RESUMO
Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.
Assuntos
Proteína Fosfatase 2 , Proteína Fosfatase 2/metabolismo , Jordânia , Fosforilação , Mutação , Holoenzimas/genética , Holoenzimas/metabolismoRESUMO
Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for â¼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.
Assuntos
Anormalidades Múltiplas , Humanos , Transtorno do Espectro Autista , Células HEK293 , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteômica , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologiaRESUMO
OBJECTIVE: We have previously shown that transient coronary artery occlusion stimulated coronary collateral growth (CCG) in healthy (Sprague Dawley) but not in metabolic syndrome (JCR:LA-cp [JCR] ) rats. Here, we sought to determine whether matrix metalloproteinases (MMPs) negatively regulate CCG in the metabolic syndrome via release of endostatin and angiostatin. APPROACH AND RESULTS: Rats underwent transient, repetitive left anterior descending occlusion and resultant myocardial ischemia (RI) for 0 to 10 days. CCG was measured in the collateral-dependent and normal zones using microspheres, MMP activation by Western blot, and endostatin and angiostatin by ELISA on days 0, 3, 6, 9, or 10 of RI. Endostatin and angiostatin were increased in JCR but not in Sprague Dawley rats on days 6 and 9 of RI. Increased endostatin and angiostatin correlated with increased MMP12 (≈ 4-fold) activation in JCR but not in Sprague Dawley rats on days 6 and 9 of RI. Inhibition of MMP12 in JCR rats nearly completely blocked endostatin (≈ 85%) and angiostatin (≈ 90%) generation and significantly improved CCG (collateral-dependent zone flow was ≈ 66% of normal zone flow versus ≈ 12% for JCR RI). CONCLUSIONS: Compromised CCG in the metabolic syndrome is, in large part, because of increased MMP12 activation and consequent increased generation of endostatin and angiostatin, which inhibits late-stage collateral remodeling.
Assuntos
Angiostatinas/metabolismo , Circulação Colateral/fisiologia , Oclusão Coronária/metabolismo , Endostatinas/metabolismo , Metaloproteinase 12 da Matriz/metabolismo , Síndrome Metabólica/metabolismo , Angiostatinas/análise , Animais , Western Blotting , Circulação Coronária/fisiologia , Oclusão Coronária/fisiopatologia , Modelos Animais de Doenças , Endostatinas/análise , Ensaio de Imunoadsorção Enzimática , Síndrome Metabólica/fisiopatologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de ReferênciaRESUMO
OBJECTIVE: Transient, repetitive occlusion stimulates coronary collateral growth (CCG) in normal animals. Vascular smooth muscle cells (VSMCs) switch to synthetic phenotype early in CCG, then return to contractile phenotype. CCG is impaired in the metabolic syndrome. We determined whether impaired CCG was attributable to aberrant VSMC phenotypic modulation by miR-145-mediated mechanisms, and whether restoration of physiological miR-145 levels in metabolic syndrome (JCR rat) improved CCG. APPROACH AND RESULTS: CCG was stimulated by transient, repetitive left anterior descending artery occlusion and evaluated after 9 days by coronary blood flow measurements (microspheres). miR-145 was delivered to JCR VSMCs via adenoviral vector (miR-145-Adv). In JCR rats, miR-145 was decreased late in CCG (≈ 2-fold day 6; ≈ 4-fold day 9 versus SD), which correlated with decreased expression of smooth muscle-specific contractile proteins (≈ 5-fold day 6; ≈ 10-fold day 9 versus SD), indicative of VSMCs' failure to return to the contractile phenotype late in CCG. miR-145 expression in JCR rats (miR-145-Adv) on days 6 to 9 of CCG completely restored VSMCs contractile phenotype and CCG (collateral/normal zone flow ratio was 0.93 ± 0.09 JCR+miR-145-Adv versus 0.12 ± 0.02 JCR versus 0.87 ± 0.02 SD). CONCLUSIONS: Restoration of VSMC contractile phenotype through miR-145 delivery is a highly promising intervention for restoration of CCG in the metabolic syndrome.
Assuntos
Circulação Colateral , Circulação Coronária , Terapia Genética , Síndrome Metabólica/terapia , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição , Adenoviridae/genética , Animais , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Síndrome Metabólica/fisiopatologia , Proteínas Musculares/biossíntese , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Fenótipo , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Ratos Zucker , Fatores de TempoRESUMO
Argonaute (Ago) plays a central role in RNA interference in metazoans, but its status in lower organisms remains ill-defined. We report on the Ago complex of the unicellular protozoan, Toxoplasma gondii (Tg), an obligatory pathogen of mammalian hosts. The PIWI-like domain of TgAgo lacked the canonical DDE/H catalytic triad, explaining its weak target RNA cleavage activity. However, TgAgo associated with a stronger RNA slicer, a Tudor staphylococcal nuclease (TSN), and with a protein Arg methyl transferase, PRMT1. Mutational analysis suggested that the N-terminal RGG-repeat domain of TgAgo was methylated by PRMT1, correlating with the recruitment of TSN. The slicer activity of TgAgo was Mg(2+)-dependent and required perfect complementarity between the guide RNA and the target. In contrast, the TSN activity was Ca(2+) -dependent and required an imperfectly paired guide RNA. Ago knockout parasites showed essentially normal growth, but in contrast, the PRMT1 knockouts grew abnormally. Chemical inhibition of Arg-methylation also had an anti-parasitic effect. These results suggest that the parasitic PRMT1 plays multiple roles, and its loss affects the recruitment of a more potent second slicer to the parasitic RNA silencing complex, the exact mechanism of which remains to be determined.
Assuntos
Proteínas Argonautas/metabolismo , Endorribonucleases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Clivagem do RNA , Toxoplasma/metabolismo , Sequência de Aminoácidos , Proteínas Argonautas/química , Proteínas Argonautas/genética , Pareamento de Bases , Sequência de Bases , Divisão Celular , Células Cultivadas , Endorribonucleases/química , Endorribonucleases/genética , Técnicas de Inativação de Genes , Humanos , Metilação , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA/química , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimentoRESUMO
An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56δ (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56δ holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56δ termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.
RESUMO
Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression.
Assuntos
Interferons/antagonistas & inibidores , Mapeamento de Interação de Proteínas , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Linhagem Celular , Núcleo Celular/química , Células Epiteliais/virologia , Humanos , Quinase I-kappa B/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/química , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Allergic conjunctivitis is characterized by itchy, watery and swollen eyes which occur in response to exposure to seasonal or environmental allergens. The early phase reaction of allergic conjunctivitis is primarily mediated by mast cell degranulation while the late phase reaction is driven by Th2 cells and eosinophils. Prostaglandin D(2) (PGD(2)), released from mast cells, is present in allergic conjunctival tears and may elicit classical allergic responses via interaction with the high-affinity DP2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells, CRTh2). Furthermore, antagonism of this receptor is well known to inhibit eosinophil chemotaxis, basophil activation and Th2 cytokine production. PGD(2), therefore, may be involved in both early and late phase reactions in response to allergen challenge. METHODS: Thus, we explored whether our novel and selective DP2 antagonist AM156 would be efficacious in animal models of allergic conjunctivitis. Furthermore, as respiratory syncytial virus (RSV) has been implicated in the pathogenesis of allergic conjunctivitis, we examined the effects of DP2 antagonism in a murine model of RSV ocular infection. RESULTS: Utilizing a guinea pig ovalbumin model and a murine ragweed model we demonstrated that AM156 reduces redness, discharge and swelling in response to allergen challenge. These effects were equal to or greater than those of current clinical treatment options for allergic conjunctivitis including topical corticosteroids and a dual-mechanism antihistamine and decongestant. AM156 significantly reduced RSV-induced ocular inflammation and IL-4 production. CONCLUSION: These results suggest that a topical DP2 antagonist such as AM156 may represent a novel therapeutic for allergic conjunctivitis.
Assuntos
Antialérgicos/uso terapêutico , Benzilaminas/uso terapêutico , Conjuntivite Alérgica/tratamento farmacológico , Conjuntivite Viral/tratamento farmacológico , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Administração Tópica , Alérgenos/imunologia , Ambrosia/imunologia , Animais , Conjuntivite Alérgica/imunologia , Conjuntivite Alérgica/metabolismo , Conjuntivite Viral/imunologia , Conjuntivite Viral/metabolismo , Modelos Animais de Doenças , Feminino , Cobaias , Interleucina-4/imunologia , Interleucina-4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/metabolismoRESUMO
Respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are two respiratory pathogens of paramount medical significance that exert high mortality. At present, there is no reliable vaccine or antiviral drug against either virus. Using an RNA interference (RNAi) approach, we show that individual as well as joint infection by RSV and PIV can be specifically prevented and inhibited by short interfering RNAs (siRNAs), instilled intranasally in the mouse, with or without transfection reagents. The degree of protection matched the antiviral activity of the siRNA in cell culture, allowing an avenue for quick screening of an efficacious siRNA. When targeting both viruses in a joint infection, excess of one siRNA moderated the inhibitory effect of the other, suggesting competition for the RNAi machinery. Our results suggest that, if properly designed, low dosages of inhaled siRNA might offer a fast, potent and easily administrable antiviral regimen against respiratory viral diseases in humans.
Assuntos
Antivirais/farmacologia , RNA Interferente Pequeno/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Infecções Respiratórias/prevenção & controle , Respirovirus/efeitos dos fármacos , Animais , Interferons/metabolismo , Pulmão/patologia , Pulmão/virologia , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismoRESUMO
A leading theory for ovarian carcinogenesis proposes that inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, nonsteroidal anti-inflammatory drugs (NSAIDs) exert promising chemopreventive activity for ovarian cancer. Unfortunately, toxicity is associated with long-term use of NSAIDs due to their cyclooxygenase (COX) inhibitory activity. Previous studies suggest the antineoplastic activity of NSAIDs is COX independent, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. Here we evaluate PDE10A as a novel therapeutic target for ovarian cancer. Analysis of The Cancer Genome Atlas (TCGA) ovarian tumors revealed PDE10A overexpression was associated with significantly worse overall survival for patients. PDE10A expression also positively correlated with the upregulation of oncogenic and inflammatory signaling pathways. Using small molecule inhibitors, Pf-2545920 and a novel NSAID-derived PDE10A inhibitor, MCI-030, we show that PDE10A inhibition leads to decreased ovarian cancer cell growth and induces cell cycle arrest and apoptosis. We demonstrate these pro-apoptotic properties occur through PKA and PKG signaling by using specific inhibitors to block their activity. PDE10A genetic knockout in ovarian cancer cells through CRISP/Cas9 editing lead to decreased cell proliferation, colony formation, migration and invasion, and in vivo tumor growth. We also demonstrate that PDE10A inhibition leads to decreased Wnt-induced ß-catenin nuclear translocation, as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. These findings implicate PDE10A as novel target for ovarian cancer chemoprevention and treatment.
Assuntos
Neoplasias Ovarianas , beta Catenina , Feminino , Humanos , Anti-Inflamatórios não Esteroides/farmacologia , beta Catenina/genética , beta Catenina/metabolismo , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteínas ras/metabolismoRESUMO
Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic ß-catenin, inhibited Wnt-induced nuclear translocation of ß-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. PREVENTION RELEVANCE: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/ß-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.
Assuntos
Neoplasias do Colo , beta Catenina , Animais , Carcinogênese , Colo/patologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/prevenção & controle , Camundongos , Inibidores de Fosfodiesterase/farmacologia , Sulindaco/farmacologiaRESUMO
As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role.
Assuntos
Actinas , Análise Mutacional de DNA , Regulação Viral da Expressão Gênica , Profilinas , RNA Viral/metabolismo , Vírus Sincicial Respiratório Humano , Actinas/química , Actinas/genética , Actinas/metabolismo , Animais , Cátions Bivalentes/metabolismo , Galinhas , Modelos Moleculares , Mutação , Profilinas/química , Profilinas/genética , Profilinas/metabolismo , Estrutura Terciária de Proteína , RNA Viral/genética , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Viruses of the Paramyxoviridae family, such as the respiratory syncytial virus (RSV), suppress cellular innate immunity represented by type I interferon (IFN) for optimal growth in their hosts. The two unique nonstructural (NS) proteins, NS1 and NS2, of RSV suppress IFN synthesis, as well as IFN function, but their exact targets are still uncharacterized. Here, we investigate if either or both of the NS proteins affect the steady-state levels of key members of the IFN pathway. We found that both NS1 and NS2 decreased the levels of TRAF3, a strategic integrator of multiple IFN-inducing signals, although NS1 was more efficient. Only NS1 reduced IKKepsilon, a key protein kinase that specifically phosphorylates and activates IFN regulatory factor 3. Loss of the TRAF3 and IKKepsilon proteins appeared to involve a nonproteasomal mechanism. Interestingly, NS2 modestly increased IKKepsilon levels. In the IFN response pathway, NS2 decreased the levels of STAT2, the essential transcription factor for IFN-inducible antiviral genes. Preliminary mapping revealed that the C-terminal 10 residues of NS1 were essential for reducing IKKepsilon levels and the C-terminal 10 residues of NS2 were essential for increasing and reducing IKKepsilon and STAT2, respectively. In contrast, deletion of up to 20 residues of the C termini of NS1 and NS2 did not diminish their TRAF3-reducing activity. Coimmunoprecipitation studies revealed that NS1 and NS2 form a heterodimer. Clearly, the NS proteins of RSV, working individually and together, regulate key signaling molecules of both the IFN activation and response pathways.
Assuntos
Regulação Viral da Expressão Gênica , Interferons/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Proteínas não Estruturais Virais/fisiologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Dimerização , Humanos , Quinase I-kappa B/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo , Células Vero , Proteínas não Estruturais Virais/químicaRESUMO
The La antigen (SS-B) associates with a wide variety of cellular and viral RNAs to affect gene expression in multiple systems. We show that La is the major cellular protein found to be associated with the abundant 44-nucleotide viral leader RNA (leRNA) early after infection with respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus. Consistent with this, La redistributes from the nucleus to the cytoplasm in RSV-infected cells. Upon RNA interference knockdown of La, leRNA is redirected to associate with the RNA-binding protein RIG-I, a known activator of interferon (IFN) gene expression, and this is accompanied by the early induction of IFN mRNA. These results suggest that La shields leRNA from RIG-I, abrogating the early viral activation of type I IFN. We mapped the leRNA binding function to RNA recognition motif 1 of La and showed that while wild-type La greatly enhanced RSV growth, a La mutant defective in RSV leRNA binding also did not support RSV growth. Comparative studies of RSV and Sendai virus and the use of IFN-negative Vero cells indicated that La supports the growth of nonsegmented negative-strand RNA viruses by both IFN suppression and a potentially novel IFN-independent mechanism.
Assuntos
Autoantígenos/fisiologia , RNA Helicases DEAD-box/química , RNA Viral , Ribonucleoproteínas/fisiologia , Motivos de Aminoácidos , Animais , Autoantígenos/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Clonagem Molecular , Citoplasma/metabolismo , Proteína DEAD-box 58 , Humanos , Modelos Genéticos , Interferência de RNA , Receptores Imunológicos , Ribonucleoproteínas/química , Células Vero , Replicação Viral , Antígeno SS-BRESUMO
MicroRNAs (miRNAs) are endogenous noncoding RNAs that down-regulate gene expression by promoting cleavage or translational arrest of target mRNAs. While most miRNAs are transcribed from their own dedicated genes, some map to introns of 'host' transcripts, the biological significance of which remains unknown. Here, we show that prostate cells are naturally devoid of EGF-like domain 7 (Egfl7) transcripts and hence also deficient in a miRNA, miR-126*, generated from splicing and processing of its ninth intron. Use of recombinant and synthetic miRNAs or a specific antagomir established a role of miR-126* in silencing prostein in non-endothelial cells. We mapped two miR-126*-binding sites in the 3'UTR of the prostein mRNA required for translational repression. Transfection of synthetic miR-126* into prostate cancer LNCaP cells strongly reduced the translation of prostein. Interestingly, loss of prostein correlated with reduction of LNCaP cell migration and invasion. Thus, the robust expression of prostein protein in the prostate cells results from a combination of transcriptional activation of the prostein gene and absence of intronic miRNA-126* due to the prostate-specific repression of the Egfl7 gene. We conclude that intronic miRNAs from tissue-specific transcripts, or their natural absence, make cardinal contributions to cellular gene expression and phenotype. These findings also open the door to tissue-specific miRNA therapy.
Assuntos
Endotélio Vascular/metabolismo , Íntrons/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/patologia , Biossíntese de Proteínas , Regiões 3' não Traduzidas , Sequência de Bases , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Proteínas de Membrana/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Invasividade Neoplásica , Especificidade de Órgãos , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente PequenoRESUMO
Stable RNA interference (RNAi) is commonly achieved by recombinant expression of short hairpin RNA (shRNA). To generate virus-resistant cell lines, we cloned a shRNA cassette against the phosphoprotein gene of respiratory syncytial virus (RSV) into a polIII-driven plasmid vector. Analysis of individual stable transfectants showed a spectrum of RSV resistance correlating with the levels of shRNA expressed from different chromosomal locations. Interestingly, resistance in a minority of clones was due to mono-allelic disruption of the cellular gene for vasodilator-stimulated phosphoprotein (VASP). Thus, pure clones of chromosomally integrated DNA-directed RNAi can exhibit gene disruption phenotypes resembling but unrelated to RNAi.
Assuntos
Moléculas de Adesão Celular/fisiologia , Imunidade , Proteínas dos Microfilamentos/fisiologia , Oligodesoxirribonucleotídeos/genética , Fosfoproteínas/fisiologia , Interferência de RNA , RNA Interferente Pequeno/fisiologia , Vírus Sinciciais Respiratórios/imunologia , Sequência de Bases , Moléculas de Adesão Celular/genética , Cromossomos Humanos , Clonagem Molecular/métodos , Vetores Genéticos , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/farmacologia , Fosfoproteínas/genética , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , TransfecçãoRESUMO
Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Cantaridina/química , Inibidores Enzimáticos/química , Proteínas Nucleares/química , Fosfoproteínas Fosfatases/química , Proteína Fosfatase 1/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Domínios Proteicos , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Geldanamycin (GA), an antibiotic of the ansamycin family and an inhibitor of heat shock protein 90 (Hsp90), was previously shown to inhibit the malarial parasite, Plasmodium falciparum. Here we report that cyclosporin A (CsA), an inhibitor of parasitic cyclophilin (Cyp) and protein phosphatase 2B (calcineurin, CN), acted synergistically with GA to inhibit the erythrocytic growth of the parasite. Parasitic calcineurin associated with Hsp90 in vivo, and GA inhibited the association, but CsA had no effect. In a number of CsA-resistant (CsA(R)) P. falciparum clones mutations were detected in functionally significant amino acid residues of the catalytic and regulatory subunits of calcineurin (CnA and CnB, respectively) and in two out of three parasitic cyclophilins, namely Cyp19A and Cyp19B. No mutation was detected in the third cyclophilin, Cyp24. Further analysis of the mutant CnA revealed that its protein phosphatase activity was highly CsA-resistant in vitro. Similarly, one of the mutant Cyp19A proteins was purified and found to be unable to inhibit parasitic CN in the presence of CsA. Together, these results underscore the importance of the proper assembly and function of CN in plasmodial biology and suggest that the inhibition of CN can be a potential mechanism behind the CsA-sensitivity of the malaria parasite.
Assuntos
Antimaláricos/farmacologia , Calcineurina/metabolismo , Ciclosporina/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Quinonas/farmacologia , Sequência de Aminoácidos , Animais , Aromatase/genética , Benzoquinonas , Inibidores de Calcineurina , Resistência a Medicamentos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Lactamas Macrocíclicas , Dados de Sequência Molecular , Mutação , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Ligação Proteica , Proteínas de Protozoários/genética , Alinhamento de SequênciaRESUMO
We have identified an immunophilin of the FKBP family in Plasmodium falciparum that contains a conserved peptidyl prolyl isomerase (PPIase) and tetratricopeptide repeat (TPR) domains. The 35 kDa protein was named FKBP35 and expressed in bacteria. Recombinant FKBP35 exhibited potent PPIase and protein folding activities against defined substrates in vitro, suggesting that it is a parasitic chaperone. Both activities were inhibited by macrolide immunosuppressant drugs, ascomycin (a FK506 derivative) and rapamycin, but not by cyclosporin A, providing biochemical evidence of its inclusion in the FKBP family. Interestingly, FKBP35 inhibited purified plasmodial calcineurin (protein phosphatase 2B) in the absence of any drug. In the parasite's cell, FKBP35 exhibited a stage-specific nucleocytoplasmic shuttling and did not co-localize with calcineurin. FKBP35 associated with plasmodial heat shock protein 90 (Hsp90), another member of the chaperone superfamily, via the TPR domain. Geldanamycin, a Hsp90 inhibitor, and ascomycin inhibited P. falciparum growth in a synergistic fashion. Extensive search of the P. falciparum genome revealed no other FKBP sequence, implicating PfFKBP35 as a highly significant antimalarial drug target. Thus, the single FKBP of Plasmodium is an essential parasitic chaperone with a novel drug-independent calcineurin-inhibitory activity.
Assuntos
Inibidores de Calcineurina , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Sequência de Aminoácidos , Animais , Antimaláricos/farmacologia , Domínio Catalítico , Núcleo Celular/química , Ciclosporina/farmacologia , Citoplasma/química , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/farmacologia , Dados de Sequência Molecular , Peso Molecular , Plasmodium falciparum/efeitos dos fármacos , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sirolimo/farmacologia , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/farmacologiaRESUMO
BACKGROUND: Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum. RESULTS: P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca+2-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca+2. We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca+2, the processed cores are constitutively active and either less responsive or unresponsive to Ca+2. The processing is extremely rapid, specific, and occurs in vivo. CONCLUSIONS: Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation.