RESUMO
BACKGROUND: Students from a range of health disciplines need to learn from people with lived experience of mental distress and recovery to develop recovery capabilities for mental health practice. AIMS: The aims of this study are to describe the co-design of a teaching resource, to explore the experience of people with lived experience during the resource development, and to evaluate the outcome of the resource on student recovery capabilities. METHOD: Using a sequential mixed method, a project group consisting of six people with lived experience and 10 academics from five health disciplines was convened to co-develop teaching resources. People with lived experience met independently without researchers on several occasions to decide on the key topics and met with the research team monthly. The teaching resource was used in mental health subjects for two health professional programmes, and the Capabilities for Recovery-Oriented Practice Questionnaire (CROP-Q) was used before and after to measure any change in student recovery capabilities. Scores were compared using the Wilcoxon signed rank test. The people with lived experience were also interviewed about their experience of being involved in constructing the teaching resources. Interviews were audiotaped, transcribed, and analysed thematically. RESULTS: The finished resource consisted of 28 short videos and suggested teaching plans. Occupational therapy and nursing student scores on the CROP-Q prior to using the educational resource (n = 33) were 68 (median) and post scores (n = 28) were 74 (median), indicating a statistically significant improvement in recovery capability (P = 0.04). Lived experience interview themes were (i) the importance of lived experience in education; (ii) personal benefits of participating; (iii) co-design experience; and (iv) creating the resource. CONCLUSION: Co-design of teaching resources with people with lived experience was pivotal to the success and quality of the final product, and people with lived experience described personal benefits of participating in resource development. More evidence to demonstrate the use of the CROP-Q in teaching and practice is needed.
Assuntos
Transtornos Mentais , Recuperação da Saúde Mental , Terapia Ocupacional , Humanos , Estudantes , Transtornos Mentais/psicologia , Saúde MentalRESUMO
A range of social and structural barriers continue to impede timely diagnosis and consistent access to care for Latinos living with HIV in the U.S. Navigation programs have helped other populations overcome comparable barriers to care. This qualitative paper examined nine navigation programs that were culturally tailored for Mexicans or Puerto Ricans, using a transnational framework that situated clients in the context of lives that bridge the U.S. and their countries of origin. We completed in-depth semi-structured interviews with 48 clients and 27 intervention providers. A framework approach guided analysis. We identified two overarching themes: developing trusting and supportive relationships between navigators and clients and empowering clients to connect and stay in primary care, which summarized the impact of the interventions on participants' lives and the approaches used to increase their care engagement. Our findings highlight the importance of tailoring intervention strategies to the unique experiences of specific Latino populations.
Assuntos
Infecções por HIV/etnologia , Hispânico ou Latino , Navegação de Pacientes/métodos , Adulto , Emigrantes e Imigrantes , Feminino , Infecções por HIV/terapia , Humanos , Entrevistas como Assunto , Masculino , México/etnologia , Navegação de Pacientes/organização & administração , Participação do Paciente/métodos , Porto Rico/etnologia , Estados UnidosRESUMO
Interventions aiming to improve access to and retention in HIV care are optimized when they are tailored to clients' needs. This paper describes an initiative of interventions implemented by ten demonstration sites using a transnational framework to tailor services for Mexicans and Puerto Ricans living with HIV. Transnationalism describes how immigrants (and their children) exist in their "receiving" place (e.g., continental U.S.) while simultaneously maintaining connections to their country or place of origin (e.g., Mexico). We describe interventions in terms of the strategies used, the theory informing design and the tailoring, and the integration of transnationalism. We argue how applying the transnational framework may improve the quality and effectiveness of services in response to the initiative's overall goal, which is to produce innovative, robust, evidence-informed strategies that go beyond traditional tailoring approaches for HIV interventions with Latino/as populations.
Assuntos
Assistência à Saúde Culturalmente Competente/organização & administração , Emigrantes e Imigrantes/estatística & dados numéricos , Infecções por HIV/prevenção & controle , Comportamentos Relacionados com a Saúde/etnologia , Hispânico ou Latino/estatística & dados numéricos , Aceitação pelo Paciente de Cuidados de Saúde/etnologia , Serviços de Saúde Comunitária/organização & administração , Aconselhamento , Feminino , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Humanos , MasculinoRESUMO
BACKGROUND: Electroconvulsive therapy (ECT) is one of the most controversial treatments in psychiatry. This controversy and diverse and often strongly held opinions can make decision making processes around ECT more complex. METHOD: This consumer-led project explored the experiences of individuals who had received ECT in terms of the information they received, their experience of ECT and suggestions for ways that decision making processes and experiences of ECT can be improved. Interviews were conducted by consumer researchers who had also received ECT and transcripts were analysed using constant comparative techniques. RESULTS: Seventeen individuals participated. Four overarching categories were identified from participant interviews: Information matters; Preparation and decisions before ECT; Experience of ECT; and Suggestions for improvement. Most participants suggested that more information was required and that this information should be made available more regularly to support decision making. Additional suggestions included greater involvement of family and friends (including having a family member or friend present during the ECT procedure), opportunities to gain information from individuals who had received ECT and more support for managing memory and cognitive side effects. CONCLUSION: This study provides valuable consumer-provided insights and recommendations for psychiatrists and mental health clinicians working within ECT clinics and with consumers considering or preparing for ECT.
Assuntos
Tomada de Decisões , Eletroconvulsoterapia/psicologia , Aceitação pelo Paciente de Cuidados de Saúde/psicologia , Adulto , Feminino , Humanos , Masculino , Educação de Pacientes como Assunto , Satisfação do Paciente , Pesquisa QualitativaRESUMO
The Vif protein of human immunodeficiency virus-1 (HIV-1) interacts with members of the APOBEC family of cytidine deaminases. In this study, we isolated RNA from renal cortex as well as from isolated glomeruli and tubulointerstitial fractions from two pigtailed macaques that were exsanguinated and perfused with saline. RT-PCR results indicate that APOBEC3G was detected in the tubule fractions but not in the glomerular fractions. Immunoblot analysis using lysates prepared from these same fractions and a monoclonal antibody to APOBEC3G confirmed the RT-PCR findings. To determine which cell types express APOBEC3G, immunohistochemical studies were performed using this monoclonal antibody on renal cortical sections. Our results clearly show that the glomeruli do not express APOBEC3G but that select tubules within the cortex express APOBEC3G at high levels. To further differentiate the distribution of APOBEC3G expression, serial sections were stained with the lectins Dolichos biflorus agglutinin (DBA) and Phaseolus vulgaris erythroagglutinin (PHA-E), which differentially bind to epithelial cells of the tubules and glomeruli. Our results indicate that APOBEC3G expression was restricted to PHA-E-staining tubules and not DBA-staining tubules, suggesting that APOBEC3G expression was restricted to proximal convoluted tubules. These findings suggest that infection of epithelial cells of proximal renal tubules could suppress Vif-defective HIV-1 replication, whereas infection of cells of the glomeruli, a major target of HIV-associated nephropathy, could act as a reservoir for the replication of Vif-defective HIV-1.
Assuntos
Citidina Desaminase/biossíntese , Células Epiteliais/enzimologia , Glomérulos Renais/enzimologia , Túbulos Renais/enzimologia , Animais , Citidina Desaminase/genética , Immunoblotting , Imuno-Histoquímica , Túbulos Renais/citologia , Macaca nemestrina , Fito-Hemaglutininas , Lectinas de Plantas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Central to organization of signaling pathways are scaffolding, anchoring and adaptor proteins that mediate localized assembly of multi-protein complexes containing receptors, second messenger-generating enzymes, kinases, phosphatases, and substrates. At the postsynaptic density (PSD) of excitatory synapses, AMPA (AMPAR) and NMDA (NMDAR) glutamate receptors are linked to signaling proteins, the actin cytoskeleton, and synaptic adhesion molecules on dendritic spines through a network of scaffolding proteins that may play important roles regulating synaptic structure and receptor functions in synaptic plasticity underlying learning and memory. AMPARs are rapidly recruited to dendritic spines through NMDAR activation during induction of long-term potentiation (LTP) through pathways that also increase the size and F-actin content of spines. Phosphorylation of AMPAR-GluR1 subunits by the cAMP-dependent protein kinase (PKA) helps stabilize AMPARs recruited during LTP. In contrast, induction of long-term depression (LTD) leads to rapid calcineurin-protein phosphatase 2B (CaN) mediated dephosphorylation of PKA-phosphorylated GluR1 receptors, endocytic removal of AMPAR from synapses, and a reduction in spine size. However, mechanisms for coordinately regulating AMPAR localization, phosphorylation, and synaptic structure by PKA and CaN are not well understood. A kinase-anchoring protein (AKAP) 79/150 is a PKA- and CaN-anchoring protein that is linked to NMDARs and AMPARs through PSD-95 and SAP97 membrane-associated guanylate kinase (MAGUK) scaffolds. Importantly, disruption of PKA-anchoring in neurons and functional analysis of