RESUMEN
BACKGROUND: Vascular surgical patients have a high rate of readmission, and the cost of readmission for these patients has not been described. Herein, we characterize and compare institutional index hospitalization and 30-day readmission cost following open and endovascular vascular procedures. METHODS: The American College of Surgeons National Surgical Quality Improvement Program database was used to identify inpatient open and endovascular procedures at a single institution, from January 2011 through June 2012. Variable and fixed costs for index hospitalization and unplanned 30-day readmissions were obtained using SAP BusinessObjects. Patient characteristics and outcome variables were analyzed using Student t tests or Wilcoxon rank-sum nonparametric tests for continuous variables and Fisher exact tests for categorical variables. RESULTS: One thousand twenty-six inpatient procedures were included in the analysis. There were 605 (59%) open and 421 (41%) endovascular procedures with a 30-day unplanned readmission rate of 16.9% and 17.8%, respectively (P = .679). The mean index hospitalization costs for open and endovascular procedures were US$27 653 and US$23 999, respectively (P = .146). The mean costs for 30-day unplanned readmission for open and endovascular procedures were US$19 117 and US$17 887, respectively (P = .635). Among open procedures, the mean cost for patients not readmitted was US$28 321 compared to US$31 115 for those readmitted (P = .003). Among endovascular procedures, the mean cost for patients not readmitted was US$26 908 compared to US$32 262 for those readmitted (P = .028). CONCLUSION: The cost of index hospitalization and 30-day unplanned readmission are similar for open and endovascular procedures. Readmitted patients had a higher mean index hospitalization cost irrespective of open or endovascular procedure.
Asunto(s)
Implantación de Prótesis Vascular/economía , Procedimientos Endovasculares/economía , Costos de Hospital , Readmisión del Paciente/economía , Anciano , Anciano de 80 o más Años , Implantación de Prótesis Vascular/efectos adversos , Bases de Datos Factuales , Procedimientos Endovasculares/efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Económicos , Modelos Estadísticos , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Estados UnidosRESUMEN
BACKGROUND: Lung transplantation outcomes are among the least favorable, with most recipients eventually developing bronchiolitis obliterans syndrome (BOS) and subsequent graft failure. The presence of human leukocyte antigen (HLA)-DR has been implicated in the pathogenesis of BOS and may play a role in these poor outcomes. METHODS: Lung transplant donor and recipient data were retrospectively gathered from the United Network for Organ Sharing database from January 2006 to June 2013. Donor and recipient characteristics, proportion of recipients treated for first year rejection, and 5-y rates of survival and freedom from BOS were determined according to HLA-DR1, -DR7, -DR13, and -DR15 status in both donor and recipient. Each HLA-DR allele was stratified by donor-recipient pair positivity status. RESULTS: A total of 7402 lung transplant recipients met the inclusion and exclusion criteria. There were significant but small differences in donor and recipient characteristics for each HLA-DR group. The recipients in the D(-)R(+) pairing for HLA-DR13 and those in the D(+)R(-) pairing for HLA-DR15 had significantly higher rates of receiving treatment for rejection within the first year after transplant (P = 0.024 and P = 0.001, respectively). There were no differences in 5-y survival or freedom from BOS for any of the four HLA-DR alleles studied. CONCLUSIONS: There are higher rates of patients treated for rejection within the first year who are either negative for the HLA-DR15 allele but received a donor-positive lung or positive for the HLA-DR13 allele but received a donor-negative lung for that allele. However, these differences do not appear to affect long-term outcomes.
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Bronquiolitis Obliterante/inmunología , Rechazo de Injerto/inmunología , Subtipos Serológicos HLA-DR/metabolismo , Trasplante de Pulmón , Complicaciones Posoperatorias/inmunología , Adulto , Anciano , Biomarcadores/metabolismo , Bronquiolitis Obliterante/etiología , Femenino , Humanos , Modelos Logísticos , Trasplante de Pulmón/mortalidad , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
The abdominal aortic aneurysm (AAA) is a disease process that carries significant morbidity and mortality in the absence of early identification and treatment. While current management includes surveillance and surgical treatment of low- and high-risk aneurysms, respectively, our narrow understanding of the pathophysiology of AAAs limits our ability to more effectively manage and perhaps even prevent the occurrence of this highly morbid disease. Over the past couple of decades, there has been considerable interest in exploring the role of autoimmunity as an etiological component of AAA. This review covers the current literature pertaining to this immunological process, focusing on research that highlights the local and systemic immune components found in both human patients and murine models. A better understanding of the autoimmune mechanisms in the pathogenesis of AAAs can pave the way to novel and improved treatment strategies in this patient population.
Asunto(s)
Aneurisma de la Aorta Abdominal/inmunología , Autoinmunidad , Animales , Aneurisma de la Aorta Abdominal/etiología , HumanosRESUMEN
Although recipient body mass index (BMI) and age are known risk factors for mortality after heart transplantation, how they interact to influence survival is unknown. Our study utilized the UNOS registry from 1997 to 2012 to define the interaction between BMI and age and its impact on survival after heart transplantation. Recipients were stratified by BMI: underweight (<18.5), normal weight (18.5-24.99), overweight (25-29.99), and either moderate (30-34.99), severe (35-39.99), or very severe (≥40) obesity. Recipients were secondarily stratified based on age: 18-40 (younger recipients), 40-65 (reference group), and ≥65 (advanced age recipients). Among younger recipients, being underweight was associated with improved adjusted survival (HR 0.902; p = 0.010) while higher mortality was seen in younger overweight recipients (HR 1.260; p = 0.005). However, no differences in adjusted survival were appreciated in underweight and overweight advanced age recipients. Obesity (BMI ≥ 30) was associated with increased adjusted mortality in normal age recipients (HR 1.152; p = 0.021) and even more so with young (HR 1.576; p < 0.001) and advanced age recipients (HR 1.292; p = 0.001). These results demonstrate that BMI and age interact to impact survival as age modifies BMI-mortality curves, particularly with younger and advanced age recipients.
Asunto(s)
Factores de Edad , Índice de Masa Corporal , Insuficiencia Cardíaca/mortalidad , Insuficiencia Cardíaca/cirugía , Trasplante de Corazón/mortalidad , Adulto , Anciano , Bases de Datos Factuales , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Tasa de SupervivenciaRESUMEN
Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation, where more than half of donor allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high-mobility group box-1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE-sufficient (wild-type) or RAGE-deficient (RAGE(-/-)) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardiopulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia, and decreased compliance (a measure of the lungs' ability to expand), all of which were attenuated in RAGE(-/-) mice. Neutralizing systemic HMGB1 induced by TBI reversed hypoxia and improved lung compliance. Compared to wild-type donors, lungs from RAGE(-/-) TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung before transplantation and predicted impaired oxygenation after transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway before transplant may improve recipient outcomes after lung transplantation.
Asunto(s)
Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/fisiopatología , Proteína HMGB1/metabolismo , Trasplante de Pulmón , Pulmón/fisiopatología , Receptores Inmunológicos/metabolismo , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/fisiopatología , Adulto , Animales , Anticuerpos Neutralizantes/farmacología , Lesiones Encefálicas/complicaciones , Gasto Cardíaco/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Interleucina-10/metabolismo , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Péptidos/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/deficiencia , Donantes de Tejidos , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/metabolismoRESUMEN
Despite significant medical advances since the advent of lung transplantation, improvements in long-term survival have been largely unrealized. Chronic lung allograft dysfunction, in particular obliterative bronchiolitis, is the primary limiting factor. The predominant etiology of obliterative bronchiolitis involves the recipient's innate and adaptive immune response to the transplanted allograft. Current therapeutic strategies have failed to provide a definitive treatment paradigm to improve long-term outcomes. Inducing immune tolerance is an emerging therapeutic strategy that abrogates allograft rejection, avoids immunosuppression, and improves long-term graft function. The aim of this review is to discuss the key immunologic components of obliterative bronchiolitis, describe the state of establishing immune tolerance in transplantation, and highlight those strategies being evaluated in lung transplantation.
