RESUMO
Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.
Assuntos
Retículo Endoplasmático , Processamento de Proteína Pós-Traducional , Subunidades Ribossômicas Maiores de Eucariotos , Ubiquitina-Proteína Ligases , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Homeostase , Membranas Intracelulares/metabolismo , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Peptidil Transferases/ultraestrutura , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , RNA de Transferência/metabolismo , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo , Canais de Translocação SEC/ultraestrutura , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestruturaRESUMO
Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signaling. Failure of phospho-control accounts for many human diseases. While a kinase phosphorylates multiple substrates, a substrate is often phosphorylated by multiple kinases. This renders phospho-control at the substrate level challenging, as it requires inhibition of multiple kinases, which would thus affect other kinase substrates. Here, we describe the development and application of the affinity-directed phosphatase (AdPhosphatase) system for targeted dephosphorylation of specific phospho-substrates. By deploying the Protein Phosphatase 1 or 2A catalytic subunits conjugated to an antigen-stabilized anti-GFP nanobody, we can promote the dephosphorylation of two independent phospho-proteins, FAM83D or ULK1, knocked in with GFP-tags using CRISPR-Cas9, with exquisite specificity. By redirecting protein phosphatases to neo-substrates through nanobody-mediated proximity, AdPhosphatase can alter the phospho-status and function of target proteins and thus, offers a new modality for potential drug discovery approaches.
Assuntos
Proteínas Quinases , Proteína Fosfatase 2 , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/metabolismo , Especificidade por Substrato , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
The ARID1A subunit of SWI/SNF chromatin remodeling complexes is a potent tumor suppressor. Here, a degron is applied to detect rapid loss of chromatin accessibility at thousands of loci where ARID1A acts to generate accessible minidomains of nucleosomes. Loss of ARID1A also results in the redistribution of the coactivator EP300. Co-incident EP300 dissociation and lost chromatin accessibility at enhancer elements are highly enriched adjacent to rapidly downregulated genes. In contrast, sites of gained EP300 occupancy are linked to genes that are transcriptionally upregulated. These chromatin changes are associated with a small number of genes that are differentially expressed in the first hours following loss of ARID1A. Indirect or adaptive changes dominate the transcriptome following growth for days after loss of ARID1A and result in strong engagement with cancer pathways. The identification of this hierarchy suggests sites for intervention in ARID1A-driven diseases.
Assuntos
Proteínas de Ligação a DNA/deficiência , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/metabolismo , Lesões Pré-Cancerosas/metabolismo , Fatores de Transcrição/deficiência , Transcrição Gênica , Ativação Transcricional , Animais , Sítios de Ligação , Linhagem Celular , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Nucleossomos/genética , Lesões Pré-Cancerosas/genética , Proteólise , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
This paper examines the case of Ebola, ça Suffit trial which was conducted in Guinea during Ebola Virus Disease (EVD) outbreak in 2015. I demonstrate that various non-epistemic considerations may legitimately influence the criteria for evaluating the efficacy and effectiveness of a candidate vaccine. Such non-epistemic considerations, which are social, ethical, and pragmatic, can be better placed and addressed in scientific research by appealing to non-epistemic values. I consider two significant features any newly developed vaccine should possess; (1) the duration of immunity the vaccine provides; and (2) safety with respect to the side effects of the vaccine. Then, I argue that social and ethical values are relevant and desirable in setting the parameters for evaluating these two features of vaccines. The parameters that are employed for setting up the criteria for assessing the features might have far-reaching implications on the well-being of society in general, and the health conditions of several thousand people in particular. The reason is that these features can play a decisive role during the evaluation of the efficacy and effectiveness of the vaccine. I conclude by showing why it is necessary to reject the concept of epistemic priority, at least when scientists engage in policy-oriented research.
Assuntos
Vacinas contra Ebola/administração & dosagem , Ética , Doença pelo Vírus Ebola/prevenção & controle , Fatores Sociais , Vacinas contra Ebola/normas , Guiné , Doença pelo Vírus Ebola/psicologia , HumanosRESUMO
The self-renewal efficiency of mouse embryonic stem cells (ESCs) is determined by the concentration of the transcription factor NANOG. While NANOG binds thousands of sites in chromatin, the regulatory systems that control DNA binding are poorly characterised. Here, we show that NANOG is phosphorylated by casein kinase I, and identify target residues. Phosphomimetic substitutions at phosphorylation sites within the homeodomain (S130 and S131) have site-specific functional effects. Phosphomimetic substitution of S130 abolishes DNA binding by NANOG and eliminates LIF-independent self-renewal. In contrast, phosphomimetic substitution of S131 enhances LIF-independent self-renewal, without influencing DNA binding. Modelling the DNA-homeodomain complex explains the disparate effects of these phosphomimetic substitutions. These results indicate how phosphorylation may influence NANOG homeodomain interactions that underpin ESC self-renewal.
