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1.
J Cell Biol ; 223(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38578285

RESUMO

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge ß-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.


Assuntos
Toxina da Cólera , Endorribonucleases , Proteínas Serina-Treonina Quinases , Resposta a Proteínas não Dobradas , Toxina da Cólera/genética , Toxina da Cólera/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Humanos , Animais , Camundongos , Linhagem Celular
2.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34251416

RESUMO

Epithelial cells lining mucosal surfaces distinctively express the inflammatory bowel disease risk gene INAVA. We previously found that INAVA has dual and competing functions: one at lateral membranes where it affects mucosal barrier function and the other in the cytosol where INAVA enhances IL-1ß signal transduction and protein ubiquitination and forms puncta. We now find that IL-1ß-induced INAVA puncta are biomolecular condensates that rapidly assemble and physiologically resolve. The condensates contain ubiquitin and the E3 ligase ßTrCP2, and their formation correlates with amplified ubiquitination, suggesting function in regulation of cellular proteostasis. Accordingly, a small-molecule screen identified ROS inducers, proteasome inhibitors, and inhibitors of the protein folding chaperone HSP90 as potent agonists for INAVA condensate formation. Notably, inhibitors of the p38α and mTOR pathways enhanced resolution of the condensates, and inhibitors of the Rho-ROCK pathway induced INAVA's competing function by recruiting INAVA to newly assembled intercellular junctions in cells where none existed before.


Assuntos
Proteínas de Transporte/genética , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Junções Intercelulares/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Contendo Repetições de beta-Transducina/genética , Células CACO-2 , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteostase/efeitos dos fármacos , Proteostase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/classificação , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo
3.
Oncogene ; 39(33): 5570-5587, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641858

RESUMO

The ARHGAP35 gene encoding p190A RhoGAP (p190A) is significantly altered by both mutation and allelic deletion in human cancer, but the functional implications of such alterations are not known. Here, we demonstrate for the first time that p190A is a tumor suppressor using a xenograft mouse model with carcinoma cells harboring defined ARHGAP35 alterations. In vitro, restoration of p190A expression in carcinoma cells promotes contact inhibition of proliferation (CIP) through activation of LATS kinases and phosphorylation of the proto-oncogenic transcriptional co-activator YAP. In contrast, p190A forms harboring recurrent cancer mutations exhibit loss of function in modulating the Hippo pathway, inducing CIP, as well as attenuated suppression of tumor growth in mice. We determine that p190A promotes mesenchymal to epithelial transition (MET) and elicits expression of a cassette of epithelial adherens junction-associated genes in a cell density-dependent manner. This cassette includes CDH1 encoding E-cadherin, which amplifies p190A-mediated LATS activation and is necessary for CIP. Oppositely, we establish that p190A is obligatory for E-cadherin to activate LATS kinases and induce CIP. Collectively, this work defines a novel mechanism by which p190A and E-cadherin cooperate in modulating Hippo signaling to suppress tumor cell growth.


Assuntos
Antígenos CD/biossíntese , Caderinas/biossíntese , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Proteínas Repressoras/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Cell Biol ; 219(2)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31985747

RESUMO

IRE1ß is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1ß have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1ß diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1ß can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1ß has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1ß to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host-environment interface.


Assuntos
Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Células CACO-2 , Endorribonucleases/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas Serina-Treonina Quinases/genética , Proteostase , Análise de Sequência de Proteína , Transdução de Sinais , Estresse Fisiológico , Resposta a Proteínas não Dobradas
5.
Bio Protoc ; 10(15): e3707, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33659371

RESUMO

The organization and distribution of proteins, lipids, and nucleic acids in eukaryotic cells is an essential process for cell function. Retrograde trafficking from the plasma membrane to the Golgi and endoplasmic reticulum can greatly modify cell membrane composition and intracellular protein dynamics, and thus typifies a key sorting step. However, methods to efficiently quantify the extent or kinetics of these events are currently limited. Here, we describe a novel quantitative and effectively real-time single-cell flow cytometry assay to directly measure retrograde membrane transport. The assay takes advantage of the well-known retrograde trafficking of cholera toxin engineered with split-fluorescent proteins to generate novel tools for immediate monitoring of intracellular trafficking. This approach will greatly extend the ability to study the underlying biology of intracellular membrane trafficking, and how trafficking systems can adapt to the physiologic needs of different cell types and cell states.

