INS, INSR, IRS1, IRS2, PIK3CA, Akt2, and GLUT4. Using these 7 principal proteins, multiple sequences alignment has been created. The scores between sequences also have been developed. We have constructed a phylogenetic tree and modified it with node and distance. Besides, we have generated sequence logos and ultimately developed the protein-protein interaction network. The small insulin signal transduction protein arrangement shows complex network between the functional proteins.

  4. Antiviral Drugs: Seasonal Flu

    Centers for Disease Control (CDC) Podcasts

    2010-09-29

    In this podcast, Dr. Joe Bresee explains the nature of antiviral drugs and how they are used for seasonal flu.  Created: 9/29/2010 by National Center for Immunization and Respiratory Diseases (NCIRD).   Date Released: 9/29/2010.

  5. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor

    DEFF Research Database (Denmark)

    Chen, Y; Grall, D; Salcini, A E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have...... kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors....... recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation...

  6. Engineering Synthetic Proteins to Generate Ca2+Signals in Mammalian Cells.

    Science.gov (United States)

    Qudrat, Anam; Truong, Kevin

    2017-03-17

    The versatility of Ca 2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca 2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca 2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca 2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca 2+ ). By coupling these chimeric proteins that generate Ca 2+ signals with proteins that respond to Ca 2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca 2+ . Thus, using this design strategy, it is possible to engineer proteins to generate a Ca 2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca 2+ -activated processes.

  7. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  8. High expression of G-protein signaling modulator 2 in hepatocellular carcinoma facilitates tumor growth and metastasis by activating the PI3K/AKT signaling pathway.

    Science.gov (United States)

    He, Xiao-Qin; Zhang, Yue-Feng; Yu, Jia-Jun; Gan, Yuan-Yuan; Han, Na-Na; Zhang, Mei-Xia; Ge, Wei; Deng, Jun-Jian; Zheng, Yong-Fa; Xu, Xi-Ming

    2017-03-01

    The aim of this study was to investigate the role of G-protein signaling modulator 2 in the carcinogenesis and progression of hepatocellular carcinoma. We previously showed that G-protein signaling modulator 2 was upregulated in hepatitis B virus-related hepatocellular carcinoma tissues through a hierarchical clustering analysis. With this study, we first assessed the expression pattern of G-protein signaling modulator 2 in hepatocellular carcinoma specimens and adjacent noncancerous tissues; clinical data were analyzed, along survival times, utilizing the Kaplan-Meier method. Moreover, the functions of G-protein signaling modulator 2 were examined using small-interfering RNAs in vitro. The results showed that G-protein signaling modulator 2 was clearly overexpressed in hepatocellular carcinoma tissues and cell lines and that the G-protein signaling modulator 2 expression level was related to tumor size and hepatitis B virus infection. Furthermore, G-protein signaling modulator 2 knockdown studies suggested that G-protein signaling modulator 2 accelerates cell growth, cell cycle, migration, and invasion and inhibits apoptosis, acting as an oncogene in hepatocellular carcinoma. Western blotting indicated that silencing of G-protein signaling modulator 2 in HepG2 and SMMC-7721 cells increased the expression levels of Bax, caspase-3, and E-cadherin, while notably suppressing the cyclin-dependent kinase 4, cyclin-dependent kinase 6, CyclinD1, Snail1, Vimentin, and matrix metallopeptidase 9 expression levels, compared with that in the control groups. In addition, we found that G-protein signaling modulator 2 can affect the expression of key proteins involved in protein kinase B activation. In conclusion, high expression of G-protein signaling modulator 2 was involved in the pathological processes of hepatocellular carcinoma through activation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which may provide an attractive potential diagnostic

  9. HIV-1 proteins in infected cells determine the presentation of viral peptides by HLA class I and class II molecules and the nature of the cellular and humoral antiviral immune responses--a review.

    Science.gov (United States)

    Becker, Y

    1994-07-01

    The goals of molecular virology and immunology during the second half of the 20th century have been to provide the conceptual approaches and the tools for the development of safe and efficient virus vaccines for the human population. The success of the vaccination approach to prevent virus epidemics was attributed to the ability of inactivated and live virus vaccines to induce a humoral immune response and to produce antiviral neutralizing antibodies in the vaccinees. The successful development of antiviral vaccines and their application to most of the human population led to a marked decrease in virus epidemics around the globe. Despite this remarkable achievement, the developing epidemics of HIV-caused AIDS (accompanied by activation of latent herpesviruses in AIDS patients), epidemics of Dengue fever, and infections with respiratory syncytial virus may indicate that conventional approaches to the development of virus vaccines that induce antiviral humoral responses may not suffice. This may indicate that virus vaccines that induce a cellular immune response, leading to the destruction of virus-infected cells by CD8+ cytotoxic T cells (CTLs), may be needed. Antiviral CD8+ CTLs are induced by viral peptides presented within the peptide binding grooves of HLA class I molecules present on the surface of infected cells. Studies in the last decade provided an insight into the presentation of viral peptides by HLA class I molecules to CD8+ T cells. These studies are here reviewed, together with a review of the molecular events of virus replication, to obtain an overview of how viral peptides associate with the HLA class I molecules. A similar review is provided on the molecular pathway by which viral proteins, used as subunit vaccines or inactivated virus particles, are taken up by endosomes in the endosome pathway and are processed by proteolytic enzymes into peptides that interact with HLA class II molecules during their transport to the plasma membrane of antigen

  10. Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain

    Science.gov (United States)

    Kakar, Mudit; Davis, James R.; Kern, Steve E.; Lim, Carol S.

    2007-01-01

    Ligand regulated localization controllable protein constructs were optimized in this study. Several constructs were made from a classical nuclear export signal (HIV-rev, MAPKK, or progesterone receptor) in combination with a SV40 T-antigen type nuclear import signal. Different ligand binding domains (LBDs from glucocorticoid receptor or progesterone receptor) were also tested for their ability to impart control over localization of proteins. This study was designed to create constructs which are cytoplasmic in the absence of ligand and nuclear in the presence of ligand, and also to regulate the amount of protein translocating to the nucleus on ligand induction. The balance between the strengths of import and export signals was critical for overall localization of proteins. The amount of protein entering the nucleus was also affected by the dose of ligand (10-100nM). However, the overall import characteristics were determined by the strengths of localization signals and the inherent localization properties of the LBD used. This study established that the amount of protein present in a particular compartment can be regulated by the use of localization signals of various strengths. These optimized localization controllable protein constructs can be used to correct for diseases due to aberrant localization of proteins. PMID:17574289

  11. PACRG, a protein linked to ciliary motility, mediates cellular signaling

    NARCIS (Netherlands)

    Loucks, C.M. (Catrina M.); N.J. Bialas (Nathan); M.P.J. Dekkers (Martijn); Walker, D.S. (Denise S.); Grundy, L.J. (Laura J.); Li, C. (Chunmei); Inglis, P.N. (P. Nick); Kida, K. (Katarzyna); W.R. Schafer (William); O.E. Blacque (Oliver); G. Jansen (Gert); M.R. Leroux (Michel)

    2016-01-01

    textabstractCilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary

  12. Mitogen-activated protein kinase and abscisic acid signal transduction

    NARCIS (Netherlands)

    Heimovaara-Dijkstra, S.; Testerink, C.; Wang, M.

    1998-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C),

  13. The impact of RGS and other G-protein regulatory proteins on Gαi-mediated signaling in immunity.

    Science.gov (United States)

    Kehrl, John H

    2016-08-15

    Leukocyte chemoattractant receptors are members of the G-protein coupled receptor (GPCR) family. Signaling downstream of these receptors directs the localization, positioning and homeostatic trafficking of leukocytes; as well as their recruitment to, and their retention at, inflammatory sites. Ligand induced changes in the molecular conformation of chemoattractant receptors results in the engagement of heterotrimeric G-proteins, which promotes α subunits to undergo GTP/GDP exchange. This results in the functional release of βγ subunits from the heterotrimers, thereby activating downstream effector molecules, which initiate leukocyte polarization, gradient sensing, and directional migration. Pertussis toxin ADP ribosylates Gαi subunits and prevents chemoattractant receptors from triggering Gαi nucleotide exchange. The use of pertussis toxin revealed the essential importance of Gαi subunit nucleotide exchange for chemoattractant receptor signaling. More recent studies have identified a range of regulatory mechanisms that target these receptors and their associated heterotrimeric G-proteins, thereby helping to control the magnitude, kinetics, and duration of signaling. A failure in these regulatory pathways can lead to impaired receptor signaling and immunopathology. The analysis of mice with targeted deletions of Gαi isoforms as well as some of these G-protein regulatory proteins is providing insights into their roles in chemoattractant receptor signaling. Published by Elsevier Inc.

  14. Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton.

    NARCIS (Netherlands)

    Luchtenborg, A.M.; Solis, G.P.; Egger-Adam, D.; Koval, A.; Lin, C.; Blanchard, M.G.; Kellenberger, S.; Katanaev, V.L.

    2014-01-01

    Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of

  15. Emergency Spatiotemporal Shift: The Response of Protein Kinase D to Stress Signals in the Cardiovascular System.

    Science.gov (United States)

    Wood, Brent M; Bossuyt, Julie

    2017-01-01

    Protein Kinase D isoforms (PKD 1-3) are key mediators of neurohormonal, oxidative, and metabolic stress signals. PKDs impact a wide variety of signaling pathways and cellular functions including actin dynamics, vesicle trafficking, cell motility, survival, contractility, energy substrate utilization, and gene transcription. PKD activity is also increasingly linked to cancer, immune regulation, pain modulation, memory, angiogenesis, and cardiovascular disease. This increasing complexity and diversity of PKD function, highlights the importance of tight spatiotemporal control of the kinase via protein-protein interactions, post-translational modifications or targeting via scaffolding proteins. In this review, we focus on the spatiotemporal regulation and effects of PKD signaling in response to neurohormonal, oxidant and metabolic signals that have implications for myocardial disease. Precise targeting of these mechanisms will be crucial in the design of PKD-based therapeutic strategies.

  16. Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

    Science.gov (United States)

    Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

    2011-01-01

    Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

  17. Cell signaling, post-translational protein modifications and NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Theillet, Francois-Xavier [In-cell NMR Group, Department of NMR-Supported Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin) (Germany); Smet-Nocca, Caroline [Universite Lille Nord de France, CNRS UMR 8576 (France); Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas [In-cell NMR Group, Department of NMR-Supported Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin) (Germany); Yoon, Mi-Kyung; Kriwacki, Richard W. [St. Jude Children' s Research Hospital, Department of Structural Biology (United States); Landrieu, Isabelle; Lippens, Guy [Universite Lille Nord de France, CNRS UMR 8576 (France); Selenko, Philipp, E-mail: selenko@fmp-berlin.de [In-cell NMR Group, Department of NMR-Supported Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin) (Germany)

    2012-11-15

    Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.

  18. RO 90-7501 enhances TLR3 and RLR agonist induced antiviral response.

    Directory of Open Access Journals (Sweden)

    Fang Guo

    Full Text Available Recognition of virus infection by innate pattern recognition receptors (PRRs, including membrane-associated toll-like receptors (TLR and cytoplasmic RIG-I-like receptors (RLR, activates cascades of signal transduction pathways leading to production of type I interferons (IFN and proinflammatory cytokines that orchestrate the elimination of the viruses. Although it has been demonstrated that PRR-mediated innate immunity plays an essential role in defending virus from infection, it also occasionally results in overwhelming production of proinflammatory cytokines that cause severe inflammation, blood vessel leakage and tissue damage. In our efforts to identify small molecules that selectively enhance PRR-mediated antiviral, but not the detrimental inflammatory response, we discovered a compound, RO 90-7501 ('2'-(4-Aminophenyl-[2,5'-bi-1H-benzimidazol]-5-amine, that significantly promoted both TLR3 and RLR ligand-induced IFN-β gene expression and antiviral response, most likely via selective activation of p38 mitogen-activated protein kinase (MAPK pathway. Our results thus imply that pharmacological modulation of PRR signal transduction pathways in favor of the induction of a beneficial antiviral response can be a novel therapeutic strategy.

  19. Presenilin dependence of phospholipase C and protein kinase C signaling

    DEFF Research Database (Denmark)

    Dehvari, Nodi; Cedazo-Minguez, Angel; Isacsson, Ola

    2007-01-01

    -stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha...... and PKCgamma activations were significantly lower in PS1 and PS2 double knockout MEFs after PLC stimulation. Protein levels of PKCalpha and PKCgamma were lower in PS1 and PS2 double knockout MEFs. In contrast, PKCdelta levels were significantly elevated in PS1 and PS2 double knockout as well as in PS1 knockout...

  20. Tubulin, actin and heterotrimeric G proteins: coordination of signaling and structure.

    Science.gov (United States)

    Schappi, Jeffrey M; Krbanjevic, Aleksandar; Rasenick, Mark M

    2014-02-01

    G proteins mediate signals from membrane G protein coupled receptors to the cell interior, evoking significant regulation of cell physiology. The cytoskeleton contributes to cell morphology, motility, division, and transport functions. This review will discuss the interplay between heterotrimeric G protein signaling and elements of the cytoskeleton. Also described and discussed will be the interplay between tubulin and G proteins that results in atypical modulation of signaling pathways and cytoskeletal dynamics. This will be extended to describe how tubulin and G proteins act in concert to influence various aspects of cellular behavior. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters.This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé. © 2013.

  1. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    International Nuclear Information System (INIS)

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Wang, Yu; Zhu, Yan-Li; Jiang, Shi-Jin

    2013-01-01

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1–17 and 18–36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  2. Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells.

    NARCIS (Netherlands)

    Colak, D.; Mori, T.; Brill, M.S; Pfeifer, A.; Falk, S.; Deng, C.; Monteiro, R.; Mummery, C.L.; Sommer, L.; Gotz, M.

    2008-01-01

    In the mammalian brain, neurogenesis continues only in few regions of the forebrain. The molecular signals governing neurogenesis in these unique neurogenic niches, however, are still ill defined. Here, we show that bone morphogenic protein (BMP)-mediated signaling is active in adult neural stem

  3. MAVS protein is attenuated by rotavirus nonstructural protein 1.

    Directory of Open Access Journals (Sweden)

    Satabdi Nandi

    Full Text Available Rotavirus is the single, most important agent of infantile gastroenteritis in many animal species, including humans. In developing countries, rotavirus infection attributes approximately 500,000 deaths annually. Like other viruses it establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Among various pattern recognition receptors (PAMPs of the host, the cytosolic RNA helicases interact with viral RNA to activate the Mitochondrial Antiviral Signaling protein (MAVS, which regulates cellular interferon response. With an aim to identify the role of different PAMPs in rotavirus infected cell, MAVS was found to degrade in a time dependent and strain independent manner. Rotavirus non-structural protein 1 (NSP1 which is a known IFN antagonist, interacted with MAVS and degraded it in a strain independent manner, resulting in a complete loss of RNA sensing machinery in the infected cell. To best of our knowledge, this is the first report on NSP1 functionality where a signaling protein is targeted unanimously in all strains. In addition NSP1 inhibited the formation of detergent resistant MAVS aggregates, thereby averting the antiviral signaling cascade. The present study highlights the multifunctional role of rotavirus NSP1 and reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit by various back up strategies.

  4. Smallpox Antiviral Drug

    Science.gov (United States)

    2007-01-01

    phogenic proteolysis is crucial for simple RNA viruses such as poliovirus and HIV, and also appears to play a central role in the assembly of more...al particles [14]; unidirectional packaging of bacteriophage T4 DNA [15]; completion of the infectious poliovirus virion in a flexible configuration...effects of an antiviral both in vitro and in vivo. Some viruses have not been adapted to grow in tissue culture cells or due to their genetic makeup are

  5. Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

    Science.gov (United States)

    Hackenberg, Dieter; McKain, Michael R; Lee, Soon Goo; Roy Choudhury, Swarup; McCann, Tyler; Schreier, Spencer; Harkess, Alex; Pires, J Chris; Wong, Gane Ka-Shu; Jez, Joseph M; Kellogg, Elizabeth A; Pandey, Sona

    2017-10-01

    Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  6. Role of PY Motif Containing Protein, WBP-2 in ER, PR Signaling and Breast Tumorigenesis

    Science.gov (United States)

    2009-09-01

    of Runx2 family (24) and TEAD /TEF family (25), the proapoptotic protein p73 (26, 27), and is involved in ErbB4 signaling (28). In the present study...A, Kaneko KJ, Shu H, Zhao Y, DePamphilis ML 2001 TEAD /TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein

  7. Protein Phosphatase 2A Signaling in Human Prostate Cancer

    Science.gov (United States)

    2014-08-01

    phosphatidylinositol 3’-kinase and Akt/protein kinase B. Cancer Res 1999;59:1449-53. (14) Grethe S, Porn -Ares MI. p38 MAPK regulates phosphorylation of Bad...growth and sig- nalling. Biochem J 2001;353:417–39. 15. Grethe S, Porn -Ares MI. p38 MAPK regulates phosphorylation of Bad via PP2A-dependent suppression of

  8. Indispensable roles of mammalian Cbl family proteins as negative regulators of protein tyrosine kinase signaling: Insights from in vivo models

    OpenAIRE

    Naramura, Mayumi; Band, Vimla; Band, Hamid

    2011-01-01

    All higher eukaryotes utilize protein tyrosine kinases (PTKs) as molecular switches to control a variety of cellular signals. Notably, many PTKs have been identified as proto-oncogenes whose aberrant expression, mutations or co-option by pathogens can lead to human malignancies. Thus, it is obvious that PTK functions must be precisely regulated in order to maintain homeostasis of an organism. Investigations over the past fifteen years have revealed that members of the Cbl family proteins can ...

  9. Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Tomoyo Ujisawa

    Full Text Available Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron.

  10. Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans

    Science.gov (United States)

    Ujisawa, Tomoyo; Ohta, Akane; Uda-Yagi, Misato

    2016-01-01

    Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron. PMID:27788246

  11. Exercise and Glycemic Control: Focus on Redox Homeostasis and Redox-Sensitive Protein Signaling

    Science.gov (United States)

    Parker, Lewan; Shaw, Christopher S.; Stepto, Nigel K.; Levinger, Itamar

    2017-01-01

    Physical inactivity, excess energy consumption, and obesity are associated with elevated systemic oxidative stress and the sustained activation of redox-sensitive stress-activated protein kinase (SAPK) and mitogen-activated protein kinase signaling pathways. Sustained SAPK activation leads to aberrant insulin signaling, impaired glycemic control, and the development and progression of cardiometabolic disease. Paradoxically, acute exercise transiently increases oxidative stress and SAPK signaling, yet postexercise glycemic control and skeletal muscle function are enhanced. Furthermore, regular exercise leads to the upregulation of antioxidant defense, which likely assists in the mitigation of chronic oxidative stress-associated disease. In this review, we explore the complex spatiotemporal interplay between exercise, oxidative stress, and glycemic control, and highlight exercise-induced reactive oxygen species and redox-sensitive protein signaling as important regulators of glucose homeostasis. PMID:28529499

  12. Proximity-dependent protein detection based on enzyme-assisted fluorescence signal amplification.

    Science.gov (United States)

    Tan, Yuyu; Guo, Qiuping; Zhao, Xiayu; Yang, Xiaohai; Wang, Kemin; Huang, Jin; Zhou, Yu

    2014-01-15

    In this paper, we develop a sensitive fluorescence method for protein detection based on proximity extension and enzyme-assisted signal amplification. In this novel method, pairs of proximity probes are designed, and the recognition elements are integrated into the proximity probes. Then proteins are detected by transforming aptamer or antibody-protein binding signals into DNA detection based on proximity effect. In addition, nick sites are introduced into the proximity probes to amplify the detectable signal. As proof of concept, detection of human α-thrombin and human IgG are demonstrated in this study. The aptamers and antibodies are coupled in the proximity probes as recognition elements for human α-thrombin and human IgG respectively. In the presence of target protein, aptamer or antibody-protein binding signals are transformed into detectable signals by the proximity effect, and can be further amplified by enzyme-assisted strand displacement. The above mentioned strategies consequently bring the limit of detection (LOD) to as low as 1 pM for human α-thrombin and 6 pM for human IgG. Furthermore, this method might be extended to sensitive detection of other proteins by changing recognition elements of proximity probes. © 2013 Elsevier B.V. All rights reserved.

  13. Analysing protein-protein interactions of the Myxococcus xanthus Dif signalling pathway using the yeast two-hybrid system.

    Science.gov (United States)

    Lancero, Hope L; Castaneda, Schryl; Caberoy, Nora B; Ma, Xiaoyuan; Garza, Anthony G; Shi, Wenyuan

    2005-05-01

    The dif operon is essential for fruiting body formation, fibril (exopolysaccharide) production and social motility of Myxococcus xanthus. The dif locus contains a gene cluster homologous to chemotaxis genes such as mcp (difA), cheW (difC), cheY (difD), cheA (difE) and cheC (difF), as well as an unknown ORF called difB. This study used yeast two-hybrid analysis to investigate possible interactions between Dif proteins, and determined that DifA, C, D and E interact in a similar fashion to chemotaxis proteins of Escherichia coli and Bacillus subtilis. It also showed that DifF interacted with DifD, and that the novel protein DifB did not interact with Dif proteins. Furthermore, DifA-F proteins were used to determine other possible protein-protein interactions in the M. xanthus genomic library. The authors not only confirmed the specific interactions among known Dif proteins, but also discovered two novel interactions between DifE and Nla19, and DifB and YidC, providing some new information about the Dif signalling pathway. Based on these findings, a model for the Dif signalling pathway is proposed.

  14. Brain expressed and X-linked (Bex proteins are intrinsically disordered proteins (IDPs and form new signaling hubs.

    Directory of Open Access Journals (Sweden)

    Eva M Fernandez

    Full Text Available Intrinsically disordered proteins (IDPs are abundant in complex organisms. Due to their promiscuous nature and their ability to adopt several conformations IDPs constitute important points of network regulation. The family of Brain Expressed and X-linked (Bex proteins consists of 5 members in humans (Bex1-5. Recent reports have implicated Bex proteins in transcriptional regulation and signaling pathways involved in neurodegeneration, cancer, cell cycle and tumor growth. However, structural and biophysical data for this protein family is almost non-existent. We used bioinformatics analyses to show that Bex proteins contain long regions of intrinsic disorder which are conserved across all members. Moreover, we confirmed the intrinsic disorder by circular dichroism spectroscopy of Bex1 after expression and purification in E. coli. These observations strongly suggest that Bex proteins constitute a new group of IDPs. Based on these findings, together with the demonstrated promiscuity of Bex proteins and their involvement in different signaling pathways, we propose that Bex family members play important roles in the formation of protein network hubs.

  15. A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C elegans.

    Science.gov (United States)

    Srinivasan, Dayalan G; Fisk, Ridgely M; Xu, Huihong; van den Heuvel, Sander

    2003-05-15

    The Caenorhabditis elegans coiled-coil protein LIN-5 mediates several processes in cell division that depend on spindle forces, including alignment and segregation of chromosomes and positioning of the spindle. Here, we describe two closely related proteins, GPR-1 and GPR-2 (G protein regulator), which associate with LIN-5 in vivo and in vitro and depend on LIN-5 for localization to the spindle and cell cortex. GPR-1/GPR-2 contain a GoLoco/GPR motif that mediates interaction with GDP-bound Galpha(i/o). Inactivation of lin-5, gpr-1/gpr-2, or the Galpha(i/o) genes goa-1 and gpa-16 all cause highly similar chromosome segregation and spindle positioning defects, indicating a positive role for the LIN-5 and GPR proteins in G protein signaling. The lin-5 and gpr-1/gpr-2 genes appear to act downstream of the par polarity genes in the one- and two-cell stages and downstream of the tyrosine kinase-related genes mes-1 and src-1 at the four-cell stage. Together, these results indicate that GPR-1/GPR-2 in association with LIN-5 activate G protein signaling to affect spindle force. Polarity determinants may regulate LIN-5/GPR/Galpha locally to create the asymmetric forces that drive spindle movement. Results in C. elegans and other species are consistent with a novel model for receptor-independent activation of Galpha(i/o) signaling.

  16. Neuronal signaling modulates protein homeostasis in Caenorhabditis elegans post-synaptic muscle cells.

    Science.gov (United States)

    Garcia, Susana M; Casanueva, M Olivia; Silva, M Catarina; Amaral, Margarida D; Morimoto, Richard I

    2007-11-15

    Protein homeostasis maintains proper intracellular balance by promoting protein folding and clearance mechanisms while minimizing the stress caused by the accumulation of misfolded and damaged proteins. Chronic expression of aggregation-prone proteins is deleterious to the cell and has been linked to a wide range of conformational disorders. The molecular response to misfolded proteins is highly conserved and generally studied as a cell-autonomous process. Here, we provide evidence that neuronal signaling is an important modulator of protein homeostasis in post-synaptic muscle cells. In a forward genetic screen in Caenorhabditis elegans for enhancers of polyglutamine aggregation in muscle cells, we identified unc-30, a neuron-specific transcription factor that regulates the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We used additional sensors of protein conformational states to show that defective GABA signaling or increased acetylcholine (ACh) signaling causes a general imbalance in protein homeostasis in post-synaptic muscle cells. Moreover, exposure to GABA antagonists or ACh agonists has a similar effect, which reveals that toxins that act at the neuromuscular junction are potent modifiers of protein conformational disorders. These results demonstrate the importance of intercellular communication in intracellular homeostasis.

  17. [Antiviral properties of basidiomycetes metabolites].

    Science.gov (United States)

    Avtonomova, A V; Krasnopolskaya, L M

    2014-01-01

    The data on the antiviral action of the Ganoderma lucidum, Lentinus edodes, Grifola frondosa, Agaricus brasiliensis and other basidiomycetes metabolites are summurized. The metabolites of these species of basidiomycetes exhibit a direct antiviral effect on herpes simplex virus types I and II, human immunodeficiency virus (HIV), hepatitis B virus, vesicular stomatitis virus, influenza virus, Epstein-Barr virus, and others. Moreover, metabolites of basidiomycetes increased antiviral immunity.

  18. Effect of resistance exercise contraction mode and protein supplementation on members of the STARS signalling pathway

    Science.gov (United States)

    Vissing, Kristian; Rahbek, Stine K; Lamon, Severine; Farup, Jean; Stefanetti, Renae J; Wallace, Marita A; Vendelbo, Mikkel H; Russell, Aaron

    2013-01-01

    The striated muscle activator of Rho signalling (STARS) pathway is suggested to provide a link between external stress responses and transcriptional regulation in muscle. However, the sensitivity of STARS signalling to different mechanical stresses has not been investigated. In a comparative study, we examined the regulation of the STARS signalling pathway in response to unilateral resistance exercise performed as either eccentric (ECC) or concentric (CONC) contractions as well as prolonged training; with and without whey protein supplementation. Skeletal muscle STARS, myocardian-related transcription factor-A (MRTF-A) and serum response factor (SRF) mRNA and protein, as well as muscle cross-sectional area and maximal voluntary contraction, were measured. A single-bout of exercise produced increases in STARS and SRF mRNA and decreases in MRTF-A mRNA with both ECC and CONC exercise, but with an enhanced response occurring following ECC exercise. A 31% increase in STARS protein was observed exclusively after CONC exercise (P protein levels increased similarly by 48% with both CONC and ECC exercise (P hypertrophy and produced increases in MRTF-A protein of 125% and 99%, respectively (P protein. There was no effect of whey protein supplementation. These results show that resistance exercise provides an acute stimulation of the STARS pathway that is contraction mode dependent. The responses to acute exercise were more pronounced than responses to accumulated training, suggesting that STARS signalling is primarily involved in the initial phase of exercise-induced muscle adaptations. PMID:23753523

  19. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    Science.gov (United States)

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    2017-12-01

    Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

  1. Multistep Current Signal in Protein Translocation through Graphene Nanopores

    KAUST Repository

    Bonome, Emma Letizia

    2015-05-07

    © 2015 American Chemical Society. In nanopore sensing experiments, the properties of molecules are probed by the variation of ionic currents flowing through the nanopore. In this context, the electronic properties and the single-layer thickness of graphene constitute a major advantage for molecule characterization. Here we analyze the translocation pathway of the thioredoxin protein across a graphene nanopore, and the related ionic currents, by integrating two nonequilibrium molecular dynamics methods with a bioinformatic structural analysis. To obtain a qualitative picture of the translocation process and to identify salient features we performed unsupervised structural clustering on translocation conformations. This allowed us to identify some specific and robust translocation intermediates, characterized by significantly different ionic current flows. We found that the ion current strictly anticorrelates with the amount of pore occupancy by thioredoxin residues, providing a putative explanation of the multilevel current scenario observed in recently published translocation experiments.

  2. Redox modification of caveolar proteins in the cardiovascular system- role in cellular signalling and disease.

    Science.gov (United States)

    Bubb, Kristen J; Birgisdottir, Asa Birna; Tang, Owen; Hansen, Thomas; Figtree, Gemma A

    2017-08-01

    Rapid and coordinated release of a variety of reactive oxygen species (ROS) such as superoxide (O 2 .- ), hydrogen peroxide (H 2 O 2 ) and peroxynitrite, in specific microdomains, play a crucial role in cell signalling in the cardiovascular system. These reactions are mediated by reversible and functional modifications of a wide variety of key proteins. Dysregulation of this oxidative signalling occurs in almost all forms of cardiovascular disease (CVD), including at the very early phases. Despite the heavily publicized failure of "antioxidants" to improve CVD progression, pharmacotherapies such as those targeting the renin-angiotensin system, or statins, exert at least part of their large clinical benefit via modulating cellular redox signalling. Over 250 proteins, including receptors, ion channels and pumps, and signalling proteins are found in the caveolae. An increasing proportion of these are being recognized as redox regulated-proteins, that reside in the immediate vicinity of the two major cellular sources of ROS, nicotinamide adenine dinucleotide phosphate oxidase (Nox) and uncoupled endothelial nitric oxide synthase (eNOS). This review focuses on what is known about redox signalling within the caveolae, as well as endogenous protective mechanisms utilized by the cell, and new approaches to targeting dysregulated redox signalling in the caveolae as a therapeutic strategy in CVD. Copyright © 2017. Published by Elsevier Inc.

  3. Atypical Diabetic Foot Ulcer Keratinocyte Protein Signaling Correlates with Impaired Wound Healing

    Directory of Open Access Journals (Sweden)

    Glenn D. Hoke

    2016-01-01

    Full Text Available Diabetes mellitus is associated with chronic diabetic foot ulcers (DFUs and wound infections often resulting in lower extremity amputations. The protein signaling architecture of the mechanisms responsible for impaired DFU healing has not been characterized. In this preliminary clinical study, the intracellular levels of proteins involved in signal transduction networks relevant to wound healing were non-biasedly measured using reverse-phase protein arrays (RPPA in keratinocytes isolated from DFU wound biopsies. RPPA allows for the simultaneous documentation and assessment of the signaling pathways active in each DFU. Thus, RPPA provides for the accurate mapping of wound healing pathways associated with apoptosis, proliferation, senescence, survival, and angiogenesis. From the study data, we have identified potential diagnostic, or predictive, biomarkers for DFU wound healing derived from the ratios of quantified signaling protein expressions within interconnected pathways. These biomarkers may allow physicians to personalize therapeutic strategies for DFU management on an individual basis based upon the signaling architecture present in each wound. Additionally, we have identified altered, interconnected signaling pathways within DFU keratinocytes that may help guide the development of therapeutics to modulate these dysregulated pathways, many of which parallel the therapeutic targets which are the hallmarks of molecular therapies for treating cancer.

  4. Priming GPCR signaling through the synergistic effect of two G proteins.

    Science.gov (United States)

    Gupte, Tejas M; Malik, Rabia U; Sommese, Ruth F; Ritt, Michael; Sivaramakrishnan, Sivaraj

    2017-04-04

    Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D 1 dopamine receptor (D 1 -R). Reciprocally, Gs enhances IP 1 through vasopressin receptor (V 1A -R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving ( i ) sequential G-protein interactions at the cognate site, ( ii ) G-protein interactions at distinct allosteric and cognate sites on the GPCR, and ( iii ) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.

  5. Protein kinase D1 signaling in angiogenic gene expression and VEGF-mediated angiogenesis

    Directory of Open Access Journals (Sweden)

    Bin eRen MD, Phd, FAHA

    2016-05-01

    Full Text Available Protein kinase D 1 (PKD-1 is a signaling kinase important in fundamental cell functions including migration, proliferation and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis.

  6. Discovery of intramolecular signal transduction network based on a new protein dynamics model of energy dissipation.

    Directory of Open Access Journals (Sweden)

    Cheng-Wei Ma

    Full Text Available A novel approach to reveal intramolecular signal transduction network is proposed in this work. To this end, a new algorithm of network construction is developed, which is based on a new protein dynamics model of energy dissipation. A key feature of this approach is that direction information is specified after inferring protein residue-residue interaction network involved in the process of signal transduction. This enables fundamental analysis of the regulation hierarchy and identification of regulation hubs of the signaling network. A well-studied allosteric enzyme, E. coli aspartokinase III, is used as a model system to demonstrate the new method. Comparison with experimental results shows that the new approach is able to predict all the sites that have been experimentally proved to desensitize allosteric regulation of the enzyme. In addition, the signal transduction network shows a clear preference for specific structural regions, secondary structural types and residue conservation. Occurrence of super-hubs in the network indicates that allosteric regulation tends to gather residues with high connection ability to collectively facilitate the signaling process. Furthermore, a new parameter of propagation coefficient is defined to determine the propagation capability of residues within a signal transduction network. In conclusion, the new approach is useful for fundamental understanding of the process of intramolecular signal transduction and thus has significant impact on rational design of novel allosteric proteins.

  7. Lack of beta-arrestin signaling in the absence of active G proteins.

    Science.gov (United States)

    Grundmann, Manuel; Merten, Nicole; Malfacini, Davide; Inoue, Asuka; Preis, Philip; Simon, Katharina; Rüttiger, Nelly; Ziegler, Nicole; Benkel, Tobias; Schmitt, Nina Katharina; Ishida, Satoru; Müller, Ines; Reher, Raphael; Kawakami, Kouki; Inoue, Ayumi; Rick, Ulrike; Kühl, Toni; Imhof, Diana; Aoki, Junken; König, Gabriele M; Hoffmann, Carsten; Gomeza, Jesus; Wess, Jürgen; Kostenis, Evi

    2018-01-23

    G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

  8. Systematic analysis of essential genes reveals important regulators of G protein signaling.

    Science.gov (United States)

    Cappell, Steven D; Baker, Rachael; Skowyra, Dorota; Dohlman, Henrik G

    2010-06-11

    The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify additional signaling components, we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly represented. Included in this group are members of the Skp, Cullin, F box (SCF) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCF(Cdc4) acts together with the Cdc34 ubiquitin-conjugating enzyme at the level of the G protein; promotes degradation of the G protein alpha subunit, Gpa1, in vivo; and catalyzes Gpa1 ubiquitination in vitro. These insights to the G protein signaling network reveal the essential genome as an untapped resource for identifying new components and regulators of signal transduction pathways. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  9. Significance of expression of suppressor of cytokine signaling proteins: Suppressor of cytokine signaling-1, suppressor of cytokine signaling-2, and suppressor of cytokine signaling-3 in papillary thyroid cancer

    Directory of Open Access Journals (Sweden)

    Toral Pundrik Kobawala

    2017-01-01

    Conclusion: Expression of the studied SOCS proteins may be a consequence of activation of Janus kinase-signal transducers and activators of transcription and other pathways supporting growth and survival of cancer cells that are sustained by several cytokines. Thus, SOCS-1, SOCS-2, and SOCS-3 proteins may directly or indirectly, have important roles in development and pathogenesis of papillary thyroid cancer.

  10. CRM1 Inhibitors for Antiviral Therapy

    Directory of Open Access Journals (Sweden)

    Cynthia Mathew

    2017-06-01

    Full Text Available Infectious diseases are a major global concern and despite major advancements in medical research, still cause significant morbidity and mortality. Progress in antiviral therapy is particularly hindered by appearance of mutants capable of overcoming the effects of drugs targeting viral components. Alternatively, development of drugs targeting host proteins essential for completion of viral lifecycle holds potential as a viable strategy for antiviral therapy. Nucleocytoplasmic trafficking pathways in particular are involved in several pathological conditions including cancer and viral infections, where hijacking or alteration of function of key transporter proteins, such as Chromosome Region Maintenance1 (CRM1 is observed. Overexpression of CRM1-mediated nuclear export is evident in several solid and hematological malignancies. Interestingly, CRM1-mediated nuclear export of viral components is crucial in various stages of the viral lifecycle and assembly. This review summarizes the role of CRM1 in cancer and selected viruses. Leptomycin B (LMB is the prototypical inhibitor of CRM1 potent against various cancer cell lines overexpressing CRM1 and in limiting viral infections at nanomolar concentrations in vitro. However, the irreversible shutdown of nuclear export results in high cytotoxicity and limited efficacy in vivo. This has prompted search for synthetic and natural CRM1 inhibitors that can potentially be developed as broadly active antivirals, some of which are summarized in this review.

  11. Evasion and subversion of interferon-mediated antiviral immunity by Kaposi's sarcoma-associated herpesvirus: an overview.

    Science.gov (United States)

    Sathish, Narayanan; Yuan, Yan

    2011-11-01

    Viral invasion of a host cell triggers immune responses with both innate and adaptive components. The innate immune response involving the induction of type I interferons (alpha and beta interferons [IFN-α and -β]) constitutes the first line of antiviral defenses. The type I IFNs signal the transcription of a group of antiviral effector proteins, the IFN-stimulated genes (ISGs), which target distinct viral components and distinct stages of the viral life cycle, aiming to eliminate invading viruses. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma (KS), a sudden upsurge of type I IFN-mediated innate antiviral signals is seen immediately following both primary de novo infection and viral lytic reactivation from latency. Potent subversion of these responses thus becomes mandatory for the successful establishment of a primary infection following viral entry as well as for efficient viral assembly and egress. This review gives a concise overview of the induction of the type I IFN signaling pathways in response to viral infection and provides a comprehensive understanding of the antagonizing effects exerted by KSHV on type I IFN pathways wielded at various stages of the viral life cycle. Information garnered from this review should result in a better understanding of KSHV biology essential for the development of immunotherapeutic strategies targeted toward KSHV-associated malignancies.

  12. Evasion and Subversion of Interferon-Mediated Antiviral Immunity by Kaposi's Sarcoma-Associated Herpesvirus: an Overview ▿

    Science.gov (United States)

    Sathish, Narayanan; Yuan, Yan

    2011-01-01

    Viral invasion of a host cell triggers immune responses with both innate and adaptive components. The innate immune response involving the induction of type I interferons (alpha and beta interferons [IFN-α and -β]) constitutes the first line of antiviral defenses. The type I IFNs signal the transcription of a group of antiviral effector proteins, the IFN-stimulated genes (ISGs), which target distinct viral components and distinct stages of the viral life cycle, aiming to eliminate invading viruses. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma (KS), a sudden upsurge of type I IFN-mediated innate antiviral signals is seen immediately following both primary de novo infection and viral lytic reactivation from latency. Potent subversion of these responses thus becomes mandatory for the successful establishment of a primary infection following viral entry as well as for efficient viral assembly and egress. This review gives a concise overview of the induction of the type I IFN signaling pathways in response to viral infection and provides a comprehensive understanding of the antagonizing effects exerted by KSHV on type I IFN pathways wielded at various stages of the viral life cycle. Information garnered from this review should result in a better understanding of KSHV biology essential for the development of immunotherapeutic strategies targeted toward KSHV-associated malignancies. PMID:21775463

  13. Signaling by Small GTPases at Cell-Cell junctions: Protein Interactions Building Control and Networks.

    Science.gov (United States)

    Braga, Vania

    2017-09-11

    A number of interesting reports highlight the intricate network of signaling proteins that coordinate formation and maintenance of cell-cell contacts. We have much yet to learn about how the in vitro binding data is translated into protein association inside the cells and whether such interaction modulates the signaling properties of the protein. What emerges from recent studies is the importance to carefully consider small GTPase activation in the context of where its activation occurs, which upstream regulators are involved in the activation/inactivation cycle and the GTPase interacting partners that determine the intracellular niche and extent of signaling. Data discussed here unravel unparalleled cooperation and coordination of functions among GTPases and their regulators in supporting strong adhesion between cells. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  14. The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip compartment.

    Science.gov (United States)

    He, Mu; Subramanian, Radhika; Bangs, Fiona; Omelchenko, Tatiana; Liem, Karel F; Kapoor, Tarun M; Anderson, Kathryn V

    2014-07-01

    Mammalian Hedgehog (Hh) signal transduction requires a primary cilium, a microtubule-based organelle, and the Gli-Sufu complexes that mediate Hh signalling, which are enriched at cilia tips. Kif7, a kinesin-4 family protein, is a conserved regulator of the Hh signalling pathway and a human ciliopathy protein. Here we show that Kif7 localizes to the cilium tip, the site of microtubule plus ends, where it limits cilium length and controls cilium structure. Purified recombinant Kif7 binds the plus ends of growing microtubules in vitro, where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. Kif7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of Kif7 in the mammalian Hh pathway is to control cilium architecture and to create a single cilium tip compartment, where Gli-Sufu activity can be correctly regulated.

  15. Energy transduction and signal averaging of fluctuating electric fields by a single protein ion channel.

    Science.gov (United States)

    Verdia-Baguena, C; Gomez, V; Cervera, J; Ramirez, P; Mafe, S

    2016-12-21

    We demonstrate the electrical rectification and signal averaging of fluctuating signals using a biological nanostructure in aqueous solution: a single protein ion channel inserted in the lipid bilayer characteristic of cell membranes. The conversion of oscillating, zero time-average potentials into directional currents permits charging of a load capacitor to significant steady-state voltages within a few minutes in the case of the outer membrane porin F (OmpF) protein, a bacterial channel of Escherichia coli. The experiments and simulations show signal averaging effects at a more fundamental level than the traditional cell and tissue scales, which are characterized by ensembles of many ion channels operating simultaneously. The results also suggest signal transduction schemes with bio-electronic interfaces and ionic circuits where soft matter nanodiodes can be coupled to conventional electronic elements.

  16. Molecular signal networks and regulating mechanisms of the unfolded protein response.

    Science.gov (United States)

    Gong, Jing; Wang, Xing-Zhi; Wang, Tao; Chen, Jiao-Jiao; Xie, Xiao-Yuan; Hu, Hui; Yu, Fang; Liu, Hui-Lin; Jiang, Xing-Yan; Fan, Han-Dong

    Within the cell, several mechanisms exist to maintain homeostasis of the endoplasmic reticulum (ER). One of the primary mechanisms is the unfolded protein response (UPR). In this review, we primarily focus on the latest signal webs and regulation mechanisms of the UPR. The relationships among ER stress, apoptosis, and cancer are also discussed. Under the normal state, binding immunoglobulin protein (BiP) interacts with the three sensors (protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1α (IRE1α)). Under ER stress, misfolded proteins interact with BiP, resulting in the release of BiP from the sensors. Subsequently, the three sensors dimerize and autophosphorylate to promote the signal cascades of ER stress. ER stress includes a series of positive and negative feedback signals, such as those regulating the stabilization of the sensors/BiP complex, activating and inactivating the sensors by autophosphorylation and dephosphorylation, activating specific transcription factors to enable selective transcription, and augmenting the ability to refold and export. Apart from the three basic pathways, vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR)-phospholipase C-γ (PLCγ)-mammalian target of rapamycin complex 1 (mTORC1) pathway, induced only in solid tumors, can also activate ATF6 and PERK signal cascades, and IRE1α also can be activated by activated RAC-alpha serine/threonine-protein kinase (AKT). A moderate UPR functions as a pro-survival signal to return the cell to its state of homeostasis. However, persistent ER stress will induce cells to undergo apoptosis in response to increasing reactive oxygen species (ROS), Ca 2+ in the cytoplasmic matrix, and other apoptosis signal cascades, such as c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription 3 (STAT3), and P38, when cellular damage exceeds the capacity of this adaptive response.

  17. Improved Protein Arrays for Quantitative Systems Analysis of the Dynamics of Signaling Pathway Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chin-Rang [National Inst. of Health (NIH), Bethesda, MD (United States). National Heart, Lung and Blood Inst.

    2013-12-11

    Astronauts and workers in nuclear plants who repeatedly exposed to low doses of ionizing radiation (IR, <10 cGy) are likely to incur specific changes in signal transduction and gene expression in various tissues of their body. Remarkable advances in high throughput genomics and proteomics technologies enable researchers to broaden their focus from examining single gene/protein kinetics to better understanding global gene/protein expression profiling and biological pathway analyses, namely Systems Biology. An ultimate goal of systems biology is to develop dynamic mathematical models of interacting biological systems capable of simulating living systems in a computer. This Glue Grant is to complement Dr. Boothman’s existing DOE grant (No. DE-FG02-06ER64186) entitled “The IGF1/IGF-1R-MAPK-Secretory Clusterin (sCLU) Pathway: Mediator of a Low Dose IR-Inducible Bystander Effect” to develop sensitive and quantitative proteomic technology that suitable for low dose radiobiology researches. An improved version of quantitative protein array platform utilizing linear Quantum dot signaling for systematically measuring protein levels and phosphorylation states for systems biology modeling is presented. The signals are amplified by a confocal laser Quantum dot scanner resulting in ~1000-fold more sensitivity than traditional Western blots and show the good linearity that is impossible for the signals of HRP-amplification. Therefore this improved protein array technology is suitable to detect weak responses of low dose radiation. Software is developed to facilitate the quantitative readout of signaling network activities. Kinetics of EGFRvIII mutant signaling was analyzed to quantify cross-talks between EGFR and other signaling pathways.