Assuntos
Caseína Quinase I/metabolismo , Autorrenovação Celular , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/metabolismo , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Camundongos , Proteína Homeobox Nanog/química , Proteína Homeobox Nanog/genética , FosforilaçãoRESUMO
Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. The Ser/Arg (SR)-rich splicing factor (SRSF) protein kinase (SRPK), which is implicated in splicing regulation, is one such conserved eukaryotic kinase. Surprisingly, we show that SRPK has acquired the capacity to control a neurodevelopmental ubiquitin signaling pathway. In mammalian embryonic stem cells and cultured neurons, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of nuclear transcription factor substrates, thereby acting to restrain a neural gene expression program that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signaling that ensures correct regulation of neurodevelopmental gene expression.
Assuntos
Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Deficiência Intelectual/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Mutação/genética , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteólise , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The majority of mutations identified in patients with amelogenesis imperfecta have been mapped to FAM83H. As FAM83H expression is not limited to the enamel, how FAM83H contributes to amelogenesis is still largely unknown. We previously reported that members of the FAM83 family of proteins interact with and regulate the subcellular distribution of the promiscuous serine-threonine protein kinase CK1 family, through their shared N-terminal DUF1669 domains. FAM83H co-localises with CK1 isoforms to speckle-like structures in both the cytoplasm and nucleus. In this report, we show FAM83H, unlike other FAM83 proteins, interacts and colocalises with NCK1/2 tyrosine kinase adaptor proteins. This interaction is mediated by proline-rich motifs within the C-terminus of FAM83H, specifically interacting with the second and third SH3 domains of NCK1/2. Moreover, FAM83H pathogenic AI mutant proteins, which trigger C-terminal truncations of FAM83H, retain their interactions with CK1 isoforms but lose interaction with NCK1/2. These AI mutant FAM83H proteins acquire a nuclear localisation, and recruit CK1 isoforms to the nucleus where CK1 retains its kinase activity. As understanding the constituents of the FAM83H-localised speckles may hold the key to unravelling potential substrates of FAM83H-associated CK1 substrates, we employed a TurboID-based proximity labelling approach and uncovered several proteins including Iporin and BAG3 as potential constituents of the speckles.
Assuntos
Amelogênese Imperfeita/genética , Mutação/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas/química , Proteínas/metabolismoRESUMO
Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.
Assuntos
Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/genética , Animais , Sistemas CRISPR-Cas , Glicosilação , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , N-Acetilglucosaminiltransferases/química , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
Background: Two recessive mutations in the FAM83G gene, causing A34E and R52P amino acid substitutions in the DUF1669 domain of the PAWS1 protein, are associated with palmoplantar keratoderma (PPK) in humans and dogs respectively. We have previously reported that PAWS1 associates with the Ser/Thr protein kinase CK1α through the DUF1669 domain to mediate canonical Wnt signalling. Methods: Co-immunoprecipitation was used to investigate possible changes to PAWS1 interactors caused by the mutations. We also compared the stability of wild-type and mutant PAWS1 in cycloheximide-treated cells. Effects on Wnt signalling were determined using the TOPflash luciferase reporter assay in U2OS cells expressing PAWS1 mutant proteins. The ability of PAWS1 to induce axis duplication in Xenopus embryos was also tested. Finally, we knocked-in the A34E mutation at the native gene locus and measured Wnt-induced AXIN2 gene expression by RT-qPCR. Results: We show that these PAWS1 A34E and PAWS1 R52P mutants fail to interact with CK1α but, like the wild-type protein, do interact with CD2AP and SMAD1. Like cells carrying a PAWS1 F296A mutation, which also abolishes CK1α binding, cells carrying the A34E and R52P mutants respond poorly to Wnt signalling to an extent resembling that observed in FAM83G gene knockout cells. Consistent with this observation, these mutants, in contrast to the wild-type protein, fail to induce axis duplication in Xenopus embryos. We also found that the A34E and R52P mutant proteins are less abundant than the native protein and appear to be less stable, both when overexpressed in FAM83G-knockout cells and when knocked-in at the native FAM83G locus. Ala 34 of PAWS1 is conserved in all FAM83 proteins and mutating the equivalent residue in FAM83H (A31E) also abolishes interaction with CK1 isoforms. Conclusions: We propose that mutations in PAWS1 cause PPK pathogenesis through disruption of the CK1α interaction and attenuation of Wnt signalling.
