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1.
Int J Neurosci ; 129(10): 1013-1023, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31092102

RESUMO

The effective treatment of many diseases requires the use of multiple treatment strategies among which neuromodulation is playing an increasingly important role. Neuromodulation devices that act to normalize or modulate nerve activity through the targeted delivery of electrical stimuli will be the focus of this review. These devices encompass deep brain stimulators, vagus nerve stimulators, spinal cord simulators and sacral nerve stimulators. Already neuromodulation has proven successful in the treatment of a broad range of conditions from Parkinson's disease to chronic pain and urinary incontinence. Many of these approaches seek to exploit the activities of the autonomic nervous system, which influences organ function through the release of neurotransmitters and associated signalling cascades. This review will outline existing and emerging applications for each of these neuromodulation devices, proposed mechanisms of action and clinical studies evaluating both their safety and therapeutic efficacy.


Assuntos
Terapia por Estimulação Elétrica/métodos , Terapia por Estimulação Elétrica/tendências , Estimulação Elétrica Nervosa Transcutânea/métodos , Estimulação Elétrica Nervosa Transcutânea/tendências , Ensaios Clínicos como Assunto/métodos , Estimulação Encefálica Profunda/métodos , Estimulação Encefálica Profunda/tendências , Epilepsia/fisiopatologia , Epilepsia/terapia , Humanos , Doença de Parkinson/fisiopatologia , Doença de Parkinson/terapia , Estimulação da Medula Espinal/métodos , Estimulação da Medula Espinal/tendências , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapia , Resultado do Tratamento , Estimulação do Nervo Vago/métodos , Estimulação do Nervo Vago/tendências
2.
Heliyon ; 4(12): e01065, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30603699

RESUMO

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600E BRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.

3.
Cell Signal ; 28(6): 561-71, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26898828

RESUMO

The (V600E)BRAF oncogenic mutation is detected in a wide range of human cancers and induces hyperactivation of the downstream MEK-ERK signalling cascade. Although output of the BRAF-MEK-ERK pathway is regulated by feed-forward RAF activity, feedback control also plays an important role. One such feedback pathway has been identified in Caenorhabditis elegans and involves ERK-mediated phosphorylation of BRAF within a CDC4 phosphodegron (CPD), targeting BRAF for degradation via CDC4 (also known as FBXW7), a component of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex. Here we investigate this pathway in mammalian cells. Short-term expression of autochthonous (V600E)BRAF in mouse embryonic fibroblasts (MEFs) leads to down-regulation of BRAF protein levels in a proteasome-dependent manner and (V600E)BRAF has a reduced half-life compared to (WT)BRAF in HEK293(T) cells. These effects were reversed by treatment with the MEK inhibitor PD184352. We have identified the equivalent CPD at residues 400-405 in human BRAF and have found that mutation of ERK phosphorylation sites at residues T401 and S405 in (V600E)BRAF increases the half-life of the protein. While BRAF and FBXW7 co-immunoprecipitated, the overexpression of FBXW7 did not influence the half-life of either (WT)BRAF or (V600E)BRAF. Furthermore, disruption of the substrate-binding site of mouse FBXW7 using the R482Q mutation did not affect the interaction with BRAF and the expression levels of (WT)BRAF and (V600E)BRAF were not altered in MEFs derived from mice with the homozygous knockin (R482Q)FBXW7 mutation. Overall these data confirm the existence of a negative feedback pathway by which BRAF protein stability is regulated by ERK. However, unlike the situation in C. elegans, FBXW7 does not play a unique role in mediating subsequent BRAF degradation.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas F-Box/fisiologia , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Células Cultivadas , Senescência Celular , Regulação para Baixo , Proteína 7 com Repetições F-Box-WD , Retroalimentação Fisiológica , Células HEK293 , Humanos , Camundongos , Mutação , Estabilidade Proteica , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Supressoras de Tumor/fisiologia
4.
Med Eng Phys ; 37(11): 1047-52, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26391067