  18. Estimation of kinetic parameters related to biochemical interactions between hydrogen peroxide and signal transduction proteins

    OpenAIRE

    Brito, Paula M.; Antunes, Fernando

    2014-01-01

    The lack of kinetic data concerning the biological effects of reactive oxygen species is slowing down the development of the field of redox signaling. Herein, we deduced and applied equations to estimate kinetic parameters from typical redox signaling experiments. H2O2-sensing mediated by the oxidation of a protein target and the switch-off of this sensor, by being converted back to its reduced form, are the two processes for which kinetic parameters are determined. The experimental data requ...

  19. Regulator of G-protein signaling 5 controls blood pressure homeostasis and vessel wall remodeling.

    Science.gov (United States)

    Holobotovskyy, Vasyl; Manzur, Mitali; Tare, Marianne; Burchell, Jennifer; Bolitho, Erin; Viola, Helena; Hool, Livia C; Arnolda, Leonard F; McKitrick, Douglas J; Ganss, Ruth

    2013-03-01

    Regulator of G-protein signaling 5 (RGS5) modulates G-protein-coupled receptor signaling and is prominently expressed in arterial smooth muscle cells. Our group first reported that RGS5 is important in vascular remodeling during tumor angiogenesis. We hypothesized that RGS5 may play an important role in vessel wall remodeling and blood pressure regulation. To demonstrate that RGS5 has a unique and nonredundant role in the pathogenesis of hypertension and to identify crucial RGS5-regulated signaling pathways. We observed that arterial RGS5 expression is downregulated with chronically elevated blood pressure after angiotensin II infusion. Using a knockout mouse model, radiotelemetry, and pharmacological inhibition, we subsequently showed that loss of RGS5 results in profound hypertension. RGS5 signaling is linked to the renin-angiotensin system and directly controls vascular resistance, vessel contractility, and remodeling. RGS5 deficiency aggravates pathophysiological features of hypertension, such as medial hypertrophy and fibrosis. Moreover, we demonstrate that protein kinase C, mitogen-activated protein kinase/extracellular signal-regulated kinase, and Rho kinase signaling pathways are major effectors of RGS5-mediated hypertension. Loss of RGS5 results in hypertension. Loss of RGS5 signaling also correlates with hyper-responsiveness to vasoconstrictors and vascular stiffening. This establishes a significant, distinct, and causal role of RGS5 in vascular homeostasis. RGS5 modulates signaling through the angiotensin II receptor 1 and major Gαq-coupled downstream pathways, including Rho kinase. So far, activation of RhoA/Rho kinase has not been associated with RGS molecules. Thus, RGS5 is a crucial regulator of blood pressure homeostasis with significant clinical implications for vascular pathologies, such as hypertension.

  20. DMPD: G-protein-coupled receptor expression, function, and signaling in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17456803 G-protein-coupled receptor expression, function, and signaling in macropha...2007 Apr 24. (.png) (.svg) (.html) (.csml) Show G-protein-coupled receptor expression, function, and signali...ng in macrophages. PubmedID 17456803 Title G-protein-coupled receptor expression, function, and sign

  1. DMPD: Pellino proteins: novel players in TLR and IL-1R signalling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17635639 Pellino proteins: novel players in TLR and IL-1R signalling. Schauvliege R..., Janssens S, Beyaert R. J Cell Mol Med. 2007 May-Jun;11(3):453-61. (.png) (.svg) (.html) (.csml) Show Pellino proteins...: novel players in TLR and IL-1R signalling. PubmedID 17635639 Title Pellino proteins: novel play

  2. Regulation of the Host Antiviral State by Intercellular Communications

    Directory of Open Access Journals (Sweden)

    Sonia Assil

    2015-08-01

    Full Text Available Viruses usually induce a profound remodeling of host cells, including the usurpation of host machinery to support their replication and production of virions to invade new cells. Nonetheless, recognition of viruses by the host often triggers innate immune signaling, preventing viral spread and modulating the function of immune cells. It conventionally occurs through production of antiviral factors and cytokines by infected cells. Virtually all viruses have evolved mechanisms to blunt such responses. Importantly, it is becoming increasingly recognized that infected cells also transmit signals to regulate innate immunity in uninfected neighboring cells. These alternative pathways are notably mediated by vesicular secretion of various virus- and host-derived products (miRNAs, RNAs, and proteins and non-infectious viral particles. In this review, we focus on these newly-described modes of cell-to-cell communications and their impact on neighboring cell functions. The reception of these signals can have anti- and pro-viral impacts, as well as more complex effects in the host such as oncogenesis and inflammation. Therefore, these “broadcasting” functions, which might be tuned by an arms race involving selective evolution driven by either the host or the virus, constitute novel and original regulations of viral infection, either highly localized or systemic.

  3. Understanding the GPCR biased signaling through G protein and arrestin complex structures.

    Science.gov (United States)

    Zhou, X Edward; Melcher, Karsten; Xu, H Eric

    2017-08-01

    G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are important drug targets for many human diseases. The determination of the 3-D structure of GPCRs and their signaling complexes has promoted our understanding of GPCR biology and provided templates for structure-based drug discovery. In this review, we focus on the recent structure work on GPCR signaling complexes, the β2-adrenoreceptor-Gs and the rhodopsin-arrestin complexes in particular, and highlight the structural features of GPCR complexes involved in G protein- and arrestin-mediated signal transduction. The crystal structures reveal distinct structural mechanisms by which GPCRs recruit a G protein and an arrestin. A comparison of the two complex structures provides insight into the molecular mechanism of functionally selective GPCR signaling, and a structural basis for the discovery of G protein- and arrestin-biased treatments of human diseases related to GPCR signal transduction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer.

    Science.gov (United States)

    Thompson, Joshua J; Williams, Christopher S

    2018-02-26

    Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a heterotrimeric serine-threonine phosphatase-composed of a structural, regulatory, and catalytic subunit-that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components-especially the multitude of regulatory subunits-has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis.

  5. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer

    Directory of Open Access Journals (Sweden)

    Joshua J. Thompson

    2018-02-01

    Full Text Available Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A is a heterotrimeric serine-threonine phosphatase—composed of a structural, regulatory, and catalytic subunit—that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components—especially the multitude of regulatory subunits—has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis.

  6. Jatropha curcas Protein Concentrate Stimulates Insulin Signaling, Lipogenesis, Protein Synthesis and the PKCα Pathway in Rat Liver.

    Science.gov (United States)

    León-López, Liliana; Márquez-Mota, Claudia C; Velázquez-Villegas, Laura A; Gálvez-Mariscal, Amanda; Arrieta-Báez, Daniel; Dávila-Ortiz, Gloria; Tovar, Armando R; Torres, Nimbe

    2015-09-01

    Jatropha curcas is an oil seed plant that belongs to the Euphorbiaceae family. Nontoxic genotypes have been reported in Mexico. The purpose of the present work was to evaluate the effect of a Mexican variety of J. curcas protein concentrate (JCP) on weight gain, biochemical parameters, and the expression of genes and proteins involved in insulin signaling, lipogenesis, cholesterol and protein synthesis in rats. The results demonstrated that short-term consumption of JCP increased serum glucose, insulin, triglycerides and cholesterol levels as well as the expression of transcription factors involved in lipogenesis and cholesterol synthesis (SREBP-1 and LXRα). Moreover, there was an increase in insulin signaling mediated by Akt phosphorylation and mTOR. JCP also increased PKCα protein abundance and the activation of downstream signaling pathway targets such as the AP1 and NF-κB transcription factors typically activated by phorbol esters. These results suggested that phorbol esters are present in JCP, and that they could be involved in the activation of PKC which may be responsible for the high insulin secretion and consequently the activation of insulin-dependent pathways. Our data suggest that this Mexican Jatropha variety contains toxic compounds that produce negative metabolic effects which require caution when using in the applications of Jatropha-based products in medicine and nutrition.

  7. G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

    Science.gov (United States)

    Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

    2015-03-13

    Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

    Directory of Open Access Journals (Sweden)

    Michael P Grant

    Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

  9. Neuronal RING finger protein 11 (RNF11 regulates canonical NF-κB signaling

    Directory of Open Access Journals (Sweden)

    Pranski Elaine L

    2012-04-01

    Full Text Available Abstract Background The RING domain-containing protein RING finger protein 11 (RNF11 is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases. Methods and results Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11’s association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and

  10. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  11. Hormone response element binding proteins: novel regulators of vitamin D and estrogen signaling.

    Science.gov (United States)

    Lisse, Thomas S; Hewison, Martin; Adams, John S

    2011-03-01

    Insights from vitamin D-resistant New World primates and their human homologues as models of natural and pathological insensitivity to sterol/steroid action have uncovered a family of novel intracellular vitamin D and estrogen regulatory proteins involved in hormone action. The proteins, known as "vitamin D or estrogen response element-binding proteins", behave as potent cis-acting, transdominant regulators to inhibit steroid receptor binding to DNA response elements and is responsible for vitamin D and estrogen resistances. This set of interactors belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family of previously known pre-mRNA-interacting proteins. This review provides new insights into the mechanism by which these novel regulators of signaling and metabolism can act to regulate responses to vitamin D and estrogen. In addition the review also describes other molecules that are known to influence nuclear receptor signaling through interaction with hormone response elements. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Perturbation waves in proteins and protein networks: applications of percolation and game theories in signaling and drug design.

    Science.gov (United States)

    Antal, Miklós A; Böde, Csaba; Csermely, Peter

    2009-04-01

    The network paradigm is increasingly used to describe the dynamics of complex systems. Here we review the current results and propose future development areas in the assessment of perturbation waves, i.e. propagating structural changes in amino acid networks building individual protein molecules and in protein-protein interaction networks (interactomes). We assess the possibilities and critically review the initial attempts for the application of game theory to the often rather complicated process, when two protein molecules approach each other, mutually adjust their conformations via multiple communication steps and finally, bind to each other. We also summarize available data on the application of percolation theory for the prediction of amino acid network- and interactome-dynamics. Furthermore, we give an overview of the dissection of signals and noise in the cellular context of various perturbations. Finally, we propose possible applications of the reviewed methodologies in drug design.

  13. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling

    DEFF Research Database (Denmark)

    Blagoev, B.; Kratchmarova, I.; Ong, S.E.

    2003-01-01

    Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we em...

  14. Bone morphogenetic protein 2 signaling negatively modulates lymphatic development in vertebrate embryos

    DEFF Research Database (Denmark)

    Dunworth, William P; Cardona-Costa, Jose; Bozkulak, Esra Cagavi

    2014-01-01

    : Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. METHODS AND RESULTS: BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2......RATIONALE: The emergence of lymphatic endothelial cells (LECs) seems to be highly regulated during development. Although several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown. OBJECTIVE...... signaling in zebrafish embryos and mouse embryonic stem cell-derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox...

  15. Inflammatory signalling regulates eccentric contraction-induced protein synthesis in cachectic skeletal muscle.

    Science.gov (United States)

    Hardee, Justin P; Counts, Brittany R; Gao, Song; VanderVeen, Brandon N; Fix, Dennis K; Koh, Ho-Jin; Carson, James A

    2017-12-07

    Skeletal muscle responds to eccentric contractions (ECC) with an anabolic response that involves the induction of protein synthesis through the mechanistic target of rapamycin complex 1. While we have reported that repeated ECC bouts after cachexia initiation attenuated muscle mass loss and inflammatory signalling, cachectic muscle's capacity to induce protein synthesis in response to ECC has not been determined. Therefore, we examined cachectic muscle's ability to induce mechano-sensitive pathways and protein synthesis in response to an anabolic stimulus involving ECC and determined the role of muscle signal transducer and activator of transcription 3 (STAT3)/nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signalling on ECC-induced anabolic signalling. Mechano-sensitive pathways and anabolic signalling were examined immediately post or 3 h after a single ECC bout in cachectic male Apc Min/+ mice (n = 17; 16 ± 1% body weight loss). Muscle STAT3/NFκB regulation of basal and ECC-induced anabolic signalling was also examined in an additional cohort of Apc Min/+ mice (n = 10; 16 ± 1% body weight loss) that received pyrrolidine dithiocarbamate 24 h prior to a single ECC bout. In all experiments, the left tibialis anterior performed ECC while the right tibialis anterior served as intra-animal control. Data were analysed by Student's t-test or two-way repeated measures analysis of variance with Student-Newman-Keuls post-hoc when appropriate. The accepted level of significance was set at P < 0.05 for all analysis. Apc Min/+ mice exhibited a cachectic muscle signature demonstrated by perturbed proteostasis (Ribosomal Protein S6 (RPS6), P70S6K, Atrogin-1, and Muscle RING-finger protein-1 (MuRF1)), metabolic (adenosine monophosphate-activated protein kinase, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and Cytochrome c oxidase subunit IV (COXIV)), and inflammatory (STAT3, NFκB, extracellular signal

  16. Displacement and hybridization reactions in aptamer-functionalized hydrogels for biomimetic protein release and signal transduction.

    Science.gov (United States)

    Lai, Jinping; Li, Shihui; Shi, Xuechen; Coyne, James; Zhao, Nan; Dong, Fengping; Mao, Yingwei; Wang, Yong

    2017-11-01

    A variety of hydrogels have been synthesized for controlling the release of signaling molecules in applications such as drug delivery and regenerative medicine. However, it remains challenging to synthesize hydrogels with the ability to control the release of signaling molecules sequentially or periodically under physiological conditions as living cells do in response to the variation of metabolism. The purpose of this work was to study a novel biomimetic hydrogel system with the ability of recapitulating the procedure of cellular signal transduction and controlling the sequential release of signaling molecules under physiological conditions. In the presence of a small chemical, the signaling molecule is regulated to change from a DNA-bound state to a free state and the freed signaling molecule is able to regulate intracellular signal transduction and cell migration. Moreover, periodic exposure of the hydrogel system to the small chemical leads to sequential protein release. Since signaling molecules are important for every activity of the cell, this hydrogel system holds potential as a metabolism-responsive platform for controlled release of signaling molecules and cell regulation in various applications.

  17. A synthetic peptide derived from the animo acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. Carlson; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractSynthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP'2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific

  18. Wnt Signaling Translocates Lys48-Linked Polyubiquitinated Proteins to the Lysosomal Pathway

    Directory of Open Access Journals (Sweden)

    Hyunjoon Kim

    2015-05-01

    Full Text Available Cellular proteins are degraded in either proteasomes or lysosomes depending on the types of ubiquitin chains that covalently modify them. It is not known whether the choice between these two pathways is physiologically regulated. The Lys48-polyubiquitin chain is the major signal directing proteins for degradation in proteasomes. Here, we report the unexpected finding that canonical Wnt signaling translocates some K48-linked polyubiquitinated proteins to the endolysosomal pathway. Proteasomal target proteins, such as β-catenin, Smad1, and Smad4, were targeted into endolysosomes in a process dependent on GSK3 activity. Relocalization was also dependent on Axin1 and the multivesicular body (MVB proteins HRS/Vps27 and Vps4. The Wnt-induced accumulation of K48-linked polyubiquitinated proteins in endolysosomal organelles was accompanied by a transient decrease in cellular levels of free mono-ubiquitin, which may contribute to Wnt-regulated stabilization of proteins (Wnt/STOP. We conclude that Wnt redirects Lys48-polyubiquitinated proteins that are normally degraded in proteasomes to endolysosomes.

  19. Posttranslational protein S-palmitoylation and the compartmentalization of signaling molecules in neurons

    Directory of Open Access Journals (Sweden)

    SEAN I PATTERSON

    2002-01-01

    Full Text Available Protein domains play a fundamental role in the spatial and temporal organization of intracellular signaling systems. While protein phosphorylation has long been known to modify the interactions that underlie this organization, the dynamic cycling of lipids should now be included amongst the posttranslational processes determining specificity in signal transduction. The characteristics of this process are reminiscent of the properties of protein and lipid phosphorylation in determining compartmentalization through SH2 or PH domains. Recent studies have confirmed the functional importance of protein S-palmitoylation in the compartmentalization of signaling molecules that support normal physiological function in cell division and apoptosis, and synaptic transmission and neurite outgrowth. In neurons, S-palmitoylation and targeting of proteins to rafts are regulated differentially in development by a number of processes, including some related to synaptogenesis and synaptic plasticity. Alterations in the S-palmitoylation state of proteins substantially affect their cellular function, raising the possibility of new therapeutic targets in cancer and nervous system injury and disease.

  20. Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

    Science.gov (United States)

    Cui, Yanbing; Meng, Yiwei; Zhang, Juan; Cheng, Bin; Yin, Huijia; Gao, Chao; Xu, Ping; Yang, Chunyu

    2017-01-01

    In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. The Battle between Rotavirus and Its Host for Control of the Interferon Signaling Pathway

    Science.gov (United States)

    Arnold, Michelle M.; Sen, Adrish; Greenberg, Harry B.; Patton, John T.

    2013-01-01

    Viral pathogens must overcome innate antiviral responses to replicate successfully in the host organism. Some of the mechanisms viruses use to interfere with antiviral responses in the infected cell include preventing detection of viral components, perturbing the function of transcription factors that initiate antiviral responses, and inhibiting downstream signal transduction. RNA viruses with small genomes and limited coding space often express multifunctional proteins that modulate several aspects of the normal host response to infection. One such virus, rotavirus, is an important pediatric pathogen that causes severe gastroenteritis, leading to ∼450,000 deaths globally each year. In this review, we discuss the nature of the innate antiviral responses triggered by rotavirus infection and the viral mechanisms for inhibiting these responses. PMID:23359266

  2. Antiviral immunity in amphibians.

    Science.gov (United States)

    Chen, Guangchun; Robert, Jacques

    2011-11-01

    Although a variety of virus species can infect amphibians, diseases caused by ranaviruses ([RVs]; Iridoviridae) have become prominent, and are a major concern for biodiversity, agriculture and international trade. The relatively recent and rapid increase in prevalence of RV infections, the wide range of host species infected by RVs, the variability in host resistance among population of the same species and among different developmental stages, all suggest an important involvement of the amphibian immune system. Nevertheless, the roles of the immune system in the etiology of viral diseases in amphibians are still poorly investigated. We review here the current knowledge of antiviral immunity in amphibians, focusing on model species such as the frog Xenopus and the salamander (Ambystoma tigrinum), and on recent progress in generating tools to better understand how host immune defenses control RV infections, pathogenicity, and transmission.

  3. Aciclovir: nuevo antiviral

    Directory of Open Access Journals (Sweden)

    G. Repetto

    2017-05-01

    Full Text Available El aciclovir es un antiviral útil en infecciones graves causadas por el virus varicela-zoster. Es bien tolerado con escasas reacciones adversas. En pacientes deshidratados, en insuficiencia renal o si la infusión endovenosa es muy rápida, puede ocacionar una "nefropatía obstructiva" transitoria. Existen preparados de uso tópico, oftálmico, endovenoso y oral; esta última vía constituye una ventaja sobre la vidarabina con la que tiene en común el espectro de actividad. En razón de su selectividad, riesgo de resistencia y número reducido de antivirales, su prescripción debe restringirse a infecciones graves causadas por los agentes inmunodeprimidos; excluyendo por lo tanto las comunes y autolimitadas, frecuentes en el individuo normal.

  4. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis*

    Science.gov (United States)

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E.; Trimble, William S.; Foster, Leonard J.; Finlay, B. Brett

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  5. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    Science.gov (United States)

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  6. Interactions between Trypanosoma cruzi secreted proteins and host cell signaling pathways

    Directory of Open Access Journals (Sweden)

    Renata Watanabe Costa

    2016-03-01

    Full Text Available Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6-7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion.

  7. Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

    DEFF Research Database (Denmark)

    Lund, I K; Hansen, J A; Andersen, H S

    2005-01-01

    Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators of this sign...

  8. Spatiotemporal aspects of G protein signaling : Where GPCRs and Rho GTPases meet

    NARCIS (Netherlands)

    van Unen, J.

    2017-01-01

    The experimental work conducted for this thesis is aimed towards a better understanding of the fundamental aspects of G protein signaling at the plasma membrane and beyond. The use of advanced microscopy techniques in living cells allows the collection of quantitative information on reaction

  9. Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation

    Czech Academy of Sciences Publication Activity Database

    Liu, Q.; Vain, T.; Viotti, C.; Doyle, S. M.; Tarkowská, Danuše; Novák, Ondřej; Zipfel, C.; Sitbon, F.; Robert, S.; Hofius, D.

    2018-01-01

    Roč. 11, č. 4 (2018), s. 553-567 ISSN 1674-2052 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis * brassinosteroid signaling * DUF300 proteins * tonoplast * vacuole integrity Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 8.827, year: 2016

  10. Spatial Organization in Protein Kinase A Signaling Emerged at the Base of Animal Evolution

    NARCIS (Netherlands)

    Peng, Mao; Aye, Thin Thin; Snel, Berend; Van Breukelen, Bas; Scholten, Arjen; Heck, Albert J R

    2015-01-01

    In phosphorylation-directed signaling, spatial and temporal control is organized by complex interaction networks that diligently direct kinases toward distinct substrates to fine-tune specificity. How these protein networks originate and evolve into complex regulatory machineries are among the most

  11. Canonical signal recognition particle components can be bypassed for posttranslational protein targeting in chloroplasts

    NARCIS (Netherlands)

    Tzvetkova-Chevolleau, Tzvetelina; Hutin, Claire; Noël, Laurent D; Goforth, Robyn; Carde, Jean-Pierre; Caffarri, Stephano; Sinning, Irmgard; Groves, Matthew; Teulon, Jean-Marie; Hoffman, Neil E; Henry, Ralph; Havaux, Michel; Nussaume, Laurent

    The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) target proteins both cotranslationally and posttranslationally to the thylakoids. This dual function enables cpSRP to utilize its posttranslational activities for targeting a family of nucleus-encoded light-harvesting

  12. Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins

    NARCIS (Netherlands)

    Welsh, M.; Songyang, Z.; Frantz, J. D.; Trüb, T.; Reedquist, K. A.; Karlsson, T.; Miyazaki, M.; Cantley, L. C.; Band, H.; Shoelson, S. E.

    1998-01-01

    Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of

  13. Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

    Science.gov (United States)

    Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

    2018-02-15

    The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Enhanced endogenous bone morphogenetic protein signaling protects against bleomycin induced pulmonary fibrosis.

