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1.
Am J Physiol Gastrointest Liver Physiol ; 327(1): G25-G35, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38713618

RESUMEN

Cholesterol is essential for the stability and architecture of the plasma membrane and a precursor of bile acids and steroid hormones in mammals. Excess dietary cholesterol uptake leads to hypercholesterolemia and atherosclerosis and plays a role in cancer development. The role of actin-binding scaffolding protein LIM and SH3 protein 1 (LASP1) in cholesterol trafficking has not been investigated previously. Cholesterol levels, its uptake, and excretion were studied in mice deficient for low-density lipoprotein receptor and Lasp1 (Ldlr-/-Lasp1-/- mice) upon feeding a high-fat diet, and in LASP1-knockdown, differentiated human intestinal epithelial CaCo-2 cells. When compared with diet-fed Ldlr-/- control mice, Ldlr-/-Lasp1-/- mice displayed a reduction in serum cholesterol levels. Mechanistically, we identified a new role of LASP1 in controlling the translocation of the intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) to the apical cell surface, which was limited in LASP1-knockdown human CaCo-2 enterocytes and in the intestine of Ldlr-/- Lasp1-/- compared with Ldlr-/- mice, linked to LASP1-pAKT signaling but not CDC42 activation. In line, a reduction in cholesterol reabsorption was noted in LASP1-knockdown CaCo-2 cells in vitro, and an enhanced cholesterol excretion via the feces was observed in Ldlr-/- Lasp1-/- mice. These data uncover a novel function of Lasp1 in cholesterol trafficking, promoting cholesterol reabsorption in the intestine. Targeting LASP1 locally could thus represent a novel targeting strategy to ameliorate hypercholesterolemia and associated diseases.NEW & NOTEWORTHY We here uncovered LASP1 as a novel regulator of the shuttling of the sterol transporter NPC1L1 to the cell surface in enterocytes to control cholesterol absorption. Accordingly, LASP1-deficient mice displayed lowered serum cholesterol levels under dietary cholesterol supplementation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Colesterol , Proteínas del Citoesqueleto , Proteínas con Dominio LIM , Proteínas de Transporte de Membrana , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Células CACO-2 , Humanos , Proteínas con Dominio LIM/metabolismo , Proteínas con Dominio LIM/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones , Colesterol/metabolismo , Colesterol/sangre , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Receptores de LDL/metabolismo , Receptores de LDL/genética , Mucosa Intestinal/metabolismo , Enterocitos/metabolismo , Absorción Intestinal , Dieta Alta en Grasa , Proteínas de Homeodominio
2.
J Theor Biol ; 550: 111222, 2022 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-35843440

RESUMEN

BACKGROUND: The cyclic nucleotides cAMP and cGMP inhibit platelet activation. Different platelet signaling modules work together. We develop here a modelling framework to integrate different signaling modules and apply it to platelets. RESULTS: We introduce a novel standardized bilinear coupling mechanism allowing sub model debugging and standardization of coupling with optimal data driven modelling by methods from optimization. Besides cAMP signaling our model considers specific cGMP effects including external stimuli by drugs. Moreover, the output of the cGMP module serves as input for a modular model of VASP phosphorylation and for the activity of cAMP and cGMP pathways in platelets. Experimental data driven modeling allows us to design models with quantitative output. We use the condensed information about involved regulation and system responses for modeling drug effects and obtaining optimal experimental settings. Stepwise further validation of our model is given by direct experimental data. CONCLUSIONS: We present a general framework for model integration using modules and their stimulus responses. We demonstrate it by a multi-modular model for platelet signaling focusing on cGMP and VASP phosphorylation. Moreover, this allows to estimate drug action on any of the inhibitory cyclic nucleotide pathways (cGMP, cAMP) and is supported by experimental data.


Asunto(s)
Plaquetas , AMP Cíclico , GMP Cíclico , Nucleótidos Cíclicos , Fosfoproteínas , Fosforilación
3.
Int J Mol Sci ; 23(3)2022 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-35163387

RESUMEN

Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed-neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets.


Asunto(s)
Plaquetas/metabolismo , Ciclofilina A/metabolismo , Activación Plaquetaria , Acetilación , Animales , Humanos , Lisina , Ratones
4.
J Neurosci ; 40(3): 526-541, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31754010

RESUMEN

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.


Asunto(s)
Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/fisiología , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Dominios Homologos src/genética , Dominios Homologos src/fisiología , Actinas/metabolismo , Animales , Dendritas/ultraestructura , Espinas Dendríticas/fisiología , Espinas Dendríticas/ultraestructura , Potenciales Postsinápticos Excitadores/genética , Técnicas de Silenciamiento del Gen , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/fisiología , Red Nerviosa/citología , Red Nerviosa/crecimiento & desarrollo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas
5.
FASEB J ; 34(4): 5453-5464, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32086849

RESUMEN

The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane-associated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASP-1, an actin-binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP-1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP-1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton-bound protein. We identify CD2AP as a novel LASP-1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin-angiotensin-aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP-1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.


Asunto(s)
Citoesqueleto de Actina/fisiología , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Riñón/fisiología , Proteínas de Microfilamentos/metabolismo , Podocitos/fisiología , Animales , Proteínas de Drosophila/genética , Proteínas de Microfilamentos/genética , Fosforilación
6.
J Cell Mol Med ; 24(5): 2942-2955, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31957290

RESUMEN

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR-ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR-ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1-mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell-mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Resistencia a Antineoplásicos , Técnicas de Inactivación de Genes , Proteínas con Dominio LIM/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Inhibidores de Proteínas Quinasas/farmacología , Receptores CXCR4/metabolismo , Adenosina Trifosfato/metabolismo , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Biosíntesis de Proteínas/efectos de los fármacos , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Transcripción Genética/efectos de los fármacos , Resultado del Tratamiento
7.
Int J Cancer ; 144(4): 859-867, 2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30267407

RESUMEN

Soft-tissue sarcomas are rare, heterogeneous, and often aggressive mesenchymal cancers. Many of them are associated with poor outcome, partially because biomarkers that can identify high-risk patients are lacking. Studies on sarcomas are often limited by small sample-sizes rendering the identification of biomarkers difficult when focusing on individual cohorts. However, the increasing number of publicly available 'omics' data opens inroads to overcome this obstacle. Here, we combine transcriptome analyses, immunohistochemistry, and functional assays to show that high adenosine monophosphate deaminase 2 (AMPD2) is a robust prognostic biomarker for worse outcome in undifferentiated pleomorphic sarcoma (UPS). Gene expression and survival data for UPS from two independent studies were subjected to survival association-testing. Genes, whose high expression was significantly correlated with worse outcome in both cohorts, were considered as biomarker candidates. The best candidate, AMPD2, was validated in a tissue microarray. Analysis of DNA copy-number data and matched transcriptomes indicated that high AMPD2 expression is significantly correlated with gains at the AMPD2 locus. Gene set enrichment analyses of AMPD2 co-expressed genes in both transcriptome datasets suggested that AMPD2-high UPS are enriched in tumorigenic signatures. Consistently, knockdown of AMPD2 by RNA interference in an UPS cell line inhibited proliferation in vitro and tumorigenicity in vivo. Collectively, we provide evidence that AMPD2 may serve as a biomarker for outcome prediction in UPS. Our study exemplifies how the integration of 'omics' data, immunohistochemistry, and functional experiments can identify novel biomarkers even in a rare sarcoma, which may serve as a blueprint for biomarker identification for other rare cancers.


Asunto(s)
AMP Desaminasa/genética , Biomarcadores de Tumor/genética , Genómica/métodos , Histiocitoma Fibroso Maligno/genética , AMP Desaminasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histiocitoma Fibroso Maligno/metabolismo , Histiocitoma Fibroso Maligno/patología , Humanos , Estimación de Kaplan-Meier , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Pronóstico , Tratamiento con ARN de Interferencia/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Adulto Joven
8.
Drug Metab Dispos ; 46(11): 1827-1835, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30154105