RESUMO
The concerted action of many protein kinases helps orchestrate the error-free progression through mitosis of mammalian cells. The roles and regulation of some prominent mitotic kinases, such as cyclin-dependent kinases, are well established. However, these and other known mitotic kinases alone cannot account for the extent of protein phosphorylation that has been reported during mammalian mitosis. Here we demonstrate that CK1α, of the casein kinase 1 family of protein kinases, localises to the spindle and is required for proper spindle positioning and timely cell division. CK1α is recruited to the spindle by FAM83D, and cells devoid of FAM83D, or those harbouring CK1α-binding-deficient FAM83DF283A/F283A knockin mutations, display pronounced spindle positioning defects, and a prolonged mitosis. Restoring FAM83D at the endogenous locus in FAM83D-/- cells, or artificially delivering CK1α to the spindle in FAM83DF283A/F283A cells, rescues these defects. These findings implicate CK1α as new mitotic kinase that orchestrates the kinetics and orientation of cell division.
Assuntos
Caseína Quinase I/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Caseína Quinase I/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Citometria de Fluxo , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Mitose/fisiologiaRESUMO
In this essay, I argue that at least in two phases of pharmaceutical research, especially while assessing the adequacy of the accumulated data and its interpretation, the influence of non-epistemic values is necessary. I examine a specific case from the domain of pharmaceutical research and demonstrate that there are multiple competing sets of values which may legitimately or illegitimately influence different phases of the inquiry. In such cases, the choice of the appropriate set of values-epistemic as well as non-epistemic-should be made on the basis of the set's viability to promote specific epistemic and social aims of the research, in the case at hand, rather than other competing aims. I put forth an account which addresses two philosophically significant and interrelated questions in the context of values in science debates: (i) which aim should be prioritized when there are different stakeholders who try to achieve competing but, sometimes, conflicting goals; and (ii) what should be the criteria according to which the prioritization of a particular goal(s) over other competing goals can be determined? I argue that both these questions can be addressed by invoking the principle of non-arbitrariness and the mere means principle and self-determination right. The philosophical import of raising and addressing these questions from an ethical perspective is that this approach leads to a better understanding of problems in today's pharmaceutical research and guides us in efforts to alleviate these problems.
Assuntos
Ensaios Clínicos como Assunto/estatística & dados numéricos , Interpretação Estatística de Dados , Conhecimento , FilosofiaRESUMO
OBJECTIVE: Immediate extubation may have outcome benefits when judiciously instituted after neonatal congenital cardiac surgery. We sought to evaluate the outcomes of immediate extubation specifically in neonates undergoing stage 1 Norwood palliation of hypoplastic left heart syndrome. METHODS: Consecutive neonates undergoing stage 1 Norwood (January 2010 to December 2016) for hypoplastic left heart syndrome were retrospectively studied. Immediate extubation was defined as successful extubation before termination of anesthetic care. Preoperative and intraoperative variables were compared between immediate extubation and nonimmediate extubation groups, and bivariate analyses and descriptive methods were used to express the association of outcome variables with immediate extubation. Data were expressed as number and percent for categoric variables, and median and interquartile range for continuous variables. RESULTS: Of 23 patients who underwent stage 1 palliation, 5 had immediate extubation (22%). There were no differences in preoperative or intraoperative factors between patients who did and did not undergo immediate extubation. There were no deaths in the immediate extubation group. In the nonimmediate extubation group, 3 patients died before hospital discharge. One patient who had immediate extubation and 4 patients among those who did not have immediate extubation had to be reintubated in the 96 hours that followed extubation (P = 1). Intensive care unit length of stay was 8 (3-17) and 8 (5-18) (days) for the immediate extubation group and nonimmediate extubation groups, respectively (P = .71). CONCLUSIONS: Immediate extubation strategy was safely accomplished in one-fifth of this cohort of hypoplastic left heart syndrome. A larger cohort may delineate the determinants of immediate extubation and its benefits in infants undergoing stage 1 single ventricle palliation.
Assuntos
Extubação , Síndrome do Coração Esquerdo Hipoplásico/cirurgia , Procedimentos de Norwood , Cuidados Paliativos , Tempo para o Tratamento , Extubação/efeitos adversos , Extubação/mortalidade , Feminino , Mortalidade Hospitalar , Humanos , Síndrome do Coração Esquerdo Hipoplásico/diagnóstico , Síndrome do Coração Esquerdo Hipoplásico/mortalidade , Síndrome do Coração Esquerdo Hipoplásico/fisiopatologia , Recém-Nascido , Intubação Intratraqueal , Masculino , Procedimentos de Norwood/efeitos adversos , Procedimentos de Norwood/mortalidade , Complicações Pós-Operatórias/mortalidade , Complicações Pós-Operatórias/terapia , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.
Assuntos
Caseína Quinase I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Neoplasias/química , Domínios Proteicos , Caseína Quinase I/química , Caseína Quinase I/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas , Transdução de SinaisRESUMO
The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co-localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.
Assuntos
Caseína Quinase Ialfa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Via de Sinalização Wnt , Animais , Proteína Axina/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular , Expressão Ectópica do Gene , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismoRESUMO
Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells.This article has an associated First Person interview with the first authors of the paper.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Adesões Focais/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transdução de SinaisRESUMO
Objectives: We sought to identify preoperative, intraoperative and anatomical factors associated with immediate extubation (IE) after arterial switch operation for d-transposition of great arteries (dTGA). Methods: This was a single-centre retrospective study performed from 1 January 2010 to 30 June 2015. IE was defined as successful extubation in the operating room (OR). Univariate/bivariate regression of preoperative, intraoperative and anatomical variables was used to determine associations with IE. Results: Of 32 patients in the dTGA spectrum (age at operation 6 days), 18 (56%) underwent IE. Twelve (71%) of the 17 patients with an intact ventricular septum and 6 (43%) of the 14 patients with ventricular septal defect (VSD) underwent IE, whereas none of the patients with double outlet right ventricle or aortic arch obstruction ( n = 4) did. Patients who had cardiopulmonary bypass time (CPB) >173 min ( P = 0.01), lowest temperature on CPB (T min) ≤ 30.4°C ( P = 0.04) and aortic cross-clamp time >86 min ( P = 0.04) were more likely to be left intubated at the end of the procedure. There was no significant difference in patient's chronological age, gestational age, post-conceptual age, weight, coronary anatomy or prevalence of VSD between those who did and did not undergo IE. There was a median increase in intensive care unit (ICU) length of stay (LOS) by 1 day (33%, P = 0.03) and ICU costs by $12 338 (15%, P = 0.06) in non-IE patients. The OR turnover time ( P = 0.09) and reintubation rate ( P = 1) at 24 h post-extubation did not differ between those who did and did not have IE. There was no myocardial dysfunction evident on predismissal echocardiography in either group. Conclusions: In this cohort of infants, post repair for TGA, 56% were extubated immediately in the OR. Greater CPB and cross-clamp times and T min ≤ 30.4°C were associated with a lesser likelihood of IE. IE was associated with shorter ICU length of stay.
Assuntos
Extubação/métodos , Transposição das Grandes Artérias/métodos , Transposição dos Grandes Vasos/cirurgia , Anestesia Geral/métodos , Cuidados Críticos , Comunicação Interventricular/patologia , Humanos , Recém-Nascido , Tempo de Internação/estatística & dados numéricos , Cuidados Pós-Operatórios/métodos , Período Pós-Operatório , Estudos Retrospectivos , Transposição dos Grandes Vasos/patologia , Resultado do TratamentoRESUMO
Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation - a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl--sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl- uptake and stimulation of KCC3-mediated Cl- extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought "Cl-/volume-sensitive kinase" of the cation-Cl- cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.
Assuntos
Encéfalo/metabolismo , Cloretos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores/metabolismo , Animais , Barreira Hematoencefálica , Tamanho Celular , Células HEK293 , Humanos , Transporte de Íons , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pressão Osmótica , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteômica , Interferência de RNA , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/deficiência , Simportadores/genéticaRESUMO
Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagia , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Relacionadas à Autofagia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Proteína Huntingtina/metabolismo , Masculino , Camundongos , Doenças Neurodegenerativas/patologia , Agregados Proteicos , Dobramento de Proteína , ProteóliseRESUMO
BACKGROUND: We sought to identify preoperative and intraoperative predictors of immediate extubation (IE) after open heart surgery in neonates. The effect of IE on the postoperative intensive care unit (ICU) length of stay (LOS), cost of postoperative ICU care, operating room turnover, and reintubation rates was assessed. METHODS: Patients younger than 31 days who underwent cardiac surgery with cardiopulmonary bypass (January 2010 to December 2013) at a tertiary-care children's hospital were studied. Immediate extubation was defined as successful extubation before termination of anesthetic care. Data on preoperative and intraoperative variables were compared using descriptive, bivariate, and multivariate statistics to identify the predictors of IE. Propensity scores were used to assess effects of IE on ICU LOS, the cost of ICU care, reintubation rates, and operating room turnover time. RESULTS: One hundred forty-eight procedures done at a median age of 7 days resulted in 45 IEs (30.4%). The IE rate was 22.2% with single-ventricle heart disease. Independent predictors of IE were the absence of the need for preoperative ventilatory assistance, higher gestational age, anesthesiologist, and shorter cardiopulmonary bypass. Immediate extubation was associated with shorter ICU LOS (8.3 versus 12.7 days; p < 0.0001) and lower cost of ICU care (mean postoperative ICU charges, $157,449 versus $198,197; p < 0.0001) with no significant difference in the probability of reintubation (p = 0.7). Immediate extubation was associated with longer operating room turnover time (38.4 versus 46.7 minutes; p = 0.009). CONCLUSIONS: Immediate extubation was accomplished in 30.4% of neonates undergoing open heart surgery involving cardiopulmonary bypass. Immediate extubation was associated with lesser ICU LOS, postoperative ICU costs, and minimal increase in operating room turnover time, but without an increase in reintubation rates. Low gestational age, preoperative ventilatory support requirement, and prolonged cardiopulmonary bypass time were inversely associated with the ability to accomplish IE.
Assuntos
Extubação , Procedimentos Cirúrgicos Cardíacos , Cuidados Pós-Operatórios/estatística & dados numéricos , Extubação/economia , Extubação/estatística & dados numéricos , Anestesia/economia , Anestesia/métodos , Anestesia/estatística & dados numéricos , Procedimentos Cirúrgicos Cardíacos/economia , Ponte Cardiopulmonar , Feminino , Idade Gestacional , Custos Hospitalares , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Pediátrica/economia , Unidades de Terapia Intensiva Pediátrica/estatística & dados numéricos , Intubação Intratraqueal/economia , Intubação Intratraqueal/estatística & dados numéricos , Tempo de Internação/economia , Tempo de Internação/estatística & dados numéricos , Masculino , Salas Cirúrgicas/economia , Duração da Cirurgia , Cuidados Pós-Operatórios/economia , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/terapia , Curva ROC , Sala de Recuperação/economia , Sala de Recuperação/estatística & dados numéricos , Reoperação/estatística & dados numéricos , Respiração Artificial/estatística & dados numéricos , Estudos RetrospectivosRESUMO
Introduction: The development of multiarticulating hands holds the potential to restore lost function for upper-limb amputees. However, access to the full potential of commercialized devices is limited due to conventional control strategies for switching prosthesis modes, such as hand grips. For example, to switch grips in one conventional strategy, the prosthesis user must generate electromyogram (EMG) triggers (such as a cocontraction), which are cumbersome and nonintuitive. For this reason, alternative control strategies have emerged, which seek to facilitate grip switching. One specific application uses radio frequency identification (RFID) tags programmed with grip information. These tags can be placed on objects in the environment or carried on person. Upon approaching an RFID tag, the user's prosthesis reads the grip programmed on the tag and commands the hand into that grip. The purpose of this study was to compare the conventional strategy (using EMG triggers) with the alternative strategy (using RFID tags). Methods: The study evaluated three subjects: two users who actively use multiarticulating hands ("experienced" users) and one user who had never worn a multiarticulating hand ("new" user). Subjects were evaluated on two performance metrics: trigger completion time and the percentage of triggers that were successful on first attempt (first attempt success rate). Subjects also rated the difficulty, effort, and frustration with each strategy. Results: Results suggested faster trigger completion times with the EMG strategy for the experienced users and mixed results for the new user. Overall, the three subjects rated the RFID strategy as less difficult, tiring, and frustrating than the EMG strategy. Discussion and Conclusions: Continued studies with a larger subject pool are necessary to determine factors influencing performance and patient preference. This would allow identification of best strategies to access the full potential of new commercial devices. Still, the authors suggest that the synergistic use of both strategies can yield great benefits for both experienced and new multiarticulating hand users.