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1.
Anal Chim Acta ; 1317: 342916, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-39030013

RESUMO

BACKGROUND: Protein misfolding and aggregation can lead to various diseases. Recent studies have shed light on the aggregated protein in breast cancer pathology, which suggests that it is crucial to design chemical sensors that visualize protein aggregates in breast cancer, especially in clinical patient-derived samples. However, most reported sensors are constrained in cultured cell lines. RESULTS: In this work, we present the development of two isophorone-based crystallization-induced-emission fluorophores for detecting proteome aggregation in breast cancer cell line and tissues biopsied from diseased patients, designated as A1 and A2. These probes exhibited viscosity sensitivity and recovered their fluorescence strongly at crystalline state. Moreover, A1 and A2 exhibit selective binding capacity and strong fluorescence for various aggregated proteins. Utilizing these probes, we detect protein aggregation in stressed breast cancer cells, xenograft mouse model of human breast cancer and clinical patient-derived samples. Notably, the fluorescence intensity of both probes light up in tumor tissues. SIGNIFICANCE: The synthesized isophorone-based crystallization-induced-emission fluorophores, A1 and A2, enable sensitive detection of protein aggregation in breast cancer cells and tissues. In the future, aggregated proteins are expected to become indicators for early diagnosis and clinical disease monitoring of breast cancer.


Assuntos
Neoplasias da Mama , Cristalização , Corantes Fluorescentes , Proteoma , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Animais , Feminino , Corantes Fluorescentes/química , Proteoma/análise , Proteoma/química , Camundongos , Agregados Proteicos , Linhagem Celular Tumoral , Camundongos Nus
2.
Bioorg Chem ; 148: 107491, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38788365

RESUMO

As a consequence of somatosensory nervous system injury or disease, neuropathic pain is commonly associated with chemotherapies, known as chemotherapy-induced peripheral neuropathy (CIPN). However, the mechanisms underlying CIPN-induced proteome aggregation in neuronal cells remain elusive due to limited detection tools. Herein, we present series sensors for fluorescence imaging (AggStain) and proteomics analysis (AggLink) to visualize and capture aggregated proteome in CIPN neuronal cell model. The environment-sensitive AggStain imaging sensor selectively binds and detects protein aggregation with 12.3 fold fluorescence enhancement. Further, the covalent AggLink proteomic sensor captures cellular aggregated proteins and profiles their composition via LC-MS/MS analysis. This integrative sensor platform reveals the presence of proteome aggregation in CIPN cell model and highlights its potential for broader applications in assessing proteome stability under various cellular stress conditions.


Assuntos
Antineoplásicos , Doenças do Sistema Nervoso Periférico , Proteoma , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/metabolismo , Humanos , Proteoma/análise , Proteoma/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Estrutura Molecular , Agregados Proteicos/efeitos dos fármacos , Imagem Óptica , Relação Dose-Resposta a Droga , Proteômica , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia
3.
Anal Chem ; 96(12): 4978-4986, 2024 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-38471057

RESUMO

Bioaccumulation of nanoplastic particles has drawn increasing attention regarding environmental sustainability and biosafety. How nanoplastic particles interact with the cellular milieu still remains elusive. Herein, we exemplify a general approach to profile the composition of a "protein corona" interacting with nanoparticles via the photocatalytic protein proximity labeling method. To enable photocatalytic proximity labeling of the proteome interacting with particles, iodine-substituted BODIPY (I-BODIPY) is selected as the photosensitizer and covalently conjugated onto amino-polystyrene nanoparticles as a model system. Next, selective proximity labeling of interacting proteins is demonstrated using I-BODIPY-labeled nanoplastic particles in both Escherichia coli lysate and live alpha mouse liver 12 cells. Mechanistic studies reveal that the covalent modifications of proteins by an aminoalkyne substrate are conducted via a reactive oxygen species photosensitization pathway. Further proteomic analysis uncovers that mitochondria-related proteins are intensively involved in the protein corona, indicating substantial interactions between nanoplastic particles and mitochondria. In addition, proteostasis network components are also identified, accompanied by consequent cellular proteome aggregation confirmed by fluorescence imaging. Together, this work exemplifies a general strategy to interrogate the composition of the protein corona of nanomaterials by endowing them with photooxidation properties to enable photocatalytic protein proximity labeling function.


Assuntos
Compostos de Boro , Nanopartículas , Coroa de Proteína , Animais , Camundongos , Microplásticos , Proteoma , Proteômica , Poliestirenos
4.
Chem Rev ; 124(4): 1738-1861, 2024 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-38354333

RESUMO

The microenvironment is indispensable for functionality of various biomacromolecules, subcellular compartments, living cells, and organisms. In particular, physical properties within the biological microenvironment could exert profound effects on both the cellular physiology and pathology, with parameters including the polarity, viscosity, pH, and other relevant factors. There is a significant demand to directly visualize and quantitatively measure the fluctuation in the cellular microenvironment with spatiotemporal resolution. To satisfy this need, analytical methods based on fluorescence probes offer great opportunities due to the facile, sensitive, and dynamic detection that these molecules could enable in varying biological settings from in vitro samples to live animal models. Herein, we focus on various types of small molecule fluorescent probes for the detection and measurement of physical parameters of the microenvironment, including pH, polarity, viscosity, mechanical force, temperature, and electron potential. For each parameter, we primarily describe the chemical mechanisms underlying how physical properties are correlated with changes of various fluorescent signals. This review provides both an overview and a perspective for the development of small molecule fluorescent probes to visualize the dynamic changes in the cellular environment, to expand the knowledge for biological process, and to enrich diagnostic tools for human diseases.


Assuntos
Corantes Fluorescentes , Fenômenos Mecânicos , Animais , Humanos , Corantes Fluorescentes/química , Microambiente Celular
5.
Chem Commun (Camb) ; 59(66): 10008-10011, 2023 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-37522834

RESUMO

Tau protein aggregation into neurofibrillary tangles often causes tauopathies. Herein, we report fluorene based sensors with fluorogenicity upon binding to tau proteins. Intriguingly, these sensors possess triplet state properties to inhibit tau fibrillation upon photo-induced crosslinking.


Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Proteínas tau/metabolismo , Tauopatias/metabolismo , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/metabolismo , Fluorenos , Doença de Alzheimer/metabolismo , Fosforilação
6.
ACS Sens ; 7(7): 1919-1925, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35776067

RESUMO

Deterioration of protein homeostasis (proteostasis) often induces aberrant proteome aggregation. Visualization and dissection of the stressed proteome are of particular interest given their association with numerous degenerative diseases. Recent progress in chemical cellular stress sensors allows for direct visualization of aggregated proteome. Beyond its localization and morphology, the physicochemical nature and the dynamics of the aggregated proteome have been challenging to explore. Herein, we developed a series of solvatochromic fluorene-based D-π-A probes that can selectively and noncovalently bind to a misfolded and aggregated proteome and report on their compactness heterogeneity upon cellular stresses. We achieved this goal by variation of the heterocyclic acceptors to modulate their solvatochromism and binding affinity to amorphous aggregated proteins. The optimized sensor P6 was capable of sensing the polarity differences among different aggregated proteins via its fluorescence emission wavelength. In live cells, P6 revealed the cellular compactness heterogeneity in the aggregated proteome upon cellular stresses. Given the combinative solvatochromic and noncovalent properties, our probe can reversibly monitor the dynamic changes in the aggregated proteome compactness upon stress and after stress recovery, suggesting its potential applications in search of therapeutics to counteract disease-causing proteome stresses.


Assuntos
Dobramento de Proteína , Proteoma , Proteostase
7.
Chem Commun (Camb) ; 58(35): 5407-5410, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35415726

RESUMO

Protein aggregation in the cell is often manifested by the formation of subcellular punctate structures. Herein, we modulated the solvatochromism and solubility of Nile Red fluorophore derivatives to quantitatively study the polarity inside pathogenic protein aggregates, revealing structure- and protein-dependent polarity heterogeneity.


Assuntos
Oxazinas , Agregados Proteicos , Corantes Fluorescentes/química , Ionóforos , Oxazinas/química
8.
Chem Sci ; 12(24): 8468-8476, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34221329

RESUMO

Co-aggregation of multiple pathogenic proteins is common in neurodegenerative diseases but deconvolution of such biochemical process is challenging. Herein, we developed a dual-color fluorogenic thermal shift assay to simultaneously report on the aggregation of two different proteins and quantitatively study their thermodynamic stability during co-aggregation. Expansion of spectral coverage was first achieved by developing multi-color fluorogenic protein aggregation sensors. Orthogonal detection was enabled by conjugating sensors of minimal fluorescence crosstalk to two different proteins via sortase-tag technology. Using this assay, we quantified shifts in melting temperatures in a heterozygous model protein system, revealing that the thermodynamic stability of wild-type proteins was significantly compromised by the mutant ones but not vice versa. We also examined how small molecule ligands selectively and differentially interfere with such interplay. Finally, we demonstrated these sensors are suited to visualize how different proteins exert influence on each other upon their co-aggregation in live cells.

9.
Angew Chem Int Ed Engl ; 60(20): 11335-11343, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33655618

RESUMO

Covalent chemical reactions to modify aggregated proteins are rare. Here, we reported covalent Michael addition can generally occur upon protein aggregation. Such reactivity was initially discovered by a bioinspired fluorescent color-switch probe mimicking the photo-conversion mechanism of Kaede fluorescent protein. This probe was dark with folded proteins but turned on red fluorescence (620 nm) when it non-covalently bound to misfolded proteins. Supported by the biochemical and mass spectrometry results, the probe chemoselectively reacted with the reactive cysteines of aggregated proteins via covalent Michael addition and gradually switched to green fluorescence (515 nm) upon protein aggregation. Exploiting this Michael addition chemistry in the malachite green dye derivatives demonstrated its general applicability and chemical tunability, resulting in different fluorescence color-switch responses. Our work may offer a new avenue to explore other chemical reactions upon protein aggregation and design covalent probes for imaging, chemical proteomics, and therapeutic purposes.


Assuntos
Corantes Fluorescentes/química , Proteínas Luminescentes/química , Estrutura Molecular , Agregados Proteicos
10.
Anal Chem ; 93(3): 1717-1724, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33382253

RESUMO

Stress-induced intracellular proteome aggregation is a hallmark and a biomarker of various human diseases. Current sensors requiring either cellular fixation or covalent modification of the entire proteome are not suitable for live-cell applications and dynamics study. Herein, we report a noncovalent, cell-permeable, and fluorogenic sensor that can reversibly bind to proteome amorphous aggregates and monitor their formation, transition, and clearance in live cells. This sensor was structurally optimized from previously reported fluorescent protein chromophores to enable noncovalent and reversible binding to aggregated proteins. Unlike all previous sensors, the noncovalent and reversible nature of this probe allows for dynamic detection of both the formation and clearance of aggregated proteome in one live-cell sample. Under different cellular stresses, this sensor reveals drastic differences in the morphology and location of aggregated proteome. Furthermore, we have shown that this sensor can detect the transition from proteome liquid-to-liquid phase separation to liquid-to-solid phase separation in a two-color imaging experiment. Overall, the sensor reported here can serve as a facile tool to screen therapeutic drugs and identify cellular pathways that ameliorate pathogenic proteome aggregation in live-cell models.


Assuntos
Corantes Fluorescentes/química , Proteoma/química , Técnicas Biossensoriais , Células HEK293 , Humanos , Estrutura Molecular , Imagem Óptica , Agregados Proteicos , Solubilidade , Espectrometria de Fluorescência
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