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1.
Proc Natl Acad Sci U S A ; 120(49): e2307371120, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38032933

RESUMO

There has been considerable progress in the development of computational methods for designing protein-protein interactions, but engineering high-affinity binders without extensive screening and maturation remains challenging. Here, we test a protein design pipeline that uses iterative rounds of deep learning (DL)-based structure prediction (AlphaFold2) and sequence optimization (ProteinMPNN) to design autoinhibitory domains (AiDs) for a PD-L1 antagonist. With the goal of creating an anticancer agent that is inactive until reaching the tumor environment, we sought to create autoinhibited (or masked) forms of the PD-L1 antagonist that can be unmasked by tumor-enriched proteases. Twenty-three de novo designed AiDs, varying in length and topology, were fused to the antagonist with a protease-sensitive linker, and binding to PD-L1 was measured with and without protease treatment. Nine of the fusion proteins demonstrated conditional binding to PD-L1, and the top-performing AiDs were selected for further characterization as single-domain proteins. Without any experimental affinity maturation, four of the AiDs bind to the PD-L1 antagonist with equilibrium dissociation constants (KDs) below 150 nM, with the lowest KD equal to 0.9 nM. Our study demonstrates that DL-based protein modeling can be used to rapidly generate high-affinity protein binders.


Assuntos
Antígeno B7-H1 , Aprendizado Profundo , Neoplasias , Humanos , Antígeno B7-H1/antagonistas & inibidores , Peptídeo Hidrolases , Proteínas
2.
bioRxiv ; 2023 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-37205527

RESUMO

There has been considerable progress in the development of computational methods for designing protein-protein interactions, but engineering high-affinity binders without extensive screening and maturation remains challenging. Here, we test a protein design pipeline that uses iterative rounds of deep learning (DL)-based structure prediction (AlphaFold2) and sequence optimization (ProteinMPNN) to design autoinhibitory domains (AiDs) for a PD-L1 antagonist. Inspired by recent advances in therapeutic design, we sought to create autoinhibited (or masked) forms of the antagonist that can be conditionally activated by proteases. Twenty-three de novo designed AiDs, varying in length and topology, were fused to the antagonist with a protease sensitive linker, and binding to PD-L1 was tested with and without protease treatment. Nine of the fusion proteins demonstrated conditional binding to PD-L1 and the top performing AiDs were selected for further characterization as single domain proteins. Without any experimental affinity maturation, four of the AiDs bind to the PD-L1 antagonist with equilibrium dissociation constants (KDs) below 150 nM, with the lowest KD equal to 0.9 nM. Our study demonstrates that DL-based protein modeling can be used to rapidly generate high affinity protein binders.

3.
ACS Chem Biol ; 15(11): 2848-2853, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33074647

RESUMO

Bacterial photoactivated adenylyl cyclase (bPAC) has been widely used in signal transduction research. However, due to its low two-photon absorption, bPAC cannot be efficiently activated by two-photon (2P) excitation. Taking advantage of the high two-photon absorption of monomeric teal fluorescent protein 1 (mTFP1), we herein developed 2P-activatable bPAC (2pabPAC), a fusion protein consisting of bPAC and mTFP1. In 2pabPAC, the energy absorbed by mTFP1 excites bPAC by Fürster resonance energy transfer (FRET) at ca. 43% efficiency. The light-induced increase in cAMP was monitored by a red-shifted FRET biosensor for PKA. In 3D MDCK cells and mouse liver, PKA was activated at single-cell resolution under a 2P microscope. We found that PKA activation in a single hepatocyte caused PKA activation in neighboring cells, indicating the propagation of PKA activation. Thus, 2pabPAC will provide a versatile platform for controlling the cAMP signaling pathway and investigating cell-to-cell communication in vivo.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Transdução de Sinais , Análise de Célula Única/métodos , Adenilil Ciclases/metabolismo , Animais , Bactérias/enzimologia , Técnicas Biossensoriais/métodos , Comunicação Celular , Cães , Ativação Enzimática , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Fígado/metabolismo , Células Madin Darby de Rim Canino , Camundongos
4.
Nat Methods ; 16(10): 1029-1036, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501546

RESUMO

Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Förster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2. Based on structure-guided engineering, a chimeric protein with 40% FRET efficiency was developed and named 2P-activatable CRY2, or 2paCRY2. 2paCRY2 was employed to develop a RAF1 activation system named 2paRAF. In three-dimensionally cultured cells expressing 2paRAF, extracellular signal-regulated kinase (ERK) was efficiently activated by 2P excitation at single-cell resolution. Photoactivation of ERK was also accomplished in the epidermal cells of 2paRAF-expressing mice. We further developed an mTFP1-fused LOV domain that exhibits efficient response to 2P excitation. Collectively, FRAPA will pave the way to single-cell optical control of signaling pathways in vivo.


Assuntos
Flavoproteínas/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Optogenética , Fótons , Animais , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos
5.
Ann Thorac Surg ; 98(3): e63-5, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25193223

RESUMO

We report a rare case of multicentric Castleman disease treated successfully with single-lung transplantation. A 12-year-old patient developed increasing dyspnea. Elevated serum interleukin-6 (177.0 pg/mL) and immunoglobulin G (IgG; 13,900 mg/dL) levels were observed. Steroid therapy was effective but the respiratory condition gradually deteriorated. He underwent single-lung transplantation at 36 years of age. Preoperative interleukin-6 and IgG levels were 0.3 pg/mL and 5,260 mg/dL, respectively. After 6 months he is alive without symptoms. Postoperative IgG levels were restored to normal limits (1,624 mg/dL) and interleukin-6 levels remained within normal limits (1.4 pg/mL). Overinflation of the native left lung also improved.


Assuntos
Hiperplasia do Linfonodo Gigante/cirurgia , Transplante de Pulmão/métodos , Criança , Humanos , Masculino , Indução de Remissão
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