Asunto(s)
Bronquiolitis Obliterante/prevención & control , Rechazo de Injerto/prevención & control , Isoantígenos/administración & dosificación , Trasplante de Pulmón , Tolerancia al Trasplante/efectos de los fármacos , Inmunidad Adaptativa , Administración Oral , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/patología , Bronquiolitis Obliterante/inmunología , Bronquiolitis Obliterante/patología , Complejo CD3/genética , Complejo CD3/inmunología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Humanos , Inmunidad Innata , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Quimera por Trasplante , Trasplante HomólogoRESUMEN
BACKGROUND: Data are limited regarding the influence of donor age on outcomes after heart transplantation. We sought to determine if advanced donor age is associated with differences in survival after heart transplantation and how this compares to waitlist survival. METHODS: All adult heart transplants from 2000 to 2012 were identified using the United Network for Organ Sharing database. Donors were stratified into four age groups: 18-39 (reference group), 40-49, 50-54, and 55 and above. Propensity scoring was used to compare status IA waitlist patients who did not undergo transplantation with IA recipients who received hearts from advanced age donors. The primary outcome of interest was recipient survival and this was analyzed with multivariate Cox regression analysis and the Kaplan-Meier method. RESULTS: A total of 22,960 adult heart transplant recipients were identified. Recipients of hearts from all three older donor groups had significantly increased risk of mortality (HR, 1.187-1.426, all p < 0.001) compared to recipients from donors age 18 to 39. Additionally, propensity-matched status IA patients managed medically without transplantation had significantly worse adjusted survival than status IA recipients who received hearts from older donors age ≥55 (HR, 1.362, p < 0.001). CONCLUSIONS: Compared to donors aged 18-39, age 40 and above is associated with worse adjusted recipient survival in heart transplantation. This survival difference becomes more pronounced as age increases to above 55. However, the survival rate among status IA patients who receive hearts from advanced age donors (≥55) is significantly better compared to similar status IA patients who are managed without transplantation.
Asunto(s)
Trasplante de Corazón/mortalidad , Sistema de Registros , Donantes de Tejidos/estadística & datos numéricos , Obtención de Tejidos y Órganos/organización & administración , Obtención de Tejidos y Órganos/estadística & datos numéricos , Adolescente , Adulto , Factores de Edad , Anciano , Bases de Datos Factuales , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Puntaje de Propensión , Modelos de Riesgos Proporcionales , Riesgo , Tasa de Supervivencia , Adulto JovenRESUMEN
The nuclear lamins are type V intermediate filament proteins that form meshworks at the inner aspect of the nuclear envelope and are also present throughout the nuclear interior. Through these meshwork structures, lamins regulate the shape, size, and mechanical properties of the nucleus. During the last 25 years, the Goldman laboratory has studied the organization and dynamic properties of the lamins in the nucleus. These studies have characterized the role of lamin phosphorylation in nuclear lamina assembly and disassembly during mitosis. Furthermore, our studies have demonstrated a role for the lamins in chromatin modification and epigenetics, transcription, and DNA replication. Recently, the discovery of numerous mutations in the gene encoding A-type lamins causing the collection of diseases known as laminopathies has provided new insights into the roles of lamins in cellular regulation and differentiation.
Asunto(s)
Núcleo Celular/metabolismo , Enfermedad , Laminas/metabolismo , Animales , Núcleo Celular/ultraestructura , Replicación del ADN , Epigénesis Genética , Humanos , Transcripción GenéticaRESUMEN
Nuclear pore complexes, the conduits for information exchange between the nucleus and cytoplasm, appear broadly similar in eukaryotes from yeast to human. Precisely how nuclear pore complexes regulate macromolecular and ionic traffic remains unknown, but recent advances in the identification and characterization of components of the complex by proteomics and genomics have provided new insights.
