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1.
J Surg Res ; 295: 493-504, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38071779

RESUMO

INTRODUCTION: While intravenous fluid therapy is essential to re-establishing volume status in children who have experienced trauma, aggressive resuscitation can lead to various complications. There remains a lack of consensus on whether pediatric trauma patients will benefit from a liberal or restrictive crystalloid resuscitation approach and how to optimally identify and transition between fluid phases. METHODS: A panel was comprised of physicians with expertise in pediatric trauma, critical care, and emergency medicine. A three-round Delphi process was conducted via an online survey, with each round being followed by a live video conference. Experts agreed or disagreed with each aspect of the proposed fluid management algorithm on a five-level Likert scale. The group opinion level defined an algorithm parameter's acceptance or rejection with greater than 75% agreement resulting in acceptance and greater than 50% disagreement resulting in rejection. The remaining were discussed and re-presented in the next round. RESULTS: Fourteen experts from five Level 1 pediatric trauma centers representing three subspecialties were included. Responses were received from 13/14 participants (93%). In round 1, 64% of the parameters were accepted, while the remaining 36% were discussed and re-presented. In round 2, 90% of the parameters were accepted. Following round 3, there was 100% acceptance by all the experts on the revised and final version of the algorithm. CONCLUSIONS: We present a validated algorithm for intavenous fluid management in pediatric trauma patients that focuses on the de-escalation of fluids. Focusing on this time point of fluid therapy will help minimize iatrogenic complications of crystalloid fluids within this patient population.


Assuntos
Estado Terminal , Ressuscitação , Humanos , Criança , Estado Terminal/terapia , Ressuscitação/métodos , Hidratação/métodos , Cuidados Críticos , Soluções Cristaloides , Técnica Delphi
3.
Proc Natl Acad Sci U S A ; 112(37): 11530-5, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26324893

RESUMO

The ability to deliver drugs to specific sites in the lung could radically improve therapeutic outcomes of a variety of lung diseases, including cystic fibrosis, severe bronchopneumonia, chronic obstructive pulmonary disease, and lung cancer. Using conventional methods for pulmonary drug administration, precise, localized delivery of exact doses of drugs to target regions remains challenging. Here we describe a more controlled delivery of soluble reagents (e.g., drugs, enzymes, and radionuclides) in microvolume liquid plugs to targeted branches of the pulmonary airway tree: upper airways, small airways (bronchioles), or the most distal alveoli. In this approach, a soluble liquid plug of very small volume (<1 mL) is instilled into the upper airways, and with programmed air ventilation of the lungs, the plug is pushed into a specific desired (more distal) airway to achieve deposition of liquid film onto the lung epithelium. The plug volume and ventilation conditions were determined by mathematical modeling of plug transport in a tubular geometry, and targeted liquid film deposition was demonstrated in rat lungs by three different in vivo imaging modalities. The experimental and modeling data suggest that instillation of microvolumes of liquid into a ventilated pulmonary airway could be an effective strategy to deliver exact doses of drugs to targeted pathologic regions of the lung, especially those inaccessible by bronchoscopy, to increase in situ efficacy of the drug and minimize systemic side effects.


Assuntos
Sistemas de Liberação de Medicamentos , Pulmão/fisiologia , Surfactantes Pulmonares/administração & dosagem , Animais , Masculino , Microscopia de Fluorescência , Modelos Teóricos , Alvéolos Pulmonares/fisiologia , Ventilação Pulmonar , Ratos , Ratos Sprague-Dawley , Mecânica Respiratória/fisiologia , Sistema Respiratório/efeitos dos fármacos
4.
Nature ; 452(7185): 365-9, 2008 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-18354482

