RESUMEN
Rubisco, the key enzyme of CO2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.
Asunto(s)
Cianobacterias/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Orgánulos/metabolismo , Fotosíntesis/fisiología , Ribulosa-Bifosfato Carboxilasa/fisiología , Activador de Tejido Plasminógeno/química , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (KD = 1.8 µM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.
Asunto(s)
Calreticulina , Plasminógeno , Animales , Bovinos , Humanos , Calreticulina/genética , Calreticulina/aislamiento & purificación , Calreticulina/metabolismo , Fibrinolisina/metabolismo , Plasminógeno/genética , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Dominios Proteicos/genética , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas de Inactivación de Genes , Línea Celular Tumoral , Neoplasias/fisiopatologíaRESUMEN
Physical activity-induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating ß-Catenin. Silencing Wnt1 impairs mechanically induced ß-Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/ß-Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)-induced and age-related osteoporotic mouse models compared with non-OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post-menopausal and age-related osteoporosis, respectively.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal , Osteogénesis , Animales , Femenino , Ratones , beta Catenina/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Osteoblastos , ARN Interferente Pequeño , Vía de Señalización Wnt , Activador de Tejido Plasminógeno/metabolismoRESUMEN
How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Rhodobacter sphaeroides/enzimología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Dominio Catalítico , Reactivos de Enlaces Cruzados/química , Medición de Intercambio de Deuterio , Estabilidad de Enzimas , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Simulación del Acoplamiento Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Rhodobacter sphaeroides/genética , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/genética , Relación Estructura-Actividad , Factores de Tiempo , Activador de Tejido Plasminógeno/química , Activador de Tejido Plasminógeno/genéticaRESUMEN
PURPOSE OF REVIEW: The angiopoietin-like (ANGPTL) proteins ANGPTL3 and ANGPTL4 are critical lipoprotein lipase (LPL) inhibitors. This review discusses the unique ability of the insulin-responsive protein ANGPTL8 to regulate triglyceride (TG) metabolism by forming ANGPTL3/8 and ANGPTL4/8 complexes that control tissue-specific LPL activities. RECENT FINDINGS: After feeding, ANGPTL4/8 acts locally in adipose tissue, has decreased LPL-inhibitory activity compared to ANGPTL4, and binds tissue plasminogen activator (tPA) and plasminogen to generate plasmin, which cleaves ANGPTL4/8 and other LPL inhibitors. This enables LPL to be fully active postprandially to promote efficient fatty acid (FA) uptake and minimize ectopic fat deposition. In contrast, liver-derived ANGPTL3/8 acts in an endocrine manner, has markedly increased LPL-inhibitory activity compared to ANGPTL3, and potently inhibits LPL in oxidative tissues to direct TG toward adipose tissue for storage. Circulating ANGPTL3/8 levels are strongly correlated with serum TG, and the ANGPTL3/8 LPL-inhibitory epitope is blocked by the TG-lowering protein apolipoprotein A5 (ApoA5). SUMMARY: ANGPTL8 plays a crucial role in TG metabolism by forming ANGPTL3/8 and ANGPTL4/8 complexes that differentially modulate LPL activities in oxidative and adipose tissues respectively. Selective ANGPTL8 inhibition in the context of the ANGPTL3/8 complex has the potential to be a promising strategy for treating dyslipidemia.
Asunto(s)
Proteína 8 Similar a la Angiopoyetina , Hormonas Peptídicas , Humanos , Proteínas Similares a la Angiopoyetina/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Transporte Biológico , Lipoproteína Lipasa/metabolismo , Triglicéridos/metabolismo , Proteína 3 Similar a la Angiopoyetina , Hormonas Peptídicas/metabolismoRESUMEN
We modify a three-dimensional multiscale model of fibrinolysis to study the effect of plasmin-mediated degradation of fibrin on tissue plasminogen activator (tPA) diffusion and fibrinolysis. We propose that tPA is released from a fibrin fiber by simple kinetic unbinding, as well as by "forced unbinding," which occurs when plasmin degrades fibrin to which tPA is bound. We show that, if tPA is bound to a small-enough piece of fibrin that it can diffuse into the clot, then plasmin can increase the effective diffusion of tPA. If tPA is bound to larger fibrin degradation products (FDPs) that can only diffuse along the clot, then plasmin can decrease the effective diffusion of tPA. We find that lysis rates are fastest when tPA is bound to fibrin that can diffuse into the clot, and slowest when tPA is bound to FDPs that can only diffuse along the clot. Laboratory experiments confirm that FDPs can diffuse into a clot, and they support the model hypothesis that forced unbinding of tPA results in a mix of FDPs, such that tPA bound to FDPs can diffuse both into and along the clot. Regardless of how tPA is released from a fiber, a tPA mutant with a smaller dissociation constant results in slower lysis (because tPA binds strongly to fibrin), and a tPA mutant with a larger dissociation constant results in faster lysis.
