HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

PubMed

Bruno, Anna Paola; De Simone, Francesca Isabella; Iorio, Vittoria; De Marco, Margot; Khalili, Kamel; Sariyer, Ilker Kudret; Capunzo, Mario; Nori, Stefania Lucia; Rosati, Alessandra

2014-01-01

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

Nucleolar protein trafficking in response to HIV-1 Tat: rewiring the nucleolus.

PubMed

Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

2012-01-01

The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these

Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus

PubMed Central

Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.

2012-01-01

The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these

The h-region of twin-arginine signal peptides supports productive binding of bacterial Tat precursor proteins to the TatBC receptor complex.

PubMed

Ulfig, Agnes; Fröbel, Julia; Lausberg, Frank; Blümmel, Anne-Sophie; Heide, Anna Katharina; Müller, Matthias; Freudl, Roland

2017-06-30

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in their binding to the Tat translocase, but some facets of this interaction remain unclear. Here, we investigated the role of the hydrophobic (h-) region of the Escherichia coli trimethylamine N -oxide reductase (TorA) signal peptide in TatBC receptor binding in vivo and in vitro We show that besides the RR motif, a minimal, functional h-region in the signal peptide is required for Tat-dependent export in Escherichia coli Furthermore, we identified mutations in the h-region that synergistically suppressed the export defect of a TorA[KQ]-30aa-MalE Tat reporter protein in which the RR motif was replaced with a lysine-glutamine pair. Strikingly, all suppressor mutations increased the hydrophobicity of the h-region. By systematically replacing a neutral residue in the h-region with various amino acids, we detected a positive correlation between the hydrophobicity of the h-region and the translocation efficiency of the resulting reporter variants. In vitro cross-linking of residues located in the periplasmically-oriented part of the TatBC receptor to TorA[KQ]-30aa-MalE reporter variants harboring a more hydrophobic h-region in their signal peptides confirmed that unlike in TorA[KQ]-30aa-MalE with an unaltered h-region, the mutated reporters moved deep into the TatBC-binding cavity. Our results clearly indicate that, besides the Tat motif, the h-region of the Tat signal peptides is another important binding determinant that significantly contributes to the productive interaction of Tat precursor proteins with the TatBC receptor complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors

PubMed Central

Kehn-Hall, Kylene; Guendel, Irene; Carpio, Lawrence; Skaltsounis, Leandros; Meijer, Laurent; Al-Harthi, Lena; Steiner, Joseph P.; Nath, Avindra; Kutsch, Olaf; Kashanchi, Fatah

2013-01-01

The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC50 of 40 nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC50 of 4 nM in primary macrophages and 0.5 nM in astrocytes infected with HIV-1. 6BIOder displayed an IC50 value of 0.03 nM through in vitro GSK-3β kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity. PMID:21514616

Exosomal miR-9 Released from HIV Tat Stimulated Astrocytes Mediates Microglial Migration.

PubMed

Yang, Lu; Niu, Fang; Yao, Honghong; Liao, Ke; Chen, Xufeng; Kook, Yeonhee; Ma, Rong; Hu, Guoku; Buch, Shilpa

2018-03-01

Chronic neuroinflammation still remains a common underlying feature of HIV-infected patients on combined anti-retroviral therapy (cART). Previous studies have reported that despite near complete suppression of virus replication by cART, cytotoxic viral proteins such as HIV trans-activating regulatory protein (Tat) continue to persist in tissues such as the brain and the lymph nodes, thereby contributing, in part, to chronic glial activation observed in HIV-associated neurological disorders (HAND). Understanding how the glial cells cross talk to mediate neuropathology is thus of paramount importance. MicroRNAs (miR) also known as regulators of gene expression, have emerged as key paracrine signaling mediators that regulate disease pathogenesis and cellular crosstalk, through their transfer via the extracellular vesicles (EV). In the current study we have identified a novel function of miR-9, that of mediating microglial migration. We demonstrate that miR-9 released from Tat-stimulated astrocytes can be taken up by microglia resulting in their migratory phenotype. Exposure of human astrocytoma (A172) cells to HIV Tat resulted in induction and release of miR-9 in the EVs, which, was taken up by microglia, leading in turn, increased migration of the latter cells, a process that could be blocked by both an exosome inhibitor GW4869 or a specific target protector of miR-9. Furthermore, it was also demonstrated that EV miR-9 mediated inhibition of the expression of target PTEN, via its binding to the 3'UTR seed sequence of the PTEN mRNA, was critical for microglial migration. To validate the role of miR-9 in this process, microglial cells were treated with EVs loaded with miR-9, which resulted in significant downregulation of PTEN expression with a concomitant increase in microglial migration. These findings were corroborated by transfecting microglia with a specific target protector of PTEN, that blocked miR-9-mediated downregulation of PTEN as well as microglial

Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

PubMed Central

Liu, Xiaoyan; Hu, Wenhui; Wang, Yinye

2014-01-01

Stroke is the major cause of death and disability worldwide, and the thrombolytic therapy currently available was unsatisfactory. 14-3-3ε is a well characterized member of 14-3-3 family, and has been reported to protect neurons against apoptosis in cerebral ischemia. However, it cannot transverse blood brain barrier (BBB) due to its large size. A protein transduction domain (PTD) of HIV TAT protein, is capable of delivering a large variety of proteins into the brain. In this study, we generated a fusion protein TAT-14-3-3ε, and evaluated its potential neuroprotective effect in rat focal ischemia/reperfusion (I/R) model. Western blot analysis validated the efficient transduction of TAT-14-3-3ε fusion protein into brain via a route of intravenous injection. TAT-14-3-3ε pre-treatment 2 h before ischemia significantly reduced cerebral infarction volume and improved neurologic score, while post-treatment 2 h after ischemia was less effective. Importantly, pre- or post-ischemic treatment with TAT-14-3-3ε significantly increased the number of surviving neurons as determined by Nissl staining, and attenuated I/R-induced neuronal apoptosis as showed by the decrease in apoptotic cell numbers and the inhibition of caspase-3 activity. Moreover, the introduction of 14-3-3ε into brain by TAT-mediated delivering reduced the formation of autophagosome, attenuated LC3B-II upregulation and reversed p62 downregulation induced by ischemic injury. Such inhibition of autophagy was reversed by treatment with an autophagy inducer rapamycin (RAP), which also attenuated the neuroprotective effect of TAT-14-3-3ε. Conversely, autophagy inhibitor 3-methyladenine (3-MA) inhibited I/R-induced the increase in autophagic activity, and attenuated I/R-induced brain infarct. These results suggest that TAT-14-3-3ε can be efficiently transduced into brain and exert significantly protective effect against brain ischemic injury through inhibiting neuronal apoptosis and autophagic activation. PMID

Modelling the metabolism of protein secretion through the Tat route in Streptomyces lividans.

PubMed

Valverde, José R; Gullón, Sonia; Mellado, Rafael P

2018-06-14

Streptomyces lividans has demonstrated its value as an efficient host for protein production due to its ability to secrete functional proteins directly to the media. Secretory proteins that use the major Sec route need to be properly folded outside the cell, whereas secretory proteins using the Tat route appear outside the cell correctly folded. This feature makes the Tat system very attractive for the production of natural or engineered Tat secretory proteins. S. lividans cells are known to respond differently to overproduction and secretion of Tat versus Sec proteins. Increased understanding of the impact of protein secretion through the Tat route can be obtained by a deeper analysis of the metabolic impact associated with protein production, and its dependence on protein origin, composition, secretion mechanisms, growth phases and nutrients. Flux Balance Analysis of Genome-Scale Metabolic Network models provides a theoretical framework to investigate cell metabolism under different constraints. We have built new models for various S. lividans strains to better understand the mechanisms associated with overproduction of proteins secreted through the Tat route. We compare models of an S. lividans Tat-dependent agarase overproducing strain with those of the S. lividans wild-type, an S. lividans strain carrying the multi-copy plasmid vector and an α-amylase Sec-dependent overproducing strain. Using updated genomic, transcriptomic and experimental data we could extend existing S. lividans models and produce a new model which produces improved results largely extending the coverage of S. lividans strains, the number of genes and reactions being considered, the predictive behaviour and the dependence on specification of exchange constraints. Comparison of the optimized solutions obtained highlights numerous changes between Tat- and Sec-dependent protein secreting strains affecting the metabolism of carbon, amino acids, nucleotides, lipids and cofactors, and

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

PubMed

Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

2010-10-01

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

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

Sugden, Scott, E-mail: scott.sugden@ircm.qc.ca

HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process bymore » recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.« less

Efficient mucosal delivery of the HIV-1 Tat protein using the synthetic lipopeptide MALP-2 as adjuvant.

PubMed

Borsutzky, Stefan; Fiorelli, Valeria; Ebensen, Thomas; Tripiciano, Antonella; Rharbaoui, Faiza; Scoglio, Arianna; Link, Claudia; Nappi, Filomena; Morr, Michael; Buttó, Stefano; Cafaro, Aurelio; Mühlradt, Peter F; Ensoli, Barbara; Guzmán, Carlos A

2003-06-01

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell-mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV-1 Tat protein as antigen and the synthetic lipopeptide, macrophage-activating lipopeptide-2 (MALP-2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti-Tat antibody responses, and Tat-specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freund's adjuvant. Major linear B cell epitopes mapped within aa 1-20 and 46-60, whereas T cell epitopes were identified within aa 36-50 and 56-70. These epitopes have also been described in vaccinated primates and in HIV-1-infected individuals with better prognosis. Analysis of the anti-Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP-2, as opposed to the Th2 response observed with Tat plus incomplete Freund's adjuvant. Tat-specific IFN-gamma-producing cells were significantly increased only in response to Tat plus MALP-2. These data suggest that Malp-2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.

HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production.

PubMed

El-Amine, Rawan; Germini, Diego; Zakharova, Vlada V; Tsfasman, Tatyana; Sheval, Eugene V; Louzada, Ruy A N; Dupuy, Corinne; Bilhou-Nabera, Chrystèle; Hamade, Aline; Najjar, Fadia; Oksenhendler, Eric; Lipinski, Marс; Chernyak, Boris V; Vassetzky, Yegor S

2018-05-01

Human immunodeficiency virus (HIV) infection is associated with B-cell malignancies in patients though HIV-1 is not able to infect B-cells. The rate of B-cell lymphomas in HIV-infected individuals remains high even under the combined antiretroviral therapy (cART) that reconstitutes the immune function. Thus, the contribution of HIV-1 to B-cell oncogenesis remains enigmatic. HIV-1 induces oxidative stress and DNA damage in infected cells via multiple mechanisms, including viral Tat protein. We have detected elevated levels of reactive oxygen species (ROS) and DNA damage in B-cells of HIV-infected individuals. As Tat is present in blood of infected individuals and is able to transduce cells, we hypothesized that it could induce oxidative DNA damage in B-cells promoting genetic instability and malignant transformation. Indeed, incubation of B-cells isolated from healthy donors with purified Tat protein led to oxidative stress, a decrease in the glutathione (GSH) levels, DNA damage and appearance of chromosomal aberrations. The effects of Tat relied on its transcriptional activity and were mediated by NF-κB activation. Tat stimulated oxidative stress in B-cells mostly via mitochondrial ROS production which depended on the reverse electron flow in Complex I of respiratory chain. We propose that Tat-induced oxidative stress, DNA damage and chromosomal aberrations are novel oncogenic factors favoring B-cell lymphomas in HIV-1 infected individuals. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

PubMed Central

Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

2013-01-01

Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial

Protective effects of transduced Tat-DJ-1 protein against oxidative stress and ischemic brain injury.

PubMed

Jeong, Hoon Jae; Kim, Dae Won; Kim, Mi Jin; Woo, Su Jung; Kim, Hye Ri; Kim, So Mi; Jo, Hyo Sang; Hwang, Hyun Sook; Kim, Duk Soo; Cho, Sung Woo; Won, Moo Ho; Han, Kyu Hyung; Park, Jin Seu; Eum, Won Sik; Choi, Soo Young

2012-10-31

Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a doseand time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.

RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

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

Dabney-Smith, Carole

Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembledmore » proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cpTat

Extensive interactions between HIV TAT and TAF(II)250.

PubMed

Weissman, J D; Hwang, J R; Singer, D S

2001-03-09

The HIV transactivator, Tat, has been shown to be capable of potent repression of transcription initiation. Repression is mediated by the C-terminal segment of Tat, which binds the TFIID component, TAF(II)250, although the site(s) of interaction were not defined previously. We now report that the interaction between Tat and TAF(II)250 is extensive and involves multiple contacts between the Tat protein and TAF(II)250. The C-terminal domain of Tat, which is necessary for repression of transcription initiation, binds to a segment of TAF(II)250 that encompasses its acetyl transferase (AT) domain (885-1034 amino acids (aa)). Surprisingly, the N-terminal segment of Tat, which contains its activation domains, also binds to TAF(II)250 and interacts with two discontinuous segments of TAF(II)250 located between 885 and 984 aa and 1120 and 1279 aa. Binding of Tat to the 885-984 aa segment of TAF(II)250 requires the cysteine-rich domain of Tat, but not the acidic or glutamine-rich domains. Binding by the N-terminal domain of Tat to the 1120-1279 aa TAF(II)250 segment does not involve the acidic, cysteine- or glutamine-rich domains. Repression of transcription initiation by Tat requires functional TAF(II)250. We now demonstrate that transcription of the HIV LTR does not depend on TAF(II)250 which may account for its resistance to Tat mediated repression.

FBI-1 can stimulate HIV-1 Tat activity and is targeted to a novel subnuclear domain that includes the Tat-P-TEFb-containing nuclear speckles.

PubMed

Pendergrast, P Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

2002-03-01

FBI-1 is a cellular POZ-domain-containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor-rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.

FBI-1 Can Stimulate HIV-1 Tat Activity and Is Targeted to a Novel Subnuclear Domain that Includes the Tat-P-TEFb—containing Nuclear Speckles

PubMed Central

Pendergrast, P. Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

2002-01-01

FBI-1 is a cellular POZ-domain–containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor–rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription. PMID:11907272

HIV-1 Tat protein promotes formation of more-processive elongation complexes.

PubMed Central

Marciniak, R A; Sharp, P A

1991-01-01

The Tat protein of HIV-1 trans-activates transcription in vitro in a cell-free extract of HeLa nuclei. Quantitative analysis of the efficiency of elongation revealed that a majority of the elongation complexes generated by the HIV-1 promoter were not highly processive and terminated within the first 500 nucleotides. Tat trans-activation of transcription from the HIV-1 promoter resulted from an increase in processive character of the elongation complexes. More specifically, the analysis suggests that there exist two classes of elongation complexes initiating from the HIV promoter: a less-processive form and a more-processive form. Addition of purified Tat protein was found to increase the abundance of the more-processive class of elongation complex. The purine nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits transcription in this reaction by decreasing the efficiency of elongation. Surprisingly, stimulation of transcription elongation by Tat was preferentially inhibited by the addition of DRB. Images PMID:1756726

Synthesis and characterization of tat-mediated O-CMC magnetic nanoparticles having anticancer function

NASA Astrophysics Data System (ADS)

Zhao, Aijie; Yao, Peng; Kang, Chunshang; Yuan, Xubo; Chang, Jin; Pu, Peiyu

2005-08-01

This paper describes a new formulation of magnetic nanoparticles coated by a novel polymer matrix—O-carboxylmethylated chitosan (O-CMC) as drug/gene carrier. The O-CMC magnetic nanoparticles were derivatized with a peptide sequence from the HIV-tat protein to improve the translocational property and cellar uptake of the nanoparticles. To evaluate the O-MNPs-tat as drug carriers, MTX was incorporated as a model drug and MTX-loaded O-MNPs-tat with an average diameter of 45-60 nm were prepared and characterized by TEM, AFM and VSM. The cytotoxicity of MTX-loaded O-MNPs-tat was investigated with U-937 tumor cells. The results showed that the MTX-loaded O-MNPs-tat retained significant antitumor toxicity; additionally, sustained release of MTX from O-CMC nanoparticles was observed in vitro, suggesting that the tat-O-MNPs could be a novel magnetic targeting carrier.

Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

NASA Astrophysics Data System (ADS)

Sun, Xiao-Hong; Baltimore, David

1989-04-01

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

Twin-arginine signal peptide of Bacillus subtilis YwbN can direct Tat-dependent secretion of methyl parathion hydrolase.

PubMed

Liu, Ruihua; Zuo, Zhenqiang; Xu, Yingming; Song, Cunjiang; Jiang, Hong; Qiao, Chuanling; Xu, Ping; Zhou, Qixing; Yang, Chao

2014-04-02

The twin-arginine translocation (Tat) pathway exports folded proteins across the cytoplasmic membranes of bacteria and archaea. Two parallel Tat pathways (TatAdCd and TatAyCy systems) with distinct substrate specificities have previously been discovered in Bacillus subtilis. In this study, to secrete methyl parathion hydrolase (MPH) into the growth medium, the twin-arginine signal peptide of B. subtilis YwbN was used to target MPH to the Tat pathway of B. subtilis. Western blot analysis and MPH assays demonstrated that active MPH was secreted into the culture supernatant of wild-type cells. No MPH secretion occurred in a total-tat2 mutant, indicating that the observed export in wild-type cells was mediated exclusively by the Tat pathway. Export was fully blocked in a tatAyCy mutant. In contrast, the tatAdCd mutant was still capable of secreting MPH. These results indicated that the MPH secretion directed by the YwbN signal peptide was specifically mediated by the TatAyCy system. The N-terminal sequence of secreted MPH was determined as AAPQVR, demonstrating that the YwbN signal peptide had been processed correctly. This is the first report of functional secretion of a heterologous protein via the B. subtilis TatAyCy system. This study highlights the potential of the TatAyCy system to be used for secretion of other heterologous proteins in B. subtilis.

Anti-inflammatory effects of Tat-Annexin protein on ovalbumin-induced airway inflammation in a mouse model of asthma

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

Lee, Sun Hwa; Kim, Dae Won; Kim, Hye Ri

Highlights: Black-Right-Pointing-Pointer We construct a cell permeable Tat-ANX1 fusion protein. Black-Right-Pointing-Pointer We examined the protective effects of Tat-ANX1 protein on OVA-induced asthma in animal models. Black-Right-Pointing-Pointer Transduced Tat-ANX1 protein protects from the OVA-induced production of cytokines and eosinophils in BAL fluid. Black-Right-Pointing-Pointer Tat-ANX1 protein markedly reduced OVA-induced MAPK in lung tissues. Black-Right-Pointing-Pointer Tat-ANX1 protein could be useful as a therapeutic agent for lung disorders including asthma. -- Abstract: Chronic airway inflammation is a key feature of bronchial asthma. Annexin-1 (ANX1) is an anti-inflammatory protein that is an important modulator and plays a key role in inflammation. Although the precise actionmore » of ANX1 remains unclear, it has emerged as a potential drug target for inflammatory diseases such as asthma. To examine the protective effects of ANX1 protein on ovalbumin (OVA)-induced asthma in animal models, we used a cell-permeable Tat-ANX1 protein. Mice sensitized and challenged with OVA antigen had an increased amount of cytokines and eosinophils in their bronchoalveolar lavage (BAL) fluid. However, administration of Tat-ANX1 protein before OVA challenge significantly decreased the levels of cytokines (interleukin (IL)-4, IL-5, and IL-13) and BAL fluid in lung tissues. Furthermore, OVA significantly increased the activation of mitogen-activated protein kinase (MAPK) in lung tissues, whereas Tat-ANX1 protein markedly reduced phosphorylation of MAPKs such as extracellular signal-regulated protein kinase, p38, and stress-activated protein kinase/c-Jun N-terminal kinase. These results suggest that transduced Tat-ANX1 protein may be a potential protein therapeutic agent for the treatment of lung disorders including asthma.« less

Oxidative Stress Is Associated with Neuroinflammation in Animal Models of HIV-1 Tat Neurotoxicity

PubMed Central

Louboutin, Jean-Pierre; Agrawal, Lokesh; Reyes, Beverly A. S.; Van Bockstaele, Elisabeth J.; Strayer, David S.

2014-01-01

HIV-1 trans-acting protein Tat, an essential protein for viral replication, is a key mediator of neurotoxicity. If Tat oxidant injury and neurotoxicity have been described, consequent neuroinflammation is less understood. Rat caudate-putamens (CPs) were challenged with Tat, with or without prior rSV40-delivered superoxide dismutase or glutathione peroxidase. Tat injection caused oxidative stress. Administration of Tat in the CP induced an increase in numbers of Iba-1- and CD68-positive cells, as well as an infiltration of astrocytes. We also tested the effect of more protracted Tat exposure on neuroinflammation using an experimental model of chronic Tat exposure. SV(Tat): a recombinant SV40-derived gene transfer vector was inoculated into the rat CP, leading to chronic expression of Tat, oxidative stress, and ongoing apoptosis, mainly located in neurons. Intra-CP SV(Tat) injection induced an increase in microglia and astrocytes, suggesting that protracted Tat production increased neuroinflammation. SV(SOD1) or SV(GPx1) significantly reduced neuroinflammation following Tat administration into the CP. Thus, Tat-induced oxidative stress, CNS injury, neuron loss and inflammation may be mitigated by antioxidant gene delivery. PMID:26784879

Genetic Evidence for a Tight Cooperation of TatB and TatC during Productive Recognition of Twin-Arginine (Tat) Signal Peptides in Escherichia coli

PubMed Central

Lausberg, Frank; Fleckenstein, Stefan; Kreutzenbeck, Peter; Fröbel, Julia; Rose, Patrick; Müller, Matthias; Freudl, Roland

2012-01-01

The twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K) that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE) by aspartate blocked export of the corresponding TorA(D+2)-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D+2) signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D+2)-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment. PMID:22761916

Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli.

PubMed

Lausberg, Frank; Fleckenstein, Stefan; Kreutzenbeck, Peter; Fröbel, Julia; Rose, Patrick; Müller, Matthias; Freudl, Roland

2012-01-01

The twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K) that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE) by aspartate blocked export of the corresponding TorA(D(+2))-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D(+2)) signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2))-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.

Novel mechanism and factor for regulation by HIV-1 Tat.

PubMed Central

Zhou, Q; Sharp, P A

1995-01-01

Tat regulation of human immunodeficiency virus (HIV) transcription is unique because of its specificity for an RNA target, TAR, and its ability to increase the efficiency of elongation by polymerase. A reconstituted reaction that is Tat-specific and TAR-dependent for activation of HIV transcription has been used to identify and partially purify a cellular activity that is required for trans-activation by Tat, but not by other activators. In the reaction, Tat stimulates the efficiency of elongation by polymerase, whereas Sp1 and other DNA sequence-specific transcription factors activate the rate of initiation. Furthermore, while TATA binding protein (TBP)-associated factors (TAFs) in the TFIID complex are required for activation by transcription factors, they are dispensable for Tat function. Thus, Tat acts through a novel mechanism, which is mediated by a specific host cellular factor, to stimulate HIV-1 gene expression. Images PMID:7835343

The HIV-1 associated protein, Tat1–86, impairs dopamine transporters and interacts with cocaine to reduce nerve terminal function: a no-net-flux microdialysis study

PubMed Central

Ferris, Mark J.; Frederick-Duus, Danielle; Fadel, Jim; Mactutus, Charles F.; Booze, Rosemarie M.

2009-01-01

Injection drug use accounts for approximately one-third of HIV-infections in the United States. HIV associated proteins have been shown to interact with various drugs of abuse to incite concerted neurotoxicity. One common area for their interaction is the nerve terminal, including dopamine transporter (DAT) systems. However, results regarding DAT function and regulation in HIV-infection, regardless of drug use, are mixed. Thus, the present experiments were designed to explicitly control Tat and cocaine administration in an in vivo model in order to reconcile differences that exist in the literature to date. We examined Tat plus cocaine-induced alterations using no-net-flux microdialysis, which is sensitive to alterations in DAT function, in order to test the potential for DAT as an early mediator of HIV-induced oxidative stress and neurodegeneration in vivo. Within 5 hours of intra-accumbal administration of the HIV-associated protein, Tat, we noted a significant reduction in local DAT efficiency with little change in DA overflow/release dynamics. Further, at 48 hrs post-Tat administration, we demonstrated a concerted effect of the HIV-protein Tat with cocaine on both uptake and release function. Finally, we discuss the extent to which DAT dysfunction may be considered a predecessor to generalized nerve terminal dysfunction. Characterization of DAT dysfunction in vivo may provide an early pharamacotherapeutic target, which in turn may prevent or attenuate downstream mediators of neurotoxicity (i.e., reactive species) to DA systems occurring in NeuroAIDS. PMID:19344635

Clinicopathologic significance of minichromosome maintenance protein 2 and Tat-interacting protein 30 expression in benign and malignant lesions of the gallbladder.

PubMed

Liu, Dong-cai; Yang, Zhu-lin

2011-11-01

Gallbladder cancers are aggressive tumors with a poor prognosis and high mortality rate. To find specific biological markers for early diagnosis and prognosis and to develop possible alternative treatment strategies, we examined minichromosome maintenance protein 2 (MCM2) and Tat-interacting protein 30 (TIP30) expression in 108 gallbladder adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. Expression of MCM2 was significantly higher in adenocarcinomas than in peritumoral tissues (χ² = 8.41; P < .01), adenomatous polyps (χ² = 6.81; P < .01), and chronic cholecystitis (χ² = 21.00; P < .01). In contrast, Tat-interacting protein 30 expression was significantly less in adenocarcinomas than in peritumoral tissues (χ² = 13.26; P < .01), adenomatous polyps (χ² = 4.76; P < .05), and chronic cholecystitis (χ² = 18.93; P < .01). The benign lesions in gallbladder epithelium with positive MCM2 or negative Tat-interacting protein 30 expression showed moderate to severe atypical hyperplasia. Expression of MCM2 and absence of Tat-interacting protein 30 were significantly associated with poor differentiation, large tumor mass, lymph node metastasis, and invasion of adenocarcinoma. Univariate Kaplan-Meier analysis showed that either elevated MCM2 (P = .006) or lowered Tat-interacting protein 30 (P = .006) expression was closely associated with shorter overall survival. Multivariate Cox regression analysis revealed that expression of MCM2 (P = .007) or nonexpression of Tat-interacting protein 30 (P = .009) was an independent predictor of a poor prognosis in adenocarcinoma. Our results suggest that overexpression of MCM2 or loss of expression of Tat-interacting protein 30 is closely related to carcinogenesis, progression, biological behavior, and prognosis of gallbladder adenocarcinoma. Copyright © 2011 Elsevier Inc. All rights reserved.

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

PubMed Central

Roy, Chandra Nath; Promjunyakul, Warunya; Hatakeyama, Hiroyasu; Gonda, Kohsuke; Imamura, Junji; Vasudevanpillai, Biju; Ohuchi, Noriaki; Kanzaki, Makoto; Higuchi, Hideo; Kaku, Mitsuo

2013-01-01

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines. PMID:23732912

Identification of amino acids that promote specific and rigid TAR RNA-tat protein complex formation.

PubMed

Edwards, Thomas E; Robinson, Bruce H; Sigurdsson, Snorri Th

2005-03-01

The Tat protein and the transactivation responsive (TAR) RNA form an essential complex in the HIV lifecycle, and mutations in the basic region of the Tat protein alter this RNA-protein molecular recognition. Here, EPR spectroscopy was used to identify amino acids, flanking an essential arginine of the Tat protein, which contribute to specific and rigid TAR-Tat complex formation by monitoring changes in the mobility of nitroxide spin-labeled TAR RNA nucleotides upon binding. Arginine to lysine N-terminal mutations did not affect TAR RNA interfacial dynamics. In contrast, C-terminal point mutations, R56 in particular, affected the mobility of nucleotides U23 and U38, which are involved in a base-triple interaction in the complex. This report highlights the role of dynamics in specific molecular complex formation and demonstrates the ability of EPR spectroscopy to study interfacial dynamics of macromolecular complexes.

A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

PubMed

Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J

2016-06-30

DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans.

Pressure-induced endocytic degradation of the Saccharomyces cerevisiae low-affinity tryptophan permease Tat1 is mediated by Rsp5 ubiquitin ligase and functionally redundant PPxY motif proteins.

PubMed

Suzuki, Asaha; Mochizuki, Takahiro; Uemura, Satoshi; Hiraki, Toshiki; Abe, Fumiyoshi

2013-07-01

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1(K29R-K31R)-GFP remained. The HPG1-1 (Rsp5(P514T)) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.

Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

PubMed Central

Suzuki, Asaha; Mochizuki, Takahiro; Uemura, Satoshi; Hiraki, Toshiki

2013-01-01

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1K29R-K31R-GFP remained. The HPG1-1 (Rsp5P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure. PMID:23666621

Human immunodeficiency virus-1 protein Tat induces excitotoxic loss of presynaptic terminals in hippocampal cultures.

PubMed

Shin, Angela H; Thayer, Stanley A

2013-05-01

Human immunodeficiency virus (HIV) infection of the CNS produces dendritic damage that correlates with cognitive decline in patients with HIV-associated neurocognitive disorders (HAND). HIV-induced neurotoxicity results in part from viral proteins shed from infected cells, including the HIV transactivator of transcription (Tat). We previously showed that Tat binds to the low density lipoprotein receptor-related protein (LRP), resulting in overactivation of NMDA receptors, activation of the ubiquitin-proteasome pathway, and subsequent loss of postsynaptic densities. Here, we show that Tat also induces a loss of presynaptic terminals. The number of presynaptic terminals was quantified using confocal imaging of synaptophysin fused to green fluorescent protein (Syn-GFP). Tat-induced loss of presynaptic terminals was secondary to excitatory postsynaptic mechanisms because treatment with an LRP antagonist or an NMDA receptor antagonist inhibited this loss. Treatment with nutlin-3, an E3 ligase inhibitor, prevented Tat-induced loss of presynaptic terminals. These data suggest that Tat-induced loss of presynaptic terminals is a consequence of excitotoxic postsynaptic activity. We previously found that ifenprodil, an NR2B subunit-selective NMDA receptor antagonist, induced recovery of postsynaptic densities. Here we show that Tat-induced loss of presynaptic terminals was reversed by ifenprodil treatment. Thus, Tat-induced loss of presynaptic terminals is reversible, and this recovery can be initiated by inhibiting a subset of postsynaptic NMDA receptors. Understanding the dynamics of synaptic changes in response to HIV infection of the CNS may lead to the design of improved pharmacotherapies for HAND patients. Copyright © 2012 Elsevier Inc. All rights reserved.

HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

PubMed

Kesby, James P; Najera, Julia A; Romoli, Benedetto; Fang, Yiding; Basova, Liana; Birmingham, Amanda; Marcondes, Maria Cecilia G; Dulcis, Davide; Semenova, Svetlana

2017-10-01

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

PubMed Central

Park, Jung-Sun; Park, Soo-Young; Cho, Hyun-Il; Sohn, Hyun-Jung

2011-01-01

Background Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-γ ELISPOT assay, cytotoxicity assay and tetramer staining. Results DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines. PMID:21860612

The effects of HIV-1 regulatory TAT protein expression on brain reward function, response to psychostimulants and delay-dependent memory in mice.

PubMed

Kesby, James P; Markou, Athina; Semenova, Svetlana

2016-10-01

Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport

PubMed Central

Quizon, Pamela M.; Sun, Wei-Lun; Yuan, Yaxia; Midde, Narasimha M.; Zhan, Chang-Guo; Zhu, Jun

2016-01-01

Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission. PMID:27966610

Oral Administration of TAT-PTD-Diapause Hormone Fusion Protein Interferes With Helicoverpa armigera (Lepidoptera: Noctuidae) Development.

PubMed

Zhou, Zhou; Li, Yongli; Yuan, Chunyan; Zhang, Yongan; Qu, Liangjian

2015-01-01

Diapause hormone (DH), which can terminate diapause in Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), has shown promise as a pest control method. However, the main challenge in using DH as an insecticide lies in achieving effective oral delivery, since the peptide may be degraded by digestive enzymes in the gut. To improve the efficacy of oral DH application, the Clostera anastomosis (L.) (Lepidoptera: Notodontidae) diapause hormone (caDH) was fused to the Protein Transduction Domain (PTD) of the human immunodeficiency virus-1 transactivator of transcription (TAT). Cellular transduction of TAT-caDH was verified with the use of a green fluorescent protein fusion, and its ability to terminate diapause was verified by injection into diapausing H. armigera pupae. Orally administered TAT-caDH resulted in larval growth inhibition. In TAT-caDH-treated insects, larval duration was delayed and the pupation rates were decreased at both development promoting conditions [27 °C, a photoperiod of 14:10(L:D) h] and diapause inducing conditions [20 °C, a photoperiod of 10:14(L:D) h]. No significant difference in diapause rate was observed between the TAT-caDH-treated and caDH-treated or control pupae maintained at diapause inducing conditions. Our results show that treatment with a recombinant TAT-caDH protein can affect larval development in H. armigera, and it suggest that TAT-DH treatment may be useful for controlling pests. This study is the first record of oral DH application in insect. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

PPAR agonist-mediated protection against HIV Tat-induced cerebrovascular toxicity is enhanced in MMP-9-deficient mice

PubMed Central

Huang, Wen; Chen, Lei; Zhang, Bei; Park, Minseon; Toborek, Michal

2014-01-01

The strategies to protect against the disrupted blood–brain barrier (BBB) in HIV-1 infection are not well developed. Therefore, we investigated the potential of peroxisome proliferator-activated receptor (PPAR) agonists to prevent enhanced BBB permeability induced by HIV-1-specific protein Tat. Exposure to Tat via the internal carotid artery (ICA) disrupted permeability across the BBB; however, this effect was attenuated in mice treated with fenofibrate (PPARα agonist) or rosiglitazone (PPARγ agonist). In contrast, exposure to GW9662 (PPARγ antagonist) exacerbated Tat-induced disruption of the BBB integrity. Increased BBB permeability was associated with decreased tight junction (TJ) protein expression and activation of ERK1/2 and Akt in brain microvessels; these effects were attenuated by cotreatment with fenofibrate but not with rosiglitazone. Importantly, both PPAR agonists also protected against Tat-induced astrogliosis and neuronal loss. Because disruption of TJ integrity has been linked to matrix metalloproteinase (MMP) activity, we also evaluated Tat-induced effects in MMP-9-deficient mice. Tat-induced cerebrovascular toxicity, astrogliosis, and neuronal loss were less pronounced in MMP-9-deficient mice as compared with wild-type controls and were further attenuated by PPAR agonists. These results indicate that enhancing PPAR activity combined with targeting MMPs may provide effective therapeutic strategies in brain infection by HIV-1. PMID:24424383

Detection of human immunodeficiency virus type 1 (HIV-1) Tat protein by aptamer-based biosensors

NASA Astrophysics Data System (ADS)

Hashim, Uda; Fatin, M. F.; Ruslinda, A. R.; Gopinath, Subash C. B.; Uda, M. N. A.

2017-03-01

A study was conducted to detect the human immunodeficiency virus (HIV-1) Tat protein using interdigitated electrodes. The measurements and images of the IDEs' finger gaps and the images of chitosan-carbon nanotubes deposited on top of the interdigitated electrodes were taken using the Scanning Electron Microscope. The detection of HIV-1 Tat protein was done using split aptamers and aptamer tail. Biosensors were chosen as diagnostic equipment due to their rapid diagnostic capabilities.

Phase I therapeutic trial of the HIV-1 Tat protein and long term follow-up.

PubMed

Longo, Olimpia; Tripiciano, Antonella; Fiorelli, Valeria; Bellino, Stefania; Scoglio, Arianna; Collacchi, Barbara; Alvarez, Maria Josè Ruiz; Francavilla, Vittorio; Arancio, Angela; Paniccia, Giovanni; Lazzarin, Adriano; Tambussi, Giuseppe; Din, Chiara Tassan; Visintini, Raffaele; Narciso, Pasquale; Antinori, Andrea; D'Offizi, Gianpiero; Giulianelli, Marina; Carta, Maria; Di Carlo, Aldo; Palamara, Guido; Giuliani, Massimo; Laguardia, Maria Elena; Monini, Paolo; Magnani, Mauro; Ensoli, Fabrizio; Ensoli, Barbara

2009-05-26

A randomized, double blind, placebo-controlled phase I vaccine trial based on the native Tat protein was conducted in HIV-infected asymptomatic individuals. The vaccine was administered five times subcute with alum or intradermally without adjuvant at 7.5microg, 15microg or 30microg doses, respectively. The Tat vaccine was well tolerated both locally and systemically and induced and/or maintained Tat-specific T helper (Th)-1 T-cell responses and Th-2 responses in all subjects with a wide spectrum of functional anti-Tat antibodies, rarely seen in HIV-infected subjects. The data indicate the achievement of both the primary (safety) and secondary (immunogenicity) endpoints of the study.

Regulation of the Human Endogenous Retrovirus K (HML-2) Transcriptome by the HIV-1 Tat Protein

PubMed Central

Gonzalez-Hernandez, Marta J.; Cavalcoli, James D.; Sartor, Maureen A.; Contreras-Galindo, Rafael; Meng, Fan; Dai, Manhong; Dube, Derek; Saha, Anjan K.; Gitlin, Scott D.; Omenn, Gilbert S.; Kaplan, Mark H.

2014-01-01

ABSTRACT Approximately 8% of the human genome is made up of endogenous retroviral sequences. As the HIV-1 Tat protein activates the overall expression of the human endogenous retrovirus type K (HERV-K) (HML-2), we used next-generation sequencing to determine which of the 91 currently annotated HERV-K (HML-2) proviruses are regulated by Tat. Transcriptome sequencing of total RNA isolated from Tat- and vehicle-treated peripheral blood lymphocytes from a healthy donor showed that Tat significantly activates expression of 26 unique HERV-K (HML-2) proviruses, silences 12, and does not significantly alter the expression of the remaining proviruses. Quantitative reverse transcription-PCR validation of the sequencing data was performed on Tat-treated PBLs of seven donors using provirus-specific primers and corroborated the results with a substantial degree of quantitative similarity. IMPORTANCE The expression of HERV-K (HML-2) is tightly regulated but becomes markedly increased following infection with HIV-1, in part due to the HIV-1 Tat protein. The findings reported here demonstrate the complexity of the genome-wide regulation of HERV-K (HML-2) expression by Tat. This work also demonstrates that although HERV-K (HML-2) proviruses in the human genome are highly similar in terms of DNA sequence, modulation of the expression of specific proviruses in a given biological situation can be ascertained using next-generation sequencing and bioinformatics analysis. PMID:24872592

The intracellular delivery of TAT-aequorin reveals calcium-mediated sensing of environmental and symbiotic signals by the arbuscular mycorrhizal fungus Gigaspora margarita.

PubMed

Moscatiello, Roberto; Sello, Simone; Novero, Mara; Negro, Alessandro; Bonfante, Paola; Navazio, Lorella

2014-08-01

Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Impact of Genetic Variations in HIV-1 Tat on LTR-Mediated Transcription via TAR RNA Interaction.

