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1.
Sci Rep ; 11(1): 7815, 2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837238

RESUMO

Polyglutamine (polyQ) expansion of proteins can trigger protein misfolding and amyloid-like aggregation, which thus lead to severe cytotoxicities and even the respective neurodegenerative diseases. However, why polyQ aggregation is toxic to cells is not fully elucidated. Here, we took the fragments of polyQ-expanded (PQE) ataxin-7 (Atx7) and huntingtin (Htt) as models to investigate the effect of polyQ aggregates on the cellular proteostasis of endogenous ataxin-3 (Atx3), a protein that frequently appears in diverse inclusion bodies. We found that PQE Atx7 and Htt impair the cellular proteostasis of Atx3 by reducing its soluble as well as total Atx3 level but enhancing formation of the aggregates. Expression of these polyQ proteins promotes proteasomal degradation of endogenous Atx3 and accumulation of its aggregated form. Then we verified that the co-chaperone HSJ1 is an essential factor that orchestrates the balance of cellular proteostasis of Atx3; and further discovered that the polyQ proteins can sequester HSJ1 into aggregates or inclusions in a UIM domain-dependent manner. Thereby, the impairment of Atx3 proteostasis may be attributed to the sequestration and functional loss of cellular HSJ1. This study deciphers a potential mechanism underlying how PQE protein triggers proteinopathies, and also provides additional evidence in supporting the hijacking hypothesis that sequestration of cellular interacting partners by protein aggregates leads to cytotoxicity or neurodegeneration.

2.
EMBO Rep ; : e51649, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33855783

RESUMO

Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. Here, we show that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. We further show that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. We finally demonstrate that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis.

3.
Drug Deliv ; 28(1): 454-462, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33620010

RESUMO

This study aimed to construct a transdermal iontophoresis delivery system for terazosin hydrochloride (IDDS-TEH), which included a positive and negative electrode hydrogel prescription. Intact guinea pig skin was used as a model for the skin barrier function, and the current intensity, terazosin hydrochloride (TEH) concentration, pH, competitive salt, and transdermal enhancer properties were studied. The blood drug concentration was determined in Sprague-Dawley (SD) rats using HPLC, and the antihypertensive effects of IDDS-TEH were evaluated in spontaneously hypertensive rats (SHRs). The results showed that the steady-state penetration rate of TEH increased (from 80.36 µg·cm-2·h-1 to 304.93 µg·cm-2·h-1), followed by an increase in the current intensity (from 0.10 mA·cm-2 to 0.49 mA·cm-2). The pH values also had a significant influence on percutaneous penetration. The blood concentration of IDDS-TEH was significantly higher (p < .05) than with passive diffusion, which could not be detected. The main pharmacokinetic parameters of the high current group (0.17 mA·cm-2) and the low current group (0.09 mA·cm-2) were AUC0-t: 5873.0 ng·mL-1·h and 2493.7 ng·mL-1·h, respectively. Meanwhile, the pharmacodynamic results showed that IDDS-TEH significantly decreased the blood pressure of SHRs compared with the TEH hydrogel without loading current. Therefore, TEH could be successfully delivered by the transdermal iontophoresis system in vitro and in vivo, and further clinical studies should be explored to develop a therapeutically useful protocol.

4.
Sci Total Environ ; : 142690, 2020 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-33071127

RESUMO

Regional acclimatisation and microbial interactions significantly influence the resilience of reef-building corals facing anthropogenic climate change, allowing them to adapt to environmental stresses. However, the connections between community structure and microbial interactions of the endemic coral microbiome and holobiont acclimatisation remain unclear. Herein, we used generation sequencing of internal transcribed spacer (ITS2) and 16S rRNA genes to investigate the microbiome composition (Symbiodiniaceae and bacteria) and associated potential interactions of endemic dominant coral holobionts (Pocillopora verrucosa and Turbinaria peltata) in the South China Sea (SCS). We found that shifts in Symbiodiniaceae and bacterial communities of P. verrucosa were associated with latitudinal gradient and climate zone changes, respectively. The C1 sub-clade consistently dominated the Symbiodiniaceae community in T. peltata; yet, the bacterial community structure was spatially heterogeneous. The relative abundance of the core microbiome among P. verrucosa holobionts was reduced in the biogeographical transition zone, while bacterial taxa associated with anthropogenic activity (Escherichia coli and Sphingomonas) were identified in the core microbiomes. Symbiodiniaceae and bacteria potentially interact in microbial co-occurrence networks. Further, increased bacterial, and Symbiodiniaceae α-diversity was associated with increased and decreased network complexity, respectively. Hence, Symbiodiniaceae and bacteria demonstrated different flexibility in latitudinal or climatic environmental regimes, which correlated with holobiont acclimatisation. Core microbiome analysis has indicated that the function of core bacterial microbiota might have changed in distinct environmental regimes, implying potential human activity in the coral habitats. Increased bacterial α diversity may lead to a decline in the stability of coral-microorganism symbioses, whereas rare Symbiodiniaceae may help to retain symbioses. Cladocopium, γ-proteobacteria, while α-proteobacteria may have been the primary drivers in the Symbiodiniaceae-bacterial interactions (SBIs). Our study highlights the association between microbiome shift in distinct environmental regimes and holobiont acclimatisation, while providing insights into the impact of SBIs on holobiont health and acclimatisation during climate change.

5.
Biochem J ; 477(21): 4295-4312, 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33094816

RESUMO

Ubiquitin-specific protease 19 (USP19) is a member of the deubiquitinating (DUB) enzymes that catalyze removing the ubiquitin signals from target proteins. Our previous research has demonstrated that USP19 up-regulates the protein level and aggregation of polyQ-expanded huntingtin through the involvement of heat shock protein 90 (HSP90). Here, we present solution structures of the CS1, CS2 and UbL domains of USP19 and structural insights into their domain interactions. We found that the tandem CS domains fold back to interact with the C-terminal USP domain (USPD) intra-molecularly that leads to inhibition of the catalytic core of USP19, especially CS1 interacts with the embedded UbL domain and CS2 does with the CH2 catalytic core. Moreover, CS2 specifically interacts with the NBD domain of HSP90, which can activate the DUB enzyme. A mechanism of auto-inhibition of USP19 and activation by HSP90 is proposed, on which USP19 modulates the protein level of polyQ-expanded huntingtin in cells. This study provides structural and mechanistic insights into the modulation of protein level and aggregation by USP19 with the assistance of HSP90.

6.
Soft Matter ; 16(25): 5981-5989, 2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32543634

RESUMO

Complex emulsions are used to fabricate new morphologies of multiple Janus droplets, evolving from non-engulfing to complete engulfing core/shell configuration. The produced droplets contain an aqueous phase of dextran (DEX) solution and an oil phase, which is mixed with ethoxylated trimethylolpropane triacrylate (ETPTA) and poly(ethylene glycol) diacrylate (PEGDA). The PEGDA in the oil phase is transferred into the aqueous phase to form complex morphologies due to the phase separation of PEGDA and DEX. The effects are investigated including the ratio of oil to aqueous phase, the content of initial PEGDA, DEX and surfactants, and the type of surfactants. DEX/PEGDA-ETPTA core/shell-single phase Janus droplets are formed with an increasing engulfed oil droplet into the aqueous droplet while the ratio of oil to aqueous phase increases or the initial PEGDA content increases. The high DEX content leads to the DEX-PEGDA-ETPTA doublet Janus. The use of surfactants polyglycerol polyricinoleate (PGPR) and Span 80 results in the formation of DEX/PEGDA/ETPTA single core/double shell and DEX/PEGDA-ETPTA core/shell-single phase Janus droplets, respectively. These complex emulsions are utilized to fabricate solid particles of complex shapes. This method contributes to new material design underpinned by mass transfer and phase separation, which can be extended to other complex emulsion systems.