Asunto(s)
Genómica , Poro Nuclear/química , Poro Nuclear/genética , Proteoma/genética , Animales , Humanos , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteoma/química , Proteoma/metabolismoRESUMEN
The importin alpha family of transport factors mediates the nuclear import of classical nuclear localization signal-containing proteins. In order to understand how multiple importin alpha proteins are regulated both in individual cells and in a whole organism, the three importin alpha (ima) genes of Caenorhabditis elegans have been identified and studied. All three IMAs are expressed in the germline; however, only IMA-3 is expressed in the soma. RNA interference (RNAi) experiments demonstrate that IMA-3 is required for the progression of meiotic prophase I during oocyte development. Loss of IMA-3 expression leads also to a disruption of the nuclear pore complex accompanied by the mis-localization of P granules. A range of defects occurring in ima-3(RNAi) F1 progeny further supports a role for IMA-3 during embryonic and larval development. The functional association of IMA-3 with distinct cellular events, its expression pattern and intracellular localization indicate that regulation of the nuclear transport machinery is involved in the control of developmental pathways.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/fisiología , Proteínas del Helminto/fisiología , Proteínas Nucleares/fisiología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Caenorhabditis elegans/genética , ADN Complementario/genética , ADN de Helmintos/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/genética , Carioferinas , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Oocitos/crecimiento & desarrollo , Oogénesis/genética , Fenotipo , ARN de Helminto/genética , Homología de Secuencia de AminoácidoRESUMEN
The guanosine triphosphatase Ran stimulates assembly of microtubule asters and spindles in mitotic Xenopus egg extracts. A carboxyl-terminal region of the nuclear-mitotic apparatus protein (NuMA), a nuclear protein required for organizing mitotic spindle poles, mimics Ran's ability to induce asters. This NuMA fragment also specifically interacted with the nuclear transport factor, importin-beta. We show that importin-beta is an inhibitor of microtubule aster assembly in Xenopus egg extracts and that Ran regulates the interaction between importin-beta and NuMA. Importin-beta therefore links NuMA to regulation by Ran. This suggests that similar mechanisms regulate nuclear import during interphase and spindle assembly during mitosis.
Asunto(s)
Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Huso Acromático/metabolismo , Proteínas de Xenopus , Proteína de Unión al GTP ran/metabolismo , Animales , Extractos Celulares , Núcleo Celular/metabolismo , Guanosina Trifosfato/metabolismo , Interfase , Carioferinas , Microtúbulos/efectos de los fármacos , Mitosis , Modelos Biológicos , Proteínas Nucleares/farmacología , Óvulo , Paclitaxel/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Huso Acromático/efectos de los fármacos , XenopusRESUMEN
Fixed-dose combination therapy has received increased interest since publication of JNC-VI report and WHO/ISH guidelines 1999. We compared in a randomized, double-blind study the efficacy and tolerability of valsartan 80 mg combined with hydrochlorothiazide (HCTZ) 12.5 mg to monotherapy with either HCTZ 12.5 mg or 25 mg in patients with essential hypertension inadequately controlled by previous HCTZ 12.5 mg monotherapy. Two hundred and seventeen patients whose blood pressure (BP) control remained poor (95 mmHg < or = sitting diastolic BP < 115 mmHg) after a 4-week single-blind period with HCTZ 12.5 mg were randomized to receive either combination therapy with valsartan 80 mg plus HCTZ 12.5 mg (V/HCTZ) or monotherapy with HCTZ 12.5 mg or HCTZ 25 mg for 8 weeks. Reduction of sitting trough diastolic BP between baseline and week 8 as well as tolerability was evaluated. Reduction in trough diastolic BP was most pronounced in the V/HCTZ group (-11.3 mmHg) and significantly greater than in the HCTZ 12.5 mg group (-2.9 mmHg, p < 0.001) and the HCTZ 25 mg group (-5.7 mmHg, p < 0.001). Tolerability of study medication was comparable between all three groups. In conclusion, switching to V/HCTZ combination therapy provides an additional lowering of BP compared to dosage increase of the thiazide in patients with BP insufficiently controlled by HCTZ 12.5 mg monotherapy.