PubMed

Ronsard, Larance; Ganguli, Nilanjana; Singh, Vivek K; Mohankumar, Kumaravel; Rai, Tripti; Sridharan, Subhashree; Pajaniradje, Sankar; Kumar, Binod; Rai, Devesh; Chaudhuri, Suhnrita; Coumar, Mohane S; Ramachandran, Vishnampettai G; Banerjea, Akhil C

2017-01-01

HIV-1 evades host defense through mutations and recombination events, generating numerous variants in an infected patient. These variants with an undiminished virulence can multiply rapidly in order to progress to AIDS. One of the targets to intervene in HIV-1 replication is the trans -activator of transcription (Tat), a major regulatory protein that transactivates the long terminal repeat promoter through its interaction with trans -activation response (TAR) RNA. In this study, HIV-1 infected patients ( n = 120) from North India revealed Ser46Phe (20%) and Ser61Arg (2%) mutations in the Tat variants with a strong interaction toward TAR leading to enhanced transactivation activities. Molecular dynamics simulation data verified that the variants with this mutation had a higher binding affinity for TAR than both the wild-type Tat and other variants that lacked Ser46Phe and Ser61Arg. Other mutations in Tat conferred varying affinities for TAR interaction leading to differential transactivation abilities. This is the first report from North India with a clinical validation of CD4 counts to demonstrate the influence of Tat genetic variations affecting the stability of Tat and its interaction with TAR. This study highlights the co-evolution pattern of Tat and predominant nucleotides for Tat activity, facilitating the identification of genetic determinants for the attenuation of viral gene expression.

HIV-tat alters Connexin43 expression and trafficking in human astrocytes: role in NeuroAIDS.

PubMed

Berman, Joan W; Carvallo, Loreto; Buckner, Clarisa M; Luers, Aimée; Prevedel, Lisa; Bennett, Michael V; Eugenin, Eliseo A

2016-03-02

HIV-associated neurocognitive disorders (HAND) are a major complication in at least half of the infected population despite effective antiretroviral treatment and immune reconstitution. HIV-associated CNS damage is not correlated with active viral replication but instead is associated with mechanisms that regulate inflammation and neuronal compromise. Our data indicate that one of these mechanisms is mediated by gap junction channels and/or hemichannels. Normally, gap junction channels shutdown under inflammatory conditions, including viral diseases. However, HIV infection upregulates Connexin43 (Cx43) expression and maintains gap junctional communication by unknown mechanism(s). Human primary astrocytes were exposed to several HIV proteins as well as to HIV, and expression and function of Connexin43- and Connexin30-containing channels were determined by western blot, immunofluorescence, microinjection of a fluorescent tracer and chromatin immunoprecipitation (ChIP). Here, we demonstrate that HIV infection increases Cx43 expression in vivo. HIV-tat, the transactivator of the virus, and no other HIV proteins tested, increases Cx43 expression and maintains functional gap junctional communication in human astrocytes. Cx43 upregulation is mediated by binding of the HIV-tat protein to the Cx43 promoter, but not to the Cx30 promoter, resulting in increased Cx43 messenger RNA (mRNA) and protein as well as gap junctional communication. We propose that HIV-tat contributes to the spread of intracellular toxic signals generated in a few HIV-infected cells into surrounding uninfected cells by upregulating gap junctional communication. In the current antiretroviral era, where HIV replication is often completely suppressed, viral factors such as HIV-tat are still produced and released from infected cells. Thus, blocking the effects of HIV-tat could result in new strategies to reduce the damaging consequences of HIV infection of the CNS.

CAVEOLIN-1 REGULATES HIV-1 TAT-INDUCED ALTERATIONS OF TIGHT JUNCTION PROTEIN EXPRESSION VIA MODULATION OF THE RAS SIGNALING

PubMed Central

Zhong, Yu; Smart, Eric J.; Weksler, Babette; Couraud, Pierre-Olivier; Hennig, Bernhard; Toborek, Michal

2009-01-01

The blood-brain barrier (BBB) is the critical structure for preventing HIV trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1 infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMEC). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMEC. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions upon Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients. PMID:18667611

Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

NASA Astrophysics Data System (ADS)

Filikov, Anton V.; James, Thomas L.

1998-05-01

A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli.

PubMed

Caldinelli, Laura; Albani, Diego; Pollegioni, Loredano

2013-04-04

Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.

Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice

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

Kim, Young Nam; Kim, Dae Won; Jo, Hyo Sang

Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB)more » and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases. - Highlights: • Transduced Tat-CBR1 reduces LPS-induced inflammatory mediators and cytokines. • Tat-CBR1 inhibits MAPK and NF-κB activation. • Tat-CBR1 ameliorates inflammation response in vitro and in vivo. • Tat-CBR1 may be useful as potential therapeutic agent for inflammation.« less

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

PubMed

Sosic, Alice; Saccone, Irene; Carraro, Caterina; Kenderdine, Thomas; Gamba, Elia; Caliendo, Giuseppe; Corvino, Angela; Di Vaio, Paola; Fiorino, Ferdinando; Magli, Elisa; Perissutti, Elisa; Santagada, Vincenzo; Severino, Beatrice; Spada, Valentina; Fabris, Dan; Frecentese, Francesco; Gatto, Barbara

2018-06-12

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

Progesterone protects normative anxiety-like responding among ovariectomized female mice that conditionally express the HIV-1 regulatory protein, Tat, in the CNS.

PubMed

Paris, Jason J; Fenwick, Jason; McLaughlin, Jay P

2014-05-01

Increased anxiety is co-morbid with human immunodeficiency virus (HIV) infection. Actions of the neurotoxic HIV-1 regulatory protein, Tat, may contribute to affective dysfunction. We hypothesized that Tat expression would increase anxiety-like behavior of female GT-tg bigenic mice that express HIV-1 Tat protein in the brain in a doxycycline-dependent manner. Furthermore, given reports that HIV-induced anxiety may occur at lower rates among women, and that the neurotoxic effects of Tat are ameliorated by sex steroids in vitro, we hypothesized that 17β-estradiol and/or progesterone would ameliorate Tat-induced anxiety-like effects. Among naturally-cycling proestrous and diestrous mice, Tat-induction via 7days of doxycycline treatment significantly increased anxiety-like responding in an open field, elevated plus maze and a marble-burying task, compared to treatment with saline. Proestrous mice demonstrated less anxiety-like behavior than diestrous mice in the open field and elevated plus maze, but these effects did not significantly interact with Tat-induction. Among ovariectomized mice, doxycycline-induced Tat protein significantly increased anxiety-like behavior in an elevated plus maze and a marble burying task compared to saline-treated mice, but not an open field (where anxiety-like responding was already maximal). Co-administration of progesterone (4mg/kg), but not 17β-estradiol (0.09mg/kg), with doxycycline significantly ameliorated anxiety-like responding in the elevated plus maze and marble burying tasks. When administered together, 17β-estradiol partially antagonized the protective effects of progesterone on Tat-induced anxiety-like behavior. These findings support evidence of steroid-protection over HIV-1 proteins, and extend them by demonstrating the protective capacity of progesterone on Tat-induced anxiety-like behavior of ovariectomized female mice. Copyright © 2014 Elsevier Inc. All rights reserved.

Efficient induction of anti-tumor immunity by a TAT-CEA fusion protein vaccine with poly(I:C) in a murine colorectal tumor model.

PubMed

Park, Jung-Sun; Kim, Hye-Sung; Park, Hye-Mi; Kim, Chang-Hyun; Kim, Tai-Gyu

2011-11-03

Protein vaccines may be a useful strategy for cancer immunotherapy because recombinant tumor antigen proteins can be produced on a large scale at relatively low cost and have been shown to be safe for clinical application. However, protein vaccines have historically exhibited poor immunogenicity; thus, an improved strategy is needed for successful induction of immune responses. TAT peptide is a protein transduction domain composed of an 11-amino acid peptide (TAT(47-57): YGRKKRRQRRR). The positive charge of this peptide allows protein antigen fused with it to improve cell penetration. Poly(I:C) is a synthetic double-stranded RNA that is negatively charged and favors interaction with the cationic TAT peptide. Poly(I:C) has been reported on adjuvant role in tumor vaccine through promotion of immune responses. Therefore, we demonstrated that vaccine with a mixture of TAT-CEA fusion protein and poly(I:C) can induce anti-tumor immunity in a murine colorectal tumor model. Splenocytes from mice vaccinated with a mixture of TAT-CEA fusion protein and poly(I:C) effectively induced CEA-specific IFN-γ-producing T cells and showed cytotoxic activity specific for MC-38-cea2 tumor cells expressing CEA. Vaccine with a mixture of TAT-CEA fusion protein and poly(I:C) delayed tumor growth in MC-38-cea-2 tumor-bearing mice. Depletion of CD8(+) T cells and NK cells reversed the inhibition of tumor growth in an MC-38-cea2-bearing mice, indicating that CD8(+) T cells and NK cells are responsible for anti-tumor immunity by vaccine with a mixture of TAT-CEA fusion protein and poly(I:C). Taken together, these results suggest that poly(I:C) could be used as a potent adjuvant to induce the anti-tumor immunity of a TAT-CEA fusion protein vaccine in a murine colorectal tumor model. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular Dynamics Simulation and Experimental Verification of the Interaction between Cyclin T1 and HIV-1 Tat Proteins

PubMed Central

Asamitsu, Kaori; Hibi, Yurina

2015-01-01

The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2’ helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1’ and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound. PMID:25781978

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

PubMed

Lin, Min-Hsuan; Sivakumaran, Haran; Jones, Alun; Li, Dongsheng; Harper, Callista; Wei, Ting; Jin, Hongping; Rustanti, Lina; Meunier, Frederic A; Spann, Kirsten; Harrich, David

2014-12-14

Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased

Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

PubMed Central

Sonnemann, Kevin J.; Heun-Johnson, Hanke; Turner, Amy J.; Baltgalvis, Kristen A.; Lowe, Dawn A.; Ervasti, James M.

2009-01-01

Background The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or ΔR4-21 “micro” utrophin (μUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice. Methods and Findings Recombinant TAT-Utr and TAT-μUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-μUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290±920 U versus 5,950±1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%±5% versus 37%±4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%±5% versus 40%±8% drop; PBS versus TAT), and increased specific force production (9.7±1.1 N/cm2 versus 12.8±0.9 N/cm2; PBS versus TAT). Conclusions These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin. PMID:19478831

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

PubMed

Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

2012-11-02

HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

A mini-review of TAT-MyoD fused proteins: state of the art and problems to solve.

PubMed

Patruno, Marco; Melotti, Luca; Gomiero, Chiara; Sacchetto, Roberta; Topel, Ohad; Martinello, Tiziana

2017-12-05

The transcriptional activator TAT is a small peptide essential for viral replication and possesses the property of entering the cells from the extracellular milieu, acting as a membrane shuttle. In order to safely differentiate cells an innovative methodology, based on the fusion of transcription factors and the TAT sequence, is discussed in this short review. In several studies, it has been demonstrated that TAT protein can be observed in the cell nucleus after few hours from the inoculation although its way of action is not fully understood. However, further studies will be necessary to develop this methodology for clinical purposes.

HIV-1 Proteins, Tat and gp120, Target the Developing Dopamine System

PubMed Central

Fitting, Sylvia; Booze, Rosemarie M.; Mactutus, Charles F.

2015-01-01

In 2014, 3.2 million children (< 15 years of age) were estimated to be living with HIV and AIDS worldwide, with the 240,000 newly infected children in the past year, i.e., another child infected approximately every two minutes [1]. The primary mode of HIV infection is through mother-to-child transmission (MTCT), occurring either in utero, intrapartum, or during breastfeeding. The effects of HIV-1 on the central nervous system (CNS) are putatively accepted to be mediated, in part, via viral proteins, such as Tat and gp120. The current review focuses on the targets of HIV-1 proteins during the development of the dopamine (DA) system, which appears to be specifically susceptible in HIV-1-infected children. Collectively, the data suggest that the DA system is a clinically relevant target in chronic HIV-1 infection, is one of the major targets in pediatric HIV-1 CNS infection, and may be specifically susceptible during development. The present review discusses the development of the DA system, follows the possible targets of the HIV-1 proteins during the development of the DA system, and suggests potential therapeutic approaches. By coupling our growing understanding of the development of the CNS with the pronounced age-related differences in disease progression, new light may be shed on the neurological and neurocognitive deficits that follow HIV-1 infection. PMID:25613135

Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3.

PubMed

Kim, Jiyoung; Kim, Yeon-Soo

2018-06-06

Human immunodeficiency virus type 1 (HIV-1) Tat, an important regulator of viral transcription, interacts with diverse cellular proteins and promotes or inhibits cell proliferation. Here, we show that ribosomal protein S3 (RPS3) plays an important role in mitosis through an interaction with α-tubulin and that Tat binds to and inhibits the localization of RPS3 in the mitotic spindle during mitosis. RPS3 colocalized with α-tubulin around chromosomes in the mitotic spindle. Depletion of RPS3 promoted α-tubulin assembly, while overexpression of RPS3 impaired α-tubulin assembly. Depletion of RPS3 resulted in aberrant mitotic spindle formation, segregation failure, and defective abscission. Moreover, ectopic expression of RPS3 rescued the cell proliferation defect in RPS3-knockdown cells. HIV-1 Tat interacted with RPS3 through its basic domain and increased the level of RPS3 in the nucleus. Expression of Tat caused defects in mitotic spindle formation and chromosome assembly in mitosis. Moreover, the localization of RPS3 in the mitotic spindle was disrupted when HIV-1 Tat was expressed in HeLa and Jurkat cells. These results suggest that Tat inhibits cell proliferation via an interaction with RPS3 and thereby disrupts mitotic spindle formation during HIV-1 infection. These results might provide insight into the mechanism underlying lymphocyte pathogenesis during HIV-1 infection.

HIV-1 Tat-based vaccines: from basic science to clinical trials.

PubMed

Fanales-Belasio, Emanuele; Cafaro, Aurelio; Cara, Andrea; Negri, Donatella R M; Fiorelli, Valeria; Butto, Stefano; Moretti, Sonia; Maggiorella, Maria Teresa; Baroncelli, Silvia; Michelini, Zuleika; Tripiciano, Antonella; Sernicola, Leonardo; Scoglio, Arianna; Borsetti, Alessandra; Ridolfi, Barbara; Bona, Roberta; Ten Haaft, Peter; Macchia, Iole; Leone, Pasqualina; Pavone-Cossut, Maria Rosaria; Nappi, Filomena; Vardas, Eftyhia; Magnani, Mauro; Laguardia, Elena; Caputo, Antonella; Titti, Fausto; Ensoli, Barbara

2002-09-01

Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.

Transduced Tat-DJ-1 Protein Protects against Oxidative Stress-Induced SH-SY5Y Cell Death and Parkinson Disease in a Mouse Model

PubMed Central

Jeong, Hoon Jae; Kim, Dae Won; Woo, Su Jung; Kim, Hye Ri; Kim, So Mi; Jo, Hyo Sang; Park, Meeyoung; Kim, Duk-Soo; Kwon, Oh-Shin; Hwang, In Koo; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

2012-01-01

Parkinson’s disease (PD) is a well known neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compact (SN). Although the exact mechanism remains unclear, oxidative stress plays a critical role in the pathogenesis of PD. DJ-1 is a multifunctional protein, a potent antioxidant and chaperone, the loss of function of which is linked to the autosomal recessive early onset of PD. Therefore, we investigated the protective effects of DJ-1 protein against SH-SY5Y cells and in a PD mouse model using a cell permeable Tat-DJ-1 protein. Tat-DJ-1 protein rapidly transduced into the cells and showed a protective effect on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death by reducing the reactive oxygen species (ROS). In addition, we found that Tat-DJ-1 protein protects against dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-induced PD mouse models. These results suggest that Tat-DJ-1 protein provides a potential therapeutic strategy for against ROS related human diseases including PD. PMID:22526393

TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells.

PubMed

Erlich-Hadad, Tal; Hadad, Rita; Feldman, Anat; Greif, Hagar; Lictenstein, Michal; Lorberboum-Galski, Haya

2018-03-01

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Antibodies to the HIV-1 Tat protein correlated with nonprogression to AIDS: a rationale for the use of Tat toxoid as an HIV-1 vaccine.

PubMed

Zagury, J F; Sill, A; Blattner, W; Lachgar, A; Le Buanec, H; Richardson, M; Rappaport, J; Hendel, H; Bizzini, B; Gringeri, A; Carcagno, M; Criscuolo, M; Burny, A; Gallo, R C; Zagury, D

1998-01-01

To investigate which immune parameters, such as antibodies against HIV-1 specificities, or viral parameters, such as p24 antigenemia, are predictive of disease progression. We performed studies on serum collected from individuals exhibiting two extremes of disease evolution--67 fast progressors (FP) and 182 nonprogressors (NP)--at their enrollment. After a 1- to 2-year clinical follow-up of 104 nonprogressors after their enrollment, we could determine the best serologic predictors for disease progression. We investigated levels of antibodies to tetanus toxoid and to HIV antigens including Env, Gag, Nef, and Tat proteins, as well as p24 antigenemia, viremia, CD4 cell count, and interferon-alpha (IFN-alpha) titers in FPs and NPs, and we correlated these data with clinical and biologic signs of progression. p24 Antigenemia, a marker of viral replication, and anti-Tat antibodies were highly and inversely correlated in both groups (P < .001). Furthermore, anti-p24 antibodies and low serum IFN-alpha levels were correlated to the NP versus the FP cohort. Finally, among NPs, only antibodies to Tat and not to the other HIV specificities (Env, Nef, Gag) were significantly predictive of clinical stability during their follow-up. Antibodies toward HIV-1 Tat, which are inversely correlated to p24 antigenemia, appear as a critical marker for a lack of disease progression. This study strongly suggests that rising anti-Tat antibodies through active immunization may be beneficial in AIDS vaccine development to control viral replication.

A Role for p38 Mitogen-activated Protein Kinase-mediated Threonine 30-dependent Norepinephrine Transporter Regulation in Cocaine Sensitization and Conditioned Place Preference*

PubMed Central

Mannangatti, Padmanabhan; NarasimhaNaidu, Kamalakkannan; Damaj, Mohamad Imad; Ramamoorthy, Sammanda; Jayanthi, Lankupalle Damodara

2015-01-01

The noradrenergic and p38 mitogen-activated protein kinase (p38 MAPK) systems are implicated in cocaine-elicited behaviors. Previously, we demonstrated a role for p38 MAPK-mediated norepinephrine transporter (NET) Thr30 phosphorylation in cocaine-induced NET up-regulation (Mannangatti, P., Arapulisamy, O., Shippenberg, T. S., Ramamoorthy, S., and Jayanthi, L. D. (2011) J. Biol. Chem. 286, 20239–20250). The present study explored the functional interaction between p38 MAPK-mediated NET regulation and cocaine-induced behaviors. In vitro cocaine treatment of mouse prefrontal cortex synaptosomes resulted in enhanced NET function, surface expression, and phosphorylation. Pretreatment with PD169316, a p38 MAPK inhibitor, completely blocked cocaine-mediated NET up-regulation and phosphorylation. In mice, in vivo administration of p38 MAPK inhibitor SB203580 completely blocked cocaine-induced NET up-regulation and p38 MAPK activation in the prefrontal cortex and nucleus accumbens. When tested for cocaine-induced locomotor sensitization and conditioned place preference (CPP), mice receiving SB203580 on cocaine challenge day or on postconditioning test day exhibited significantly reduced cocaine sensitization and CPP. A transactivator of transcription (TAT) peptide strategy was utilized to test the involvement of the NET-Thr30 motif. In vitro treatment of synaptosomes with TAT-NET-Thr30 (wild-type peptide) completely blocked cocaine-mediated NET up-regulation and phosphorylation. In vivo administration of TAT-NET-Thr30 peptide but not TAT-NET-T30A (mutant peptide) completely blocked cocaine-mediated NET up-regulation and phosphorylation. In the cocaine CPP paradigm, mice receiving TAT-NET-Thr30 but not TAT-NET-T30A on postconditioning test day exhibited significantly reduced cocaine CPP. Following extinction, TAT-NET-Thr30 when given prior to cocaine challenge significantly reduced reinstatement of cocaine CPP. These results demonstrate that the direct inhibition of p38

Sequence conservation and antibody cross-recognition of clade B human immunodeficiency virus (HIV) type 1 Tat protein in HIV-1-infected Italians, Ugandans, and South Africans.

PubMed

Buttò, Stefano; Fiorelli, Valeria; Tripiciano, Antonella; Ruiz-Alvarez, Maria J; Scoglio, Arianna; Ensoli, Fabrizio; Ciccozzi, Massimo; Collacchi, Barbara; Sabbatucci, Michela; Cafaro, Aurelio; Guzmán, Carlos A; Borsetti, Alessandra; Caputo, Antonella; Vardas, Eftyhia; Colvin, Mark; Lukwiya, Matthew; Rezza, Giovanni; Ensoli, Barbara

2003-10-15

We determined immune cross-recognition and the degree of Tat conservation in patients infected by local human immunodeficiency virus (HIV) type 1 strains. The data indicated a similar prevalence of total and epitope-specific anti-Tat IgG in 578 serum samples from HIV-infected Italian (n=302), Ugandan (n=139), and South African (n=137) subjects, using the same B clade Tat protein that is being used in vaccine trials. In particular, anti-Tat antibodies were detected in 13.2%, 10.8%, and 13.9% of HIV-1-infected individuals from Italy, Uganda, and South Africa, respectively. Sequence analysis results indicated a high similarity of Tat from the different circulating viruses with BH-10 Tat, particularly in the 1-58 amino acid region, which contains most of the immunogenic epitopes. These data indicate an effective cross-recognition of a B-clade laboratory strain-derived Tat protein vaccine by individuals infected with different local viruses, owing to the high similarity of Tat epitopes.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

PubMed

Cappelletti, Pamela; Binda, Elisa; Tunesi, Marta; Albani, Diego; Giordano, Carmen; Molla, Gianluca; Pollegioni, Loredano

2017-10-01

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Imaging HIV-1 Tat Trafficking and Interactions by Engineered Green-Fluorescent-Protein Tagging

NASA Astrophysics Data System (ADS)

Beltram, Fabio

2002-03-01

The direct monitoring of protein function in live cells under physiologically relevant conditions is one of the most powerful and innovative methodologies for proteomics. Efficient florescent probes fully compatible with human-cell expression are the fundamental tools for these studies and their optimization opens the way to resolution at the single-protein level. Biological events involving protein pairs are also directly accessible thanks to tuning of protein-tag spectral properties and production of complementary pairs. Such pairs are characterized by overlapping absorption (for the acceptor tag) and emission (for the donor tag) spectra. By tagging the proteins of interest with acceptor and donor molecules, protein interaction can be directly visualized by FRET, fluorescent resonant energy transfer. In this talk we shall present the design by molecular dynamics calculations and the application of optimized green fluorescent proteins to the study of the human immunodeficiency virus HIV-1 proteomics. In particular trafficking and cellular interactions of HIV-1 transactivator protein Tat in live human cells will be presented. Tat localization and complex internalization pathways of exogenous molecules will be presented thanks to the peculiar optical properties of mutated GFPs. Cellular protein partners and subcellular interaction sites will be identified and directly visualized. The relevance of such results and of advanced spectroscopic and imaging techniques for a new level of understanding of biological processes and its significance for advancement in molecular biology will be underlined. A. Marcello et al., J. Biol. Chem. 276, 39220 (2001). R. Cinelli et al., Appl. Phys. Lett. 79, 3353 (2001).

Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities.

PubMed

Kreutzenbeck, Peter; Kröger, Carsten; Lausberg, Frank; Blaudeck, Natascha; Sprenger, Georg A; Freudl, Roland

2007-03-16

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.

Protective Effect of Tat PTD-Hsp27 Fusion Protein on Tau Hyperphosphorylation Induced by Okadaic Acid in the Human Neuroblastoma Cell Line SH-SY5Y.

PubMed

Choi, Sunghyun; Oh, Jae Hoon; Kim, Hyeseon; Nam, So Hee; Shin, Jeehae; Park, Jong-Sang

2015-10-01

Alzheimer's disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2 h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

PubMed

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

2017-07-04

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells

PubMed Central

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A.S.; Zhou, Pengbo; Mesuraca, Maria

2017-01-01

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery. Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes. PMID:28187462

The evolution of subtype B HIV-1 tat in the Netherlands during 1985-2012.

PubMed

van der Kuyl, Antoinette C; Vink, Monique; Zorgdrager, Fokla; Bakker, Margreet; Wymant, Chris; Hall, Matthew; Gall, Astrid; Blanquart, François; Berkhout, Ben; Fraser, Christophe; Cornelissen, Marion

2018-05-02

For the production of viral genomic RNA, HIV-1 is dependent on an early viral protein, Tat, which is required for high-level transcription. The quantity of viral RNA detectable in blood of HIV-1 infected individuals varies dramatically, and a factor involved could be the efficiency of Tat protein variants to stimulate RNA transcription. HIV-1 virulence, measured by set-point viral load, has been observed to increase over time in the Netherlands and elsewhere. Investigation of tat gene evolution in clinical isolates could discover a role of Tat in this changing virulence. A dataset of 291 Dutch HIV-1 subtype B tat genes, derived from full-length HIV-1 genome sequences from samples obtained between 1985-2012, was used to analyse the evolution of Tat. Twenty-two patient-derived tat genes, and the control Tat HXB2 were analysed for their capacity to stimulate expression of an LTR-luciferase reporter gene construct in diverse cell lines, as well as for their ability to complement a tat-defective HIV-1 LAI clone. Analysis of 291 historical tat sequences from the Netherlands showed ample amino acid (aa) variation between isolates, although no specific mutations were selected for over time. Of note, however, the encoded protein varied its length over the years through the loss or gain of stop codons in the second exon. In transmission clusters, a selection against the shorter Tat86 ORF was apparent in favour of the more common Tat101 version, likely due to negative selection against Tat86 itself, although random drift, transmission bottlenecks, or linkage to other variants could also explain the observation. There was no correlation between Tat length and set-point viral load; however, the number of non-intermediate variants in our study was small. In addition, variation in the length of Tat did not significantly change its capacity to stimulate transcription. From 1985 till 2012, variation in the length of the HIV-1 subtype B tat gene is increasingly found in the Dutch

The grafting of universal T-helper epitopes enhances immunogenicity of HIV-1 Tat concurrently improving its safety profile.

PubMed

Kashi, Venkatesh P; Jacob, Rajesh A; Shamanna, Raghavendra A; Menon, Malini; Balasiddaiah, Anangi; Varghese, Rebu K; Bachu, Mahesh; Ranga, Udaykumar

2014-01-01

Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol711 into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.

Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae).

PubMed

Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M; Zhang, Shuyi

2014-01-01

Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.

PubMed

Kim, Aeyung; Shin, Tae-Hwan; Shin, Seung-Min; Pham, Chuong D; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

2012-01-01

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

PubMed Central

Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

2012-01-01

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

Intracellular transduction of TAT-Hsp27 fusion protein enhancing cell survival and regeneration capacity of cardiac stem cells in acute myocardial infarction.

PubMed

Kim, Hye Jung; Kim, Myoung-Hun; Kim, Jong Tae; Lee, Won-Jin; Kim, Eunjung; Lim, Kwang Suk; Kim, Jang Kyoung; Yang, Young Il; Park, Ki Dong; Kim, Yong-Hee

2015-10-10

Myocardial infarction (MI) results in the substantial loss of functional cardiomyocytes, which frequently leads to intractable heart disorders. Cardiac stem cells (CSCs) that retain the capacity to replace all cardiac cells might be a promising strategy for providing a source of new functional cardiomyocytes; however, the poor survival and engraftment of transplanted CSCs in the hostile environment of MI critically mitigate their therapeutic benefits. To capitalize their therapeutic potential, an ex vivo strategy in which CSCs were introduced to the recombinant heat shock protein 27 (Hsp27) through a TAT protein transduction domain for increasing the viability and engraftment in the infarcted myocardium was designed. A recombinant TAT fused Hsp27 (TAT-Hsp27) was able to enter CSCs in a dose-dependent manner. CSCs transduced with TAT-Hsp27 expressed not only endogenous Hsp27 but externally introduced Hsp27, resulting in substantial increase of their anti-oxidative and anti-apoptotic properties via suppressing reactive oxygen species production, the MAPKs signaling pathway, and caspase activation. TAT-Hsp27 enabled CSCs to be protected from apoptotic- and hypoxic-induced cell death during in vitro cardiomyogenic differentiation. In vivo studies demonstrated that CSCs transduced TAT-Hsp27 significantly increased the survival and engraftment in the acutely infarcted myocardium, which is closely related to caspase activity suppression. Finally, CSCs transduced TAT-Hsp27 improved cardiac function and attenuated cardiac remodeling in comparison with non-transduced CSCs. Overall, our approach, which is based on the ex vivo intracellular transduction of TAT-Hsp27 into CSCs before myocardial delivery, might be effective in treating MI. Copyright © 2015 Elsevier B.V. All rights reserved.

A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

PubMed Central

Fujinaga, Koh; Irwin, Dan; Taube, Ran; Zhang, Fan; Geyer, Matthias; Peterlin, B. Matija

2002-01-01

The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5′ end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn2+-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn2+-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex. PMID:12438619

Impaired plant growth and development caused by human immunodeficiency virus type 1 Tat.

PubMed

Cueno, Marni E; Hibi, Yurina; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

2010-10-01

Previous attempts to express the human immunodeficiency virus 1 (HIV-1) Tat (trans-activator of transcription) protein in plants resulted in a number of physiological abnormalities, such as stunted growth and absence of seed formation, that could not be explained. In the study reported here, we expressed Tat in tomato and observed phenotypic abnormalities, including stunted growth, absence of root formation, chlorosis, and plant death, as a result of reduced cytokinin levels. These reduced levels were ascribed to a differentially expressed CKO35 in Tat-bombarded tomato. Of the two CKO isoforms that are naturally expressed in tomato, CKO43 and CKO37, only the expression of CKO37 was affected by Tat. Our analysis of the Tat confirmed that the Arg-rich and RGD motifs of Tat have functional relevance in tomato and that independent mutations at these motifs caused inhibition of the differentially expressed CKO isoform and the extracellular secretion of the Tat protein, respectively, in our Tat-bombarded tomato samples.

Relaxed Evolution in the Tyrosine Aminotransferase Gene Tat in Old World Fruit Bats (Chiroptera: Pteropodidae)

PubMed Central

Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M.; Zhang, Shuyi

2014-01-01

Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet. PMID:24824435

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

PubMed

Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-06-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

PubMed Central

Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-01-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994

Caffeine Blocks HIV-1 Tat-Induced Amyloid Beta Production and Tau Phosphorylation.

PubMed

Soliman, Mahmoud L; Geiger, Jonathan D; Chen, Xuesong

2017-03-01

The increased life expectancy of people living with HIV-1 who are taking effective anti-retroviral therapeutics is now accompanied by increased Alzheimer's disease (AD)-like neurocognitive problems and neuropathological features such as increased levels of amyloid beta (Aβ) and phosphorylated tau proteins. Others and we have shown that HIV-1 Tat promotes the development of AD-like pathology. Indeed, HIV-1 Tat once endocytosed into neurons can alter morphological features and functions of endolysosomes as well as increase Aβ generation. Caffeine has been shown to have protective actions against AD and based on our recent findings that caffeine can inhibit endocytosis in neurons and can prevent neuronal Aβ generation, we tested the hypothesis that caffeine blocks HIV-1 Tat-induced Aβ generation and tau phosphorylation. In SH-SY5Y cells over-expressing wild-type amyloid beta precursor protein (AβPP), we demonstrated that HIV-1 Tat significantly increased secreted levels and intracellular levels of Aβ as well as cellular protein levels of phosphorylated tau. Caffeine significantly decreased levels of secreted and cellular levels of Aβ, and significantly blocked HIV-1 Tat-induced increases in secreted and cellular levels of Aβ. Caffeine also blocked HIV-1 Tat-induced increases in cellular levels of phosphorylated tau. Furthermore, caffeine blocked HIV-1 Tat-induced endolysosome dysfunction as indicated by decreased protein levels of vacuolar-ATPase and increased protein levels of cathepsin D. These results further implicate endolysosome dysfunction in the pathogenesis of AD and HAND, and by virtue of its ability to prevent and/or block neuropathological features associated with AD and HAND caffeine might find use as an effective adjunctive therapeutic agent.

HIV proteins (gp120 and Tat) and methamphetamine in oxidative stress-induced damage in the brain: Potential role of the thiol antioxidant N-acetylcysteine amide

PubMed Central

Banerjee, Atrayee; Zhang, Xinsheng; Manda, Kalyan Reddy; Banks, William A; Ercal, Nuran

2010-01-01

An increased risk of HIV-1 associated dementia (HAD) has been observed in patients abusing methamphetamine (METH). Since both HIV viral proteins (gp120, Tat) and METH induce oxidative stress, drug abusing patients are at a greater risk of oxidative stress-induced damage. The objective of this study was to determine if N-acetylcysteine amide (NACA) protects the blood brain barrier (BBB) from oxidative stress-induced damage in animals exposed to gp120, Tat and METH. To study this, CD-1 mice pre-treated with NACA/saline, received injections of gp120, Tat, gp120 + Tat or saline for 5 days, followed by three injections of METH/saline on the fifth day, and sacrificed 24 h after the final injection. Various oxidative stress parameters were measured, and animals treated with gp120+Tat+Meth were found to be the most challenged group, as indicated by their GSH and MDA levels. Treatment with NACA significantly rescued the animals from oxidative stress. Further, NACA-treated animals had significantly higher expression of TJ proteins and BBB permeability as compared to the group treated with gp120+Tat+METH alone, indicating that NACA can protect the BBB from oxidative stress-induced damage in gp120, Tat and METH exposed animals, and thus could be a viable therapeutic option for patients with HAD. PMID:20188164

Effects of human chromosome 12 on interactions between Tat and TAR of human immunodeficiency virus type 1.

PubMed Central

Alonso, A; Cujec, T P; Peterlin, B M

1994-01-01

Rates of transcriptions of the human immunodeficiency virus are greatly increased by the viral trans activator Tat. In vitro, Tat binds to the 5' bulge of the trans-activation response (TAR) RNA stem-loop, which is present in all viral transcripts. In human cells, the central loop in TAR and its cellular RNA-binding proteins are also critical for the function of Tat. Previously, we demonstrated that in rodent cells (CHO cells), but not in those which contain the human chromosome 12 (CHO12 cells), Tat-TAR interactions are compromised. In this study, we examined the roles of the bulge and loop in TAR in Tat trans activation in these cells. Whereas low levels of trans activation depended solely on interactions between Tat and the bulge in CHO cells, high levels of trans activation depended also on interactions between Tat and the loop in CHO12 cells. Since the TAR loop binding proteins in these two cell lines were identical and different from their human counterpart, the human chromosome 12 does not encode TAR loop binding proteins. In vivo binding competition studies with TAR decoys confirmed that the binding of Tat to TAR is more efficient in CHO12 cells. Thus, the protein(s) encoded on human chromosome 12 helps to tether Tat to TAR via its loop, which results in high levels of trans activation. Images PMID:8083988

Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1.

PubMed Central

Chun, R; Glabe, C G; Fan, H

1990-01-01

Full-length (86-residue) polypeptide corresponding to the human immunodeficiency virus type 1 tat trans-activating protein was chemically synthesized on a semiautomated apparatus, using an Fmoc amino acid continuous-flow strategy. The bulk material was relatively homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, and it showed trans-activating activity when scrape loaded into cells containing a human immunodeficiency virus long terminal repeat-chloramphenicol acetyl-transferase reporter plasmid. Reverse-phase high-pressure liquid chromatography yielded a rather broad elution profile, and assays across the column for biological activity indicated a sharper peak. Thus, high-pressure liquid chromatography provided for enrichment of biological activity. Fast atom bombardment-mass spectrometry of tryptic digests of synthetic tat identified several of the predicted tryptic peptides, consistent with accurate chemical synthesis. Images PMID:2186178

HIV-1 Tat reduces nephrin in human podocytes: a potential mechanism for enhanced glomerular permeability in HIV-associated nephropathy.

PubMed

Doublier, Sophie; Zennaro, Cristina; Spatola, Tiziana; Lupia, Enrico; Bottelli, Antonella; Deregibus, Maria Chiara; Carraro, Michele; Conaldi, Pier Giulio; Camussi, Giovanni

2007-02-19

To determine whether HIV-1 Tat may directly alter glomerular permeability in HIV-associated nephropathy (HIVAN). Heavy proteinuria is a hallmark of HIVAN. The slit diaphragm is the ultimate glomerular filtration barrier critical for maintaining the efficiency of the ultrafiltration unit of the kidney. In this study, we evaluated the direct effect of Tat protein on the permeability of isolated glomeruli and on the expression of nephrin, the main slit diaphragm component, by human cultured podocytes. Permeability was studied by measuring the permeability to albumin in isolated rat glomeruli. We also evaluated the expression of nephrin in human cultured podocytes by using immunofluorescence and Western blot. We found that Tat increased albumin permeability in isolated glomeruli, and rapidly induced the redistribution and loss of nephrin in cultured podocytes. Pretreatment of glomeruli and podocytes with blocking antibodies showed that Tat reduced nephrin expression by engaging vascular endothelial growth factor receptors types 2 and 3 and the integrin alphavbeta3. Pre-incubation of podocytes with two platelet-activating factor (PAF) receptor antagonists prevented the loss and redistribution of nephrin induced by Tat, suggesting that PAF is an intracellular mediator of Tat action. Tat induced a rapid PAF synthesis by podocytes. When podocytes transfected to overexpress PAF-acetylhydrolase, the main catabolic enzyme of PAF, were stimulated with Tat, the redistribution and loss of nephrin was abrogated. The present results define a mechanism by which Tat may reduce nephrin expression in podocytes, thus increasing glomerular permeability. This provides new insights in the understanding of HIVAN pathogenesis.

Evidence that a sequence similar to TAR is important for induction of the JC virus late promoter by human immunodeficiency virus type 1 Tat.

PubMed Central

Chowdhury, M; Taylor, J P; Chang, C F; Rappaport, J; Khalili, K

1992-01-01

A specific RNA sequence located in the leader of all human immunodeficiency virus type 1 (HIV-1) mRNAs termed the transactivation response element, or TAR, is a primary target for induction of HIV-1 long terminal repeat activity by the HIV-1-derived trans-regulatory protein, Tat. Human neurotropic virus, JC virus (JCV), a causative agent of the degenerative demyelinating disease progressive multifocal leukoencephalopathy, contains sequences in the 5' end of the late RNA species with an extensive homology to HIV-1 TAR. In this study, we examined the possible role of the JCV-derived TAR-homologous sequence in Tat-mediated activation of the JCV late promoter (Tada et al., Proc. Natl. Acad. Sci. USA 87:3479-3483, 1990). Results from site-directed mutagenesis revealed that critical G residues required for the function of HIV-1 TAR that are conserved in the JCV TAR homolog play an important role in Tat activation of the JCV promoter. In addition, in vivo competition studies suggest that shared regulatory components mediate Tat activation of the JCV late and HIV-1 long terminal repeat promoters. Furthermore, we showed that the JCV-derived TAR sequence behaves in the same way as HIV-1 TAR in response to two distinct Tat mutants, one of which that has no ability to bind to HIV-1 TAR and another that lacks transcriptional activity on a responsive promoter. These results suggest that the TAR homolog of the JCV late promoter is responsive to HIV-1 Tat induction and thus may participate in the overall activation of the JCV late promoter mediated by this transactivation. Images PMID:1331525

Safety and immunogenicity of HIV-1 Tat toxoid in immunocompromised HIV-1-infected patients.