6.
Mol Biol Cell ; 31(7): 511-519, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31774722

RESUMO

Retrograde membrane trafficking from plasma membrane to Golgi and endoplasmic reticulum typifies one of the key sorting steps emerging from the early endosome that affects cell surface and intracellular protein dynamics underlying cell function. While some cell surface proteins and lipids are known to sort retrograde, there are few effective methods to quantitatively measure the extent or kinetics of these events. Here we took advantage of the well-known retrograde trafficking of cholera toxin and newly defined split fluorescent protein technology to develop a quantitative, sensitive, and effectively real-time single-cell flow cytometry assay for retrograde membrane transport. The approach can be applied in high throughput to elucidate the underlying biology of membrane traffic and how endosomes adapt to the physiologic needs of different cell types and cell states.


Assuntos
Bioensaio/métodos , Membrana Celular/metabolismo , Análise de Célula Única/métodos , Transporte Biológico , Toxina da Cólera/metabolismo , Doença , Retículo Endoplasmático/metabolismo , Fluorescência , Células HEK293 , Humanos , Células K562
7.
JAMA Neurol ; 77(2): 215-224, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710340

RESUMO

Importance: Basket-design clinical trials that allow investigation of treatment effects on different clinical syndromes that share the same molecular pathophysiology have not previously been attempted in neurodegenerative disease. Objective: To assess the safety, tolerability, and pharmacodynamics of the microtubule stabilizer TPI-287 (abeotaxane) in Alzheimer disease (AD) or the 4-repeat tauopathies (4RT) progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS). Design, Setting, and Participants: Two parallel-design, double-blind, placebo-controlled phase 1 randomized clinical trials in AD and 4RT were conducted from December 20, 2013, through May 4, 2017, at the University of California, San Francisco, and University of Alabama at Birmingham. A total of 94 patients with clinically diagnosed AD (n = 39) and 4RT (n = 55) were screened; of these, 3 refused to participate, and 10 with AD and 11 with 4RT did not meet inclusion criteria. A total of 29 patients with AD, 14 with PSP, and 30 with ß-amyloid-negative CBS (determined on positron emission tomography findings) were enrolled. Data were analyzed from December 20, 2013, through May 4, 2017, based on modified intention to treat. Interventions: Randomization was 8:3 drug to placebo in 3 sequential dose cohorts receiving 2.0, 6.3, or 20.0 mg/m2 of intravenous TPI-287 once every 3 weeks for 9 weeks, with an optional 6-week open-label extension. Main Outcomes and Measures: Primary end points were safety and tolerability (maximal tolerated dose) of TPI-287. Secondary and exploratory end points included TPI-287 levels in cerebrospinal fluid (CSF) and changes on biomarker, clinical, and neuropsychology measures. Results: A total of 68 participants (38 men [56%]; median age, 65 [range, 50-85] years) were included in the modified intention-to-treat analysis, of whom 26 had AD (14 women [54%]; median age, 63 [range, 50-76] years), and 42 had 4RT (16 women [38%]; median age, 69 [range, 54-83] years). Three severe anaphylactoid reactions occurred in TPI-287-treated patients with AD, whereas none were seen in patients with 4RT, leading to a maximal tolerated dose of 6.3 mg/m2 for AD and 20.0 mg/m2 for 4RT. More falls (3 in the placebo group vs 11 in the TPI-287 group) and a dose-related worsening of dementia symptoms (mean [SD] in the CDR plus NACC FTLD-SB [Clinical Dementia Rating scale sum of boxes with frontotemporal dementia measures], 0.5 [1.8] in the placebo group vs 0.7 [1.6] in the TPI-287 group; median difference, 1.5 [95% CI, 0-2.5]; P = .03) were seen in patients with 4RT. Despite undetectable TPI-287 levels in CSF, CSF biomarkers demonstrated decreased chitinase-3-like protein-1 (YKL-40) levels in the 4RT treatment arm (mean [SD], -8.4 [26.0] ng/mL) compared with placebo (mean [SD], 10.4 [42.3] ng/mL; median difference, -14.6 [95% CI, -30.0 to 0.2] ng/mL; P = .048, Mann-Whitney test). Conclusions and Relevance: In this randomized clinical trial, TPI-287 was less tolerated in patients with AD than in those with 4RT owing to the presence of anaphylactoid reactions. The ability to reveal different tau therapeutic effects in various tauopathy syndromes suggests that basket trials are a valuable approach to tau therapeutic early clinical development. Trial Registration: ClinicalTrials.gov identifiers: NCT019666666 and NCT02133846.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Paralisia Supranuclear Progressiva/tratamento farmacológico , Taxoides/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/líquido cefalorraquidiano , Paralisia Supranuclear Progressiva/líquido cefalorraquidiano , Taxoides/efeitos adversos , Taxoides/uso terapêutico , Resultado do Tratamento
8.
Elife ; 72018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30355448

RESUMO

Homeostasis at mucosal surfaces requires cross-talk between the environment and barrier epithelial cells. Disruption of barrier function typifies mucosal disease. Here we elucidate a bifunctional role in coordinating this cross-talk for the inflammatory bowel disease risk-gene INAVA. Both activities require INAVA's DUF3338 domain (renamed CUPID). CUPID stably binds the cytohesin ARF-GEF ARNO to effect lateral membrane F-actin assembly underlying cell-cell junctions and barrier function. Unexpectedly, when bound to CUPID, ARNO affects F-actin dynamics in the absence of its canonical activity as a guanine nucleotide-exchange factor. Upon exposure to IL-1ß, INAVA relocates to form cytosolic puncta, where CUPID amplifies TRAF6-dependent polyubiquitination and inflammatory signaling. In this case, ARNO binding to CUPID negatively-regulates polyubiquitination and the inflammatory response. INAVA and ARNO act similarly in primary human macrophages responding to IL-1ß and to NOD2 agonists. Thus, INAVA-CUPID exhibits dual functions, coordinated directly by ARNO, that bridge epithelial barrier function with extracellular signals and inflammation.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Mucosa/metabolismo , Mucosa/patologia , Transdução de Sinais , Actinas/metabolismo , Proteínas de Transporte/química , Membrana Celular/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Junções Intercelulares/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação
10.
J Cell Biol ; 217(9): 3183-3201, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-29934311

RESUMO

ARHGAP35 encoding p190A RhoGAP is a cancer-associated gene with a mutation spectrum suggestive of a tumor-suppressor function. In this study, we demonstrate that loss of heterozygosity for ARHGAP35 occurs in human tumors. We sought to identify tumor-suppressor capacities for p190A RhoGAP (p190A) and its paralog p190B in epithelial cells. We reveal an essential role for p190A and p190B to promote contact inhibition of cell proliferation (CIP), a function that relies on RhoGAP activity. Unbiased mRNA sequencing analyses establish that p190A and p190B modulate expression of genes associated with the Hippo pathway. Accordingly, we determine that p190A and p190B induce CIP by repressing YAP-TEAD-regulated gene transcription through activation of LATS kinases and inhibition of the Rho-ROCK pathway. Finally, we demonstrate that loss of a single p190 paralog is sufficient to elicit nuclear translocation of YAP and perturb CIP in epithelial cells cultured in Matrigel. Collectively, our data reveal a novel mechanism consistent with a tumor-suppressor function for ARHGAP35.


Assuntos
Proliferação de Células/fisiologia , Inibição de Contato/fisiologia , Células Epiteliais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias/patologia , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Cães , Proteínas Ativadoras de GTPase/genética , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Via de Sinalização Hippo , Humanos , Células Madin Darby de Rim Canino , Neoplasias/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAP , Quinases Associadas a rho/metabolismo
12.
Sleep ; 40(11)2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29029214

RESUMO

Objectives: To elucidate the unique sleep and waking characteristics in progressive supranuclear palsy (PSP), a neurodegenerative disease associated with motor deficits and dementia that largely affects the brainstem and thalamic regions. Methods: A total of 20 PSP and 16 healthy older adult controls participated in this study. The participants underwent an overnight polysomnography and multiple sleep latency test (MSLT) the following day. Prior to the MSLT last trial, they were asked to complete the Stanford Sleepiness Scale. Data were assessed for measures of latency to sleep onset, sleep duration, waking, and sleep staging during the night. Mean sleep latency, a measure of daytime sleepiness, sleep onset rapid eye movement (REM) periods, and microsleeps were studied with the MSLT. Spectral analysis of wake electroencephalogram (EEG) was performed for 30-second periods at the start of each MSLT trial. Results: PSP took significantly longer time to fall asleep (p < .001), slept less during the night (p ≤ .001), and had more wake after sleep onset than controls (p ≤ .001). PSP had less N2 sleep (p < .05) and N3 sleep (p < .05), and REM sleep (p < .001) than controls. During the MSLT, PSP took significantly longer to fall asleep (p < .001), did not have microsleeps when they remained awake throughout the assessment periods, but were subjectively sleepier than controls (p < .05). Gamma power was increased during wake EEG in PSP (p < .01). Conclusions: Sleep/waking regulation and REM sleep regulation are disrupted in PSP, leading to profound sleep deprivation without recuperation. Our findings suggest a diminished homeostatic sleep drive in PSP. This hyperaroused state is unique and is a severely disabling feature of PSP.


Assuntos
Latência do Sono , Paralisia Supranuclear Progressiva/fisiopatologia , Vigília , Idoso , Escuridão , Demência/complicações , Eletroencefalografia , Feminino , Humanos , Masculino , Polissonografia , Privação do Sono/fisiopatologia , Sono REM , Paralisia Supranuclear Progressiva/complicações , Fatores de Tempo
13.
Neurology ; 87(19): 2016-2025, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27742814

RESUMO

OBJECTIVE: To examine the utility and reliability of volumetric MRI in measuring disease progression in the 4 repeat tauopathies, progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS), to support clinical development of new tau-directed therapeutic agents. METHODS: Six- and 12-month changes in regional MRI volumes and PSP Rating Scale scores were examined in 55 patients with PSP and 33 patients with CBS (78% amyloid PET negative) compared to 30 normal controls from a multicenter natural history study. Longitudinal voxel-based morphometric analyses identified patterns of volume loss, and region-of-interest analyses examined rates of volume loss in brainstem (midbrain, pons, superior cerebellar peduncle), cortical, and subcortical regions based on previously validated atlases. Results were compared to those in a replication cohort of 226 patients with PSP with MRI data from the AL-108-231 clinical trial. RESULTS: Patients with CBS exhibited greater baseline atrophy and greater longitudinal atrophy rates in cortical and basal ganglia regions than patients with PSP; however, midbrain and pontine atrophy rates were similar. Voxel-wise analyses showed distinct patterns of regional longitudinal atrophy in each group as compared to normal controls. The midbrain/pons volumetric ratio differed between diagnoses but remained stable over time. In both patient groups, brainstem atrophy rates were correlated with disease progression measured using the PSP Rating Scale. CONCLUSIONS: Volume loss is quantifiable over a period of 6 months in CBS and PSP. Future clinical trials may be able to combine CBS and PSP to measure therapeutic effects.


Assuntos
Gânglios da Base/patologia , Córtex Cerebral/patologia , Paralisia Supranuclear Progressiva/complicações , Idoso , Atrofia/diagnóstico por imagem , Atrofia/patologia , Gânglios da Base/diagnóstico por imagem , Córtex Cerebral/diagnóstico por imagem , Progressão da Doença , Feminino , Seguimentos , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estatísticas não Paramétricas , Paralisia Supranuclear Progressiva/diagnóstico por imagem
14.
PLoS One ; 11(6): e0157218, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27310132

RESUMO

Progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS) are both 4 microtubule binding repeat tauopathy related disorders. Clinical trials need new biomarkers to assess the effectiveness of tau-directed therapies. This study investigated the regional distribution of longitudinal diffusion tensor imaging changes, measured by fractional anisotropy, radial and axial diffusivity over 6 months median interval, in 23 normal control subjects, 35 patients with PSP, and 25 patients with CBS. A mixed-effects framework was used to test longitudinal changes within and between groups. Correlations between changes in diffusion variables and clinical progression were also tested. The study found that over a 6 month period and compared to controls, the most prominent changes in PSP were up to 3±1% higher rates of FA reduction predominantly in superior cerebellar peduncles, and up to 18±6% higher rates of diffusivity increases in caudate nuclei. The most prominent changes in CBS compared to controls were up to 4±1% higher rates of anisotropy reduction and 18±6% higher rates of diffusivity increase in basal ganglia and widespread white matter regions. Compared to PSP, CBS was mainly associated with up to 3±1% greater rates of anisotropy reduction around the central sulci, and 11±3% greater rates of diffusivity increase in superior fronto-occipital fascicules. Rates of diffusivity increases in the superior cerebellar peduncle correlated with rates of ocular motor decline in PSP patients. This study demonstrated that longitudinal diffusion tensor imaging measurement is a promising surrogate marker of disease progression in PSP and CBS over a relatively short period.


Assuntos
Núcleo Caudado/diagnóstico por imagem , Córtex Cerebral/diagnóstico por imagem , Pedúnculo Cerebelar Médio/diagnóstico por imagem , Paralisia Supranuclear Progressiva/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , Idoso , Anisotropia , Estudos de Casos e Controles , Núcleo Caudado/patologia , Núcleo Caudado/fisiopatologia , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Imagem de Tensor de Difusão , Progressão da Doença , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Pedúnculo Cerebelar Médio/patologia , Pedúnculo Cerebelar Médio/fisiopatologia , Paralisia Supranuclear Progressiva/patologia , Paralisia Supranuclear Progressiva/fisiopatologia , Síndrome , Substância Branca/patologia , Substância Branca/fisiopatologia
15.
Mov Disord ; 31(5): 742-7, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26948290

RESUMO

BACKGROUND: Two recent randomized, placebo-controlled trials of putative disease-modifying agents (davunetide, tideglusib) in progressive supranuclear palsy (PSP) failed to show efficacy, but generated data relevant for future trials. METHODS: We provide sample size calculations based on data collected in 187 PSP patients assigned to placebo in these trials. A placebo effect was calculated. RESULTS: The total PSP-Rating Scale required the least number of patients per group (N = 51) to detect a 50% change in the 1-year progression and 39 when including patients with ≤ 5 years disease duration. The Schwab and England Activities of Daily Living required 70 patients per group and was highly correlated with the PSP-Rating Scale. A placebo effect was not detected in these scales. CONCLUSIONS: We propose the 1-year PSP-Rating Scale score change as the single primary readout in clinical neuroprotective or disease-modifying trials. The Schwab and England Activities of Daily Living could be used as a secondary outcome. © 2016 International Parkinson and Movement Disorder Society.


Assuntos
Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Efeito Placebo , Ensaios Clínicos Controlados Aleatórios como Assunto/estatística & dados numéricos , Projetos de Pesquisa/estatística & dados numéricos , Índice de Gravidade de Doença , Paralisia Supranuclear Progressiva/tratamento farmacológico , Atividades Cotidianas , Humanos , Oligopeptídeos/farmacologia , Tamanho da Amostra , Tiadiazóis/farmacologia
16.
Mol Cell Proteomics ; 13(11): 3164-76, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25073739

RESUMO

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.


Assuntos
Monofosfato de Adenosina/química , Proteínas de Bactérias/metabolismo , Química Click/métodos , Reação de Cicloadição , Processamento de Proteína Pós-Traducional/fisiologia , Sequência de Bases , Cobre/química , Interações Hospedeiro-Patógeno , Humanos , Pasteurellaceae/metabolismo , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Vibrioses/patologia , Vibrio parahaemolyticus/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Proteína RAC2 de Ligação ao GTP
17.
Plant Physiol ; 164(1): 424-39, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24198318

RESUMO

MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Transporte/metabolismo , Ácido Abscísico/genética , Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Secas , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Mutação , Estômatos de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/crescimento & desenvolvimento , Transdução de Sinais/genética , Estresse Fisiológico
18.
Cell Host Microbe ; 13(5): 558-569, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23684307

RESUMO

The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1α (IRE1α), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1α-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1α to degrade endogenous mRNA, a process termed regulated IRE1α-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cytosolic sensor of RNA viruses, to activate NF-κB and interferon pathways. We propose IRE1α provides for a generalized mechanism of innate immune surveillance originating within the ER lumen.


Assuntos
Toxina da Cólera/imunologia , Toxina da Cólera/metabolismo , RNA Helicases DEAD-box/imunologia , Endorribonucleases/imunologia , Endorribonucleases/metabolismo , Imunidade Inata , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Humanos , Ligação Proteica , Receptores Imunológicos
19.
J Am Chem Soc ; 133(43): 17103-5, 2011 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-21942216

RESUMO

Protein AMPylation is an emerging post-translational modification, which plays key roles in bacterial pathogenesis and cell biology. Enzymes with AMPylation activity, referred to as AMPylators, have been identified in several bacterial pathogens and eukaryotes. To facilitate the study of this unique modification, we developed an alkynyl chemical reporter for detection and identification of protein AMPylation substrates. Covalent functionalization of AMPylation substrates with the alkynyl reporter in lieu of adenylyl 5'-monophosphate (AMP) allows their subsequent bioorthogonal ligation with azide-fluorescent dyes or affinity enrichment tags. We show that this chemical reporter is transferred by a range of AMPylators onto their cognate protein substrates and allows rapid detection and identification of AMPylated substrates.


Assuntos
Monofosfato de Adenosina/metabolismo , Alcinos/metabolismo , Proteínas/metabolismo , Monofosfato de Adenosina/química , Alcinos/química , Proteínas/química , Proteômica
20.
J Biotechnol ; 151(3): 251-4, 2011 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-21185336

RESUMO

An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.


Assuntos
Monofosfato de Adenosina/imunologia , Anticorpos/química , Treonina/imunologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Western Blotting , Clonagem Molecular , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Soros Imunes , Processamento de Proteína Pós-Traducional , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Treonina/química , Treonina/metabolismo , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/imunologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo
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