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1.
Proc Natl Acad Sci U S A ; 117(12): 6540-6549, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32161136

RESUMO

The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.


Assuntos
Membranas Intracelulares/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Retroalimentação Fisiológica , Reguladores de Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Membranas Intracelulares/química , Modelos Biológicos , Prenilação de Proteína , Transporte Proteico , Transdução de Sinais , Processos Estocásticos , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/química
2.
Mol Ther ; 25(1): 102-119, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28129106

RESUMO

Inflammation, which is a highly regulated host response against danger signals, may be harmful if it is excessive and deregulated. Ideally, anti-inflammatory therapy should autonomously commence as soon as possible after the onset of inflammation, should be controllable by a physician, and should not systemically block beneficial immune response in the long term. We describe a genetically encoded anti-inflammatory mammalian cell device based on a modular engineered genetic circuit comprising a sensor, an amplifier, a "thresholder" to restrict activation of a positive-feedback loop, a combination of advanced clinically used biopharmaceutical proteins, and orthogonal regulatory elements that linked modules into the functional device. This genetic circuit was autonomously activated by inflammatory signals, including endogenous cecal ligation and puncture (CLP)-induced inflammation in mice and serum from a systemic juvenile idiopathic arthritis (sIJA) patient, and could be reset externally by a chemical signal. The microencapsulated anti-inflammatory device significantly reduced the pathology in dextran sodium sulfate (DSS)-induced acute murine colitis, demonstrating a synthetic immunological approach for autonomous anti-inflammatory therapy.


Assuntos
Anti-Inflamatórios , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Inflamação/genética , Inflamação/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Inflamação/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Camundongos , Engenharia de Proteínas , Biologia Sintética
3.
Radiol Oncol ; 56(1): 1-13, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35148469

RESUMO

BACKGROUND: Since the advent of viral vector gene therapy in 1990s, cancer treatment with viral vectors promised to revolutionize the field of oncology. Notably, viral vectors offer a unique combination of efficient gene delivery and engagement of the immune system for anti-tumour response. Despite the early potential, viral vector-based cancer treatments are only recently making a big impact, most prominently as gene delivery devices in approved CAR-T cell therapies, cancer vaccines and targeted oncolytic therapeutics. To reach this broad spectrum of applications, a number of challenges have been overcome - from our understanding of cancer biology to vector design, manufacture and engineering. Here, we take an overview of viral vector usage in cancer therapy and discuss the latest advancements. We also consider production platforms that enable mainstream adoption of viral vectors for cancer gene therapy. CONCLUSIONS: Viral vectors offer numerous opportunities in cancer therapy. Recent advances in vector production platforms open new avenues in safe and efficient viral therapeutic strategies, streamlining the transition from lab bench to bedside. As viral vectors come of age, they could become a standard tool in the cancer treatment arsenal.


Assuntos
Vacinas Anticâncer , Neoplasias , Vacinas Anticâncer/genética , Vacinas Anticâncer/uso terapêutico , Genes Neoplásicos , Terapia Genética , Vetores Genéticos , Humanos , Neoplasias/genética , Neoplasias/terapia
4.
Nat Commun ; 5: 5007, 2014 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-25264186

RESUMO

Bistable switches are fundamental regulatory elements of complex systems, ranging from electronics to living cells. Designed genetic toggle switches have been constructed from pairs of natural transcriptional repressors wired to inhibit one another. The complexity of the engineered regulatory circuits can be increased using orthogonal transcriptional regulators based on designed DNA-binding domains. However, a mutual repressor-based toggle switch comprising DNA-binding domains of transcription-activator-like effectors (TALEs) did not support bistability in mammalian cells. Here, the challenge of engineering a bistable switch based on monomeric DNA-binding domains is solved via the introduction of a positive feedback loop composed of activators based on the same TALE domains as their opposing repressors and competition for the same DNA operator site. This design introduces nonlinearity and results in epigenetic bistability. This principle could be used to employ other monomeric DNA-binding domains such as CRISPR for applications ranging from reprogramming cells to building digital biological memory.


Assuntos
DNA/química , Engenharia Genética/métodos , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epigênese Genética , Células HEK293 , Humanos , Luciferases/metabolismo , Microscopia Confocal , Modelos Teóricos , Ligação Proteica , Estrutura Terciária de Proteína , Processos Estocásticos
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