GluR1-MAGUK-AKAP79 complexes in heterologous cells suggests that AKAP79/150-anchored PKA and CaN may regulate AMPARs in LTD. In the work presented at the "First International Meeting on Anchored cAMP Signaling Pathways" (Berlin-Buch, Germany, October 15-16, 2005), we demonstrate that AKAP79/150 is targeted to dendritic spines by an N-terminal basic region that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), F-actin, and actin-linked cadherin adhesion molecules. Thus, anchoring of PKA and CaN as well as physical linkage of the AKAP to both cadherin-cytoskeletal and MAGUK-receptor complexes could play roles in coordinating changes in synaptic structure and receptor signaling functions underlying plasticity. Importantly, we provide evidence showing that NMDAR-CaN signaling pathways implicated in AMPAR regulation during LTD lead to a disruption of AKAP79/150 interactions with actin, MAGUKs, and cadherins and lead to a loss of the AKAP and anchored PKA from postsynapses. Our studies thus far indicate that this AKAP79/150 translocation depends on activation of CaN, F-actin reorganization, and possibly Ca(2+)-CaM binding to the N-terminal basic regions. Importantly, this tranlocation of the AKAP79/150-PKA complex from spines may shift the balance of PKA kinase and CaN/PP1 phosphatase activity at the postsynapse in favor of the phosphatases. This loss of PKA could then promote actions of CaN and PP1 during induction of LTD including maintaining AMPAR dephosphorylation, promoting AMPAR endocytosis, and preventing AMPAR recycling. Overall, these findings challenge the accepted notion that AKAPs are static anchors that position signaling proteins near fixed target substrates and instead suggest that AKAPs can function in more dynamic manners to regulate local signaling events.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Proteínas de Ancoragem à Quinase A , Actinas/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Humanos , Potenciação de Longa Duração , Depressão Sináptica de Longo Prazo , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Neurotoxinas/efeitos adversos , Proteínas Associadas à Matriz Nuclear/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Distribuição TecidualRESUMO
Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV(KU-1bMC33) in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV(M2)) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV(KU-1bMC33)) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV(VpuA19H) replicated with similar kinetics as the parental SHIV(KU-1bMC33) and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV(KU-1bMC33). This SHIV(VpuA19H) virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV(M2). Electron microscopic examination of SHIV(VpuA19H)-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV(M2)-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the alteration of one amino acid and provide additional evidence that drugs targeting the Vpu TM/ion channel can be effective anti-HIV-1 drugs.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Rimantadina/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico Ativo , Antígenos CD4/metabolismo , DNA Viral/genética , Farmacorresistência Viral/genética , Genes vpu , HIV-1/química , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Hibridização Genética , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Vírus da Imunodeficiência Símia/química , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
The Vpu protein of human immunodeficiency virus type 1 has been shown to shunt the CD4 receptor molecule to the proteasome for degradation and to enhance virus release from infected cells. The exact mechanism by which the Vpu protein enhances virus release is currently unknown but some investigators have shown that this function is associated with the transmembrane domain and potential ion channel properties. In this study, we determined if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A virus. We constructed chimeric vpu gene in which the transmembrane domain of Vpu was replaced with that of the M2 protein of influenza. This chimeric vpu gene was substituted for the vpu gene in the genome of a pathogenic simian human immunodeficiency virus, SHIVKU-1bMC33. The resulting virus, SHIVM2, synthesized a Vpu protein that had a slightly different Mr compared to the parental SHIVKU-1bMC33, reflecting the different sizes of the two Vpu proteins. The SHIVM2 was shown to replicate with slightly reduced kinetics when compared to the parental SHIVKU-1bMC33 but electron microscopy revealed that the site of maturation was similar to the parental virus SHIVKU1bMC33. We show that the replication and spread of SHIVM2 could be blocked with the antiviral drug rimantadine, which is known to target the M2 ion channel. Our results indicate a dose dependent inhibition of SHIVM2 with 100 microM rimantadine resulting in a >95% decrease in p27 released into the culture medium. Rimantadine did not affect the replication of the parental SHIVKU-1bMC33. Examination of SHIVM2-infected cells treated with 50 microM rimantadine revealed numerous viral particles associated with the cell plasma membrane and within intracytoplasmic vesicles, which is similar to HIV-1 mutants lacking a functional vpu. To determine if SHIVM2 was as pathogenic as the parental SHIVKU-1bMC33 virus, two pig-tailed macaques were inoculated and followed for up to 8 months. Both pig-tailed macaques developed severe CD4+ T cell loss within 1 month of inoculation, high viral loads, and histological lesions consistent with lymphoid depletion similar to the parental SHIVKU-1bMC33. Taken together, these results indicate for the first time that the TM domain of the Vpu protein can be functionally substituted with the TM of M2 of influenza A virus, and shows that compounds that target the TM domain of Vpu protein of HIV-1 could serve as novel anti-HIV-1 drugs.
Assuntos
Macaca nemestrina/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/química , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD4/metabolismo , Linhagem Celular , Regulação Viral da Expressão Gênica , Proteínas do Vírus da Imunodeficiência Humana , Linfócitos/ultraestrutura , Linfócitos/virologia , Dados de Sequência Molecular , Engenharia de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , RNA Viral/sangue , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Imunodeficiência Símia/genética , Carga Viral , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
The structure of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) is composed of a short Nterminal domain (NTD), a transmembrane domain (TM), and a cytoplasmic domain (CD). Previous studies have shown that the Vpu protein from subtype B HIV-1 is transported predominantly to the rough endoplasmic reticulum (RER)/Golgi complex compartments of the cell and is not incorporated into virions. Using a previously described VpuEGFP reporter system in which the Vpu protein was fused to the gene for enhanced green fluorescent protein (EGFP), we showed that the subtype B Vpu fusion protein was localized to the RER/Golgi region of the cell, similar to the native protein. In the present study, we show that fusion of the subtype C Vpu to EGFP results in a fusion protein that is transported to the cell surface. Using this reporter system, chimeric Vpu proteins in which the CD of the subtype B and C proteins were exchanged showed that the CD was sufficient for targeting the subtype B protein to the Golgi complex of the cell. Following identification of the cytoplasmic domain as being responsible for intracellular targeting, we then generated a series of mutants in which 13, 23, 31, 38, 51, and 56 amino acids were deleted from the cytoplasmic domain of subtype B Vpu. These deletion mutants were analyzed by SDS-PAGE for size, for membrane localization, and intracellular localization by confocal fluorescence microscopy. Our results indicate that the mutant with the carboxyl-terminal 13 amino acids deleted was still localized to the Golgi complex but mutants with 23, 31, 38, 51, and 56 amino acids from the carboxyl-terminus of the subtype B Vpu were transported to the cell surface. These results suggest that a signal for the retention of the subtype B Vpu within the Golgi complex resides in the second alpha-helical domain.
Assuntos
Regulação Viral da Expressão Gênica , Complexo de Golgi/metabolismo , HIV-1/classificação , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Sequência de Aminoácidos , Linhagem Celular , Deleção de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
A-kinase-anchoring protein (AKAP) 79/150 organizes a scaffold of cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and protein phosphatase 2B/calcineurin that regulates phosphorylation pathways underlying neuronal long-term potentiation and long-term depression (LTD) synaptic plasticity. AKAP79/150 postsynaptic targeting requires three N-terminal basic domains that bind F-actin and acidic phospholipids. Here, we report a novel interaction of these domains with cadherin adhesion molecules that are linked to actin through beta-catenin (beta-cat) at neuronal synapses and epithelial adherens junctions. Mapping the AKAP binding site in cadherins identified overlap with beta-cat binding; however, no competition between AKAP and beta-cat binding to cadherins was detected in vitro. Accordingly, AKAP79/150 exhibited polarized localization with beta-cat and cadherins in epithelial cell lateral membranes, and beta-cat was present in AKAP-cadherin complexes isolated from epithelial cells, cultured neurons, and rat brain synaptic membranes. Inhibition of epithelial cell cadherin adhesion and actin polymerization redistributed intact AKAP-cadherin complexes from lateral membranes to intracellular compartments. In contrast, stimulation of neuronal pathways implicated in LTD that depolymerize postsynaptic F-actin disrupted AKAP-cadherin interactions and resulted in loss of the AKAP, but not cadherins, from synapses. This neuronal regulation of AKAP79/150 targeting to cadherins may be important in functional and structural synaptic modifications underlying plasticity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caderinas/metabolismo , Células Epiteliais/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Proteínas de Ancoragem à Quinase A , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sítios de Ligação , Calcineurina/metabolismo , Adesão Celular , Polaridade Celular , Células Cultivadas , Cães , Células Epiteliais/citologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Neurônios/citologia , Ligação Proteica , Ratos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIVKU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIVTM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (VpuTMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIVTM, three pig-tailed macaques were inoculated with the SHIVTM and monitored for 6-8 months for CD4+ T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIVKU-1bMC33, inoculation of macaques with SHIVTM did not cause a severe CD4+ T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIVKU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIVKU-1bMC33 in pig-tailed macaques.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , HIV-1/genética , Vírus Reordenados/fisiologia , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas Virais Reguladoras e Acessórias/fisiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Regulação para Baixo , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Linfonodos/virologia , Macaca nemestrina , Estrutura Terciária de Proteína/fisiologia , Vírus Reordenados/patogenicidade , Vírus da Imunodeficiência Símia/genética , Virulência , Replicação ViralRESUMO
Human immunodeficiency virus type 1 (HIV-1) along with simian immunodeficiency viruses from chimpanzees (SIV(cpz)) and three species of Old World monkeys from the genus Cercopithecus have been shown to encode a Vpu protein. To date, the functional characterization of Vpu has been limited to a small number of subtype B and more recently subtype C Vpu proteins. Using a recently developed VpuEGFP reporter system, we have shown that the subtype B and C Vpus are capable of preventing CD4 from being expressed on the cell surface. Using the same reporter system, we report here on the expression and functional analysis of Vpu protein from four SIV(cpz) isolates (CAM13, ANT, TAN1, and GAB1). All four SIV Vpu fusion proteins were efficiently expressed and prevented CD4 expression on the cell surface and induced CD4 degradation. This was surprising as three of the SIV(cpz) Vpu fusion proteins had only one canonical casein kinase II (CK-II) site (CAM13, ANT, TAN1) while previous studies with laboratory adapted HXB2 had indicated that both CK-II sites were required for CD4 degradation. Both ANT and TAN1 Vpu sequences encoded five consecutive negatively charged amino acids residues following the only CKII site (SAIEEDEE for ANT; SGVEEDEE for TAN1). We thus explored the possibility that this stretch of negatively charged amino acids might substitute for the lack of second CK-II site. Substitution of the aspartic acid at position 61 and glutamic acid at position 63 in the SIV(cpz) ANT Vpu within with lysine residues abolished the ability of this protein to down-modulate cell surface expression of CD4. Similarly, change of a serine to an alanine residue following the single consensus CK-II site of the CAM13 Vpu (SGNESDGGEEE) abolished CD4-down-regulation, suggesting that this serine was phosphorylated in the absence of a canonical CK-II site. Our results indicate that the serine was required, suggesting that this serine was phosphorylated by CK-II or possibly another cellular kinase. Taken together, these results show for the first time that Vpu proteins from SIV(cpz) isolates, although quite diverse in sequence and predicted secondary structure from the HIV-1 subtype B protein, are capable of down-regulating CD4, which is one of the major functions of the HIV-1 protein.
Assuntos
Antígenos CD4/metabolismo , Regulação para Baixo , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pan troglodytes/virologia , Filogenia , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (alpha/beta), whereas three gene families (B, B', and B'') with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aalpha subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aalpha RNAi decreased total PP2A activity as well as protein levels of C, B, and B' but not B'' subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aalpha triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aalpha cDNAs rescued RNAi-induced loss of the C subunit, and Aalpha point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Abeta and Aalpha mutants capable of binding to at least one family of regulatory subunits were able to delay Aalpha RNAi-induced death of PC12 cells. However, only the expression of wild-type Aalpha restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aalpha subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.
Assuntos
Sobrevivência Celular , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Estrutura Quaternária de Proteína , Animais , Apoptose/fisiologia , Humanos , Células PC12 , Fosfoproteínas Fosfatases/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteína Fosfatase 2 , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Interferência de RNA , Ratos , Transdução de Sinais/fisiologiaRESUMO
The Vpu protein is the smallest of the proteins encoded by human immunodeficiency virus type 1 (HIV-1). This transmembrane protein interacts with the CD4 molecule in the rough endoplasmic reticulum (RER), resulting in its degradation via the proteasome pathway. Vpu also has been shown to enhance virion release from infected cells. While much has been learned about the function of Vpu in cell culture systems, its exact role in HIV-1 pathogenesis is still unknown. This has been primarily due to the lack of a suitable primate model system since vpu is found only in HIV-1 and simian immunodeficiency viruses isolated from chimpanzees (SIVcpz), and three species of old world monkeys within the genus Cercopithecus. Several laboratories have developed pathogenic molecular clones of simian-human immunodeficiency virus (SHIV) in which the tat, rev, vpu and env genes of HIV-1 are expressed in the genetic background of SIV. The availability of such clones has allowed investigators to assess the role of Vpu in pathogenesis using a relevant animal model. This review will focus on the current understanding of the structure-function relationships of Vpu protein and recent advances using the SHIV model to assess the role of Vpu in HIV-1 pathogenesis.
Assuntos
Genes vpu/fisiologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Proteínas Virais Reguladoras e Acessórias/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD4/imunologia , Membrana Celular/virologia , Modelos Animais de Doenças , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Macaca , Dados de Sequência Molecular , Vírus Reordenados , Alinhamento de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/imunologia , Virulência , Replicação ViralRESUMO
Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5' to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIV(Vpenv)) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIV(Vpenv)-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV(Vpenv)-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIV(KU-1bMC33). Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIV(Vpenv)-infected cells compared to cultures inoculated with parental SHIV(KU-1bMC33). Furthermore, virus was observed maturing into intracellular vesicles of SHIV(Vpenv)-infected cells. To assess the pathogenicity of SHIV(Vpenv), three pig-tailed macaques were inoculated with the SHIV(Vpenv) and monitored for 6 months for CD4(+) T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIV(Vpenv) caused a severe CD4(+) T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4(+) T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIV(Vpenv) with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.
Assuntos
Produtos do Gene env/metabolismo , HIV-1/patogenicidade , Precursores de Proteínas/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas Virais Reguladoras e Acessórias/metabolismo , Vírion/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Proteína gp160 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Linfócitos/virologia , Macaca/virologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Calcineurina/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Ancoragem à Quinase A , Animais , Células COS , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Transferência de Energia , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
At the postsynaptic membrane of glutamatergic synapses, the cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and calcineurin (CaN) anchoring protein AKAP79/150 is recruited to NMDA and AMPA glutamate receptors by postsynaptic density (PSD)-95 family membrane-associated guanylate kinase (MAGUK) scaffold proteins. These signaling scaffold complexes may function to regulate receptor phosphorylation in synaptic plasticity. Thus, it is important to understand regulation of AKAP79/150 targeting to synapses and recruitment to PSD-MAGUK complexes. AKAP79 is targeted to the plasma membrane by an N-terminal basic domain that binds phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) and is regulated by PKC phosphorylation and calmodulin binding. Here we demonstrate that this same domain also binds F-actin in a calmodulin- and PKC-regulated manner, targets to membrane ruffles enriched in F-actin and PI-4,5-P(2) in COS7 cells, and localizes to dendritic spines with F-actin and PSD-MAGUKs in hippocampal neurons. Inhibition of actin polymerization disrupted AKAP79 targeting of PKA and CaN to ruffles in COS7 cells and endogenous AKAP79/150 dendritic spine localization with PKA, CaN, and PSD-MAGUKs in neurons. AKAP79/150 postsynaptic localization was rapidly regulated by NMDA receptors through CaN activation and F-actin remodeling, further suggesting that AKAP79/150 signaling scaffold targeting depends on actin dynamics. NMDA receptor activation also regulated dendritic spine localization of PKA and CaN and association of the AKAP79/150-PKA complex with PSD-MAGUKs. Because AMPA receptor PKA phosphorylation and synaptic localization are regulated by similar NMDA receptor-CaN signaling pathways linked to hippocampal long-term depression, this regulation of AKAP79/150 postsynaptic targeting might be important for synaptic plasticity.