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Antihipertensivos/administración & dosificación , Hidroclorotiazida/administración & dosificación , Hipertensión/tratamiento farmacológico , Tetrazoles/administración & dosificación , Valina/análogos & derivados , Valina/administración & dosificación , Anciano , Antihipertensivos/farmacología , Antihipertensivos/toxicidad , Presión Sanguínea/efectos de los fármacos , Método Doble Ciego , Quimioterapia Combinada , Femenino , Humanos , Hidroclorotiazida/farmacología , Hidroclorotiazida/toxicidad , Hipertensión/fisiopatología , Cinética , Masculino , Persona de Mediana Edad , Tetrazoles/farmacología , Tetrazoles/toxicidad , Equivalencia Terapéutica , Resultado del Tratamiento , Valina/farmacología , Valina/toxicidad , ValsartánRESUMEN
The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in the control of cell growth. eIF4E binds to the mRNA 5' cap structure m(7)GpppN (where N is any nucleotide), and promotes ribosome binding to the mRNA in the cytoplasm. However, a fraction of eIF4E localizes to the nucleus. Here we describe the cloning and functional characterization of a new eIF4E-binding protein, referred to as 4E-T (eIF4E-Transporter). We demonstrate that 4E-T is a nucleocytoplasmic shuttling protein that contains an eIF4E-binding site, one bipartite nuclear localization signal and two leucine-rich nuclear export signals. eIF4E forms a complex with the importin alphabeta heterodimer only in the presence of 4E-T. Overexpression of wild-type 4E-T, but not of a mutant defective for eIF4E binding, causes the nuclear accumulation of HA-eIF4E in cells treated with leptomycin B. Taken together, these results demonstrate that the novel nucleocytoplasmic shuttling protein 4E-T mediates the nuclear import of eIF4E via the importin alphabeta pathway by a piggy-back mechanism.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Factores de Iniciación de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Compartimento Celular , Fraccionamiento Celular , Núcleo Celular/metabolismo , Clonación Molecular , Secuencia Conservada , Citoplasma/metabolismo , Factor 4E Eucariótico de Iniciación , Biblioteca de Genes , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Señales de Localización Nuclear , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Unión Proteica , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Transport between the nucleus and cytoplasm involves both stationary components and mobile factors acting in concert to move macromolecules through the nuclear pore complex. Multiple transport pathways requiring both unique and shared components have been identified. In the past 18 months, new findings have shed light on the nature of some of the mobile components of these pathways. New receptor-cargo pairs for both import and export pathways have been identified extending the breadth of known transport pathways. Surprising findings on the role of Ran and energy in transport have changed our way of thinking about the mechanism of movement through the nuclear pore.
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Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas/metabolismo , ARN/metabolismo , Animales , Transporte Biológico , Humanos , Membrana Nuclear/metabolismo , Señales de Localización Nuclear , Proteínas Nucleares/fisiologíaRESUMEN
DNA can enter intact mammalian nuclei with varying degrees of efficiency in both transfected and microinjected cells, yet very little is known about the mechanism by which it crosses the nuclear membrane. Nucleocytoplasmic transport of fluorescently labeled DNA was studied using a digitonin-permeabilized cell system. DNA accumulated in the nucleus with a punctate staining pattern in over 80% of the permeabilized HeLa cells. Nuclear localization of the labeled DNA was energy dependent and occurred through the nuclear pore, but did not require the addition of soluble cytoplasmic protein factors necessary for protein import.
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Permeabilidad de la Membrana Celular/fisiología , Núcleo Celular/metabolismo , ADN/farmacocinética , Digitonina , Indicadores y Reactivos , Unión Competitiva/fisiología , Extractos Celulares/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Núcleo Celular/química , Citoplasma/química , ADN/química , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Colorantes Fluorescentes/farmacocinética , Células HeLa , Humanos , Lectinas/farmacología , Microinyecciones , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/farmacocinética , Fosfoproteínas/química , Fosfoproteínas/farmacocinética , Ficocianina/farmacocinética , Transfección , Xantenos/farmacocinéticaRESUMEN
Alternative splicing of the T-cell protein tyrosine phosphatase (TCPTP) transcript generates two forms of the enzyme that differ at their extreme C termini: a 48-kDa endoplasmic reticulum-associated form and a 45-kDa nuclear form. By affinity chromatography, using GST-TCPTP fusion proteins, we have isolated three cytoplasmic proteins of 120, 116, and 97 kDa that interact with TCPTP. The p120 protein associated with residues 377-415 from the C terminus of the 48-kDa form of TCPTP, whereas the recognition site for p97 and p116 was mapped to residues 350-381 encompassing the TCPTP nuclear localization sequence (NLS). The TCPTP NLS was shown to be bipartite, requiring basic residues 350-358 (basic cluster I) and 377-381 (basic cluster II), the sites of interaction with p97 and p116, for efficient nuclear translocation. The interaction between p97, p116, and the TCPTP NLS appeared unique in that these proteins did not form a stable interaction with the classical NLS of SV40 large T antigen or the standard bipartite NLS of nucleoplasmin. Sequence analysis of p97 identified it as the nuclear import factor p97 (importin-beta), which is an essential component of the nuclear import machinery. In assays in vitro in permeabilized cells, p97 was necessary but not sufficient for optimal nuclear import of TCPTP. We found that TCPTP co-immunoprecipitated with the nuclear import factor p97 from cell lysates and that purified recombinant p97 and TCPTP interacted directly in vitro. These results indicate selectivity in the binding of p97 and p116 to the TCPTP NLS and suggest that p97 may mediate events that are distinct from the classical nuclear import process. Moreover, these results demonstrate that the C-terminal segment of TCPTP contains docking sites for interaction with proteins that may function to target the enzyme to defined intracellular locations and in the process regulate TCPTP function.
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Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Células 3T3 , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Compartimento Celular , Células HeLa , Humanos , Carioferinas , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Fracciones Subcelulares/metabolismo , beta CarioferinasRESUMEN
The interaction of the nuclear protein import factor p97 with the nuclear localization sequence (NLS) receptor, the nuclear pore complex, and Ran/TC4 is important for coordinating the events of protein import to the nucleus. We have mapped the binding domains on p97 for the NLS receptor and the nuclear pore. The NLS receptor-binding domain of p97 maps to the C-terminal 60% of the protein between residues 356 and 876. The pore complex-binding domain of p97 maps to residues 152-352. The pore complex-binding domain overlaps the Ran-GTP- and Ran-GDP-binding domains on p97, but only Ran-GTP competes for docking in permeabilized cells. The N-ethylmaleimide sensitivity of the p97 for docking was investigated and found to be due to inhibition of p97 binding to the pore complex and to the NLS receptor. Site-directed mutagenesis of conserved cysteine residues in the pore- and receptor-binding domains identified two cysteines, C223 and C228, that were required for p97 to bind the nuclear pore. Inhibition studies on docking and accumulation of a NLS protein provided additional evidence that the domains identified biochemically are the functional domains involved in protein import. Together, these results suggest that Ran-GTP dissociates the receptor complex and prevents p97 binding to the pore by inducing a conformational change in the structure of p97 rather than simple competition for binding sites.
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Núcleo Celular/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Sitios de Unión , Transporte Biológico , Compartimento Celular , Cisteína/fisiología , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Mutación Puntual , alfa Carioferinas , Proteína de Unión al GTP ranRESUMEN
Nucleolar phosphoprotein B23 is a putative ribosome assembly factor with a relatively high affinity for peptides containing sequences of nuclear localization signals (NLSs) of the SV40 T-antigen type [Szebeni, A., Herrera, J. E., & Olson, O. J. (1995) Biochemistry 34, 8037-8042]. The effects of protein B23 on nuclear import were determined by an in vitro assay [Dean, D. A., & Kasamatsu, H. (1994) J. Biol. Chem. 269, 4910-4916] using NLS peptide-conjugated bovine serum albumin (NLS-BSA) or the HIV-1 Rev protein as substrates for import into isolated rat liver nuclei. The import was ATP-dependent and inhibited by wheat germ agglutinin or by an antibody against p97, a component of the nuclear import system. The rate of import of either substrate was increased if protein B23 was added to the incubation medium. Similar enhancements of import were seen with both isoforms (B23.1 and B23.2). The stimulatory effect on Rev protein import was saturable with maximum stimulation (2-3-fold) at a molar ratio of protein B23:Rev of approximately 1:1. Phosphorylation of protein B23.1 by casein kinase II produced an additional doubling of the import rate. This effect was not seen if protein B23.1 was phosphorylated with a cdc2 type protein kinase. Mutant forms of protein B23.1 in which the nuclear localization signal was either deleted or altered did not stimulate import of the substrates. These results suggest that protein B23 plays a role as an accessory factor in the nuclear import of the NLS-containing proteins and that phosphorylation at sites in the highly acidic segments of the protein enhances the stimulatory effect.
Asunto(s)
Núcleo Celular/metabolismo , Productos del Gen rev/metabolismo , VIH-1 , Proteínas Nucleares/metabolismo , Albúmina Sérica/metabolismo , Adenosina Trifosfato/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Transformadores de Poliomavirus/metabolismo , Quinasa de la Caseína II , Reactivos de Enlaces Cruzados/metabolismo , Fluoresceínas , Colorantes Fluorescentes/metabolismo , Hígado/metabolismo , Microscopía Fluorescente , Mutación , Señales de Localización Nuclear , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Nucleares/farmacología , Nucleofosmina , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Aglutininas del Germen de Trigo/farmacología , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Several proteins are required for the transport of nuclear proteins from the cytoplasm to the nucleus, including the nuclear location sequence receptor (NLS receptor), p97, the small nuclear GTPase Ran/TC4, and several nucleoporins. The interaction of Ran with p97 is thought to regulate the interaction of these transport components. Ran-GTP alone binds p97, but Ran-GDP binds p97 only in conjunction with RanBP1. Using site-directed mutagenesis and deletion analysis, we have identified two distinct but overlapping binding domains for Ran-GTP and Ran-GDP/RanBP1 on p97. A short acidic sequence in p97 is part of the Ran-GDP/RanBP1 binding domain, possibly functioning in a similar manner as the C-terminal acidic sequence in Ran. A conserved cysteine residue in p97, Cys-158, is required for binding Ran-GDP/RanBP1, but not for binding of Ran-GTP to p97. In a permeabilized cell protein import assay, a mutant p97 with alanine substituted for Cys-158 is unable to support import in the presence of NLS receptor and Ran. These results support a direct active role for Ran-GDP in the receptor complex and provide evidence that the activity of downstream effectors of small GTPases may be regulated by both GTP- and GDP-bound forms of the protein.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Nucleares/metabolismo , Alanina , Sitios de Unión , Transporte Biológico , Compartimento Celular , Cisteína , Humanos , Mutación Puntual , Proteínas Recombinantes , Relación Estructura-Actividad , beta Carioferinas , Proteína de Unión al GTP ranRESUMEN
Macromolecules that are imported into the nucleus can be divided into classes according to their nuclear import signals. The best characterized class consists of proteins which carry a basic nuclear localization signal (NLS), whose transport requires the importin alpha/beta heterodimer. U snRNP import depends on both the trimethylguanosine cap of the snRNA and a signal formed when the Sm core proteins bind the RNA. Here, factor requirements for U snRNP nuclear import are studied using an in vitro system. Depletion of importin alpha, the importin subunit that binds the NLS, is found to stimulate rather than inhibit U snRNP import. This stimulation is shown to be due to a common requirement for importin beta in both U snRNP and NLS protein import. Saturation of importin beta-mediated transport with the importin beta-binding domain of importin alpha blocks U snRNP import both in vitro and in vivo. Immunodepletion of importin beta inhibits both NLS-mediated and U snRNP import. While the former requires re-addition of both importin alpha and importin beta, re-addition of importin beta alone to immunodepleted extracts was sufficient to restore efficient U snRNP import. Thus importin beta is required for U snRNP import, and it functions in this process without the NLS-specific importin alpha.