Asunto(s)
Fibrinolisina , Fibrinólisis , Fibrinolisina/metabolismo , Fibrinolisina/farmacología , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacología , Fibrina/metabolismo , Cinética , Plasminógeno/metabolismoRESUMEN
Epithelial-mesenchymal transition (EMT) is an important biological process contributing to kidney fibrosis and chronic kidney disease. This process is characterized by decreased epithelial phenotypes/markers and increased mesenchymal phenotypes/markers. Tubular epithelial cells (TECs) are commonly susceptible to EMT by various stimuli, for example, transforming growth factor-ß (TGF-ß), cellular communication network factor 2, angiotensin-II, fibroblast growth factor-2, oncostatin M, matrix metalloproteinase-2, tissue plasminogen activator (t-PA), plasmin, interleukin-1ß, and reactive oxygen species. Similarly, glomerular podocytes can undergo EMT via these stimuli and by high glucose condition in diabetic kidney disease. EMT of TECs and podocytes leads to tubulointerstitial fibrosis and glomerulosclerosis, respectively. Signaling pathways involved in EMT-mediated kidney fibrosis are diverse and complex. TGF-ß1/Smad and Wnt/ß-catenin pathways are the major venues triggering EMT in TECs and podocytes. These two pathways thus serve as the major therapeutic targets against EMT-mediated kidney fibrosis. To date, a number of EMT inhibitors have been identified and characterized. As expected, the majority of these EMT inhibitors affect TGF-ß1/Smad and Wnt/ß-catenin pathways. In addition to kidney fibrosis, these EMT-targeted antifibrotic inhibitors are expected to be effective for treatment against fibrosis in other organs/tissues.
Asunto(s)
Factor de Crecimiento Transformador beta1 , beta Catenina , Humanos , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , beta Catenina/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/farmacología , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacología , Células Epiteliales/metabolismo , Vía de Señalización Wnt , Transición Epitelial-Mesenquimal , Riñón , FibrosisRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) impedes brain plasmin synthesis. Reduced plasmin activity facilitates cumulation of amyloid beta (Aß) in Alzheimer's disease (AD). Since plasmin also regulates the synaptic activity, it is possible that altered PAI-1 is present in other neurodegenerative disorders. We investigated whether PAI-1 and its counter-regulatory tissue plasminogen activator (tPA) are altered in serum of patients with dementia due to frontotemporal lobar degeneration (FTLD). Thirty five FTLD patients (21 in mild cognitive impairment stage (MCI) and 14 in dementia stage) and 10 cognitively healthy controls were recruited. Serum tPA and PAI-1 protein levels were measured by anova. Correlation between biochemical and demographic data were explored by measuring Pearson correlation coefficient. Serum PAI-1 levels were elevated in the FTLD dementia group as compared to FTLD MCI and controls. tPA serum levels and PAI-1/tPA ratio did not significantly differ among groups. There was a negative correlation between PAI-1 serum levels and disease severity measured by MMSE score. No correlations of tPA serum levels and PAI-1/tPA ratio with MMSE were found. Increased PAI-1 serum levels may serve as a marker of dementia in FTLD, suggesting that, besides Aß pathway, the plasmin system may affect cognition through synaptic activity.
Asunto(s)
Degeneración Lobar Frontotemporal , Inhibidor 1 de Activador Plasminogénico , Humanos , Inhibidor 1 de Activador Plasminogénico/sangre , Femenino , Masculino , Degeneración Lobar Frontotemporal/sangre , Anciano , Persona de Mediana Edad , Activador de Tejido Plasminógeno/sangre , Activador de Tejido Plasminógeno/metabolismo , Disfunción Cognitiva/sangre , Biomarcadores/sangre , Estudios de Casos y ControlesRESUMEN
BACKGROUND: Intravenous injection of alteplase, a recombinant tPA (tissue-type plasminogen activator) as a thrombolytic agent has revolutionized ischemic stroke management. However, tPA is a more complex enzyme than expected, being for instance able to promote thrombolysis, but at the same time, also able to influence neuronal survival and to affect the integrity of the blood-brain barrier. Accordingly, the respective impact of endogenous tPA expressed/present in the brain parenchyma versus in the circulation during stroke remains debated. METHODS: To address this issue, we used mice with constitutive deletion of tPA (tPANull [tPA-deficient mice]) or conditional deletion of endothelial tPA (VECad [vascular endothelial-Cadherin-Cre-recombinase]-Cre∆tPA). We also developed parabioses between tPANull and wild-type mice (tPAWT), anticipating that a tPAWT donor would restore levels of tPA to normal ones, in the circulation but not in the brain parenchyma of a tPANull recipient. Stroke outcomes were investigated by magnetic resonance imaging in a thrombo-embolic or a thrombotic stroke model, induced by local thrombin injection or FeCl3 application on the endothelium, respectively. RESULTS: First, our data show that endothelial tPA, released into the circulation after stroke onset, plays an overall beneficial role following thrombo-embolic stroke. Accordingly, after 24 hours, tPANull/tPANull parabionts displayed less spontaneous recanalization and reperfusion and larger infarcts compared with tPAWT/tPAWT littermates. However, when associated to tPAWT littermates, tPANull mice had similar perfusion deficits, but less severe brain infarcts. In the thrombotic stroke model, homo- and hetero-typic parabionts did not differ in the extent of brain damages and did not differentially recanalize and reperfuse. CONCLUSIONS: Together, our data reveal that during thromboembolic stroke, endogenous circulating tPA from endothelial cells sustains a spontaneous recanalization and reperfusion of the tissue, thus, limiting the extension of ischemic lesions. In this context, the impact of endogenous parenchymal tPA is limited.
Asunto(s)
Accidente Cerebrovascular , Accidente Cerebrovascular Trombótico , Animales , Ratones , Modelos Animales de Enfermedad , Células Endoteliales , Endotelio , Ratones Noqueados , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/patología , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismoRESUMEN
The combined stress of drought and salinity is prevalent in various regions of the world, affects several physiological and biochemical processes in crops, and causes their yield to decrease. Photosynthesis is one of the main processes that are disturbed by combined stress. Therefore, improving the photosynthetic efficiency of crops is one of the most promising strategies to overcome environmental stresses, making studying the molecular basis of regulation of photosynthesis a necessity. In this study, we sought a potential mechanism that regulated a major component of the combined stress response in the important crop barley (Hordeum vulgare L.), namely the Rubisco activase A (RcaA) gene. Promoter analysis of the RcaA gene led to identifying Jasmonic acid (JA)-responsive elements with a high occurrence. Specifically, a Myelocytomatosis oncogenes 2 (MYC2) transcription factor binding site was highlighted as a plausible functional promoter motif. We conducted a controlled greenhouse experiment with an abiotic stress-susceptible barley genotype and evaluated expression profiling of the RcaA and MYC2 genes, photosynthetic parameters, plant water status, and cell membrane damages under JA, combined drought and salinity stress (CS) and JA + CS treatments. Our results showed that applying JA enhances barley's photosynthetic efficiency and water relations and considerably compensates for the adverse effects of combined stress. Significant association was observed among gene expression profiles and evaluated physiochemical characteristics. The results showed a plausible regulatory route through the JA-dependent MYC2-RcaA module involved in photosynthesis regulation and combined stress tolerance. These findings provide valuable knowledge for further functional studies of the regulation of photosynthesis under abiotic stresses toward the development of multiple-stress-tolerant crops.
Asunto(s)
Ciclopentanos , Hordeum , Oxilipinas , Hordeum/genética , Hordeum/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacología , Sequías , Fotosíntesis/genética , Estrés Salino , Estrés Fisiológico , Agua/metabolismo , SalinidadRESUMEN
The current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurologic outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy, and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase Cß (PKCß) phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We show that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased phosphorylation of occludin serine 490 (S490) in the ischemic penumbra in a tPA-dependent manner, as tPA-/- mice were significantly protected from MCAO-induced occludin phosphorylation. Intraventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCß-dependent manner. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCß, reduced MCAO-induced permeability and improved functional outcome. Furthermore, inhibiting PKCß after MCAO prevented ICH associated with delayed thrombolysis. These results show that PKCß phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCß inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.
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Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Hemorragia Cerebral/tratamiento farmacológico , Hemorragia Cerebral/etiología , Fibrinolíticos/uso terapéutico , Humanos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Ratones , Ocludina/genética , Ocludina/metabolismo , Fosforilación , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/etiología , Terapia Trombolítica/efectos adversos , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Reactive oxygen species (ROS) are constantly generated in a living organism. An imbalance between the amount of generated reactive species in the body and their destruction leads to the development of oxidative stress. Proteins are extremely vulnerable targets for ROS molecules, which can cause oxidative modifications of amino acid residues, thus altering structure and function of intra- and extracellular proteins. The current review considers the effect of oxidation on the structural rearrangements and functional activity of hemostasis proteins: coagulation system proteins such as fibrinogen, prothrombin/thrombin, factor VII/VIIa; anticoagulant proteins - thrombomodulin and protein C; proteins of the fibrinolytic system such as plasminogen, tissue plasminogen activator and plasminogen activator inhibitor-1. Structure and function of the proteins, oxidative modifications, and their detrimental consequences resulting from the induced oxidation or oxidative stress in vivo are described. Possible effects of oxidative modifications of proteins in vitro and in vivo leading to disruption of the coagulation and fibrinolysis processes are summarized and systematized, and the possibility of a compensatory mechanism in maintaining hemostasis under oxidative stress is analyzed.
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Hemostasis , Activador de Tejido Plasminógeno , Activador de Tejido Plasminógeno/metabolismo , Especies Reactivas de Oxígeno , Coagulación Sanguínea , Factores de Coagulación Sanguínea/metabolismo , Estrés OxidativoRESUMEN
Rationale: Sonographic septations are assumed to be important clinical predictors of outcome in pleural infection, but the evidence for this is sparse. The inflammatory and fibrinolysis-associated intrapleural pathway(s) leading to septation formation have not been studied in a large cohort of pleural fluid (PF) samples with confirmed pleural infection matched with ultrasound and clinical outcome data. Objectives: To assess the presence and severity of septations against baseline PF PAI-1 (Plasminogen-Activator Inhibitor-1) and other inflammatory and fibrinolysis-associated proteins as well as to correlate these with clinically important outcomes. Methods: We analyzed 214 pleural fluid samples from PILOT (Pleural Infection Longitudinal Outcome Study), a prospective observational pleural infection study, for inflammatory and fibrinolysis-associated proteins using the Luminex platform. Multivariate regression analyses were used to assess the association of pleural biological markers with septation presence and severity (on ultrasound) and clinical outcomes. Measurements and Main Results: PF PAI-1 was the only protein independently associated with septation presence (P < 0.001) and septation severity (P = 0.003). PF PAI-1 concentrations were associated with increased length of stay (P = 0.048) and increased 12-month mortality (P = 0.003). Sonographic septations alone had no relation to clinical outcomes. Conclusions: In a large and well-characterized cohort, this is the first study to associate pleural biological parameters with a validated sonographic septation outcome in pleural infection. PF PAI-1 is the first biomarker to demonstrate an independent association with mortality. Although PF PAI-1 plays an integral role in driving septation formation, septations themselves are not associated with clinically important outcomes. These novel findings now require prospective validation.
Asunto(s)
Infecciones , Inhibidor 1 de Activador Plasminogénico , Enfermedades Pleurales , Humanos , Fibrinólisis , Infecciones/metabolismo , Inhibidor 1 de Activador Plasminogénico/análisis , Inhibidor 1 de Activador Plasminogénico/metabolismo , Pleura/diagnóstico por imagen , Pleura/metabolismo , Enfermedades Pleurales/diagnóstico por imagen , Enfermedades Pleurales/metabolismo , Derrame Pleural/genética , Estudios Prospectivos , Activador de Tejido Plasminógeno/análisis , Activador de Tejido Plasminógeno/metabolismo , UltrasonografíaRESUMEN
Understanding how stress hormones induce apoptosis in oviductal epithelial cells (OECs) and mural granulosa cells (MGCs) can reveal the mechanisms by which female stress impairs embryonic development and oocyte competence. A recent study showed that tissue plasminogen activator (tPA) ameliorates corticosterone-induced apoptosis in MGCs and OECs by acting on its receptors low-density lipoprotein receptor-related protein 1 (LRP1) and Annexin A2 (ANXA2), respectively. However, whether tPA is involved in corticotropin-releasing hormone (CRH)-induced apoptosis and whether it uses the same or different receptors to inhibit apoptosis induced by different hormones in the same cell type remains unknown. This study showed that CRH triggered apoptosis in both OECs and MGCs and significantly downregulated tPA expression. Moreover, tPA inhibits CRH-induced apoptosis by acting on ANXA2 in both OECs and MGCs. While ANXA2 inhibits apoptosis via phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling, LRP1 reduces apoptosis via mitogen-activated protein kinase (MAPK) signaling. Thus, tPA used the same receptor to inhibit CRH-induced apoptosis in both OECs and MGCs, however used different receptors to inhibit corticosterone-induced apoptosis in MGCs and OECs. These data helps understand the mechanism by which female stress impairs embryo/oocyte competence and proapoptotic factors trigger apoptosis in different cell types.
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Apoptosis , Hormona Liberadora de Corticotropina , Células Epiteliales , Células de la Granulosa , Activador de Tejido Plasminógeno , Animales , Femenino , Apoptosis/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Ratones , Activador de Tejido Plasminógeno/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Hormona Liberadora de Corticotropina/metabolismo , Transducción de Señal/efectos de los fármacos , Oviductos/metabolismo , Oviductos/efectos de los fármacos , Anexina A2/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Trompas Uterinas/metabolismo , Trompas Uterinas/efectos de los fármacosRESUMEN
Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.
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Adenocarcinoma de Células Claras , Neoplasias Ováricas , Serina Proteasas , Adenocarcinoma de Células Claras/enzimología , Adenocarcinoma de Células Claras/patología , Femenino , Humanos , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Serina Proteasas/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Tripsina , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Alpha-tocopherol (vitamin E) is an essential nutrient that functions as a major lipid-soluble antioxidant in humans. The alpha-tocopherol transfer protein (TTP) binds α-tocopherol with high affinity and selectivity and regulates whole-body distribution of the vitamin. Heritable mutations in the TTPA gene result in familial vitamin E deficiency, elevated indices of oxidative stress, and progressive neurodegeneration that manifest primarily in spinocerebellar ataxia. Although the essential role of vitamin E in neurological health has been recognized for over 50 years, the mechanisms by which this essential nutrient is transported in the central nervous system are poorly understood. Here we found that, in the murine cerebellum, TTP is selectively expressed in glial fibrillary acidic protein-positive astrocytes, where it facilitates efflux of vitamin E to neighboring neurons. We also show that induction of oxidative stress enhances the transcription of the TtpA gene in cultured cerebellar astrocytes. Furthermore, secretion of vitamin E from astrocytes is mediated by an ABC-type transporter, and uptake of the vitamin into neurons involves the low-density lipoprotein receptor-related protein 1. Taken together, our data indicate that TTP-expressing astrocytes control the delivery of vitamin E from astrocytes to neurons, and that this process is homeostatically responsive to oxidative stress. These are the first observations that address the detailed molecular mechanisms of vitamin E transport in the central nervous system, and these results have important implications for understanding the molecular underpinnings of oxidative stress-related neurodegenerative diseases.
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Astrocitos , Proteínas Portadoras , Cerebelo , Neuronas , Vitamina E , alfa-Tocoferol , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Proteínas Portadoras/metabolismo , Cerebelo/citología , Cerebelo/metabolismo , Humanos , Ratones , Neuronas/citología , Neuronas/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Tocoferoles , Vitamina E/metabolismo , Vitaminas , alfa-Tocoferol/metabolismoRESUMEN
The transcription factor Ghd2 increases rice yield potential under normal conditions and accelerates leaf senescence under drought stress. However, its mechanism on the regulation of leaf senescence under drought stress remains unclear. In the present study, to unveil the mechanism, one target of Ghd2, the Rubisco activase gene RCA, was identified through the combined analysis of Ghd2-CRISPR transcriptome data and Ghd2-overexpression microarray data. Ghd2 binds to the 'CACA' motif in the RCA promoter by its CCT domain and upregulates RCA expression. RCA has alternative transcripts, RCAS and RCAL, which are predominantly expressed under normal conditions and drought stress, respectively. Similar to Ghd2-overexpressing plants, RCAL-overexpressing plants were more sensitive to drought stress than the wild-type. However, the plants overexpressing RCAS showed a weak drought-sensitive phenotype. Moreover, RCAL knockdown and knockout plants did not show yield loss under normal conditions, but exhibited enhanced drought tolerance and delayed leaf senescence. The chlorophyll content, the free amino acid content and the expression of senescence-related genes in the RCAL mutant were lower than those in the wild-type plants under drought stress. In summary, Ghd2 induces leaf senescence by upregulating RCAL expression under drought stress, and the RCAL mutant has important values in breeding drought-tolerant varieties.
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Oryza , Sequías , Regulación de la Expresión Génica de las Plantas/genética , Oryza/metabolismo , Fitomejoramiento , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Estrés Fisiológico , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The acute ischemic stroke therapy of choice is the application of Alteplase, a drug containing the enzyme tissue-type plasminogen activator (tPa) which rapidly destabilizes blood clots. A central hallmark of stroke pathology is blood-brain barrier (BBB) breakdown associated with tight junction (TJ) protein degradation, which seems to be significantly more severe under therapeutic conditions. The exact mechanisms how tPa facilitates BBB breakdown are not entirely understood. There is evidence that an interaction with the lipoprotein receptor-related protein 1 (LRP1), allowing tPa transport across the BBB into the central nervous system, is necessary for this therapeutic side effect. Whether tPa-mediated disruption of BBB integrity is initiated directly on microvascular endothelial cells or other brain cell types is still elusive. In this study we could not observe any changes of barrier properties in microvascular endothelial cells after tPa incubation. However, we present evidence that tPa causes changes in microglial activation and BBB breakdown after LRP1-mediated transport across the BBB. Using a monoclonal antibody targeting the tPa binding sites of LRP1 decreased tPa transport across an endothelial barrier. Our results indicate that limiting tPa transport from the vascular system into the brain by coapplication of a LRP1-blocking monoclonal antibody might be a novel approach to minimize tPa-related BBB damage during acute stroke therapy.
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Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Activador de Tejido Plasminógeno/efectos adversos , Activador de Tejido Plasminógeno/metabolismo , Células Endoteliales/metabolismo , Accidente Cerebrovascular Isquémico/inducido químicamente , Accidente Cerebrovascular Isquémico/complicaciones , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patología , Anticuerpos Monoclonales/uso terapéutico , Lipoproteínas LDLRESUMEN
Rubisco activase (RCA) catalyzes the release of inhibitory sugar phosphates from ribulose-1,6-biphosphate carboxylase/oxygenase (Rubisco) and can play an important role in biochemical limitations of photosynthesis under dynamic light and elevated temperatures. There is interest in increasing RCA activity to improve crop productivity, but a lack of understanding about the regulation of photosynthesis complicates engineering strategies. In this review, we discuss work relevant to improving RCA with a focus on advances in understanding the structural cause of RCA instability under heat stress and the regulatory interactions between RCA and components of photosynthesis. This reveals substantial variation in RCA thermostability that can be influenced by single amino acid substitutions, and that engineered variants can perform better in vitro and in vivo under heat stress. In addition, there are indications RCA activity is controlled by transcriptional, post-transcriptional, post-translational, and spatial regulation, which may be important for balancing between carbon fixation and light capture. Finally, we provide an overview of findings from recent field experiments and consider the requirements for commercial validation as part of efforts to increase crop yields in the face of global climate change.
Asunto(s)
Ribulosa-Bifosfato Carboxilasa , Activador de Tejido Plasminógeno , Ribulosa-Bifosfato Carboxilasa/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Fotosíntesis/fisiología , Proteínas de Plantas/metabolismoRESUMEN
Vitreomacular traction syndrome results from persistent vitreoretinal adhesions in the setting of partial posterior vitreous detachment (PVD). Vitrectomy and reattachment of retina is an effective therapeutic approach. The adhesion between vitreous cortex and internal limiting membrane (ILM) of the retina is stronger in youth, which brings difficulties to induce PVD in vitrectomy. Several clinical investigations demonstrated that intravitreous injection of plasmin before vitrectomy could reduce the risk of detachment. In our study, a novel recombinant human microplasminogen (rhµPlg) was expressed by Pichia pastoris. Molecular docking showed that the binding of rhµPlg with tissue plasminogen activator (t-PA) was similar to plasminogen, suggesting rh µPlg could be activated by t-PA to generate microplasmin (µPlm). Moreover, rhµPlg had higher catalytic activity than plasminogen in amidolytic assays. Complete PVD was found at vitreous posterior pole of 125 µg rhµPlg-treated eyes without morphological change of retina in juvenile rabbits via intraocular injection. Our results demonstrate that rhµPlg has a potential value in the treatment of vitreoretinopathy.