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1.
Proc Natl Acad Sci U S A ; 121(12): e2313236121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38466837

RESUMO

Phase separation drives compartmentalization of intracellular contents into various biomolecular condensates. Individual condensate components are thought to differentially contribute to the organization and function of condensates. However, how intermolecular interactions among constituent biomolecules modulate the phase behaviors of multicomponent condensates remains unclear. Here, we used core components of the inhibitory postsynaptic density (iPSD) as a model system to quantitatively probe how the network of intra- and intermolecular interactions defines the composition and cellular distribution of biomolecular condensates. We found that oligomerization-driven phase separation of gephyrin, an iPSD-specific scaffold, is critically modulated by an intrinsically disordered linker region exhibiting minimal homotypic attractions. Other iPSD components, such as neurotransmitter receptors, differentially promote gephyrin condensation through distinct binding modes and affinities. We further demonstrated that the local accumulation of scaffold-binding proteins at the cell membrane promotes the nucleation of gephyrin condensates in neurons. These results suggest that in multicomponent systems, the extent of scaffold condensation can be fine-tuned by scaffold-binding factors, a potential regulatory mechanism for self-organized compartmentalization in cells.


Assuntos
Proteínas de Transporte , Proteínas de Membrana , Proteínas de Membrana/metabolismo , Proteínas de Transporte/metabolismo , Sinapses/metabolismo , Termodinâmica
2.
Int J Mol Sci ; 22(19)2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34639077

RESUMO

Biomolecular phase separation denotes the demixing of a specific set of intracellular components without membrane encapsulation. Recent studies have found that biomolecular phase separation is involved in a wide range of cellular processes. In particular, phase separation is involved in the formation and regulation of chromosome structures at various levels. Here, we review the current understanding of biomolecular phase separation related to chromosomes. First, we discuss the fundamental principles of phase separation and introduce several examples of nuclear/chromosomal biomolecular assemblies formed by phase separation. We also briefly explain the experimental and computational methods used to study phase separation in chromosomes. Finally, we discuss a recent phase separation model, termed bridging-induced phase separation (BIPS), which can explain the formation of local chromosome structures.


Assuntos
Cromossomos Humanos/química , Substâncias Macromoleculares/química , Transição de Fase , Humanos
3.
Biotechnol Bioeng ; 117(6): 1904-1908, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32068245

RESUMO

Complement component 3a (C3a) plays a crucial role in the immune response and host defense, but it is also involved in pro-inflammatory responses, causing many inflammatory disorders. Blockade of C3a has been regarded as a potent therapeutic strategy for inflammatory diseases. Here, we present the development of a human C3a (hC3a)-specific protein binder, which effectively inhibits pro-inflammatory responses. The protein binder, which is composed of leucine-rich repeat modules, was selected against hC3a through phage display, and its binding affinity was matured up to 600 pM by further expanding the binding interface in a module-by-module manner. The developed protein binder was shown to have more than 10-fold higher specificity to hC3a compared with human C5a, exhibiting a remarkable suppression effect on pro-inflammatory response in monocyte, by blocking the interaction between hC3a and its receptor. The hC3a-specific protein binder is likely to have a therapeutic potential for C3a-mediated inflammatory diseases.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Complemento C3a/antagonistas & inibidores , Inflamação/tratamento farmacológico , Leucina/análogos & derivados , Leucina/farmacologia , Células Cultivadas , Ativação do Complemento/efeitos dos fármacos , Complemento C3a/imunologia , Humanos , Inflamação/imunologia , Modelos Moleculares
4.
Biochem Biophys Res Commun ; 477(4): 1072-1077, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27416759

RESUMO

The strongest anaphylatoxin, C5a, plays a critical role in the proinflammatory responses, causing the pathogenesis of a number of inflammatory diseases including sepsis, asthma, and rheumatoid arthritis. Inhibitors of C5a thus have great potential as therapeutics for various inflammatory disorders. Herein, we present the development of a high-affinity repebody against human C5a (hC5a), which effectively suppresses the proinflammatory response. A repebody scaffold composed of leucine-rich repeat (LRR) modules was previously developed as an alternative protein scaffold. A repebody specifically binding to hC5a was selected through a phage display, and its affinity was increased up to 5 nM using modular engineering. The repebody was shown to effectively inhibit the production of C5a-induced proinflammatory cytokines by human monocytes. To obtain insight into a mode of action by the repebody, we determined its crystal structure in complex with hC5a. A structural analysis revealed that the repebody binds to the D1 and D3 regions of hC5a, overlapping several epitope residues with the hC5a receptor (hC5aR). It is thus likely that the repebody suppresses the hC5a-mediated immune response in monocytes by blocking the binding of hC5a to its receptor. The anti-hC5a repebody can be developed as a potential therapeutic for C5a-involved inflammatory diseases.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Complemento C5a/química , Complemento C5a/imunologia , Mediadores da Inflamação/imunologia , Receptor da Anafilatoxina C5a/química , Receptor da Anafilatoxina C5a/imunologia , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Células Cultivadas , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Mediadores da Inflamação/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ligação Proteica , Conformação Proteica
5.
Biotechnol Bioeng ; 113(12): 2544-2552, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27241141

RESUMO

A polymerase chain reaction (PCR) using a thermostable DNA polymerase is the most widely applied method in many areas of research, including life sciences, biotechnology, and medical sciences. However, a conventional PCR incurs an amplification of undesired genes mainly owing to non-specifically annealed primers and the formation of a primer-dimer complex. Herein, we present the development of a Taq DNA polymerase-specific repebody, which is a small-sized protein binder composed of leucine rich repeat (LRR) modules, as a thermolabile inhibitor for a precise and accurate gene amplification by PCR. We selected a repebody that specifically binds to the DNA polymerase through a phage display, and increased its affinity to up to 10 nM through a modular evolution approach. The repebody was shown to effectively inhibit DNA polymerase activity at low temperature and undergo thermal denaturation at high temperature, leading to a rapid and full recovery of the polymerase activity, during the initial denaturation step of the PCR. The performance and utility of the repebody was demonstrated through an accurate and efficient amplification of a target gene without nonspecific gene products in both conventional and real-time PCRs. The repebody is expected to be effectively utilized as a thermolabile inhibitor in a PCR. Biotechnol. Bioeng. 2016;113: 2544-2552. © 2016 Wiley Periodicals, Inc.


Assuntos
DNA/genética , Amplificação de Genes/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Aminoácidos/genética , Taq Polimerase/genética , Taq Polimerase/antagonistas & inibidores
6.
Biotechnol Bioeng ; 113(8): 1639-46, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26773973

RESUMO

The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc.


Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Exotoxinas/metabolismo , Espaço Intracelular/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Toxinas Shiga/metabolismo , Fatores de Virulência/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exotoxinas/química , Exotoxinas/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Toxinas Shiga/química , Toxinas Shiga/genética , Fatores de Virulência/química , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosa
7.
Biochem Biophys Res Commun ; 464(4): 1282-1289, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26220340

RESUMO

The cell-specific cytosolic delivery of functional macromolecules with high efficiency is of great significance in molecular medicine and biotechnology. Herein, we present a Shiga-like toxin II-based high-efficiency and receptor-specific intracellular delivery system. We designed and constructed the Shiga-like toxin-based carrier (STC) to comprise the targeting and translocation domains, and used it for delivering a protein cargo. The STC was shown to deliver a protein cargo into the cytosol with high efficiency in a receptor-specific manner, exhibiting much higher efficiency than the most widely used cell-penetrating peptide. The general utility of the STC was demonstrated by modulating the targeting domain. The present delivery platform can be widely used for the intracellular delivery of diverse biomolecules in a receptor-specific and genetically encodable manner.


Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/farmacocinética , Nanocápsulas/química , Toxina Shiga II/farmacocinética , Animais , Proteínas de Fluorescência Verde/genética , Humanos , Nanocápsulas/ultraestrutura , Toxina Shiga II/química , Toxina Shiga II/genética
8.
Mol Ther ; 22(7): 1254-1265, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24682171

RESUMO

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses for host defense and tumorigenic process. Upregulation of IL-6 is known to constitutively phosphorylate signal transducer and activator of transcription 3 (STAT3), leading to activation of multiple oncogene pathways and inflammatory cascade. Here, we present the development of a high-affinity protein binder, termed repebody, which effectively suppresses non-small cell lung cancer in vivo by blocking the IL-6/STAT3 signaling. We selected a repebody that prevents human IL-6 (hIL-6) from binding to its receptor by a competitive immunoassay, and modulated its binding affinity for hIL-6 up to a picomolar range by a modular approach that mimics the combinatorial assembly of diverse modules to form antigen-specific receptors in nature. The resulting repebody was highly specific for hIL-6, effectively inhibiting the STAT3 phosphorylation in a dose- and binding affinity-response manner in vitro. The repebody was shown to have a remarkable suppression effect on the growth of tumors and STAT3 phosphorylation in xenograft mice with non-small cell lung cancer by blocking the hIL-6/STAT3 signaling. Structural analysis of the repebody and IL-6 complex revealed that the repebody binds the site 2a of hIL-6, overlapping a number of epitope residues at site 2a with gp130, and consequently causes a steric hindrance to the formation of IL-6/IL-6Rα complex. Our results suggest that high-affinity repebody targeting the IL-6/STAT3 pathway can be developed as therapeutics for non-small cell lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
9.
BMB Rep ; 55(8): 363-369, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35880435

RESUMO

Biomolecular phase separation has recently attracted broad interest, due to its role in the spatiotemporal compartmentalization of living cells. It governs the formation, regulation, and dissociation of biomolecular condensates, which play multiple roles in vivo, from activating specific biochemical reactions to organizing chromatin. Interestingly, biomolecular phase separation seems to be a mainly passive process, which can be explained by relatively simple physical principles and reproduced in vitro with a minimal set of components. This Mini review focuses on our current understanding of the fundamental principles of biomolecular phase separation and the recent progress in the research on this topic. [BMB Reports 2022; 55(8): 363-369].

10.
PLoS One ; 11(3): e0152522, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27015541

RESUMO

Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among people over the age of 60. Vascular endothelial growth factor (VEGF) plays a major role in pathological angiogenesis in AMD. Herein, we present the development of an anti- human VEGF repebody, which is a small-sized protein binder consisting of leucine-rich repeat (LRR) modules. The anti-VEGF repebody selected through a phage-display was shown to have a high affinity and specificity for human VEGF. We demonstrate that this repebody effectively inhibits in vitro angiogenic cellular processes, such as proliferation and migration, by blocking the VEGF-mediated signaling pathway. The repebody was also shown to have a strong suppression effect on choroidal neovascularization (CNV) and vascular leakage in vivo. Our results indicate that the anti-VEGF repebody has a therapeutic potential for treating neovascular AMD as well as other VEGF-involved diseases including diabetic retinopathy and metastatic cancers.


Assuntos
Anticorpos/química , Neovascularização de Coroide/imunologia , Leucina/química , Fator A de Crescimento do Endotélio Vascular/imunologia , Sequência de Aminoácidos , Inibidores da Angiogênese/uso terapêutico , Animais , Movimento Celular , Proliferação de Células , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Degeneração Macular/imunologia , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Neoplasias/tratamento farmacológico , Biblioteca de Peptídeos , Transdução de Sinais , Ressonância de Plasmônio de Superfície
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