RESUMO
Monocytes and macrophages are important effectors and regulators of inflammation, and both their differentiation and activation are regulated strictly in response to environmental cues. Angiopoietin-like protein 2 (Angptl2) is a multifaceted protein, displaying many physiological and pathological functions in inflammation, angiogenesis, hematopoiesis, and tumor development. Although recent studies implicate Angptl2 in chronic inflammation, the mechanisms of inflammation caused by Angptl2 remain unclear. The purpose of the present study was to elucidate the role of Angptl2 in inflammation by understanding the effects of Angptl2 on monocytes/macrophages. We showed that Angptl2 directly activates resident murine peritoneal monocytes and macrophages and induces a drastic upregulation of the transcription of several inflammatory genes including nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, and several proinflammatory cytokine genes such as interleukin (IL)-1ß, IL-6, TNFα, and CSF2, along with activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. Concordantly, proinflammatory cytokines IL-1ß, IL-6, TNFα, and GM-CSF, were rapidly elevated from murine peritoneal monocytes and macrophages. These results demonstrate a novel role for Angptl2 in inflammation via the direct activation of peritoneal monocytes and macrophages.
Assuntos
Angiopoietinas/imunologia , Macrófagos Peritoneais/imunologia , Monócitos/imunologia , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Células HEK293 , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/patologia , NF-kappa B/genética , NF-kappa B/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Cultura Primária de Células , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
The level of TGF-ß/bone morphogenetic protein (BMP) signaling through Smad is tightly regulated to ensure proper embryonic patterning and homeostasis. Here we show that Smad activation by TGF-ß/BMP is blocked by a highly conserved phosphorylation event in the α-helix 1 region of Smad [T312 in Drosophila Smad1 (MAD)]. α-helix 1 phosphorylation reduces Smad interaction with TGF-ß/BMP receptor kinase and affects all receptor-activated Smads except Smad3. Tissue culture and transgenic studies in Drosophila further demonstrate that the biological activity of MAD is repressed by T312 phosphorylation in vivo. Through RNAi screening of the kinome, we have identified Misshapen (Msn) and the mammalian orthologs TNIK, MINK1, and MAP4K4 as the kinases responsible for α-helix 1 phosphorylation. Targeted expression of an active form of Msn in the wing imaginal disk disrupted activation of endogenous MAD by Dpp and expression of the Dpp/MAD target gene. Msn kinases belong to the Ste20 kinase family that has been shown to act as MAP kinase kinase kinase kinase (MAP4K). Our findings thus reveal a function of Msn independent of its impact on MAP kinase cascades. This Smad inhibition mechanism by Msn likely has important implications for development and disease.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Smad/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Genes de Insetos , Humanos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Estrutura Secundária de Proteína , Interferência de RNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas Smad/química , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cutaneous squamous cell carcinoma (cSCC) results from transformation of epidermal keratinocytes. Invasion of transformed keratinocytes through the basement membrane into the dermis results in invasive cSCC with substantial metastatic potential. To better understand the mechanisms for invasion and metastasis, we compared the protein expression profiles of a non-metastatic transformed mouse keratinocyte line and its metastatic derivative. Keratin 8 (Krt8) and Krt18, not seen in normal keratinocytes, were coexpressed and formed Krt8/18 filaments in the metastatic line. The metastatic line efficiently invaded an artificial basement membrane in vitro owing to the Krt8/18-coexpression, since coexpression of exogenous Krt8/18 in the non-invasive parental line conferred invasiveness. To test whether the Krt8/18-coexpression is induced and is involved in cSCC invasion, we examined specimens from 21 pre-invasive and 24 invasive cSCC patients by immunohistochemistry, and the ectopic Krt8/18-coexpression was almost exclusively found in invasive cSCC. Further studies are needed to examine the clinical significance of ectopic Krt8/18-coexpression in cSCC.
Assuntos
Carcinoma de Células Escamosas/patologia , Queratina-18/biossíntese , Queratina-8/biossíntese , Queratinócitos/patologia , Neoplasias Cutâneas/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Humanos , Queratinócitos/metabolismo , Camundongos , Invasividade NeoplásicaRESUMO
Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rap2 effector. Here we report that, in HEK293T cells, Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for membrane-association and TNIK activation, whereas all Rap2 proteins, including Rap2B, require palmitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rap2 proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rap2 proteins.
Assuntos
Endossomos/enzimologia , Lipoilação , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/enzimologia , Ativação Enzimática , Quinases do Centro Germinativo , Complexo de Golgi/enzimologia , Humanos , Dados de Sequência Molecular , Fenótipo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Rap1 and Rap2 are similar Ras-like G proteins but perform distinct functions. By the affinity chromatography/mass-spectrometry approach and the yeast two-hybrid screening, we identified Misshapen/NIKs-related kinase (MINK) as a novel Rap2-interacting protein that does not interact with Rap1 or Ras. MINK is a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases. The interaction between MINK and Rap2 was GTP-dependent and required Phe39 within the effector region of Rap2; the corresponding residue in Rap1 and Ras is Ser. MINK was enriched in the brain, and both MINK and its close relative, Traf2- and Nck-interacting kinase (TNIK), interacted with a postsynaptic scaffold protein containing tetratricopeptide repeats, ankyrin repeats and a coiled-coil region (TANC1) and induced its phosphorylation, under control of Rap2 in cultured cells. These are novel actions of MINK and TNIK, and consistent with a role of MINK as a Rap2 effector in the brain.
Assuntos
Encéfalo/metabolismo , Venenos de Crotalídeos/metabolismo , Lectinas Tipo C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Pareamento Cromossômico , Venenos de Crotalídeos/genética , Quinases do Centro Germinativo , Humanos , Lectinas Tipo C/genética , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RatosRESUMO
PURPOSE: To investigate global protein expression profiles in the retinas of normal and glucocorticoid-induced ocular hypertensive rats by proteomic analysis. METHODS: Ocular hypertension was induced by topical application of dexamethasone (DEX) for 4 weeks. Age-matched untreated rats served as controls. Intraocular pressure (IOP) was monitored by an electronic tonometer. Retinal protein expression profiling was carried out by two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. RESULTS: In DEX-treated rats, average IOP was elevated significantly compared with controls. With DEX treatment, levels of four proteins were altered, as revealed by 2-D DIGE and MALDI-TOF mass spectrometry: apolipoprotein A1 (apoA1), a lipid-binding protein, upregulated 1.9-fold, P < 0.05; alpha A crystallin (CRYAA), a molecular chaperone, downregulated 2.7-fold, P < 0.01; superoxide dismutase 1 (SOD1), an antioxidant enzyme, downregulated 2.3-fold, P < 0.05; and triosephosphate isomerase 1 (TPI1), a glycolytic enzyme, downregulated 2.3-fold, P < 0.01. CONCLUSIONS: Downregulation of CRYAA, SOD1, and TPI1, observed here after a short period of DEX-induced ocular hypertension, may be involved in the onset of neural damage in steroid-induced glaucoma.
Assuntos
Modelos Animais de Doenças , Hipertensão Ocular/metabolismo , Estresse Oxidativo , Retina/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Dexametasona , Regulação para Baixo , Eletroforese em Gel Bidimensional , Glucocorticoides , Pressão Intraocular , Hipertensão Ocular/induzido quimicamente , Proteômica , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Triose-Fosfato Isomerase/metabolismo , Cadeia A de alfa-Cristalina/metabolismoRESUMO
MicroRNAs (miRs) are expected to serve as prognostic tools for cancer. However, many miRs have been reported as prognostic markers of recurrence or metastasis in oral squamous cell carcinoma patients. We aimed to determine the prognostic markers in early-stage tongue squamous cell carcinoma (TSCC). Based on previous studies, we hypothesized that miR-10a, 10b, 196a-5p, 196a-3p, and 196b were prognostic markers and we retrospectively performed miR expression analyses using formalin-fixed paraffin-embedded sections of surgical specimens. Total RNA was isolated from cancer tissues and adjacent normal tissue as control, and samples were collected by laser-capture microdissection. After cDNA synthesis, reverse transcription-quantitative polymerase chain reaction was performed. Statistical analyses for patient clinicopathological characteristics, recurrence/metastasis, and survival rates were performed to discern their relationships with miR expression levels, and the 2-ΔΔCq method was used. miR-196a-5p levels were significantly upregulated in early-stage TSCC, particularly in the lymph node metastasis (LNM) group. The LNM-free survival rate in the low miR-196a-5p ΔΔCq value regulation group was found to be lower than that in the high ΔΔCq value regulation group (P=0.0079). Receiver operating characteristic analysis of ΔΔCq values revealed that miR-196a-5p had a P-value=0.0025, area under the curve=0.740, and a cut-off value=-0.875 for distinguishing LNM. To our knowledge, this is the first study to examine LNM-related miRs in early-stage TSCC as well as miRs and 'delayed LNM' in head and neck cancer. miR-196a-5p upregulation may predict delayed LNM. Our data serve as a foundation for future studies to evaluate miR levels and facilitate the prediction of delayed LNM during early-stage TSCC, which prevent metastasis when combined with close follow-up and aggressive adjuvant therapy or elective neck dissection. Moreover, our data will serve as a foundation for future studies to evaluate whether miR-196a-5p can serve as a therapeutic marker for preventing metastasis.
RESUMO
PURPOSE: To investigate global protein expression profiles in the trabecular meshwork (TM) of normal and glucocorticoid-induced ocular hypertensive rat eyes by proteomic analysis, which has not yet been conducted to date. MATERIALS AND METHODS: A rat ocular hypertension model was produced by topical application of dexamethasone (DEX) for 4 weeks. Age-matched untreated rats served as controls. Intraocular pressure (IOP) was monitored by an electronic tonometer. TM protein expression profiling and protein identification was carried out by a two-dimensional fluorescence differential gel electrophoresis (2-D DIGE) system and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, respectively. RESULTS: In DEX-treated rats, average IOP was elevated significantly, as compared with controls. By the DEX treatment, 14 TM protein spots were up- or downregulated consistently in 2-D DIGE analyses. Proteins exhibiting more than 2-fold statistically significant change were identified by MALDI-TOF mass spectrometry. alpha A-Crystallin and beta A(3)-crystallin were upregulated, while the C-propeptides of type I collagen were downregulated. CONCLUSION: Relatively short-term glucocorticoid application induced alteration in the expression of a number of proteins, including downregulation of type I collagen C-propeptides. This could reflect impaired collagen turnover in the TM of glucocorticoid-treated eyes.
Assuntos
Colágeno Tipo I/metabolismo , Hipertensão Ocular/metabolismo , Precursores de Proteínas/metabolismo , Proteômica , Malha Trabecular/metabolismo , Animais , Cristalinas/metabolismo , Dexametasona , Regulação para Baixo , Eletroforese em Gel Bidimensional , Glucocorticoides , Pressão Intraocular , Masculino , Espectrometria de Massas , Hipertensão Ocular/induzido quimicamente , Hipertensão Ocular/fisiopatologia , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para Cima , Cadeia A de alfa-CristalinaRESUMO
Bullous congenital ichthyosiform erythroderma (BCIE) is an autosomally dominant inherited disorder characterized by erythematous, erosive, and bullous skin lesions over the entire body at birth and abnormal hyperkeratosis on the palmoplantar sufaces as the patient grows older. BCIE is caused by a mutation in the keratin 1 (K1) and/or keratin 10 (K10) genes, and most pathogenic mutations are found within the helix initiation and termination motifs of the central helical rod domain (K1 and K10) or the upstream H1 homology domain (K10). In addition to inherited cases, sporadic cases due to a new mutation account for approximately half the total cases of BCIE. We report herein a typical sporadic case of BCIE with erythroderma, erosion, and blisters on the entire body surface at birth and palmoplantar and flexuaral areas of hyperkeratosis in the later stage. We found in this case a novel mutation, 559C to T, at amino acid position 187, which resulted in a leucine to phenylalanine substitution within the helix initiation motif of K1.
Assuntos
Hiperceratose Epidermolítica/genética , Queratinas/genética , Mutação , Substituição de Aminoácidos , Feminino , Humanos , Hiperceratose Epidermolítica/patologia , Lactente , Recém-Nascido , Queratina-1 , Reação em Cadeia da Polimerase , Pele/patologiaRESUMO
Genetic studies on Schizosaccharomyces pombe adenylyl cyclase (cyr1) have shown that its activity is positively regulated by a heterotrimetric G protein a subunit gpa2 and that the resulting increase in intracellular cAMP concentration causes inhibition of sexual development including mating and meiosis. However, molecular mechanism underlying this gpa2-dependent regulation of cyr1 remains to be clarified. Here, we show that gpa2 exhibits a direct and GTP-dependent binding to the Ras-associating domain (RAD) of cyr1, which is identified by a computer algorithm-based search of the cyr1 amino acid sequence. Overexpression of this RAD results in acceleration of the sexual development of fission yeast cells presumably by competitive sequestration of gpa2. Furthermore, cyr1 is activated in vitro by the addition of purified gpa2, which is converted to the active state by treatment with AlF4-. These results indicate a crucial role of the RAD as a direct binding site of gpa2 in activation of cyr1. Thus, RADs, which have been defined as a conserved motif shared among the Ras-family small G protein-associating domains, are for the first time shown to exhibit a functional association with a member of the heterotrimeric G proteins.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Genes ras/fisiologia , Dados de Sequência Molecular , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Diferenciação Sexual/genéticaRESUMO
To determine the clinical implications and prognostic value of the human papillomavirus (HPV) genotype, we evaluated the various HPV types in patients receiving radiotherapy for squamous cell carcinoma of the cervix. The study population included 113 invasive squamous cell carcinoma patients treated with radiation or chemoradiation between 1993 and 2002. The median age of the patients was 61 years. Tumors were classified by the International Federation of Gynecology and Obstetrics staging as stage IB in 11 patients, stage II in 39, stage III in 57 and stage IVA in 6 patients. To investigate HPV infection and its genotypes in the tumor specimens, L1 consensus PCR was performed followed by the direct nucleotide sequencing of the PCR products. Ninety-five samples (84.1%) were positive for HPV DNA. The most prevalent type was HPV-16 (34.7%). Poorer response to radiotherapy was observed in the patients with the HPV-16 genotype, in which 7 of the 33 patients had persistent disease. Only 1 of the 10 patients with HPV-58, 1 of the 5 with HPV-31 and 5 of the 10 patients with HPV-33 had a recurrence. The 5-year survival rate was 90, 80, 69.4 and 39% in the HPV-58, HPV-31, HPV-16 and HPV-33 type groups, respectively. Patients with HPV-31 and HPV-58 types were found to have better survival, whereas patients with the HPV-33 type experienced a higher risk of death. HPV genotyping may serve as a potential biomarker of response to radiation and prognosis in cervical carcinoma patients undergoing radio- or chemoradiotherapy.
RESUMO
Nedd4-1 is a "neuronal precursor cell expressed and developmentally downregulated protein" and among the most abundant E3 ubiquitin ligases in mammalian neurons. In analyses of conventional and conditional Nedd4-1-deficient mice, we found that Nedd4-1 plays a critical role in dendrite formation. Nedd4-1, the serine/threonine kinase TNIK, and Rap2A form a complex that controls Nedd4-1-mediated ubiquitination of Rap2A. Ubiquitination by Nedd4-1 inhibits Rap2A function, which reduces the activity of Rap2 effector kinases of the TNIK family and promotes dendrite growth. We conclude that a Nedd4-1/Rap2A/TNIK signaling pathway regulates neurite growth and arborization in mammalian neurons.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neuritos/fisiologia , Neurônios/citologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Técnicas de Cocultura/métodos , Complexos Endossomais de Distribuição Requeridos para Transporte/deficiência , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Quinases do Centro Germinativo , Proteínas de Fluorescência Verde/genética , Hipocampo , Humanos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Microglia/fisiologia , Proteína Proteolipídica de Mielina/metabolismo , Ubiquitina-Proteína Ligases Nedd4 , Neuritos/efeitos dos fármacos , Técnicas de Patch-Clamp , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Coloração pela Prata/métodos , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Transfecção/métodos , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Proteínas rap de Ligação ao GTP/genéticaAssuntos
Glucocorticoides/farmacologia , Interferon beta/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico Sintase/genética , RNA Mensageiro/antagonistas & inibidores , Animais , Linhagem Celular , Resistência a Medicamentos , Macrófagos/fisiologia , Camundongos , Óxido Nítrico Sintase Tipo IIRESUMO
Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecal-specific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , Regulação da Expressão Gênica , Ovulação/fisiologia , Fosfoinositídeo Fosfolipase C/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Montagem e Desmontagem da Cromatina , Inositol 1,4,5-Trifosfato/metabolismo , Mutação , Fosfoinositídeo Fosfolipase C/genética , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
There have been no large-scale epidemiological studies of human papillomavirus (HPV) genotype distribution of common warts in Japan. A total of 213 patients with common warts (104 males and 109 females) in Japan were studied to detect HPV genotype distribution by polymerase chain reaction (PCR) and direct sequencing analysis. The results were as follows: 94 HPV-1a (44.1%), 35 HPV-4 (16.4%), 30 HPV-65 (14.1%), 13 HPV-27 (6.1%), 13 HPV-2a (6.1%), 9 HPV-57b (4.22%), 3 HPV-16 (1.41%), 2 HPV-6a (0.94%), 2 HPV-63 (0.94%), and 1 case for each of HPV-3, -5, -5b, -7, -10, -21, -29, -47, -56, -57, -62, and -92 (0.47%, respectively). Four cases (1.88%) were found in which two different HPV types were detected within the lesions: one case of HPV-1a with HPV-16, one case of HPV-1a with HPV-65, one case of HPV-6a with HPV-8, and one case of HPV-65 with HPV-16. There were seven cases of mucosal types (3.3%), that is, two HPV-6a, three HPV-16, one HPV-56, and one HPV-62, and three cases of epidermodysplasia verruciformis (EV)-related types (1.41%), that is, one HPV-5, one HPV-5b (both of which belonged to a high-risk group), and one HPV-47 (which belonged to a low-risk group). To date, this is the largest sequencing-based study of HPV for common warts in Japan. It is said that common warts are induced predominantly by HPV-2, -27, and -57 in European population. However, the present results showed that in Japan they were induced mostly by HPV-1, -4, and -65. This suggests that regional differences in HPV genotype distribution may exist between European and Japanese populations.
Assuntos
Papillomaviridae/classificação , Infecções por Papillomavirus/virologia , Infecções Tumorais por Vírus/virologia , Verrugas/virologia , Adolescente , Adulto , Idoso , Criança , DNA Viral/análise , Feminino , Genoma Viral , Genótipo , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Reação em Cadeia da Polimerase , Análise de SequênciaRESUMO
Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific signaling role is unclear. By yeast two-hybrid screening, we have found that the Caenorhabditis elegans ortholog of Rap2 interacts with a protein containing a Rho-GTPase-activating protein (RhoGAP) domain, ZK669.1a, whose human ortholog PARG1 exhibits RhoGAP activity in vitro. ZK669.1a and PARG1 share a homology region with previously unknown function, designated the ZK669.1a and PARG1 homology (ZPH) region. Here we show that the ZPH region of PARG1 mediates interaction with Rap2. PARG1 interacted with Rap2 in a GTP-dependent manner but not with Ras or Rap1. We also show that PARG1 and its mutant lacking the ZPH region induce typical cytoskeletal changes for Rho inactivation in fibroblasts. Rap2 suppressed this in vivo action of PARG1 but not that of the mutant PARG1. These results suggest that PARG1 is a putative specific effector of Rap2 to regulate Rho.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Células NIH 3T3 , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 13 , Técnicas do Sistema de Duplo-HíbridoRESUMO
Phospholipase Cepsilon (PLCepsilon) is a novel class of phosphoinositide-specific PLC with unknown physiological functions. Here, we present the first genetic analysis of PLCepsilon in an intact organism, the nematode Caenorhabditis elegans. Ovulation in C. elegans is dependent on an inositol 1,4,5-trisphosphate (IP(3)) signaling pathway activated by the receptor tyrosine kinase LET-23. We generated deletion mutants of the gene, plc-1, encoding C. elegans PLCepsilon. We observed a novel ovulation phenotype whereby oocytes are trapped in the spermatheca due to delayed dilation of the spermatheca-uterine valve. The expression of plc-1 in the adult spermatheca is consistent with its involvement in regulation of ovulation. On the other hand, we failed to observe genetic interaction of plc-1 with let-23-mediated IP(3) signaling pathway genes, suggesting a complex mechanism for control of ovulation.
Assuntos
Caenorhabditis elegans/fisiologia , Ovulação/fisiologia , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/fisiologia , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Receptores ErbB/metabolismo , Componentes do Gene , Proteínas de Fluorescência Verde , Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Luminescentes , Microinjeções , Microscopia de Vídeo , Mutação/genética , Fosfoinositídeo Fosfolipase C , Transformação Genética , Fosfolipases Tipo C/metabolismoRESUMO
Using nucleotide sequencing-based genotyping, we conducted a case-control study to examine cervical cancer risk associated with human papillomavirus (HPV) infection in a Japanese population. A consensus primer pair was used to amplify DNA from the L1 region of HPV by polymerase chain reaction (PCR). By PCR, 311 of 356 patients with cervical cancer and 333 of 3249 control individuals were positive for HPV. By the direct sequencing of PCR products, nucleotide sequences of 30 genotypes were obtained. A high incidence of type 52 and a low incidence of type 16 were characteristic of the control group. Odds ratios were estimated for 18 genotypes. Types 71, 90, and 91, previously uncharacterized, were classified as low-risk genotypes, which is consistent with predictions made on the basis of phylogeny. The present study is the first large case-control study of its kind to use nucleotide sequencing-based genotyping.
Assuntos
Carcinoma de Células Escamosas/virologia , Papillomaviridae/genética , Infecções por Papillomavirus/epidemiologia , Infecções Tumorais por Vírus/epidemiologia , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/epidemiologia , Estudos de Casos e Controles , DNA Viral/química , DNA Viral/genética , Feminino , Genótipo , Humanos , Japão/epidemiologia , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/epidemiologiaRESUMO
Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cepsilon, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase gamma (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site of Saccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674-1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclase in vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2(Val-19))-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals.
Assuntos
Adenilil Ciclases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenilil Ciclases/química , Sequência de Aminoácidos , Sítios de Ligação , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Cinética , Leucina , Dados de Sequência Molecular , Mutagênese Insercional , Fragmentos de Peptídeos/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVE: This study was undertaken to examine a possible correlation between clearance or persistence of human papillomavirus (HPV) infection and radiation response in carcinoma of the cervix. STUDY DESIGN: We reviewed 97 patients with HPV-positive cervical squamous cell carcinoma (International Federation of Gynecology and Obstetrics [FIGO] stage IB-IVA) treated with radiotherapy. Examination of HPV DNA was performed by the polymerase chain reaction-based assay. RESULTS: Cervical HPV DNA cleared in 42 patients (43.3%) and persisted in 55 patients (56.7%) at the end of irradiation. All of 42 HPV-cleared patients (100.0%) and 50 of 55 HPV-persistent patients (90.9%) had complete response. Of 92 patients with complete response, 20 (21.7%) had local recurrence develop. The recurrence rate was significantly higher in HPV-persisted patients (34.0%) than in HPV-cleared patients (7.1%) ( P = .0016). Univariate analysis demonstrated the significant differences for both 5-year local disease-free survival (LDFS) and overall survival (OAS) between HPV-cleared and HPV-persisted groups. Multivariate analysis showed that persistence of HPV DNA was the most powerful independent predictor for LDFS and OAS compared with other prognostic factors, such as FIGO stage or node swelling. CONCLUSION: In HPV-positive cervical carcinoma, persistence of HPV DNA in the cervix at the end of irradiation was highly predictive of LDFS and OAS.