7.
Sci Rep ; 10(1): 9519, 2020 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518289

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

8.
Sci Total Environ ; 723: 138026, 2020 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-32213418

RESUMO

Global warming has degraded coral reef ecosystems worldwide. Some corals develop thermal tolerance by associating with heat-tolerant Symbiodiniaceae. Here, we studied the mechanisms surrounding the dispersal, genetic variation and symbionts interaction of heat-tolerant Durusdinium trenchii across 13° latitudes in the South China Sea (SCS), to explore the possible mechanisms underlying these changes. Our results showed that Durusdinium trenchii are widely distributed in the seawater from the SCS. Our analyses of microsatellite loci revealed that D. trenchii has a high genetic diversity in the SCS; STRUCTURE analysis indicated that D. trenchii can be divided into four populations within the SCS; There exist positive correlations between genetic variation and geographic isolation, average sea surface temperature (SST) and variations in SST. Network modelling inferences showed that D. trenchii is a key species in the Symbiodiniaceae communities in the tropical SCS and contributes the greatest number of co-exclusion relationships. These results indicated that D. trenchii can affect the rare Symbiodiniaceae community. The long lifespan and the monsoon-driven ocean currents have shaped the wide distribution of D. trenchii. But low SST limits the ability of D. trenchii to establish stable symbioses with coral in the subtropical habitats. Geographical isolation and SST have shaped significant genetic variation of D.trenchii around the SCS. Our data reveals the biogeography and genetic population characteristics of D. trenchii in the Indo-Pacific region, and suggests that heat-tolerance and high genetic diversity of D. trenchii aid the corals with their adaptation to climate change.


Assuntos
Antozoários , Animais , China , Mudança Climática , Recifes de Corais , Ecossistema , Variação Genética , Temperatura Alta
9.
J Am Chem Soc ; 142(7): 3412-3421, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32003979

RESUMO

TDP-43 is a primary pathological hallmark protein of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, which may exist in the form of amyloid inclusions in the cells of patients. In addition to serving as a biomarker for these diseases, TDP-43 can also directly trigger neurodegeneration. We previously determined the amyloidogenic core region of TDP-43 (residues 311-360) and showed by solution NMR that this region includes two α-helices [(321-330) and (335-343)] in solution. We suggested that the helix-to-sheet structural transformation initiates TDP-43 aggregation. In the present study, X-ray diffraction shows that TDP-43 (311-360) aggregates adopt a cross-ß structure. Thioredoxin (Trx)-fused TDP-43 (311-360) can undergo liquid-liquid phase separation (LLPS) before fibrillation, suggesting that phase separation is an intermediate step before amyloid formation. Solid-state NMR (SSNMR), carried out to elucidate the structural changes of TDP-43 (311-360) at the atomic level, indicates five ß-strands of the amyloids formed, with the major two ß-strands contributed by the first helical region in the solution structure. The NMR evidence is also in support of the fibril having a parallel in-register conformation, implying a mechanism in which the helix-helix interactions in LLPS are converted into ß-strand parallel lateral association upon fibrillation. Our studies have assigned many key interresidue interactions that contribute to the stability of the fibril, including F316 with I318 and Q327 and W334 with A325, A326, A329, and S332. SSNMR with 1H detection reveals a unique close interaction between the indole Nε1-Hε1 of W334 and the side-chain carbonyl of Q343. This interaction could be a very important factor in initiating TDP-43 (311-360) folding/misfolding in LLPS.


Assuntos
Proteínas Amiloidogênicas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas Amiloidogênicas/química , Proteínas de Ligação a DNA/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Transição de Fase , Conformação Proteica , Multimerização Proteica
10.
Thorac Cancer ; 11(3): 754-761, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32012484

RESUMO

BACKGROUND: There is no consensus on the definition or recommended radiotherapy treatment of ultracentral non-small cell lung cancer (NSCLC). Here, we report our institution's experience in treating ultracentral lung cancer patients with stereotactic ablative radiotherapy (SABR) of 60 Gy in eight fractions. METHODS: We retrospectively reviewed the outcomes of 21 ultracentral NSCLC patients treated with 60 Gy SABR in eight fractions. We defined ultracentral lung cancer as the planning target volume (PTV) directly abutting or overlapping central structures, including the proximal bronchial tree, heart, and great vessels but not the esophagus. The Kaplan-Meier method was used to estimate overall survival (OS), progression-free survival (PFS) and local control (LC). Toxicity was scored per the CTCAE v4.03. RESULTS: The median follow-up time was 15 months, and the median OS was 15 months. The one- and two-year OS rates were 87.5% and 76.6%, respectively. The one- and two-year PFS rates were 71.1% and 64.0%, respectively. The one- and two-year LC rates were 92.9% and 92.9%, respectively. The rate of grade 2 treatment-related toxicities was 19.1%. There was no grade ≥ 3 treatment-related toxicity. CONCLUSION: SABR of 60 Gy in eight fractions is feasible for ultracentral NSCLC.

11.
Langmuir ; 36(2): 576-584, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31877048

RESUMO

In the present work, a novel microchannel device was developed and used for the preparation of core-shell microspheres combining with a dextran/poly(ethylene glycol) diacrylate (DEX/PEGDA) aqueous two-phase system. Silica@silica core-shell microspheres were prepared as a model material. Silica@silica core-shell microspheres with different sizes of cores and thicknesses of shells were prepared by using different flowrate ratios of DEX/silica and PEGDA/silica aqueous solutions. The content of colloidal silica and the calcination temperature have a significant effect on the texture properties of the prepared core-shell microspheres. The surface area decreased from 199 to 177 m2/g with an increase in the colloidal silica content from 30 to 60 wt %. For a specific colloidal silica content (50 wt %), with the increase in calcination temperature from room temperature to 650 °C, the total pore volume went through a maximum of 0.7 cm3 g-1 with a surface area of 178 m2 g-1 and pore size of 7.32 nm at 450 °C. Due to the accumulation of metal nanoparticles in DEX, different metal nanoparticles (Ni and Pd) were successfully introduced into the core of the core-shell microspheres for the preparation of silica/metal nanoparticles@silica core-shell microsphere catalysts. The catalysts showed similar catalytic performance as the metal nanoparticles for hydrogenation of 4-nitrophenol with a conversion higher than 95%. However, the core-shell microsphere catalyst is much easier to recover. The reuse experiments indicated that the core-shell catalyst has high stability.

12.
Zhongguo Fei Ai Za Zhi ; 22(11): 696-701, 2019 Nov 20.
Artigo em Chinês | MEDLINE | ID: mdl-31771738

RESUMO

BACKGROUND: The standard treatment for locally advanced non-small cell lung cancer (NSCLC) is radiotherapy concurrent with chemotherapy, but the survival was not satisfied. With the development of intensity modulated radiotherapy, simultaneous integrated boost technique (SIB) becomes the research direction of locally advanced NSCLC. The aim of this study is to investigate the efficacy and safety of SIB intensity modulated radiotherapy technique for locally advanced NSCLC. METHODS: We retrospectively reviewed the clinical data of locally advanced NSCLC who were treated with radiotherapy by SIB technique in Peking University Cancer Hospital from June 2015 to December 2018. Kaplan-Meier method was used for analysis. RESULTS: Ninty-three patients were included in the analysis. After a median follow-up of 34.23 months, 3-year overall survival (OS), progression-free survival (PFS), local-recurrence free survival (LRFS) and metastasis free survival (MFS) rates were 53.0%, 37.0%, 50.5% and 50.5%, respectively. The incidence of grade ≥3 esophagitis was 5.4%. There were 2 (2.2%) patients experiencing grade ≥3 radiation-related pneumonia. CONCLUSIONS: Radiation with SIB intensity modulated radiotherapy technique is effective and safe for patients with locally advanced NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Radioterapia de Intensidade Modulada/métodos , Adulto , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Radioterapia de Intensidade Modulada/efeitos adversos , Estudos Retrospectivos , Segurança , Análise de Sobrevida
13.
Sci Rep ; 9(1): 7481, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097749

RESUMO

Ataxin-7 (Atx7) is a disease-related protein associated with the pathogenesis of spinocerebellar ataxia 7, while its polyglutamine (polyQ) tract in N-terminus is the causative source of aggregation and proteinopathy. We investigated the structure, dynamics and aggregation properties of the N-terminal 62-residue fragment of Atx7 (Atx7-N) by biochemical and biophysical approaches. The results showed that the normal Atx7-N with a tract of 10 glutamines (10Q) overall adopts a flexible and disordered structure, but it may contain a short or small population of helical structure in solution. PolyQ expansion increases the α-helical propensity of the polyQ tract and consequently enhances its transformation into ß-sheet structures during amyloid aggregation. An alanine-rich region (ARR) just ahead of the polyQ tract forms a local and relatively stable α-helix. The ARR α-helix can initiate and stabilize helical formation of the following polyQ tract, but it may suppress aggregation of the polyQ-expanded Atx7-N both in vitro and in cell. Thus, the preceding ARR segment in Atx7-N may influence the dynamic structure and aggregation property of the polyQ tract and even determine the threshold of the pathogenic polyQ lengths. This study may gain structural and dynamic insights into amyloid aggregation of Atx7 and help us further understand the Atx7 proteinopathy based on polyQ expansion.


Assuntos
Amiloide/química , Ataxina-7/química , Simulação de Dinâmica Molecular , Multimerização Proteica , Amiloide/metabolismo , Ataxina-7/metabolismo , Células HEK293 , Humanos , Peptídeos/química , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta
14.
Clin Lung Cancer ; 19(4): e399-e404, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29519614

RESUMO

PURPOSE: Stereotactic ablative body radiotherapy (SABR) represents an exciting, tolerable, and highly effective form of radiotherapy. Ongoing investigations into the interactions between radiotherapy and the immune system have uncovered new mechanisms that can be exploited to improve efficacy. We determined whether baseline or posttreatment immune parameters could predict disease control and toxicity in stage I non-small-cell lung cancer (NSCLC) patients treated with SABR. PATIENTS AND METHODS: Peripheral blood samples were collected from 62 patients 24 hours before treatment and within 4 weeks after treatment for lymphocyte subset count analysis. All peripheral blood samples were analyzed by flow cytometry. Associated parameters were evaluated to determine their association with progression-free survival (PFS) and symptomatic radiation pneumonitis (grade 2 or higher). The survival rates were estimated with Kaplan-Meier and multivariable analyses using binary logistic regression analysis or a Cox proportional hazards model. RESULTS: At a median follow-up time of 36.0 months, the PFS rates for years 1, 2, and 3 were 91.0%, 82.5%, and 48.9%, respectively. The multivariable logistic regression analysis showed that only proportion of lung receiving 20 Gy of radiotherapy (odds ratio = 1.41; 95% confidence interval, 1.05-1.87; P = .023) and mean lung dose (odds ratio = 2.02; 95% confidence interval, 1.16-3.53; P = .016) were associated with symptomatic radiation pneumonitis (grade 2 or higher). Moreover, the immune parameters had no predictive value. In the multivariable Cox regression analysis, an elevated posttreatment cytotoxic CD8+ T-cell level was an independent prognostic factor for longer PFS in stage I NSCLC (hazard ratio, 1.16; 95% confidence interval, 1.01-1.28; P = .01). CONCLUSION: A higher posttreatment cytotoxic CD8+ T-cell level was predictive of better PFS in stage I NSCLC patients receiving SABR. Thus, enhancing tumor antigen-specific cellular immunity by combining radiotherapy and immunotherapy might be a crucial strategy for improving survival in these patients.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/radioterapia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Pneumonite por Radiação/imunologia , Radiocirurgia/efeitos adversos , Radiocirurgia/métodos , Estudos Retrospectivos , Subpopulações de Linfócitos T/imunologia , Resultado do Tratamento
15.
FASEB J ; 32(6): 2923-2933, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29401586

RESUMO

The components of ubiquitin (Ub)-proteasome system, such as Ub, Ub adaptors, or proteasome subunits, are commonly accumulated with the aggregated proteins in inclusions, but how protein aggregates sequester Ub-related proteins remains elusive. Using N-terminal huntingtin (Htt-N552) and ataxin (Atx)-3 as model proteins, we investigated the molecular mechanism underlying sequestration of Ub adaptors by polyQ-expanded proteins. We found that polyQ-expanded Htt-N552 and Atx-3 sequester endogenous Ub adaptors, human RAD23 homolog B (hHR23B) and ubiquilin (UBQLN)-2, into inclusions. This sequestration effect is dependent on the UBA domains of Ub adaptors and the conjugated Ub of the aggregated proteins. Moreover, polyQ-expanded Htt-N552 and Atx-3 reduce the protein level of xeroderma pigmentosum group C (XPC) by sequestration of hHR23B, suggesting that this process may cut down the available quantity of hHR23B and thus affect its normal function in stabilizing XPC. Our findings demonstrate that polyQ-expanded proteins sequester Ub adaptors or other Ub-related proteins into aggregates or inclusions through ubiquitination of the pathogenic proteins. This study may also provide a common mechanism for the formation of Ub-positive inclusions in cells.-Yang, H., Yue, H.-W., He, W.-T., Hong, J.-Y., Jiang, L.-L., Hu, H.-Y. PolyQ-expanded huntingtin and ataxin-3 sequester ubiquitin adaptors hHR23B and UBQLN2 into aggregates via conjugated ubiquitin.


Assuntos
Ataxina-3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Huntingtina/metabolismo , Peptídeos/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Ataxina-3/genética , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteína Huntingtina/genética , Peptídeos/genética , Domínios Proteicos , Estabilidade Proteica , Proteínas Repressoras/genética , Ubiquitinas/genética
16.
Sci Rep ; 7(1): 14797, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29093475

RESUMO

Huntington's disease (HD) is caused by aberrant expansion of polyglutamine (polyQ) in the N-terminus of huntingtin (Htt). Our previous study has demonstrated that HSP90 is involved in the triage decision of Htt, but how HSP90 recognizes and regulates Htt remains elusive. We investigated the interaction between HSP90 and the N-terminal fragments of Htt (Htt-N), such as the N-terminal 90-residue fragment (Htt-N90). Our results showed that HSP90 binds to the N-terminal extreme of Htt-N in a sequence just ahead of the polyQ tract. Structural integration of the middle and C-terminal domains of HSP90 is essential for interacting with Htt-N90, and the dimerization mediated by the C-terminal domain facilitates this interaction. Moreover, ubiquitin-specific protease 19 (USP19), a deubiquitinating enzyme interacting with HSP90, up-regulates the protein level of Htt-N90 and consequently promotes its aggregation, whereas disruption of the interaction between Htt-N90 and HSP90 attenuates the effect of USP19 on Htt-N90. Thus, HSP90 interacts with Htt-N90 on the N-terminal amphipathic α-helix, and then recruits USP19 to modulate the protein level and aggregation of Htt-N90. This study provides mechanistic insights into the recognition between HSP90 and the N-terminus of Htt, and the triage decision for the Htt protein by the HSP90 chaperone system.


Assuntos
Endopeptidases , Proteínas de Choque Térmico HSP90 , Proteína Huntingtina , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Domínios Proteicos
17.
Sci Rep ; 7(1): 6196, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28733604

RESUMO

TDP-43 is a nuclear factor that functions in promoting pre-mRNA splicing. Deletion of the N-terminal domain (NTD) and nuclear localization signal (NLS) (i.e., TDP-35) results in mislocalization to cytoplasm and formation of inclusions. However, how the NTD functions in TDP-43 activity and proteinopathy remains largely unknown. Here, we studied the structure and function of the NTD in inclusion formation and pre-mRNA splicing of TDP-43 by using biochemical and biophysical approaches. We found that TDP-43 NTD forms a homodimer in solution in a concentration-dependent manner, and formation of intermolecular disulfide results in further tetramerization. Based on the NMR structure of TDP-43 NTD, the dimerization interface centered on Leu71 and Val72 around the ß7-strand was defined by mutagenesis and size-exclusion chromatography. Cell experiments revealed that the N-terminal dimerization plays roles in protecting TDP-43 against formation of cytoplasmic inclusions and enhancing pre-mRNA splicing activity of TDP-43 in nucleus. This study may provide mechanistic insights into the physiological function of TDP-43 and its related proteinopathies.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Processamento de RNA , Cromatografia em Gel , Citoplasma/metabolismo , Dissulfetos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Multimerização Proteica , Estrutura Secundária de Proteína
18.
Curr Protein Pept Sci ; 18(1): 100-103, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27396751

RESUMO

Protein aggregation and amyloidogenesis are closely associated with the pathogenesis of neurodegenerative diseases. Elucidating the morphology and structure of the amyloid aggregates or fibrils is important for understanding the molecular mechanisms of these proteinopathies. This review article describes the general principle and establishment of solid-state circular dichroism (ssCD) spectroscopy, and discusses its application for the analysis of secondary structures of proteins or peptides in amyloids and structural transformation of these proteins or peptides during their amyloidogenic aggregation.


Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Dicroísmo Circular , Agregados Proteicos , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Dicroísmo Circular/métodos , Humanos , Conformação Proteica , Relação Estrutura-Atividade
19.
Sci Rep ; 6: 23928, 2016 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-27030292

RESUMO

TDP-43 is a DNA/RNA binding protein associated with TDP-43 proteinopathies. Many mutations have been identified in the flexible C-terminal region, which is implicated in the disease pathology. We investigated four point mutations in the amyloidogenic core region (residues 311-360) of TDP-43 by biochemical and spectroscopic methods. We found that the G335D mutation enhances the aggregation and inclusion formation of TDP-43 and this mutant in TDP-35 (the C-terminal fragment of 35 kDa) exaggerates the antagonist effect on RNA processing by endogenous TDP-43; whereas Q343R gives an opposite effect. As a comparison, M337V and Q331K have very little impact on the aggregation and inclusion formation of TDP-43 or TDP-35. NMR structural analysis showed that the G335D mutant in the core region forms a loop linker between the two α-helices and promotes α-to-ß transition, but Q343R loses the second helix and consequently the structural transformation. Thus, the propensity of structural transformation in the amyloidogenic core of TDP-43 determines its aggregation and inclusion formation. This study may provide a molecular mechanism of the TDP-43 proteinopathies caused by genetic mutations.


Assuntos
Proteínas Amiloidogênicas/química , Proteínas de Ligação a DNA/química , Mutação , Agregados Proteicos/genética , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Proteínas Amiloidogênicas/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética
20.
Thorac Cancer ; 6(3): 269-74, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26273372

RESUMO

BACKGROUND: The purpose of this study was to investigate the prevalence, distribution, and prognostic role of v-raf murine sarcoma viral oncogene homolog B (BRAF) V600E mutations in Chinese patients with lung adenocarcinoma (ADC), and to explore the possibility of BRAF V600E mutation detection in plasma DNA. METHODS: Data from 190 patients with lung ADCs treated at the Peking University Cancer Hospital from July 2011 to March 2012 were collected. The amplification refractory mutation system was used for BRAF V600E testing and denaturing high-performance liquid chromatography for epidermal growth factor receptor (EGFR) mutation detection. In BRAF V600E-mutant cases, paired plasma DNA was tested for mutation status of BRAF V600E and EGFR. The distribution and prognostic role of BRAF V600E mutations were analyzed using SPSS 13.0. RESULTS: Among 190 patients with advanced lung ADC, eight (4.2%) cases carried BRAF V600E mutations. V600E mutations presented more frequently in women than in men (6 of 96, 6.3% vs. P = 0.1). BRAF and EGFR mutations were concomitantly presented in three patients. Five of the eight patients with BRAF V600E mutations had matched plasma DNA samples and V600E mutations were found in three plasma samples. CONCLUSION: The prevalence of BRAF V600E mutations in Chinese patients with lung ADC is 4.2%. Circulating plasma DNA may be used for BRAF V600E mutation analysis in lung adenocarcinoma.

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