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1.
Adv Sci (Weinh) ; 7(2): 1901165, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31993280

RESUMO

Finding an effective therapeutic regimen is an urgent demand for various neurodegenerative disorders including Huntington's disease (HD). For the difficulties in observing the dynamic aggregation and oligomerization process of mutant Huntingtin (mHtt) in vivo, the evaluation of potential drugs at the molecular protein level is usually restricted. By combing lifetime-based fluorescence microscopies and biophysical tools, it is showcased that a designed amphiphilic peptide, which targets the mHtt at an early stage, can perturb the oligomer assembly process nanoscopically, suppress the amyloid property of mHtt, conformationally transform the oligomers and/or aggregates of mHtt, and ameliorate mHtt-induced neurological damage and aggregation in cell and HD mouse models. It is also found that this amphiphilic peptide is able to transport to the brain and rescue the memory deficit through intranasal administration, indicating its targeting specificity in vivo. In summary, a biophotonic platform is provided to investigate the oligomerization/aggregation process in detail that offers insight into the design and effect of a targeted therapeutic agent for Huntington's disease.

3.
ACS Nano ; 11(7): 6795-6807, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28653830

RESUMO

The abnormal assembly of misfolded proteins into neurotoxic aggregates is the hallmark associated with neurodegenerative diseases. Herein, we establish a photocontrollable platform to trigger amyloidogenesis to recapitulate the pathogenesis of amyotrophic lateral sclerosis (ALS) by applying a chemically engineered probe as a "switch" in live cells. This probe is composed of an amyloidogenic peptide from TDP-43, a photolabile linker, a polycationic sequence both to mask amyloidogenicity and for cell penetration, and a fluorophore for visualization. The photocontrollable probe can self-assemble into a spherical vesicle but rapidly develops massive nanofibrils with amyloid properties upon photoactivation. The photoinduced in vitro fibrillization process is characterized by biophysical techniques. In cellular experiments, this cell-penetrable vesicle was retained in the cytoplasm, seeded the mislocalized endogenous TDP-43 into aggregates upon irradiation, and consequently initiated apoptosis. In addition, this photocontrollable vesicle interfered with nucleocytoplasmic protein transport and triggered cortical neuron degeneration. Our developed strategy provides in vitro and in vivo spatiotemporal control of neurotoxic fibrillar aggregate formation, which can be readily applied in the studies of protein misfolding, aggregation-induced protein mislocalization, and amyloid-induced pathogenesis in different diseases.

4.
Sci Rep ; 5: 14992, 2015 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-26450664

RESUMO

The abundant accumulation of inclusion bodies containing polyglutamine-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in Huntington's disease (HD). Here, we demonstrate that FKBP12, an isomerase that exhibits reduced expression in HD, decreases the amyloidogenicity of mHTT, interrupts its oligomerization process, and structurally promotes the formation of amorphous deposits. By combining fluorescence-activated cell sorting with multiple biophysical techniques, we confirm that FKBP12 reduces the amyloid property of these ultrastructural-distinct mHTT aggregates within cells. Moreover, the neuroprotective effect of FKBP12 is demonstrated in both cellular and nematode models. Finally, we show that FKBP12 also inhibit the fibrillization process of other disease-related and aggregation-prone peptides. Our results suggest a novel function of FKBP12 in ameliorating the proteotoxicity in mHTT, which may shed light on unraveling the roles of FKBP12 in different neurodegenerative diseases and developing possible therapeutic strategies.


Assuntos
Mutação , Proteínas do Tecido Nervoso/genética , Peptídeos/genética , Proteína 1A de Ligação a Tacrolimo/genética , Expansão das Repetições de Trinucleotídeos/genética , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Animais , Animais Geneticamente Modificados , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Agregados Proteicos/genética , Conformação Proteica , Proteína 1A de Ligação a Tacrolimo/metabolismo
5.
Chem Commun (Camb) ; 51(41): 8652-5, 2015 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-25905771

RESUMO

We identify a new amyloidogenic peptide from the glutamine/asparagine-rich region of the FTLD-related protein (TDP-43), which can seed both the full-length and N-terminus-truncated TDP-43. Through the microinjection and real-time fluorescence imaging, we also found that this novel peptide could trigger cell apoptosis and initiate TDP-43 aggregation in the cytosol.


Assuntos
Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/farmacologia , Proteínas de Ligação a DNA/química , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/induzido quimicamente , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Citosol/metabolismo , Fluorescência , Corantes Fluorescentes/química , Degeneração Lobar Frontotemporal , Humanos , Estrutura Molecular , Ratos , Espectrometria de Fluorescência , Fatores de Tempo
6.
Bioengineering (Basel) ; 2(3): 139-159, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-28952475

RESUMO

Haptotaxis, i.e., cell migration in response to adhesive gradients, has been previously implicated in cancer metastasis. A better understanding of cell migration dynamics and their regulation could ultimately lead to new drug targets, especially for cancers with poor prognoses, such as ovarian cancer. Haptotaxis has not been well-studied due to the lack of biomimetic, biocompatible models, where, for example, microcontact printing and microfluidics approaches are primarily limited to 2D surfaces and cannot produce the 3D submicron features to which cells respond. Here we used multiphoton excited (MPE) phototochemistry to fabricate nano/microstructured gradients of laminin (LN) as 2.5D models of the ovarian basal lamina to study the haptotaxis dynamics of a series of ovarian cancer cells. Using these models, we found that increased LN concentration increased migration speed and also alignment of the overall cell morphology and their cytoskeleton along the linear axis of the gradients. Both these metrics were enhanced on LN compared to BSA gradients of the same design, demonstrating the importance of both topographic and ECM cues on the adhesion/migration dynamics. Using two different gradient designs, we addressed the question of the roles of local concentration and slope and found that the specific haptotactic response depends on the cell phenotype and not simply the gradient design. Moreover, small changes in concentration strongly affected the migration properties. This work is a necessary step in studying haptotaxis in more complete 3D models of the tumor microenvironment for ovarian and other cancers.

7.
PLoS One ; 9(8): e103644, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25090004

RESUMO

TAR DNA-binding protein (TDP-43) was identified as the major ubiquitinated component deposited in the inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) in 2006. Later on, numerous ALS-related mutations were found in either the glycine or glutamine/asparagine-rich region on the TDP-43 C-terminus, which hinted on the importance of mutations on the disease pathogenesis. However, how the structural conversion was influenced by the mutations and the biological significance of these peptides remains unclear. In this work, various peptides bearing pathogenic or de novo designed mutations were synthesized and displayed their ability to form twisted amyloid fibers, cause liposome leakage, and mediate cellular toxicity as confirmed by transmission electron microscopy (TEM), circular dichroism (CD), Thioflavin T (ThT) assay, Raman spectroscopy, calcein leakage assay, and cell viability assay. We have also shown that replacing glycines with prolines, known to obstruct ß-sheet formation, at the different positions in these peptides may influence the amyloidogenesis process and neurotoxicity. In these cases, GGG308PPP mutant was not able to form beta-amyloid, cause liposome leakage, nor jeopardized cell survival, which hinted on the importance of the glycines (308-310) during amyloidogenesis.


Assuntos
Substituição de Aminoácidos , Amiloide/metabolismo , Proteínas de Ligação a DNA/genética , Glicina/metabolismo , Mutação/genética , Peptídeos/toxicidade , Prolina/genética , Sequência de Aminoácidos , Amiloide/ultraestrutura , Animais , Benzotiazóis , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/toxicidade , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidade , Peptídeos/química , Peptídeos/metabolismo , Agregados Proteicos/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Análise Espectral Raman , Tiazóis/metabolismo , Fatores de Tempo
8.
PLoS One ; 8(5): e64002, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23737961

RESUMO

The aggregation of TAR DNA-binding protein (TDP-43) has been shown as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. While evidence has suggested that mutation or truncation in TDP-43 influences its aggregation process, nevertheless, the correlation between the TDP-43 aggregation propensity and its binding substrates has not been fully established in TDP-43 proteinopathy. To address this question, we have established a platform based on the in vitro protein expression system to evaluate the solubility change of TDP-43 in response to factors such as nucleotide binding and temperature. Our results suggest that the solubility of TDP-43 is largely influenced by its cognate single-strand DNA (ssDNA) or RNA (ssRNA) rather than hnRNP, which is known to associate with TDP-43 C-terminus. The direct interaction between the refolded TDP-43, purified from E.coli, and ssDNA were further characterized by Circular Dichroism (CD) as well as turbidity and filter binding assay. In addition, ssDNA or ssRNA failed to prevent the aggregation of the F147L/F149L double mutant or truncated TDP-43 (TDP208-414). Consistently, these two mutants form aggregates, in contrast with the wild-type TDP-43, when expressed in Neuro2a cells. Our results demonstrate an intimate relationship between the solubility of TDP-43 and its DNA or RNA binding affinity, which may shed light on the role of TDP-43 in ALS and FTLD.


Assuntos
DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Multimerização Proteica/efeitos dos fármacos , RNA/metabolismo , RNA/farmacologia , Animais , Linhagem Celular Tumoral , Sistema Livre de Células , Proteínas de Ligação a DNA/genética , Escherichia coli/citologia , Humanos , Camundongos , Mutação , Estrutura Quaternária de Proteína , Transporte Proteico/efeitos dos fármacos , Coelhos , Reticulócitos/citologia , Solubilidade
9.
Exp Biol Med (Maywood) ; 235(7): 796-804, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20542955

RESUMO

The family of WW domain-containing proteins contains over 2000 members. The small WW domain module is responsible, in part, for protein/protein binding interactions and signaling. Many of these proteins are located at the membrane/cytoskeleton area, where they act as adaptors to receive signals from the cell surface. In this review, we provide molecular insights regarding recent novel findings on signaling from the cell surface toward WW domain-containing oxidoreductase, known as WWOX, FOR or WOX1. More specifically, transforming growth factor beta 1 utilizes cell surface hyaluronidase Hyal-2 (hyaluronoglucosaminidase 2) as a cognate receptor for signaling with WWOX and Smad4 to control gene transcription, growth and death. Complement C1q alone, bypassing the activation of classical pathway, signals a novel event of apoptosis by inducing microvillus formation and WWOX activation. Deficiency in these signaling events appears to favorably support cancer growth.


Assuntos
Oxirredutases/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Moléculas de Adesão Celular/fisiologia , Complemento C1q/fisiologia , Proteínas Ligadas por GPI , Humanos , Hialuronoglucosaminidase/fisiologia , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Proteína Smad4/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Oxidorredutase com Domínios WW
10.
Opt Express ; 18(4): 3649-59, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20389375

RESUMO

This paper demonstrates the first combination for wide-field surface plasmon (SP) phase microscopy and SP-enhanced fluorescence microscopy to image living cells' contacts on the surface of a bio-substrate simultaneously. The phase microscopy with a phase-shift interferometry and common-path optical setup can provide high-sensitivity phase information in long-term stability. Simultaneously, the fluorescence microscopy with the enhancement of a local electromagnetic field can supply bright fluorescent images. The combined microscope imposes a high numerical aperture objective upon the excitation of surface plasmon through a silver film with a thickness of 30 nm. The developed SP microscope is successfully applied to the real-time bright observation of the transfected fluorescence of living cells localized near the cell membrane on the bio-substrate and the high-sensitivity phase image of the cell-substrate contacts at the same time.


Assuntos
Membrana Celular/ultraestrutura , Aumento da Imagem/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Contraste de Fase/instrumentação , Técnica de Subtração/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento
11.
PLoS One ; 4(6): e5755, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19484134

RESUMO

BACKGROUND: Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells. METHODOLOGY/PRINCIPAL FINDINGS: DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues. CONCLUSIONS/SIGNIFICANCE: We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.


Assuntos
Apoptose , Complemento C1q/fisiologia , Regulação Neoplásica da Expressão Gênica , Oxirredutases/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Complemento C1q/metabolismo , Regulação para Baixo , Genes Dominantes , Humanos , Masculino , Microscopia de Fluorescência/métodos , Modelos Biológicos , Fosforilação , Proteína Supressora de Tumor p53/metabolismo , Oxidorredutase com Domínios WW
12.
Opt Express ; 17(8): 5987-97, 2009 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-19365417

RESUMO

A surface plasmon-enhanced two-photon total-internal-reflection fluorescence (TIRF) microscope has been developed to provide fluorescent images of living cell membranes. The proposed microscope with the help of surface plasmons (SPs) not only provides brighter fluorescent images based on the mechanism of local electromagnetic field enhancement, but also reduces photobleaching due to having a shorter fluorophore lifetime. In comparison with a one-photon TIRF, the two-photon TIRF can achieve higher signal-to-noise ratio cell membrane imaging due its smaller excitation volume and lower scattering. By combining the SP enhancement and two-photon excitation TIRF, the microscope has demonstrated it's capability for brighter and more contrasted fluorescence membrane images of living monkey kidney COS-7 fibroblasts transfected with an EYFP-MEM or EGFP-WOX1 construct.


Assuntos
Aumento da Imagem/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Animais , Células COS , Chlorocebus aethiops , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
13.
Opt Express ; 14(20): 9307-16, 2006 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19529314

RESUMO

Using a total internal reflection fluorescence microscopy (TIRFM) technique to image live cells on a biosurface not only provides an enhanced understanding of cellular functions, but also improves the signal-to-noise ratio of the images. However, the intensity of the fluorescence signal must be increased if a more dynamic biomolecular imaging capability is required. Accordingly, this study presents a surface plasmon-enhanced TIRFM technique in which the fluorescence signals are enhanced via surface plasmons offered by a silver nanolayer. The developed microscopy technique is successfully applied to the real-time observation of the thrombomodulin proteins of live cell membranes. The experimental results and the simulation results demonstrate that the live cell membrane images obtained in the proposed surface plasmon-enhanced TIRFM technique are brighter by approximately one order of magnitude than those provided by conventional TIRFM.

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