    Science.gov (United States)

    De Langhe, Ellen; Cailotto, Frederic; De Vooght, Vanessa; Aznar-Lopez, Carolina; Vanoirbeek, Jeroen Alfons; Luyten, Frank Prosper; Lories, Rik Jozef Urbain

    2015-03-15

    Effective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFβ) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFβ superfamily of growth factors, modulate fibrogenesis driven by TGFβ. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis. Lung fibrosis was induced in wild-type or noggin haploinsufficient (Nog +/LacZ ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis. Nog +/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFβ and BMP signaling followed an inverse course, with dynamic activation of TGFβ signaling and repression of BMP signaling activity. Upon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog +/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFβ, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.

  15. Insights into biological information processing: structural and dynamical analysis of a human protein signalling network

    Energy Technology Data Exchange (ETDEWEB)

    Fuente, Alberto de la; Fotia, Giorgio; Maggio, Fabio; Mancosu, Gianmaria; Pieroni, Enrico [CRS4 Bioinformatica, Parco Tecnologico POLARIS, Ed.1, Loc Piscinamanna, Pula (Italy)], E-mail: alf@crs4.it

    2008-06-06

    We present an investigation on the structural and dynamical properties of a 'human protein signalling network' (HPSN). This biological network is composed of nodes that correspond to proteins and directed edges that represent signal flows. In order to gain insight into the organization of cell information processing this network is analysed taking into account explicitly the edge directions. We explore the topological properties of the HPSN at the global and the local scale, further applying the generating function formalism to provide a suitable comparative model. The relationship between the node degrees and the distribution of signals through the network is characterized using degree correlation profiles. Finally, we analyse the dynamical properties of small sub-graphs showing high correlation between their occurrence and dynamic stability.

  16. FGF signal interpretation is directed by Sprouty and Spred proteins during mesoderm formation.

    Science.gov (United States)

    Sivak, Jeremy M; Petersen, Lars F; Amaya, Enrique

    2005-05-01

    Vertebrate gastrulation requires coordination of mesoderm specification with morphogenetic movements. While both of these processes require FGF signaling, it is not known how mesoderm specification and cell movements are coordinated during gastrulation. The related Sprouty and Spred protein families are recently discovered regulators of receptor tyrosine kinase signaling. We identified two genes for each family in Xenopus tropicalis: Xtsprouty1, Xtsprouty2, Xtspred1, and Xtspred2. In gain- and loss-of-function experiments we show that XtSprouty and XtSpred proteins modulate different signaling pathways downstream of the FGF receptor (FGFR), and consequently different developmental processes. Notably, XtSproutys inhibit morphogenesis and Ca(2+) and PKCdelta signaling, leaving MAPK activation and mesoderm specification intact. In contrast, XtSpreds inhibit MAPK activation and mesoderm specification, with little effect on Ca(2+) or PKCdelta signaling. These differences, combined with the timing of their developmental expression, suggest a mechanism to switch FGFR signal interpretation to coordinate mesoderm formation and cell movements during gastrulation.

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

    Science.gov (United States)

    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.

  18. Increased signaling entropy in cancer requires the scale-free property of protein interaction networks

    Science.gov (United States)

    Teschendorff, Andrew E.; Banerji, Christopher R. S.; Severini, Simone; Kuehn, Reimer; Sollich, Peter

    2015-01-01

    One of the key characteristics of cancer cells is an increased phenotypic plasticity, driven by underlying genetic and epigenetic perturbations. However, at a systems-level it is unclear how these perturbations give rise to the observed increased plasticity. Elucidating such systems-level principles is key for an improved understanding of cancer. Recently, it has been shown that signaling entropy, an overall measure of signaling pathway promiscuity, and computable from integrating a sample's gene expression profile with a protein interaction network, correlates with phenotypic plasticity and is increased in cancer compared to normal tissue. Here we develop a computational framework for studying the effects of network perturbations on signaling entropy. We demonstrate that the increased signaling entropy of cancer is driven by two factors: (i) the scale-free (or near scale-free) topology of the interaction network, and (ii) a subtle positive correlation between differential gene expression and node connectivity. Indeed, we show that if protein interaction networks were random graphs, described by Poisson degree distributions, that cancer would generally not exhibit an increased signaling entropy. In summary, this work exposes a deep connection between cancer, signaling entropy and interaction network topology. PMID:25919796

  19. Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states.

    Science.gov (United States)

    LeBon, Lauren; Lee, Tom V; Sprinzak, David; Jafar-Nejad, Hamed; Elowitz, Michael B

    2014-09-25

    The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation.

  20. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors...

  1. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors...

  2. A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu–Gli protein complexes

    OpenAIRE

    Tukachinsky, Hanna; Lopez, Lyle V.; Salic, Adrian

    2010-01-01

    In vertebrates, Hedgehog (Hh) signaling initiated in primary cilia activates the membrane protein Smoothened (Smo) and leads to activation of Gli proteins, the transcriptional effectors of the pathway. In the absence of signaling, Gli proteins are inhibited by the cytoplasmic protein Suppressor of Fused (SuFu). It is unclear how Hh activates Gli and whether it directly regulates SuFu. We find that Hh stimulation quickly recruits endogenous SuFu–Gli complexes to cilia, suggesting a model in wh...

  3. Practical aspects of NMR signal assignment in larger and challenging proteins

    Science.gov (United States)

    Frueh, Dominique P.

    2014-01-01

    NMR has matured into a technique routinely employed for studying proteins in near physiological conditions. However, applications to larger proteins are impeded by the complexity of the various correlation maps necessary to assign NMR signals. This article reviews the data analysis techniques traditionally employed for resonance assignment and describes alternative protocols necessary for overcoming challenges in large protein spectra. In particular, simultaneous analysis of multiple spectra may help overcome ambiguities or may reveal correlations in an indirect manner. Similarly, visualization of orthogonal planes in a multidimensional spectrum can provide alternative assignment procedures. We describe examples of such strategies for assignment of backbone, methyl, and nOe resonances. We describe experimental aspects of data acquisition for the related experiments and provide guidelines for preliminary studies. Focus is placed on large folded monomeric proteins and examples are provided for 37, 48, 53, and 81 kDa proteins. PMID:24534088

  4. Hepatitis C Virus Core Protein Modulates Endoglin (CD105) Signaling Pathway for Liver Pathogenesis.

    Science.gov (United States)

    Kwon, Young-Chan; Sasaki, Reina; Meyer, Keith; Ray, Ranjit

    2017-11-01

    Endoglin is part of the TGF-β receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In a previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition (EMT) state and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV-associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of an HCV core-expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV-infected cells or viral core gene-transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase 1 (ALK1) Fc blocking peptide and endoglin small interfering RNA (siRNA). Further, follow-up from in vitro analysis suggested that the antiapoptosis Bcl2 protein, proliferation-related cyclin D1 protein, and CSC-associated Hes1, Notch1, Nanog, and Sox2 proteins are enhanced during infection or ectopic expression of HCV core protein. IMPORTANCE Endoglin plays a crucial role in fibrogenesis and angiogenesis and is an important protein for tumor growth, survival, and cancer cell metastasis. Endoglin enhances ALK1-SMAD1/5 signaling in different cell types, leading to increased proliferation and migration responses. We have observed endoglin expression on the HCV core-expressing cell surface of human hepatocyte origin and activation of phospho-SMAD1/5 and ID1 downstream signaling molecules. ID1 protein plays a role in CSC properties, and we found that

  5. Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Heenan, Erin J.; Vanhooke, Janeen L.; Temple, Brenda R.; Betts, Laurie; Sondek, John E.; Dohlman, Henrik G.; (UNC)

    2009-09-11

    G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light, and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein {alpha} subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein {beta} subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical G{beta} subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential G{gamma} protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 G{beta}-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.

  6. Mitogen-activated protein kinase signaling pathways of the tangerine pathotype of Alternaria alternata

    Directory of Open Access Journals (Sweden)

    Kuang-Ren Chung

    2013-06-01

    Full Text Available Mitogen-activated protein kinase (MAPK- mediated signaling pathways have been known to have important functions in eukaryotic organisms. The mechanisms by which the filamentous fungus Alternaria alternata senses and responds to environmental signals have begun to be elucidated. Available data indicate that A. alternata utilizes the Fus3, Hog1 and Slt2 MAPK-mediated signaling pathways, either separately or in a cooperative manner, for conidia formation, resistance to oxidative and osmotic stress, and pathogenesis to citrus. This review provides an overview of our current knowledge of MAPK signaling pathways, in conjunction with the two-component histidine kinase and the Skn7 response regulator, in the tangerine pathotype of A. alternata.

  7. TUNABLE TENSOR VOTING FOR REGULARIZING PUNCTATE PATTERNS OFMEMBRANE-BOUND PROTEIN SIGNALS

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro; Bebis, George; Parvin, Bahram

    2009-04-29

    Membrane-bound protein, expressed in the basal-lateral region, is heterogeneous and an important endpoint for understanding biological processes. At the optical resolution, membrane-bound protein can be visualized as being diffused (e.g., E-cadherin), punctate (e.g., connexin), or simultaneously diffused and punctate as a result of sample preparation or conditioning. Furthermore, there is a significant amount of heterogeneity as a result of technical and biological variations. This paper aims at enhancing membrane-bound proteins that are expressed between epithelial cells so that quantitative analysis can be enabled on a cell-by-cell basis. We propose a method to detect and enhance membrane-bound protein signal from noisy images. More precisely, we build upon the tensor voting framework in order to produce an efficient method to detect and refine perceptually interesting linear structures in images. The novelty of the proposed method is in its iterative tuning of the tensor voting fields, which allows the concentration of the votes only over areas of interest. The method is shown to produce high quality enhancements of membrane-bound protein signals with combined punctate and diffused characteristics. Experimental results demonstrate the benefits of using tunable tensor voting for enhancing and differentiating cell-cell adhesion mediated by integral cell membrane protein.

  8. cAMP-dependent Protein Kinase (PKA) Signaling Is Impaired in the Diabetic Heart.

    Science.gov (United States)

    Bockus, Lee B; Humphries, Kenneth M

    2015-12-04

    Diabetes mellitus causes cardiac dysfunction and heart failure that is associated with metabolic abnormalities and autonomic impairment. Autonomic control of ventricular function occurs through regulation of cAMP-dependent protein kinase (PKA). The diabetic heart has suppressed β-adrenergic responsiveness, partly attributable to receptor changes, yet little is known about how PKA signaling is directly affected. Control and streptozotocin-induced diabetic mice were therefore administered 8-bromo-cAMP (8Br-cAMP) acutely to activate PKA in a receptor-independent manner, and cardiac hemodynamic function and PKA signaling were evaluated. In response to 8Br-cAMP treatment, diabetic mice had impaired inotropic and lusitropic responses, thus demonstrating postreceptor defects. This impaired signaling was mediated by reduced PKA activity and PKA catalytic subunit content in the cytoplasm and myofilaments. Compartment-specific loss of PKA was reflected by reduced phosphorylation of discrete substrates. In response to 8Br-cAMP treatment, the glycolytic activator PFK-2 was robustly phosphorylated in control animals but not diabetics. Control adult cardiomyocytes cultured in lipid-supplemented media developed similar changes in PKA signaling, suggesting that lipotoxicity is a contributor to diabetes-induced β-adrenergic signaling dysfunction. This work demonstrates that PKA signaling is impaired in diabetes and suggests that treating hyperlipidemia is vital for proper cardiac signaling and function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. cAMP-dependent Protein Kinase (PKA) Signaling Is Impaired in the Diabetic Heart*

    Science.gov (United States)

    Bockus, Lee B.; Humphries, Kenneth M.

    2015-01-01

    Diabetes mellitus causes cardiac dysfunction and heart failure that is associated with metabolic abnormalities and autonomic impairment. Autonomic control of ventricular function occurs through regulation of cAMP-dependent protein kinase (PKA). The diabetic heart has suppressed β-adrenergic responsiveness, partly attributable to receptor changes, yet little is known about how PKA signaling is directly affected. Control and streptozotocin-induced diabetic mice were therefore administered 8-bromo-cAMP (8Br-cAMP) acutely to activate PKA in a receptor-independent manner, and cardiac hemodynamic function and PKA signaling were evaluated. In response to 8Br-cAMP treatment, diabetic mice had impaired inotropic and lusitropic responses, thus demonstrating postreceptor defects. This impaired signaling was mediated by reduced PKA activity and PKA catalytic subunit content in the cytoplasm and myofilaments. Compartment-specific loss of PKA was reflected by reduced phosphorylation of discrete substrates. In response to 8Br-cAMP treatment, the glycolytic activator PFK-2 was robustly phosphorylated in control animals but not diabetics. Control adult cardiomyocytes cultured in lipid-supplemented media developed similar changes in PKA signaling, suggesting that lipotoxicity is a contributor to diabetes-induced β-adrenergic signaling dysfunction. This work demonstrates that PKA signaling is impaired in diabetes and suggests that treating hyperlipidemia is vital for proper cardiac signaling and function. PMID:26468277

  10. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Science.gov (United States)

    Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

    2011-01-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  11. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Directory of Open Access Journals (Sweden)

    Andrea Cerutti

    Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS, but no nuclear export signal (NES has yet been identified.We show here that the aa(109-133 region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126 in the identified NES or in the sequence encoding the mature core aa(1-173 significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  12. Effects of protein-energy malnutrition on NF-kappaB signalling in murine peritoneal macrophages.

    Science.gov (United States)

    Fock, Ricardo Ambrósio; Rogero, Marcelo Macedo; Vinolo, Marco Aurélio Ramirez; Curi, Rui; Borges, Maria Carolina; Borelli, Primavera

    2010-04-01

    Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-kappaB is kept from binding to its consensus sequence by the inhibitor I kappaB alpha, which retains NF-kappaB in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), I kappaB alpha is rapidly degraded and NF-kappaB is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-alpha. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-kappaB. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-alpha by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, I kappaB alpha and NF-kappaB, NF-kappaB activation and TNF-alpha mRNA and protein synthesis in macrophages. Two-month-old male BALB/C mice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-alpha mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-kappaB activation after LPS stimulation. These results led us to conclude that PEM changes NF-kB signalling pathway in macrophages to LPS stimulus.

  13. ANTIVIRAL EFFECTS OF PLASMA AND MILK-PROTEINS - LACTOFERRIN SHOWS POTENT ACTIVITY AGAINST BOTH HUMAN-IMMUNODEFICIENCY-VIRUS AND HUMAN CYTOMEGALOVIRUS REPLICATION IN-VITRO

    NARCIS (Netherlands)

    HARMSEN, MC; SWART, PJ; DEBETHUNE, MP; PAUWELS, R; DECLERCQ, E; THE, TH; MEIJER, DKF

    Native and chemically derivatized proteins purified from serum and milk were assayed in vitro to assess their inhibiting capacity on the cytopathic effect of human immunodeficiency virus (HIV)-1 and human cytomegalovirus (HCMV) on MT4 cells and fibroblasts, respectively. Only native and

  14. Novel fused tetrathiocines as antivirals that target the nucleocapsid zinc finger containing protein of the feline immunodeficiency virus (FIV) as a model of HIV infection.

    Science.gov (United States)

    Asquith, Christopher R M; Meli, Marina L; Konstantinova, Lidia S; Laitinen, Tuomo; Poso, Antti; Rakitin, Oleg A; Hofmann-Lehmann, Regina; Allenspach, Karin; Hilton, Stephen T

    2015-03-15

    A novel series of fused tetrathiocines were prepared for evaluation of activity against the nucleocapsid protein of the feline immunodeficiency virus (FIV) in an in vitro cell culture approach. The results demonstrated that the compounds display potent nanomolar activity and low toxicity against this key model of HIV infection. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  15. Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, Debra J.; Morse, Robert; Todd, Adrian G. [Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom); Eggleton, Paul [Inflammation and Musculoskeletal Disease, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom); MRC Immunochemistry Unit, University of Oxford, Oxford OX1 3QU (United Kingdom); Lorson, Christian L. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Young, Philip J., E-mail: philip.young@pms.ac.uk [Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom)

    2009-12-25

    The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.

  16. The effect of exercise-intensity on skeletal muscle stress kinase and insulin protein signaling.

    Directory of Open Access Journals (Sweden)

    Lewan Parker

    Full Text Available Stress and mitogen activated protein kinase (SAPK signaling play an important role in glucose homeostasis and the physiological adaptation to exercise. However, the effects of acute high-intensity interval exercise (HIIE and sprint interval exercise (SIE on activation of these signaling pathways are unclear.Eight young and recreationally active adults performed a single cycling session of HIIE (5 x 4 minutes at 75% Wmax, SIE (4 x 30 second Wingate sprints, and continuous moderate-intensity exercise work-matched to HIIE (CMIE; 30 minutes at 50% of Wmax, separated by a minimum of 1 week. Skeletal muscle SAPK and insulin protein signaling were measured immediately, and 3 hours after exercise.SIE elicited greater skeletal muscle NF-κB p65 phosphorylation immediately after exercise (SIE: ~40%; HIIE: ~4%; CMIE; ~13%; p 0.05, remained lower 3 hours after HIIE (~-34%; p < 0.05, and decreased 3 hours after CMIE (~-33%; p < 0.05.Despite consisting of less total work than CMIE and HIIE, SIE proved to be an effective stimulus for the activation of stress protein kinase signaling pathways linked to exercise-mediated adaptation of skeletal muscle. Furthermore, post-exercise AS160Ser588 phosphorylation decreased in an exercise-intensity and post-exercise time-course dependent manner.

  17. Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

    International Nuclear Information System (INIS)

    Shaw, Debra J.; Morse, Robert; Todd, Adrian G.; Eggleton, Paul; Lorson, Christian L.; Young, Philip J.

    2009-01-01

    The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.

  18. Effect of resistance exercise contraction mode and protein supplementation on members of the STARS signalling pathway.

    Science.gov (United States)

    Vissing, Kristian; Rahbek, Stine K; Lamon, Severine; Farup, Jean; Stefanetti, Renae J; Wallace, Marita A; Vendelbo, Mikkel H; Russell, Aaron

    2013-08-01

    The striated muscle activator of Rho signalling (STARS) pathway is suggested to provide a link between external stress responses and transcriptional regulation in muscle. However, the sensitivity of STARS signalling to different mechanical stresses has not been investigated. In a comparative study, we examined the regulation of the STARS signalling pathway in response to unilateral resistance exercise performed as either eccentric (ECC) or concentric (CONC) contractions as well as prolonged training; with and without whey protein supplementation. Skeletal muscle STARS, myocardian-related transcription factor-A (MRTF-A) and serum response factor (SRF) mRNA and protein, as well as muscle cross-sectional area and maximal voluntary contraction, were measured. A single-bout of exercise produced increases in STARS and SRF mRNA and decreases in MRTF-A mRNA with both ECC and CONC exercise, but with an enhanced response occurring following ECC exercise. A 31% increase in STARS protein was observed exclusively after CONC exercise (P STARS pathway that is contraction mode dependent. The responses to acute exercise were more pronounced than responses to accumulated training, suggesting that STARS signalling is primarily involved in the initial phase of exercise-induced muscle adaptations.

  19. Protein degradation mechanisms modulate abscisic acid signaling and responses during abiotic stress.

    Science.gov (United States)

    Jurkiewicz, Pawel; Batoko, Henri

    2018-02-01

    Abiotic stresses such as salinity, drought, high temperature or freezing can be perceived, in part, as a transient or permanent hyperosmotic stress by the plant cell. As sessile organisms, the detrimental effects of these environmental insults limit plants productivity but also their geographical distribution. Sensing and signaling events that detect the hyperosmotic (or simply osmotic) stress involve the cellular increase of active abscisic acid (ABA). The stress phytohormone ABA regulates fundamental growth and developmental processes in the plant by marshalling metabolic and gene-expression reprogramming. Among the ABA-responsive genes, some are strictly ABA-dependent in that their expression is almost undetectable in absence of elevated levels of cellular ABA, thus their physiological role may be required only transiently. In addition, ABA-dependent modulation of some of the signaling effectors can be irreversible. In this review, without any pretention to being exhaustive, we use specific examples to illustrate how mechanistically conserved eukaryotic cell proteolytic pathways affect ABA-dependent signaling. We describe how defined proteolysis mechanisms in the plant cell, including Regulated Intramembrane Proteolysis (RIP), the Ubiquitin 26S Proteasomal System (UPS), the endocytic and autophagy pathways, contribute to regulate the spatiotemporal level and activity of PP2Cs (protein phosphatases 2C), and how an intriguing ABA-induced protein, the plant Translocator protein (TSPO), is targeted for degradation. Degradation of regulatory or effector molecules modulates or desensitizes ABA-dependent signaling and reestablishes cellular homeostasis. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling.

    Science.gov (United States)

    Ota, Mitsunori; Sasaki, Hiroshi

    2008-12-01

    Regulation of organ size is important for development and tissue homeostasis. In Drosophila, Hippo signaling controls organ size by regulating the activity of a TEAD transcription factor, Scalloped, through modulation of its co-activator protein Yki. Here, we show that mouse Tead proteins regulate cell proliferation by mediating Hippo signaling. In NIH3T3 cells, cell density and Hippo signaling regulated the activity of endogenous Tead proteins by modulating nuclear localization of a Yki homolog, Yap1, and the resulting change in Tead activity altered cell proliferation. Tead2-VP16 mimicked Yap1 overexpression, including increased cell proliferation, reduced cell death, promotion of EMT, lack of cell contact inhibition and promotion of tumor formation. Growth-promoting activities of various Yap1 mutants correlated with their Tead-co-activator activities. Tead2-VP16 and Yap1 regulated largely overlapping sets of genes. However, only a few of the Tead/Yap1-regulated genes in NIH3T3 cells were affected in Tead1(-/-);Tead2(-/-) or Yap1(-/-) embryos. Most of the previously identified Yap1-regulated genes were not affected in NIH3T3 cells or mutant mice. In embryos, levels of nuclear Yap1 and Tead1 varied depending on cell type. Strong nuclear accumulation of Yap1 and Tead1 were seen in myocardium, correlating with requirements of Tead1 for proliferation. However, their distribution did not always correlate with proliferation. Taken together, mammalian Tead proteins regulate cell proliferation and contact inhibition as a transcriptional mediator of Hippo signaling, but the mechanisms by which Tead/Yap1 regulate cell proliferation differ depending on the cell type, and Tead, Yap1 and Hippo signaling may play multiple roles in mouse embryos.

  1. R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

    Directory of Open Access Journals (Sweden)

    Zhang Jian-Hua

    2007-09-01

    Full Text Available Abstract Background Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS proteins of the R7 subfamily (R7-RGS. The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP, a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. Results We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. Conclusion A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of

  2. The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity.

    Science.gov (United States)

    Wang, Dashan

    2018-02-12

    The aim of this paper is to clarify the critical role of GPCR signaling in T cell immunity. The G protein-coupled receptors (GPCRs) are the most common targets in current pharmaceutical industry, and represent the largest and most versatile family of cell surface communicating molecules. GPCRs can be activated by a diverse array of ligands including neurotransmitters, chemokines as well as sensory stimuli. Therefore, GPCRs are involved in many key cellular and physiological processes, such as sense of light, taste and smell, neurotransmission, metabolism, endocrine and exocrine secretion. In recent years, GPCRs have been found to play an important role in immune system. T cell is an important type of immune cell, which plays a central role in cell-mediated immunity. A variety of GPCRs and their signaling mediators (RGS proteins, GRKs and β-arrestin) have been found to express in T cells and involved T cell-mediated immunity. We will summarize the role of GPCR signaling and their regulatory molecules in T cell activation, homeostasis and function in this article. GPCR signaling plays an important role in T cell activation, homeostasis and function. GPCR signaling is critical in regulating T cell immunity.

  3. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling

    NARCIS (Netherlands)

    Chu, D. H.; Spits, H.; Peyron, J. F.; Rowley, R. B.; Bolen, J. B.; Weiss, A.

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs.

  4. Phase diagrams map the properties of antiviral agents directed against hepatitis B virus core assembly.

    Science.gov (United States)

    Li, Lichun; Chirapu, Srinivas Reddy; Finn, M G; Zlotnick, Adam

    2013-03-01

    Assembly effectors are small molecules that induce inappropriate virus capsid assembly to antiviral effect. To identify attributes of hepatitis B virus (HBV) assembly effectors, assembly reaction products (normal capsid, noncapsid polymer, intermediates, and free dimeric core protein) were quantified in the presence of three experimental effectors: HAP12, HAP13, and AT-130. Effectors bound stoichiometrically to capsid protein polymers, but not free protein. Thermodynamic and kinetic effects, not aberrant assembly, correlate with maximal antiviral activity.

  5. Light-load resistance exercise increases muscle protein synthesis and hypertrophy signaling in elderly men

    DEFF Research Database (Denmark)

    Agergaard, Jakob; Bülow, Jacob; Jensen, Jacob K

    2017-01-01

    INTRODUCTION: The present study investigated whether well-tolerated light-load resistance exercise (LL-RE) affects skeletal muscle fractional synthetic rate (FSR) and anabolic intracellular signaling as a way to counteract age-related loss of muscle mass. METHODS: Untrained healthy men (age: +65...... and 12g whey protein at 7 hours post-exercise; N=10) or placebo (4g maltodextrin/hour; N=10). Quadriceps muscle biopsies were taken at 0, 3, 7 and 10 hours post-exercise from both the resting and exercised leg. Myofibrillar-FSR and activity of select targets from the mTORC1-signalling cascade were...

  6. Locating proteins in the cell using TargetP, SignalP and related tools

    DEFF Research Database (Denmark)

    Emanuelsson, O.; Brunak, Søren; von Heijne, G.

    2007-01-01

    of methods to predict subcellular localization based on these sorting signals and other sequence properties. We then outline how to use a number of internet-accessible tools to arrive at a reliable subcellular localization prediction for eukaryotic and prokaryotic proteins. In particular, we provide detailed...... step-by-step instructions for the coupled use of the amino-acid sequence-based predictors TargetP, SignalP, ChloroP and TMHMM, which are all hosted at the Center for Biological Sequence Analysis, Technical University of Denmark. In addition, we describe and provide web references to other useful...

  7. Regulation and evasion of antiviral immune responses by porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Huang, Chen; Zhang, Qiong; Feng, Wen-hai

    2015-04-16

    Virus infection of mammalian cells triggers host innate immune responses to restrict viral replication and induces adaptive immunity for viral elimination. In order to survive and propagate, viruses have evolved sophisticated mechanisms to subvert host defense system by encoding proteins that target key components of the immune signaling pathways. Porcine reproductive and respiratory syndrome virus (PRRSV), a RNA virus, impairs several processes of host immune responses including interfering with interferon production and signaling, modulating cytokine expression, manipulating apoptotic responses and regulating adaptive immunity. In this review, we highlight the molecular mechanisms of how PRRSV interferes with the different steps of initial antiviral host responses to establish persistent infection in pigs. Dissection of the PRRSV-host interaction is the key in understanding PRRSV pathogenesis and will provide a basis for the rational design of vaccines. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Directory of Open Access Journals (Sweden)

    Andrea Kerekes

    Full Text Available Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  9. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Science.gov (United States)

    Kerekes, Andrea; Hoffmann, Orsolya Ivett; Iski, Gergely; Lipták, Nándor; Gócza, Elen; Kues, Wilfried A; Bősze, Zsuzsanna; Hiripi, László

    2017-01-01

    Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  10. The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ian G Woods

    2005-03-01

    Full Text Available Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.

  11. Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase.

    Science.gov (United States)

    John, M; Röhrig, H; Schmidt, J; Wieneke, U; Schell, J

    1993-01-15

    The common nodulation genes nodABC are conserved in all rhizobia and are involved in synthesis of a lipooligosaccharide signal molecule. This bacterial signal consists of a chitooligosaccharide backbone, which carries at the nonreducing end a fatty acyl chain. The modified chitooligosaccharide molecule triggers development of nodules on the roots of the leguminous host plant. To elucidate the specific role of the NodB protein in nodulation factor synthesis, we have purified recombinant NodB and determined its biochemical role by direct assays. Our data show that the NodB protein of Rhizobium meliloti deacetylates the nonreducing N-acetylglucosamine residue of chitooligosaccharides. The monosaccharide N-acetylglucosamine is not deacetylated by NodB. In the pathway of Nod factor synthesis, deacetylation at the nonreducing end of the oligosaccharide backbone may be a necessary requirement for attachment of the fatty acyl chain.

  12. Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion

    DEFF Research Database (Denmark)

    Finnie, Christine; Andersen, Birgit; Shahpiri, Azar

    2011-01-01

    to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling...... molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted...... analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms....

  13. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    Sabrina M de Munnik

    2015-03-01

    Full Text Available Human herpesviruses are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, human herpesviruses have pirated genes encoding viral G protein-coupled receptors (vGPCRs, which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies.

  14. Henipaviruses Employ a Multifaceted Approach to Evade the Antiviral Interferon Response

    Directory of Open Access Journals (Sweden)

    Megan L. Shaw

    2009-12-01

    Full Text Available Hendra and Nipah virus, which constitute the genus Henipavirus, are zoonotic paramyxoviruses that have been associated with sporadic outbreaks of severe disease and mortality in humans since their emergence in the late 1990s. Similar to other paramyxoviruses, their ability to evade the host interferon (IFN response is conferred by the P gene. The henipavirus P gene encodes four proteins; the P, V, W and C proteins, which have all been described to inhibit the antiviral response. Further studies have revealed that these proteins have overlapping but unique properties which enable the virus to block multiple signaling pathways in the IFN response. The best characterized of these is the JAK-STAT signaling pathway which is targeted by the P, V and W proteins via an interaction with the transcription factor STAT1. In addition the V and W proteins can both limit virus-induced induction of IFN but they appear to do this via distinct mechanisms that rely on unique sequences in their C-terminal domains. The ability to generate recombinant Nipah viruses now gives us the opportunity to determine the precise role for each of these proteins and address their contribution to pathogenicity. Additionally, the question of whether these multiple anti-IFN strategies are all active in the different mammalian hosts for henipaviruses, particularly the fruit bat reservoir, warrants further exploration.

  15. Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children' s of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Robbins, David J. [Department of Surgery, Molecular Oncology Program, Miller School of Medicine, University of Miami, Miami (United States); Matalon, Sadis [Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL (United States); Deshane, Jessy S. [Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL (United States); Afaq, Farrukh [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Bickers, David R. [Department of Dermatology, Columbia University Medical Center, New York (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States)

    2013-11-01

    Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions.

  16. Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

    International Nuclear Information System (INIS)

    Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C.; Ballestas, Mary E.; Elmets, Craig A.; Robbins, David J.; Matalon, Sadis; Deshane, Jessy S.; Afaq, Farrukh; Bickers, David R.; Athar, Mohammad

    2013-01-01

    Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions

  17. Rapamycin and Glucose-Target of Rapamycin (TOR) Protein Signaling in Plants*

    Science.gov (United States)

    Xiong, Yan; Sheen, Jen

    2012-01-01

    Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs. PMID:22134914

  18. Endocrinology of pregnancy in the cow: embryonic signals, placental hormones and proteins

    OpenAIRE

    Ayad, A.; Melo de Sousa, Noelita; Hornick, Jean-Luc; Touati, Kamal; Iguer-Ouada, M.; Beckers, Jean-François

    2006-01-01

    The development, the establishment and the embryonic survival at early stages of gestation are depending on an intimate dialogue between the embryo and his mother. For the embryo part, it is especially the trophoblast, or the future placenta, which plays a key role in initializing pregnancy. The placenta emits many signals of various chemical natures ( steroids, prostaglandins, peptides, proteins), some of them, e. g. the interferon tau, determine the maintenance of the corpus luteum at the b...

  19. G Protein-Linked Signaling Pathways in Bipolar and Major Depressive Disorders

    Directory of Open Access Journals (Sweden)

    Hiroaki eTomita

    2013-12-01

    Full Text Available The G-protein linked signaling system (GPLS comprises a large number of G-proteins, G protein-coupled receptors (GPCRs, GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP, phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD and bipolar disorder (BPD. This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC and anterior cingulate (ACC were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, ‘activated’ cAMP signaling activity in BPD and ‘blunted’ cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects.

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

    OpenAIRE

    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 speci...

  1. The Antiviral Effect of Baicalin on Enterovirus 71 In Vitro

    Directory of Open Access Journals (Sweden)

    Xiang Li

    2015-08-01

    Full Text Available Baicalin is a flavonoid compound extracted from Scutellaria roots that has been reported to possess antibacterial, anti-inflammatory, and antiviral activities. However, the antiviral effect of baicalin on enterovirus 71 (EV71 is still unknown. In this study, we found that baicalin showed inhibitory activity on EV71 infection and was independent of direct virucidal or prophylactic effect and inhibitory viral absorption. The expressions of EV71/3D mRNA and polymerase were significantly blocked by baicalin treatment at early stages of EV71 infection. In addition, baicalin could decrease the expressions of FasL and caspase-3, as well as inhibit the apoptosis of EV71-infected human embryonal rhabdomyosarcoma (RD cells. Altogether, these results indicate that baicalin exhibits potent antiviral effect on EV71 infection, probably through inhibiting EV71/3D polymerase expression and Fas/FasL signaling pathways.

  2. A Survey of Strategies to Modulate the Bone Morphogenetic Protein Signaling Pathway: Current and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Jonathan W. Lowery

    2016-01-01

    Full Text Available Bone morphogenetic proteins (BMPs constitute the largest subdivision of the TGF-β family of ligands and are unequivocally involved in regulating stem cell behavior. Appropriate regulation of canonical BMP signaling is critical for the development and homeostasis of numerous human organ systems, as aberrations in the BMP pathway or its regulation are increasingly associated with diverse human pathologies. In this review, we provide a wide-perspective on strategies that increase or decrease BMP signaling. We briefly outline the current FDA-approved approaches, highlight emerging next-generation technologies, and postulate prospective avenues for future investigation. We also detail how activating other pathways may indirectly modulate BMP signaling, with a particular emphasis on the relationship between the BMP and Activin/TGF-β pathways.

  3. Are suppressors of cytokine signaling proteins recently identified in atherosclerosis possible therapeutic targets?

    Science.gov (United States)

    Tang, Jingjing; Raines, Elaine W

    2005-10-01

    Atherosclerosis is a slowly progressing chronic inflammatory disease characterized by focal arterial lesions that can ultimately occlude the entire blood vessel and lead to sudden death. Lesions associated with cardiovascular events are those enriched in macrophages and other inflammatory cells. Activation of inflammatory cells within lesions induces the release of cytokines which promotes more inflammation and associated tissue damage if cytokine signaling pathways remain unregulated. Thus, pathways capable of suppressing proinflammatory cytokine signaling hold the potential to limit life-threatening cardiovascular events caused by atherogenesis. This review focuses on suppressors of cytokine signaling proteins recently identified in the atherosclerosis-prone ApoE(-/-) mouse and provides perspectives of their potential for intervention in atherosclerotic lesion progression.

  4. Protein-tyrosine phosphatase H1 controls growth hormone receptor signaling and systemic growth.

    Science.gov (United States)

    Pilecka, Iwona; Patrignani, Claudia; Pescini, Rosanna; Curchod, Marie-Laure; Perrin, Dominique; Xue, Yingzi; Yasenchak, Jason; Clark, Ann; Magnone, Maria Chiara; Zaratin, Paola; Valenzuela, David; Rommel, Christian; Hooft van Huijsduijnen, Rob

    2007-11-30

    Several protein-tyrosine phosphatases (PTPs) have been implicated in the control of growth hormone receptor (GHR) signaling, but none have been shown to affect growth in vivo. We have applied a battery of molecular and cellular approaches to test a family-wide panel of PTPs for interference with GHR signaling. Among the subset of PTPs that showed activity in multiple readouts, we selected PTP-H1/PTPN3 for further in vivo studies and found that mice lacking the PTP-H1 catalytic domain show significantly enhanced growth over their wild type littermates. In addition, PTP-H1 mutant animals had enhanced plasma and liver mRNA expression of insulin-like growth factor 1, as well as increased bone density and mineral content. These observations point to a controlling role for PTP-H1 in modulating GHR signaling and systemic growth through insulin-like growth factor 1 secretion.

  5. ANTI-VIRAL ACTIVITY OF GLYCIRRHETINIC AND GLYCIRRHIZIC ACIDS

    Directory of Open Access Journals (Sweden)

    V. V. Zarubaev

    2016-01-01

    Full Text Available Influenza is a highly contagious human disease. In the course of use of antiviral drugs drug-resistant strains of the virus are formed, resulting in reduced efficiency of the chemotherapy. The review describes the biological activity of glycirrhetinic (GLA and glycirrhizic (GA acids in terms of their use as a therapeutic agent for viral infections. So, these compounds are against a broad spectrum of viruses, including herpes, corona-, alphaand flaviviruses, human immunodeficiency virus, vaccinia virus, poliovirus type I, vesicular stomatitis virus and influenza A virus. These data indicate that anti-viral effect of these compounds is due to several types of activity — direct antiviral effects, effects on cellular proand anti-viral and immunomodulating pathways, in particular by activation of innate immunity system. GA interferes with early steps of the viral reproductive cycle such as virus binding to its receptor, the absorption of the virus by endocytosis or virus decapsidation in the cytoplasm. This is due to the effect of GA-induced reduction of membrane fluidity. Thus, one mechanism for the antiviral activity of GA is that GA molecule increases the rigidity of cellular and viral membranes after incorporation in there. This results in increasing of energy threshold required for the formation of negative curvature at the fusion zones, as well as difficult lateral migration of the virus-receptor complexes. In addition, glycyrrhizin prevents interaction of viral nucleoprotein with cellular protein HMGB1, which is necessary for the viral life cycle. Glycyrrhizin also inhibits the induction of oxidative stress during influenza infection, exhibiting antioxidant properties, which leads to a reduction of virus-induced production of cytokines/chemokines, without affecting the replication of the virus. A wide spectrum of biological activity and effect on various aspects of the viral pathogenesis substantiate the effect of GA and GLA as a component

  6. Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane.

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    Salanenka, Yuliya; Verstraeten, Inge; Löfke, Christian; Tabata, Kaori; Naramoto, Satoshi; Glanc, Matouš; Friml, Jiří

    2018-02-20

    The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses. Copyright © 2018 the Author(s). Published by PNAS.

  7. Presence of a functional but dispensable Nuclear Export Signal in the HTLV-2 Tax protein

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    Kiemer Lars

    2005-11-01

    Full Text Available Abstract Background Human T-cell leukemia virus type 1 and type 2 are related human retroviruses. HTLV-1 is the etiological agent of the Adult T-cell Leukemia/Lymphoma and of the Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy, whereas, HTLV-2 infection has not been formally associated with any T-cell malignancy. HTLV-1 and 2 genomes encode, respectively, the Tax1 and Tax2 proteins whose role is to transactivate the viral promoter. HTLV-1 and HTLV-2 Tax sequences display 28% divergence at the amino acid level. Tax1 is a shuttling protein that possesses both a non canonical nuclear import (NLS and a nuclear export (NES signal. We have recently demonstrated that Tax1 and Tax2 display different subcellular localization and that residues 90–100 are critical for this process. We investigate in the present report, whether Tax2 also possesses a functional NES. Results We first used a NES prediction method to determine whether the Tax2 protein might contain a NES and the results do suggest the presence of a NES sequence in Tax2. Using Green Fluorescent Protein-NES (GFP-NES fusion proteins, we demonstrate that the Tax2 sequence encompasses a functional NES (NES2. As shown by microscope imaging, NES2 is able to mediate translocation of GFP from the nucleus, without the context of a full length Tax protein. Furthermore, point mutations or leptomycin B treatment abrogate NES2 function. However, within the context of full length Tax2, similar point mutations in the NES2 leucine rich stretch do not modify Tax2 localization. Finally, we also show that Tax1 NES function is dependent upon the positioning of the nuclear export signal "vis-à-vis" GFP. Conclusion HTLV-2 Tax NES is functional but dispensable for the protein localization in vitro.

  8. Soybean NDR1-like proteins bind pathogen effectors and regulate resistance signaling.

    Science.gov (United States)

    Selote, Devarshi; Shine, M B; Robin, Guillaume P; Kachroo, Aardra

    2014-04-01

    Nonrace specific disease resistance 1 (NDR1) is a conserved downstream regulator of resistance (R) protein-derived signaling. We identified two NDR1-like sequences (GmNDR1a, b) from soybean, and investigated their roles in R-mediated resistance and pathogen effector detection. Silencing GmNDR1a and b in soybean shows that these genes are required for resistance derived from the Rpg1-b, Rpg3, and Rpg4 loci, against Pseudomonas syringae (Psg) expressing avrB, avrB2 and avrD1, respectively. Immunoprecipitation assays show that the GmNDR1 proteins interact with the AvrB2 and AvrD1 Psg effectors. This correlates with the enhanced virulence of Psg avrB2 and Psg avrD1 in GmNDR1-silenced rpg3 rpg4 plants, even though these strains are not normally more virulent on plants lacking cognate R loci. The GmNDR1 proteins interact with GmRIN4 proteins, but not with AvrB, or its cognate R protein Rpg1-b. However, the GmNDR1 proteins promote AvrB-independent activation of Rpg1-b when coexpressed with a phosphomimic derivative of GmRIN4b. The role of GmNDR1 proteins in Rpg1-b activation, their direct interactions with AvrB2/AvrD1, and a putative role in the virulence activities of Avr effectors, provides the first experimental evidence in support of the proposed role for NDR1 in transducing extracellular pathogen-derived signals. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  9. LIMPIC: a computational method for the separation of protein MALDI-TOF-MS signals from noise

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    Di Nicola Marta

    2007-03-01

    Full Text Available Abstract Background Mass spectrometry protein profiling is a promising tool for biomarker discovery in clinical proteomics. However, the development of a reliable approach for the separation of protein signals from noise is required. In this paper, LIMPIC, a computational method for the detection of protein peaks from linear-mode MALDI-TOF data is proposed. LIMPIC is based on novel techniques for background noise reduction and baseline removal. Peak detection is performed considering the presence of a non-homogeneous noise level in the mass spectrum. A comparison of the peaks collected from multiple spectra is used to classify them on the basis of a detection rate parameter, and hence to separate the protein signals from other disturbances. Results LIMPIC preprocessing proves to be superior than other classical preprocessing techniques, allowing for a reliable decomposition of the background noise and the baseline drift from the MALDI-TOF mass spectra. It provides lower coefficient of variation associated with the peak intensity, improving the reliability of the information that can be extracted from single spectra. Our results show that LIMPIC peak-picking is effective even in low protein concentration regimes. The analytical comparison with commercial and freeware peak-picking algorithms demonstrates its superior performances in terms of sensitivity and specificity, both on in-vitro purified protein samples and human plasma samples. Conclusion The quantitative information on the peak intensity extracted with LIMPIC could be used for the recognition of significant protein profiles by means of advanced statistic tools: LIMPIC might be valuable in the perspective of biomarker discovery.

  10. Holophytochrome-interacting proteins in Physcomitrella: putative actors in phytochrome cytoplasmic signaling

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    Anna Lena eErmert

    2016-05-01

    Full Text Available Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically-functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We discuss the roles these putative holophytochrome-interacting proteins (HIP's might have in signaling.

  11. Regulation of hedgehog signaling by Myc-interacting zinc finger protein 1, Miz1.

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    Jiuyi Lu

    Full Text Available Smoothened (Smo mediated Hedgehog (Hh signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1 as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

  12. Uncovering molecular structural mechanisms of signaling mediated by the prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Romano, Sebastian A.; Linden, Rafael [Universidade Federal do Rio de Janeiro (IBCCF/UFRl), RJ (Brazil). Inst. de Biofisica Carlos Chagas Filho; Cordeiro, Yraima; Rocha e Lima, Luis M.T. da [Universidade Federal do Rio de Janeiro (FF/UFRl), RJ (Brazil). Fac. de Farmacia; Lopes, Marilene H. [Instituto Ludwig de Pesquisa de Cancer, Sao Paulo, SP (Brazil); Silva, Jerson L.; Foguel, Debora [Universidade Federal do Rio de Janeiro (IBqM/UFRl), RJ (Brazil). Inst. de Bioquimica Medica

    2009-07-01

    The glycosyl phosphatidylinositol (GPI) - anchored prion protein (PrP{sup c}), usually associated with neurodegenerative diseases, modulates various cellular responses and may scaffold multiprotein cell surface signaling complexes. Engagement of PrP{sup c} with the secretable cochaperone hop/STI 1 induces neurotrophic transmembrane signals through unknown molecular mechanisms. We addressed whether interaction of Pr P{sup c} and hop STI 1 entails structural rearrangements relevant for signaling. Circular dichroism and fluorescence spectroscopy showed that PrP{sup c}:hop/STI 1 interaction triggers loss of PrP helical structures, involving at least a perturbation of the Pr P{sup c}{sub 143-153} beta-helix. Novel SAXS models revealed a significant C-terminal compaction of hop/STI 1 when bound to PrP{sup c}. Differing from a recent dimeric model of human hop/STI 1, both size exclusion chromatography and SAXS data support a monomeric form of free murine hop/STI 1. Changes in the Pr P{sup c}{sub 143-153} beta-helix may engage the transmembrane signaling protein laminin receptor precursor and neural cell adhesion molecule, both of which bind that domain of Pr P{sup c}, and further ligands may be engaged by the tertiary structural changes of hop/STI 1. These reciprocal structural modifications indicate a versatile mechanism for signaling mediated by Pr P{sup c}:hop/STI 1 interaction, consistent with the hypothesis that Pr P{sup c} scaffolds multiprotein signaling complexes at the cell surface. (author)

  13. Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila

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    Samuel H. Friedman

    2013-11-01

    Fragile X syndrome (FXS, the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1 gene product (FMRP, an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1 null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs: GPI-anchored glypican Dally-like protein (Dlp and transmembrane Syndecan (Sdc. Our recent work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg ligand abundance and downstream Frizzled-2 (Fz2 receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb, and downstream ERK phosphorylation (dpERK are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb and downstream signaling via phosphorylation of the transcription factor MAD (pMAD seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1 Wg and Jeb trans-synaptic signaling, and (2 synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state.