RESUMO

REST/NRSF (repressor-element-1-silencing transcription factor/neuron-restrictive silencing factor) negatively regulates the transcription of genes containing RE1 sites. REST is expressed in non-neuronal cells and stem/progenitor neuronal cells, in which it inhibits the expression of neuron-specific genes. Overexpression of REST is frequently found in human medulloblastomas and neuroblastomas, in which it is thought to maintain the stem character of tumour cells. Neural stem cells forced to express REST and c-Myc fail to differentiate and give rise to tumours in the mouse cerebellum. Expression of a splice variant of REST that lacks the carboxy terminus has been associated with neuronal tumours and small-cell lung carcinomas, and a frameshift mutant (REST-FS), which is also truncated at the C terminus, has oncogenic properties. Here we show, by using an unbiased screen, that REST is an interactor of the F-box protein beta-TrCP. REST is degraded by means of the ubiquitin ligase SCF(beta-TrCP) during the G2 phase of the cell cycle to allow transcriptional derepression of Mad2, an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind beta-TrCP, inhibited Mad2 expression and resulted in a phenotype analogous to that observed in Mad2(+/-) cells. In particular, we observed defects that were consistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chromosome bridges and mis-segregation in anaphase, tetraploidy, and faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant, which does not bind beta-TrCP. Thus, SCF(beta-TrCP)-dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis. The high levels of REST or its truncated variants found in certain human tumours may contribute to cellular transformation by promoting genomic instability.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Instabilidade Cromossômica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Fase G2 , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Proteínas Mad2 , Mitose , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Fuso Acromático/fisiologia , Fatores de Transcrição/genética , Proteínas Contendo Repetições de beta-Transducina/deficiência , Proteínas Contendo Repetições de beta-Transducina/genética
5.
Res Sq ; 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39315275

RESUMO

Childhood interstitial lung disease (chILD) secondary to pulmonary surfactant deficiency is a devastating chronic lung disease in children. Clinical presentation includes mild to severe respiratory failure and fibrosis. There is no specific treatment, except lung transplantation, which is hampered by a severe shortage of donor organs, especially for young patients. Repair of lungs with chILD represents a longstanding therapeutic challenge but cell therapy is a promising strategy. As surfactant is produced by alveolar type II epithelial (ATII) cells, engraftment with normal or gene-corrected ATII cells might provide an avenue to cure. Here we used a chILD disease-like model, Sftpc -/- mice, to provide proof-of-principle for this approach. Sftpc -/- mice developed chronic interstitial lung disease with age and were hypersensitive to bleomycin. We could engraft wild-type ATII cells after low dose bleomycin conditioning. Transplanted ATII cells produced mature SPC and attenuated bleomycin-induced lung injury up to two months post-transplant. This study demonstrates that partial replacement of mutant ATII cells can promote lung repair in a mouse model of chILD-like disease.

6.
bioRxiv ; 2023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38077087

RESUMO

Although lung disease is a major cause of mortality, the mechanisms involved in human lung regeneration are unclear because of the lack of experimental models. Here we report a novel model where human pluripotent stem cell-derived expandable cell lines sharing features of airway secretory and basal cells engraft in the distal rat lung after conditioning by locoregional de-epithelialization followed by irradiation and immunosuppression. The engrafting cells, which we named distal lung epithelial progenitors (DLEPs), contributed to alveolar epithelial cells and generated 'KRT5-pods', structures involved in distal lung repair after severe injury, but only rarely to distal airways. Most strikingly, however, injury induced by the conditioning regimen was largely prevented by the engrafting DLEPs. The approach described here provides a model to study mechanisms involved in human lung regeneration, and potentially lays the foundation for the preclinical development of cell therapy to treat lung injury and disease.

7.
Dis Model Mech ; 15(10)2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35976034

RESUMO

Despite over 30 years of intensive research for targeted therapies, treatment of acute respiratory distress syndrome (ARDS) remains supportive in nature. With mortality upwards of 30%, a high-fidelity pre-clinical model of ARDS, on which to test novel therapeutics, is urgently needed. We used the Yorkshire breed of swine to induce a reproducible model of ARDS in human-sized swine to allow the study of new therapeutics, from both mechanistic and clinical standpoints. For this, animals were anesthetized, intubated and mechanically ventilated, and pH-standardized gastric contents were delivered bronchoscopically, followed by intravenous infusion of Escherichia coli-derived lipopolysaccharide. Once the ratio of arterial oxygen partial pressure (PaO2) to fractional inspired oxygen (FIO2) had decreased to <150, the animals received standard ARDS treatment for up to 48 h. All swine developed moderate to severe ARDS. Chest radiographs taken at regular intervals showed significantly worse lung edema after induction of ARDS. Quantitative scoring of lung injury demonstrated time-dependent increases in interstitial and alveolar edema, neutrophil infiltration, and mild to moderate alveolar membrane thickening. This pre-clinical model of ARDS in human-sized swine recapitulates the clinical, radiographic and histopathologic manifestations of ARDS, providing a tool to study therapies for this highly morbid lung disease.


Assuntos
Lesão Pulmonar , Síndrome do Desconforto Respiratório , Animais , Humanos , Lipopolissacarídeos/farmacologia , Oxigênio , Suínos
8.
J Cyst Fibros ; 21(6): 1027-1035, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35525782

RESUMO

BACKGROUND: Manifestations of cystic fibrosis, although well-characterized in the proximal airways, are understudied in the distal lung. Characterization of the cystic fibrosis lung 'matrisome' (matrix proteome) has not been previously described, and could help identify biomarkers and inform therapeutic strategies. METHODS: We performed liquid chromatography-mass spectrometry, gene ontology analysis, and multi-modal imaging, including histology, immunofluorescence, and electron microscopy for a comprehensive evaluation of distal human lung extracellular matrix (matrix) structure and composition in end-stage cystic fibrosis. RESULTS: Quantitative proteomic profiling identified sixty-eight (68) matrix constituents with significantly altered expression in end-stage cystic fibrosis. Over 90% of significantly different matrix peptides detected, including structural and basement membrane proteins, were expressed at lower levels in cystic fibrosis. However, the total abundance of matrix in cystic fibrosis lungs was not significantly different from control lungs, suggesting that cystic fibrosis leads to loss of diversity among lung matrix proteins rather than an absolute loss of matrix. Visualization of distal lung matrix via immunofluorescence and electron microscopy revealed pathological remodeling of distal lung tissue architecture and loss of alveolar basement membrane, consistent with significantly altered pathways identified by gene ontology analysis. CONCLUSIONS: Dysregulation of matrix organization and aberrant wound healing pathways are associated with loss of matrix protein diversity and obliteration of distal lung tissue structure in end-stage cystic fibrosis. While many therapeutics aim to functionally restore defective cystic fibrosis transmembrane conductance regulator (CFTR), drugs that target dysregulated matrix pathways may serve as adjunct interventions to support lung recovery.


Assuntos
Fibrose Cística , Humanos , Fibrose Cística/terapia , Proteômica , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Pulmão/metabolismo
9.
mBio ; 12(3): e0079921, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34061592

RESUMO

Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. IMPORTANCE Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.


Assuntos
Encéfalo/virologia , Vírus do Sarampo/genética , Proteínas Virais de Fusão/genética , Substituição de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/patologia , Doenças do Sistema Nervoso Central/virologia , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/virologia , Masculino , Sarampo/virologia , Vírus do Sarampo/patogenicidade , Metagenômica , Camundongos , Neurônios/virologia , Organoides/citologia , Organoides/virologia , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/classificação , Proteínas Virais de Fusão/metabolismo
10.
Science ; 371(6536): 1379-1382, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33597220

RESUMO

Containment of the COVID-19 pandemic requires reducing viral transmission. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is initiated by membrane fusion between the viral and host cell membranes, which is mediated by the viral spike protein. We have designed lipopeptide fusion inhibitors that block this critical first step of infection and, on the basis of in vitro efficacy and in vivo biodistribution, selected a dimeric form for evaluation in an animal model. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour cohousing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and thus may readily translate into safe and effective intranasal prophylaxis to reduce transmission of SARS-CoV-2.


Assuntos
COVID-19/transmissão , Lipopeptídeos/administração & dosagem , Fusão de Membrana/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Inibidores de Proteínas Virais de Fusão/administração & dosagem , Internalização do Vírus/efeitos dos fármacos , Administração Intranasal , Animais , COVID-19/prevenção & controle , COVID-19/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Desenho de Fármacos , Furões , Lipopeptídeos/química , Lipopeptídeos/farmacocinética , Lipopeptídeos/farmacologia , Camundongos , Profilaxia Pré-Exposição , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Distribuição Tecidual , Células Vero , Inibidores de Proteínas Virais de Fusão/química , Inibidores de Proteínas Virais de Fusão/farmacocinética , Inibidores de Proteínas Virais de Fusão/farmacologia
11.
Nature ; 428(6979): 190-3, 2004 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-15014502

RESUMO

Skp2 and its cofactor Cks1 are the substrate-targeting subunits of the SCF(Skp2-Cks1) (Skp1/Cul1/F-box protein) ubiquitin ligase complex that regulates entry into S phase by inducing the degradation of the cyclin-dependent kinase inhibitors p21 and p27 (ref. 1). Skp2 is an oncoprotein that often shows increased expression in human cancers; however, the mechanism that regulates its cellular abundance is not well understood. Here we show that both Skp2 and Cks1 proteins are unstable in G1 and that their degradation is mediated by the ubiquitin ligase APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its activator Cdh1). Silencing of Cdh1 by RNA interference in G1 cells stabilizes Skp2 and Cks1, with a consequent increase in p21 and p27 proteolysis. Depletion of Cdh1 also increases the percentage of cells in S phase, whereas concomitant downregulation of Skp2 reverses this effect, showing that Skp2 is an essential target of APC/C(Cdh1). Expression of a stable Skp2 mutant that cannot bind APC/C(Cdh1) induces premature entry into S phase. Thus, the induction of Skp2 and Cks1 degradation in G1 represents a principal mechanism by which APC/C(Cdh1) prevents the unscheduled degradation of SCF(Skp2-Cks1) substrates and maintains the G1 state.


Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas de Transporte/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Linhagem Celular , Proteínas Culina/antagonistas & inibidores , Proteínas Culina/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Quinases Ciclina-Dependentes , Humanos , Camundongos , Mutação , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Interferência de RNA , Ratos , Proteínas Quinases Associadas a Fase S/genética , Proteínas Ligases SKP Culina F-Box/genética , Complexos Ubiquitina-Proteína Ligase/genética
12.
Nature ; 426(6962): 87-91, 2003 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-14603323

RESUMO

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.


Assuntos
Dano ao DNA , Fase S , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Fosfatases cdc25/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Dano ao DNA/efeitos da radiação , Células HeLa , Humanos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Radiação Ionizante , Ubiquitina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Fosfatases cdc25/química
13.
Artigo em Inglês | MEDLINE | ID: mdl-32351946

RESUMO

The shortage of transplantable donor organs directly affects patients with end-stage lung disease, for which transplantation remains the only definitive treatment. With the current acceptance rate of donor lungs of only 20%, rescuing even one half of the rejected donor lungs would increase the number of transplantable lungs threefold, to 60%. We review recent advances in lung bioengineering that have potential to repair the epithelial and vascular compartments of the lung. Our focus is on the long-term support and recovery of the lung ex vivo, and the replacement of defective epithelium with healthy therapeutic cells. To this end, we first review the roles of the lung epithelium and vasculature, with focus on the alveolar-capillary membrane, and then discuss the available and emerging technologies for ex vivo bioengineering of the lung by decellularization and recellularization. While there have been many meritorious advances in these technologies for recovering marginal quality lungs to the levels needed to meet the standards for transplantation - many challenges remain, motivating further studies of the extended ex vivo support and interventions in the lung. We propose that the repair of injured epithelium with preservation of quiescent vasculature will be critical for the immediate blood supply to the lung and the lung survival and function following transplantation.

14.
Sci Rep ; 7(1): 13082, 2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29026127

RESUMO

Intratracheal delivery of stem cells into injured or diseased lungs can provide a variety of therapeutic and immunomodulatory effects for the treatment of acute lung injury and chronic lung disease. While the efficacy of this approach depends on delivering the proper cell dosage into the target region of the airway, tracking and analysis of the cells have been challenging, largely due to the limited understanding of cell transport and lack of suitable cell monitoring techniques. We report on the transport and deposition of intratracheally delivered stem cells as well as strategies to modulate the number of cells (e.g., dose), topographic distribution, and region-specific delivery in small (rodent) and large (porcine and human) lungs. We also developed minimally invasive imaging techniques for real-time monitoring of intratracheally delivered cells. We propose that this approach can facilitate the implementation of patient-specific cells and lead to enhanced clinical outcomes in the treatment of lung disease with cell-based therapies.


Assuntos
Diagnóstico por Imagem/métodos , Pulmão/citologia , Células-Tronco/citologia , Animais , Células Cultivadas , Humanos , Hidrodinâmica , Pulmão/diagnóstico por imagem , Ratos , Ratos Sprague-Dawley , Suínos
15.
Sci Adv ; 3(8): e1700521, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28875163

RESUMO

End-stage lung disease is the third leading cause of death worldwide, accounting for 400,000 deaths per year in the United States alone. To reduce the morbidity and mortality associated with lung disease, new therapeutic strategies aimed at promoting lung repair and increasing the number of donor lungs available for transplantation are being explored. Because of the extreme complexity of this organ, previous attempts at bioengineering functional lungs from fully decellularized or synthetic scaffolds lacking functional vasculature have been largely unsuccessful. An intact vascular network is critical not only for maintaining the blood-gas barrier and allowing for proper graft function but also for supporting the regenerative cells. We therefore developed an airway-specific approach to removing the pulmonary epithelium, while maintaining the viability and function of the vascular endothelium, using a rat model. The resulting vascularized lung grafts supported the attachment and growth of human adult pulmonary cells and stem cell-derived lung-specified epithelial cells. We propose that de-epithelialization of the lung with preservation of intact vasculature could facilitate cell therapy of pulmonary epithelium and enable bioengineering of functional lungs for transplantation.


Assuntos
Bioengenharia , Transplante de Pulmão , Pulmão/irrigação sanguínea , Pulmão/fisiologia , Regeneração , Animais , Bioengenharia/métodos , Sobrevivência Celular , Matriz Extracelular , Imunofluorescência , Pulmão/ultraestrutura , Músculo Liso , Ratos , Medicina Regenerativa , Mucosa Respiratória , Alicerces Teciduais
17.
Blood Adv ; 4(18): 4358-4361, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32926122
18.
Mol Cell ; 23(3): 319-29, 2006 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-16885022

RESUMO

During replicative stress, Claspin mediates the phosphorylation and consequent activation of Chk1 by ATR. We found that during recovery from the DNA replication checkpoint response, Claspin is degraded in a betaTrCP-dependent manner. In vivo, Claspin is phosphorylated in a canonical DSGxxS degron sequence, which is typical of betaTrCP substrates. Phosphorylation of Claspin is mediated by Plk1 and is essential for binding to betaTrCP. In vitro ubiquitylation of Claspin requires betaTrCP, Plk1, and an intact DSGxxS degron. Significantly, expression of a stable Claspin mutant unable to bind betaTrCP prolongs the activation of Chk1, thereby attenuating the recovery from the DNA replication stress response and significantly delaying entry into mitosis. Thus, the SCFbetaTrCP-dependent degradation of Claspin is necessary for the efficient and timely termination of the DNA replication checkpoint. Importantly, in response to DNA damage in G2, Claspin proteolysis is inhibited to allow the prompt reestablishment of the checkpoint.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Replicação do DNA/fisiologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/fisiologia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Histonas/metabolismo , Humanos , Hidroxiureia/farmacologia , Modelos Biológicos , Mutação/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas Ligases SKP Culina F-Box/genética , Homologia de Sequência de Aminoácidos , Transfecção , Ubiquitina/metabolismo , Raios Ultravioleta
19.
Science ; 314(5798): 467-71, 2006 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-17053147

RESUMO

The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCF(betaTRCP). Expression in cultured cells of a stable PDCD4 mutant that is unable to bind betaTRCP inhibited translation of an mRNA with a structured 5'UTR, resulted in smaller cell size, and slowed down cell cycle progression. We propose that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proliferação de Células , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Regiões 5' não Traduzidas , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Tamanho Celular , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Mitógenos/farmacologia , Fosforilação , RNA Interferente Pequeno , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Serina/metabolismo , Soro , Transdução de Sinais , Proteínas Contendo Repetições de beta-Transducina/genética
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