RESUMO

The biomechanical performance of internal fracture fixation depends on several factors. One measure of performance is the strength of the construct. The objective of this biomechanical study was to identify the effect of load obliquity on the strength of locking and nonlocking plate and screw constructs. For this study, plates and screws were fixed to synthetic osteoporotic bone that had a 1 mm thick synthetic cortical shell. An 8-hole, 3.5 mm thick hybrid plate was fixed with either two 3.5 mm major diameter locking screws or two 4.0 mm major diameter cancellous screws. Forces were applied at 0, 45, and 90 degrees to the plate normal. Eight specimens were loaded to failure for each group. When loads were applied normal to the plate, the nonlocking construct failed initially at higher loads (123.2 ± 13.2 N) than the locking construct (108.7 ± 7.6 N, P = 0.020). For oblique loads, the locking construct failed at higher mean loads but the difference of means was not statistically significant (167.7 ± 14.9 N compared to 154.2 ± 9.4 N, P = 0.052). For loads parallel to the plate, the locking construct was much stronger than the nonlocking construct (1591 ± 227 N compared to 913 ± 237 N, P < 0.001). Stiffness and Energy outcomes are also compared.


Assuntos
Placas Ósseas , Parafusos Ósseos , Substitutos Ósseos , Osteoporose/cirurgia , Fenômenos Biomecânicos , Elasticidade , Desenho de Equipamento , Análise de Falha de Equipamento/instrumentação , Análise de Falha de Equipamento/métodos , Humanos , Falha de Prótese
5.
EMBO Mol Med ; 7(9): 1119-37, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26183450

RESUMO

The tumour microenvironment is known to play an integral role in facilitating cancer progression at advanced stages, but its function in some pre-cancerous lesions remains elusive. We have used the (V600) (E)BRAF-driven mouse lung model that develop premalignant lesions to understand stroma-tumour interactions during pre-cancerous development. In this model, we have found that immature macrophage-lineage cells (IMCs) producing PDGFA, TGFß and CC chemokines are recruited to the stroma of premalignant lung adenomas through CC chemokine receptor 1 (CCR1)-dependent mechanisms. Stromal IMCs promote proliferation and transcriptional alterations suggestive of epithelial-mesenchymal transition in isolated premalignant lung tumour cells ex vivo, and are required for the maintenance of early-stage lung tumours in vivo. Furthermore, we have found that IMC recruitment to the microenvironment is restrained by the cholesterol-binding protein, Niemann-Pick type C2 (NPC2). Studies on isolated cells ex vivo confirm that NPC2 is secreted from tumour cells and is taken up by IMCs wherein it suppresses secretion of the CCR1 ligand CC chemokine 6 (CCL6), at least in part by facilitating its lysosomal degradation. Together, these findings show that NPC2 secreted by premalignant lung tumours suppresses IMC recruitment to the microenvironment in a paracrine manner, thus identifying a novel target for the development of chemopreventive strategies in lung cancer.


Assuntos
Adenoma/patologia , Proteínas de Transporte/metabolismo , Tolerância Imunológica , Neoplasias Pulmonares/patologia , Macrófagos/imunologia , Elastase Pancreática/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Transição Epitelial-Mesenquimal , Camundongos , Proteínas Proto-Oncogênicas B-raf/genética
6.
Ayu ; 35(4): 452-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-26195912

RESUMO

BACKGROUND: Hypertension is an incurable pathological condition and lifelong therapy is required. Long term use of conventional synthetic anti-hypertensive drugs is associated with a spectrum of toxic effects. However, therapeutic interventions using herbal drugs for hypertension have gained considerable attention worldwide. AIM: To evaluate the anti-hypertensive activity of polyherbal formulation (SJT-HT-03). MATERIALS AND METHODS: The polyherbal formulation (SJT-HT-03) comprises of leaves of Aegle marmelos L., fruits of Benincasa hispida Thunb., Garcinia indica Thouars, and flowers of Musa paradiasica L., Rosa indica L., Hibiscus rosa sinensis L. Selected plants as mentioned above were collected, dried and extracted with different solvents. Formulation SJT-HT-03 (250 mg/kg, p.o.), was evaluated using two kidney one clip (2K1C) model and deoxycorticosterone acetate (DOCA)-salt-induced hypertension model using the enalapril (10 mg/kg, p.o.) and hydrochlorothiazide (5 mg/kg, p.o.) as a reference standard drug in respective models. RESULTS: SJT-HT-03 significantly reduced (P < 0.001, one-way analysis of variance followed by Turkey's multiple comparison tests) systolic as well as diastolic blood pressure (BP) in 2K1C and DOCA-salt model. Further, SJT-HT-03 has shown a significant reduction (P < 0.01) in angiotensin converting enzyme (ACE) activity in serum, clipped kidney as well as in lungs in 2K1C model, whereas significant reduction (P < 0.05) in serum Na(+) and increase in serum K(+) level in DOCA model. CONCLUSION: Polyherbal formulation SJT-HT-03 possess significant anti-hypertensive activity by producing direct depressant effect on heart, inhibition of ACE, aldosterone antagonistic as well as diuretic effect and thereby act on multiple targets to achieve optimal effect.

7.
J Struct Biol ; 184(1): 21-32, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23726984

RESUMO

Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1-R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length talin at a resolution of approximately 2.5nm using EM reconstruction of full-length talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two talin rods form a donut-shaped structure, with the two talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that talin must unravel before it can support integrin activation and engage the actin cytoskeleton.


Assuntos
Talina/química , Talina/metabolismo , Actinas/química , Actinas/metabolismo , Sítios de Ligação , Citoesqueleto/química , Citoesqueleto/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
8.
Genes Dev ; 26(17): 1945-58, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22892241

RESUMO

(L597V)BRAF mutations are acquired somatically in human cancer samples and are frequently coincident with RAS mutations. Germline (L597V)BRAF mutations are also found in several autosomal dominant developmental conditions known as RASopathies, raising the important question of how the same mutation can contribute to both pathologies. Using a conditional knock-in mouse model, we show that endogenous expression of (L597V)Braf leads to approximately twofold elevated Braf kinase activity and weak activation of the Mek/Erk pathway. This is associated with induction of RASopathy hallmarks including cardiac abnormalities and facial dysmorphia but is not sufficient for tumor formation. We combined (L597V)Braf with (G12D)Kras and found that (L597V)Braf modified (G12D)Kras oncogenesis such that fibroblast transformation and lung tumor development were more reminiscent of that driven by the high-activity (V600E)Braf mutant. Mek/Erk activation levels were comparable with those driven by (V600E)Braf in the double-mutant cells, and the gene expression signature was more similar to that induced by (V600E)Braf than (G12D)Kras. However, unlike (V600E)Braf, Mek/Erk pathway activation was mediated by both Craf and Braf, and ATP-competitive RAF inhibitors induced paradoxical Mek/Erk pathway activation. Our data show that weak activation of the Mek/Erk pathway underpins RASopathies, but in cancer, (L597V)Braf epistatically modifies the transforming effects of driver oncogenes.


Assuntos
Epistasia Genética , Sistema de Sinalização das MAP Quinases/fisiologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Quinases raf/metabolismo , Animais , Transformação Celular Neoplásica/genética , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Pulmão/patologia , Camundongos , Análise Serial de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fator 3 Associado a Receptor de TNF
9.
PLoS One ; 7(4): e34461, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22496808

RESUMO

Talin is a large (∼2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs), where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell.


Assuntos
Calpaína/metabolismo , Comunicação Celular , Adesões Focais/fisiologia , Talina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Calpaína/genética , Células Cultivadas , Citocinese , Citoesqueleto/metabolismo , Humanos , Integrinas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Talina/química , Talina/genética
10.
Eur J Cell Biol ; 91(3): 180-91, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22306379

RESUMO

Talins are adaptor proteins that connect the integrin family of cell adhesion receptors to cytoskeletal actin. Vertebrates express two closely related talins encoded by separate genes, and while it is well established that talin1 plays a key role in cell adhesion and spreading, little is known about the role of talin2. To facilitate such studies, we report the characterisation of 4 new isoform-specific talin mouse monoclonal antibodies that work in Western blotting, immuno-precipitation, immuno-fluorescence and immuno-histochemistry. Using these antibodies, we show that talin1 and talin2 do not form heterodimers, and that they are differentially localised within the cell. Talin1 was concentrated in peripheral focal adhesions while talin2 was observed in both focal and fibrillar adhesions, and knock-down of talin2 compromised fibronectin fibrillogenesis. Although differentiated human macrophages express both isoforms, only talin1 showed discrete staining and was localised to the ring structure of podosomes. However, siRNA-mediated knock-down of macrophage talin2 led to a significant reduction in podosomal matrix degradation. We have also used the antibodies to localise each isoform in tissue sections using both cryostat and paraffin-embedded material. In skeletal muscle talin2 was localised to both myotendinous junctions and costameres while talin1 was restricted to the former structure. In contrast, both isoforms co-localised in kidney with staining of the glomerulus, and the tubular epithelial and interstitial cells of the cortex and medulla. We anticipate that these antibodies will form a valuable resource for future studies on the function of the two major talin isoforms.


Assuntos
Anticorpos Monoclonais , Fibronectinas/metabolismo , Macrófagos/ultraestrutura , Isoformas de Proteínas/análise , Talina/metabolismo , Animais , Especificidade de Anticorpos , Adesões Focais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno , Ratos
11.
Biochem Soc Trans ; 40(1): 67-72, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22260667

RESUMO

The strength and duration of intracellular signalling pathway activation is a key determinant of the biological outcome of cells in response to extracellular cues. This has been particularly elucidated for the Ras/Raf/MEK [mitogen-activated growth factor/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling pathway with a number of studies in fibroblasts showing that sustained ERK signalling is a requirement for S-phase entry, whereas transient ERK signalling does not have this capability. A major unanswered question, however, is how a cell can sustain ERK activation, particularly when ERK-specific phosphatases are transcriptionally up-regulated by the pathway itself. A major point of ERK regulation is at the level of Raf, and, to sustain ERK activation in the presence of ERK phosphatases, sustained Raf activation is a requirement. Three Raf proteins exist in mammals, and the activity of all three is induced following growth factor stimulation of cells, but only B-Raf activity is maintained at later time points. This observation points to B-Raf as a regulator of sustained ERK activation. In the present review, we consider evidence for a link between B-Raf and sustained ERK activation, focusing on a potential role for the subcellular localization of B-Raf in this key physiological event.


Assuntos
Sistema de Sinalização das MAP Quinases , Transporte Proteico , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas B-raf/química
12.
Clin Biomech (Bristol, Avon) ; 27(5): 436-42, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22226074

RESUMO

BACKGROUND: Clavicle malunion affects the biomechanics of the shoulder joint. The purpose of this study is to establish the abduction, flexion, and internal (medial) rotation biomechanics of the shoulder after clavicle malunion. METHODS: A computational study was performed utilizing a three-dimensional, validated computational model of the upper extremity. Sequential shortening of the clavicle up to 20% was simulated. Muscle forces, moment arms, and moments were calculated for the surrounding musculature through a range of flexion, abduction, and internal rotation during the simulated shortening. FINDINGS: Shortening of the clavicle decreases the shoulder elevation moments of the upper extremity muscles during abduction. Internal rotation moments are also decreased with shortening. Flexion moments were affected less through physiologic range of motion. The observed effects are due to a combination of changes in moment arms of the individual muscles as well as a decrease in the force generating capacity of the muscles. Additionally, shortening of the clavicle increases coronal angulation of the clavicle at the sternoclavicular joint. INTERPRETATION: Shortening causes a decrease in the moment generating capacity as well as the total force generating capacity of the shoulder girdle muscles. The clinical significance of these computational results, which are consistent with recent clinical studies, is validation of the proposed functional deficit caused by clavicle malunion.


Assuntos
Clavícula/lesões , Clavícula/fisiopatologia , Fraturas Mal-Unidas/fisiopatologia , Modelos Biológicos , Amplitude de Movimento Articular , Articulação do Ombro/fisiopatologia , Adulto , Simulação por Computador , Humanos
13.
J Clin Epidemiol ; 64(2): 182-90, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20609562

RESUMO

OBJECTIVES: To evaluate the validity, reliability, responsiveness, and mode preference of electronic data capture (EDC) using the Western Ontario and McMaster (WOMAC) numerical rating scale (NRS) 3.1 Osteoarthritis (OA) Index on Motorola V3 mobile phones. STUDY DESIGN AND SETTING: Patients with OA undergoing hip or knee joint replacement were assessed preoperatively and 3-4 months postoperatively, completing the WOMAC Index in paper (p-WOMAC) and electronic (m-WOMAC) format in random order. RESULTS: Data were successfully and securely transmitted from patients in Australia to a server in the United States. Pearson correlations between the summated total index scores (TISs) for the p-WOMAC and m-WOMAC pre- and postsurgery were 0.98 and 0.99 (P<0.0001). There were no clinically important or statistically significant between-method differences in the adjusted total summated scores, pre- and postsurgery (adjusted mean differences=4.44, P=0.474 and 1.73, P=0.781, respectively). Internal consistency estimates of m-WOMAC reliability were 0.87-0.98. The m-WOMAC detected clinically important, statistically significant (P<0.0001) improvements in pain, stiffness, function, and TIS. No statistically significant differences in mode preference were detected. CONCLUSIONS: There was close agreement and no significant differences between m-WOMAC and p-WOMAC scores. This study confirms the validity, reliability, and responsiveness of the Exco InTouch-engineered, Java-based m-WOMAC Index application. EDC with the m-WOMAC Index provides unique opportunities for using quantitative measurement in clinical research and practice.


Assuntos
Artroplastia de Quadril/reabilitação , Artroplastia do Joelho/reabilitação , Telefone Celular , Sistemas Computadorizados de Registros Médicos/instrumentação , Osteoartrite/reabilitação , Medição da Dor/métodos , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril/psicologia , Artroplastia do Joelho/psicologia , Austrália , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/psicologia , Osteoartrite/cirurgia , Medição da Dor/instrumentação , Satisfação do Paciente , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Inquéritos e Questionários , Estados Unidos
14.
EMBO Mol Med ; 2(11): 458-71, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20941790

RESUMO

The majority of human colorectal cancers (CRCs) are initiated by mutations arising in the adenomatous polyposis coli (APC) tumour suppressor gene. However, a new class of non-APC mutated CRCs has been defined that have a serrated histopathology and carry the (V600E)BRAF oncogene. Here we have investigated the pathogenesis of serrated CRCs by expressing (V600E)Braf in the proliferative cells of the mouse gastrointestinal tract. We show that the oncogene drives an initial burst of Mek-dependent proliferation, leading to the formation of hyperplastic crypts. This is associated with ß-catenin nuclear localization by a mechanism involving Mapk/Erk kinase (Mek)-dependent, Akt-independent phosphorylation of Gsk3ß. However, hyperplastic crypts remain dormant for prolonged periods due to the induction of crypt senescence accompanied by upregulation of senescence-associated ß-galactosidase and p16(Ink4a). We show that tumour progression is associated with down-regulation of p16(Ink4a) through enhanced CpG methylation of exon 1 and knockout of Cdkn2a confirms this gene is a barrier to tumour progression. Our studies identify (V600E)BRAF as an early genetic driver mutation in serrated CRCs and indicate that, unlike APC-mutated cancers, this subtype arises by the bypassing of a (V600E)Braf driven oncogene-induced senescence programme.


Assuntos
Envelhecimento , Neoplasias Colorretais/fisiopatologia , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Trato Gastrointestinal/fisiopatologia , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Substituição de Aminoácidos/genética , Animais , Núcleo Celular/química , Neoplasias Colorretais/patologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , Trato Gastrointestinal/patologia , Perfilação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hiperplasia/patologia , Camundongos , Regulação para Cima , beta Catenina/metabolismo
15.
J Biol Chem ; 285(38): 29577-87, 2010 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-20610383

RESUMO

Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359-1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel ß-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.


Assuntos
Actinas/química , Actinas/metabolismo , Talina/química , Talina/metabolismo , Vinculina/química , Vinculina/metabolismo , Actinas/genética , Animais , Sítios de Ligação , Galinhas , Dicroísmo Circular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Células NIH 3T3 , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Talina/genética , Vinculina/genética
16.
EMBO J ; 29(6): 1069-80, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-20150896

RESUMO

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for beta-integrin tails, F0 and F1 are also required for activation of beta1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of beta1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.


Assuntos
Integrinas/metabolismo , Talina/química , Ubiquitina/química , Sequência de Aminoácidos , Sítios de Ligação , Adesão Celular , Integrinas/química , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Talina/metabolismo , Ubiquitina/metabolismo
17.
J Mol Biol ; 394(5): 944-56, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19804783

RESUMO

The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the beta-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced alphaIIbbeta3 integrin activation and for the localization of kindlin-1 to focal adhesions.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Sequência de Aminoácidos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas de Neoplasias/metabolismo , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Talina/química , Talina/genética , Talina/metabolismo
18.
J Biol Chem ; 284(22): 15097-106, 2009 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-19297334

RESUMO

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655-1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316-326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with beta3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.


Assuntos
Talina/química , Talina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Integrina beta3/química , Integrina beta3/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções
19.
J Biol Chem ; 284(13): 8866-76, 2009 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-19176533

RESUMO

The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin beta-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, "IBS2-A" (1974-2139) and "IBS2-B" (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.


Assuntos
Integrinas/química , Talina/química , Animais , Sítios de Ligação/fisiologia , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Integrinas/genética , Integrinas/metabolismo , Camundongos , Mapeamento de Peptídeos/métodos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Talina/genética , Talina/metabolismo , Vinculina/química , Vinculina/genética , Vinculina/metabolismo
20.
Biochemistry ; 45(6): 1805-17, 2006 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-16460027

RESUMO

Talin is a key protein involved in linking integrins to the actin cytoskeleton. The long flexible talin rod domain contains a number of binding sites for vinculin, a cytoskeletal protein important in stabilizing integrin-mediated cell-matrix junctions. Here we report the solution structure of a talin rod polypeptide (residues 1843-1973) which contains a single vinculin binding site (VBS; residues 1944-1969). Like other talin rod polypeptides, it consists of a helical bundle, in this case a four-helix bundle with a right-handed topology. The residues in the VBS important for vinculin binding were identified by studying the binding of a series of VBS-related peptides to the vinculin Vd1 domain. The key binding determinants are buried in the interior of the helical bundle, suggesting that a substantial structural change in the talin polypeptide is required for vinculin binding. Direct evidence for this was obtained by NMR and EPR spectroscopy. [1H,15N]-HSQC spectra of the talin fragment indicate that vinculin binding caused approximately two-thirds of the protein to adopt a flexible random coil. For EPR spectroscopy, nitroxide spin labels were attached to the talin polypeptide via appropriately located cysteine residues. Measurements of inter-nitroxide distances in doubly spin-labeled protein showed clearly that the helical bundle is disrupted and the mobility of the helices, except for the VBS helix, is markedly increased. Binding of vinculin to talin is thus a clear example of the unusual phenomenon of protein unfolding being required for protein/protein interaction.


Assuntos
Talina/química , Vinculina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cisteína/química , Proteínas do Citoesqueleto/química , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Talina/metabolismo
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