  14. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis.

    Science.gov (United States)

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E; Trimble, William S; Foster, Leonard J; Finlay, B Brett

    2015-07-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  15. Antiviral Activity of Resveratrol against Human and Animal Viruses.

    Science.gov (United States)

    Abba, Yusuf; Hassim, Hasliza; Hamzah, Hazilawati; Noordin, Mohamed Mustapha

    2015-01-01

    Resveratrol is a potent polyphenolic compound that is being extensively studied in the amelioration of viral infections both in vitro and in vivo. Its antioxidant effect is mainly elicited through inhibition of important gene pathways like the NF-κβ pathway, while its antiviral effects are associated with inhibitions of viral replication, protein synthesis, gene expression, and nucleic acid synthesis. Although the beneficial roles of resveratrol in several viral diseases have been well documented, a few adverse effects have been reported as well. This review highlights the antiviral mechanisms of resveratrol in human and animal viral infections and how some of these effects are associated with the antioxidant properties of the compound.

  16. Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response

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    Hiroyuki eOshiumi

    2016-05-01

    Full Text Available Type I interferon (IFN induces many antiviral factors in host cells. RIG-I-like receptors (RLRs are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and thus cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  17. Recently duplicated plant heterotrimeric Gα proteins with subtle biochemical differences influence specific outcomes of signal-response coupling.

    Science.gov (United States)

    Roy Choudhury, Swarup; Pandey, Sona

    2017-09-29

    Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, regulate key signaling processes in eukaryotes. The Gα subunit determines the status of signaling by switching between inactive GDP-bound and active GTP-bound forms. Unlike animal systems, in which multiple Gα proteins with variable biochemical properties exist, plants have fewer, highly similar Gα subunits that have resulted from recent genome duplications. These proteins exhibit subtle differences in their GTP-binding, GDP/GTP-exchange, and GTP-hydrolysis activities, but the extent to which these differences contribute to affect plant signaling and development remains unknown. To evaluate this, we expressed native and engineered Gα proteins from soybean in an Arabidopsis Gα-null background and studied their effects on modulating a range of developmental and hormonal signaling phenotypes. Our results indicated that inherent biochemical differences in these highly similar Gα proteins are biologically relevant, and some proteins are more flexible than others in influencing the outcomes of specific signals. These observations suggest that alterations in the rate of the G-protein cycle itself may contribute to the specificity of response regulation in plants by affecting the duration of active signaling and/or by the formation of distinct protein-protein complexes. In species such as Arabidopsis having a single canonical Gα, this rate could be affected by regulatory proteins in the presence of specific signals, whereas in plants with multiple Gα proteins, an even more complex regulation may exist, which likely contributes to the specificity of signal-response coupling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Dickkopf-related protein 1 inhibits the WNT signaling pathway and improves pig oocyte maturation.

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    Lee D Spate

    Full Text Available The ability to mature oocytes in vitro provides a tool for creating embryos by parthenogenesis, fertilization, and cloning. Unfortunately the quality of oocytes matured in vitro falls behind that of in vivo matured oocytes. To address this difference, transcriptional profiling by deep sequencing was conducted on pig oocytes that were either matured in vitro or in vivo. Alignment of over 18 million reads identified 1,316 transcripts that were differentially represented. One pathway that was overrepresented in the oocytes matured in vitro was for Wingless-type MMTV integration site (WNT signaling. In an attempt to inhibit the WNT pathway, Dickkopf-related protein 1 was added to the in vitro maturation medium. Addition of Dickkopf-related protein 1 improved the percentage of oocytes that matured to the metaphase II stage, increased the number of nuclei in the resulting blastocyst stage embryos, and reduced the amount of disheveled segment polarity protein 1 protein in oocytes. It is concluded that transcriptional profiling is a powerful method for detecting differences between in vitro and in vivo matured oocytes, and that the WNT signaling pathway is important for proper oocyte maturation.

  19. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    LENUS (Irish Health Repository)

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  20. The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides.

    Science.gov (United States)

    Tsirigos, Konstantinos D; Peters, Christoph; Shu, Nanjiang; Käll, Lukas; Elofsson, Arne

    2015-07-01

    TOPCONS (http://topcons.net/) is a widely used web server for consensus prediction of membrane protein topology. We hereby present a major update to the server, with some substantial improvements, including the following: (i) TOPCONS can now efficiently separate signal peptides from transmembrane regions. (ii) The server can now differentiate more successfully between globular and membrane proteins. (iii) The server now is even slightly faster, although a much larger database is used to generate the multiple sequence alignments. For most proteins, the final prediction is produced in a matter of seconds. (iv) The user-friendly interface is retained, with the additional feature of submitting batch files and accessing the server programmatically using standard interfaces, making it thus ideal for proteome-wide analyses. Indicatively, the user can now scan the entire human proteome in a few days. (v) For proteins with homology to a known 3D structure, the homology-inferred topology is also displayed. (vi) Finally, the combination of methods currently implemented achieves an overall increase in performance by 4% as compared to the currently available best-scoring methods and TOPCONS is the only method that can identify signal peptides and still maintain a state-of-the-art performance in topology predictions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Insulin resistance enhances the mitogen-activated protein kinase signaling pathway in ovarian granulosa cells.

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    Linghui Kong

    Full Text Available The ovary is the main regulator of female fertility. Granulosa cell dysfunction may be involved in various reproductive endocrine disorders. Here we investigated the effect of insulin resistance on the metabolism and function of ovarian granulosa cells, and dissected the functional status of the mitogen-activated protein kinase signaling pathway in these cells. Our data showed that dexamethasone-induced insulin resistance in mouse granulosa cells reduced insulin sensitivity, accompanied with an increase in phosphorylation of p44/42 mitogen-activated protein kinase. Furthermore, up-regulation of cytochrome P450 subfamily 17 and testosterone and down-regulation of progesterone were observed in insulin-resistant mouse granulosa cells. Inhibition of p44/42 mitogen-activated protein kinase after induction of insulin resistance in mouse granulosa cells decreased phosphorylation of p44/42 mitogen-activated protein kinase, downregulated cytochrome P450 subfamily 17 and lowered progesterone production. This insulin resistance cell model can successfully demonstrate certain mechanisms such as hyperandrogenism, which may inspire a new strategy for treating reproductive endocrine disorders by regulating cell signaling pathways.

  2. The photosensor protein Ppr of Rhodocista centenaria is linked to the chemotaxis signalling pathway

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    Kiefer Dorothee

    2010-11-01

    Full Text Available Abstract Background Rhodocista centenaria is a phototrophic α-proteobacterium exhibiting a phototactic behaviour visible as colony movement on agar plates directed to red light. As many phototrophic purple bacteria R. centenaria possesses a soluble photoactive yellow protein (Pyp. It exists as a long fusion protein, designated Ppr, consisting of three domains, the Pyp domain, a putative bilin binding domain (Bbd and a histidine kinase domain (Pph. The Ppr protein is involved in the regulation of polyketide synthesis but it is still unclear, how this is connected to phototaxis and chemotaxis. Results To elucidate the possible role of Ppr and Pph in the chemotactic network we studied the interaction with chemotactic proteins in vitro as well as in vivo. Matrix-assisted coelution experiments were performed to study the possible communication of the different putative binding partners. The kinase domain of the Ppr protein was found to interact with the chemotactic linker protein CheW. The formation of this complex was clearly ATP-dependent. Further results indicated that the Pph histidine kinase domain and CheW may form a complex with the chemotactic kinase CheAY suggesting a role of Ppr in the chemotaxis signalling pathway. In addition, when Ppr or Pph were expressed in Escherichia coli, the chemotactic response of the cells was dramatically affected. Conclusions The Ppr protein of Rhodocista centenaria directly interacts with the chemotactic protein CheW. This suggests a role of the Ppr protein in the regulation of the chemotactic response in addition to its role in chalcone synthesis.

  3. Evolutionary gradient of predicted nuclear localization signals (NLS)-bearing proteins in genomes of family Planctomycetaceae.

    Science.gov (United States)

    Guo, Min; Yang, Ruifu; Huang, Chen; Liao, Qiwen; Fan, Guangyi; Sun, Chenghang; Lee, Simon Ming-Yuen

    2017-04-04

    The nuclear envelope is considered a key classification marker that distinguishes prokaryotes from eukaryotes. However, this marker does not apply to the family Planctomycetaceae, which has intracellular spaces divided by lipidic intracytoplasmic membranes (ICMs). Nuclear localization signal (NLS), a short stretch of amino acid sequence, destines to transport proteins from cytoplasm into nucleus, and is also associated with the development of nuclear envelope. We attempted to investigate the NLS motifs in Planctomycetaceae genomes to demonstrate the potential molecular transition in the development of intracellular membrane system. In this study, we identified NLS-like motifs that have the same amino acid compositions as experimentally identified NLSs in genomes of 11 representative species of family Planctomycetaceae. A total of 15 NLS types and 170 NLS-bearing proteins were detected in the 11 strains. To determine the molecular transformation, we compared NLS-bearing protein abundances in the 11 representative Planctomycetaceae genomes with them in genomes of 16 taxonomically varied microorganisms: nine bacteria, two archaea and five fungi. In the 27 strains, 29 NLS types and 1101 NLS-bearing proteins were identified, principal component analysis showed a significant transitional gradient from bacteria to Planctomycetaceae to fungi on their NLS-bearing protein abundance profiles. Then, we clustered the 993 non-redundant NLS-bearing proteins into 181 families and annotated their involved metabolic pathways. Afterwards, we aligned the ten types of NLS motifs from the 13 families containing NLS-bearing proteins among bacteria, Planctomycetaceae or fungi, considering their diversity, length and origin. A transition towards increased complexity from non-planctomycete bacteria to Planctomycetaceae to archaea and fungi was detected based on the complexity of the 10 types of NLS-like motifs in the 13 NLS-bearing proteins families. The results of this study reveal that

  4. Antiviral Efficacy and Host Immune Response Induction during Sequential Treatment with SB 9200 Followed by Entecavir in Woodchucks.

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    Manasa Suresh

    Full Text Available SB 9200, an orally bioavailable dinucleotide, activates the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I and nucleotide-binding oligomerization domain-containing protein 2 (NOD2 causing the induction of the interferon (IFN signaling cascade for antiviral defense. The present study evaluated the overall antiviral response in woodchucks upon induction of immune response, first with SB 9200 followed by Entecavir (ETV versus reduction of viral burden with ETV followed by SB 9200 immunomodulation. Woodchucks chronically infected with woodchuck hepatitis virus (WHV were treated orally with SB 9200 (30 mg/kg/day and ETV (0.5 mg/kg/day. Group 1 received ETV for 4 weeks followed by SB 9200 for 12 weeks. Group 2 received SB 9200 for 12 weeks followed by ETV for 4 weeks. At the end of treatment in Group 2, average reductions of 6.4 log10 in serum WHV DNA and 3.3 log10 in WHV surface antigen were observed whereas in Group 1, average reductions of 4.2 log10 and 1.1 log10 in viremia and antigenemia were noted. Both groups demonstrated marked reductions in hepatic WHV nucleic acid levels which were more pronounced in Group 2. Following treatment cessation and the 8-week follow-up, recrudescence of viral replication was observed in Group 1 while viral relapse in Group 2 was significantly delayed. The antiviral effects observed in both groups were associated with temporally different induction of IFN-α, IFN-β, and IFN-stimulated genes in blood and liver. These results suggest that the induction of host immune responses by pretreatment with SB 9200 followed by ETV resulted in antiviral efficacy that was superior to that obtained using the strategy of viral reduction with ETV followed by immunomodulation.

  5. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling.

    Science.gov (United States)

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-03-07

    Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Dopamine D2L receptor-interacting proteins regulate dopaminergic signaling

    Directory of Open Access Journals (Sweden)

    Norifumi Shioda

    2017-10-01

    Full Text Available Dopamine receptor family proteins include seven transmembrane and trimeric GTP-binding protein-coupled receptors (GPCRs. Among them, the dopamine D2 receptor (D2R is most extensively studied. All clinically used antipsychotic drugs serve as D2R antagonists in the mesolimbic dopamine system, and their ability to block D2R signaling is positively correlated with antipsychotic efficiency. Human genetic studies also show a significant association of DRD2 polymorphisms with disorders including schizophrenia and Parkinson's disease. D2R exists as two alternatively spliced isoforms, the long isoform (D2LR and the short isoform (D2SR, which differ in a 29-amino acid (AA insert in the third cytoplasmic loop. Importantly, previous reports demonstrate functional diversity between the two isoforms in humans. In this review, we focus on binding proteins that specifically interact with the D2LR 29AA insert. We discuss how D2R activities are mediated not only by heterotrimeric G proteins but by D2LR-interacting proteins, which in part regulate diverse D2R activities. Keywords: Dopamine D2L receptor, Antipsychotic drugs, DRD2 polymorphisms, Alternatively spliced isoforms, D2LR-interacting proteins

  7. Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins.

    Science.gov (United States)

    Verrastro, Ivan; Tveen-Jensen, Karina; Woscholski, Rudiger; Spickett, Corinne M; Pitt, Andrew R

    2016-01-01

    Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Sequencing and Characterization of Novel PII Signaling Protein Gene in Microalga Haematococcus pluvialis

    Directory of Open Access Journals (Sweden)

    Ruijuan Ma

    2017-10-01

    Full Text Available The PII signaling protein is a key protein for controlling nitrogen assimilatory reactions in most organisms, but little information is reported on PII proteins of green microalga Haematococcus pluvialis. Since H. pluvialis cells can produce a large amount of astaxanthin upon nitrogen starvation, its PII protein may represent an important factor on elevated production of Haematococcus astaxanthin. This study identified and isolated the coding gene (HpGLB1 from this microalga. The full-length of HpGLB1 was 1222 bp, including 621 bp coding sequence (CDS, 103 bp 5′ untranslated region (5′ UTR, and 498 bp 3′ untranslated region (3′ UTR. The CDS could encode a protein with 206 amino acids (HpPII. Its calculated molecular weight (Mw was 22.4 kDa and the theoretical isoelectric point was 9.53. When H. pluvialis cells were exposed to nitrogen starvation, the HpGLB1 expression was increased 2.46 times in 48 h, concomitant with the raise of astaxanthin content. This study also used phylogenetic analysis to prove that HpPII was homogeneous to the PII proteins of other green microalgae. The results formed a fundamental basis for the future study on HpPII, for its potential physiological function in Haematococcus astaxanthin biosysthesis.

  9. Non Linear Programming (NLP formulation for quantitative modeling of protein signal transduction pathways.

    Directory of Open Access Journals (Sweden)

    Alexander Mitsos

    Full Text Available Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i excessive CPU time requirements and ii loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms.

  10. Non Linear Programming (NLP) formulation for quantitative modeling of protein signal transduction pathways.

    Science.gov (United States)

    Mitsos, Alexander; Melas, Ioannis N; Morris, Melody K; Saez-Rodriguez, Julio; Lauffenburger, Douglas A; Alexopoulos, Leonidas G

    2012-01-01

    Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms.

  11. In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes.

    Science.gov (United States)

    Mata, Alberto Ramírez; Pacheco, César Millán; Cruz Pérez, José F; Sáenz, Martha Minjárez; Baca, Beatriz E

    2018-03-09

    The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences. The analyzed sequences revealed that the genomes of A. humicireducens SgZ-5 T , A. lipoferum 4B, Azospirillum sp. B510, A. thiophilum BV-S T , A. halopraeferens DSM3675, A. oryzae A2P, and A. brasilense Sp7, Sp245 and Az39 encode for 29 to 41 of these predicted proteins. Notably, only 15 proteins were conserved in all nine genomes: eight GGDEF, three EAL and four GGDEF-EAL hybrid domain proteins, all of which corresponded to core genes in the genomes. The predicted proteins exhibited variable lengths, architectures and sensor domains. In addition, the predicted cellular localizations showed that some of the proteins to contain transmembrane domains, suggesting that these proteins are anchored to the membrane. Therefore, as reported in other soil bacteria, the Azospirillum genomes encode a large number of proteins that are likely involved in c-di-GMP metabolism. In addition, the data obtained here strongly suggest host specificity and environment specific adaptation. Bacteria of the Azospirillum genus cope with diverse environmental conditions to survive in soil and aquatic habitats and, in certain cases, to

  12. The Role of Unfolded Protein Response and Mitogen-Activated Protein Kinase Signaling in Neurodegenerative Diseases with Special Focus on Prion Diseases

    Directory of Open Access Journals (Sweden)

    Lifeng Yang

    2017-05-01

    Full Text Available Prion diseases are neurodegenerative pathologies characterized by the accumulation of a protease-resistant form of the cellular prion protein named prion protein scrapie (PrPSc in the brain. PrPSc accumulation in the endoplasmic reticulum (ER result in a dysregulated calcium (Ca2+ homeostasis and subsequent initiation of unfolded protein response (UPR leading to neuronal dysfunction and apoptosis. The molecular mechanisms for the transition between adaptation to ER stress and ER stress-induced apoptosis are still unclear. Mitogen-activated protein kinases (MAPKs are serine/threonine protein kinases that rule the signaling of many extracellular stimuli from plasma membrane to the nucleus. However the identification of numerous points of cross talk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress in prion diseases. Indeed the MAPK signaling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses including misfolded protein response stress. In this article, we review the UPR signaling in prion diseases and discuss the triad of MAPK signaling pathways. We also describe the role played by MAPK signaling cascades in Alzheimer’s (AD and Parkinson’s disease (PD. We will also overview the mechanisms of cell death and the role of MAPK signaling in prion disease progression and highlight potential avenues for therapeutic intervention.

  13. Putrescine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells.

    Science.gov (United States)

    Kong, Xiangfeng; Wang, Xiaoqiu; Yin, Yulong; Li, Xilong; Gao, Haijun; Bazer, Fuller W; Wu, Guoyao

    2014-11-01

    Insufficient placental growth is a major factor contributing to intrauterine growth retardation in mammals. There is growing evidence that putrescine produced from arginine (Arg) and proline via ornithine decarboxylase is a key regulator of angiogenesis, embryogenesis, as well as placental and fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that putrescine stimulates protein synthesis by activating the mechanistic target of rapamycin (mTOR) signaling pathway in porcine trophectoderm cell line 2 cells. The cells were cultured for 2 to 4 days in customized Arg-free Dulbecco modified Eagle Ham medium containing 0, 10, 25, or 50 μM putrescine or 100 μM Arg. Cell proliferation, protein synthesis, and degradation, as well as the abundance of total and phosphorylated mTOR, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. Our results indicate that putrescine promotes cell proliferation and protein synthesis in a dose- and time-dependent manner, which was inhibited by difluoro-methylornithine (an inhibitor of ornithine decarboxylase). Moreover, supplementation of culture medium with putrescine increased the abundance of phosphorylated mTOR and its downstream targets, 4EBP1 and p70 S6K1 proteins. Collectively, these findings reveal a novel and important role for putrescine in regulating the mTOR signaling pathway in porcine placental cells. We suggest that dietary supplementation with or intravenous administration of putrescine may provide a new and effective strategy to improve survival and growth of embryos/fetuses in mammals. © 2014 by the Society for the Study of Reproduction, Inc.

  14. Proteins involved in platelet signaling are differentially regulated in acute coronary syndrome: a proteomic study.

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    Andrés Fernández Parguiña

    Full Text Available BACKGROUND: Platelets play a fundamental role in pathological events underlying acute coronary syndrome (ACS. Because platelets do not have a nucleus, proteomics constitutes an optimal approach to follow platelet molecular events associated with the onset of the acute episode. METHODOLOGY/PRINCIPAL FINDINGS: We performed the first high-resolution two-dimensional gel electrophoresis-based proteome analysis of circulating platelets from patients with non-ST segment elevation ACS (NSTE-ACS. Proteins were identified by mass spectrometry and validations were by western blotting. Forty protein features (corresponding to 22 unique genes were found to be differentially regulated between NSTE-ACS patients and matched controls with chronic ischemic cardiopathy. The number of differences decreased at day 5 (28 and 6 months after the acute event (5. Interestingly, a systems biology approach demonstrated that 16 of the 22 differentially regulated proteins identified are interconnected as part of a common network related to cell assembly and organization and cell morphology, processes very related to platelet activation. Indeed, 14 of those proteins are either signaling or cytoskeletal, and nine of them are known to participate in platelet activation by αIIbβ3 and/or GPVI receptors. Several of the proteins identified participate in platelet activation through post-translational modifications, as shown here for ILK, Src and Talin. Interestingly, the platelet-secreted glycoprotein SPARC was down-regulated in NSTE-ACS patients compared to stable controls, which is consistent with a secretion process from activated platelets. CONCLUSIONS/SIGNIFICANCE: The present study provides novel information on platelet proteome changes associated with platelet activation in NSTE-ACS, highlighting the presence of proteins involved in platelet signaling. This investigation paves the way for future studies in the search for novel platelet-related biomarkers and drug targets

  15. Orf virus 002 protein targets ovine protein S100A4 and inhibits NF-kappa B signaling

    Directory of Open Access Journals (Sweden)

    Daxiang Chen

    2016-09-01

    Full Text Available Orf virus (ORFV, a member of Parapoxvirus, has evolved various strategies to modulate the immune responses of host cells. The ORFV-encoded protein ORFV002, a regulator factor, has been found to inhibit the acetylation of NF-κB-p65 by blocking phosphorylation of NF-kB-p65 at Ser276 and also to disrupt the binding of NF-kB-p65 and p300. To explore the mechanism by which ORFV002 regulates NF-κB signaling, the understanding of ORFV002 potential binding partners in host cells is critical. In this study, ovine S100 calcium binding protein A4 (S100A4, prolylendopeptidase-like (PREPL and NADH dehydrogenase (ubiquinone 1 alpha subcomplex 8 (NDUFA8 were found to interact with ORFV002 based on the yeast two-hybrid (Y2H assay using a cDNA library derived from primary ovine fetal turbinate cells (OFTu. GST pull-down and bidirectional co-immunoprecipitation assay results demonstrate that ORFV002 interacts with S100A4 directly. Following the pEGFP-ORFV002 (p002GFP transfection, we found that cytoplasmic S100A4 translocates into the nucleus and co-localizes with ORFV002. Furthermore, the inhibitory effect of ORFV002 on NF-κB signaling was significantly restored by S100A4 knock-down phenotype, suggesting ovine S100A4 participating in the ORFV002-mediated NF-κB signaling. These data demonstrate that ORFV002 inhibits the NF-κB activation through its interaction with S100A4 along with its nucleus translocation.

  16. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    DEFF Research Database (Denmark)

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

    2008-01-01

    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

  17. The role of small adaptor proteins in the control of oncogenic signaling driven by tyrosine kinases in human cancer

    Science.gov (United States)

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology. PMID:26788993

  18. Transfer functions for protein signal transduction: application to a model of striatal neural plasticity.

    Science.gov (United States)

    Scheler, Gabriele

    2013-01-01

    We present a novel formulation for biochemical reaction networks in the context of protein signal transduction. The model consists of input-output transfer functions, which are derived from differential equations, using stable equilibria. We select a set of "source" species, which are interpreted as input signals. Signals are transmitted to all other species in the system (the "target" species) with a specific delay and with a specific transmission strength. The delay is computed as the maximal reaction time until a stable equilibrium for the target species is reached, in the context of all other reactions in the system. The transmission strength is the concentration change of the target species. The computed input-output transfer functions can be stored in a matrix, fitted with parameters, and even recalled to build dynamical models on the basis of state changes. By separating the temporal and the magnitudinal domain we can greatly simplify the computational model, circumventing typical problems of complex dynamical systems. The transfer function transformation of biochemical reaction systems can be applied to mass-action kinetic models of signal transduction. The paper shows that this approach yields significant novel insights while remaining a fully testable and executable dynamical model for signal transduction. In particular we can deconstruct the complex system into local transfer functions between individual species. As an example, we examine modularity and signal integration using a published model of striatal neural plasticity. The modularizations that emerge correspond to a known biological distinction between calcium-dependent and cAMP-dependent pathways. Remarkably, we found that overall interconnectedness depends on the magnitude of inputs, with higher connectivity at low input concentrations and significant modularization at moderate to high input concentrations. This general result, which directly follows from the properties of individual transfer

  19. Transfer functions for protein signal transduction: application to a model of striatal neural plasticity.

    Directory of Open Access Journals (Sweden)

    Gabriele Scheler

    Full Text Available We present a novel formulation for biochemical reaction networks in the context of protein signal transduction. The model consists of input-output transfer functions, which are derived from differential equations, using stable equilibria. We select a set of "source" species, which are interpreted as input signals. Signals are transmitted to all other species in the system (the "target" species with a specific delay and with a specific transmission strength. The delay is computed as the maximal reaction time until a stable equilibrium for the target species is reached, in the context of all other reactions in the system. The transmission strength is the concentration change of the target species. The computed input-output transfer functions can be stored in a matrix, fitted with parameters, and even recalled to build dynamical models on the basis of state changes. By separating the temporal and the magnitudinal domain we can greatly simplify the computational model, circumventing typical problems of complex dynamical systems. The transfer function transformation of biochemical reaction systems can be applied to mass-action kinetic models of signal transduction. The paper shows that this approach yields significant novel insights while remaining a fully testable and executable dynamical model for signal transduction. In particular we can deconstruct the complex system into local transfer functions between individual species. As an example, we examine modularity and signal integration using a published model of striatal neural plasticity. The modularizations that emerge correspond to a known biological distinction between calcium-dependent and cAMP-dependent pathways. Remarkably, we found that overall interconnectedness depends on the magnitude of inputs, with higher connectivity at low input concentrations and significant modularization at moderate to high input concentrations. This general result, which directly follows from the properties of

  20. Mechanisms of virus resistance and antiviral activity of snake venoms

    Directory of Open Access Journals (Sweden)

    JVR Rivero

    2011-01-01

    Full Text Available Viruses depend on cell metabolism for their own propagation. The need to foster an intimate relationship with the host has resulted in the development of various strategies designed to help virus escape from the defense mechanisms present in the host. Over millions of years, the unremitting battle between pathogens and their hosts has led to changes in evolution of the immune system. Snake venoms are biological resources that have antiviral activity, hence substances of significant pharmacological value. The biodiversity in Brazil with respect to snakes is one of the richest on the planet; nevertheless, studies on the antiviral activity of venom from Brazilian snakes are scarce. The antiviral properties of snake venom appear as new promising therapeutic alternative against the defense mechanisms developed by viruses. In the current study, scientific papers published in recent years on the antiviral activity of venom from various species of snakes were reviewed. The objective of this review is to discuss the mechanisms of resistance developed by viruses and the components of snake venoms that present antiviral activity, particularly, enzymes, amino acids, peptides and proteins.

  1. Smallpox Antiviral Drug

    National Research Council Canada - National Science Library

    Hruby, Dennis E; Bolken, Tove C

    2005-01-01

    ...) as a model system, the goal of our currently funded work is to determine whether the 17L cysteine proteinase or the 17L metalloproteinase encoded by VV is the pox virus core protein proteinase (vCPP...

  2. Viral ancestors of antiviral systems.

    Science.gov (United States)

    Villarreal, Luis P

    2011-10-01

    All life must survive their corresponding viruses. Thus antiviral systems are essential in all living organisms. Remnants of virus derived information are also found in all life forms but have historically been considered mostly as junk DNA. However, such virus derived information can strongly affect host susceptibility to viruses. In this review, I evaluate the role viruses have had in the origin and evolution of host antiviral systems. From Archaea through bacteria and from simple to complex eukaryotes I trace the viral components that became essential elements of antiviral immunity. I conclude with a reexamination of the 'Big Bang' theory for the emergence of the adaptive immune system in vertebrates by horizontal transfer and note how viruses could have and did provide crucial and coordinated features.

  3. Viral Ancestors of Antiviral Systems

    Directory of Open Access Journals (Sweden)

    Luis P. Villarreal

    2011-10-01

    Full Text Available All life must survive their corresponding viruses. Thus antiviral systems are essential in all living organisms. Remnants of virus derived information are also found in all life forms but have historically been considered mostly as junk DNA. However, such virus derived information can strongly affect host susceptibility to viruses. In this review, I evaluate the role viruses have had in the origin and evolution of host antiviral systems. From Archaea through bacteria and from simple to complex eukaryotes I trace the viral components that became essential elements of antiviral immunity. I conclude with a reexamination of the ‘Big Bang’ theory for the emergence of the adaptive immune system in vertebrates by horizontal transfer and note how viruses could have and did provide crucial and coordinated features.

  4. PKA RIα/A-kinase anchoring proteins 10 signaling pathway and the prognosis of colorectal cancer.

    Science.gov (United States)

    Wang, Mojin; Li, Yuan; Wang, Rui; Wang, Ziqiang; Chen, Keling; Zhou, Bin; Zhou, Zongguang; Sun, Xiaofeng

    2015-03-01

    Previously study showed that the loss of the control of cAMP-dependent protein kinase A RIα (PKA RIα)/ A-kinase anchoring proteins 10 (AKAP10) signaling pathway initiate dysregulation of cellular healthy physiology leading to tumorigenesis. The aim of this study was to investigate the role of PKA RIα/AKAP10 signaling pathway in colorectal cancer (CRC). The AKAP10 expression at the mRNA and protein level have been analyzed in colon cancer cell lines, primary CRCs and matched normal mucosa samples, and compared in accordance with specific clinicopathological features of CRC. The correlation between expression of AKAP10 and PKA RIα were also analyzed. Compared with HCT116 and SW480 cells, the AKAP10 was significantly upregulated in the colon cell line KM12C and its metastatic counterparts, KM12SM and KM12L4A. Moreover, the KM12SM and KM12L4A having high metastatic potentials displayed the elevated levels of AKAP10 compared with KM12C having poor metastatic potential. A notably higher level of AKAP10 expression was found in CRC tissues at both mRNA and protein levels. Increased expression of AKAP10 in CRC patients was positively associated with the depth of invasion and the grade of differentiation. Univariate survival analysis showed that the increased expression of AKAP10 was related to poorer survival. Cox multivariate regression analysis confirmed that AKAP10 was an independent predictor of the overall survival of CRC patients. PKA RIα mRNA was also expressed at high levels in CRC. The correlation coefficient between mRNA expression of AKAP10 and PKA RIα in CRC was 0.417. AKAP10 mRNA overexpression was correlated significantly with PKA RIα. Our data indicated that PKA RIα/AKAP10 signaling pathway is associated with the progression and prognosis of CRC. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  5. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    International Nuclear Information System (INIS)

    Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A

    2008-01-01

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property

  6. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A [Technische Universitaet Muenchen, Technische Mikrobiologie, Weihenstephaner Steig 16, 85350 Freising (Germany)], E-mail: rudi.vogel@wzw.tum.de

    2008-07-15

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  7. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    Science.gov (United States)

    Vogel, R. F.; Linke, K.; Teichert, H.; Ehrmann, M. A.

    2008-07-01

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  8. [Role of G protein-mediated signal transduction in molecular pharmacodynamics].

    Science.gov (United States)

    Schütz, W; Freissmuth, M; Nanoff, C; Selzer, E; Tuisl, E

    1990-10-26

    Hormones, neurotransmitter and autacoid receptors, localized on the plasma membrane, do not interact directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger), but control their target systems via activation of an intermediary guanine nucleotide binding protein on G protein, which serves as signal transducer. Traffic of these pathways is regulated via a GTP (on)-GDP (off) switch, which is triggered by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. Receptor desensitization is a main criterion of G protein-coupled receptors with important pharmacological implications. Multiple mechanisms are responsible for the loss of sensitivity that follows against exposure. The process is initiated by uncoupling the receptor from its G protein, which is due to receptor phosphorylation by specific kinases. In the case of the beta-adrenergic receptor, two particular kinases - beta-adrenergic receptor kinase (beta ARK) and protein kinase A--are involved. Further steps of desensitization are receptor sequestration or internalization, an event as rapid and transient as receptor uncoupling, and receptor downregulation, which requires more prolonged agonist exposure. Finally, antagonists are able to induce a receptor-G protein interaction in a reverse manner to agonists. Whereas agonists stimulate both, the GDP dissociation from the G protein and the association of GTP, antagonists markedly decrease GTP association. Moreover, in the turkey erythrocyte adenylyl cyclase system antagonists decrease the GTP-stimulated adenylyl cyclase activity almost at basal levels.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion.

    Science.gov (United States)

    Masroori, Nasser; Merindol, Natacha; Berthoux, Lionel

    2016-03-22

    The promyelocytic leukemia (PML) protein, a type I interferon (IFN-I)-induced gene product and a member of the tripartite motif (TRIM) family, modulates the transcriptional activity of viruses belonging to various families. Whether PML has an impact on the replication of HIV-1 has not been fully addressed, but recent studies point to its possible involvement in the restriction of HIV-1 in human cells and in the maintenance of transcriptional latency in human cell lines in which HIV-1 is constitutively repressed. We investigated further the restriction of HIV-1 and a related lentivirus, SIVmac, by PML in murine cells and in a lymphocytic human cell line. In particular, we studied the relevance of PML to IFN-I-mediated inhibition and the role of individual human isoforms. We demonstrate that both human PML (hPML) and murine PML (mPML) inhibit the early post-entry stages of the replication of HIV-1 and a related lentivirus, SIVmac. In addition, HIV-1 was transcriptionally silenced by mPML and by hPML isoforms I, II, IV and VI in MEFs. This PML-mediated transcriptional repression was attenuated in presence of the histone deacetylase inhibitor SAHA. In contrast, depletion of PML had no effect on HIV-1 gene expression in a human T cell line. PML was found to contribute to the inhibition of HIV-1 by IFN-I. Specifically, IFN-α and IFN-β treatments of MEFs enhanced the PML-dependent inhibition of HIV-1 early replication stages. We show that PML can inhibit HIV-1 and other lentiviruses as part of the IFN-I-mediated response. The restriction takes place at two distinct steps, i.e. reverse transcription and transcription, and in an isoform-specific, cellular context-specific fashion. Our results support a model in which PML activates innate immune antilentiviral effectors. These data are relevant to the development of latency reversal-inducing pharmacological agents, since PML was previously proposed as a pharmacological target for such inhibitors. This study also has

  10. Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development.

    Science.gov (United States)

    Bhattacharyya, Sohinee; Rainey, Mark A; Arya, Priyanka; Dutta, Samikshan; George, Manju; Storck, Matthew D; McComb, Rodney D; Muirhead, David; Todd, Gordon L; Gould, Karen; Datta, Kaustubh; Gelineau-van Waes, Janee; Band, Vimla; Band, Hamid

    2016-02-17

    Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling.

  11. The Zn finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis.

    Science.gov (United States)

    Glazer, Andrew M; Wilkinson, Alex W; Backer, Chelsea B; Lapan, Sylvain W; Gutzman, Jennifer H; Cheeseman, Iain M; Reddien, Peter W

    2010-01-01

    Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.

  12. The Zn Finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis

    Science.gov (United States)

    Glazer, Andrew; Wilkinson, Alex; Backer, Chelsea B.; Lapan, Sylvain; Gutzman, Jennifer H.; Cheeseman, Iain M.; Reddien, Peter W.

    2009-01-01

    Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and that utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian S. mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates. PMID:19852954

  13. Contribution of autophagy to antiviral immunity.

    Science.gov (United States)

    Rey-Jurado, Emma; Riedel, Claudia A; González, Pablo A; Bueno, Susan M; Kalergis, Alexis M

    2015-11-14

    Although identified in the 1960's, interest in autophagy has significantly increased in the past decade with notable research efforts oriented at understanding as to how this multi-protein complex operates and is regulated. Autophagy is commonly defined as a "self-eating" process evolved by eukaryotic cells to recycle senescent organelles and expired proteins, which is significantly increased during cellular stress responses. In addition, autophagy can also play important roles during human diseases, such as cancer, neurodegenerative and autoimmune disorders. Furthermore, novel findings suggest that autophagy contributes to the host defense against microbial infections. In this article, we review the role of macroautophagy in antiviral immune responses and discuss molecular mechanisms evolved by viral pathogens to evade this process. A role for autophagy as an effector mechanism used both, by innate and adaptive immunity is also discussed. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  14. Highlights in antiviral drug research: antivirals at the horizon.

    Science.gov (United States)

    De Clercq, Erik

    2013-11-01

    This review highlights ten "hot topics" in current antiviral research: (i) new nucleoside derivatives (i.e., PSI-352938) showing high potential as a direct antiviral against hepatitis C virus (HCV); (ii) cyclopropavir, which should be further pursued for treatment of human cytomegalovirus (HCMV) infections; (iii) North-methanocarbathymidine (N-MCT), with a N-locked conformation, showing promising activity against both α- and γ-herpesviruses; (iv) CMX001, an orally bioavailable prodrug of cidofovir with broad-spectrum activity against DNA viruses, including polyoma, adeno, herpes, and pox; (v) favipiravir, which is primarily pursued for the treatment of influenza virus infections, but also inhibits the replication of other RNA viruses, particularly (-)RNA viruses such as arena, bunya, and hanta; (vi) newly emerging antiarenaviral compounds which should be more effective (and less toxic) than the ubiquitously used ribavirin; (vii) antipicornavirus agents in clinical development (pleconaril, BTA-798, and V-073); (viii) natural products receiving increased attention as potential antiviral drugs; (ix) antivirals such as U0126 targeted at specific cellular kinase pathways [i.e., mitogen extracellular kinase (MEK)], showing activity against influenza and other viruses; and (x) two structurally unrelated compounds (i.e., LJ-001 and dUY11) with broad-spectrum activity against virtually all enveloped RNA and DNA viruses. © 2012 Wiley Periodicals, Inc.

  15. Chicken MDA5 senses short double-stranded RNA with implications for antiviral response against avian influenza viruses in chicken.

    Science.gov (United States)

    Hayashi, Tsuyoshi; Watanabe, Chiaki; Suzuki, Yasushi; Tanikawa, Taichiro; Uchida, Yuko; Saito, Takehiko

    2014-01-01

    Mammalian melanoma differentiation-associated gene-5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) selectively sense double-stranded RNA (dsRNA) according to length, as well as various RNA viruses to induce an antiviral response. RIG-I, which plays a predominant role in the induction of antiviral responses against influenza virus infection, has been considered to be lacking in chicken, putting the function of chicken MDA5 (chMDA5) under the spotlight. Here, we show that chMDA5, unlike mammalian MDA5, preferentially senses shorter dsRNA synthetic analogues, poly(I:C), in chicken DF-1 fibroblasts. A requirement for caspase activation and recruitment domains for chMDA5-mediated chicken interferon beta (chIFNβ) induction and its interaction with mitochondrial antiviral signaling proteins were demonstrated. We also found that chMDA5 is involved in chIFNβ induction against avian influenza virus infection. Our findings imply that chMDA5 compensates in part the function of RIG-I in chicken, and highlights the importance of chMDA5 in the innate immune response in chicken. © 2013 S. Karger AG, Basel.

  16. Systematic differences in signal emitting and receiving revealed by PageRank analysis of a human protein interactome.

    Directory of Open Access Journals (Sweden)

    Donglei Du

    Full Text Available Most protein PageRank studies do not use signal flow direction information in protein interactions because this information was not readily available in large protein databases until recently. Therefore, four questions have yet to be answered: A What is the general difference between signal emitting and receiving in a protein interactome? B Which proteins are among the top ranked in directional ranking? C Are high ranked proteins more evolutionarily conserved than low ranked ones? D Do proteins with similar ranking tend to have similar subcellular locations? In this study, we address these questions using the forward, reverse, and non-directional PageRank approaches to rank an information-directional network of human proteins and study their evolutionary conservation. The forward ranking gives credit to information receivers, reverse ranking to information emitters, and non-directional ranking mainly to the number of interactions. The protein lists generated by the forward and non-directional rankings are highly correlated, but those by the reverse and non-directional rankings are not. The results suggest that the signal emitting/receiving system is characterized by key-emittings and relatively even receivings in the human protein interactome. Signaling pathway proteins are frequent in top ranked ones. Eight proteins are both informational top emitters and top receivers. Top ranked proteins, except a few species-related novel-function ones, are evolutionarily well conserved. Protein-subunit ranking position reflects subunit function. These results demonstrate the usefulness of different PageRank approaches in characterizing protein networks and provide insights to protein interaction in the cell.

  17. Systematic differences in signal emitting and receiving revealed by PageRank analysis of a human protein interactome.

    Science.gov (United States)

    Du, Donglei; Lee, Connie F; Li, Xiu-Qing

    2012-01-01

    Most protein PageRank studies do not use signal flow direction information in protein interactions because this information was not readily available in large protein databases until recently. Therefore, four questions have yet to be answered: A) What is the general difference between signal emitting and receiving in a protein interactome? B) Which proteins are among the top ranked in directional ranking? C) Are high ranked proteins more evolutionarily conserved than low ranked ones? D) Do proteins with similar ranking tend to have similar subcellular locations? In this study, we address these questions using the forward, reverse, and non-directional PageRank approaches to rank an information-directional network of human proteins and study their evolutionary conservation. The forward ranking gives credit to information receivers, reverse ranking to information emitters, and non-directional ranking mainly to the number of interactions. The protein lists generated by the forward and non-directional rankings are highly correlated, but those by the reverse and non-directional rankings are not. The results suggest that the signal emitting/receiving system is characterized by key-emittings and relatively even receivings in the human protein interactome. Signaling pathway proteins are frequent in top ranked ones. Eight proteins are both informational top emitters and top receivers. Top ranked proteins, except a few species-related novel-function ones, are evolutionarily well conserved. Protein-subunit ranking position reflects subunit function. These results demonstrate the usefulness of different PageRank approaches in characterizing protein networks and provide insights to protein interaction in the cell.

  18. A macrophage inflammatory protein homolog encoded by guinea pig cytomegalovirus signals via CC chemokine receptor 1

    International Nuclear Information System (INIS)

    Penfold, Mark; Miao Zhenhua; Wang Yu; Haggerty, Shannon; Schleiss, Mark R.

    2003-01-01

    Cytomegaloviruses encode homologs of cellular immune effector proteins, including chemokines (CKs) and CK receptor-like G protein-coupled receptors (GPCRs). Sequence of the guinea pig cytomegalovirus (GPCMV) genome identified an open reading frame (ORF) which predicted a 101 amino acid (aa) protein with homology to the macrophage inflammatory protein (MIP) subfamily of CC (β) CKs, designated GPCMV-MIP. To assess functionality of this CK, recombinant GPCMV-MIP was expressed in HEK293 cells and assayed for its ability to bind to and functionally interact with a variety of GPCRs. Specific signaling was observed with the hCCR1 receptor, which could be blocked with hMIP -1α in competition experiments. Migration assays revealed that GPCMV-MIP was able to induce chemotaxis in hCCR1-L1.2 cells. Antisera raised against a GST-MIP fusion protein immunoprecipitated species of ∼12 and 10 kDa from GPCMV-inoculated tissue culture lysates, and convalescent antiserum from GPCMV-infected animals was immunoreactive with GST-MIP by ELISA assay. These results represent the first substantive in vitro characterization of a functional CC CK encoded by a cytomegalovirus

  19. O-GlcNAcylation and oxidation of proteins: is signalling in the cardiovascular system becoming sweeter?

    Science.gov (United States)

    Lima, Victor V.; Spitler, Kathryn; Choi, Hyehun; Webb, R. Clinton; Tostes, Rita C.

    2012-01-01

    O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury. PMID:22757958

  20. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    Directory of Open Access Journals (Sweden)

    Allison Marie Barbaglia

    2016-04-01

    Full Text Available Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho- lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012. Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I a putative GDSL-motif lipase (II a PIG-P-like protein, with a possible receptor-like function; (III and PLAFP (phloem lipid-associated family protein, a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH, which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while

  1. A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

    Science.gov (United States)

    Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

    2017-09-15

    Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Regulation and function of bone morphogenetic protein signaling in colonic injury and inflammation.