RESUMEN

Hepatic induction in response to drugs and environmental chemicals affects drug therapies and energy metabolism. We investigated whether the induction is transmitted to the offspring. We injected 3-day- and 6-week-old F0 female mice with TCPOBOP, an activator of the nuclear receptor constitutive androstane receptor (CAR, NR1I3), and mated them 1-6 weeks afterward. We detected in the offspring long-lasting alterations of CAR-mediated drug disposition, energy metabolism, and lipid profile. The transmission to the first filial generation (F1) was mediated by TCPOBOP transfer from the F0 adipose tissue via milk, as revealed by embryo transfer, crossfostering experiments, and liquid chromatography-mass spectrometry analyses. The important environmental pollutant PCB153 activated CAR in the F1 generation in a manner similar to TCPOBOP. Our findings indicate that chemicals accumulating and persisting in adipose tissue may exert liver-mediated, health-relevant effects on F1 offspring simply via physical transmission in milk. Such effects may occur even if treatment has been terminated far ahead of conception. This should be considered in assessing developmental toxicity and in the long-term follow-up of offspring of mothers exposed to both approved and investigational drugs, and to chemicals with known or suspected accumulation in adipose tissue.


Asunto(s)
Receptores Citoplasmáticos y Nucleares/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Receptor de Androstano Constitutivo , Femenino , Hígado , Ratones , Ratones Endogámicos C57BL , Fenotipo , Embarazo , Piridinas/farmacología
9.
Biol Cell ; 105(8): 317-33, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23611148

RESUMEN

Lysophosphatidic acid (LPA) is a ubiquitously present signalling molecule involved in diverse cellular processes such as cell migration, proliferation and differentiation. LPA acts as an autocrine and/or paracrine signalling molecule via different G-protein-coupled LPA receptors (LPARs) that trigger a broad range of intracellular signalling cascades, especially the RHOA pathway. Mounting evidence suggests a crucial role of the LPA/LPAR-axis in cancer cell metastasis and promising studies are underway to investigate the therapeutic potential of LPAR-antagonists. This review summarises current knowledge on how LPA promotes cytoskeletal remodelling to enhance the migratory and invasive properties of cells, which may ultimately contribute to cancer metastasis. Furthermore, we provide comprehensive transcriptome analyses of published microarrays of more than 350 normal tissues and more than 1700 malignant tissues to define the expression signatures of LPARs and the LPA-generating enzymes autotaxin (ATX) and lipase member 1 (LIPI). These analyses demonstrate that ATX is highly expressed in a variety of carcinomas and sarcomas, whereas LIPI is almost exclusively overexpressed in highly aggressive Ewing's sarcomas, which underscores the potential contribution of LPA in metastatic disease. In addition, these analyses show that different cancer entities display distinct expression signatures of LPARs that distinguish them from one another. Finally, we discuss current approaches to specifically target the LPA/LPAR circuits in experimental cancer therapy.


Asunto(s)
Movimiento Celular , Lisofosfolípidos/metabolismo , Neoplasias/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Transducción de Señal , Animales , Humanos , Invasividad Neoplásica , Neoplasias/genética , Neoplasias/patología , Neoplasias/fisiopatología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores del Ácido Lisofosfatídico/metabolismo
10.
Biol Cell ; 105(11): 535-47, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24033704

RESUMEN

BACKGROUND INFORMATION: Ewing's sarcoma (ES) is the second most common bone-associated malignancy in children and is driven by the fusion oncogene EWS/FLI1 and characterised by rapid growth and early metastasis. Here, we explored the role of the Zyxin-related protein thyroid receptor interacting protein 6 (TRIP6) in ES. The Zyxin family comprises seven homologous proteins involved in migration and proliferation of many cell types of which Zyxin has been described as a tumour suppressor in ES. RESULTS: By interrogation of published microarray data (n = 1254), we observed that of all Zyxin proteins, only TRIP6 is highly overexpressed in primary ES compared with normal tissues. Re-analysis of published EWS/FLI1 gain- and loss-of-function microarray experiments as well as chromatin-immunoprecipitation assays revealed that TRIP6 overexpression is not mediated by EWS/FLI1. Microarray and subsequent gene-set enrichment analyses of ES cells with and without RNA interference-mediated TRIP6 knockdown demonstrated that TRIP6 expression confers a pro-proliferative and pro-invasive transcriptional signature to ES cells. While short-term proliferation was not considerably affected by TRIP6 knockdown, silencing of the protein significantly reduced migration, invasion, long-term proliferation and clonogenicity of ES cells in vitro as well as tumourigenicity in vivo. CONCLUSIONS: Taken together, our data indicate that TRIP6 acts, in contrast to Zyxin, as an oncogene that partially accounts for the autonomous migratory, invasive and proliferative properties of ES cells independent of EWS/FLI1.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular , Proteínas con Dominio LIM/metabolismo , Sarcoma de Ewing/patología , Factores de Transcripción/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Células Clonales , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Complejo de la Endopetidasa Proteasomal , Sarcoma de Ewing/genética
11.
Cardiovasc Res ; 120(4): 385-402, 2024 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-38175781

RESUMEN

AIMS: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. METHODS AND RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. CONCLUSION: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.


Asunto(s)
Ciclofilina A , Trombosis , Ratones , Humanos , Animales , Ciclofilina A/genética , Ciclofilina A/metabolismo , Productos Finales de Glicación Avanzada , Ligandos , Inflamación , Basigina/metabolismo , Trombosis/genética
12.
Pharmaceuticals (Basel) ; 17(1)2023 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-38256878

RESUMEN

Kisspeptins (KPs, KISS1) and their receptor (KISS1R) play a pivotal role as metastasis suppressor for many cancers. Low or lost KP expression is associated with higher tumor grade, increased metastatic potential, and poor prognosis. Therefore, KP expression has prognostic relevance and correlates with invasiveness in cancers. Furthermore, KISS1R represents a very promising target for molecular imaging and therapy for KISS1R-expressing tumors. The goal of this study was to evaluate the developed KISS1-54 derivative, [68Ga]KISS1-54, as a PET-imaging probe for KISS1R-expressing tumors. The NODAGA-KISS1-54 peptide was labeled by Gallium-68, and the stability of the resulting [68Ga]KISS1-54 evaluated in injection solution and human serum, followed by an examination in different KISS1R-expressing tumor cell lines, including HepG2, HeLa, MDA-MB-231, MCF7, LNCap, SK-BR-3, and HCT116. Finally, [68Ga]KISS1-54 was tested in LNCap- and MDA-MB-231-bearing mice, using µ-PET, assessing its potential as an imaging probe for PET. [68Ga]KISS1-54 was obtained in a 77 ± 7% radiochemical yield and at a >99% purity. The [68Ga]KISS1-54 cell uptake amounted to 0.6-4.4% per 100,000 cells. Moreover, the accumulation of [68Ga]KISS1-54 was effectively inhibited by nonradioactive KISS1-54. In [68Ga]KISS1-54-PET, KISS1R-positive LNCap-tumors were clearly visualized as compared to MDA-MB-231-tumor implant with predominantly intracellular KISS1R expression. Our first results suggest that [68Ga]KISS1-54 is a promising candidate for a radiotracer for targeting KISS1R-expressing tumors via PET.

13.
Biol Cell ; 103(12): 573-91, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-22054418

RESUMEN

Integrating signals from the ECM (extracellular matrix) via the cell surface into the nucleus is an essential feature of multicellular life and often malfunctions in cancer. To date many signal transducers known as shuttle proteins have been identified that act as both: a cytoskeletal and a signalling protein. Here, we highlight the interesting member of the Zyxin family TRIP6 [thyroid receptor interactor protein 6; also designated ZRP-1 (zyxin-related protein 1)] and review current literature to define its role in cell physiology and cancer. TRIP6 is a versatile scaffolding protein at FAs (focal adhesions) involved in cytoskeletal organization, coordinated cell migration and tissue invasion. Via its LIM and TDC domains TRIP6 interacts with different components of the LPA (lysophosphatidic acid), NF-κB (nuclear factor κB), glucocorticoid and AMPK (AMP-activated protein kinase) signalling pathway and thereby modulates their activity. Within the nucleus TRIP6 acts as a transcriptional cofactor regulating the transcriptional responses of these pathways. Moreover, intranuclear TRIP6 associates with proteins ensuring telomere protection and hence may contribute to genome stability. Accordingly, TRIP6 is engaged in key cellular processes such as cell proliferation, differentiation and survival. These diverse functions of TRIP6 are found to be dysregulated in various cancers and may have pleiotropic roles in tumour initiation, tumour growth and metastasis, which turn TRIP6 into an attractive candidate for cancer diagnosis and targeted therapy.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas con Dominio LIM/metabolismo , Neoplasias/metabolismo , Factores de Transcripción/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular , Proliferación Celular , Humanos , Proteínas con Dominio LIM/genética , Neoplasias/genética , Neoplasias/fisiopatología , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Transducción de Señal , Factores de Transcripción/genética
14.
Cells ; 11(23)2022 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-36497077

RESUMEN

LIM and SH3 protein 1 was originally identified as a structural cytoskeletal protein with scaffolding function. However, recent data suggest additional roles in cell signaling and gene expression, especially in tumor cells. These novel functions are primarily regulated by the site-specific phosphorylation of LASP1. This review will focus on specific phosphorylation-dependent interaction between LASP1 and cellular proteins that orchestrate primary tumor progression and metastasis. More specifically, we will describe the role of LASP1 in chemokine receptor, and PI3K/AKT signaling. We outline the nuclear role for LASP1 in terms of epigenetics and transcriptional regulation and modulation of oncogenic mRNA translation. Finally, newly identified roles for the cytoskeletal function of LASP1 next to its known canonical F-actin binding properties are included.


Asunto(s)
Proteínas con Dominio LIM , Fosfatidilinositol 3-Quinasas , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Expresión Génica
15.
Circ Res ; 104(1): 87-94, 2009 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-19023132

RESUMEN

The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein-protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.


Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Insuficiencia Cardíaca Diastólica/metabolismo , Ventrículos Cardíacos/metabolismo , Proteínas Musculares/metabolismo , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Conectina , Secuencia de Consenso , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , GMP Cíclico/fisiología , Perros , Elasticidad , Humanos , Datos de Secuencia Molecular , Proteínas Musculares/genética , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Miofibrillas/efectos de los fármacos , Miofibrillas/ultraestructura , Óxido Nítrico/fisiología , Fosforilación , Isoformas de Proteínas/metabolismo , Proteínas Quinasas/genética , Proteínas Recombinantes de Fusión/fisiología , Sarcómeros/metabolismo , Sarcómeros/ultraestructura , Relación Estructura-Actividad , Remodelación Ventricular/fisiología
16.
Cancers (Basel) ; 12(9)2020 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-32825729

RESUMEN

The CXCL12-CXCR4 axis plays a vital role in many steps of breast cancer metastasis, but the molecular mechanisms have not been fully elucidated. We previously reported that activation of CXCR4 by CXCL12 promotes the nuclear localization of LASP1 (LIM and SH3 protein 1). The nuclear LASP1 then interacts with Snail1 in triple-negative breast cancer (TNBC) cell lines. In this study, we report that the nuclear accumulation and retention of Snail1 was dependent on an increase in nuclear LASP1 levels driven by active CXCR4. The CXCR4-LASP1 axis may directly regulate the stabilization of nuclear Snail1, by upregulating nuclear levels of pS473-Akt, pS9-GSK-3ß, A20, and LSD1. Furthermore, the activation of CXCR4 induced association of LASP1 with Snail1, A20, GSK-3ß, and LSD1 endogenously. Thus, nuclear LASP1 may also regulate protein-protein interactions that facilitate the stability of Snail1. Genetic ablation of LASP1 resulted in the mislocalization of nuclear Snail1, loss of the ability of TNBC cells to invade Matrigel and a dysregulated expression of both epithelial and mesenchymal markers, including an increased expression of ALDH1A1, a marker for epithelial breast cancer stem-like cells. Our findings reveal a novel role for the CXCR4-LASP1 axis in facilitating the stability of nuclear localized Snail1.

17.
Cells ; 9(2)2020 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-32075106

RESUMEN

The serine/threonine protein kinase AKT1 is a downstream target of the chemokine receptor 4 (CXCR4), and both proteins play a central role in the modulation of diverse cellular processes, including proliferation and cell survival. While in chronic myeloid leukemia (CML) the CXCR4 is downregulated, thereby promoting the mobilization of progenitor cells into blood, the receptor is highly expressed in breast cancer cells, favoring the migratory capacity of these cells. Recently, the LIM and SH3 domain protein 1 (LASP1) has been described as a novel CXCR4 binding partner and as a promoter of the PI3K/AKT pathway. In this study, we uncovered a direct binding of LASP1, phosphorylated at S146, to both CXCR4 and AKT1, as shown by immunoprecipitation assays, pull-down experiments, and immunohistochemistry data. In contrast, phosphorylation of LASP1 at Y171 abrogated these interactions, suggesting that both LASP1 phospho-forms interact. Finally, findings demonstrating different phosphorylation patterns of LASP1 in breast cancer and chronic myeloid leukemia may have implications for CXCR4 function and tyrosine kinase inhibitor treatment.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas con Dominio LIM/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Fosforilación , Transfección
18.
Thromb Haemost ; 102(3): 520-8, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19718473

RESUMEN

During platelet adhesion, the complex cytoskeletal structure is rearranged resulting in the formation of F-actin-based filopodia and lamellipodia. Stimulatory platelet signalling pathways include binding of integrin alpha(IIb)beta(3) to fibrinogen followed by activation of protein tyrosine kinases (PTK) and phosphorylation of downstream signalling proteins. In this study, we demonstrate that the scaffolding and F-actin binding protein LASP-1 undergoes tyrosine phosphorylation in thrombin-stimulated human platelets. By means of specific inhibitors we identified Src-kinase as the primary enzyme phosphorylating LASP-1 in intact cells. These data were confirmed in platelet model cells (A5-CHO cells), constitutively expressing integrin alpha(IIb)beta(3). Fibrinogen-mediated cell stimulation resulted in a similar tyrosine phosphorylation of transiently transfected LASP-1. Site directed mutagenesis identified tyrosine 171 as the Src-kinase phosphorylation site. Immunofluorescence microscopic analysis of these cells revealed a relocation of LASP-1 to focal contacts and the leading edge of the membrane upon fibrinogen activation and tyrosine 171 phosphorylation. This translocation was also seen in adherent platelets. Concomitant with adhesion, LASP-1 translocated from the cytosol along the arms of the pseudopodia into the leading lamellae of the spreading platelets, indicating a crucial role of the protein in platelet cytoskeleton rearrangement.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Plaquetas/metabolismo , Proteínas del Citoesqueleto/fisiología , Citoesqueleto/metabolismo , Integrinas/metabolismo , Adhesividad Plaquetaria , Tirosina/química , Familia-src Quinasas/metabolismo , Actinas/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas con Dominio LIM , Fosforilación , Transporte de Proteínas
19.
Handb Exp Pharmacol ; (191): 409-21, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19089338

RESUMEN

The first cGMP-dependent protein kinase (PKG) modulators were described nearly 30 years ago and since then more than 200 compounds have been synthesized and tested, but only a small subset of these compounds has found widespread application. The aim of this review is to suggest a framework for evaluating and using PKG activators and inhibitors and to explore and interpret PKG signal transduction in cell culture-based model systems. Therefore, cross-reactivity of cGMP-analogs with other classes of cyclic nucleotide binding proteins, as well as the advantages and problems of newly designed PKG inhibitors, are discussed. Additional information and a search option are available at www.cyclic-nucleotides.org


Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/efectos de los fármacos , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Animales , Células Cultivadas , GMP Cíclico/análogos & derivados , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Humanos , ARN Interferente Pequeño/metabolismo , Transducción de Señal
20.
Mol Cancer ; 7: 31, 2008 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-18419822

RESUMEN

LIM and SH3 Protein 1 (LASP-1) was initially identified from a cDNA library of metastatic axillary lymph nodes (MLN) more than a decade ago. It was found to be overexpressed in human breast and ovarian cancer and became the first member of a newly defined LIM-protein subfamily of the nebulin group characterized by the combined presence of LIM and SH3 domains. LASP2, a novel LASP1-related gene was first identified and characterized in silico. Subsequently it proved to be a splice variant of the Nebulin gene and therefore was also termed LIM/nebulette. LASP-1 and -2 are highly conserved in their LIM, nebulin-like and SH3 domains but differ significantly at their linker regions. Both proteins are ubiquitously expressed and involved in cytoskeletal architecture, especially in the organization of focal adhesions. Here we present the first systematic review to summarize all relevant data concerning their domain organization, expression profiles, regulating factors and function. We compile evidence that both, LASP-1 and LASP-2, are important during early embryo- and fetogenesis and are highly expressed in the central nervous system of the adult. However, only LASP-1 seems to participate significantly in neuronal differentiation and plays an important functional role in migration and proliferation of certain cancer cells while the role of LASP-2 is more structural. The increased expression of LASP-1 in breast tumours correlates with high rates of nodal-metastasis and refers to a possible relevance as a prognostic marker.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transducción de Señal , Dominios Homologos src , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas con Dominio LIM , Neoplasias/metabolismo , Unión Proteica
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