PubMed

Gringeri, A; Santagostino, E; Muça-Perja, M; Mannucci, P M; Zagury, J F; Bizzini, B; Lachgar, A; Carcagno, M; Rappaport, J; Criscuolo, M; Blattner, W; Burny, A; Gallo, R C; Zagury, D

1998-01-01

To antagonize the deleterious effects of the HIV-1 toxin extracellular Tat on uninfected immune cells, we developed a new strategy of anti-HIV-1 vaccine using an inactivated but immunogenic Tat (Tat toxoid). Tat toxoid has been assayed for safety and immunogenicity in seropositive patients. The phase I vaccine clinical trial testing Tat toxoid preparation in Seppic Isa 51 oil adjuvant was performed on 14 HIV-1-infected asymptomatic although biologically immunocompromised individuals (500-200 CD4+ cells/mm3). Following as many as 8 injections, no clinical defects were observed. All patients exhibited an antibody (Ab) response to Tat, and some had cell-mediated immunity (CMI) as evaluated by skin test in vivo and T-cell proliferation in vitro. These results provide initial evidence of safety and potency of Tat toxoid vaccination in HIV-1-infected individuals.

The Taming of the Cell Penetrating Domain of the HIV Tat: Myths and Realities

PubMed Central

Chauhan, Ashok; Tikoo, Akshay; Kapur, Arvinder K.; Singh, Mahavir

2007-01-01

Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV-Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. Inspite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences. PMID:17196289

In vitro assessment of TAT — Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration

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

Barbon, Silvia, E-mail: silvia.barbon@yahoo.it

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in themore » future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6 h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury. - Highlights: • TAT-CNTF is an optimized fusion protein that preserves neurotrophic activity. • In neural cell lines, TAT-CNTF triggers the activation of signal transduction. • Fast cellular uptake of TAT

EZH2 phosphorylation regulates Tat-induced HIV-1 transactivation via ROS/Akt signaling pathway.

PubMed

Zhang, Hong-Sheng; Liu, Yang; Wu, Tong-Chao; Du, Guang-Yuan; Zhang, Feng-Juan

2015-12-21

EZH2 plays a major role in HIV-1 latency, however, the molecular linkage between Tat-induced HIV-1 transactivation and EZH2 activity is not fully understood. It was shown Tat induced HIV-1 transactivation through inhibiting EZH2 activity. Tat decreased the levels of H3K27me3 and EZH2 occupy at the long terminal repeat (LTR) of HIV-1. We further showed for the first time that transfected with Tat construct resulted in an increase in phosphorylated EZH2 (p-EZH2), mediated by active Akt. ROS/Akt-dependent p-EZH2 was correlated with Tat-induced transactivation. Our study reveals that novel mechanisms allow Tat-induced HIV-1 transactivation by ROS/Akt-dependent downregulating the EZH2 epigenetic silencing machinery. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Tat transport of a Sec passenger leads to both completely translocated as well as membrane-arrested passenger proteins.

PubMed

Dittmar, Julia; Schlesier, René; Klösgen, Ralf Bernd

2014-02-01

We have studied the membrane transport of the chimeric precursor protein 16/33, which is composed of the Tat(1)-specific transport signal of OEC16 and the Sec passenger protein OEC33, both subunits of the oxygen-evolving system associated with photosystem II. Protein transport experiments performed with isolated pea thylakoids show that the 16/33 chimera is transported in a strictly Tat-dependent manner into the thylakoid vesicles yielding mature OEC33 (mOEC33) in two different topologies. One fraction accumulates in the thylakoid lumen and is thus resistant to externally added protease. A second fraction is arrested during transport in an N-in/C-out topology within the membrane. Chase experiments demonstrate that this membrane-arrested mOEC33 moiety does not represent a translocation intermediate but instead an alternative end product of the transport process. Transport arrest of mOEC33, which is embedded in the membrane with a mildly hydrophobic protein segment, requires more than 26 additional and predominantly hydrophilic residues C-terminal of the membrane-embedded segment. Furthermore, it is stimulated by mutations which potentially affect the conformation of mOEC33 suggesting that at least partial folding of the passenger protein is required for complete membrane translocation. Copyright © 2013 Elsevier B.V. All rights reserved.

SjTat-TPI facilitates adaptive T-cell responses and reduces hepatic pathology during Schistosoma japonicum infection in BALB/c mice.

PubMed

Zhang, Wenyue; Luo, Xiaofeng; Zhang, Fan; Zhu, Yuxiao; Yang, Bingya; Hou, Min; Xu, Zhipeng; Yu, Chuanxin; Chen, Yingying; Chen, Lin; Ji, Minjun

2015-12-30

Schistosomiasis is a kind of parasitic zoonoses which causes serious damage to public health and social development. China is one of the countries most affected by Schistosoma japonicum and an effective vaccine is still needed. In this study, we adopted Tat-mediated protein transduction technology to investigate the impact of different antigen presented approaches on host's immune response and the potential protection against Schistosoma japonicum infection. We successfully constructed the recombinant S. japonicum triosephosphate isomerase, Tat-TPI, as a vaccine candidate. Whether injected with Tat-TPI in foot pad or vaccinated with Tat-TPI in the back subcutaneously for three times, the draining popliteal lymph nodes and spleen both developed a stronger CD8(+)T response (Tc1) in mice. Not only that, but it also helped CD4(+)T cells to produce more IFN-γ than TPI immunisation. In addition, it could boost IgG production, especially IgG1 subclass. Most importantly, Tat-TPI immunisation led to the significant smaller area of a single egg granuloma in the livers as compared with TPI-vaccinated or control groups. However, the anti-infection efficiency induced by Tat-TPI was still restricted. This study indicated that immunisation with Tat-fused TPI could contribute to enhance CD4(+)T-cell response and decrease hepatic egg granulomatous area after S. japonicum infection though it did not achieve our expected protection against Schistosoma japonicum infection. The optimal vaccine strategy warrants further research.

TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

2012-06-01

Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

Biological activity of Tat (47-58) peptide on human pathogenic fungi.

PubMed

Jung, Hyun Jun; Park, Yoonkyung; Hahm, Kyung-Soo; Lee, Dong Gun

2006-06-23

Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol; 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.

Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae.

PubMed

Uemura, Satoshi; Mochizuki, Takahiro; Kurosaka, Goyu; Hashimoto, Takanori; Masukawa, Yuki; Abe, Fumiyoshi

2017-10-01

Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional characterization of a human cyclin T1 mutant reveals a different binding surface for Tat and HEXIM1.

PubMed

Kuzmina, Alona; Hadad, Uzi; Fujinaga, Koh; Taube, Ran

2012-05-10

HIV transcription is regulated at the step of elongation by the viral Tat protein and the cellular positive transcription elongation factor b (P-TEFb; Cdk9/cyclin T1). Herein, a human cyclin T1 mutant, cyclin T1-U7, which contains four substitutions and one deletion in the N-terminal cyclin box, was stably expressed in HeLa cells. HIV transcription was efficiently inhibited in HeLa-HA-CycT1-U7 stable cells. Cyclin T1-U7 bound Tat but did not modulate its expression levels, which remained high. Importantly cyclin T1-U7 failed to interact with Cdk9 or HEXIM1 and did not interfere with endogenous P-TEFb activity to stimulate MEF2C or NFkB mediated transcription. In a T cell line and primary CD4+ cells, cyclin T1-U7 also inhibited HIV transcription. We conclude that cyclin T1-U7 sequesters Tat from P-TEFb and inhibits HIV transcription. Importantly, N-terminal residues in cyclin T1 are specifically involved in the binding of cyclin T1 to HEXIM1 but not to Tat. Copyright © 2012 Elsevier Inc. All rights reserved.

Herpes Simplex Virus Type 2 Triggers Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency and Collaborates with HIV-1 Tat

PubMed Central

Zhu, Xiaolei; Ma, Xinting; Yan, Qin; Zeng, Yi; Guo, Yuanyuan; Feng, Ninghan; Lu, Chun

2012-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) infection was necessary but not sufficient for Kaposi's sarcoma (KS) development without other cofactors. Previously, we identified that both human immunodeficiency type 1 (HIV-1) Tat and herpes simplex virus 1 (HSV-1) were important cofactors reactivating KSHV from latency. Here, we further investigated the potential of herpes simplex virus 2 (HSV-2) to influence KSHV replication and examined the role of Tat in this procedure. We demonstrated that HSV-2 was a potentially important factor in the pathogenesis of KS, as determined by production of lytic phase mRNA transcripts, viral proteins and infectious viral particles in BCBL-1 cells. These results were further confirmed by an RNA interference experiment using small interfering RNA targeting KSHV Rta and a luciferase reporter assay testing Rta promoter-driven luciferase activity. Mechanistic studies showed that HSV-2 infection activated nuclear factor-kappa B (NF-κB) signaling pathway. Inhibition of NF-κB pathway enhanced HSV-2-mediated KSHV activation, whereas activation of NF-κB pathway suppressed KSHV replication in HSV-2-infected BCBL-1 cells. Additionally, ectopic expression of Tat enhanced HSV-2-induced KSHV replication. These novel findings suggest a role of HSV-2 in the pathogenesis of KS and provide the first laboratory evidence that Tat may participate HSV-2-mediated KSHV activation, implying the complicated pathogenesis of acquired immunodeficiency syndrome (AIDS)-related KS (AIDS-KS) patients. PMID:22347501

Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

PubMed

Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

2014-01-01

HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

Biological activity of Tat (47-58) peptide on human pathogenic fungi

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

Jung, Hyun Jun; Park, Yoonkyung; Department of Biotechnology, Chosun University, 375 Seosuk-dong, Kwangju 501-750

2006-06-23

Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol;more » 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.« less

The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

PubMed

Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

2014-01-01

Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.

Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

PubMed

Feriotto, Giordana; Marchetti, Nicola; Costa, Valentina; Beninati, Simone; Tagliati, Federico; Mischiati, Carlo

2018-02-01

New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

The anti-cancer drug Sunitinib promotes autophagy and protects from neurotoxicity in an HIV-1 Tat model of neurodegeneration

PubMed Central

Fields, Jerel A.; Metcalf, Jeff; Overk, Cassia; Adame, Anthony; Spencer, Brian; Wrasidlo, Wolfgang; Florio, Jazmin; Rockenstein, Edward; He, Johnny J.; Masliah, Eliezer

2017-01-01

Despite the success of antiretroviral therapies to control systemic HIV-1 infection, the prevalence of HIV-associated neurocognitive disorders (HAND) has not decreased among aging patients with HIV. Autophagy pathway alterations, triggered by HIV-1 proteins including gp120, Tat, and Nef, might contribute to the neurodegenerative process in aging patients with HAND. Although no treatments are currently available to manage HAND, we have previously shown that Sunitinib, an anti-cancer drug that blocks receptor tyrosine-kinase and cyclin kinase pathways, might be of interest. Studies in cancer models suggest that sunitinib might also modulate autophagy, which is dysregulated in our models of Tat-induced neurotoxicity. We evaluated the efficacy of sunitinib to promote autophagy in the CNS and ameliorate neurodegeneration using LC3-GFP expressing neuronal cells challenged with low concentrations of Tat and using inducible Tat transgenic mice. In neuronal cultures challenged with low levels of Tat, sunitinib increased markers of autophagy such as LC3-II and reduced p62 accumulation in a dose-dependent manner. In vivo, sunitinib treatment restored LC3-II, p62, and Endophilin B1 (EndoB1) levels in doxycycline-induced Tat transgenic mice. Moreover, in these animals sunitinib reduced the hyperactivation of CDK5, tau hyper-phosphorylation and p35 cleavage to p25. Restoration of CDK5 and autophagy were associated with reduced neurodegeneration and behavioral alterations. Alterations in autophagy in the Tat tg mice were associated with reduced levels of a CDK5 substrate, EndoB1, and levels of total EndoB1 were normalized by sunitinib treatment. We conclude that sunitinib might ameliorate Tat-mediated autophagy alterations and may decrease neurodegeneration in aging patients with HAND. PMID:28105557

The preventive phase I trial with the HIV-1 Tat-based vaccine.

PubMed

Ensoli, Barbara; Fiorelli, Valeria; Ensoli, Fabrizio; Lazzarin, Adriano; Visintini, Raffaele; Narciso, Pasquale; Di Carlo, Aldo; Tripiciano, Antonella; Longo, Olimpia; Bellino, Stefania; Francavilla, Vittorio; Paniccia, Giovanni; Arancio, Angela; Scoglio, Arianna; Collacchi, Barbara; Ruiz Alvarez, Maria Josè; Tambussi, Giuseppe; Tassan Din, Chiara; Palamara, Guido; Latini, Alessandra; Antinori, Andrea; D'Offizi, Gianpiero; Giuliani, Massimo; Giulianelli, Marina; Carta, Maria; Monini, Paolo; Magnani, Mauro; Garaci, Enrico

2009-12-11

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials based on its role in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune response with the asymptomatic stage as well as on its sequence conservation among HIV clades. A randomized, double blind, placebo-controlled phase I study (ISS P-001) was conducted in healthy adult volunteers without identifiable risk of HIV infection. Tat was administered 5 times monthly, subcute in alum or intradermic alone at 7.5 microg, 15 microg or 30 microg, respectively (ClinicalTrials.gov identifier: NCT00529698). Vaccination with Tat resulted to be safe and well tolerated (primary endpoint) both locally and systemically. In addition, Tat induced both Th1 and Th2 type specific immune responses in all subjects (secondary endpoint) with a wide spectrum of functional antibodies that are rarely seen in natural infection, providing key information for further clinical development of the Tat vaccine candidate.

Homonuclear 1H NMR and circular dichroism study of the HIV-1 Tat Eli variant.

PubMed

Watkins, Jennifer D; Campbell, Grant R; Halimi, Hubert; Loret, Erwann P

2008-09-22

The HIV-1 Tat protein is a promising target to develop AIDS therapies, particularly vaccines, due to its extracellular role that protects HIV-1-infected cells from the immune system. Tat exists in two different lengths, 86 or 87 residues and 99 or 101 residues, with the long form being predominant in clinical isolates. We report here a structural study of the 99 residue Tat Eli variant using 2D liquid-state NMR, molecular modeling and circular dichroism. Tat Eli was obtained from solid-phase peptide synthesis and the purified protein was proven biologically active in a trans-activation assay. Circular dichroism spectra at different temperatures up to 70 degrees C showed that Tat Eli is not a random coil at 20 degrees C. Homonuclear 1H NMR spectra allowed us to identify 1639 NMR distance constraints out of which 264 were interresidual. Molecular modeling satisfying at least 1474 NMR constraints revealed the same folding for different model structures. The Tat Eli model has a core region composed of a part of the N-terminus including the highly conserved Trp 11. The extra residues in the Tat Eli C-terminus protrude from a groove between the basic region and the cysteine-rich region and are well exposed to the solvent. We show that active Tat variants share a similar folding pattern whatever their size, but mutations induce local structural changes.

Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.

PubMed Central

Sumner-Smith, M; Roy, S; Barnett, R; Reid, L S; Kuperman, R; Delling, U; Sonenberg, N

1991-01-01

The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat. Images PMID:1895380

The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222

PubMed Central

Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

2014-01-01

Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells. PMID:24717285

Mapping the architecture of the HIV-lTat circuit: A decision-making circuit that lacks bistability and exploits stochastic noise

PubMed Central

Razooky, Brandon S.; Weinberger, Leor S.

2014-01-01

Upon infection of a CD4+ T cell, HIV-l appears to ‘choose’ between two alternate fates: active replication or a long-lived dormant statetermed proviral latency. A transcriptional positive-feedback loop generated by the HIV-l Tat protein appears sufficient to mediate this decision. Here, we describea coupled wet-lab and computational approach that uses mathematical modeling and live-cell time-lapse microscopy to map the architecture of the HIV-l Tat transcriptional regulatorycircuit and generate predictive models of HIV-l latency. This approach provided the first characterization of a ‘decision-making’ circuit that lacks bistability andinstead exploits stochastic fluctuations in cellular molecules (i.e. noise) to generate a decision between an on or off transcriptional state. PMID:21167940

Homonuclear 1H NMR and circular dichroism study of the HIV-1 Tat Eli variant

PubMed Central

Watkins, Jennifer D; Campbell, Grant R; Halimi, Hubert; Loret, Erwann P

2008-01-01

Background The HIV-1 Tat protein is a promising target to develop AIDS therapies, particularly vaccines, due to its extracellular role that protects HIV-1-infected cells from the immune system. Tat exists in two different lengths, 86 or 87 residues and 99 or 101 residues, with the long form being predominant in clinical isolates. We report here a structural study of the 99 residue Tat Eli variant using 2D liquid-state NMR, molecular modeling and circular dichroism. Results Tat Eli was obtained from solid-phase peptide synthesis and the purified protein was proven biologically active in a trans-activation assay. Circular dichroism spectra at different temperatures up to 70°C showed that Tat Eli is not a random coil at 20°C. Homonuclear 1H NMR spectra allowed us to identify 1639 NMR distance constraints out of which 264 were interresidual. Molecular modeling satisfying at least 1474 NMR constraints revealed the same folding for different model structures. The Tat Eli model has a core region composed of a part of the N-terminus including the highly conserved Trp 11. The extra residues in the Tat Eli C-terminus protrude from a groove between the basic region and the cysteine-rich region and are well exposed to the solvent. Conclusion We show that active Tat variants share a similar folding pattern whatever their size, but mutations induce local structural changes. PMID:18808674

Protective effects of intraperitoneal injection of TAT-SOD against focal cerebral ischemia/reperfusion injury in rats.

PubMed

Ye, Nanhui; Liu, Shutao; Lin, Yanyun; Rao, Pingfan

2011-12-05

The intracellular superoxide anion has been shown to be involved in brain injury. TAT-Superoxide dismutase (TAT-SOD) can be transduced across the cell membrane to scavenge superoxide. This protein's unique properties make it a promising therapeutic candidate to attenuate cerebral damage. In this study, we sought further the understanding of the fusion protein's cerebral protective effects and the mechanism which is exerted in these effects. Male Sprague Dawley rats (n=100, 230±20 g) were divided randomly into five experimental groups: a sham group, a cerebral Ischemia/Reperfusion (I/R) group treated with saline (20 ml/Kg, i.p.), and three cerebral I/R groups treated with TAT-SOD (25 KU/ml/Kg, i.p.) at either 2h before I/R, 2h after I/R or 4h after I/R. Cerebral I/R injury was facilitated by inducing ischemia for two hours followed by 24h reperfusion. The levels of SOD, Malondialdehyde (MDA), and ATPase in cerebral tissues were determined. The apoptotic indexes were evaluated, and apoptosis genes were analyzed immunohistochemically. TAT-SOD treatment significantly increased cerebral SOD and ATPase activities, decreased MDA content, and remarkably reduced apoptosis indexes. TAT-SOD treatments 2h before or after I/R significantly reduced caspase-3 and bax proteins and boosted bcl-2 protein, while the treatment at 4h after I/R showed no influence on the three proteins. TAT-SOD treatment effectively enhanced cerebral antioxidant ability, reduced lipid peroxidation, preserved mitochondrial ATPase and thus inhibited nerve cell apoptosis. The effective treatment window extended from 2h before to 2h after I/R. Copyright © 2011 Elsevier B.V. All rights reserved.

Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

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

Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides formore » calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein

Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

NASA Astrophysics Data System (ADS)

Hiraki, Toshiki; Usui, Keiko; Abe, Fumiyoshi

2010-12-01

Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp-) strains under pressures of 15-25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp- cells to grow at 15-25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses

PubMed Central

Col, Edwige; Caron, Cécile; Chable-Bessia, Christine; Legube, Gaelle; Gazzeri, Sylvie; Komatsu, Yasuhiko; Yoshida, Minoru; Benkirane, Monsef; Trouche, Didier; Khochbin, Saadi

2005-01-01

HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination. PMID:16001085

The Tat Inhibitor Didehydro-Cortistatin A Prevents HIV-1 Reactivation from Latency

PubMed Central

Mousseau, Guillaume; Kessing, Cari F.; Fromentin, Rémi; Trautmann, Lydie; Chomont, Nicolas

2015-01-01

ABSTRACT Antiretroviral therapy (ART) inhibits HIV-1 replication, but the virus persists in latently infected resting memory CD4+ T cells susceptible to viral reactivation. The virus-encoded early gene product Tat activates transcription of the viral genome and promotes exponential viral production. Here we show that the Tat inhibitor didehydro-cortistatin A (dCA), unlike other antiretrovirals, reduces residual levels of viral transcription in several models of HIV latency, breaks the Tat-mediated transcriptional feedback loop, and establishes a nearly permanent state of latency, which greatly diminishes the capacity for virus reactivation. Importantly, treatment with dCA induces inactivation of viral transcription even after its removal, suggesting that the HIV promoter is epigenetically repressed. Critically, dCA inhibits viral reactivation upon CD3/CD28 or prostratin stimulation of latently infected CD4+ T cells from HIV-infected subjects receiving suppressive ART. Our results suggest that inclusion of a Tat inhibitor in current ART regimens may contribute to a functional HIV-1 cure by reducing low-level viremia and preventing viral reactivation from latent reservoirs. PMID:26152583

A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

PubMed

Bozue, Joel; Cote, Christopher K; Chance, Taylor; Kugelman, Jeffrey; Kern, Steven J; Kijek, Todd K; Jenkins, Amy; Mou, Sherry; Moody, Krishna; Fritz, David; Robinson, Camenzind G; Bell, Todd; Worsham, Patricia

2014-01-01

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

A Yersinia pestis tat Mutant Is Attenuated in Bubonic and Small-Aerosol Pneumonic Challenge Models of Infection but Not As Attenuated by Intranasal Challenge

PubMed Central

Bozue, Joel; Cote, Christopher K.; Chance, Taylor; Kugelman, Jeffrey; Kern, Steven J.; Kijek, Todd K.; Jenkins, Amy; Mou, Sherry; Moody, Krishna; Fritz, David; Robinson, Camenzind G.; Bell, Todd; Worsham, Patricia

2014-01-01

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge. PMID:25101850

[Biological characteristics of an enteroinvasive Escherichia coli strain with tatABC deletion].

PubMed

Gong, Zhaolong; Ye, Changyun; Liu, Xiaobing; Zhang, Min; Zhuo, Qin

2013-05-04

To study the relationship between twin-arginine translocation system (Tat) system with the biological characteristics of enteroinvasive Escherichia coli (EIEC). Through homologous recombination, we constructed EIEC's tatABC gene deletion strain and complementary strain, and explored their impact on bacterial form, substrate transport function as well as on HeLa cells and guinea pig's corneal invasion force. The tatABC gene deletion strain had apparent changes in bacterial form, loss of substrate transporter function, and significant weakened bacterial invasion force (the number of the deletion strain invading into HeLa cells was decreased significantly, and the ability of its corneal lesion capacity of the guinea pig was significantly weakened), while the complementary strain was similar to the wild strain in the above respects. EIEC's Tat protein transport system is closely related with the biological characteristics of EIEC.

Recent advances in the identification of Tat-mediated transactivation inhibitors: progressing toward a functional cure of HIV.

PubMed

Tabarrini, Oriana; Desantis, Jenny; Massari, Serena

2016-01-01

The current anti-HIV combination therapy does not eradicate the virus that persists mainly in quiescent infected CD4(+) T cells as a latent integrated provirus that resumes after therapy interruption. The Tat-mediated transactivation (TMT) is a critical step in the HIV replication cycle that could give the opportunity to reduce the size of latent reservoirs. More than two decades of research led to the identification of various TMT inhibitors. While none of them met the criteria to reach the market, the search for a suitable TMT inhibitor is still actively pursued. Really promising compounds, including one in a Phase III clinical trial, have been recently identified, thus warranting an update.

Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys.

PubMed

Maggiorella, Maria Teresa; Baroncelli, Silvia; Michelini, Zuleika; Fanales-Belasio, Emanuele; Moretti, Sonia; Sernicola, Leonardo; Cara, Andrea; Negri, Donatella R M; Buttò, Stefano; Fiorelli, Valeria; Tripiciano, Antonella; Scoglio, Arianna; Caputo, Antonella; Borsetti, Alessandra; Ridolfi, Barbara; Bona, Roberta; ten Haaft, Peter; Macchia, Iole; Leone, Pasqualina; Pavone-Cossut, Maria Rosaria; Nappi, Filomena; Ciccozzi, Massimo; Heeney, Jonathan; Titti, Fausto; Cafaro, Aurelio; Ensoli, Barbara

2004-09-03

Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.

Fruit-specific expression of the human immunodeficiency virus type 1 tat gene in tomato plants and its immunogenic potential in mice.

PubMed

Ramírez, Yuri Jorge Peña; Tasciotti, Ennio; Gutierrez-Ortega, Abel; Donayre Torres, Alberto J; Olivera Flores, María Teresa; Giacca, Mauro; Gómez Lim, Miguel Angel

2007-06-01

The human immunodeficiency virus type 1 (HIV-1) Tat protein is considered a potential candidate vaccine antigen. In an effort to design a strategy for noninvasive vaccination against HIV-1, we developed transgenic tomatoes expressing the Tat protein. Two independent plants testing positive in transgene detection analysis were selected and grown to maturity. Monoclonal antibodies against Tat recognized a protein of the expected size. Interestingly, expression of Tat seemed to be toxic to the plant, as in all cases the fruit exhibited underdeveloped reproductive structures and no seeds. Nine groups of 10 pathogen-free BALB/c male mice were primed either orally, intraperitoneally, or intramuscularly with 10 mg of tomato fruit extract derived from transgenic or wild-type plants and with 10 microg of Tat86 recombinant protein. Mice were immunized at days 0, 14, and 28, and given boosters after 15 weeks; sera were drawn 7 days after each booster, and the antibody titer was determined by enzyme-linked immunosorbent assay. All three immunization approaches induced the development of a strong anti-Tat immunological response, which increased over time. Isotype subclass determination showed the presence of mucosal (immunoglobulin A) immunity soon after the beginning of the oral immunization protocol, and the data were confirmed by the presence of anti-Tat antibodies in fecal pellets and in vaginal washes. We also demonstrated that sera from immunized mice inhibited with high efficiency recombinant Tat-dependent transactivation of the HIV-1 long terminal repeat promoter. This neutralization activity might be relevant for the suppression of extracellular Tat activities, which play an important role in HIV disease development.

Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro

PubMed Central

Gruber, Andreas; Lell, Claudia P; Speth, Cornelia; Stoiber, Heribert; Lass-Flörl, Cornelia; Sonneborn, Anja; Ernst, Joachim F; Dierich, Manfred P; Würzner, Reinhard

2001-01-01

Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, binds through its RGD-motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV-1, also possesses RGD-binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease-promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis. PMID:11899432

HIV Tat/P-TEFb Interaction: A Potential Target for Novel Anti-HIV Therapies.

PubMed

Asamitsu, Kaori; Fujinaga, Koh; Okamoto, Takashi

2018-04-17

Transcription is a crucial step in the life cycle of the human immunodeficiency virus type 1 (HIV 1) and is primarily involved in the maintenance of viral latency. Both viral and cellular transcription factors, including transcriptional activators, suppressor proteins and epigenetic factors, are involved in HIV transcription from the proviral DNA integrated within the host cell genome. Among them, the virus-encoded transcriptional activator Tat is the master regulator of HIV transcription. Interestingly, unlike other known transcriptional activators, Tat primarily activates transcriptional elongation and initiation by interacting with the cellular positive transcriptional elongation factor b (P-TEFb). In this review, we describe the molecular mechanism underlying how Tat activates viral transcription through interaction with P-TEFb. We propose a novel therapeutic strategy against HIV replication through blocking Tat action.

Role of Tat-interacting protein of 110 kDa and microRNAs in the regulation of hematopoiesis.

PubMed

Liu, Ying; He, Johnny J

2016-07-01

Hematopoiesis is regulated by cellular factors including transcription factors, microRNAs, and epigenetic modifiers. Understanding how these factors regulate hematopoiesis is pivotal for manipulating them to achieve their desired potential. In this review, we will focus on HIV-1 Tat-interacting protein of 110 kDa (Tip110) and its regulation of hematopoiesis. There are several pathways in hematopoiesis that involve Tip110 regulation. Tip110 is expressed in human cord blood CD34 cells; its expression decreases when CD34 cells begin to differentiate. Tip110 is also expressed in mouse marrow hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC). Tip110 expression increases the number, survival, and cell cycling of HPC. Tip110-mediated regulation of hematopoiesis has been linked to its reciprocal control of proto-oncogene expression. Small noncoding microRNAs (miRs) have been shown to play important roles in regulation of hematopoiesis. miR-124 specifically targets 3'-untranslated region of Tip110 and subsequently regulates Tip110 expression in HSC. Our recent findings for manipulating expression levels of Tip110 in HSC and HPC could be useful for expanding HSC and HPC and for improving engraftment of cord blood HSC/HPC.

Effect of arginine methylation on the RNA recognition and cellular uptake of Tat-derived peptides.

PubMed

Li, Jhe-Hao; Chiu, Wen-Chieh; Yao, Yun-Chiao; Cheng, Richard P

2015-05-01

Arginine (Arg) methylation is a common post-translational modification that regulates gene expression and viral infection. The HIV-1 Tat protein is an essential regulatory protein for HIV proliferation, and is methylated in the cell. The basic region (residues 47-57) of the Tat protein contains six Arg residues, and is responsible for two biological functions: RNA recognition and cellular uptake. In this study, we explore the effect of three different methylation states at each Arg residue in Tat-derived peptides on the two biological functions. The Tat-derived peptides were synthesized by solid phase peptide synthesis. TAR RNA binding of the peptides was assessed by electrophoresis mobility shift assays. The cellular uptake of the peptides into Jurkat cells was determined by flow cytometry. Our results showed that RNA recognition was affected by both methylation state and position. In particular, asymmetric dimethylation at position 53 decreased TAR RNA binding affinity significantly, but unexpectedly less so upon asymmetric dimethylation at position 52. The RNA binding affinity even slightly increased upon methylation at some of the flanking Arg residues. Upon Arg methylation, the cellular uptake of Tat-derived peptides mostly decreased. Interestingly, cellular uptake of Tat-derived peptides with a single asymmetrically dimethylated Arg residue was similar to the native all Arg peptide (at 120 μM). Based on our results, TAR RNA binding apparently required both guanidinium terminal NH groups on Arg53, whereas cellular uptake apparently required guanidinium terminal NH₂ groups instead. These results should provide insight into how nature uses arginine methylation to regulate different biological functions, and should be useful for the development of functional molecules with methylated arginines. Copyright © 2015. Published by Elsevier Ltd.

The HIV-1 viral protein Tat increases glutamate and decreases GABA exocytosis from human and mouse neocortical nerve endings.

PubMed

Musante, Veronica; Summa, Maria; Neri, Elisa; Puliti, Aldamaria; Godowicz, Tomasz T; Severi, Paolo; Battaglia, Giuseppe; Raiteri, Maurizio; Pittaluga, Anna

2010-08-01

Human immunodeficiency virus-1 (HIV-1)-encoded transactivator of transcription (Tat) potentiated the depolarization-evoked exocytosis of [(3)H]D-aspartate ([(3)H]D-ASP) from human neocortical terminals. The metabotropic glutamate (mGlu) 1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) prevented this effect, whereas the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was ineffective. Western blot analysis showed that human neocortex synaptosomes possess mGlu1 and mGlu5 receptors. Tat potentiated the K(+)-evoked release of [(3)H]D-ASP or of endogenous glutamate from mouse neocortical synaptosomes in a CPCCOEt-sensitive and MPEP-insensitive manner. Deletion of mGlu1 receptors (crv4/crv4 mice) or mGlu5 receptors (mGlu5(-/-)mouse) silenced Tat effects. Tat enhanced inositol 1,4,5-trisphosphate production in human and mouse neocortical synaptosomes, consistent with the involvement of group I mGlu receptors. Tat inhibited the K(+)-evoked release of [(3)H]gamma-aminobutyric acid ([(3)H]GABA) from human synaptosomes and that of endogenous GABA or [(3)H]GABA from mouse nerve terminals; the inhibition was insensitive to CPCCOEt or MPEP. Tat-induced effects were retained by Tat(37-72) but not by Tat(48-85). In mouse neocortical slices, Tat facilitated the K(+)- and the veratridine-induced release of [(3)H]D-ASP in a CPCCOEt-sensitive manner and was ineffective in crv4/crv4 mouse slices. These observations are relevant to the comprehension of the pathophysiological effects of Tat in central nervous system and may suggest new potential therapeutic approaches to the cure of HIV-1-associated dementia.

HIV-1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats

PubMed Central

Brailoiu, Eugen; Deliu, Elena; Sporici, Romeo A.; Benamar, Khalid

2014-01-01

The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 transactivator of transcription (Tat, fragment 1–86) produced a concentration-dependent increase in cytosolic Ca2+ in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using store-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca2+ elevation: 1) lysosomal Ca2+ mobilization, 2) Ca2+ release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca2+ influx via transient receptor potential vanilloid type 2 (TRPV2) channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca2+ entry. In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies, indicating that Tat promotes bradycardia and, consequently, may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy. PMID:24694382

Parallel conduction of the phase I preventive and therapeutic trials based on the Tat vaccine candidate.

PubMed

Bellino, S; Francavilla, V; Longo, O; Tripiciano, A; Paniccia, G; Arancio, A; Fiorelli, V; Scoglio, A; Collacchi, B; Campagna, M; Lazzarin, A; Tambussi, G; Din, C Tassan; Visintini, R; Narciso, P; Antinori, A; D'Offizi, G; Giulianelli, M; Carta, M; Di Carlo, A; Palamara, G; Giuliani, M; Laguardia, M E; Monini, P; Magnani, M; Ensoli, F; Ensoli, B

2009-09-01

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials in both uninfected (ClinicalTrials.gov identifier: NCT00529698) and infected volunteers (ClinicalTrials.gov identifier: NCT00505401). The rationale was based on the role of Tat in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune responses with the asymptomatic stage and slow-progression rate as well as on its sequence conservation among HIV clades (http://www.hiv1tat-vaccines.info/). The parallel conduction in the same clinical centers of randomized, double blind, placebo-controlled phase I studies both in healthy, immunologically competent adults and in HIV-infected, clinically asymptomatic, individuals represents a unique occasion to compare the vaccine-induced immune response in both the preventive and therapeutic setting. In both studies, the same lot of the native Tat protein was administered 5 times, every four weeks, subcute (SC) with alum adjuvant or intradermic (ID), in the absence of adjuvant, at 7.5 microg, 15 microg or 30 microg doses, respectively. The primary and secondary endpoints of these studies were the safety and immunogenicity of the vaccine candidate, respectively. The study lasted 52 weeks and monitoring was conducted for on additional 3 years. The results of both studies indicated that the Tat vaccine is safe and well tolerated both locally and systemically and it is highly immunogenic at all the dosages and by both routes of administration. Vaccination with Tat induced a balanced immune response in uninfected and infected individuals. In particular, therapeutic immunization induced functional antibodies and partially reverted the marked Th1 polarization of anti-Tat immunity seen in natural infection, and elicited a more balanced Th1/Th2 immune response. Further, the number of CD4 T cells correlated positively with anti-Tat antibody titers. Based on these results, a phase II study is ongoing in infected drug

Neuroimaging abnormalities in clade C HIV are independent of Tat genetic diversity.

PubMed

Paul, Robert H; Phillips, Sarah; Hoare, Jacqueline; Laidlaw, David H; Cabeen, Ryan; Olbricht, Gayla R; Su, Yuqing; Stein, Dan J; Engelbrecht, Susan; Seedat, Soraya; Salminen, Lauren E; Baker, Laurie M; Heaps, Jodi; Joska, John

2017-04-01

Controversy remains regarding the neurotoxicity of clade C human immunodeficiency virus (HIV-C). When examined in preclinical studies, a cysteine to serine substitution in the C31 dicysteine motif of the HIV-C Tat protein (C31S) results in less severe brain injury compared to other viral clades. By contrast, patient cohort studies identify significant neuropsychological impairment among HIV-C individuals independent of Tat variability. The present study clarified this discrepancy by examining neuroimaging markers of brain integrity among HIV-C individuals with and without the Tat substitution. Thirty-seven HIV-C individuals with the Tat C31S substitution, 109 HIV-C individuals without the Tat substitution (C31C), and 34 HIV- controls underwent 3T structural magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI). Volumes were determined for the caudate, putamen, thalamus, corpus callosum, total gray matter, and total white matter. DTI metrics included fractional anisotropy (FA), radial diffusivity (RD), and axial diffusivity (AD). Tracts of interest included the anterior thalamic radiation (ATR), cingulum bundle (CING), uncinate fasciculus (UNC), and corpus callosum (CC). HIV+ individuals exhibited smaller volumes in subcortical gray matter, total gray matter and total white matter compared to HIV- controls. HIV+ individuals also exhibited DTI abnormalities across multiple tracts compared to HIV- controls. By contrast, neither volumetric nor diffusion indices differed significantly between the Tat C31S and C31C groups. Tat C31S status is not a sufficient biomarker of HIV-related brain integrity in patient populations. Clinical attention directed at brain health is warranted for all HIV+ individuals, independent of Tat C31S or clade C status.

TAT [Training and Technology.

ERIC Educational Resources Information Center

Oak Ridge Associated Universities, TN. Manpower Development Div.

The Oak Ridge Associated Universities (ORAU) of Tennessee and the Nuclear Division of the Union Carbide Corporation established an industrial training program called Training and Technology (TAT) which was conducted at the Oak Ridge Y-12 plant. TAT instructors were provided by the regular work force of Union Carbide while ORAU provided the…

Synergistic Enhancement of Antitumor Efficacy by PEGylated Multi-walled Carbon Nanotubes Modified with Cell-Penetrating Peptide TAT

NASA Astrophysics Data System (ADS)

Hu, Shanshan; Wang, Tong; Pei, Xibo; Cai, He; Chen, Junyu; Zhang, Xin; Wan, Qianbing; Wang, Jian

2016-10-01

In the present study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to PEGylated multi-walled carbon nanotubes (MWCNTs) to develop a highly effective antitumor drug delivery system. FITC was conjugated on MWCNTs-polyethylene glycol (PEG) and MWCNTs-PEG-TAT to provide fluorescence signal for tracing the cellular uptake of the nanocarrier. After loaded with an anticancer agent, doxorubicin (DOX) via π - π stacking interaction, the physicochemical characteristics, release profile and biological evaluation of the obtained nano-sized drug carrier were investigated. The DOX loaded MWCNTs-PEG and MWCNTs-PEG-TAT drug carriers both displayed appropriate particle size, excellent stability, high drug loading, and pH-dependent drug release profile. Nevertheless, compared with DOX-MWCNTs-PEG, DOX-MWCNTs-PEG-TAT showed improved cell internalization, intracellular distribution and potentiated anticancer efficacy due to the TAT-mediated membrane translocation, endosomal escape and nuclear targeting. Furthermore, the therapeutic efficacy of DOX was not compromised after being conjugated with MWCNTs-PEG-TAT and the proposed nanocarrier was also confirmed to have a good biocompatibility. In conclusion, our results suggested that the unique combination of TAT and MWCNTs as a multifunctional drug delivery system might be a powerful tool for improved anticancer drug development.

Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

NASA Astrophysics Data System (ADS)

Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

2006-02-01

Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

Human Immunodeficiency Virus Type 1 Tat Protein Inhibits the SIRT1 Deacetylase and Induces T-Cell Hyperactivation

PubMed Central

Kwon, Hye-Sook; Brent, Michael M.; Getachew, Ruth; Jayakumar, Prerana; Chen, Lin-Feng; Schnolzer, Martina; McBurney, Michael W.; Marmorstein, Ronen; Greene, Warner C.; Ott, Melanie

2009-01-01

Summary Symptoms of T-cell hyperactivation shape the course and outcome of HIV-1 infection, but the mechanism(s) underlying this chronic immune activation are not well understood. We find that the viral transactivator Tat promotes hyperactivation of T cells by blocking the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of NF-κB. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-κB-responsive genes, a function lost in SIRT1−/− cells. These results support a model where the normal function of SIRT1 as a negative regulator of T-cell activation is suppressed by Tat during HIV infection. These events likely contribute to the state of immune cell hyperactivation found in HIV-infected individuals. PMID:18329615

Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication.

PubMed

Malina, Jaroslav; Hannon, Michael J; Brabec, Viktor

2016-07-12

The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat-TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

Initial assembly steps of a translocase for folded proteins

PubMed Central

Blümmel, Anne-Sophie; Haag, Laura A.; Eimer, Ekaterina; Müller, Matthias; Fröbel, Julia

2015-01-01

The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors. PMID:26068441

Membrane-Mediated Cooperativity of Proteins

NASA Astrophysics Data System (ADS)

Weikl, Thomas R.

2018-04-01

Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.

An attenuated herpes simplex virus type 1 (HSV1) encoding the HIV-1 Tat protein protects mice from a deadly mucosal HSV1 challenge.

PubMed

Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C

2014-01-01

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and

An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

PubMed Central

Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C.

2014-01-01

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and

HIV-Tat immunization induces cross-clade neutralizing antibodies and CD4(+) T cell increases in antiretroviral-treated South African volunteers: a randomized phase II clinical trial.

PubMed

Ensoli, Barbara; Nchabeleng, Maphoshane; Ensoli, Fabrizio; Tripiciano, Antonella; Bellino, Stefania; Picconi, Orietta; Sgadari, Cecilia; Longo, Olimpia; Tavoschi, Lara; Joffe, Daniel; Cafaro, Aurelio; Francavilla, Vittorio; Moretti, Sonia; Pavone Cossut, Maria Rosaria; Collacchi, Barbara; Arancio, Angela; Paniccia, Giovanni; Casabianca, Anna; Magnani, Mauro; Buttò, Stefano; Levendal, Elise; Ndimande, John Velaphi; Asia, Bennett; Pillay, Yogan; Garaci, Enrico; Monini, Paolo

2016-06-09

Although combined antiretroviral therapy (cART) has saved millions of lives, it is incapable of full immune reconstitution and virus eradication. The transactivator of transcription (Tat) protein is a key human immunodeficiency virus (HIV) virulence factor required for virus replication and transmission. Tat is expressed and released extracellularly by infected cells also under cART and in this form induces immune dysregulation, and promotes virus reactivation, entry and spreading. Of note, anti-Tat antibodies are rare in natural infection and, when present, correlate with asymptomatic state and reduced disease progression. This suggested that induction of anti-Tat antibodies represents a pathogenesis-driven intervention to block progression and to intensify cART. Indeed Tat-based vaccination was safe, immunogenic and capable of immune restoration in an open-label, randomized phase II clinical trial conducted in 168 cART-treated volunteers in Italy. To assess whether B-clade Tat immunization would be effective also in patients with different genetic background and infecting virus, a phase II trial was conducted in South Africa. The ISS T-003 was a 48-week randomised, double-blinded, placebo-controlled trial to evaluate immunogenicity (primary endpoint) and safety (secondary endpoint) of B-clade Tat (30 μg) given intradermally, three times at 4-week intervals, in 200 HIV-infected adults on effective cART (randomised 1:1) with CD4(+) T-cell counts ≥200 cells/µL. Study outcomes also included cross-clade anti-Tat antibodies, neutralization, CD4(+) T-cell counts and therapy compliance. Immunization was safe and well-tolerated and induced durable, high titers anti-Tat B-clade antibodies in 97 % vaccinees. Anti-Tat antibodies were cross-clade (all vaccinees tested) and neutralized Tat-mediated entry of oligomeric B-clade and C-clade envelope in dendritic cells (24 participants tested). Anti-Tat antibody titers correlated positively with neutralization. Tat

HIV-1 Tat addresses dendritic cells to induce a predominant Th1-type adaptive immune response that appears prevalent in the asymptomatic stage of infection.

PubMed

Fanales-Belasio, Emanuele; Moretti, Sonia; Fiorelli, Valeria; Tripiciano, Antonella; Pavone Cossut, Maria R; Scoglio, Arianna; Collacchi, Barbara; Nappi, Filomena; Macchia, Iole; Bellino, Stefania; Francavilla, Vittorio; Caputo, Antonella; Barillari, Giovanni; Magnani, Mauro; Laguardia, Maria Elena; Cafaro, Aurelio; Titti, Fausto; Monini, Paolo; Ensoli, Fabrizio; Ensoli, Barbara

2009-03-01

Tat is an early regulatory protein that plays a major role in human HIV-1 replication and AIDS pathogenesis, and therefore, it represents a key target for the host immune response. In natural infection, however, Abs against Tat are produced only by a small fraction (approximately 20%) of asymptomatic individuals and are rarely seen in progressors, suggesting that Tat may possess properties diverting the adaptive immunity from generating humoral responses. Here we show that a Th1-type T cell response against Tat is predominant over a Th2-type B cell response in natural HIV-1 infection. This is likely due to the capability of Tat to selectively target and very efficiently enter CD1a-expressing monocyte-derived dendritic cells (MDDC), which represent a primary target for the recognition and response to virus Ag. Upon cellular uptake, Tat induces MDDC maturation and Th1-associated cytokines and beta-chemokines production and polarizes the immune response in vitro to the Th1 pattern through the transcriptional activation of TNF-alpha gene expression. This requires the full conservation of Tat transactivation activity since neither MDDC maturation nor TNF-alpha production are found with either an oxidized Tat, which does not enter MDDC, or with a Tat protein mutated in the cysteine-rich region (cys22 Tat), which enters MDDC as the wild-type Tat but is transactivation silent. Consistently with these data, inoculation of monkeys with the native wild-type Tat induced a predominant Th1 response, whereas cys22 Tat generated mostly Th2 responses, therefore providing evidence that Tat induces a predominant Th1 polarized adaptive immune response in the host.

SHMT2 and the BRCC36/BRISC deubiquitinase regulate HIV-1 Tat K63-ubiquitylation and destruction by autophagy.

PubMed

Xu, Muyu; Moresco, James J; Chang, Max; Mukim, Amey; Smith, Davey; Diedrich, Jolene K; Yates, John R; Jones, Katherine A

2018-05-23

HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.

Sources of male chauvinism in the TAT.

PubMed

Potkay, C R; Merrens, M R

1975-10-01

Potential sources of antifemale bias in TAT stimuli were evaluated by having 358 undergraduate subjects rate 17 male and 17 female TAT figures on 7-point anchored scales. Data from the five independent rating conditions were examined by 2 x 2 ANOVA. Biases toward greater Mental Health and Intelligence for female figures were seen to be insufficient counterbalancers of biases toward greater Cultural Favorability and Identification for male figures. Achievement status was rated equivalently. TAT stimuli appeared to show a "built in" source of male chauvinism systematically "pulling" male-sex identification. Potential for unfavorable clinical evaluation was seen to be greater for female TAT subjects compared with male subjects.

The presence of anti-Tat antibodies is predictive of long-term nonprogression to AIDS or severe immunodeficiency: findings in a cohort of HIV-1 seroconverters.

PubMed

Rezza, Giovanni; Fiorelli, Valeria; Dorrucci, Maria; Ciccozzi, Massimo; Tripiciano, Antonella; Scoglio, Arianna; Collacchi, Barbara; Ruiz-Alvarez, Maria; Giannetto, Concettina; Caputo, Antonella; Tomasoni, Lina; Castelli, Francesco; Sciandra, Mauro; Sinicco, Alessandro; Ensoli, Fabrizio; Buttò, Stefano; Ensoli, Barbara

2005-04-15

The human immunodeficiency virus (HIV) type 1 Tat protein plays a key role in the life cycle of the virus and in pathogenesis and is highly conserved among HIV subtypes. On the basis of this and of safety, immunogenicity, and efficacy findings in monkeys, Tat is being tested as a vaccine in phase 1 trials. Here, we evaluated the incidence and risk of progression to advanced HIV disease by anti-Tat serostatus in a cohort of 252 HIV-1 seroconverters. The risk of progression was lower in the anti-Tat-positive subjects than in the anti-Tat-negative subjects. Progression was faster in the persistently anti-Tat-negative subjects than in the transiently anti-Tat-positive subjects, and no progression was observed in the persistently anti-Tat-positive subjects.

Ketone bodies protection against HIV-1 Tat-induced neurotoxicity.

PubMed

Hui, Liang; Chen, Xuesong; Bhatt, Dhaval; Geiger, Nicholas H; Rosenberger, Thad A; Haughey, Norman J; Masino, Susan A; Geiger, Jonathan D

2012-07-01

HIV-1-associated neurocognitive disorder (HAND) is a syndrome that ranges clinically from subtle neuropsychological impairments to profoundly disabling HIV-associated dementia. Not only is the pathogenesis of HAND unclear, but also effective treatments are unavailable. The HIV-1 transactivator of transcription protein (HIV-1 Tat) is strongly implicated in the pathogenesis of HAND, in part, because of its well-characterized ability to directly excite neurons and cause neurotoxicity. Consistent with previous findings from others, we demonstrate here that HIV-1 Tat induced neurotoxicity, increased intracellular calcium, and disrupted a variety of mitochondria functions, such as reducing mitochondrial membrane potential, increasing levels of reactive oxygen species, and decreasing bioenergetic efficiency. Of therapeutic importance, we show that treatment of cultured neurons with ketone bodies normalized HIV-1 Tat induced changes in levels of intracellular calcium, mitochondrial function, and neuronal cell death. Ketone bodies are normally produced in the body and serve as alternative energy substrates in tissues including brain and can cross the blood-brain barrier. Ketogenic strategies have been used clinically for treatment of neurological disorders and our current results suggest that similar strategies may also provide clinical benefits in the treatment of HAND. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Tat-functionalized liposomes for the treatment of meningitis: an in vitro study

PubMed Central

Bartomeu Garcia, Caterina; Shi, Di; Webster, Thomas J

2017-01-01

Bacterial meningitis has become a global concern, because of the emergence of antibiotic-resistant bacteria. It has been demonstrated that liposomes can enter bacteria, thus providing a possible treatment for numerous infections, including meningitis. Fusogenic liposomes are pH-sensitive with a high capacity to fuse with the bacteria membrane and promote intracellular drug release. Moreover, this ability can be improved by using cell-penetrating peptides (such as Tat47–57, which is a peptide derived from the Tat protein of HIV). The purpose of this in vitro study was to demonstrate for the first time the ability of the presently prepared fusogenic liposomes, which were spherical particles with a diameter of 100 nm loaded with antibiotics and functionalized with-cell penetrating peptides (Tat47–57), to fight the main bacteria that cause meningitis. For this, vancomycin, methicillin, and ampicillin antibiotics were loaded inside fusogenic liposomes to fight Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, and Escherichia coli. Antibacterial activity of Tat-functionalized and nonfunctionalized liposomes loaded with antibiotics was tested by determining bacteria colony-forming units and growth-curve assays coupled with live/dead assays using fluorescence microscopy. Results showed a remarkable decrease in antibiotic minimum inhibitory concentration when all of the bacteria were treated with these novel liposomes, especially for the functionalized liposomes loaded with methicillin. With antibiotic concentrations of 1.7–3 µg/mL for Tat-functionalized liposomes loaded with methicillin, the bacteria population was totally eradicated. Cytotoxicity tests with astrocytes and endothelial cells, major cellular components of the blood–brain barrier, were also performed for all of the liposomes, including free antibiotic and the Tat peptide. Results showed much promise for the further study of the presently formulated liposomes to treat meningitis

Immune Responses of HIV-1 Tat Transgenic Mice to Mycobacterium Tuberculosis W-Beijing SA161

PubMed Central

Honda, Jennifer R; Shang, Shaobin; Shanley, Crystal A; Caraway, Megan L; Henao-Tamayo, Marcela; Chan, Edward D; Basaraba, Randall J; Orme, Ian M; Ordway, Diane J; Flores, Sonia C

2011-01-01

Background: Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in the world and exacerbates disease caused by the human immunodeficiency virus (HIV). HIV infected individuals are prone to lung infections by a variety of microbial pathogens, including M. tuberculosis. While the destruction of the adaptive immune response by HIV is well understood, the actual pathogenesis of tuberculosis in co-infected individuals remains unclear. Tat is an HIV protein essential for efficient viral gene transcription, is secreted from infected cells, and is known to influence a variety of host inflammatory responses. We hypothesize Tat contributes to pathophysiological changes in the lung microenvironment, resulting in impaired host immune responses to infection by M. tuberculosis. Results: Herein, we show transgenic mice that express Tat by lung alveolar cells are more susceptible than non-transgenic control littermates to a low-dose aerosol infection of M. tuberculosis W-Beijing SA161. Survival assays demonstrate accelerated mortality rates of the Tat transgenic mice compared to non-transgenics. Tat transgenic mice also showed poorly organized lung granulomata-like lesions. Analysis of the host immune response using quantitative RT-PCR, flow cytometry for surface markers, and intracellular cytokine staining showed increased expression of pro-inflammatory cytokines in the lungs, increased numbers of cells expressing ICAM1, increased numbers of CD4+CD25+Foxp3+ T regulatory cells, and IL-4 producing CD4+ T cells in the Tat transgenics compared to infected non-tg mice. Conclusions: Our data show quantitative differences in the inflammatory response to the SA161 clinical isolate of M. tuberculosis W-Beijing between Tat transgenic and non-transgenic mice, suggesting Tat contributes to the pathogenesis of tuberculosis. PMID:22046211

Passage of Magnetic Tat-Conjugated Fe3O4@SiO2 Nanoparticles Across In Vitro Blood-Brain Barrier

NASA Astrophysics Data System (ADS)

Zhao, Xueqin; Shang, Ting; Zhang, Xiaodan; Ye, Ting; Wang, Dajin; Rei, Lei

2016-10-01

Delivery of diagnostic or therapeutic agents across the blood-brain barrier (BBB) remains a major challenge of brain disease treatment. Magnetic nanoparticles are actively being developed as drug carriers due to magnetic targeting and subsequently reduced off-target effects. In this paper, we developed a magnetic SiO2@Fe3O4 nanoparticle-based carrier bound to cell-penetrating peptide Tat (SiO2@Fe3O4 -Tat) and studied its fates in accessing BBB. SiO2@Fe3O4-Tat nanoparticles (NPs) exhibited suitable magnetism and good biocompatibility. NPs adding to the apical chamber of in vitro BBB model were found in the U251 glioma cells co-cultured at the bottom of the Transwell, indicating that particles passed through the barrier and taken up by glioma cells. Moreover, the synergistic effects of Tat and magnetic field could promote the efficient cellular internalization and the permeability across the barrier. Besides, functionalization with Tat peptide allowed particles to locate into the nucleus of U251 cells than the non-conjugated NPs. These results suggest that SiO2@Fe3O4-Tat NPs could penetrate the BBB through the transcytosis of brain endothelial cells and magnetically mediated dragging. Therefore, SiO2@Fe3O4-Tat NPs could be exploited as a potential drug delivery system for chemotherapy and gene therapy of brain disease.

A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat

PubMed Central

2014-01-01

Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. PMID:24985203

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

PubMed

Asamitsu, Kaori; Hirokawa, Takatsugu; Okamoto, Takashi

2017-01-01

In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons.

PubMed

Tan, Elaine Y M; Law, Janice W S; Wang, Chi-Hwa; Lee, Alan Y W

2007-12-01

Neurons in post-traumatized mammalian central nervous system show only limited degree of regeneration, which can be attributed to the presence of neurite outgrowth inhibitors in damaged myelin and glial scar, and to the apoptosis of severed central neurons and glial cells during secondary Wallerian degeneration. RhoA GTPase has been implicated as the common denominator in these counter-regeneration events, which shows significant and persistent up-regulation for weeks in injured spinal cord and cerebral infarct after stroke. While the exoenzyme C3 transferase is a potent RhoA inhibitor, its extremely low efficiency of cell entry and degradation in vivo has restricted the therapeutic value. This study aims to circumvent these problems by developing a membrane-permeating form of C3 transferase and a biopolymer-based microsphere depot system for sustainable controlled release of the protein. A membrane-permeating form of C3 transferase was developed by fusing a Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus to its amino terminal using standard molecular cloning techniques. After confirming efficient cell entry into epithelial and neuroblastoma cells, the resulting recombinant protein TAT-C3 was encapsulated in biocompatible polymer poly(D,L -lactide-co-glycolide) in the form of microspheres by a water-in-oil-in-water (W/O/W) emulsion method. By blending capped and uncapped form of the polymer at different ratios, TAT-C3 protein release profile was modified to suit the expression pattern of endogenous RhoA during CNS injuries. Bioactivity of TAT-C3 released from microspheres was assessed by RhoA ribosylation assay. In contrast to wild-type C3 transferase, the modified TAT-C3 protein was found to efficiently enter NIH3T3 and N1E-115 neuroblastoma cells as early as 6 hours of incubation. The fusion of TAT sequence to C3 transferase imposed no appreciable effects on its biological activity in promoting neurite outgrowth

Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids.

PubMed

Vilches, Clara; Boiadjieva-Knöpfel, Emilia; Bodoy, Susanna; Camargo, Simone; López de Heredia, Miguel; Prat, Esther; Ormazabal, Aida; Artuch, Rafael; Zorzano, Antonio; Verrey, François; Nunes, Virginia; Palacín, Manuel

2018-04-02

Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y + LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivo Methods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo , we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice). Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y + LAT1/CD98hc. Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo , and y + LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans. Copyright © 2018 by the American Society of Nephrology.

Genetic disruption of tubulin acetyltransferase, αTAT1, inhibits proliferation and invasion of colon cancer cells through decreases in Wnt1/β-catenin signaling

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

Oh, Somi; You, Eunae; Ko, Panseon

Microtubules are required for diverse cellular processes, and abnormal regulation of microtubule dynamics is closely associated with severe diseases including malignant tumors. In this study, we report that α-tubulin N-acetyltransferase (αTAT1), a regulator of α-tubulin acetylation, is required for colon cancer proliferation and invasion via regulation of Wnt1 and its downstream genes expression. Public transcriptome analysis showed that expression of ATAT1 is specifically upregulated in colon cancer tissue. A knockout (KO) of ATAT1 in the HCT116 colon cancer cell line, using the CRISPR/Cas9 system showed profound inhibition of proliferative and invasive activities of these cancer cells. Overexpression of αTAT1 ormore » the acetyl-mimic K40Q α-tubulin mutant in αTAT1 KO cells restored the invasiveness, indicating that microtubule acetylation induced by αTAT1 is critical for HCT116 cell invasion. Analysis of colon cancer-related gene expression in αTAT1 KO cells revealed that the loss of αTAT1 decreased the expression of WNT1. Mechanistically, abrogation of tubulin acetylation by αTAT1 knockout inhibited localization of β-catenin to the plasma membrane and nucleus, thereby resulting in the downregulation of Wnt1 and of its downstream genes including CCND1, MMP-2, and MMP-9. These results suggest that αTAT1-mediated Wnt1 expression via microtubule acetylation is important for colon cancer progression. - Highlights: • Ablation of αTAT1 inhibits HCT116 colon cancer cell invasion. • αTAT1/acetylated microtubules regulate expression of Wnt1/β-catenin target genes. • Acetylated microtubules regulate cellular localization of β-catenin. • Loss of αTAT1 prevents Wnt1 from inducing β-catenin-dependent and -independent pathways.« less

Sequential delivery of TAT-HSP27 and VEGF using microsphere/hydrogel hybrid systems for therapeutic angiogenesis.

PubMed

Shin, Seung-Hwa; Lee, Jangwook; Lim, Kwang Suk; Rhim, Taiyoun; Lee, Sang Kyung; Kim, Yong-Hee; Lee, Kuen Yong

2013-02-28

Ischemic disease is associated with high mortality and morbidity rates, and therapeutic angiogenesis via systemic or local delivery of protein drugs is one potential approach to treat the disease. In this study, we hypothesized that combined delivery of TAT-HSP27 (HSP27 fused with transcriptional activator) and VEGF could enhance the therapeutic efficacy in an ischemic mouse model, and that sequential release could be critical in therapeutic angiogenesis. Alginate hydrogels containing TAT-HSP27 as an anti-apoptotic agent were prepared, and porous PLGA microspheres loaded with VEGF as an angiogenic agent were incorporated into the hydrogels to prepare microsphere/hydrogel hybrid delivery systems. Sequential in vitro release of TAT-HSP27 and VEGF was achieved by the hybrid systems. TAT-HSP27 was depleted from alginate gels in 7 days, while VEGF was continually released for 28 days. The release rate of VEGF was attenuated by varying the porous structures of PLGA microspheres. Sequential delivery of TAT-HSP27 and VEGF was critical to protect against muscle degeneration and fibrosis, as well as to promote new blood vessel formation in the ischemic site of a mouse model. This approach to controlling the sequential release behaviors of multiple drugs could be useful in the design of novel drug delivery systems for therapeutic angiogenesis. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.

PubMed Central

Marciniak, R A; Garcia-Blanco, M A; Sharp, P A

1990-01-01

Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo. Images PMID:2333305

Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

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

Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi

HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrolmore » induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction.« less

Development of Tat-Conjugated Dendrimer for Transdermal DNA Vaccine Delivery.

PubMed

Bahadoran, Azadeh; Moeini, Hassan; Bejo, Mohd Hair; Hussein, Mohd Zobir; Omar, Abdul Rahman

In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed. First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry. TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-).  In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine.  The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05). The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Tat-modified leptin is more accessible to hypothalamus through brain-blood barrier with a significant inhibition of body-weight gain in high-fat-diet fed mice.

PubMed

Zhang, C; Su, Z; Zhao, B; Qu, Q; Tan, Y; Cai, L; Li, X

2010-01-01

Obesity in human was found mainly due to the poor transportation of leptin through brain-blood barrier (BBB), called as leptin resistance. To produce a leptin capable of penetrating BBB, we have added Tat-PTD(9) to the C terminal of leptin to construct a fusion protein. The fusion Tat-leptin and native leptin genes were synthesized by single-step insertion of a polymerase chain reaction and expressed in Escherichia coli BL21 (Rosseta). The expressing products were purified and renatured by Ni-NTA affinity chromatography, and identified by the molecular size in SDS-PAGE gel and by its immunoreactivity to specific antibody with Western-blotting assay. To bio-functionally evaluate the fusion protein, Balb/c mice fed with high-fat diet (HFD) were given Tat-leptin, leptin or saline for 19 days. The immunohistochemical staining showed the increases in positive stains for the leptin in the region of hypothalamus of the HFD mice with either Tat-leptin or leptin as compared to saline group, but the staining intensity and frequency in the group with Tat-leptin were stronger and higher than those in the group with leptin. Furthermore, the most efficiency in preventing the body-weight gain caused by HFD was found in Tat-leptin group among these three groups. These results suggest that Tat-modified leptin may become a great potential candidate for the prevention or therapy of obese patients. J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart , New York.

Long Noncoding RNA uc002yug.2 Activates HIV-1 Latency through Regulation of mRNA Levels of Various RUNX1 Isoforms and Increased Tat Expression.

PubMed

Huan, Chen; Li, Zhaolong; Ning, Shanshan; Wang, Hong; Yu, Xiao-Fang; Zhang, Wenyan

2018-05-01

The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4 + T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions. IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1. Copyright © 2018 American Society for Microbiology.

Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis.

PubMed

Matsubara, Teruhiko; Otani, Ryohei; Yamashita, Miki; Maeno, Haruka; Nodono, Hanae; Sato, Toshinori

2017-02-13

Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

Tat-Mediated Induction of miRs-34a & -138 Promotes Astrocytic Activation via Downregulation of SIRT1: Implications for Aging in HAND.

PubMed

Hu, Guoku; Liao, Ke; Yang, Lu; Pendyala, Gurudutt; Kook, Yeonhee; Fox, Howard S; Buch, Shilpa

2017-09-01

Astrocyte activation is a hallmark of HIV infection and aging in the CNS. In chronically infected HIV patients, prolonged activation of astrocytes has been linked to accelerated aging including but not limited to neurocognitive impairment and frailty. The current study addresses the role of HIV protein Tat in inducing a set of small noncoding microRNAs (miRNA) that play critical role in astrogliosis. In our efforts to link astrocyte activation as an indicator of aging, we assessed the brains of both wild type and HIV transgenic rats for the expression of glial fibrillary acidic protein (GFAP). As expected, in the WT animals we observed age-dependent increase in astrogliosis in the older animals compared to the younger group. Interestingly, compared to the young WT group, young HIV Tg rats exhibited higher levels of GFAP in this trend was also observed in the older HIV Tg rats compared to the older WT group. Based on the role of SIRT1 in aging and the regulation of SIRT1 by miRNAs-34a and -138, we next assessed the expression levels of these miRs in the brains of both the young an old WT and HIV Tg rats. While there were no significant differences in the young WT versus the HIV Tg rats, in the older HIV Tg rats there was a significant upregulation in the expression of miRs-34a & -138 in the brains. Furthermore, increased expression of miRs-34a & -138 in the older Tg rats, correlated with a concomitant decrease in their common anti-aging target protein SIRT1, in the brains of these animals. To delineate the mechanism of action we assessed the role of HIV-Tat (present in the Tg rats) in inducing miRs-34a & -138 in both the primary astrocytes and the astrocytoma cell line A172, thereby leading to posttranscriptional suppression of SIRT1 with a concomitant up regulation of NF-kB driven expression of GFAP.

CUS2, a Yeast Homolog of Human Tat-SF1, Rescues Function of Misfolded U2 through an Unusual RNA Recognition Motif

PubMed Central

Yan, Dong; Perriman, Rhonda; Igel, Haller; Howe, Kenneth J.; Neville, Megan; Ares, Manuel

1998-01-01

A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells. PMID:9710584

Creatine protects against mitochondrial dysfunction associated with HIV-1 Tat-induced neuronal injury

PubMed Central

Stevens, Patrick R.; Gawryluk, Jeremy W.; Hui, Liang; Chen, Xuesong; Geiger, Jonathan D.

2015-01-01

HIV-1 infected individuals are living longer but experiencing a prevalence rate of over 50% for HIV-1 associated neurocognitive disorders (HAND) for which no effective treatment is available. Viral and cellular factors secreted by HIV-1 infected cells leads to neuronal injury and HIV-1 Tat continues to be implicated in the pathogenesis of HAND. Here we tested the hypothesis that creatine protected against HIV-1 Tat-induced neuronal injury by preventing mitochondrial bioenergetic crisis and/or redox catastrophe. Creatine blocked HIV-1 Tat1-72-induced increases in neuron cell death and synaptic area loss. Creatine protected against HIV-1 Tat-induced decreases in ATP. Creatine and creatine plus HIV-1 Tat increased cellular levels of creatine, and creatine plus HIV-1 Tat further decreased ratios of phosphocreatine to creatine observed with creatine or HIV-1 Tat treatments alone. Additionally, creatine protected against HIV-1 Tat-induced mitochondrial hypopolarization and HIV-1 Tat-induced mitochondrial permeability transition pore opening. Thus, creatine may be a useful adjunctive therapy against HAND. PMID:25613139

Creatine protects against mitochondrial dysfunction associated with HIV-1 Tat-induced neuronal injury.

PubMed

Stevens, Patrick R; Gawryluk, Jeremy W; Hui, Liang; Chen, Xuesong; Geiger, Jonathan D

2014-01-01

HIV-1 infected individuals live longer but experience a prevalence rate of over 50% for HIV-1 associated neurocognitive disorders (HAND) for which no effective treatment is available. Viral and cellular factors secreted by HIV-1 infected cells lead to neuronal injury and HIV-1 Tat continues to be implicated in the pathogenesis of HAND. Here we tested the hypothesis that creatine protected against HIV-1 Tat-induced neuronal injury by preventing mitochondrial bioenergetic crisis and/or redox catastrophe. Creatine blocked HIV-1 Tat(1-72)-induced increases in neuron cell death and synaptic area loss. Creatine protected against HIV-1 Tat-induced decreases in ATP. Creatine and creatine plus HIV-1 Tat increased cellular levels of creatine, and creatine plus HIV-1 Tat further decreased ratios of phosphocreatine to creatine observed with creatine or HIV-1 Tat treatments alone. Additionally, creatine protected against HIV-1 Tat-induced mitochondrial hypopolarization and HIV-1 Tat-induced mitochondrial permeability transition pore opening. Thus, creatine may be a useful adjunctive therapy against HAND.

Oxidized LDL activates blood platelets through CD36/NOX2–mediated inhibition of the cGMP/protein kinase G signaling cascade

PubMed Central

Magwenzi, Simbarashe; Woodward, Casey; Wraith, Katie S.; Aburima, Ahmed; Raslan, Zaher; Jones, Huw; McNeil, Catriona; Wheatcroft, Stephen; Yuldasheva, Nadira; Febbriao, Maria; Kearney, Mark

2015-01-01

Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in dyslipidemic disorders. Although oxLDL stimulates activatory signaling, it is unclear how these events drive accelerated thrombosis. Here, we describe a mechanism for oxLDL-mediated platelet hyperactivity that requires generation of reactive oxygen species (ROS). Under arterial flow, oxLDL triggered sustained generation of platelet intracellular ROS, which was blocked by CD36 inhibitors, mimicked by CD36-specific oxidized phospholipids, and ablated in CD36−/− murine platelets. oxLDL-induced ROS generation was blocked by the reduced NAD phosphate oxidase 2 (NOX2) inhibitor, gp91ds-tat, and absent in NOX2−/− mice. The synthesis of ROS by oxLDL/CD36 required Src-family kinases and protein kinase C (PKC)-dependent phosphorylation and activation of NOX2. In functional assays, oxLDL abolished guanosine 3′,5′-cyclic monophosphate (cGMP)-mediated signaling and inhibited platelet aggregation and arrest under flow. This was prevented by either pharmacologic inhibition of NOX2 in human platelets or genetic ablation of NOX2 in murine platelets. Platelets from hyperlipidemic mice were also found to have a diminished sensitivity to cGMP when tested ex vivo, a phenotype that was corrected by infusion of gp91ds-tat into the mice. This study demonstrates that oxLDL and hyperlipidemia stimulate the generation of NOX2-derived ROS through a CD36-PKC pathway and may promote platelet hyperactivity through modulation of cGMP signaling. PMID:25710879

Neonatal hippocampal Tat injections: developmental effects on prepulse inhibition (PPI) of the auditory startle response

PubMed Central

Fitting, Sylvia; Booze, Rosemarie M.; Mactutus, Charles F.

2013-01-01

The current estimate of children (<15 years) living with HIV and AIDS is 2.2 million [UNAIDS/WHO, 2005. AIDS Epidemic Update. UNAIDS, Geneva]. The major source of infection occurs through vertical transmission of the virus from mother to child during delivery [UNAIDS/ WHO, 2005. AIDS Epidemic Update. UNAIDS, Geneva]. Recent studies have shown that timing of HIV-1 infection might be related to the onset and rate of progression of CNS disease [Blanche, S., Mayaux, M.-J., Rouziox, C., Teglas, J.-P., Firtion, G., Monpoux, F., Cicaru-Vigneron, N., Meier, F., Tricoire, J., Courpotin, C., Vilmer, E., Griscelli, C., Delfraissy, J.-F., 1994. Relation of the course of HIV infection in children to the severity of the disease in their mothers at delivery. N. Engl. J. Med. 330 (5), 308–312]. The effects of HIV on the brain are thought to be mediated indirectly through the viral toxins Tat and gp120. This study characterized developmental effects on PPI following intrahippocampal administration of Tat. On postnatal day (P)1, one male and one female pup from each of eight Sprague–Dawley litters were bilaterally injected with 50 µg Tat or saline (1 µl volume). Animals were tested for PPI of the auditory startle response (ASR) (ISIs of 0, 8,40, 80, 120, and 4000 ms, six trial blocks, Latin-square design) on days 30, 60 and 90. Tat altered PPI and the pattern of alterations was different for males and females. For males, a leftward shift was evident in the ISI for maximal inhibition of the response on day 30 and on day 60 (χ2(1) = 4.7,p ≤ .03, and χ2(1) = 5.3, p ≤ .02, respectively), but not on day 90. For females, Tat altered peak ASR latency across PPI trials (8–120 ms) at all days of testing (30, 60, and 90 days of age), as indexed by orthogonal component analyses, indicating less modulation of PPI by ISI. Data collected from a second group that were tested only once at 90 days of age, suggested that the observed adverse Tat effects for males and females early in

Molecular simulations of lipid-mediated protein-protein interactions.

PubMed

de Meyer, Frédérick Jean-Marie; Venturoli, Maddalena; Smit, Berend

2008-08-01

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

PubMed

Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

2001-12-13

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential

Role of water mediated interactions in protein-protein recognition landscapes.

PubMed

Papoian, Garegin A; Ulander, Johan; Wolynes, Peter G

2003-07-30

The energy landscape picture of protein folding and binding is employed to optimize a number of pair potentials for direct and water-mediated interactions in protein complex interfaces. We find that water-mediated interactions greatly complement direct interactions in discriminating against various types of trap interactions that model those present in the cell. We highlight the context dependent nature of knowledge-based binding potentials, as contrasted with the situation for autonomous folding. By performing a Principal Component Analysis (PCA) of the corresponding interaction matrixes, we rationalize the strength of the recognition signal for each combination of the contact type and reference trap states using the differential in the idealized "canonical" amino acid compositions of native and trap layers. The comparison of direct and water-mediated contact potential matrixes emphasizes the importance of partial solvation in stabilizing charged groups in the protein interfaces. Specific water-mediated interresidue interactions are expected to influence significantly the kinetics as well as thermodynamics of protein association.

Molecular simulation of the effect of cholesterol on lipid-mediated protein-protein interactions.

PubMed

de Meyer, Frédérick J-M; Rodgers, Jocelyn M; Willems, Thomas F; Smit, Berend

2010-12-01

Experiments and molecular simulations have shown that the hydrophobic mismatch between proteins and membranes contributes significantly to lipid-mediated protein-protein interactions. In this article, we discuss the effect of cholesterol on lipid-mediated protein-protein interactions as function of hydrophobic mismatch, protein diameter and protein cluster size, lipid tail length, and temperature. To do so, we study a mesoscopic model of a hydrated bilayer containing lipids and cholesterol in which proteins are embedded, with a hybrid dissipative particle dynamics-Monte Carlo method. We propose a mechanism by which cholesterol affects protein interactions: protein-induced, cholesterol-enriched, or cholesterol-depleted lipid shells surrounding the proteins affect the lipid-mediated protein-protein interactions. Our calculations of the potential of mean force between proteins and protein clusters show that the addition of cholesterol dramatically reduces repulsive lipid-mediated interactions between proteins (protein clusters) with positive mismatch, but does not affect attractive interactions between proteins with negative mismatch. Cholesterol has only a modest effect on the repulsive interactions between proteins with different mismatch. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas.

PubMed

Luzzi, Anna; Morettini, Federica; Gazaneo, Sara; Mundo, Lucia; Onnis, Anna; Mannucci, Susanna; Rogena, Emily A; Bellan, Cristiana; Leoncini, Lorenzo; De Falco, Giulia

2014-01-01

A close association between HIV infection and the development of cancer exists. Although the advent of highly active antiretroviral therapy has changed the epidemiology of AIDS-associated malignancies, a better understanding on how HIV can induce malignant transformation will help the development of novel therapeutic agents. HIV has been reported to induce the expression of DNMT1 in vitro, but still no information is available about the mechanisms regulating DNMT expression in HIV-related B-cell lymphomas. In this paper, we investigated the expression of DNMT family members (DNMT1, DNMT3a/b) in primary cases of aggressive B-cell lymphomas of HIV-positive subjects. Our results confirmed the activation of DNMT1 by HIV in vivo, and reported for the first time a marked up-regulation of DNMT3a and DNMT3b in HIV-positive aggressive B-cell lymphomas. DNMT up-regulation in HIV-positive tumors correlated with down-regulation of specific microRNAs, as the miR29 family, the miR148-152 cluster, known to regulate their expression. Literature reports the activation of DNMTs by the human polyomavirus BKV large T-antigen and adenovirus E1a, through the pRb/E2F pathway. We have previously demonstrated that the HIV Tat protein is able to bind to the pocket proteins and to inactivate their oncosuppressive properties, resulting in uncontrolled cell proliferation. Therefore, we focused on the role of Tat, due to its capability to be released from infected cells and to dysregulate uninfected ones, using an in vitro model in which Tat was ectopically expressed in B-cells. Our findings demonstrated that the ectopic expression of Tat was per se sufficient to determine DNMT up-regulation, based on microRNA down-regulation, and that this results in aberrant hypermethylation of target genes and microRNAs. These results point at a direct role for Tat in participating in uninfected B-cell lymphomagenesis, through dysregulation of the epigenetical control of gene expression.

HIV-1 Tat and opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines.

PubMed

El-Hage, Nazira; Wu, Guanghan; Wang, Juan; Ambati, Jayakrishna; Knapp, Pamela E; Reed, Janelle L; Bruce-Keller, Annadora J; Hauser, Kurt F

2006-01-15

Opiates exacerbate human immunodeficiency virus type 1 (HIV-1) Tat(1-72)-induced release of key proinflammatory cytokines by astrocytes, which may accelerate HIV neuropathogenesis in opiate abusers. The release of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), in particular, is potentiated by opiate-HIV Tat interactions in vitro. Although MCP-1 draws monocytes/macrophages to sites of CNS infection, and activated monocytes/microglia release factors that can damage bystander neurons, the role of MCP-1 in neuro-acquired immunodeficiency syndrome (neuroAIDS) progression in opiate abusers, or nonabusers, is uncertain. Using a chemotaxis assay, N9 microglial cell migration was found to be significantly greater in conditioned medium from mouse striatal astrocytes exposed to morphine and/or Tat(1-72) than in vehicle-, mu-opioid receptor (MOR) antagonist-, or inactive, mutant Tat(delta31-61)-treated controls. Conditioned medium from astrocytes treated with morphine and Tat caused the greatest increase in motility. The response was attenuated using conditioned medium immunoneutralized with MCP-1 antibodies, or medium from MCP-1(-/-) astrocytes. In the presence of morphine (time-release, subcutaneous implant), intrastriatal Tat increased the proportion of neural cells that were astroglia and F4/80+ macrophages at 7 days post-injection. This was not seen after treatment with Tat alone, or with morphine plus inactive Tat(delta31-61) or naltrexone. Glia displayed increased MOR and MCP-1 immunoreactivity after morphine and/or Tat exposure. The findings indicate that MCP-1 underlies most of the response of microglia, suggesting that one way in which opiates exacerbate neuroAIDS is by increasing astroglial-derived proinflammatory chemokines at focal sites of CNS infection and promoting macrophage entry and local microglial activation. Importantly, increased glial expression of MOR can trigger an opiate-driven amplification/positive feedback of MCP-1 production and

CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

PubMed Central

2012-01-01

Background The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822–829 (2011)]. Results and discussion Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. Conclusions Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target. PMID

Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension

PubMed Central

Dalvi, Pranjali; Sharma, Himanshu; Chinnappan, Mahendran; Sanderson, Miles; Allen, Julie; Zeng, Ruoxi; Choi, Augustine; O'Brien-Ladner, Amy; Dhillon, Navneet K.

2016-01-01

ABSTRACT Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids. PMID:27723373

Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension.

PubMed

Dalvi, Pranjali; Sharma, Himanshu; Chinnappan, Mahendran; Sanderson, Miles; Allen, Julie; Zeng, Ruoxi; Choi, Augustine; O'Brien-Ladner, Amy; Dhillon, Navneet K

2016-12-01

Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids.

Characterization of the influence of mediator complex in HIV-1 transcription.

PubMed

Ruiz, Alba; Pauls, Eduardo; Badia, Roger; Riveira-Muñoz, Eva; Clotet, Bonaventura; Ballana, Ester; Esté, José A

2014-10-03

HIV-1 exploits multiple host proteins during infection. siRNA-based screenings have identified new proteins implicated in different pathways of the viral cycle that participate in a broad range of cellular functions. The human Mediator complex (MED) is composed of 28 elements and represents a fundamental component of the transcription machinery, interacting with the RNA polymerase II enzyme and regulating its ability to express genes. Here, we provide an evaluation of the MED activity on HIV replication. Knockdown of 9 out of 28 human MED proteins significantly impaired viral replication without affecting cell viability, including MED6, MED7, MED11, MED14, MED21, MED26, MED27, MED28, and MED30. Impairment of viral replication by MED subunits was at a post-integration step. Inhibition of early HIV transcripts was observed by siRNA-mediated knockdown of MED6, MED7, MED11, MED14, and MED28, specifically affecting the transcription of the nascent viral mRNA transactivation-responsive element. In addition, MED14 and MED30 were shown to have special relevance during the formation of unspliced viral transcripts (p < 0.0005). Knockdown of the selected MED factors compromised HIV transcription induced by Tat, with the strongest inhibitory effect shown by siMED6 and siMED14 cells. Co-immunoprecipitation experiments suggested physical interaction between MED14 and HIV-1 Tat protein. A better understanding of the mechanisms and factors controlling HIV-1 transcription is key to addressing the development of new strategies required to inhibit HIV replication or reactivate HIV-1 from the latent reservoirs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Fluctuations in Tat copy number when it counts the most: a possible mechanism to battle the HIV latency

PubMed Central

2013-01-01

The HIV-1 virus can enter a dormant state and become inactive, which reduces accessibility by antiviral drugs. We approach this latency problem from an unconventional point of view, with the focus on understanding how intrinsic chemical noise (copy number fluctuations of the Tat protein) can be used to assist the activation process of the latent virus. Several phase diagrams have been constructed in order to visualize in which regions of the parameter space noise can drive the activation process. Essential to the study is the use of a hyperbolic coordinate system, which greatly facilitates quantification of how the various reaction rate combinations shape the noise behavior of the Tat protein feedback system. We have designed a mathematical manual of how to approach the problem of activation quantitatively, and introduce the notion of an “operating point” of the virus. For both noise-free and noise-based strategies we show how operating point off-sets induce changes in the number of Tat molecules. The major result of the analysis is that for every noise-free strategy there is a noise-based strategy that requires lower dosage, but achieves the same anti-latency effect. It appears that the noise-based activation is advantageous for every operating point. PMID:23497153

HIV Tat1-72 and Opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines

PubMed Central

El-Hage, Nazira; Wu, Guanghan; Wang, Juan; Ambati, Jayakrishna; Knapp, Pamela E.; Reed, Janelle L.; Bruce-Keller, Annadora J.; Hauser, Kurt F.

2011-01-01

Opiates exacerbate HIV-1 Tat1-72–induced release of key proinflammatory cytokines by astrocytes, which may accelerate HIV neuropathogenesis in opiate abusers. The release of monocyte chemoattractant protein-1 (MCP-1 or CCL2), in particular, is potentiated by opiate-HIV Tat interactions in vitro. Although MCP-1 draws monocytes/macrophages to sites of CNS infection, and activated monocytes/microglia release factors that can damage bystander neurons, its role in neuroAIDS progression in opiate abusers, or non-abusers, is uncertain. Using a chemotaxis assay, N9 microglial cell migration was significantly greater in conditioned medium from mouse striatal astrocytes exposed to morphine and/or Tat1-72 than in vehicle-, μ opioid receptor (MOR) antagonist-, or inactive, mutant TatΔ31-61-treated controls. Conditioned medium from astrocytes treated with morphine and Tat caused the greatest increase in motility. The response was attenuated using conditioned medium immunoneutralized with MCP-1 antibodies, or medium from MCP-1−/− astrocytes. In the presence of morphine (time-release, subcutaneous implant), intrastriatal Tat increased the proportion of neural cells that were astroglia and F4/80+ macrophages at 7 days post-injection. This was not seen following treatment with Tat alone, or with morphine plus inactive TatΔ31-61 or naltrexone. Glia displayed increased MOR and MCP-1 immunoreactivity following morphine and/or Tat exposure. The findings indicate that MCP-1 underlies most of the response of microglia, suggesting that one way in which opiates exacerbate neuroAIDS is by increasing astroglial-derived proinflammatory chemokines at focal sites of CNS infection and promoting macrophage entry and local microglial activation. Importantly, increased glial expression of MOR can trigger an opiate-driven amplification/positive feedback of MCP-1 production and inflammation. PMID:16206161

Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

PubMed Central

Kim, Dong-Hyun; Kim, Baek

2011-01-01

The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

Penetration of HIV-1 Tat47-57 into PC/PE Bilayers Assessed by MD Simulation and X-ray Scattering.

PubMed

Neale, Chris; Huang, Kun; García, Angel E; Tristram-Nagle, Stephanie

2015-09-22

The interactions of the basic, cell-penetrating region (Y47GRKKRRQRRR57) of the HIV-1 Tat protein with dioleoylphosphatidylcholine (DOPC) bilayers were previously assessed by comparing experimental X-ray diffuse scattering with atomistic molecular dynamics simulations. Here, we extend this investigation by evaluating the influence of phosphatidylethanolamine (PE) lipids. Using experimental bilayer form factors derivedfrom X-ray diffuse scattering data as a guide, our simulations indicate that Tat peptides localize close to the carbonyl-glycerol group in the headgroup region of bilayers composed of either DOPC or DOPC:DOPE (1:1) lipid. Our results also suggest that Tat peptides may more frequently insert into the hydrophobic core of bilayers composed of PC:PE (1:1) lipids than into bilayers composed entirely of PC lipids. PE lipids may facilitate peptide translocation across a lipid bilayer by stabilizing intermediate states in which hydrated peptides span the bilayer.

TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation.

PubMed Central

Taylor, J P; Pomerantz, R; Bagasra, O; Chowdhury, M; Rappaport, J; Khalili, K; Amini, S

1992-01-01

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1505523

Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides.

PubMed

Strauch, Eva-Maria; Georgiou, George

2007-11-23

In vitro studies have suggested that the TatBC complex serves as the receptor for signal peptides targeted for export via the twin-arginine translocation (Tat) pathway. Substitution of the hallmark twin-arginine dipeptide with two lysines abrogates export of physiological substrates in all organisms. We report the isolation and characterization of suppressor mutations that allow export of an ssTor(KK)-GFP-SsrA tripartite fusion. We identified two amino acid suppressor mutations in the first cytoplasmic loop of TatC. In addition, two other amino acids in the first cytoplasmic loop exhibit epistatic suppression. Surprisingly, we also identified a suppressor mutation predicted to lie within the second periplasmic loop of TatC, a region that is not expected to interact directly with the signal peptide. The suppressor mutations allowed export of the native Esherichia coli Tat substrate trimethylamine N-oxide reductase with a twin-lysine substitution in its signal sequence. The cytoplasmic suppressor mutations conferred SDS sensitivity and partial filamentation, indicating that Tat export of authentic substrates was impaired.

In Vivo Selection of CD4+ T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

PubMed Central

Braun, Stephen E.; Taube, Ran; Zhu, Quan; Wong, Fay Eng; Murakami, Akikazu; Kamau, Erick; Dwyer, Markryan; Qiu, Gang; Daigle, Janet; Carville, Angela; Johnson, R. Paul

2012-01-01

Abstract We evaluated the potential of an anti–human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4+ cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4+ T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4+ cell. Upon reinfusion of engineered autologous CD4+ cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4+ T cells progressively decreased, concurrent with loss of CD4+ cells and elevated viral loads in both animals. However, CD4+ T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4+ cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo. PMID:22734618

Interaction between Tat and Drugs of Abuse during HIV-1 Infection and Central Nervous System Disease

PubMed Central

Maubert, Monique E.; Pirrone, Vanessa; Rivera, Nina T.; Wigdahl, Brian; Nonnemacher, Michael R.

2016-01-01

In many individuals, drug abuse is intimately linked with HIV-1 infection. In addition to being associated with one-third of all HIV-1 infections in the United States, drug abuse also plays a role in disease progression and severity in HIV-1-infected patients, including adverse effects on the central nervous system (CNS). Specific systems within the brain are known to be damaged in HIV-1-infected individuals and this damage is similar to that observed in drug abuse. Even in the era of anti-retroviral therapy (ART), CNS pathogenesis occurs with HIV-1 infection, with a broad range of cognitive impairment observed, collectively referred to as HIV-1-associated neurocognitive disorders (HAND). A number of HIV-1 proteins (Tat, gp120, Nef, Vpr) have been implicated in the etiology of pathogenesis and disease as a result of the biologic activity of the extracellular form of each of the proteins in a number of tissues, including the CNS, even in ART-suppressed patients. In this review, we have made Tat the center of attention for a number of reasons. First, it has been shown to be synthesized and secreted by HIV-1-infected cells in the CNS, despite the most effective suppression therapies available to date. Second, Tat has been shown to alter the functions of several host factors, disrupting the molecular and biochemical balance of numerous pathways contributing to cellular toxicity, dysfunction, and death. In addition, the advantages and disadvantages of ART suppression with regard to controlling the genesis and progression of neurocognitive impairment are currently under debate in the field and are yet to be fully determined. In this review, we discuss the individual and concerted contributions of HIV-1 Tat, drug abuse, and ART with respect to damage in the CNS, and how these factors contribute to the development of HAND in HIV-1-infected patients. PMID:26793168

Morphine Tolerance and Physical Dependence Are Altered in Conditional HIV-1 Tat Transgenic Mice.

PubMed

Fitting, Sylvia; Stevens, David L; Khan, Fayez A; Scoggins, Krista L; Enga, Rachel M; Beardsley, Patrick M; Knapp, Pamela E; Dewey, William L; Hauser, Kurt F

2016-01-01

Despite considerable evidence that chronic opiate use selectively affects the pathophysiologic consequences of human immunodeficiency virus type 1 (HIV-1) infection in the nervous system, few studies have examined whether neuro-acquired immune deficiency syndrome (neuroAIDS) might intrinsically alter the pharmacologic responses to chronic opiate exposure. This is an important matter because HIV-1 and opiate abuse are interrelated epidemics, and HIV-1 patients are often prescribed opiates as a treatment of HIV-1-related neuropathic pain. Tolerance and physical dependence are inevitable consequences of frequent and repeated administration of morphine. In the present study, mice expressing HIV-1 Tat in a doxycycline (DOX)-inducible manner [Tat(+)], their Tat(-) controls, and control C57BL/6 mice were chronically exposed to placebo or 75-mg morphine pellets to explore the effects of Tat induction on morphine tolerance and dependence. Antinociceptive tolerance and locomotor activity tolerance were assessed using tail-flick and locomotor activity assays, respectively, and physical dependence was measured with the platform-jumping assay and recording of other withdrawal signs. We found that Tat(+) mice treated with DOX [Tat(+)/DOX] developed an increased tolerance in the tail-flick assay compared with control Tat(-)/DOX and/or C57/DOX mice. Equivalent tolerance was developed in all mice when assessed by locomotor activity. Further, Tat(+)/DOX mice expressed reduced levels of physical dependence to chronic morphine exposure after a 1-mg/kg naloxone challenge compared with control Tat(-)/DOX and/or C57/DOX mice. Assuming the results seen in Tat transgenic mice can be generalized to neuroAIDS, our findings suggest that HIV-1-infected individuals may display heightened analgesic tolerance to similar doses of opiates compared with uninfected individuals and show fewer symptoms of physical dependence. Copyright © 2015 by The American Society for Pharmacology and Experimental

Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

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

Pohlschroder Mechthild

2009-02-03

Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tatmore » pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.« less

A lentiviral vector that activates latent human immunodeficiency virus-1 proviruses by the overexpression of tat and that kills the infected cells.

PubMed

Macías, David; Oya, Ricardo; Saniger, Luisa; Martín, Francisco; Luque, Francisco

2009-11-01

Despite the efficient HIV-1 replication blockage achieved with current highly active antiretroviral therapy (HAART) therapies, HIV-1 persists in the body and survives in a latent state that can last for the entire life of the patient. A long-lived reservoir of latently infected CD4(+) memory T cells represents the most important sanctuary for the virus and the greatest obstacle for viral eradication. In this work, we present an initial step toward a gene therapy approach aimed at the activation of latent provirus to induce the death of latently infected T cells. Latent HIV-1 infection is characterized by the failure of viral gene expression as a consequence of uninitiated or aborted transcription. We have constructed an HIV-1-based lentiviral vector (p5p53RTAT3) that expresses the viral trans-activating protein Tat in a drug-regulated manner and p53 in a Rev-dependent manner. We have demonstrated that the Tat-expressed protein from p5p53RTAT3 vector reactivates latent HIV-1 proviruses in J1.1 and ACH-2 cell lines and promotes p53-induced apoptosis in the presence of Rev. Our system was able to trigger the trans-activation of the provirus 5' long terminal repeat (LTR), stimulate the expression of the Rev protein from a tat-defective provirus, and provoke apoptosis selectively in the cells transfected with a tat-defective HIV-1 provirus in contrast to those with no HIV-1 provirus. However, the Rev-dependent p53 killing of latently infected cells was not effective enough for complete elimination of the awakened HIV-1 viruses. In summary, we have developed a vector system that is efficient in activating latent HIV-1 proviruses but that needs further improvement to kill infected cells.

PSD-95 uncoupling from NMDA receptors by Tat- N-dimer ameliorates neuronal depolarization in cortical spreading depression.

PubMed

Kucharz, Krzysztof; Søndergaard Rasmussen, Ida; Bach, Anders; Strømgaard, Kristian; Lauritzen, Martin

2017-05-01

Cortical spreading depression is associated with activation of NMDA receptors, which interact with the postsynaptic density protein 95 (PSD-95) that binds to nitric oxide synthase (nNOS). Here, we tested whether inhibition of the nNOS/PSD-95/NMDA receptor complex formation by anti-ischemic compound, UCCB01-144 (Tat- N-dimer) ameliorates the persistent effects of cortical spreading depression on cortical function. Using in vivo two-photon microscopy in somatosensory cortex in mice, we show that fluorescently labelled Tat- N-dimer readily crosses blood-brain barrier and accumulates in nerve cells during the first hour after i.v. injection. The Tat- N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO 2 ) responses were preserved. During cortical spreading depression, the Tat- N-dimer reduced the average amplitude of the negative shift in direct current potential by 33% (4.1 mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40 min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading depression on cortical blood flow and CMRO 2 . We suggest that uncoupling of PSD-95 from NMDA receptors reduces overall neuronal excitability and the amplitude of the spreading depolarization wave. These findings may be of interest for understanding the neuroprotective effects of the nNOS/PSD-95 uncoupling in stroke.

Deciphering structure-activity relationships in a series of Tat/TAR inhibitors.

PubMed

Pascale, Lise; González, Alejandro López; Di Giorgio, Audrey; Gaysinski, Marc; Teixido Closa, Jordi; Tejedor, Roger Estrada; Azoulay, Stéphane; Patino, Nadia

2016-11-01

A series of pentameric "Polyamide Amino Acids" (PAAs) compounds derived from the same trimeric precursor have been synthesized and investigated as HIV TAR RNA ligands, in the absence and in the presence of a Tat fragment. All PAAs bind TAR with similar sub-micromolar affinities but their ability to compete efficiently with the Tat fragment strongly differs, IC50 ranging from 35 nM to >2 μM. While NMR and CD studies reveal that all PAA interact with TAR at the same site and induce globally the same RNA conformational change upon binding, a comparative thermodynamic study of PAA/TAR equilibria highlights distinct TAR binding modes for Tat competitor and non-competitor PAAs. This led us to suggest two distinct interaction modes that have been further validated by molecular modeling studies. While the binding of Tat competitor PAAs induces a contraction at the TAR bulge region, the binding of non-competitor ones widens it. This could account for the distinct PAA ability to compete with Tat fragment. Our work illustrates how comparative thermodynamic studies of a series of RNA ligands of same chemical family are of value for understanding their binding modes and for rationalizing structure-activity relationships.

Trade-Space Analysis Tool for Constellations (TAT-C)

NASA Technical Reports Server (NTRS)

Le Moigne, Jacqueline; Dabney, Philip; de Weck, Olivier; Foreman, Veronica; Grogan, Paul; Holland, Matthew; Hughes, Steven; Nag, Sreeja

2016-01-01

Traditionally, space missions have relied on relatively large and monolithic satellites, but in the past few years, under a changing technological and economic environment, including instrument and spacecraft miniaturization, scalable launchers, secondary launches as well as hosted payloads, there is growing interest in implementing future NASA missions as Distributed Spacecraft Missions (DSM). The objective of our project is to provide a framework that facilitates DSM Pre-Phase A investigations and optimizes DSM designs with respect to a-priori Science goals. In this first version of our Trade-space Analysis Tool for Constellations (TAT-C), we are investigating questions such as: How many spacecraft should be included in the constellation? Which design has the best costrisk value? The main goals of TAT-C are to: Handle multiple spacecraft sharing a mission objective, from SmallSats up through flagships, Explore the variables trade space for pre-defined science, cost and risk goals, and pre-defined metrics Optimize cost and performance across multiple instruments and platforms vs. one at a time.This paper describes the overall architecture of TAT-C including: a User Interface (UI) interacting with multiple users - scientists, missions designers or program managers; an Executive Driver gathering requirements from UI, then formulating Trade-space Search Requests for the Trade-space Search Iterator first with inputs from the Knowledge Base, then, in collaboration with the Orbit Coverage, Reduction Metrics, and Cost Risk modules, generating multiple potential architectures and their associated characteristics. TAT-C leverages the use of the Goddard Mission Analysis Tool (GMAT) to compute coverage and ancillary data, streamlining the computations by modeling orbits in a way that balances accuracy and performance.TAT-C current version includes uniform Walker constellations as well as Ad-Hoc constellations, and its cost model represents an aggregate model consisting of

Intelligent tit-for-tat in the iterated prisoner's dilemma game

NASA Astrophysics Data System (ADS)

Baek, Seung Ki; Kim, Beom Jun

2008-07-01

We seek a route to the equilibrium where all the agents cooperate in the iterated prisoner’s dilemma game on a two-dimensional plane, focusing on the role of tit-for-tat strategy. When a time horizon, within which a strategy can recall the past, is one time step, an equilibrium can be achieved as cooperating strategies dominate the whole population via proliferation of tit-for-tat. Extending the time horizon, we filter out poor strategies by simplified replicator dynamics and observe a similar evolutionary pattern to reach the cooperating equilibrium. In particular, the rise of a modified tit-for-tat strategy plays a central role, which implies how a robust strategy is adopted when provided with an enhanced memory capacity.

Detergent-mediated protein aggregation

PubMed Central

Neale, Chris; Ghanei, Hamed; Holyoake, John; Bishop, Russell E.; Privé, Gilbert G.; Pomès, Régis

2016-01-01

Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein’s hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation. PMID:23466535

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

PubMed

Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

2006-07-01

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner

PubMed Central

Puyal, Julien; Margue, Christiane; Michel, Sébastien; Kreis, Stephanie; Kulms, Dagmar; Barras, David; Nahimana, Aimable; Widmann, Christian

2016-01-01

Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment. PMID:27602963

HIV-1 Tat-mediated induction of Platelet-derived Growth Factor in Astrocytes: Role of Early Growth Response Gene 1

PubMed Central

Bethel-Brown, Crystal; Yao, Honghong; Callen, Shannon; Lee, Young Han; Dash, Prasanta K; Kumar, Anil; Buch, Shilpa

2011-01-01

HIV-associated neurological disorders (HAND) are estimated to affect almost 60% of HIV infected individuals. HIV-encephalitis (HIVE), the pathological correlate of the most severe form of HAND is often characterized by glial activation, cytokine/chemokine dysregulation, and neuronal damage and loss. However, the severity of HIVE correlates better with glial activation rather than viral load. Using the macaque model, it has been demonstrated that simian immunodeficiency virus encephalitis (SIVE) correlates with increased expression of the mitogen platelet-derived growth factor-B (PDGF-B) chain in the brain. The present study was aimed at exploring the role of PDGF-B chain in HIV-associated activation and proliferation of astrocytes. Specifically, the data herein demonstrate that exposure of rat and human astrocytes to the HIV-1 protein, Tat resulted in the induction of PDGF at both the mRNA and protein levels. Furthermore, PDGF-BB induction was regulated by activation of ERK1/2 and JNK signaling pathways and the downstream transcription factor, early growth response 1(Egr-1). Chromatin immunoprecipitation (ChIP) assays demonstrated binding of Egr-1 to the PDGF-B promoter. Exposure of astrocytes to PDGF-BB, in turn, led to both increased proliferation and release of pro-inflammatory cytokines MCP-1 and IL-1β. Since astrogliosis is linked to disease severity, understanding its regulation by PDGF-BB could aid in the development of therapeutic intervention strategies for HAND. PMID:21368226

TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

PubMed Central

Wong, J G; Smithgall, M D; Haffar, O K

1997-01-01

Complete activation of peripheral blood T cells requires both T-cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus-1 (HIV-1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28-mediated signal transduction using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV-1 tat gene but not the interleukin-2 (IL-2) gene. Viral induction did not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H-7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL. Images Figure 1 Figure 2 Figure 3 PMID:9135558

Covalent attachment of TAT peptides and thiolated alkyl molecules on GaAs surfaces.

PubMed

Cho, Youngnam; Ivanisevic, Albena

2005-07-07

Four TAT peptide fragments were used to functionalize GaAs surfaces by adsorption from solution. In addition, two well-studied alkylthiols, mercaptohexadecanoic acid (MHA) and 1-octadecanethiol (ODT) were utilized as references to understand the structure of the TAT peptide monolayer on GaAs. The different sequences of TAT peptides were employed in recognition experiments where a synthetic RNA sequence was tested to verify the specific interaction with the TAT peptide. The modified GaAs surfaces were characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS). AFM studies were used to compare the surface roughness before and after functionalization. XPS allowed us to characterize the chemical composition of the GaAs surface and conclude that the monolayers composed of different sequences of peptides have similar surface chemistries. Finally, FT-IRRAS experiments enabled us to deduce that the TAT peptide monolayers have a fairly ordered and densely packed alkyl chain structure. The recognition experiments showed preferred interaction of the RNA sequence toward peptides with high arginine content.

Serum protein mediators of dementia and aging proper.

PubMed

Royall, Donald R; Al-Rubaye, Safa; Bishnoi, Ram; Palmer, Raymond F

2016-12-03

The latent variable "δ" (for "dementia") appears to be uniquely responsible for the dementing aspects of cognitive impairment. Age, depressive symptoms, gender and the apolipoprotein E (APOE) ε4 allele are independently associated with δ. In this analysis, we explore serum proteins as potential mediators of age's specific association with δ in a large, ethnically diverse longitudinal cohort, the Texas Alzheimer's Research and Care Consortium (TARCC). 22 serum proteins were recognized as partial mediators of age's association with δ. These include Insulin-like Growth Factor-Binding Protein 2 (IGF-BP2), which we had previously associated with age-specific cognitive change, and both Pancreatic Polypeptide (PP) and von Willebrand Factor (vWF), previously associated with δ. Nine other δ-related proteins were not confirmed by this ethnicity adjusted analysis. Our findings suggest that age's association with the disabling fraction of cognitive performance is partially mediated by serum proteins, somatomedins and hormones. Those proteins may offer targets for the specific treatment of age-related effects on dementia severity and conversion risk.

Facilitated extinction of morphine conditioned place preference with Tat-GluA2(3Y) interference peptide.

PubMed

Dias, C; Wang, Y T; Phillips, A G

2012-08-01

Neuroplasticity including long-term depression (LTD) has been implicated in both learning processes and addiction. LTD can be blocked by intravenous administration of the interference peptide Tat-GluA2(3Y) that prevents regulated endocytosis of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor. In this study, Tat-GluA2(3Y) was used to assess the role of LTD in the induction, expression, extinction and reinstatement of morphine-induced conditioned place preference (CPP). CPP was established in rats by pairing morphine (5 mg/kg, i.p.) or saline with a specific environmental context using a balanced protocol. Tat-GluA2(3Y) (0; 1.5; 2.25 nmol/g; i.v.), scrambled peptide (Tat-GluA2(Sc)), or vehicle was administered during the acquisition phase or prior to the test for CPP. Tat-GluA2(3Y) had no effect on the induction or initial expression of morphine-induced CPP. Rats that received Tat-GluA2(3Y) or Tat-GluA2(Sc) during acquisition were subsequently tested for 11 consecutive days in order to extinguish morphine CPP. CPP was then reinstated by an injection of morphine (5 mg/kg, i.p.). Co-administration of morphine and Tat-GluA2(3Y) during acquisition greatly facilitated extinction of CPP without affecting morphine-induced reinstatement of CPP. Using an intermittent retest schedule with bi-weekly tests to measure the maintenance of CPP, Tat-GluA2(3Y) during the acquisition phase had no effect on the maintenance of CPP. We propose that co-administration of Tat-GluA2(3Y) with morphine during acquisition of CPP weakens the association between morphine and contextual cues leading to rapid extinction of morphine CPP with repeated daily testing. Copyright © 2012 Elsevier B.V. All rights reserved.

Enquête internationale sur l'état de l'art et l'état de la pratique en géotechnique

NASA Astrophysics Data System (ADS)

Acosta-Martinez, Hugo; Delage, Pierre; Nicks, Jennifer; Day, Peter

2018-05-01

Cet article présente une synthèse des résultats de l'enquête internationale sur l'état de l'art et l'état de la pratique en ingénierie géotechnique lancée par le Groupe présidentiel des entreprises associées et le Comité de supervision technique de la Société internationale de mécanique des sols et de géotechnique en mars 2017. Il résume également les discussions qui ont eu lieu sur le sujet durant le 19e CIMSG à Séoul, le 20 septembre 2017.

Ligand-gated purinergic receptors regulate HIV-1 Tat and morphine related neurotoxicity in primary mouse striatal neuron-glia co-cultures.

PubMed

Sorrell, Mary E; Hauser, Kurt F

2014-03-01

Emerging evidence suggests that opioid drugs, such as morphine and heroin, can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that opioid drugs acting via μ opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. Key aspects of microglial function are regulated by the P2X family of ATP activated ligand-gated ion channels. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, which prompted us to investigate whether the cation-permeable P2X receptors contribute to the neurotoxic effects of HIV and morphine. To address this question, neuron survival, as well as other endpoints including changes in dendritic length, extracellular ATP levels, and intracellular calcium levels, were assayed in primary neuron-glia co-cultures from mouse striatum. Treatment with TNP-ATP, a non-selective P2X antagonist, prevented the neurotoxic effects of exposure to morphine and/or HIV Tat, or ATP alone, suggesting P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. Although P2X7, and perhaps P2X1, receptor activation decreases neuron survival, neither P2X1, P2X3, nor P2X7 selective receptor antagonists prevented Tat and/or morphine-induced neurotoxicity. These and other experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and provide circumstantial evidence implicating P2X4 receptors in particular. Our findings reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompanies neuroAIDS and opiate abuse.

HIV-1 Tat affects the programming and functionality of human CD8⁺ T cells by modulating the expression of T-box transcription factors.

PubMed

Sforza, Fabio; Nicoli, Francesco; Gallerani, Eleonora; Finessi, Valentina; Reali, Eva; Cafaro, Aurelio; Caputo, Antonella; Ensoli, Barbara; Gavioli, Riccardo

2014-07-31

HIV infection is characterized by several immune dysfunctions of both CD8⁺ and CD4⁺ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8⁺ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4⁺ T cells; however, its effects on CD8⁺ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8⁺ T-cell functionality and programming. CD8⁺ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Tat favors the secretion of interleukin-2, interferon-γ and granzyme B in CD8⁺ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8⁺ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Tat deeply alters the programming and functionality of CD8⁺ T lymphocytes.

Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations.

PubMed

Ul Ain, Qurrat; Lee, Jong Hwan; Woo, Young Sun; Kim, Yong-Hee

2016-09-01

Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein's nature, transduction efficiency and stability.

Application of Bacillus sp. TAT105 to reduce ammonia emissions during pilot-scale composting of swine manure.

PubMed

Kuroda, Kazutaka; Tanaka, Akihiro; Furuhashi, Kenich; Nakasaki, Kiyohiko

2017-12-01

Thermophilic ammonium-tolerant bacterium Bacillus sp. TAT105 grows and reduces ammonia (NH 3 ) emissions by assimilating ammonium nitrogen during composting of swine feces. To evaluate the efficacy of a biological additive containing TAT105 at reducing NH 3 emissions, composting tests of swine manure on a pilot scale (1.8 m 3 ) were conducted. In the TAT105-added treatment, NH 3 emissions and nitrogen loss were lower than those in the control treatment without TAT105. No significant difference was detected in losses in the weight and volatile solids between the treatments. Concentration of thermophilic ammonium-tolerant bacteria in the compost increased in both treatments at the initial stage of composting. In the TAT105-added treatment, bacterial concentration reached ~10 9 colony-forming units per gram of dry matter, several-fold higher than that in the control and stayed at the same level until the end. These results suggest that TAT105 grows during composting and reduces NH 3 emissions in TAT105-added treatment.

Not Your Same Old Story: New Rules for Thematic Apperceptive Techniques (TATs).

PubMed

Jenkins, Sharon Rae

2017-01-01

Stories told about pictures have been used for both research and clinical practice since the beginning of modern personality assessment. However, with the growing science-practice gap, these thematic apperceptive techniques (TATs) have been used differently in those 2 venues. Scientific validation is presumptively general, but clinical application is idiographic and situation-specific. A bridge is needed. The manualized human-scored narrative analysis systems discussed here are valuable scientist-practitioner tools, but they require a validation literature to support further research publication, maintain their role in clinical training, and justify clinicians' reimbursement by third-party payers. To facilitate wider understanding of manualized TAT methodologies, this article addresses long-standing criticisms of TAT reliability and proposes some strategic solutions to the measurement error problem for both researchers and clinicians, including analyzing person-situation interactions, purposeful situation sampling for within-storyteller comparisons, and uses of small samples. The new rules for TATs include conceptual and methodological standards that researchers should aim to meet and report, reviewers should apply to manuscripts, and clinical assessors can use to analyze their own data and justify third-party payment.

TIT FOR TAT in sticklebacks and the evolution of cooperation

NASA Astrophysics Data System (ADS)

Milinski, Manfred

1987-01-01

The problems of achieving mutual cooperation can be formalized in a game called the Prisoner's Dilemma in which selfish defection is always more rewarding than cooperation1. If the two protagonists have a certain minimum probability of meeting again a strategy called TIT FOR TAT is very successful2. In TIT FOR TAT the player cooperates on the first move and thereafter does whatever the opponent did on the previous move. I have studied the behaviour of fish when confronting a potential predator, because conflicts can arise within pairs of fish in these circumstances which I argue resemble a series of games of Prisoner's Dilemma. Using a system of mirrors, single three-spined sticklebacks (Gasterosteus aculeatus) approaching a live predator were provided with either a simulated cooperating companion or a simulated defecting one. In both cases the test fish behaved according to TIT FOR TAT supporting the hypothesis that cooperation can evolve among egoists.

Chronic morphine and HIV-1 Tat promote differential central nervous system trafficking of CD3+ and Ly6C+ immune cells in a murine Streptococcus pneumoniae infection model.

PubMed

Dutta, Raini; Roy, Sabita

2015-06-20

Persistent systemic infection results in excessive trafficking of peripheral immune cells into the central nervous system (CNS), thereby contributing to sustained neuroinflammation that leads to neurocognitive deficits. In this study, we explored the role of opportunistic systemic infection with Streptococcus pneumoniae in the recruitment of peripheral leukocytes into the CNS and its contribution to HIV-1-associated neurocognitive disorders in opioid-dependent individuals. Wild-type B6CBAF1 (wt), μ-opioid receptor knockout (MORKO), FVB/N luciferase transgenic, and Toll-like receptor 2 and 4 knockout (TLR2KO and TLR4KO) mice were subcutaneously implanted with morphine/placebo pellet followed by HIV-1 Transactivator of transcription (Tat) protein injection intravenously and S. pneumoniae administration intraperitoneally. On postoperative day 5, brains perfused with phosphate-buffered saline were harvested and subjected to immunohistochemistry (for bacterial trafficking and chemokine ligand generation), flow cytometry (for phenotypic characterization of CNS trafficked immune cells), Western blot, and real-time PCR (for ligand expression). Our results show differential leukocyte trafficking of T lymphocytes (CD3+) and inflammatory monocytes (Ly6C+) into the CNS of mice treated with morphine, HIV-1 Tat, and/or S. pneumoniae. In addition, we demonstrate a Trojan horse mechanism for bacterial dissemination across the blood-brain barrier into the CNS by monocytes. Activation of TLRs on microglia induced a chemokine gradient that facilitated receptor-dependent trafficking of peripheral immune cells into the CNS. HIV-1 Tat induced trafficking of Ly6C+ and CD3+ cells into the CNS; infection with S. pneumoniae facilitated infiltration of only T lymphocytes into the CNS. We also observed differential chemokine secretion in the CNS, with CCL5 being the predominant chemokine following HIV-1 Tat treatment, which was potentiated further with morphine. S. pneumoniae alone led to

C-peptide inhibitors of Ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking.

PubMed

Higgins, Chelsea D; Koellhoffer, Jayne F; Chandran, Kartik; Lai, Jonathan R

2013-10-01

We previously described potent inhibition of Ebola virus entry by a 'C-peptide' based on the GP2 C-heptad repeat region (CHR) targeted to endosomes ('Tat-Ebo'). Here, we report the synthesis and evaluation of C-peptides conjugated to cholesterol, and Tat-Ebo analogs containing covalent side chain-side chain crosslinks to promote α-helical conformation. We found that the cholesterol-conjugated C-peptides were potent inhibitors of Ebola virus glycoprotein (GP)-mediated cell entry (~10(3)-fold reduction in infection at 40 μM). However, this mechanism of inhibition is somewhat non-specific because the cholesterol-conjugated peptides also inhibited cell entry mediated by vesicular stomatitis virus glycoprotein G. One side chain-side chain crosslinked peptide had moderately higher activity than the parent compound Tat-Ebo. Circular dichroism revealed that the cholesterol-conjugated peptides unexpectedly formed a strong α-helical conformation that was independent of concentration. Side chain-side chain crosslinking enhanced α-helical stability of the Tat-Ebo variants, but only at neutral pH. These result provide insight into mechanisms of C-peptide inhibiton of Ebola virus GP-mediated cell entry. Copyright © 2013 Elsevier Ltd. All rights reserved.

Selective Vulnerability of Striatal D2 versus D1 Dopamine Receptor-Expressing Medium Spiny Neurons in HIV-1 Tat Transgenic Male Mice.

PubMed

Schier, Christina J; Marks, William D; Paris, Jason J; Barbour, Aaron J; McLane, Virginia D; Maragos, William F; McQuiston, A Rory; Knapp, Pamela E; Hauser, Kurt F

2017-06-07

remains evident in specific brain regions such as the dorsal striatum. A possible explanation for the sustained neuronal injury is that the neurotoxic HIV-1 regulatory protein trans -activator of transcription (Tat) continues to be expressed in virally suppressed patients on cART. Using inducible Tat-expressing transgenic mice, we found that dopamine subtype 2 (D2) receptor-expressing medium spiny neurons (MSNs) are selectively vulnerable to Tat exposure compared with D1 receptor-expressing MSNs. This includes Tat-induced reductions in D2 MSN dendritic spine density, increased dendritic damage, and disruptions in neuronal excitability, which coincide with elevated anxiety-like behavior. These data suggest that D2 MSNs and specific circuits within the basal ganglia are preferentially vulnerable to HIV-1. Copyright © 2017 the authors 0270-6474/17/375759-12$15.00/0.

CDKL5 protein substitution therapy rescues neurological phenotypes of a mouse model of CDKL5 disorder.

PubMed

Trazzi, Stefania; De Franceschi, Marianna; Fuchs, Claudia; Bastianini, Stefano; Viggiano, Rocchina; Lupori, Leonardo; Mazziotti, Raffaele; Medici, Giorgio; Lo Martire, Viviana; Ren, Elisa; Rimondini, Roberto; Zoccoli, Giovanna; Bartesaghi, Renata; Pizzorusso, Tommaso; Ciani, Elisabetta

2018-05-01

Cyclin-dependent kinase like-5 (CDKL5) disorder is a rare neurodevelopmental disease caused by mutations in the CDKL5 gene. The consequent misexpression of the CDKL5 protein in the nervous system leads to a severe phenotype characterized by intellectual disability, motor impairment, visual deficits and early-onset epilepsy. No therapy is available for CDKL5 disorder. It has been reported that a protein transduction domain (TAT) is able to deliver macromolecules into cells and even into the brain when fused to a given protein. We demonstrate that TAT-CDKL5 fusion protein is efficiently internalized by target cells and retains CDKL5 activity. Intracerebroventricular infusion of TAT-CDKL5 restored hippocampal development, hippocampus-dependent memory and breathing pattern in Cdkl5-null mice. Notably, systemically administered TAT-CDKL5 protein passed the blood-brain-barrier, reached the CNS, and rescued various neuroanatomical and behavioral defects, including breathing pattern and visual responses. Our results suggest that CDKL5 protein therapy may be an effective clinical tool for the treatment of CDKL5 disorder.

miR-34a is a common link in both HIV- and antiretroviral therapy-induced vascular aging.

PubMed

Zhan, Jiaxin; Qin, Shanshan; Lu, Lili; Hu, Xiamin; Zhou, Jun; Sun, Yeying; Yang, Jian; Liu, Ying; Wang, Zunzhe; Tan, Ning; Chen, Jiyan; Zhang, Chunxiang

2016-11-26

Both HIV and antiretroviral therapy could induce vascular aging with unclear mechanisms. In this study, via microarray analysis, we identified, for the first time, that miR-34a expression was significantly increased in both HIV-infected, and antiretroviral agents-treated vessels and vascular endothelial cells (ECs) from these vessels. In cultured ECs, miR-34a expression was significantly increased by HIV-Tat protein and by the antiretroviral agents, lopinavir/ritonavir. Both HIV-Tat protein and antiretroviral agents could induce EC senescence, which was inhibited by miR-34a inhibition. In contrast, EC senescence was exacerbated by miR-34a overexpression. In addition, the vascular ECs isolated from miR-34a knockout mice were resistant to HIV and antiretroviral agents-mediated senescence. In vivo, miR-34a expression in mouse vascular walls and their ECs was increased by antiretroviral therapy and by HIV-1 Tat transgenic approach. miR-34a inhibition could effectively inhibit both HIV-Tat protein and antiretroviral therapy-induced vascular aging in mice. The increased miR-34a was induced via p53, whereas Sirt1 was a downstream target gene of miR-34a in both HIV-Tat protein and antiretroviral agents-treated ECs and vessels. The study has demonstrated that miR-34a is a common link in both HIV and antiretroviral therapy-mediated vascular aging.

Mutations at Tyrosine 88, Lysine 92 and Tyrosine 470 of human dopamine transporter result in an attenuation of HIV-1 Tat-induced inhibition of dopamine transport

PubMed Central

Midde, Narasimha M.; Yuan, Yaxia; Quizon, Pamela M.; Sun, Wei-Lun; Huang, Xiaoqin; Zhan, Chang-Guo; Zhu, Jun

2015-01-01

HIV-1 transactivator of transcription (Tat) protein disrupts the dopamine (DA) neurotransmission by inhibiting DA transporter (DAT) function, leading to increased neurocognitive impairment in HIV-1 infected individuals. Through integrated computational modeling and pharmacological studies, we have demonstrated that mutation of tyrosine470 (Y470H) of human DAT (hDAT) attenuates Tat-induced inhibition of DA uptake by changing the transporter conformational transitions. The present study examined the functional influences of other substitutions at tyrosine470 (Y470F and Y470A) and tyrosine88 (Y88F) and lysine92 (K92M), two other relevant residues for Tat binding to hDAT, in Tat-induced inhibitory effects on DA transport. Y88F, K92M and Y470A attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of these residues for Tat binding to hDAT. Compared to wild type hDAT, Y470A and K92M but not Y88F reduced the maximal velocity of [3H]DA uptake without changes in the Km. Y88F and K92M enhanced IC50 values for DA inhibition of [3H]DA uptake and [3H]WIN35,428 binding but decreased IC50 for cocaine and GBR12909 inhibition of [3H]DA uptake, suggesting that these residues are critical for substrate and these inhibitors. Y470F, Y470A, Y88F and K92M attenuated zinc-induced increase of [3H]WIN35,428 binding. Moreover, only Y470A and K92M enhanced DA efflux relative to wild type hDAT, suggesting mutations of these residues differentially modulate transporter conformational transitions. These results demonstrate Tyr88 and Lys92 along with Tyr470 as functional recognition residues in hDAT for Tat-induced inhibition of DA transport and provide mechanistic insights into identifying target residues on the DAT for Tat binding. PMID:25604666

The Relationship Between Behavioral Indices of Aggression and Hostile Content on the TAT

ERIC Educational Resources Information Center

Matranga, James T.

1976-01-01

Adolescent male delinquents were administered the Thematic Apperception Test (TAT) to examine the relationship between behavioral indices of aggression and hostility. The results of this investigation supported the hypothesis that an inverse relationship exists between hostility on the TAT and ratings of aggressive behavior in adolescent males.…

Business Case Analysis: Continuous Integrated Logistics Support-Targeted Allowance Technique (CILS-TAT)

DTIC Science & Technology

2013-06-01

In this research, we examine the Naval Sea Logistics Command s Continuous Integrated Logistics Support Targeted Allowancing Technique (CILS TAT) and... the feasibility of program re-implementation. We conduct an analysis of this allowancing method s effectiveness onboard U.S. Navy Ballistic Missile...Defense (BMD) ships, measure the costs associated with performing a CILS TAT, and provide recommendations concerning possible improvements to the

Prediction of virus-host protein-protein interactions mediated by short linear motifs.

PubMed

Becerra, Andrés; Bucheli, Victor A; Moreno, Pedro A

2017-03-09

Short linear motifs in host organisms proteins can be mimicked by viruses to create protein-protein interactions that disable or control metabolic pathways. Given that viral linear motif instances of host motif regular expressions can be found by chance, it is necessary to develop filtering methods of functional linear motifs. We conduct a systematic comparison of linear motifs filtering methods to develop a computational approach for predicting motif-mediated protein-protein interactions between human and the human immunodeficiency virus 1 (HIV-1). We implemented three filtering methods to obtain linear motif sets: 1) conserved in viral proteins (C), 2) located in disordered regions (D) and 3) rare or scarce in a set of randomized viral sequences (R). The sets C,D,R are united and intersected. The resulting sets are compared by the number of protein-protein interactions correctly inferred with them - with experimental validation. The comparison is done with HIV-1 sequences and interactions from the National Institute of Allergy and Infectious Diseases (NIAID). The number of correctly inferred interactions allows to rank the interactions by the sets used to deduce them: D∪R and C. The ordering of the sets is descending on the probability of capturing functional interactions. With respect to HIV-1, the sets C∪R, D∪R, C∪D∪R infer all known interactions between HIV1 and human proteins mediated by linear motifs. We found that the majority of conserved linear motifs in the virus are located in disordered regions. We have developed a method for predicting protein-protein interactions mediated by linear motifs between HIV-1 and human proteins. The method only use protein sequences as inputs. We can extend the software developed to any other eukaryotic virus and host in order to find and rank candidate interactions. In future works we will use it to explore possible viral attack mechanisms based on linear motif mimicry.

Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles

PubMed Central

Peetla, Chiranjeevi; Rao, Kavitha S.; Labhasetwar, Vinod

2009-01-01

The aim of the study was to test the hypothesis that the biophysical interactions of the trans-activating transcriptor (TAT) peptide-conjugated nanoparticles (NPs) with a model cell membrane could predict the cellular uptake of the encapsulated therapeutic agent. To test the above hypothesis, the biophysical interactions of ritonavir-loaded poly (L-lactide) nanoparticles (RNPs), either conjugated to a TAT peptide (TAT-RNPs) or scrambled TAT peptide (sc-TAT-RNPs), were studied with an endothelial cell model membrane (EMM) using a Langmuir film balance, and the corresponding human vascular endothelial cells (HUVECs) were used to study the uptake of the encapsulated therapeutic. Biophysical interactions were determined from the changes in surface pressure (SP) of the EMM as a function of time following interaction with NPs, and the compression isotherm (π–A) of the EMM lipid mixture in the presence of NPs. In addition, the EMMs were transferred onto a silicon substrate following interactions with NPs using the Langmuir–Schaeffer (LS) technique. The transferred LS films were imaged by atomic force microscopy (AFM) to determine the changes in lipid morphology and to characterize the NP–membrane interactions. TAT-RNPs showed an increase in SP of the EMM, which was dependent upon the amount of the peptide bound to NPs and the concentration of NPs, whereas sc-TAT-RNPs and RNPs did not show any significant change in SP. The isotherm experiment showed a shift towards higher mean molecular area (mmA) in the presence of TAT-RNPs, indicating their interactions with the lipids of the EMM, whereas sc-TAT-RNPs and RNPs did not show any significant change. The AFM images showed condensation of the lipids following interaction with TAT-RNPs, indicating their penetration into the EMM, whereas RNPs did not cause any change. Surface analysis and 3-D AFM images of the EMM further confirmed penetration of TAT-RNPs into the EMM whereas RNPs were seen anchored loosely to the

The Narrative Arc of TATs: Introduction to the JPA Special Section on Thematic Apperceptive Techniques.

PubMed

Jenkins, Sharon Rae

2017-01-01

The past decade has seen important developments in thematic apperceptive techniques (TATs), with the creation of new card sets having alternate pictures representing different cultures, new scoring systems becoming available, and increasing international communication of these achievements. However, continuing impediments to the development of a validational literature include lingering mistaken assumptions about the nature of story data, ongoing debates about appropriate psychometric evaluation, and continuing questions about how stimuli and scoring systems should be conceptualized and interpreted. Negotiating the publication system can impede some potential authors. Excellent work on TATs with children is not well known in the adult-focused journals. The labor burden of meeting increasingly sophisticated publication standards might be a barrier to assessors focused on clinical practice. Accumulating a focused evidence base is challenging given the diversity of criterion variables for which TATs have been used. Research on TATs by clinicians can span the science-practice gap, but the narrative arc can be a dramatic one. The articles in this special section on TATs represent important conceptual, methodological, and substantive innovations.

Business Case Analysis: Continuous Integrated Logistics Support-Targeted Allowance Technique (CILS-TAT)

DTIC Science & Technology

2013-05-30

In this research, we examine the Naval Sea Logistics Command’s Continuous Integrated Logistics Support-Targeted Allowancing Technique (CILS-TAT) and... the feasibility of program re-implementation. We conduct an analysis of this allowancing method’s effectiveness onboard U.S. Navy Ballistic Missile...Defense (BMD) ships, measure the costs associated with performing a CILS-TAT, and provide recommendations concerning possible improvements to the

Protein-mediated loops in supercoiled DNA create large topological domains

PubMed Central

Yan, Yan; Ding, Yue; Leng, Fenfei; Dunlap, David; Finzi, Laura

2018-01-01

Abstract Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites. PMID:29538766

Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.

PubMed

Ma, Xianyue; Cline, Kenneth

2013-03-01

Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

PATHOPHYSIOLOGICAL CONSEQUENCES OF TAT-HKII PEPTIDE ADMINISTRATION ARE INDEPENDENT OF IMPAIRED VASCULAR FUNCTION AND ENSUING ISCHEMIA

PubMed Central

Nederlof, Rianne; Xie, Chaoqin; Eerbeek, Otto; Koeman, Anneke; Milstein, Dan MJ; Hollmann, Markus W; Mik, Egbert G; Warley, Alice; Southworth, Richard; Akar, Fadi G.; Zuurbier, Coert J

2013-01-01

Rationale We have shown that partial dissociation of HKII from mitochondria in the intact heart using low dose (200 nM) TAT-HKII prevents the cardioprotective effects of ischemic preconditioning (IPC) whereas high-dose (10 μM) TAT-HKII administration results in rapid myocardial dysfunction, mitochondrial depolarization and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely due to vasoconstriction and ensuing ischemia. Objective To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium. Methods and Results Using a variety of complementary techniques, including mitochondrial membrane potential (ΔΨm) imaging, high-resolution optical action potential (AP) mapping, analysis of lactate production, NADH epifluorescence, lactate dehydrogenase (LDH) release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of IPC, is not associated with ischemia or ischemic-injury. Conclusions Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia; thereby, lending further credence to the role of mitochondria bound HKII as a critical regulator of cardiac function, ischemia-reperfusion (IR) injury, and cardioprotection by IPC. PMID:23329797

TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors.

PubMed

Sethuraman, Vijay A; Bae, You Han

2007-04-02

A novel drug targeting system for acidic solid tumors has been developed based on ultra pH-sensitive polymer and cell penetrating TAT. The delivery system consisted of two components: 1) A polymeric micelle that has a hydrophobic core made of poly(l-lactic acid) (PLLA) and a hydrophilic shell consisting of polyethylene glycol (PEG) conjugated to TAT (TAT micelle), 2) an ultra pH-sensitive diblock copolymer of poly(methacryloyl sulfadimethoxine) (PSD) and PEG (PSD-b-PEG). The anionic PSD is complexed with cationic TAT of the micelles to achieve the final carrier, which could systemically shield the micelles and expose them at slightly acidic tumor pH. TAT micelles had particle sizes between 20 and 45 nm and their critical micelle concentrations were 3.5 mg/l to 5.5 mg/l. The TAT micelles, upon mixing with pH-sensitive PSD-b-PEG, showed a slight increase in particle size between pH 8.0 and 6.8 (60-90 nm), indicating complexation. As the pH was decreased (pH 6.6 to 6.0) two populations were observed, one that of normal TAT micelles (45 nm) and the other of aggregated hydrophobic PSD-b-PEG. Zeta potential measurements showed similar trend substantiating the shielding/deshielding process. Flow cytometry and confocal microscopy showed significantly higher uptake of TAT micelles at pH 6.6 compared to pH 7.4 indicating shielding at normal pH and deshielding at tumor pH. The confocal microscopy indicated that the TAT not only translocates into the cells but is also seen on the surface of the nucleus. These results strongly indicate that the above micelles would be able to target any hydrophobic drug near the nucleus.

Assisted annotation of medical free text using RapTAT

PubMed Central

Gobbel, Glenn T; Garvin, Jennifer; Reeves, Ruth; Cronin, Robert M; Heavirland, Julia; Williams, Jenifer; Weaver, Allison; Jayaramaraja, Shrimalini; Giuse, Dario; Speroff, Theodore; Brown, Steven H; Xu, Hua; Matheny, Michael E

2014-01-01

Objective To determine whether assisted annotation using interactive training can reduce the time required to annotate a clinical document corpus without introducing bias. Materials and methods A tool, RapTAT, was designed to assist annotation by iteratively pre-annotating probable phrases of interest within a document, presenting the annotations to a reviewer for correction, and then using the corrected annotations for further machine learning-based training before pre-annotating subsequent documents. Annotators reviewed 404 clinical notes either manually or using RapTAT assistance for concepts related to quality of care during heart failure treatment. Notes were divided into 20 batches of 19–21 documents for iterative annotation and training. Results The number of correct RapTAT pre-annotations increased significantly and annotation time per batch decreased by ∼50% over the course of annotation. Annotation rate increased from batch to batch for assisted but not manual reviewers. Pre-annotation F-measure increased from 0.5 to 0.6 to >0.80 (relative to both assisted reviewer and reference annotations) over the first three batches and more slowly thereafter. Overall inter-annotator agreement was significantly higher between RapTAT-assisted reviewers (0.89) than between manual reviewers (0.85). Discussion The tool reduced workload by decreasing the number of annotations needing to be added and helping reviewers to annotate at an increased rate. Agreement between the pre-annotations and reference standard, and agreement between the pre-annotations and assisted annotations, were similar throughout the annotation process, which suggests that pre-annotation did not introduce bias. Conclusions Pre-annotations generated by a tool capable of interactive training can reduce the time required to create an annotated document corpus by up to 50%. PMID:24431336

Chimeric Peptide Tat-HA-NR2B9c Improves Regenerative Repair after Transient Global Ischemia.

PubMed

Zhou, Hai-Hui; Zhang, Li; Zhang, Hai-Xia; Zhang, Jin-Ping; Ge, Wei-Hong

2017-01-01

Transient global ischemia (TGI) is a major public health problem, and it heightens the need of effective treatments. The present study was undertaken to investigate whether recombinant polypeptide Tat-HA-NR2B9c improves spatial learning and memory deficits in rats after TGI. Rats were subjected to 20-min ischemia induced by four-vessel occlusion (4-VO) method and daily injected with Tat-HA-NR2B9c (1.12 mg/kg) for 1 week. Tat-HA-NR2B9c increased CREB activity, upregulated B-cell lymphoma-2 (Bcl-2) expression after treated for 24 h. There was a significant increase in dendrite spine density in hippocampal CA1 region and BrdU-positive cells and BrdU/NeuN-positive cells in the dentate gyrus after Tat-HA-NR2B9c treatment, compared with ischemia group at postischemic day 28. Inhibition of the CREB activation by recombinant lentivirus, LV-CREB133-GFP, abolished the upregulation effects of Tat-HA-NR2B9c on Bcl-2 expression. Moreover, Tat-HA-NR2B9c improved the impaired spatial learning and memory ability in Morris water maze. These results suggest that Tat-HA-NR2B9c substantially ameliorated the TGI-induced loss of dendrite spine in hippocampal CA1, increased neurogenesis in dentate gyrus, and significantly improved cognitive abilities by the CREB pathway in rats after transient global cerebral ischemia. It may be served as a treatment for TGI.

Eudragit S100-Coated Chitosan Nanoparticles Co-loading Tat for Enhanced Oral Colon Absorption of Insulin.

PubMed

Chen, Shuangxi; Guo, Feng; Deng, Tiantian; Zhu, Siqi; Liu, Wenyu; Zhong, Haijun; Yu, Hua; Luo, Rong; Deng, Zeyuan

2017-05-01

In order to improve oral absorption of insulin, especially the absorption at the colon, Eudragit S100® (ES)-coated chitosan nanoparticles loading insulin and a trans-activating transcriptional peptide (Tat) were employed as the vehicle. In vitro releases of insulin and Tat from ES-coated chitosan nanoparticles had a pH-dependant characteristic. A small amount of the contents was released from the coated nanoparticles at pH 1.2 simulated gastric fluid, while a fairly fast and complete release was observed in pH 7.4 medium. Caco-2 cell was used as the model of cellular transport and uptake studies. The results showed that the cellular transport and uptake of insulin for ES-coated chitosan nanoparticles co-loading insulin and Tat (ES-Tat-cNPs) were about 3-fold and 4-fold higher than those for the nanoparticles loading only insulin (ES-cNPs), respectively. The evaluations in vivo of ES-Tat-cNPs were conducted on diabetic rats and normal minipigs, respectively. The experimental results on rats revealed that the pharmacodynamical bioavailability of ES-Tat-cNPs had 2.16-fold increase compared with ES-cNPs. After oral administration of nanoparticle suspensions to the minipigs, insulin bioavailability of ES-Tat-cNPs was 1.73-fold higher than that of ES-cNPs, and the main absorption site of insulin was probably located in the colon for the two nanoparticles. In summary, this report provided an exploratory means for the improvement of oral absorption of insulin.

Tumor acidity-activatable TAT targeted nanomedicine for enlarged fluorescence/magnetic resonance imaging-guided photodynamic therapy.

PubMed

Gao, Meng; Fan, Feng; Li, Dongdong; Yu, Yue; Mao, Kuirong; Sun, Tianmeng; Qian, Haisheng; Tao, Wei; Yang, Xianzhu

2017-07-01

Nanoparticles simultaneously integrated the photosensitizers and diagnostic agents represent an emerging approach for imaging-guided photodynamic therapy (PDT). However, the diagnostic sensitivity and therapeutic efficacy of nanoparticles as well as the heterogeneity of tumors pose tremendous challenges for clinical imaging-guided PDT treatment. Herein, a polymeric nanoparticle with tumor acidity (pH e )-activatable TAT targeting ligand that encapsulates the photosensitizer chlorin e6 (Ce6) and chelates contrast agent Gd 3+ is successfully developed for fluorescence/magnetic resonance (MR) dual-model imaging-guided precision PDT. We show clear evidence that the resulting nanoparticle DA TAT-NP [its TAT lysine residues' amines was modified by 2,3-dimethylmaleic anhydride (DA)] efficiently avoids the rapid clearance by reticuloendothelial system (RES) by masking of the TAT peptide, resulting in the significantly prolonged circulation time in the blood. Once accumulating in the tumor tissues, DA TAT-NP is reactivated by tumor acidity to promote cellular uptake, resulting in enlarged fluorescence/MR imaging signal intensity and elevated in vivo PDT therapeutic effect. This concept provides new avenues to design tumor acidity-activatable targeted nanoparticles for imaging-guided cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protein transduction domain of transactivating transcriptional activator fused to outer membrane protein K of Vibrio parahaemolyticus to vaccinate marbled eels (Anguilla marmorata) confers protection against mortality caused by V. parahaemolyticus.

PubMed

Wang, Hang; Yang, Wei; Shen, Guoying; Zhang, Jianting; Lv, Wei; Ji, Binfeng; Meng, Chun

2015-07-01

Although immersion and oral vaccination are the most practical methods for fish farmers, their applications are very limited due to low immune stimulation effect. We used the protein transduction domain (PTD) of transactivating transcriptional factor (TAT) derived from HIV TAT protein to increase the delivery efficiency of aquatic protein vaccines. Vibrio parahaemolyticus outer membrane protein K (ompK), a reported vaccine candidate for the prevention of V. parahaemolyticus infection, was fused with TAT-PTD expressed in Escherichia coli. We found that PTD-ompK fusion protein effectively penetrated into marbled eel bodies. Analysis of ompK antibody titres demonstrated that immersion vaccination with PTD-ompK was superior to ompK alone and induced robust immune stimulation in marbled eels. Both active and passive protection analyses against immersive challenge with V. parahaemolyticus strains demonstrated that marbled eels immunized with PTD-ompK survived significantly longer than those immunized with ompK alone. Our results indicated that TAT-PTD could be served as is an efficient delivery system for aquatic immersion vaccinations against various infectious diseases commonly seen in aquatic farm industry. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Improving ED specimen TAT using Lean Six Sigma.

PubMed

Sanders, Janet H; Karr, Tedd

2015-01-01

Lean and Six Sigma are continuous improvement methodologies that have garnered international fame for improving manufacturing and service processes. Increasingly these methodologies are demonstrating their power to also improve healthcare processes. The purpose of this paper is to discuss a case study for the application of Lean and Six Sigma tools in the reduction of turnaround time (TAT) for Emergency Department (ED) specimens. This application of the scientific methodologies uncovered opportunities to improve the entire ED to lab system for the specimens. This case study provides details on the completion of a Lean Six Sigma project in a 1,000 bed tertiary care teaching hospital. Six Sigma's Define, Measure, Analyze, Improve, and Control methodology is very similar to good medical practice: first, relevant information is obtained and assembled; second, a careful and thorough diagnosis is completed; third, a treatment is proposed and implemented; and fourth, checks are made to determine if the treatment was effective. Lean's primary goal is to do more with less work and waste. The Lean methodology was used to identify and eliminate waste through rapid implementation of change. The initial focus of this project was the reduction of turn-around-times for ED specimens. However, the results led to better processes for both the internal and external customers of this and other processes. The project results included: a 50 percent decrease in vials used for testing, a 50 percent decrease in unused or extra specimens, a 90 percent decrease in ED specimens without orders, a 30 percent decrease in complete blood count analysis (CBCA) Median TAT, a 50 percent decrease in CBCA TAT Variation, a 10 percent decrease in Troponin TAT Variation, a 18.2 percent decrease in URPN TAT Variation, and a 2-5 minute decrease in ED registered nurses rainbow draw time. This case study demonstrated how the quantitative power of Six Sigma and the speed of Lean worked in harmony to improve

Morphological, Histochemical, Immunohistochemical, and Ultrastructural Characterization of Tumors and Dysplastic and Non-Neoplastic Lesions Arising in BK Virus/tat Transgenic Mice

PubMed Central

Altavilla, Giuseppe; Trabanelli, Cecilia; Merlin, Michela; Caputo, Antonella; Lanfredi, Massimo; Barbanti-Brodano, Giuseppe; Corallini, Alfredo

1999-01-01

To study the role in AIDS pathogenesis of the human immunodeficiency virus type 1 (HIV-1) Tat protein, a transactivator of viral and cellular genes, we generated transgenic mice with a recombinant DNA containing BK virus (BKV) early region and the HIV-1 tat gene, directed by its own promoter-enhancer. DNA hybridization revealed that the transgene is stably maintained in all organs of transgenic mice as a tandem insertion in a number of copies ranging from 5 to 20 per cell. In addition, tat and BKV RNA were expressed in all tissues. Transgenic mice developed three types of lesions: 1) tumors, 2) hyperplastic and dysplastic lesions, and 3) non-neoplastic lesions. Tumors of different histotypes, such as lymphomas, adenocarcinomas of skin glands, leiomyosarcomas, skin squamous cell carcinomas, hepatomas, hepatocarcinomas, and cavernous liver hemangiomas, developed in 29% of transgenic animals. The majority of tumors were malignant, invasive, and producing metastases. Conversely, tumors of only two histotypes (lymphomas and adenocarcinomas of skin glands) appeared in control mice. Hyperplastic and dysplastic lesions were more frequent in transgenic than in control mice and involved the skin or its adnexes, the liver and the rectum, indicating multiple targets for the activity of the transgene. Pyelonephritis, frequently complicated with hydronephrosis, inflammatory eye lesions, and amyloid depositions represented the most frequent non-neoplastic lesions detected in transgenic mice. Many of the pathological findings observed in this animal model are comparable to similar lesions appearing in AIDS patients, suggesting a relevant role for Tat in the pathogenesis of such lesions during the course of AIDS. PMID:10233861

Protein-mediated looping of DNA under tension requires supercoiling

PubMed Central

Yan, Yan; Leng, Fenfei; Finzi, Laura; Dunlap, David

2018-01-01

Abstract Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the HU protein, an abundant nucleoid protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of HU could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension. PMID:29365152

Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals

PubMed Central

Paulmurugan, Ramasamy; Gambhir, Sanjiv S.

2014-01-01

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule–mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction–mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGS-FACGSLSCGSF. A 9 ± 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation. PMID:16103094

Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

PubMed

Paulmurugan, Ramasamy; Gambhir, Sanjiv S

2005-08-15

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

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

Jung, Kyung oh; Biomedical Sciences, Seoul National University College of Medicine; Cancer Research Institute, Seoul National University College of Medicine

Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescencemore » signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.« less

Dual-mode enhancement of metallothionein protein with cell transduction and retention peptide fusion.

PubMed

Lim, Kwang Suk; Lim, Myoung-Hwa; Won, Young-Wook; Kim, Jang Kyoung; Kang, Young Cheol; Park, Eun Jeong; Chae, Ji-Won; Kim, So-Mi; Ryu, Seong-Eon; Pak, Youngmi Kim; Kim, Yong-Hee

2013-10-28

Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), have been developed as effective systems for delivering bio-active cargos such as proteins, genes and particles. Further improvements on cell-specific targeting, intracellular organelle targeting and intracellular retention are still necessary to enhance the therapeutic effect of PTD fusion proteins. In order to enhance the cell transduction and retention of anti-oxidative metallothionein protein (MT), MT was recombinantly fused with transcriptional activator (Tat) with or without a short peptide (sMTS) derived from mitochondria malate dehydrogenase (mMDH). Cellular uptake and retention time of fusion protein were significantly increased in the H9c2 cell by sMTS. The Tat-sMTS-MT (TMM) fusion protein protected H9c2 cells more effectively against hypoxia, hyperglycemia and combination compared with Tat-MT (TM) by reducing intracellular ROS level. It maintained the normal blood glucose level over an extended period of time in a streptozotocin-induced diabetic mouse model. PTD-sMTS-MT fusion protein has a potential to be used as a therapeutic protein for the treatment or prevention of diabetes and diabetic complications. © 2013.

The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures.

PubMed

Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W

2014-03-01

Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to

NF-κB Activation in Hypothalamic Pro-opiomelanocortin Neurons Is Essential in Illness- and Leptin-induced Anorexia*

PubMed Central

Jang, Pil-Geum; Namkoong, Cherl; Kang, Gil Myoung; Hur, Man-Wook; Kim, Seung-Whan; Kim, Geun Hyang; Kang, Yeoungsup; Jeon, Min-Jae; Kim, Eun Hee; Lee, Myung-Shik; Karin, Michael; Baik, Ja-Hyun; Park, Joong-Yeol; Lee, Ki-Up; Kim, Young-Bum; Kim, Min-Seon

2010-01-01

Anorexia and weight loss are prevalent in infectious diseases. To investigate the molecular mechanisms underlying these phenomena, we established animal models of infection-associated anorexia by administrating bacterial and viral products, lipopolysaccharide (LPS) and human immunodeficiency virus-1 transactivator protein (Tat). In these models, we found that the nuclear factor-κB (NF-κB), a pivotal transcription factor for inflammation-related proteins, was activated in the hypothalamus. In parallel, administration of LPS and Tat increased hypothalamic pro-inflammatory cytokine production, which was abrogated by inhibition of hypothalamic NF-κB. In vitro, NF-κB activation directly stimulated the transcriptional activity of pro-opiomelanocortin (POMC), a precursor of anorexigenic melanocortin, and mediated the stimulatory effects of LPS, Tat, and pro-inflammatory cytokines on POMC transcription, implying the involvement of NF-κB in controlling feeding behavior. Consistently, hypothalamic injection of LPS and Tat caused a significant reduction in food intake and body weight, which was prevented by blockade of NF-κB and melanocortin. Furthermore, disruption of IκB kinase-β, an upstream kinase of NF-κB, in POMC neurons attenuated LPS- and Tat-induced anorexia. These findings suggest that infection-associated anorexia and weight loss are mediated via NF-κB activation in hypothalamic POMC neurons. In addition, hypothalamic NF-κB was activated by leptin, an important anorexigenic hormone, and mediates leptin-stimulated POMC transcription, indicating that hypothalamic NF-κB also serves as a downstream signaling pathway of leptin. PMID:20097762

NF-kappaB activation in hypothalamic pro-opiomelanocortin neurons is essential in illness- and leptin-induced anorexia.

PubMed

Jang, Pil-Geum; Namkoong, Cherl; Kang, Gil Myoung; Hur, Man-Wook; Kim, Seung-Whan; Kim, Geun Hyang; Kang, Yeoungsup; Jeon, Min-Jae; Kim, Eun Hee; Lee, Myung-Shik; Karin, Michael; Baik, Ja-Hyun; Park, Joong-Yeol; Lee, Ki-Up; Kim, Young-Bum; Kim, Min-Seon

2010-03-26

Anorexia and weight loss are prevalent in infectious diseases. To investigate the molecular mechanisms underlying these phenomena, we established animal models of infection-associated anorexia by administrating bacterial and viral products, lipopolysaccharide (LPS) and human immunodeficiency virus-1 transactivator protein (Tat). In these models, we found that the nuclear factor-kappaB (NF-kappaB), a pivotal transcription factor for inflammation-related proteins, was activated in the hypothalamus. In parallel, administration of LPS and Tat increased hypothalamic pro-inflammatory cytokine production, which was abrogated by inhibition of hypothalamic NF-kappaB. In vitro, NF-kappaB activation directly stimulated the transcriptional activity of pro-opiomelanocortin (POMC), a precursor of anorexigenic melanocortin, and mediated the stimulatory effects of LPS, Tat, and pro-inflammatory cytokines on POMC transcription, implying the involvement of NF-kappaB in controlling feeding behavior. Consistently, hypothalamic injection of LPS and Tat caused a significant reduction in food intake and body weight, which was prevented by blockade of NF-kappaB and melanocortin. Furthermore, disruption of I kappaB kinase-beta, an upstream kinase of NF-kappaB, in POMC neurons attenuated LPS- and Tat-induced anorexia. These findings suggest that infection-associated anorexia and weight loss are mediated via NF-kappaB activation in hypothalamic POMC neurons. In addition, hypothalamic NF-kappaB was activated by leptin, an important anorexigenic hormone, and mediates leptin-stimulated POMC transcription, indicating that hypothalamic NF-kappaB also serves as a downstream signaling pathway of leptin.

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

PubMed

Tuttle, Lisa M; Pacheco, Derek; Warfield, Linda; Luo, Jie; Ranish, Jeff; Hahn, Steven; Klevit, Rachel E

2018-03-20

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.

PubMed

Liu, Jie; Berger, Christopher L; Morrical, Scott W

2013-11-12

Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.

Is the Achievement Motive Gender-Biased? The Validity of TAT/PSE in Women and Men

PubMed Central

Gruber, Nicole

2017-01-01

In picture story exercises like the Thematic Apperception Test (TAT; Heckhausen, 1963), different pictures are presented to a person with the instruction to create a story using the scenes portrayed in the image. It is assumed, that people identify themselves with the people in the images and project their unconscious motives (e.g., achievement motive) onto them. As the TAT shows only men in the pictures, critics claimed the test is gender-biased; assuming women cannot identify with men in pictures. However, it was not assessed, whether female protagonists of the picture really trigger the same achievement motive as men. Therefore, two studies were conducted to address the gender difference and validity of the TAT using a version with only men in the pictures (study 1) or only women in the pictures (study 2). The results shows that the original TAT of Heckhausen is a valid instrument for women and men, but the modified version with only women in the pictures cannot validly measure the achievement motive in the male sample. PMID:28261126

Benefit-Cost Analysis of TAT Phase I Worker Training. Training and Technology Project. Special Report.

ERIC Educational Resources Information Center

Kirby, Frederick C.; Castagna, Paul A.

The purpose of this study is to estimate costs and benefits and to compute alternative benefit-cost ratios for both the individuals and the Federal Government as a result of investing time and resources in the Training and Technology (TAT) Project. TAT is a continuing experimental program in training skilled workers for private industry. The five…

Strong G-Protein-Mediated Inhibition of Sodium Channels.

PubMed

Mattheisen, Glynis B; Tsintsadze, Timur; Smith, Stephen M

2018-05-29

Voltage-gated sodium channels (VGSCs) are strategically positioned to mediate neuronal plasticity because of their influence on action potential waveform. VGSC function may be strongly inhibited by local anesthetic and antiepileptic drugs and modestly modulated via second messenger pathways. Here, we report that the allosteric modulators of the calcium-sensing receptor (CaSR) cinacalcet, calindol, calhex, and NPS 2143 completely inhibit VGSC current in the vast majority of cultured mouse neocortical neurons. This form of VGSC current block persisted in CaSR-deficient neurons, indicating a CaSR-independent mechanism. Cinacalcet-mediated blockade of VGSCs was prevented by the guanosine diphosphate (GDP) analog GDPβs, indicating that G-proteins mediated this effect. Cinacalcet inhibited VGSCs by increasing channel inactivation, and block was reversed by prolonged hyperpolarization. Strong cinacalcet inhibition of VGSC currents was also present in acutely isolated mouse cortical neurons. These data identify a dynamic signaling pathway by which G-proteins regulate VGSC current to indirectly modulate central neuronal excitability. Published by Elsevier Inc.

Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

PubMed

Liu, Youtao; Lacal, Jesus; Firtel, Richard A; Kortholt, Arjan

2018-07-04

The directional movement toward extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

Utilization of Bacillus sp. strain TAT105 as a biological additive to reduce ammonia emissions during composting of swine feces.

PubMed

Kuroda, Kazutaka; Waki, Miyoko; Yasuda, Tomoko; Fukumoto, Yasuyuki; Tanaka, Akihiro; Nakasaki, Kiyohiko

2015-01-01

Bacillus sp. strain TAT105 is a thermophilic, ammonium-tolerant bacterium that grows assimilating ammonium nitrogen and reduces ammonia emission during composting of swine feces. To develop a practical use of TAT105, a dried solid culture of TAT105 (5.3 × 10(9) CFU/g of dry matter) was prepared as an additive. It could be stored for one year without significant reduction of TAT105. Laboratory-scale composting of swine feces was conducted by mixing the additive. When the additive, mixed with an equal weight of water one day before use, was added to obtain a TAT105 concentration of above 10(7) CFU/g of dry matter in the initial material, the ammonia concentration emitted was lower and nitrogen loss was approximately 22% lower in the treatment with the additive than in the control treatment without the additive. The colony formation on an agar medium containing high ammonium could be used for enumeration of TAT105 in the composted materials.

The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis

PubMed Central

Benjak, Andrej; Uplekar, Swapna; Rougemont, Jacques; Guilhot, Christophe; Malaga, Wladimir; Martín, Carlos; Cole, Stewart T.

2014-01-01

The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. PMID:24874799

Adaptor proteins in protein kinase C-mediated signal transduction.

PubMed

Schechtman, D; Mochly-Rosen, D

2001-10-01

Spatial and temporal organization of signal transduction is essential in determining the speed and precision by which signaling events occur. Adaptor proteins are key to organizing signaling enzymes near their select substrates and away from others in order to optimize precision and speed of response. Here, we describe the role of adaptor proteins in determining the specific function of individual protein kinase C (PKC) isozymes. These isozyme-selective proteins were called collectively RACKs (receptors for activated C-kinase). The role of RACKs in PKC-mediated signaling was determined using isozyme-specific inhibitors and activators of the binding of each isozyme to its respective RACK. In addition to anchoring activated PKC isozymes, RACKs anchor other signaling enzymes. RACK1, the anchoring protein for activated betaIIPKC, binds for example, Src tyrosine kinase, integrin, and phosphodiesterase. RACK2, the epsilonPKC-specific RACK, is a coated-vesicle protein and thus is involved in vesicular release and cell-cell communication. Therefore, RACKs are not only adaptors for PKC, but also serve as adaptor proteins for several other signaling enzymes. Because at least some of the proteins that bind to RACKs, including PKC itself, regulate cell growth, modulating their interactions with RACKs may help elucidate signaling pathways leading to carcinogenesis and could result in the identification of novel therapeutic targets.

Characterization of free radical defense system in high glucose cultured HeLa-tat cells: consequences for glucose-induced cytotoxicity.

PubMed

Bouvard, Sophie; Faure, Patrice; Roucard, Corinne; Favier, Alain; Halimi, Serge

2002-09-01

HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1 has a decreased antioxidant potential. In this work, we used this model to investigate the effect of a high glucose level (20 mM) on the glucose induced cytotoxicity and on the antioxidant system. In comparison to cell culture under control medium, HeLa-wild cell cultured under 20 mM glucose did not exhibit necrosis or apoptosis, contrary to HeLa-tat cell presenting a significant increase in necrotic or apoptotic state. Moreover after 48 h culture under high glucose level the HeLa-tat proliferation rate was not higher than the one of HeLa-wild cells. In HeLa-wild cell high glucose level resulted in an induction of glutathione reductase activity in opposition to HeLa-tat cells where no change was observed. High glucose level resulted in 20% increase in GSSG/GSH ratio in HeLa-wild cells and 38% increase in HeLa-tat cells. Moreover, high glucose level resulted in a dramatic cytosolic thiol decrease and an important lipid peroxidation in HeLa-tat cells. No significant change of these two parameters was observed in HeLa-wild cells. In both cell lines, high glucose resulted in an increase of total SOD activity, as a consequence of the increase in Cu,Zn-SOD activity. High glucose did not result in an increase of Mn-SOD activity in both cell lines. As a consequence of tat tranfection Mn-SOD activity was 50% lower in HeLa-tat cells in comparison to HeLa-wild cells. This work emphasizes the importance of the antioxidant system in the glucose induced cytotoxicity.

Therapeutic Immunization with HIV-1 Tat Reduces Immune Activation and Loss of Regulatory T-Cells and Improves Immune Function in Subjects on HAART

PubMed Central

Ensoli, Barbara; Bellino, Stefania; Tripiciano, Antonella; Longo, Olimpia; Francavilla, Vittorio; Marcotullio, Simone; Cafaro, Aurelio; Picconi, Orietta; Paniccia, Giovanni; Scoglio, Arianna; Arancio, Angela; Ariola, Cristina; Ruiz Alvarez, Maria J.; Campagna, Massimo; Scaramuzzi, Donato; Iori, Cristina; Esposito, Roberto; Mussini, Cristina; Ghinelli, Florio; Sighinolfi, Laura; Palamara, Guido; Latini, Alessandra; Angarano, Gioacchino; Ladisa, Nicoletta; Soscia, Fabrizio; Mercurio, Vito S.; Lazzarin, Adriano; Tambussi, Giuseppe; Visintini, Raffaele; Mazzotta, Francesco; Di Pietro, Massimo; Galli, Massimo; Rusconi, Stefano; Carosi, Giampiero; Torti, Carlo; Di Perri, Giovanni; Bonora, Stefano; Ensoli, Fabrizio; Garaci, Enrico

2010-01-01

Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4+ T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4+ and CD8+ cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4+ T cells and B cells with reduction of CD8+ T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4+ and CD8+ T cells were accompanied by increases of CD4+ and CD8+ T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the

Therapeutic immunization with HIV-1 Tat reduces immune activation and loss of regulatory T-cells and improves immune function in subjects on HAART.

PubMed

Ensoli, Barbara; Bellino, Stefania; Tripiciano, Antonella; Longo, Olimpia; Francavilla, Vittorio; Marcotullio, Simone; Cafaro, Aurelio; Picconi, Orietta; Paniccia, Giovanni; Scoglio, Arianna; Arancio, Angela; Ariola, Cristina; Ruiz Alvarez, Maria J; Campagna, Massimo; Scaramuzzi, Donato; Iori, Cristina; Esposito, Roberto; Mussini, Cristina; Ghinelli, Florio; Sighinolfi, Laura; Palamara, Guido; Latini, Alessandra; Angarano, Gioacchino; Ladisa, Nicoletta; Soscia, Fabrizio; Mercurio, Vito S; Lazzarin, Adriano; Tambussi, Giuseppe; Visintini, Raffaele; Mazzotta, Francesco; Di Pietro, Massimo; Galli, Massimo; Rusconi, Stefano; Carosi, Giampiero; Torti, Carlo; Di Perri, Giovanni; Bonora, Stefano; Ensoli, Fabrizio; Garaci, Enrico

2010-11-11

Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4(+) T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4(+) and CD8(+) cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4(+) T cells and B cells with reduction of CD8(+) T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4(+) and CD8(+) T cells were accompanied by increases of CD4(+) and CD8(+) T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent

Statistical models for detecting differential chromatin interactions mediated by a protein.

PubMed

Niu, Liang; Li, Guoliang; Lin, Shili

2014-01-01

Chromatin interactions mediated by a protein of interest are of great scientific interest. Recent studies show that protein-mediated chromatin interactions can have different intensities in different types of cells or in different developmental stages of a cell. Such differences can be associated with a disease or with the development of a cell. Thus, it is of great importance to detect protein-mediated chromatin interactions with different intensities in different cells. A recent molecular technique, Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET), which uses formaldehyde cross-linking and paired-end sequencing, is able to detect genome-wide chromatin interactions mediated by a protein of interest. Here we proposed two models (One-Step Model and Two-Step Model) for two sample ChIA-PET count data (one biological replicate in each sample) to identify differential chromatin interactions mediated by a protein of interest. Both models incorporate the data dependency and the extent to which a fragment pair is related to a pair of DNA loci of interest to make accurate identifications. The One-Step Model makes use of the data more efficiently but is more computationally intensive. An extensive simulation study showed that the models can detect those differentially interacted chromatins and there is a good agreement between each classification result and the truth. Application of the method to a two-sample ChIA-PET data set illustrates its utility. The two models are implemented as an R package MDM (available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM).

Statistical Models for Detecting Differential Chromatin Interactions Mediated by a Protein

PubMed Central

Niu, Liang; Li, Guoliang; Lin, Shili

2014-01-01

Chromatin interactions mediated by a protein of interest are of great scientific interest. Recent studies show that protein-mediated chromatin interactions can have different intensities in different types of cells or in different developmental stages of a cell. Such differences can be associated with a disease or with the development of a cell. Thus, it is of great importance to detect protein-mediated chromatin interactions with different intensities in different cells. A recent molecular technique, Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET), which uses formaldehyde cross-linking and paired-end sequencing, is able to detect genome-wide chromatin interactions mediated by a protein of interest. Here we proposed two models (One-Step Model and Two-Step Model) for two sample ChIA-PET count data (one biological replicate in each sample) to identify differential chromatin interactions mediated by a protein of interest. Both models incorporate the data dependency and the extent to which a fragment pair is related to a pair of DNA loci of interest to make accurate identifications. The One-Step Model makes use of the data more efficiently but is more computationally intensive. An extensive simulation study showed that the models can detect those differentially interacted chromatins and there is a good agreement between each classification result and the truth. Application of the method to a two-sample ChIA-PET data set illustrates its utility. The two models are implemented as an R package MDM (available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM). PMID:24835279

ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

PubMed

Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

2016-03-22

Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides.

PubMed

Moutal, Aubin; Li, Wennan; Wang, Yue; Ju, Weina; Luo, Shizhen; Cai, Song; François-Moutal, Liberty; Perez-Miller, Samantha; Hu, Jackie; Dustrude, Erik T; Vanderah, Todd W; Gokhale, Vijay; Khanna, May; Khanna, Rajesh

2017-02-05

N-type voltage-gated calcium (Ca v 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Ca v 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Ca v 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Ca v 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential. Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Ca v 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120. The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models. These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3. © 2017 The British Pharmacological Society.

Engineered fluorescent proteins illuminate the bacterial periplasm

PubMed Central

Dammeyer, Thorben; Tinnefeld, Philip

2012-01-01

The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation - a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP), remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat) pathway, but actively fold in the periplasm following general secretory pathway (Sec) and signal recognition particle (SRP) mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins. PMID:24688673

Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

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

Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael

2005-05-10

The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, couldmore » be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.« less

Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region.

PubMed Central

Gaynor, R; Soultanakis, E; Kuwabara, M; Garcia, J; Sigman, D S

1989-01-01

The transactivator protein, tat, encoded by the human immunodeficiency virus is a key regulator of viral transcription. Activation by the tat protein requires sequences downstream of the transcription initiation site called the transactivating region (TAR). RNA derived from the TAR is capable of forming a stable stem-loop structure and the maintenance of both the stem structure and the loop sequences located between +19 and +44 is required for complete in vivo activation by tat. Gel retardation assays with RNA from both wild-type and mutant TAR constructs generated in vitro with SP6 polymerase indicated specific binding of HeLa nuclear proteins to the TAR. To characterize this RNA-protein interaction, a method of chemical "imprinting" has been developed using photoactivated uranyl acetate as the nucleolytic agent. This reagent nicks RNA under physiological conditions at all four nucleotides in a reaction that is independent of sequence and secondary structure. Specific interaction of cellular proteins with TAR RNA could be detected by enhanced cleavages or imprints surrounding the loop region. Mutations that either disrupted stem base-pairing or extensively changed the primary sequence resulted in alterations in the cleavage pattern of the TAR RNA. Structural features of the TAR RNA stem-loop essential for tat activation are also required for specific binding of the HeLa cell nuclear protein. Images PMID:2544877

Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

PubMed

Li, Jie; Lin, Shixian; Wang, Jie; Jia, Shang; Yang, Maiyun; Hao, Ziyang; Zhang, Xiaoyu; Chen, Peng R

2013-05-15

Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

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

Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep

2010-04-25

Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNAmore » Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.« less

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

PubMed

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

2013-07-19

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. Copyright © 2013 Elsevier Inc. All rights reserved.

Exploring transduction mechanisms of protein transduction domains (PTDs) in living cells utilizing single-quantum dot tracking (SQT) technology.

PubMed

Suzuki, Yasuhiro

2012-01-01

Specific protein domains known as protein transduction domains (PTDs) can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs), we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP) in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT), to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.

Human immunodeficiency virus type 1 Tat does not transactivate mature trans-acting responsive region RNA species in the nucleus or cytoplasm of primate cells.

PubMed Central

Chin, D J; Selby, M J; Peterlin, B M

1991-01-01

Human immunodeficiency virus (HIV)-encoded transactivator Tat is essential for viral gene expression and replication. By interacting with a nascent RNA stem-loop called the trans-acting responsive region (TAR). Tat increases rates of initiation and/or elongation of HIV transcription. Several reports have also suggested that Tat has additional effects on mature HIV RNA species including modification of primary transcripts in the nucleus and their increased translation in the cytoplasm. These posttranscriptional effects are most pronounced in the Xenopus oocyte. To investigate directly whether Tat has similar effects on viral transcripts in cells that are permissive for HIV replication, we cotransfected and microinjected human and monkey cells with Tat and TAR in the form of DNA or RNA. Whereas Tat transactivated TAR DNA targets, it did not transactivate TAR RNA targets in the nucleus of microinjected cells or in the cytoplasm of transfected cells. We conclude that in cells permissive for viral replication, Tat exerts its effect primarily at the level of HIV transcription. Images PMID:1900539

TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors

PubMed Central

Sethuraman, Vijay A; Bae, You Han

2007-01-01

A novel drug targeting system for acidic solid tumors has been developed based on ultra pH sensitive polymer and cell penetrating TAT. The delivery system consisted of two components: 1) A polymeric micelle that has a hydrophobic core made of Poly(L-lactic acid) (PLLA) and a hydrophilic shell consisting of Polyethylene Glycol (PEG) conjugated to TAT (TATmicelle), 2) An ultra pH sensitive diblock copolymer of poly(methacryloyl sulfadimethoxine) (PSD) and PEG (PSD-b-PEG). The anionic PSD is complexed with cationic TAT of the micelles to achieve the final carrier, which could systemically shield the micelles and expose them at slightly acidic tumor pH. TATmicelles had particle sizes between 20 to 45 nm and their critical micelle concentrations were 3.5 mg/L to 5.5 mg/L. The TATmicelles, upon mixing with pH sensitive PSD-b-PEG, showed slight increase in particle size between pH 8.0 and 6.8 (60–90 nm), indicating complexation. As the pH was decreased (pH 6.6 to 6.0) two populations were observed, one that of normal TAT micelles (45 nm) and the other of aggregated hydrophobic PSD-b-PEG. Zeta potential measurements showed similar trend substantiating the shielding/deshielding process. Flowcytometry and confocal microscopy showed significantly higher uptake of TAT micelles at pH 6.6 compared to pH 7.4 indicating shielding at normal pH and deshielding at tumor pH. The flowcytometry indicated that the TAT not only translocates into the cells but is also seen on the surface of the nucleus. These results strongly indicate that the above drug loaded micelles would be able to target any hydrophobic drug near the nucleus. PMID:17239466

A strategy of win-stay, lose-shift that outperforms tit-for-tat in the Prisoner's Dilemma game

NASA Astrophysics Data System (ADS)

Nowak, Martin; Sigmund, Karl

1993-07-01

THE Prisoner's Dilemma is the leading metaphor for the evolution of cooperative behaviour in populations of selfish agents, especially since the well-known computer tournaments of Axelrod1 and their application to biological communities2,3. In Axelrod's simulations, the simple strategy tit-for-tat did outstandingly well and subsequently became the major paradigm for reciprocal altruism4 12. Here we present extended evolutionary simulations of heterogeneous ensembles of probabilistic strategies including mutation and selection, and report the unexpected success of another protagonist: Pavlov. This strategy is as simple as tit-for-tat and embodies the fundamental behavioural mechanism win-stay, lose-shift, which seems to be a widespread rule13. Pavlov's success is based on two important advantages over tit-for-tat: it can correct occasional mistakes and exploit unconditional cooperators. This second feature prevents Pavlov populations from being undermined by unconditional cooperators, which in turn invite defectors. Pavlov seems to be more robust than tit-for-tat, suggesting that cooperative behaviour in natural situations may often be based on win-stay, lose-shift.

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform.

PubMed

Koushika, S P; Lisbin, M J; White, K

1996-12-01

Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian. Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.

The YPR153W gene is essential for the pressure tolerance of tryptophan permease Tat2 in the yeast Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Kurosaka, Goyu; Abe, Fumiyoshi

2018-01-01

In the yeast Saccharomyces cerevisiae, hydrostatic pressure at 25 MPa is known to be nonlethal but significantly impairs the uptake of tryptophan by the permease Tat2, thereby inhibiting the growth of strains that require tryptophan from the medium. Here, we found that the lack of the YPR153W gene, so far poorly characterized for its role in yeast, caused a serious adverse effect on the growth at 10-25 MPa in the strain that required tryptophan. Deletion for YPR153W resulted in an increased rate of pressure-induced degradation of Tat2, suggesting that Tat2 is destabilized in the YPR153W deletion mutant at 25 MPa. Overexpression of the TAT2 gene enabled the deletion mutant to grow at 25 MPa. These results suggest that Ypr153w is essential for the stability and proper transport function of Tat2 under pressure at 10-25 MPa.

Thermodynamic contributions for the incorporation of GTA triplets within canonical TAT/TAT and C+GC/C+GC base-triplet stacks of DNA triplexes.

PubMed

Soto, Ana Maria; Marky, Luis A

2002-10-15

Nucleic acid triple helices may be used in the control of gene expression. One limitation of using triplex-forming oligonucleotides as therapeutic agents is that their target sequences are limited to homopurine tracts. To increase the repertoire of sequences that can be targeted, it has been postulated that a guanine can target a thymidine forming a stable GTA mismatch triplet. In this work, we have used a combination of optical and calorimetric techniques to determine thermodynamic unfolding profiles of two triplexes containing a single GTA triplet, d(A(3)TA(3)C(5)T(3)AT(3)C(5)T(3)GT(3)) (ATA) and d(AGTGAC(5)TCACTC(5)TCGCT) (GTG), and their control triplexes, d(A(7)C(5)T(7)C(5)T(7)) (TAT7) and d(AGAGAC(5)TCTCTC(5)TCTCT) (AG5T). In general, the presence of a GTA mismatch in DNA triplexes is destabilizing; however, this destabilization is greater when placed in a C(+)GC/C(+)GC base-triplet stack than between a TAT/TAT stack. These destabilizations are accompanied by a reduced unfolding enthalpy of approximately 10 kcal/mol, suggesting a decrease in the base stacking contributions surrounding the mismatch. Relative to their corresponding control triplexes, the folding of ATA is accompanied by a lower counterion uptake and a similar proton uptake, while GTG folding is accompanied by an increase in the counterion and proton uptakes. These effects are consistent with the observed decrease in stacking interactions. The overall results indicate that the main difficulty of targeting pyrimidine interruptions is that the decrease in stacking contributions, due to the incorporation of a GTA mismatch, affects the stability of the neighboring base triplets. This suggests that nucleotide analogues that increase the strength of these base-triplet stacks will result in a more effective targeting of pyrimidine interruptions.

The Tlo Proteins Are Stoichiometric Components of Candida albicans Mediator Anchored via the Med3 Subunit

PubMed Central

Zhang, Anda; Petrov, Kostadin O.; Hyun, Emily R.; Liu, Zhongle; Gerber, Scott A.

2012-01-01

The amplification of the TLO (for telomere-associated) genes in Candida albicans, compared to its less pathogenic, close relative Candida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis. PMID:22562472

Inhibition of mitochondrial fragmentation diminishes Huntington’s disease–associated neurodegeneration

PubMed Central

Guo, Xing; Disatnik, Marie-Helene; Monbureau, Marie; Shamloo, Mehrdad; Mochly-Rosen, Daria; Qi, Xin

2013-01-01

Huntington’s disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell–derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell–derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT–like inhibitor may prevent or slow the progression of HD. PMID:24231356

Emerging functions of multi-protein complex Mediator with special emphasis on plants.

PubMed

Malik, Naveen; Agarwal, Pinky; Tyagi, Akhilesh

2017-10-01

Mediator is a multi-subunit protein complex which is involved in transcriptional regulation in yeast and other eukaryotes. As a co-activator, it connects information from transcriptional activators/repressors to transcriptional machinery including RNA polymerase II and general transcription factors. It is not only involved in transcription initiation but also has important roles to play in transcription elongation and termination. Functional attributes of different Mediator subunits have been largely defined in yeast and mammalian systems earlier, while such studies in plants have gained momentum recently. Mediator regulates various processes related to plant development and is also involved in biotic and abiotic stress response. Thus, plant Mediator, like yeast and mammalian Mediator complex, is indispensable for plant growth and survival. Interaction of its multiple subunits with other regulatory proteins and their ectopic expression or knockdown in model plant like Arabidopsis and certain crop plants are paving the way to biochemical analysis and unravel molecular mechanisms of action of Mediator in plants.

[Keap1-tat peptide attenuates oxidative stress damage in hippocampal CA1 region and learning and memory deficits following global cerebral ischemia].

PubMed

Tu, Jing-yi; Zhu, Ying; Shang, Shu-ling; Zhang, Xi; Tang, Hui; Wang, Rui-min

2016-02-18

To design Keap1-tat peptide and explore its neuroprotective role on hipocampal CA1 neuron, as well as the effect on spacial learning and memory function following global cerebral ischemia. Adult male Sprague Dawley (SD) rats were subjected to global cerebral ischemia (GCI) by four-vessel occlusion for 15 min and randomly divided into five groups: sham, sham+Keap1-tat, ischemia/reperfusion (I/R), Keap1-tat peptide- and vehicle-administrated groups. For Keap1-tat or vehicle groups, the rats were treated with Keap1-tat (30, 50, 100 μg in 5 μL 0.9% saline) or the same volume vehicle by intracerebroventricular injection (icv) 30 min prior to ischemia. Cresyl violet staining was used to observe the surviving neurons and 4-hydroxy-2-noneal (4-HNE) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunostaining were used to detect the change of markers response to oxidative stress in hippocampal CA1 region. The spatial learning and memory function of the rats was evaluated using Morris water maze. Compared with sham group, the number of surviving neurons in ischemia-reperfusion and vehicle groups significantly decreased in the hippocampal CA1 region (P<0.05), while administration of Keap1-tat significantly decreased the damage following GCI (P<0.05), and the dose of 50 μg existed the most effective neuroprotective role. Furthermore, immunostaining intensity of 4-HNE and 8-OHdG, markers of oxidative stress damage attenuated by Keap1-tat peptide as compared with vehicle group in CA1 region. Of significant interest, the time of finding underwater platform in Keap1-tat group animals was significantly short, and after removing the platform, the probe time of Keap1-tat group animals in the original quadrant where the platform was significantly increased compared with that of vehicle and I/R group animals (P<0.05). Keap1-tat peptide can effectively attenuate neuronal damage in hippocampal CA1 region and improve learning and memory function, which might bedue to the attenuation of

Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins

PubMed Central

Brunger, Axel T.; Cipriano, Daniel J.; Diao, Jiajie

2015-01-01

Abstract Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed. PMID:25788028

Negotiation of intracellular membrane barriers by TAT-modified gold nanoparticles.

PubMed

Krpetić, Zeljka; Saleemi, Samia; Prior, Ian A; Sée, Violaine; Qureshi, Rumana; Brust, Mathias

2011-06-28

This paper contributes to the debate on how nanosized objects negotiate membrane barriers inside biological cells. The uptake of peptide-modified gold nanoparticles by HeLa cells has been quantified using atomic emission spectroscopy. The TAT peptide from the HIV virus was singled out as a particularly effective promoter of cellular uptake. The evolution of the intracellular distribution of TAT-modified gold nanoparticles with time has been studied in detail by TEM and systematic image analysis. An unusual trend of particles disappearing from the cytosol and the nucleus and accumulating massively in vesicular bodies was observed. Subsequent release of the particles, both by membrane rupture and by direct transfer across the membrane boundary, was frequently found. Ultimately, near total clearing of particles from the cells occurred. This work provides support for the hypothesis that cell-penetrating peptides can enable small objects to negotiate membrane barriers also in the absence of dedicated transport mechanisms.

A novel domain of amino-Nogo-A protects HT22 cells exposed to oxygen glucose deprivation by inhibiting NADPH oxidase activity.

PubMed

Guo, Fan; Wang, Huiwen; Li, Liya; Zhou, Heng; Wei, Haidong; Jin, Weilin; Wang, Qiang; Xiong, Lize

2013-04-01

This study aimed to investigate the protective effect of the M9 region (residues 290-562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia-reperfusion induced by oxygen-glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.

Abrogation of TNF-mediated cytotoxicity by space flight involves protein kinase C

NASA Technical Reports Server (NTRS)

Woods, K. M.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Experiments conducted on STS-50 indicated that space flight significantly inhibited tumor necrosis factor (TNF)-mediated killing of LM929 cells compared to ground controls. In ground-based studies, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also inhibited TNF-mediated killing of LM929 cells. Therefore, we used PKC inhibitors to determine if the inhibitory effects of spaceflight on TNF-mediated cytotoxicity involved the activation of PKC. In experiments conducted onboard space shuttle mission STS-54, we saw that in the presence of the protein kinase C inhibitors H7 and H8, TNF-mediated cytotoxicity was restored to levels of those observed in the ground controls. Subsequent experiments done during the STS-57 mission tested the dose response of two protein kinase inhibitors, H7 and HA1004. We again saw that killing was restored in a dose-dependent manner, with inhibitor concentrations known to inhibit PKC being most effective. These data suggest that space flight ameliorates the action of TNF by affecting PKC in target cells.

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

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

Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In

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-mediatedmore » 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.« less

Indian Long-term Non-Progressors Show Broad ADCC Responses with Preferential Recognition of V3 Region of Envelope and a Region from Tat Protein.

PubMed

Kulkarni, Archana; Kurle, Swarali; Shete, Ashwini; Ghate, Manisha; Godbole, Sheela; Madhavi, Vijaya; Kent, Stephen J; Paranjape, Ramesh; Thakar, Madhuri

2017-01-01

HIV-specific antibody-dependent cell cytotoxicity (ADCC) is likely to be important in governing protection from human immunodeficiency virus (HIV) and slowing disease progression. Little is known about the ADCC responses to HIV-1 subtype C. We characterized ADCC responses in HIV-1 subtype C-infected Indian subjects with slow disease progression and identified the dominant antigenic regions recognized by these antibodies. ADCC responses were measured in plasma from 34 long-term non-progressors (LTNPs), who were asymptomatic and maintained CD4 count above 500 cells/mm 3 for the last 7 years in the absence of antiretroviral therapy (ART), and 58 ART naïve progressors with CD4 count <500 cells/mm 3 against overlapping HIV-1 peptides using a flow cytometry-based antibody-dependent natural killer (NK) cell activation assay. The assay measured CD107a expression on NK cells as a marker of antibody-dependent NK cell activation and IFN-γ secretion by NK cells upon activation. The ADCC epitopes were mapped using the matrix of overlapping peptides. Indian LTNPs showed higher and broader ADCC responses compared to the progressors. The Env-C and Tat-specific ADCC responses were associated with lower plasma viral load, whereas the Env-C responses were also associated with higher CD4 counts. Five of 10 LTNP responders targeted epitopes in the V3 region (amino acids 288-330) of Env-C. Additionally, three Tat regions were targeted by ADCC antibodies from LTNPs. ADCC responses were associated with slow HIV progression in Indian subtype C-infected cohort. The frequently recognized peptides from the V3 loop of Env and the novel epitopes from Tat by the LTNPs warrants further study to understand the role of ADCC responses to these regions in control and prevention of HIV-1 infection.

Electrochemically mediated polymerization for highly sensitive detection of protein kinase activity.

PubMed

Hu, Qiong; Wang, Qiangwei; Jiang, Cuihua; Zhang, Jian; Kong, Jinming; Zhang, Xueji

2018-07-01

Protein kinases play a pivotal role in cellular regulation and signal transduction, the detection of protein kinase activity and inhibition is therefore of great importance to clinical diagnosis and drug discovery. In this work, a novel electrochemical platform using the electrochemically mediated polymerization as an efficient and cost-effective signal amplification strategy is described for the highly sensitive detection of protein kinase activity. This platform involves 1) the phosphorylation of substrate peptide by protein kinase, 2) the attachment of alkyl halide to the phosphorylated sites via the carboxylate-Zr 4+ -phosphate chemistry, and 3) the in situ grafting of electroactive polymers from the phosphorylated sites through the electrochemically mediated atom transfer radical polymerization (eATRP) at a negative potential, in the presence of the surface-attached alkyl halide as the initiator and the electroactive tag-conjugated acrylate as the monomer, respectively. Due to the electrochemically mediated polymerization, a large number of electroactive tags can be linked to each phosphorylated site, thereby greatly improving the detection sensitivity. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase (PKA) with a detection limit down to 1.63 mU mL -1 . Results also demonstrate that it is highly selective and can be used for the screening of protein kinase inhibitors. The potential application of our platform for protein kinase activity detection in complex biological samples has been further verified using normal human serum and HepG2 cell lysate. Moreover, our platform is operationally simple, highly efficient and cost-effective, thus holding great potential in protein kinase detection and inhibitor screening. Copyright © 2018 Elsevier B.V. All rights reserved.

A newly identified protein of Leptospira interrogans mediates binding to laminin.

PubMed

Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2009-10-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

Nucleation of Iron Oxide Nanoparticles Mediated by Mms6 Protein in Situ

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

Kashyap, Sanjay; Woehl, Taylor J; Liu, Xunpei

2014-09-23

Biomineralization proteins are widely used as templating agents in biomimetic synthesis of a variety of organic–inorganic nanostructures. However, the role of the protein in controlling the nucleation and growth of biomimetic particles is not well understood, because the mechanism of the bioinspired reaction is often deduced from ex situ analysis of the resultant nanoscale mineral phase. Here we report the direct visualization of biomimetic iron oxide nanoparticle nucleation mediated by an acidic bacterial recombinant protein, Mms6, during an in situ reaction induced by the controlled addition of sodium hydroxide to solution-phase Mms6 protein micelles incubated with ferric chloride. Using inmore » situ liquid cell scanning transmission electron microscopy we observe the liquid iron prenucleation phase and nascent amorphous nanoparticles forming preferentially on the surface of protein micelles. Our results provide insight into the early steps of protein-mediated biomimetic nucleation of iron oxide and point to the importance of an extended protein surface during nanoparticle formation.« less

Protein disulphide isomerase is required for signal peptide peptidase-mediated protein degradation

PubMed Central

Lee, Seong-Ok; Cho, Kwangmin; Cho, Sunglim; Kim, Ilkwon; Oh, Changhoon; Ahn, Kwangseog

2010-01-01

The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2-induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA-mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate-binding mutant of PDI, but not the catalytically inactive mutant, dominant-negatively inhibited US2-mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP-mediated degradation of CD3δ but not Derlin-1-dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP-mediated ER-associated degradation machinery. PMID:19942855

Protein disulphide isomerase is required for signal peptide peptidase-mediated protein degradation.

PubMed

Lee, Seong-Ok; Cho, Kwangmin; Cho, Sunglim; Kim, Ilkwon; Oh, Changhoon; Ahn, Kwangseog

2010-01-20

The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2-induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA-mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate-binding mutant of PDI, but not the catalytically inactive mutant, dominant-negatively inhibited US2-mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP-mediated degradation of CD3delta but not Derlin-1-dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP-mediated ER-associated degradation machinery.

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

PubMed Central

Antos, John M.; Ingram, Jessica; Fang, Tao; Pishesha, Novalia; Truttmann, Matthias C.; Ploegh, Hidde L.

2017-01-01

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five amino acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. PMID:19365788

Protein Mediated Oxidative Stress in Patients with Diabetes and its Associated Neuropathy: Correlation with Protein Carbonylation and Disease Activity Markers

PubMed Central

Almogbel, Ebtehal

2017-01-01

Introduction Free radicals have been implicated as Diabetes Mellitus (DM) contributors in type 2 DM and its associated Diabetes Mellitus Neuropathy (DMN). However, the potential for protein mediated oxidative stress to contribute disease pathogenesis remains largely unexplored. Aim To investigate the status and contribution of protein mediated oxidative stress in patients with DM or DMN and to explore whether oxidative protein modification has a role in DM progression to DM associated neuropathy. Materials and Methods Sera from 42 DM and 37 DMN patients with varying levels of disease activities biomarkers (HbA1C, patients’ age or disease duration) and 21 age- and sex-matched healthy controls were evaluated for serum levels of protein mediated oxidative stress. Results Serum analysis showed significantly higher levels of protein carbonyl contents in both DM and DMN patients compared with healthy controls. Importantly, not only was there an increased number of subjects positive for protein carbonylation, but also the levels of protein carbonyl contents were significantly higher among DM and DMN patients, whose HbA1C were ≥8.8 as compared with patients with lower HbA1C (HbA1C<8.8). Similar pattern of protein carbonyls formation was also observed with patients’ ages or with patient’s disease durations, suggesting a possible relationship between protein oxidation and disease progression. Furthermore, sera from DMN patients had higher levels of protein carbonylation compared with non-neuropathic DM patients’ sera, suggesting an involvement of protein oxidation in the progression of diabetes to diabetes neuropathy. Conclusion These findings support an association between protein oxidation and DM or DMN progression. The stronger response observed in patients with higher HbA1C or patients’ ages or disease durations suggests, that protein mediated oxidative stress may be useful in evaluating the progression of DM and its associated DMN and in elucidating the

Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

PubMed Central

Jayasena, S D; Johnston, B H

1992-01-01

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo. Images PMID:1565648

Field Trial Data Analysis and Testing (FiTAT) Tool

DTIC Science & Technology

2011-10-01

amplitude/phase pour chaque antenne du réseau) contenu dans les données acquises pourrait être faite par FiTAT et utilisé dans PAASoM pour déterminer...identity matrix, k is the loop gain, φ is the correlation matrix of the incident signals and T = [1 0 . . . 0]T . The length of vector T is equal to the

Tat peptide and hexadecylphosphocholine introduction into pegylated liposomal doxorubicin: An in vitro and in vivo study on drug cellular delivery, release, biodistribution and antitumor activity.

PubMed

Teymouri, Manouchehr; Badiee, Ali; Golmohammadzadeh, Shiva; Sadri, Kayvan; Akhtari, Javad; Mellat, Mostafa; Nikpoor, Amin Reza; Jaafari, Mahmoud Reza

2016-09-10

We have investigated the co-addition of hexadecylphosphocholine (HePC) and a Tat derived peptide (Tat), coupled to Maleimide-PEG2000-DSPE pegylated liposomal doxorubicin (PLD) in many respects, including drug and liposome cellular delivery, drug release, biodistribution, in vivo cell delivery and antitumor activity. The liposomes were HePC-free and -containing liposomes, from which liposomes with 25, 50, 100 and 200 numbers of Tat/liposome were prepared. Similarly, DiI-C18 (3)-model liposomes (DiI-L and DiI-HePC-L) were prepared. HePC and Tat increased cellular delivery of Dox and cytotoxicity in B16F0 melanoma and C26 colon carcinoma cells. Tat enhanced liposome-cell interaction and caused Dox burst release. HePC and Tat reduced the serum retention time of liposomal Dox, slightly and dramatically, respectively. In comparison, Tat-liposomes enhanced Dox delivery to liver and spleen cells 3h post-injection. Likewise, Dox content of these tissues and tumor was lower at 24h. The naïve liposomes retarded tumor growth more effectively and their related median survival time of the treated C26 bearing BALB/c mice was longer than those of Tat-liposomes (MST>45days versus MST<38days). Overall liposomes exhibiting sustained drug release and negligible cell interaction were more suitable delivery systems in targeting cancerous tumors and suppressing their growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein Corona Modulates Uptake and Toxicity of Nanoceria via Clathrin-Mediated Endocytosis.

PubMed

Mazzolini, Julie; Weber, Ralf J M; Chen, Hsueh-Shih; Khan, Abdullah; Guggenheim, Emily; Shaw, Robert K; Chipman, James K; Viant, Mark R; Rappoport, Joshua Z

2016-08-01

Particles present in diesel exhaust have been proposed as a significant contributor to the development of acute and chronic lung diseases, including respiratory infection and allergic asthma. Nanoceria (CeO2 nanoparticles) are used to increase fuel efficiency in internal combustion engines, are present in exhaust fumes, and could affect cells of the airway. Components from the environment such as biologically derived proteins, carbohydrates, and lipids can form a dynamic layer, commonly referred to as the "protein corona" which alters cellular nanoparticle interactions and internalization. Using confocal reflectance microscopy, we quantified nanoceria uptake by lung-derived cells in the presence and absence of a serum-derived protein corona. Employing mass spectrometry, we identified components of the protein corona, and demonstrated that the interaction between transferrin in the protein corona and the transferrin receptor is involved in mediating the cellular entry of nanoceria via clathrin-mediated endocytosis. Furthermore, under these conditions nanoceria does not affect cell growth, viability, or metabolism, even at high concentration. Alternatively, despite the antioxidant capacity of nanoceria, in serum-free conditions these nanoparticles induce plasma membrane disruption and cause changes in cellular metabolism. Thus, our results identify a specific receptor-mediated mechanism for nanoceria entry, and provide significant insight into the potential for nanoparticle-dependent toxicity. © 2016 Marine Biological Laboratory.

Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

PubMed Central

Liang, Jingjing; Sagum, Cari A.; Bedford, Mark T.; Sudol, Marius; Han, Ziying

2017-01-01

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles. PMID:28076420

Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress.

PubMed

Liang, Jingjing; Sagum, Cari A; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Han, Ziying; Harty, Ronald N

2017-01-01

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.

PURA, the gene encoding Pur-alpha, member of an ancient nucleic acid-binding protein family with mammalian neurological functions.

PubMed

Daniel, Dianne C; Johnson, Edward M

2018-02-15

The PURA gene encodes Pur-alpha, a 322 amino acid protein with repeated nucleic acid binding domains that are highly conserved from bacteria through humans. PUR genes with a single copy of this domain have been detected so far in spirochetes and bacteroides. Lower eukaryotes possess one copy of the PUR gene, whereas chordates possess 1 to 4 PUR family members. Human PUR genes encode Pur-alpha (Pura), Pur-beta (Purb) and two forms of Pur-gamma (Purg). Pur-alpha is a protein that binds specific DNA and RNA sequence elements. Human PURA, located at chromosome band 5q31, is under complex control of three promoters. The entire protein coding sequence of PURA is contiguous within a single exon. Several studies have found that overexpression or microinjection of Pura inhibits anchorage-independent growth of oncogenically transformed cells and blocks proliferation at either G1-S or G2-M checkpoints. Effects on the cell cycle may be mediated by interaction of Pura with cellular proteins including Cyclin/Cdk complexes and the Rb tumor suppressor protein. PURA knockout mice die shortly after birth with effects on brain and hematopoietic development. In humans environmentally induced heterozygous deletions of PURA have been implicated in forms of myelodysplastic syndrome and progression to acute myelogenous leukemia. Pura plays a role in AIDS through association with the HIV-1 protein, Tat. In the brain Tat and Pura association in glial cells activates transcription and replication of JC polyomavirus, the agent causing the demyelination disease, progressive multifocal leukoencephalopathy. Tat and Pura also act to stimulate replication of the HIV-1 RNA genome. In neurons Pura accompanies mRNA transcripts to sites of translation in dendrites. Microdeletions in the PURA locus have been implicated in several neurological disorders. De novo PURA mutations have been related to a spectrum of phenotypes indicating a potential PURA syndrome. The nucleic acid, G-rich Pura binding

Computational Model for DNA Organization Mediated by Protein Interaction in Prokaryotes

NASA Astrophysics Data System (ADS)

Garimella, Karthik; Kharel, Savan

2016-03-01

In Escherichia Coli, there are several mechanisms that drive chromosomal organization. We know through experiments that the E. Coli chromosome is condensed into highly structured regions known as macrodomains (MDs). One of the regions known as the Terminus undergoes DNA-bridging condensation that form loops between distant DNA sites and it is known to be mediated by a Terminus specific protein, which binds to specific markers within the Terminus region. In the absence of Terminus specific protein, however, the Terminus region is known to not condense nearly as much, which will likely impede several biological processes including DNA replication. In order to understand the molecular basis of protein mediation in vivo several models of Terminus specific segregation have been constructed in silico which model DNA as polymer chains.

Characterization of Mediator Complex and its Associated Proteins from Rice.

PubMed

Samanta, Subhasis; Thakur, Jitendra Kumar

2017-01-01

The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.

An approach to crystallizing proteins by metal-mediated synthetic symmetrization

PubMed Central

Laganowsky, Arthur; Zhao, Minglei; Soriaga, Angela B; Sawaya, Michael R; Cascio, Duilio; Yeates, Todd O

2011-01-01

Combining the concepts of synthetic symmetrization with the approach of engineering metal-binding sites, we have developed a new crystallization methodology termed metal-mediated synthetic symmetrization. In this method, pairs of histidine or cysteine mutations are introduced on the surface of target proteins, generating crystal lattice contacts or oligomeric assemblies upon coordination with metal. Metal-mediated synthetic symmetrization greatly expands the packing and oligomeric assembly possibilities of target proteins, thereby increasing the chances of growing diffraction-quality crystals. To demonstrate this method, we designed various T4 lysozyme (T4L) and maltose-binding protein (MBP) mutants and cocrystallized them with one of three metal ions: copper (Cu2+), nickel (Ni2+), or zinc (Zn2+). The approach resulted in 16 new crystal structures—eight for T4L and eight for MBP—displaying a variety of oligomeric assemblies and packing modes, representing in total 13 new and distinct crystal forms for these proteins. We discuss the potential utility of the method for crystallizing target proteins of unknown structure by engineering in pairs of histidine or cysteine residues. As an alternate strategy, we propose that the varied crystallization-prone forms of T4L or MBP engineered in this work could be used as crystallization chaperones, by fusing them genetically to target proteins of interest. PMID:21898649

Structure of a low-population binding intermediate in protein-RNA recognition

PubMed Central

Bardaro, Michael F.; Aprile, Francesco A.; Varani, Gabriele; Vendruscolo, Michele

2016-01-01

The interaction of the HIV-1 protein transactivator of transcription (Tat) and its cognate transactivation response element (TAR) RNA transactivates viral transcription and represents a paradigm for the widespread occurrence of conformational rearrangements in protein-RNA recognition. Although the structures of free and bound forms of TAR are well characterized, the conformations of the intermediates in the binding process are still unknown. By determining the free energy landscape of the complex using NMR residual dipolar couplings in replica-averaged metadynamics simulations, we observe two low-population intermediates. We then rationally design two mutants, one in the protein and another in the RNA, that weaken specific nonnative interactions that stabilize one of the intermediates. By using surface plasmon resonance, we show that these mutations lower the release rate of Tat, as predicted. These results identify the structure of an intermediate for RNA-protein binding and illustrate a general strategy to achieve this goal with high resolution. PMID:27286828

Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

PubMed

Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

2017-09-01

Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

Sulforaphane-induced autophagy flux prevents prion protein-mediated neurotoxicity through AMPK pathway.

PubMed

Lee, J-H; Jeong, J-K; Park, S-Y

2014-10-10

Prion diseases are neurodegenerative and infectious disorders that involve accumulation of misfolded scrapie prion protein, and which are characterized by spongiform degeneration. Autophagy, a major homeostatic process responsible for the degradation of cytoplasmic components, has garnered attention as the potential target for neurodegenerative diseases such as prion disease. We focused on protective effects of sulforaphane found in cruciferous vegetables on prion-mediated neurotoxicity and the mechanism of sulforaphane related to autophagy. In human neuroblastoma cells, sulforaphane protected prion protein (PrP) (106-126)-mediated neurotoxicity and increased autophagy flux marker microtubule-associated protein 1 light chain 3-II protein levels, following a decrease of p62 protein level. Pharmacological and genetical inhibition of autophagy by 3MA, wortmannin and knockdown of autophagy-related 5 (ATG5) led to block the effect of sulforaphane against PrP (106-126)-induced neurotoxicity. Furthermore we demonstrated that both sulforaphane-induced autophagy and protective effect of sulforaphane against PrP (106-126)-induced neurotoxicity are dependent on the AMP-activated protein kinase (AMPK) signaling. The present results indicated that sulforaphane of cruciferous vegetables enhanced autophagy flux led to the protection effects against prion-mediated neurotoxicity, which was regulated by AMPK signaling pathways in human neuron cells. Our data also suggest that sulforaphane has a potential value as a therapeutic tool in neurodegenerative disease including prion diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR

PubMed Central

Sedore, Stanley C.; Byers, Sarah A.; Biglione, Sebastian; Price, Jason P.; Maury, Wendy J.; Price, David H.

2007-01-01

Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb–HEXIM1–7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb–HEXIM1–7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5′ UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR. PMID:17576689

Protein- mediated enamel mineralization

PubMed Central

Moradian-Oldak, Janet

2012-01-01

Enamel is a hard nanocomposite bioceramic with significant resilience that protects the mammalian tooth from external physical and chemical damages. The remarkable mechanical properties of enamel are associated with its hierarchical structural organization and its thorough connection with underlying dentin. This dynamic mineralizing system offers scientists a wealth of information that allows the study of basic principals of organic matrix-mediated biomineralization and can potentially be utilized in the fields of material science and engineering for development and design of biomimetic materials. This chapter will provide a brief overview of enamel hierarchical structure and properties as well as the process and stages of amelogenesis. Particular emphasis is given to current knowledge of extracellular matrix protein and proteinases, and the structural chemistry of the matrix components and their putative functions. The chapter will conclude by discussing the potential of enamel for regrowth. PMID:22652761

Sequence patterns mediating functions of disordered proteins.

PubMed

Exarchos, Konstantinos P; Kourou, Konstantina; Exarchos, Themis P; Papaloukas, Costas; Karamouzis, Michalis V; Fotiadis, Dimitrios I

2015-01-01

Disordered proteins lack specific 3D structure in their native state and have been implicated with numerous cellular functions as well as with the induction of severe diseases, e.g., cardiovascular and neurodegenerative diseases as well as diabetes. Due to their conformational flexibility they are often found to interact with a multitude of protein molecules; this one-to-many interaction which is vital for their versatile functioning involves short consensus protein sequences, which are normally detected using slow and cumbersome experimental procedures. In this work we exploit information from disorder-oriented protein interaction networks focused specifically on humans, in order to assemble, by means of overrepresentation, a set of sequence patterns that mediate the functioning of disordered proteins; hence, we are able to identify how a single protein achieves such functional promiscuity. Next, we study the sequential characteristics of the extracted patterns, which exhibit a striking preference towards a very limited subset of amino acids; specifically, residues leucine, glutamic acid, and serine are particularly frequent among the extracted patterns, and we also observe a nontrivial propensity towards alanine and glycine. Furthermore, based on the extracted patterns we set off to infer potential functional implications in order to verify our findings and potentially further extrapolate our knowledge regarding the functioning of disordered proteins. We observe that the extracted patterns are primarily involved with regulation, binding and posttranslational modifications, which constitute the most prominent functions of disordered proteins.

Examining post-translational modification-mediated protein–protein interactions using a chemical proteomics approach

PubMed Central

Li, Xiang; Foley, Emily A; Kawashima, Shigehiro A; Molloy, Kelly R; Li, Yinyin; Chait, Brian T; Kapoor, Tarun M

2013-01-01

Post-translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM-dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross-linking-assisted and stable isotope labeling in cell culture-based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation-dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine-9 (H3K9Me3)-dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation-dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine-3 (H3T3-Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3-Phos-binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3-Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me3). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs. PMID:23281010

Butelase-mediated cyclization and ligation of peptides and proteins.

PubMed

Nguyen, Giang K T; Qiu, Yibo; Cao, Yuan; Hemu, Xinya; Liu, Chuan-Fa; Tam, James P

2016-10-01

Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn-His-Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.

Increasing spinal 5-HT2A receptor responsiveness mediates anti-allodynic effect and potentiates fluoxetine efficacy in neuropathic rats. Evidence for GABA release.

PubMed

Dupuis, Amandine; Wattiez, Anne-Sophie; Pinguet, Jérémy; Richard, Damien; Libert, Frédéric; Chalus, Maryse; Aissouni, Youssef; Sion, Benoit; Ardid, Denis; Marin, Philippe; Eschalier, Alain; Courteix, Christine

2017-04-01

Antidepressants are one of the first line treatments for neuropathic pain but their use is limited by the incidence and severity of side effects of tricyclics and the weak effectiveness of selective serotonin reuptake inhibitors (SSRIs). Serotonin type 2A (5-HT 2A ) receptors interact with PDZ proteins that regulate their functionality and SSRI efficacy to alleviate pain. We investigated whether an interfering peptide (TAT-2ASCV) disrupting the interaction between 5-HT 2A receptors and associated PDZ proteins would improve the treatment of traumatic neuropathic allodynia. Tactile allodynia was assessed in spinal nerve ligation-induced neuropathic pain in rats using von Frey filaments after acute treatment with TAT-2ASCV and/or 5-HT 2A receptor agonist, alone or in combination with repeated treatment with fluoxetine. In vivo microdialysis was performed in order to examine the involvement of GABA in TAT-2ASCV/fluoxetine treatment-associated analgesia. TAT-2ASCV (100ng, single i.t. injection) improved SNL-induced tactile allodynia by increasing 5-HT 2A receptor responsiveness to endogenous 5-HT. Fluoxetine alone (10mg/kg, five i.p. injections) slightly increased tactile thresholds and its co-administration with TAT-2ASCV (100ng, single i.t. injection) further enhanced the anti-allodynic effect. This effect depends on the integrity of descending serotonergic bulbospinal pathways and spinal release of GABA. The anti-allodynic effect of fluoxetine can be enhanced by disrupting 5-HT 2A receptor-PDZ protein interactions. This enhancement depends on 5-HT 2A receptor activation, spinal GABA release and GABAA receptor activation. Copyright © 2016 Elsevier Ltd. All rights reserved.

DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions

PubMed Central

Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

2016-01-01

Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction’s mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein–protein interactions or protein–DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1 040 000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43 000 RNA-mediated interactions, and ∼12 000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network

Fusion protein based on Grb2-SH2 domain for cancer therapy

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

Saito, Yuriko; Graduate School of Pharmaceutical Sciences, Chiba University; Furukawa, Takako, E-mail: tfuru@nirs.go.jp

2010-08-20

Research highlights: {yields} Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. {yields} We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. {yields} The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. {yields} TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylatedmore » EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.« less

A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

PubMed Central

Gonzalez, Nicola H.; Felsner, Gregor; Schramm, Frederic D.; Klingl, Andreas; Maier, Uwe-G.; Bolte, Kathrin

2011-01-01

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1. PMID:21966495

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

PubMed Central

James, Victoria; Zhang, Yining; Foxler, Daniel E.; de Moor, Cornelia H.; Kong, Yi Wen; Webb, Thomas M.; Self, Tim J.; Feng, Yungfeng; Lagos, Dimitrios; Chu, Chia-Ying; Rana, Tariq M.; Morley, Simon J.; Longmore, Gregory D.; Bushell, Martin; Sharp, Tyson V.

2010-01-01

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m7GTP cap–protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5′cap and Ago1/2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex. PMID:20616046

Combination with anti-tit-for-tat remedies problems of tit-for-tat.

PubMed

Yi, Su Do; Baek, Seung Ki; Choi, Jung-Kyoo

2017-01-07

One of the most important questions in game theory concerns how mutual cooperation can be achieved and maintained in a social dilemma. In Axelrod's tournaments of the iterated prisoner's dilemma, Tit-for-Tat (TFT) demonstrated the role of reciprocity in the emergence of cooperation. However, the stability of TFT does not hold in the presence of implementation error, and a TFT population is prone to neutral drift to unconditional cooperation, which eventually invites defectors. We argue that a combination of TFT and anti-TFT (ATFT) overcomes these difficulties in a noisy environment, provided that ATFT is defined as choosing the opposite to the opponent's last move. According to this TFT-ATFT strategy, a player normally uses TFT; turns to ATFT upon recognizing his or her own error; returns to TFT either when mutual cooperation is recovered or when the opponent unilaterally defects twice in a row. The proposed strategy provides simple and deterministic behavioral rules for correcting implementation error in a way that cannot be exploited by the opponent, and suppresses the neutral drift to unconditional cooperation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of the proteins related to SET-mediated hepatic cytotoxicity of trichloroethylene by proteomic analysis.

PubMed

Ren, Xiaohu; Yang, Xifei; Hong, Wen-Xu; Huang, Peiwu; Wang, Yong; Liu, Wei; Ye, Jinbo; Huang, Haiyan; Huang, Xinfeng; Shen, Liming; Yang, Linqing; Zhuang, Zhixiong; Liu, Jianjun

2014-05-16

Trichloroethylene (TCE) is an effective solvent for a variety of organic materials. Since the wide use of TCE as industrial degreasing of metals, adhesive paint and polyvinyl chloride production, TCE has turned into an environmental and occupational toxicant. Exposure to TCE could cause severe hepatotoxicity; however, the toxic mechanisms of TCE remain poorly understood. Recently, we reported that SET protein mediated TCE-induced cytotoxicity in L-02 cells. Here, we further identified the proteins related to SET-mediated hepatic cytotoxicity of TCE using the techniques of DIGE (differential gel electrophoresis) and MALDI-TOF-MS/MS. Among the 20 differential proteins identified, 8 were found to be modulated by SET in TCE-induced cytotoxicity and three of them (cofilin-1, peroxiredoxin-2 and S100-A11) were validated by Western-blot analysis. The functional analysis revealed that most of the identified SET-modulated proteins are apoptosis-associated proteins. These data indicated that these proteins may be involved in SET-mediated hepatic cytotoxicity of TCE in L-02 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Prostacyclin-induced hyperthermia - Implication of a protein mediator

NASA Technical Reports Server (NTRS)

Kandasamy, S. B.; Williams, B. A.

1982-01-01

The mechanism of the prostacyclin-linked hyperthermia is studied in rabbits. Results show that intracerebroventricular administration of prostacyclin (PGI2) induces dose-related hyperthermia at room temperature (21 C), as well as at low (4 C) and high (30 C) ambient temperatures. It is found that this PGI2-induced hyperthermia is not mediated by its stable metabolite 6-keto prostaglandin F-1(alpha). Only one of the three anion transport systems, the liver transport system, appears to be important to the central inactivation of pyrogen, prostaglandin E2, and PGI2. Phenoxybenzamine and pimozide have no thermolytic effect on PGI2-induced hyperthermia, while PGI2 still induces hyperthermia after norepinephrine (NE) and dopamine levels are depleted by 6-hydroxydopamine. Indomethacin and SC-19220 (a PG antagonist) do not antagonize PGI2 induced hyperthermia, while theophylline does not accentuate the PGI2-induced hyperthermia. However, the hyperthermic response to PGI2 is attenuated by central administration of the protein synthesis inhibitor, anisomycin. It is concluded that PGI2-induced hyperthermia is not induced by NE, dopamine, or cyclic AMP, but rather that a protein mediator is implicated in the induction of fever by PG12.

Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity.

PubMed

Jacobs, Y; Schnabel, C A; Cleary, M L

1999-07-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

Trimeric Association of Hox and TALE Homeodomain Proteins Mediates Hoxb2 Hindbrain Enhancer Activity

PubMed Central

Jacobs, Yakop; Schnabel, Catherine A.; Cleary, Michael L.

1999-01-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element. PMID:10373562

Hot-spot identification on a broad class of proteins and RNA suggest unifying principles of molecular recognition

PubMed Central

Kulp, John L.; Cloudsdale, Ian S.; Kulp, John L.

2017-01-01

Chemically diverse fragments tend to collectively bind at localized sites on proteins, which is a cornerstone of fragment-based techniques. A central question is how general are these strategies for predicting a wide variety of molecular interactions such as small molecule-protein, protein-protein and protein-nucleic acid for both experimental and computational methods. To address this issue, we recently proposed three governing principles, (1) accurate prediction of fragment-macromolecule binding free energy, (2) accurate prediction of water-macromolecule binding free energy, and (3) locating sites on a macromolecule that have high affinity for a diversity of fragments and low affinity for water. To test the generality of these concepts we used the computational technique of Simulated Annealing of Chemical Potential to design one small fragment to break the RecA-RecA protein-protein interaction and three fragments that inhibit peptide-deformylase via water-mediated multi-body interactions. Experiments confirm the predictions that 6-hydroxydopamine potently inhibits RecA and that PDF inhibition quantitatively tracks the water-mediated binding predictions. Additionally, the principles correctly predict the essential bound waters in HIV Protease, the surprisingly extensive binding site of elastase, the pinpoint location of electron transfer in dihydrofolate reductase, the HIV TAT-TAR protein-RNA interactions, and the MDM2-MDM4 differential binding to p53. The experimental confirmations of highly non-obvious predictions combined with the precise characterization of a broad range of known phenomena lend strong support to the generality of fragment-based methods for characterizing molecular recognition. PMID:28837642

Hot-spot identification on a broad class of proteins and RNA suggest unifying principles of molecular recognition.

PubMed

Kulp, John L; Cloudsdale, Ian S; Kulp, John L; Guarnieri, Frank

2017-01-01

Chemically diverse fragments tend to collectively bind at localized sites on proteins, which is a cornerstone of fragment-based techniques. A central question is how general are these strategies for predicting a wide variety of molecular interactions such as small molecule-protein, protein-protein and protein-nucleic acid for both experimental and computational methods. To address this issue, we recently proposed three governing principles, (1) accurate prediction of fragment-macromolecule binding free energy, (2) accurate prediction of water-macromolecule binding free energy, and (3) locating sites on a macromolecule that have high affinity for a diversity of fragments and low affinity for water. To test the generality of these concepts we used the computational technique of Simulated Annealing of Chemical Potential to design one small fragment to break the RecA-RecA protein-protein interaction and three fragments that inhibit peptide-deformylase via water-mediated multi-body interactions. Experiments confirm the predictions that 6-hydroxydopamine potently inhibits RecA and that PDF inhibition quantitatively tracks the water-mediated binding predictions. Additionally, the principles correctly predict the essential bound waters in HIV Protease, the surprisingly extensive binding site of elastase, the pinpoint location of electron transfer in dihydrofolate reductase, the HIV TAT-TAR protein-RNA interactions, and the MDM2-MDM4 differential binding to p53. The experimental confirmations of highly non-obvious predictions combined with the precise characterization of a broad range of known phenomena lend strong support to the generality of fragment-based methods for characterizing molecular recognition.

Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration

PubMed Central

Zhang, Jian-Nan; Michel, Uwe; Lenz, Christof; Friedel, Caroline C.; Köster, Sarah; d’Hedouville, Zara; Tönges, Lars; Urlaub, Henning; Bähr, Mathias; Lingor, Paul; Koch, Jan C.

2016-01-01

Axonal degeneration is a key initiating event in many neurological diseases. Focal lesions to axons result in a rapid disintegration of the perilesional axon by acute axonal degeneration (AAD) within several hours. However, the underlying molecular mechanisms of AAD are only incompletely understood. Here, we studied AAD in vivo through live-imaging of the rat optic nerve and in vitro in primary rat cortical neurons in microfluidic chambers. We found that calpain is activated early during AAD of the optic nerve and that calpain inhibition completely inhibits axonal fragmentation on the proximal side of the crush while it attenuates AAD on the distal side. A screening of calpain targets revealed that collapsin response mediator protein-2 (CRMP2) is a main downstream target of calpain activation in AAD. CRMP2-overexpression delayed bulb formation and rescued impairment of axonal mitochondrial transport after axotomy in vitro. In vivo, CRMP2-overexpression effectively protected the proximal axon from fragmentation within 6 hours after crush. Finally, a proteomic analysis of the optic nerve was performed at 6 hours after crush, which identified further proteins regulated during AAD, including several interactors of CRMP2. These findings reveal CRMP2 as an important mediator of AAD and define it as a putative therapeutic target. PMID:27845394

Insulin-mediated inhibition of p38 mitogen-activated protein kinase protects cardiomyocytes in severe burns.

PubMed

Lv, Gen-fa; Dong, Mao-long; Hu, Da-hai; Zhang, Wan-fu; Wang, Yun-chuan; Tang, Chao-wu; Zhu, Xiong-xiang

2011-01-01

Thermal injury inhibits Akt activation and upregulates p38 mitogen-activated protein kinase, which in turn induces inflammation and increases apoptosis. This study aimed to elucidate the mechanism underlying the cytoprotective role of insulin in severe burns by examining the effects of insulin on inflammation and apoptosis mediated by p38 mitogen-activated protein kinase in burn serum-challenged cardiomyocytes. Neonatal rat cardiomyocytes were exposed to burn serum for 6 hours in the presence or absence of insulin and pretreated with inhibitors to p38 mitogen-activated protein kinase (SB203580) and Akt (LY294002). The authors examined expression of myocardial tumor necrosis factor-alpha, cardiac myofilament proteins caspase-3 and Bcl2, and apoptosis. Burn serum-induced upregulation of tumor necrosis factor was inhibited by both SB203580 and insulin. LY294002 reversed insulin-mediated downregulation of tumor necrosis factor. Both SB203580 and insulin inhibited apoptosis, resulting in fewer pyknotic nuclei and inhibition of caspase-3 activation and Bcl2 downregulation. LY294002 reversed insulin-mediated inhibition of apoptosis. Insulin decreases inflammatory cytokine expression and apoptosis via PI3K/Akt-mediated inhibition of p38 mitogen-activated protein kinase. The cytoprotective role of insulin suggests that it may have a potential role in strategies for treating thermal injuries.

Systematic identification of phosphorylation-mediated protein interaction switches

PubMed Central

Wichmann, Oliver; Utz, Mathias; Andre, Timon; Minguez, Pablo; Parca, Luca; Roth, Frederick P.; Gavin, Anne-Claude; Bork, Peer; Russell, Robert B.

2017-01-01

Proteomics techniques can identify thousands of phosphorylation sites in a single experiment, the majority of which are new and lack precise information about function or molecular mechanism. Here we present a fast method to predict potential phosphorylation switches by mapping phosphorylation sites to protein-protein interactions of known structure and analysing the properties of the protein interface. We predict 1024 sites that could potentially enable or disable particular interactions. We tested a selection of these switches and showed that phosphomimetic mutations indeed affect interactions. We estimate that there are likely thousands of phosphorylation mediated switches yet to be uncovered, even among existing phosphorylation datasets. The results suggest that phosphorylation sites on globular, as distinct from disordered, parts of the proteome frequently function as switches, which might be one of the ancient roles for kinase phosphorylation. PMID:28346509

Tit for tat in heterogeneous populations

NASA Astrophysics Data System (ADS)

Nowak, Martin A.; Sigmund, Karl

1992-01-01

THE 'iterated prisoner's dilemma' is now the orthodox paradigm for the evolution of cooperation among selfish individuals. This viewpoint is strongly supported by Axelrod's computer tournaments, where 'tit for tat' (TFT) finished first1. This has stimulated interest in the role of reciprocity in biological societies1-8. Most theoretical investigations, however, assumed homogeneous populations (the setting for evolutionary stable strategies9,10) and programs immune to errors. Here we try to come closer to the biological situation by following a program6 that takes stochasticities into account and investigates representative samples. We find that a small fraction of TFT players is essential for the emergence of reciprocation in a heterogeneous population, but only paves the way for a more generous strategy. TFT is the pivot, rather than the aim, of an evolution towards cooperation.

Yarrowia lipolytica vesicle-mediated protein transport pathways

PubMed Central

Swennen, Dominique; Beckerich, Jean-Marie

2007-01-01

Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y

A plasma membrane sucrose-binding protein that mediates sucrose uptake shares structural and sequence similarity with seed storage proteins but remains functionally distinct.

PubMed

Overvoorde, P J; Chao, W S; Grimes, H D

1997-06-20

Photoaffinity labeling of a soybean cotyledon membrane fraction identified a sucrose-binding protein (SBP). Subsequent studies have shown that the SBP is a unique plasma membrane protein that mediates the linear uptake of sucrose in the presence of up to 30 mM external sucrose when ectopically expressed in yeast. Analysis of the SBP-deduced amino acid sequence indicates it lacks sequence similarity with other known transport proteins. Data presented here, however, indicate that the SBP shares significant sequence and structural homology with the vicilin-like seed storage proteins that organize into homotrimers. These similarities include a repeated sequence that forms the basis of the reiterated domain structure characteristic of the vicilin-like protein family. In addition, analytical ultracentrifugation and nonreducing SDS-polyacrylamide gel electrophoresis demonstrate that the SBP appears to be organized into oligomeric complexes with a Mr indicative of the existence of SBP homotrimers and homodimers. The structural similarity shared by the SBP and vicilin-like proteins provides a novel framework to explore the mechanistic basis of SBP-mediated sucrose uptake. Expression of the maize Glb protein (a vicilin-like protein closely related to the SBP) in yeast demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake. Thus, despite sequence and structural similarities shared by the SBP and the vicilin-like protein family, the SBP is functionally divergent from other members of this group.

C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.

PubMed

Kapur, Rick; Heitink-Pollé, Katja M J; Porcelijn, Leendert; Bentlage, Arthur E H; Bruin, Marrie C A; Visser, Remco; Roos, Dirk; Schasfoort, Richard B M; de Haas, Masja; van der Schoot, C Ellen; Vidarsson, Gestur

2015-03-12

Immune-mediated platelet destruction is most frequently caused by allo- or autoantibodies via Fcγ receptor-dependent phagocytosis. Disease severity can be predicted neither by antibody isotype nor by titer, indicating that other factors play a role. Here we show that the acute phase protein C-reactive protein (CRP), a ligand for Fc receptors on phagocytes, enhances antibody-mediated platelet destruction by human phagocytes in vitro and in vivo in mice. Without antiplatelet antibodies, CRP was found to be inert toward platelets, but it bound to phosphorylcholine exposed after oxidation triggered by antiplatelet antibodies, thereby enhancing platelet phagocytosis. CRP levels were significantly elevated in patients with allo- and autoantibody-mediated thrombocytopenias compared with healthy controls. Within a week, intravenous immunoglobulin treatment in children with newly diagnosed immune thrombocytopenia led to significant decrease of CRP levels, increased platelet numbers, and clinically decreased bleeding severity. Furthermore, the higher the level of CRP at diagnosis, the longer it took before stable platelet counts were reached. These data suggest that CRP amplifies antibody-mediated platelet destruction and may in part explain the aggravation of thrombocytopenia on infections. Hence, targeting CRP could offer new therapeutic opportunities for these patients. © 2015 by The American Society of Hematology.

Splice-mediated Variants of Proteins (SpliVaP) - data and characterization of changes in signatures among protein isoforms due to alternative splicing.

PubMed

Floris, Matteo; Orsini, Massimiliano; Thanaraj, Thangavel Alphonse

2008-10-02

It is often the case that mammalian genes are alternatively spliced; the resulting alternate transcripts often encode protein isoforms that differ in amino acid sequences. Changes among the protein isoforms can alter the cellular properties of proteins. The effect can range from a subtle modulation to a complete loss of function. (i) We examined human splice-mediated protein isoforms (as extracted from a manually curated data set, and from a computationally predicted data set) for differences in the annotation for protein signatures (Pfam domains and PRINTS fingerprints) and we characterized the differences & their effects on protein functionalities. An important question addressed relates to the extent of protein isoforms that may lack any known function in the cell. (ii) We present a database that reports differences in protein signatures among human splice-mediated protein isoform sequences. (i) Characterization: The work points to distinct sets of alternatively spliced genes with varying degrees of annotation for the splice-mediated protein isoforms. Protein molecular functions seen to be often affected are those that relate to: binding, catalytic, transcription regulation, structural molecule, transporter, motor, and antioxidant; and the processes that are often affected are nucleic acid binding, signal transduction, and protein-protein interactions. Signatures are often included/excluded and truncated in length among protein isoforms; truncation is seen as the predominant type of change. Analysis points to the following novel aspects: (a) Analysis using data from the manually curated Vega indicates that one in 8.9 genes can lead to a protein isoform of no "known" function; and one in 18 expressed protein isoforms can be such an "orphan" isoform; the corresponding numbers as seen with computationally predicted ASD data set are: one in 4.9 genes and one in 9.8 isoforms. (b) When swapping of signatures occurs, it is often between those of same functional

Identification, RNAi Knockdown and Functional Analysis of an Ejaculate Protein that Mediates a Postmating, Prezgotic Phenotype in a cricket

USDA-ARS?s Scientific Manuscript database

Male ejaculate proteins, including both sperm and seminal fluid proteins, play an important role in mediating reproductive biology. The function of ejaculate proteins can include enabling sperm-egg interactions, enhancing sperm storage, mediating female attractiveness, and even regulating female lif...

Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869▿

PubMed Central

Kikuchi, Yoshimi; Date, Masayo; Itaya, Hiroshi; Matsui, Kazuhiko; Wu, Long-Fei

2006-01-01

Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins. PMID:16997984

Overexpression of PLK3 Mediates the Degradation of Abnormal Prion Proteins Dependent on Chaperone-Mediated Autophagy.

PubMed

Wang, Hui; Tian, Chan; Sun, Jing; Chen, Li-Na; Lv, Yan; Yang, Xiao-Dong; Xiao, Kang; Wang, Jing; Chen, Cao; Shi, Qi; Shao, Qi-Xiang; Dong, Xiao-Ping

2017-08-01

Polo-like kinase 3 (PLK3) is the main cause of cell cycle reentry-related neuronal apoptosis which has been implicated in the pathogenesis of prion diseases. Previous work also showed the regulatory activity of exogenous PLK3 on the degradation of PrP (prion protein) mutants and pathogenic PrP Sc ; however, the precise mechanisms remain unknown. In this study, we identified that the overexpression of PLK3-mediated degradation of PrP mutant and PrP Sc was repressed by lysosome rather than by proteasomal and macroautophagy inhibitors. Core components of chaperone-mediated autophagy (CMA) effectors, lysosome-associated membrane protein type 2A (LAMP2a), and heat shock cognate protein 70 (Hsc70) are markedly decreased in the HEK293T cells expressing PrP mutant and scrapie-infected cell line SMB-S15. Meanwhile, PrP mutant showed ability to interact with LAMP2a and Hsc70. Overexpression of PLK3 sufficiently increased the cellular levels of LAMP2a and Hsc70, accompanying with declining the accumulations of PrP mutant and PrP Sc . The kinase domain (KD) of PLK3 was responsible for elevating LAMP2a and Hsc70. Knockdown of endogenous PLK3 enhanced the activity of macroautophagy in the cultured cells. Moreover, time-dependent reductions of LAMP2a and Hsc70 were also observed in the brain tissues of hamster-adapted scrapie agent 263K-infected hamsters, indicating an impairment of CMA during prion infection. Those data indicate that the overexpression of PLK3-mediated degradation of abnormal PrP is largely dependent on CMA pathway.

Quinone-induced protein handling changes: Implications for major protein handling systems in quinone-mediated toxicity

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

Xiong, Rui; Siegel, David; Ross, David, E-mail: david.ross@ucdenver.edu

2014-10-15

Para-quinones such as 1,4-Benzoquinone (BQ) and menadione (MD) and ortho-quinones including the oxidation products of catecholamines, are derived from xenobiotics as well as endogenous molecules. The effects of quinones on major protein handling systems in cells; the 20/26S proteasome, the ER stress response, autophagy, chaperone proteins and aggresome formation, have not been investigated in a systematic manner. Both BQ and aminochrome (AC) inhibited proteasomal activity and activated the ER stress response and autophagy in rat dopaminergic N27 cells. AC also induced aggresome formation while MD had little effect on any protein handling systems in N27 cells. The effect of NQO1more » on quinone induced protein handling changes and toxicity was examined using N27 cells stably transfected with NQO1 to generate an isogenic NQO1-overexpressing line. NQO1 protected against BQ–induced apoptosis but led to a potentiation of AC- and MD-induced apoptosis. Modulation of quinone-induced apoptosis in N27 and NQO1-overexpressing cells correlated only with changes in the ER stress response and not with changes in other protein handling systems. These data suggested that NQO1 modulated the ER stress response to potentiate toxicity of AC and MD, but protected against BQ toxicity. We further demonstrated that NQO1 mediated reduction to unstable hydroquinones and subsequent redox cycling was important for the activation of the ER stress response and toxicity for both AC and MD. In summary, our data demonstrate that quinone-specific changes in protein handling are evident in N27 cells and the induction of the ER stress response is associated with quinone-mediated toxicity. - Highlights: • Unstable hydroquinones contributed to quinone-induced ER stress and toxicity.« less

Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery

PubMed Central

Merino, Felipe; Bouvier, Benjamin; Cojocaru, Vlad

2015-01-01

Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions. PMID:26067358

Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

PubMed

Merino, Felipe; Bouvier, Benjamin; Cojocaru, Vlad

2015-06-01

Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

PubMed

Zhong, Huailing; Wade, Susan M; Woolf, Peter J; Linderman, Jennifer J; Traynor, John R; Neubig, Richard R

2003-02-28

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

Recognition of dengue virus protein using epitope-mediated molecularly imprinted film.

PubMed

Tai, Dar-Fu; Lin, Chung-Yin; Wu, Tzong-Zeng; Chen, Li-Kuang

2005-08-15

Molecularly imprinted film was fabricated in the presence of a pentadecapeptide onto a quartz crystal microbalance (QCM) chip. This 15-mer peptide has been known as the linear epitope of the dengue virus NS1 protein. Imprinting resulted in an increased polymer affinity toward the corresponding templates but also to the virus protein. Direct detection of the dengue virus protein was achieved quantitatively. The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies. The binding effect was further enhanced and confirmed using a monoclonal antibody to form a sandwich with the MIP-NS1 protein complex on the chip. No pretreatment was required.

Roles of Arenavirus Z Protein in Mediating Virion Budding, Viral Transcription-Inhibition and Interferon-Beta Suppression.

PubMed

Shao, Junjie; Liang, Yuying; Ly, Hinh

2018-01-01

The smallest arenaviral protein is the zinc-finger protein (Z) that belongs to the RING finger protein family. Z serves as a main component required for virus budding from the membrane of the infected cells through self-oligomerization, a process that can be aided by the viral nucleoprotein (NP) to form the viral matrix of progeny virus particles. Z has also been shown to be essential for mediating viral transcriptional repression activity by locking the L polymerase onto the viral promoter in a catalytically inactive state, thus limiting viral replication. The Z protein has also recently been shown to inhibit the type I interferon-induction pathway by directly binding to the intracellular pathogen-sensor proteins RIG-I and MDA5, and thus inhibiting their normal functions. This chapter describes several assays used to examine the important roles of the arenaviral Z protein in mediating virus budding (i.e., either Z self-budding or NP-Z budding activities), viral transcriptional inhibition in a viral minigenome (MG) assay, and type I IFN suppression in an IFN-β promoter-mediated luciferase reporter assay.

FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.

PubMed

Morrison, D J; Pendergrast, P S; Stavropoulos, P; Colmenares, S U; Kobayashi, R; Hernandez, N

1999-03-01

The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors.

Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface.

PubMed

Kamiya, Noriho; Doi, Satoshi; Tominaga, Jo; Ichinose, Hirofumi; Goto, Masahiro

2005-01-01

An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.

Cluster Formation of Anchored Proteins Induced by Membrane-Mediated Interaction

PubMed Central

Li, Shuangyang; Zhang, Xianren; Wang, Wenchuan

2010-01-01

Abstract Computer simulations were used to study the cluster formation of anchored proteins in a membrane. The rate and extent of clustering was found to be dependent upon the hydrophobic length of the anchored proteins embedded in the membrane. The cluster formation mechanism of anchored proteins in our work was ascribed to the different local perturbations on the upper and lower monolayers of the membrane and the intermonolayer coupling. Simulation results demonstrated that only when the penetration depth of anchored proteins was larger than half the membrane thickness, could the structure of the lower monolayer be significantly deformed. Additionally, studies on the local structures of membranes indicated weak perturbation of bilayer thickness for a shallowly inserted protein, while there was significant perturbation for a more deeply inserted protein. The origin of membrane-mediated protein-protein interaction is therefore due to the local perturbation of the membrane thickness, and the entropy loss—both of which are caused by the conformation restriction on the lipid chains and the enhanced intermonolayer coupling for a deeply inserted protein. Finally, in this study we addressed the difference of cluster formation mechanisms between anchored proteins and transmembrane proteins. PMID:20513399

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

PubMed Central

2010-01-01

Background Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level. Results A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins. Conclusions Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range. PMID:20569466

Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

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

Heintz, Udo; Meinhart, Anton; Winkler, Andreas, E-mail: andreas.winkler@mpimf-heidelberg.mpg.de

2014-03-01

Crystal structures of two truncated variants of the transcription factor PpsR from R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function. Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteinsmore » are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsR{sub Q-PAS1}) and two (PpsR{sub N-Q-PAS1}) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR{sub ΔHTH}) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR{sub ΔHTH} structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N

Nitrosative Stress and Nitrated Proteins in Trichloroethene-Mediated Autoimmunity

PubMed Central

Wang, Gangduo; Wang, Jianling; Luo, Xuemei; Ansari, G. A. Shakeel; Khan, M. Firoze

2014-01-01

Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, has been linked to a variety of autoimmune diseases (ADs) including SLE, scleroderma and hepatitis. Mechanisms involved in the pathogenesis of ADs are largely unknown. Earlier studies from our laboratory in MRL+/+ mice suggested the contribution of oxidative/nitrosative stress in TCE-induced autoimmunity, and N-acetylcysteine (NAC) supplementation provided protection by attenuating oxidative stress. This study was undertaken to further evaluate the contribution of nitrosative stress in TCE-mediated autoimmunity and to identify proteins susceptible to nitrosative stress. Groups of female MRL +/+ mice were given TCE, NAC or TCE + NAC for 6 weeks (TCE, 10 mmol/kg, i.p., every 4th day; NAC, ∼250 mg/kg/day via drinking water). TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT) in sera and livers. Proteomic analysis identified 14 additional nitrated proteins in the livers of TCE-treated mice. Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-κB. Remarkably, NAC supplementation not only ameliorated TCE-induced nitrosative stress as evident from decreased iNOS, NT, nitrated proteins, NF-κB p65 activation and increased GSH levels, but also the markers of autoimmunity, as evident from decreased levels of autoantibodies in the sera. These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for designing therapeutic strategies. PMID:24892995

Armet is an effector protein mediating aphid-plant interactions.

PubMed

Wang, Wei; Dai, Huaien; Zhang, Yi; Chandrasekar, Raman; Luo, Lan; Hiromasa, Yasuaki; Sheng, Changzhong; Peng, Gongxin; Chen, Shaoliang; Tomich, John M; Reese, John; Edwards, Owain; Kang, Le; Reeck, Gerald; Cui, Feng

2015-05-01

Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding. Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet, namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armet's physical and chemical properties and its intracellular role are comparable to those reported for mammalian Armets. Uniquely, we detected Armet in aphid watery saliva and in the phloem sap of fava beans fed on by aphids. Armet's transcript level is several times higher in the salivary gland when aphids feed on bean plants than when they feed on an artificial diet. Knockdown of the Armet transcript by RNA interference disturbs aphid feeding behavior on fava beans measured by the electrical penetration graph technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves induced a transcriptional response that included pathogen-responsive genes. The data suggest that Armet is an effector protein mediating aphid-plant interactions. © FASEB.

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

PubMed

Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

2014-08-03

Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

An Auxilin-Like J-Domain Protein, JAC1, Regulates Phototropin-Mediated Chloroplast Movement in Arabidopsis1[w

PubMed Central

Suetsugu, Noriyuki; Kagawa, Takatoshi; Wada, Masamitsu

2005-01-01

The ambient-light conditions mediate chloroplast relocation in plant cells. Under the low-light conditions, chloroplasts accumulate in the light (accumulation response), while under the high-light conditions, they avoid the light (avoidance response). In Arabidopsis (Arabidopsis thaliana), the accumulation response is mediated by two blue-light receptors, termed phototropins (phot1 and phot2) that act redundantly, and the avoidance response is mediated by phot2 alone. A mutant, J-domain protein required for chloroplast accumulation response 1 (jac1), lacks the accumulation response under weak blue light but shows a normal avoidance response under strong blue light. In dark-adapted wild-type cells, chloroplasts accumulate on the bottom of cells. Both the jac1 and phot2 mutants are defective in this chloroplast movement in darkness. Positional cloning of JAC1 reveals that this gene encodes a J-domain protein, resembling clathrin-uncoating factor auxilin at its C terminus. The amounts of JAC1 transcripts and JAC1 proteins are not regulated by light and by phototropins. A green fluorescent protein-JAC1 fusion protein showed a similar localization pattern to green fluorescent protein alone in a transient expression assay using Arabidopsis mesophyll cells and onion (Allium cepa) epidermal cells, suggesting that the JAC1 protein may be a soluble cytosolic protein. Together, these results suggest that JAC1 is an essential component of phototropin-mediated chloroplast movement. PMID:16113208

Prediction of the translocon-mediated membrane insertion free energies of protein sequences.

PubMed

Park, Yungki; Helms, Volkhard

2008-05-15

Helical membrane proteins (HMPs) play crucial roles in a variety of cellular processes. Unlike water-soluble proteins, HMPs need not only to fold but also get inserted into the membrane to be fully functional. This process of membrane insertion is mediated by the translocon complex. Thus, it is of great interest to develop computational methods for predicting the translocon-mediated membrane insertion free energies of protein sequences. We have developed Membrane Insertion (MINS), a novel sequence-based computational method for predicting the membrane insertion free energies of protein sequences. A benchmark test gives a correlation coefficient of 0.74 between predicted and observed free energies for 357 known cases, which corresponds to a mean unsigned error of 0.41 kcal/mol. These results are significantly better than those obtained by traditional hydropathy analysis. Moreover, the ability of MINS to reasonably predict membrane insertion free energies of protein sequences allows for effective identification of transmembrane (TM) segments. Subsequently, MINS was applied to predict the membrane insertion free energies of 316 TM segments found in known structures. An in-depth analysis of the predicted free energies reveals a number of interesting findings about the biogenesis and structural stability of HMPs. A web server for MINS is available at http://service.bioinformatik.uni-saarland.de/mins

The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study

PubMed Central

2014-01-01

Background Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination. Findings Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization. Conclusions Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy. PMID:24961156

Proteins mediating DNA loops effectively block transcription.

PubMed

Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

2017-07-01

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Predicting PDZ domain mediated protein interactions from structure

PubMed Central

2013-01-01

Background PDZ domains are structural protein domains that recognize simple linear amino acid motifs, often at protein C-termini, and mediate protein-protein interactions (PPIs) in important biological processes, such as ion channel regulation, cell polarity and neural development. PDZ domain-peptide interaction predictors have been developed based on domain and peptide sequence information. Since domain structure is known to influence binding specificity, we hypothesized that structural information could be used to predict new interactions compared to sequence-based predictors. Results We developed a novel computational predictor of PDZ domain and C-terminal peptide interactions using a support vector machine trained with PDZ domain structure and peptide sequence information. Performance was estimated using extensive cross validation testing. We used the structure-based predictor to scan the human proteome for ligands of 218 PDZ domains and show that the predictions correspond to known PDZ domain-peptide interactions and PPIs in curated databases. The structure-based predictor is complementary to the sequence-based predictor, finding unique known and novel PPIs, and is less dependent on training–testing domain sequence similarity. We used a functional enrichment analysis of our hits to create a predicted map of PDZ domain biology. This map highlights PDZ domain involvement in diverse biological processes, some only found by the structure-based predictor. Based on this analysis, we predict novel PDZ domain involvement in xenobiotic metabolism and suggest new interactions for other processes including wound healing and Wnt signalling. Conclusions We built a structure-based predictor of PDZ domain-peptide interactions, which can be used to scan C-terminal proteomes for PDZ interactions. We also show that the structure-based predictor finds many known PDZ mediated PPIs in human that were not found by our previous sequence-based predictor and is less dependent on

All-trans-retinoic acid inhibits collapsin response mediator protein-2 transcriptional activity during SH-SY5Y neuroblastoma cell differentiation.

PubMed

Fontán-Gabás, Lorena; Oliemuller, Erik; Martínez-Irujo, Juan José; de Miguel, Carlos; Rouzaut, Ana

2007-01-01

Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro-oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein-2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein-2 transcriptional regulation during all-trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells. All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein-2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein-2 basal promoter activity is regulated by the transcription factors AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus sequences in the collapsin response mediator protein-2 promoter. Decreased attachment of AP-2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-retinoic acid-mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all-trans-retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application.