    Science.gov (United States)

    Ji, Tuo; Takabayashi, Hidehiko; Mao, Maria; Han, Xu; Xue, Xiang; Brazil, Jennifer C; Eaton, Kathryn A; Shah, Yatrik M; Todisco, Andrea

    2017-01-01

    The bone morphogenetic proteins (BMPs) regulate gastrointestinal homeostasis. We investigated the expression of BMP-4 and the localization and function of BMP signaling during colonic injury and inflammation. Mice expressing the β-galactosidase (β-gal) gene under the control of a BMP-responsive element (BRE), BMP-4-β-gal/ mice, and animals generated by crossing villin-Cre mice to mice with floxed alleles of BMP receptor 1A (villin-Cre;Bmpr1a flox/flox ) were treated with dextran sodium sulfate (DSS) to induce colonic injury and inflammation. Expression of BMP-4, β-gal, BMPR1A, IL-8, α-smooth muscle actin, and phosphorylated Smad1, -5, and -8 was assessed by X-Gal staining, quantitative RT-PCR, and immunohistochemistry. Morphology of the colonic mucosa was examined by staining with hematoxylin and eosin. The effect of IFN-γ, TNF-α, IL-1β, and IL-6 on BMP-4 mRNA expression was investigated in human intestinal fibroblasts, whereas that of BMP-4 on IL-8 was assessed in human colonic organoids. BMP-4 was localized in α-smooth muscle actin-positive mesenchymal cells while the majority of BMP-generated signals targeted the epithelium. DSS caused injury and inflammation leading to reduced expression of BMP-4 and of BMPR1A mRNAs, and to decreased BMP signaling. Deletion of BMPR1A enhanced colonic inflammation and damage. Administration of anti-TNF-α antibodies to DSS-treated mice ameliorated colonic inflammation and increased the expression of BMP-4 and BMPR1A mRNAs. TNF-α and IL-1β inhibited both basal and IFN-γ-stimulated BMP-4 expression, whereas IL-6 had no effect. BMP-4 reduced TNF-α-stimulated IL-8 mRNA expressor IL-8 mRNA expression in the organoids. Inflammation and injury inhibit BMP-4 expression and signaling, leading to enhanced colonic damage and inflammation. These observations underscore the importance of BMP signaling in the regulation of intestinal inflammation and homeostasis. In this study we report a series of novel observations that

  3. Elafin Reverses Pulmonary Hypertension via Caveolin-1–Dependent Bone Morphogenetic Protein Signaling

    Science.gov (United States)

    Nickel, Nils P.; Spiekerkoetter, Edda; Gu, Mingxia; Li, Caiyun G.; Li, Hai; Kaschwich, Mark; Diebold, Isabel; Hennigs, Jan K.; Kim, Ki-Yoon; Miyagawa, Kazuya; Wang, Lingli; Cao, Aiqin; Sa, Silin; Jiang, Xinguo; Stockstill, Raymond W.; Nicolls, Mark R.; Zamanian, Roham T.; Bland, Richard D.

    2015-01-01

    Rationale: Pulmonary arterial hypertension is characterized by endothelial dysfunction, impaired bone morphogenetic protein receptor 2 (BMPR2) signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates hypoxic pulmonary hypertension in mice, but its potential to improve endothelial function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown. Objectives: To assess elafin-mediated regression of pulmonary vascular pathology in rats and in lung explants from patients with pulmonary hypertension. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells and to elucidate the underlying mechanism. Methods: Rats with pulmonary hypertension induced by vascular endothelial growth factor receptor blockade and hypoxia (Sugen/hypoxia) as well as lung organ cultures from patients with pulmonary hypertension were used to assess elafin-mediated reversibility of pulmonary vascular disease. Pulmonary arterial endothelial cells from patients and control subjects were used to determine the efficacy and mechanism of elafin-mediated BMPR2 signaling. Measurements and Main Results: In Sugen/hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a BMPR2 target. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and decreased neointimal lesions in lung organ culture. In normal and patient pulmonary artery endothelial cells, elafin promoted angiogenesis by increasing pSMAD-dependent and -independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via

  4. A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

    Science.gov (United States)

    Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T; Chen, Shouwen

    2017-04-24

    Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

  5. Signal regulatory protein alpha (SIRPalpha cells in the adaptive response to ESAT-6/CFP-10 protein of tuberculous mycobacteria.

    Directory of Open Access Journals (Sweden)

    W Ray Waters

    2009-07-01

    Full Text Available Early secretory antigenic target-6 (ESAT-6 and culture filtrate protein-10 (CFP-10 are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRPalpha (also referred to as macrophage fusion receptor or CD172a is essential for multi-nucleate giant cell formation.In the present study, ESAT-6/CFP-10 complex and SIRPalpha interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a(+ (SIRPalpha-expressing cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail, but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis DeltaRD1. Sorted CD172a(+ cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8alpha. Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c(+, CD172a(+, and CD3(+ cells, including CD172a-expressing multi-nucleated giant cells.These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a(+ cells, bind to CD172a(+ cells, and induce multi-nucleated giant cells expressing CD172a.

  6. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors.

    Science.gov (United States)

    Culhane, Kelly J; Liu, Yuting; Cai, Yingying; Yan, Elsa C Y

    2015-01-01

    Although family B G protein-coupled receptors (GPCRs) contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

  7. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    Kelly J Culhane

    2015-11-01

    Full Text Available Although family B G protein-coupled receptors (GPCRs contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

  8. Overexpression of protein O-fucosyltransferase 1 accelerates hepatocellular carcinoma progression via the Notch signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Lijie [Liver Surgery Department, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai (China); Dong, Pingping [Department of Gastroenterology and Hepatology, Shanghai Institute of Liver Diseases, Zhongshan Hospital of Fudan University, Shanghai (China); Liu, Longzi; Gao, Qiang; Duan, Meng [Liver Surgery Department, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai (China); Zhang, Si; Chen, She [Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Xue, Ruyi, E-mail: xue.ruyi@zs-hospital.sh.cn [Department of Gastroenterology and Hepatology, Shanghai Institute of Liver Diseases, Zhongshan Hospital of Fudan University, Shanghai (China); Wang, Xiaoying, E-mail: xiaoyingwang@fudan.edu.cn [Liver Surgery Department, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai (China)

    2016-04-29

    Aberrant activation of Notch signaling frequently occurs in liver cancer, and is associated with liver malignancies. However, the mechanisms regulating pathologic Notch activation in hepatocellular carcinoma (HCC) remain unclear. Protein O-fucosyltransferase 1 (Pofut1) catalyzes the addition of O-linked fucose to the epidermal growth factor-like repeats of Notch. In the present study, we detected the expression of Pofut1 in 8 HCC cell lines and 253 human HCC tissues. We reported that Pofut1 was overexpressed in HCC cell lines and clinical HCC tissues, and Pofut1 overexpression clinically correlated with the unfavorable survival and high disease recurrence in HCC. The in vitro assay demonstrated that Pofut1 overexpression accelerated the cell proliferation and migration in HCC cells. Furthermore, Pofut1 overexpression promoted the binding of Notch ligand Dll1 to Notch receptor, and hence activated Notch signaling pathway in HCC cells, indicating that Pofut1 overexpression could be a reason for the aberrant activation of Notch signaling in HCC. Taken together, our findings indicated that an aberrant activated Pofut1-Notch pathway was involved in HCC progression, and blockage of this pathway could be a promising strategy for the therapy of HCC. - Highlights: • Pofut1 overexpression in HCC was correlated with aggressive tumor behaviors. • Pofut1 overexpression in HCC was associated with poor prognosis. • Pofut1 promoted cell proliferation, migration and invasion in hepatoma cells. • Pofut1 activated Notch signaling pathway in hepatoma cells.

  9. Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

    Science.gov (United States)

    Luchetti, Giovanni; Sircar, Ria; Kong, Jennifer H; Nachtergaele, Sigrid; Sagner, Andreas; Byrne, Eamon FX; Covey, Douglas F; Siebold, Christian; Rohatgi, Rajat

    2016-01-01

    Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility. DOI: http://dx.doi.org/10.7554/eLife.20304.001 PMID:27705744

  10. Review: Mitogen-Activated Protein kinases in nutritional signaling in Arabidopsis

    KAUST Repository

    Chardin, Camille

    2017-04-14

    Mitogen-Activated Protein Kinase (MAPK) cascades are functional modules widespread among eukaryotic organisms. In plants, these modules are encoded by large multigenic families and are involved in many biological processes ranging from stress responses to cellular differentiation and organ development. Furthermore, MAPK pathways are involved in the perception of environmental and physiological modifications. Interestingly, some MAPKs play a role in several signaling networks and could have an integrative function for the response of plants to their environment. In this review, we describe the classification of MAPKs and highlight some of their biochemical actions. We performed an in silico analysis of MAPK gene expression in response to nutrients supporting their involvement in nutritional signaling. While several MAPKs have been identified as players in sugar, nitrogen, phosphate, iron and potassium-related signaling pathways, their biochemical functions are yet mainly unknown. The integration of these regulatory cascades in the current understanding of nutrient signaling is discussed and potential new avenues for approaches toward plants with higher nutrient use efficiencies are evoked.

  11. Overexpression of protein O-fucosyltransferase 1 accelerates hepatocellular carcinoma progression via the Notch signaling pathway

    International Nuclear Information System (INIS)

    Ma, Lijie; Dong, Pingping; Liu, Longzi; Gao, Qiang; Duan, Meng; Zhang, Si; Chen, She; Xue, Ruyi; Wang, Xiaoying

    2016-01-01

    Aberrant activation of Notch signaling frequently occurs in liver cancer, and is associated with liver malignancies. However, the mechanisms regulating pathologic Notch activation in hepatocellular carcinoma (HCC) remain unclear. Protein O-fucosyltransferase 1 (Pofut1) catalyzes the addition of O-linked fucose to the epidermal growth factor-like repeats of Notch. In the present study, we detected the expression of Pofut1 in 8 HCC cell lines and 253 human HCC tissues. We reported that Pofut1 was overexpressed in HCC cell lines and clinical HCC tissues, and Pofut1 overexpression clinically correlated with the unfavorable survival and high disease recurrence in HCC. The in vitro assay demonstrated that Pofut1 overexpression accelerated the cell proliferation and migration in HCC cells. Furthermore, Pofut1 overexpression promoted the binding of Notch ligand Dll1 to Notch receptor, and hence activated Notch signaling pathway in HCC cells, indicating that Pofut1 overexpression could be a reason for the aberrant activation of Notch signaling in HCC. Taken together, our findings indicated that an aberrant activated Pofut1-Notch pathway was involved in HCC progression, and blockage of this pathway could be a promising strategy for the therapy of HCC. - Highlights: • Pofut1 overexpression in HCC was correlated with aggressive tumor behaviors. • Pofut1 overexpression in HCC was associated with poor prognosis. • Pofut1 promoted cell proliferation, migration and invasion in hepatoma cells. • Pofut1 activated Notch signaling pathway in hepatoma cells.

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

    Science.gov (United States)

    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.

  13. "Nuclear FGF receptor-1 and CREB binding protein: an integrative signaling module".

    Science.gov (United States)

    Stachowiak, Michal K; Birkaya, B; Aletta, J M; Narla, S T; Benson, C A; Decker, B; Stachowiak, E K

    2015-05-01

    In this review we summarize the current understanding of a novel integrative function of Fibroblast Growth Factor Receptor-1 (FGFR1) and its partner CREB Binding Protein (CBP) acting as a nuclear regulatory complex. Nuclear FGFR1 and CBP interact with and regulate numerous genes on various chromosomes. FGFR1 dynamic oscillatory interactions with chromatin and with specific genes, underwrites gene regulation mediated by diverse developmental signals. Integrative Nuclear FGFR1 Signaling (INFS) effects the differentiation of stem cells and neural progenitor cells via the gene-controlling Feed-Forward-And-Gate mechanism. Nuclear accumulation of FGFR1 occurs in numerous cell types and disruption of INFS may play an important role in developmental disorders such as schizophrenia, and in metastatic diseases such as cancer. Enhancement of INFS may be used to coordinate the gene regulation needed to activate cell differentiation for regenerative purposes or to provide interruption of cancer stem cell proliferation. © 2014 Wiley Periodicals, Inc.

  14. Role of inhibitory proteins as modulators of oscillations in NFB signalling.

    Science.gov (United States)

    Nikolov, S; Vera, J; Rath, O; Kolch, W; Wolkenhauer, O

    2009-03-01

    The authors discuss the role of the Raf kinase inhibitory protein (RKIP) as a modulator of oscillations in NFB signalling. A mathematical model of the NFB signalling pathway was derived and the Lyapunov-Andronov theory was used to analyse dynamical properties of the system. The analytical results were complemented by predictive numerical simulations. Our results suggest that the nature of oscillations, emerging under sustained stimulation of the system, depends on the interplay between the IB kinase (IKK) stimulation and the inhibitory action of RKIP. The authors found a mathematical relation that defines isoclines in IKK and RKIP levels for which the properties of oscillations are conserved and changes in the stimulation can be compensated by modulating RKIP inhibition. On the other hand, the shifting from the current isocline provokes modulation in either the amplitude (for stronger stimulation) or the frequency (for weaker stimulation).

  15. Phosphatidylcholine transfer protein interacts with thioesterase superfamily member 2 to attenuate insulin signaling.

    Science.gov (United States)

    Ersoy, Baran A; Tarun, Akansha; D'Aquino, Katharine; Hancer, Nancy J; Ukomadu, Chinweike; White, Morris F; Michel, Thomas; Manning, Brendan D; Cohen, David E

    2013-07-30

    Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenced by insulin-independent IRS2 activation after knockdown, genetic ablation, or chemical inhibition of PC-TP. In addition, IRS2 was activated after knockdown of THEM2, providing support for a role for the interaction of PC-TP with THEM2 in suppressing insulin signaling. Additionally, we showed that PC-TP bound to tuberous sclerosis complex 2 (TSC2) and stabilized the components of the TSC1-TSC2 complex, which functions to inhibit mTORC1. Preventing phosphatidylcholine from binding to PC-TP disrupted interactions of PC-TP with THEM2 and TSC2, and disruption of the PC-TP-THEM2 complex was associated with increased activation of both IRS2 and mTORC1. In livers of mice with genetic ablation of PC-TP or that had been treated with a PC-TP inhibitor, steady-state amounts of IRS2 were increased, whereas those of TSC2 were decreased. These findings reveal a phospholipid-dependent mechanism that suppresses insulin signaling downstream of its receptor.

  16. Modulation of the beta-catenin signaling pathway by the dishevelled-associated protein Hipk1.

    Directory of Open Access Journals (Sweden)

    Sarah H Louie

    Full Text Available BACKGROUND: Wnts are evolutionarily conserved ligands that signal through beta-catenin-dependent and beta-catenin-independent pathways to regulate cell fate, proliferation, polarity, and movements during vertebrate development. Dishevelled (Dsh/Dvl is a multi-domain scaffold protein required for virtually all known Wnt signaling activities, raising interest in the identification and functions of Dsh-associated proteins. METHODOLOGY: We conducted a yeast-2-hybrid screen using an N-terminal fragment of Dsh, resulting in isolation of the Xenopus laevis ortholog of Hipk1. Interaction between the Dsh and Hipk1 proteins was confirmed by co-immunoprecipitation assays and mass spectrometry, and further experiments suggest that Hipk1 also complexes with the transcription factor Tcf3. Supporting a nuclear function during X. laevis development, Myc-tagged Hipk1 localizes primarily to the nucleus in animal cap explants, and the endogenous transcript is strongly expressed during gastrula and neurula stages. Experimental manipulations of Hipk1 levels indicate that Hipk1 can repress Wnt/beta-catenin target gene activation, as demonstrated by beta-catenin reporter assays in human embryonic kidney cells and by indicators of dorsal specification in X. laevis embryos at the late blastula stage. In addition, a subset of Wnt-responsive genes subsequently requires Hipk1 for activation in the involuting mesoderm during gastrulation. Moreover, either over-expression or knock-down of Hipk1 leads to perturbed convergent extension cell movements involved in both gastrulation and neural tube closure. CONCLUSIONS: These results suggest that Hipk1 contributes in a complex fashion to Dsh-dependent signaling activities during early vertebrate development. This includes regulating the transcription of Wnt/beta-catenin target genes in the nucleus, possibly in both repressive and activating ways under changing developmental contexts. This regulation is required to modulate gene

  17. GIT1/beta PIX signaling proteins and PAK1 kinase regulate microtubule nucleation

    Czech Academy of Sciences Publication Activity Database

    Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

    2016-01-01

    Roč. 1863, č. 6 (2016), s. 1282-1297 ISSN 0167-4889 R&D Projects: GA ČR GAP302/12/1673; GA ČR GA15-22194S; GA MŠk LH12050; GA MZd NT14467; GA ČR GA16-23702S Institutional support: RVO:68378050 Keywords : Centrosome * Microtubule nucleation * gamma-tubulin * GIT1/beta PIX signaling proteins * PAK1 kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.521, year: 2016

  18. Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

    Science.gov (United States)

    Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali

    2014-01-01

    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

  19. Disruption of axonal transport perturbs bone morphogenetic protein (BMP)--signaling and contributes to synaptic abnormalities in two neurodegenerative diseases.

    Science.gov (United States)

    Kang, Min Jung; Hansen, Timothy J; Mickiewicz, Monique; Kaczynski, Tadeusz J; Fye, Samantha; Gunawardena, Shermali

    2014-01-01

    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.

  20. The future of antiviral immunotoxins

    DEFF Research Database (Denmark)

    Spiess, K.; Høy Jakobsen, Mette; Kledal, Thomas N

    2016-01-01

    There is a constant need for new therapeutic interventions in a wide range of infectious diseases. Over the past few years, the immunotoxins have entered the stage as promising antiviral treatments. Immunotoxins have been extensively explored in cancer treatment and have achieved FDA approval...

  1. Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.

    Science.gov (United States)

    Selman, Colin; Tullet, Jennifer M A; Wieser, Daniela; Irvine, Elaine; Lingard, Steven J; Choudhury, Agharul I; Claret, Marc; Al-Qassab, Hind; Carmignac, Danielle; Ramadani, Faruk; Woods, Angela; Robinson, Iain C A; Schuster, Eugene; Batterham, Rachel L; Kozma, Sara C; Thomas, George; Carling, David; Okkenhaug, Klaus; Thornton, Janet M; Partridge, Linda; Gems, David; Withers, Dominic J

    2009-10-02

    Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

  2. Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

    Directory of Open Access Journals (Sweden)

    Takashi W. Ijiri

    2012-01-01

    Full Text Available In sexual reproduction, two gamete cells (i.e., egg and sperm fuse (fertilization to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization.

  3. The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins)

    NARCIS (Netherlands)

    Faber, Klaas Nico; Haima, Pieter; Gietl, Christine; Harder, Willem; Ab, Geert; Veenhuis, Marten

    1994-01-01

    Two main types of peroxisomal targeting signals have been identified that reside either at the extreme C terminus (PTS1) or the N terminus (PTS2) of the protein. In the methylotrophic yeast Hansenula polymorpha the majority of peroxisomal matrix proteins are of the PTS1 type. Thus far, for H.

  4. Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the TGEV S protein.

    Science.gov (United States)

    Trincone, Anna; Schwegmann-Weßels, Christel

    2015-04-16

    The spike protein S of transmissible gastroenteritis virus, an Alphacoronavirus, contains a tyrosine-based sorting signal that is responsible for ERGIC retention and may be important for a correct viral assembly process. To find out whether the S protein interacts with cellular proteins via this sorting signal, a pulldown assay with GST fusion proteins was performed. Filamin A has been identified as a putative interaction candidate. Immunofluorescence assays confirmed a co-localization between the TGEV S protein and filamin A. Further experiments have to be performed to prove a significant impact of filamin A on TGEV infection. Different approaches of several researchers for the identification of cellular interaction candidates relevant for coronavirus replication are summarized. These results may help in the future to identify the role of cellular proteins during coronavirus assembly at the ER-Golgi intermediate compartment. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. HOIL-1L interacting protein (HOIP as an NF-kappaB regulating component of the CD40 signaling complex.

    Directory of Open Access Journals (Sweden)

    Bruce S Hostager

    2010-06-01

    Full Text Available The tumor necrosis factor receptor (TNFR superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-kappaB activation through the ubiquitin-dependent activation of IKKgamma. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-kappaB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling.

  6. Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

    NARCIS (Netherlands)

    van Dijl, J M; Jong, de Anne; Smith, H; Bron, Sierd; Venema, G

    The effects of 25-fold overproduction of Escherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from the Bacillus subtilis chromosome and the mature part of TEM-beta-lactamase,

  7. Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

    Science.gov (United States)

    Rice, Anne M; Mahling, Ryan; Fealey, Michael E; Rannikko, Anika; Dunleavy, Katie; Hendrickson, Troy; Lohese, K Jean; Kruggel, Spencer; Heiling, Hillary; Harren, Daniel; Sutton, R Bryan; Pastor, John; Hinderliter, Anne

    2014-09-01

    Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. CREB-binding protein, p300, butyrate, and Wnt signaling in colorectal cancer.

    Science.gov (United States)

    Bordonaro, Michael; Lazarova, Darina L

    2015-07-21

    This paper reviews the distinctive roles played by the transcriptional coactivators CREB-binding protein (CBP) and p300 in Wnt/β-catenin signaling and cell physiology in colorectal cancer (CRC). Specifically, we focus on the effects of CBP- and p300-mediated Wnt activity on (1) neoplastic progression; (2) the activities of butyrate, a breakdown product of dietary fiber, on cell signaling and colonic cell physiology; (3) the development of resistance to histone deacetylase inhibitors (HDACis), including butyrate and synthetic HDACis, in colonic cells; and (4) the physiology and number of cancer stem cells. Mutations of the Wnt/β-catenin signaling pathway initiate the majority of CRC cases, and we have shown that hyperactivation of this pathway by butyrate and other HDACis promotes CRC cell apoptosis. This activity by butyrate may in part explain the preventive action of fiber against CRC. However, individuals with a high-fiber diet may still develop neoplasia; therefore, resistance to the chemopreventive action of butyrate likely contributes to CRC. CBP or p300 may modify the ability of butyrate to influence colonic cell physiology since the two transcriptional coactivators affect Wnt signaling, and likely, its hyperactivation by butyrate. Also, CBP and p300 likely affect colonic tumorigenesis, as well as stem cell pluripotency. Improvement of CRC prevention and therapy requires a better understanding of the alterations in Wnt signaling and gene expression that underlie neoplastic progression, stem cell fate, and the development of resistance to butyrate and clinically relevant HDACis. Detailed knowledge of how CBP- and p300 modulate colonic cell physiology may lead to new approaches for anti-CRC prevention and therapeutics, particularly with respect to combinatorial therapy of CBP/p300 inhibitors with HDACis.

  9. A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity.

    Science.gov (United States)

    Croft, Wayne; Hill, Claire; McCann, Eilish; Bond, Michael; Esparza-Franco, Manuel; Bennett, Jeannette; Rand, David; Davey, John; Ladds, Graham

    2013-09-20

    G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.

  10. Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction[OPEN

    Science.gov (United States)

    Shi, Jinrui; Wang, Hongyu; Habben, Jeffrey E.

    2016-01-01

    The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize. PMID:27268962

  11. Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene