RESUMO
Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.
Assuntos
Permeabilidade Capilar/fisiologia , Corpo Lúteo/irrigação sanguínea , Neovascularização Fisiológica , Serina Endopeptidases/deficiência , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/genética , Células Cultivadas , Corpo Lúteo/patologia , Corpo Lúteo/fisiopatologia , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Técnicas de Silenciamento de Genes , Hemorragia/etiologia , Hemorragia/genética , Hemorragia/fisiopatologia , Humanos , Luteinização/genética , Luteinização/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/genética , Fenótipo , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologiaRESUMO
The affiliation of Erik W. Martin is corrected in this paper.
RESUMO
Ovarian cancer is the leading cause of death among all the gynecological cancers in the USA. Ovarian cancer employs a unique mode of metastasis, as exfoliated tumor cells disseminate within the peritoneal cavity, colonizing in several sites as well as accumulating ascites. Tumor recurrence and widespread metastasis are significant factors contributing to poor prognosis. PRSS21 is a metastasis-associated ovarian cancer gene that encodes the glycosyl-phosphatidylinositol-linked serine protease, testisin. Testisin expression is increased in multiple ovarian tumor types, with relatively little expression in normal tissues, but is differentially decreased in metastatic ovarian serous carcinomas compared to primary tumors. Here we explored the function of testisin in late-stage ovarian cancer progression using a murine xenograft model of ovarian intraperitoneal tumor metastasis. Increased tumor testisin expression inhibited intra-peritoneal tumor seeding and colonization, ascites accumulation, and metastatic tumor burden that was dependent on catalytically active testisin. The known testisin substrate, protease-activated receptor-2 (PAR-2), is a target of testisin activity. Gene profiling and mechanistic studies demonstrate that testisin activity suppresses the synthesis and secretion of pro-angiogenic angiopoietins, ANG2 and ANGPTL4, which normally promote vascular leak and edema. These observations support a model wherein testisin activates PAR-2 to antagonize proangiogenic angiopoietins that modulate vascular permeability and ascites accumulation associated with ovarian tumor metastasis. KEY MESSAGES: Testisin inhibits metastatic ovarian tumor burden and ascites production. Testisin activity antagonizes ANG2 and ANGPTL4 synthesis and secretion. PAR-2 is a proteolytic target of testisin on the surface of ovarian cancer cells.
Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Neoplasias Ovarianas/metabolismo , Ribonuclease Pancreático/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Camundongos Nus , Metástase Neoplásica/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Ovarianas/patologia , Proteólise , Proteínas de Transporte Vesicular/metabolismoRESUMO
PURPOSE OF REVIEW: The endothelial cell plasma membrane is a metabolically active, dynamic, and fluid microenvironment where pericellular proteolysis plays a critical role. Membrane-anchored proteases may be expressed by endothelial cells as well as mural cells and leukocytes with distribution both inside and outside of the vascular system. Here, we will review the recent advances in our understanding of the direct and indirect roles of membrane-anchored proteases in vascular biology and the possible conservation of their extravascular functions in endothelial cell biology. RECENT FINDINGS: Membrane-anchored proteases belonging to the serine or metalloprotease families contain amino-terminal or carboxy-terminal domains, which serve to tether their extracellular protease domains directly at the plasma membrane. This architecture enables protease function and substrate repertoire to be regulated through dynamic localization in distinct areas of the cell membrane. These proteases are proving to be key components of the cell machinery for regulating vascular permeability, generation of vasoactive peptides, receptor tyrosine kinase transactivation, extracellular matrix proteolysis, and angiogenesis. SUMMARY: A complex picture of the interdependence between membrane-anchored protease localization and function is emerging that may provide a mechanism for precise coordination of extracellular signals and intracellular responses through communication with the cytoskeleton and with cellular signaling molecules.
Assuntos
Membrana Celular/enzimologia , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Serina Endopeptidases/metabolismo , Células Endoteliais/citologia , HumanosRESUMO
BACKGROUND: The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH's modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL). RESULTS: Treatment of DLBCL cells with EtOH suppressed mTORC1 complex formation while increasing AKT phosphorylation and mTORC2 complex assembly. INK128 completely abrogated AKT phosphorylation without affecting the structure of mTORC1/2 complexes. Accordingly, EtOH less profoundly suppressed cap-dependent translation and global protein synthesis, compared to a remarkable inhibitory effect of INK128 treatment. Importantly, EtOH treatment induced the formation of stress granules, while INK128 suppressed their formation. Microarray analysis of polysomal RNA revealed that although both agents primarily affected cell growth and survival, EtOH and INK128 regulated the synthesis of mostly distinct genes involved in these processes. Though both EtOH and INK128 inhibited cell cycle, proliferation and autophagy, EtOH, in contrast to INK128, did not induce cell apoptosis. CONCLUSION: Given that EtOH, similar to pharmacologic mTOR inhibitors, inhibits mTOR signaling, we systematically explored the effect of EtOH and INK128 on mTOR signal transduction, components of the mTORC1/2 interaction and their downstream effectors in DLBCL malignancy. We found that EtOH partially inhibits mTOR signaling and protein translation, compared to INK128's complete mTOR inhibition. Translatome analysis of mTOR downstream target genes established that differential inhibition of mTOR by EtOH and INK128 distinctly modulates translation of specific subsets of mRNAs involved in cell growth and survival, leading to differential cellular response and survival.
Assuntos
Benzoxazóis/farmacologia , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Autofagia/efeitos dos fármacos , Autofagia/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Human diffuse large B-cell lymphomas (DLBCLs) often aberrantly express oncogenes that generally contain complex secondary structures in their 5' untranslated region (UTR). Oncogenes with complex 5'UTRs require enhanced eIF4A RNA helicase activity for translation. PDCD4 inhibits eIF4A, and PDCD4 knockout mice have a high penetrance for B-cell lymphomas. Here, we show that on B-cell receptor (BCR)-mediated p70s6K activation, PDCD4 is degraded, and eIF4A activity is greatly enhanced. We identified a subset of genes involved in BCR signaling, including CARD11, BCL10, and MALT1, that have complex 5'UTRs and encode proteins with short half-lives. Expression of these known oncogenic proteins is enhanced on BCR activation and is attenuated by the eIF4A inhibitor Silvestrol. Antigen-experienced immunoglobulin (Ig)G(+) splenic B cells, from which most DLBCLs are derived, have higher levels of eIF4A cap-binding activity and protein translation than IgM(+) B cells. Our results suggest that eIF4A-mediated enhancement of oncogene translation may be a critical component for lymphoma progression, and specific targeting of eIF4A may be an attractive therapeutic approach in the management of human B-cell lymphomas.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Guanilato Ciclase/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Regiões 5' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 10 de Linfoma CCL de Células B , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Western Blotting , Proteínas Adaptadoras de Sinalização CARD/genética , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/genética , Guanilato Ciclase/genética , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Pessoa de Meia-Idade , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Triterpenos/farmacologiaRESUMO
Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.
Assuntos
Proliferação de Células , Fator de Iniciação Eucariótico 4G/genética , Linfoma Difuso de Grandes Células B/genética , MicroRNAs/genética , Animais , Linhagem Celular Tumoral , Senescência Celular/genética , Regulação para Baixo , Fator de Iniciação Eucariótico 4G/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/patologia , Camundongos , MicroRNAs/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONALE: G protein-coupled receptor kinase 2 (GRK2) is abundantly expressed in the heart, and its expression and activity are increased in injured or stressed myocardium. This upregulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes, and its inhibition by a peptide comprising the last 194 amino acids of GRK2 (known as carboxyl-terminus of ß-adrenergic receptor kinase [bARKct]) is cardioprotective. OBJECTIVE: The aim of this study was to elucidate the signaling mechanism that accounts for the prodeath signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the carboxyl-terminus of ß-adrenergic receptor kinase. METHODS AND RESULTS: Using in vivo mouse models of ischemic injury and also cultured myocytes, we found that GRK2 localizes to mitochondria, providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced prodeath signaling. Localization of GRK2 to mitochondria was dependent on phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal-regulated kinases, resulting in enhanced GRK2 binding to heat shock protein 90, which chaperoned GRK2 to mitochondria. Mechanistic studies in vivo and in vitro showed that extracellular signal-regulated kinase regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent prodeath signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca(2+)-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. CONCLUSIONS: We identify GRK2 as a prodeath kinase in the heart, acting in a novel manner through mitochondrial localization via extracellular signal-regulated kinase regulation.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Mitocôndrias Cardíacas/metabolismo , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Bovinos , Células Cultivadas , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Células HEK293 , Proteínas de Choque Térmico HSP90/biossíntese , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/patologia , Miócitos Cardíacos/patologia , Estresse Oxidativo/genética , Ratos , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure. The chronic adrenergic stimulation that accompanies heart failure is a signaling abnormality that leads to the upregulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part owing to uncoupling of the ß-adrenergic receptor system. In this study, we explored the possibility that enhanced GRK2 expression and activity, as seen during heart failure, can negatively affect cardiac metabolism as part of its pathogenic profile. METHODS AND RESULTS: Positron emission tomography studies revealed in transgenic mice that cardiac-specific overexpression of GRK2 negatively affected cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes heart failure development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 in cardiomyocytes, causing insulin-dependent negative signaling feedback, including inhibition of membrane translocation of the glucose transporter GLUT4. This identifies insulin receptor substrate-1 as a novel nonreceptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents postischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of heart failure. CONCLUSIONS: Our data provide novel insights into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through ß-adrenergic receptors and metabolism through the insulin receptor.
Assuntos
Glicemia/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Resistência à Insulina/genética , Isquemia Miocárdica/metabolismo , Animais , Metabolismo Energético/fisiologia , Terapia Genética/métodos , Transportador de Glucose Tipo 4/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/prevenção & controle , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Camundongos Transgênicos , Isquemia Miocárdica/diagnóstico por imagem , Isquemia Miocárdica/terapia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação/fisiologia , Tomografia por Emissão de Pósitrons , Transdução de Sinais/fisiologiaRESUMO
The reversible conjugation of ubiquitin and ubiquitin-like (UbL) proteins to protein substrates plays a critical role in the regulation of many cellular pathways. The removal of ubiquitin from target proteins is performed by ubiquitin proteases also known as deubiquitylases (DUBs). Owing to their substrate specificity and the central role ubiquitylation plays in cell signaling pathways, DUB are attractive targets for therapeutic development. The development of DUB inhibitors requires assays that are amenable to high-throughput screening and provide rapid assessment of inhibitor selectivity. Determination of inhibitor selectivity at an early stage of drug discovery will reduce drug failure in the clinic as well as reduce overall drug development costs. We have developed two novel assays, UbL-Enterokinase light chain and UbL-Granzyme B, for quantifying ubiquitin and UbL protease activity. In our quest to discover and characterize novel chemical entities, we have combined these assays with a previously developed assay in a multiplex format. This multiplex format allows for the detection of three distinct protease activities simultaneously, in a single well. We have demonstrated that the multiplex format is able to distinguish between selective and nonselective protease inhibitors. Specifically, we have used this assay format to characterize P022077, a selective ubiquitin-specific protease 7 inhibitor discovered at Progenra.
Assuntos
Corantes Fluorescentes/análise , Inibidores de Proteases/química , Ubiquitina/antagonistas & inibidores , Ubiquitinas/antagonistas & inibidores , Técnicas de Laboratório Clínico , Fluorescência , Corantes Fluorescentes/metabolismo , Pichia , Inibidores de Proteases/análise , Inibidores de Proteases/metabolismo , Especificidade por Substrato/fisiologia , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases.