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[This retracts the article DOI: 10.1039/C8RA01200H.].
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Stimulatory immune receptor NKG2D binds diverse ligands to elicit differential anti-tumor and anti-virus immune responses. Two conflicting degeneracy recognition models based on static crystal structures and in-solution binding affinities have been considered for almost two decades. Whether and how NKG2D recognizes and discriminates diverse ligands still remain unclear. Using live-cell-based single-molecule biomechanical assay, we characterized the in situ binding kinetics of NKG2D interacting with different ligands in the absence or presence of mechanical force. We found that mechanical force application selectively prolonged NKG2D interaction lifetimes with the ligands MICA and MICB, but not with ULBPs, and that force-strengthened binding is much more pronounced for MICA than for other ligands. We also integrated steered molecular dynamics simulations and mutagenesis to reveal force-induced rotational conformational changes of MICA, involving formation of additional hydrogen bonds on its binding interface with NKG2D, impeding MICA dissociation under force. We further provided a kinetic triggering model to reveal that force-dependent affinity determines NKG2D ligand discrimination and its downstream NK cell activation. Together, our results demonstrate that NKG2D has a discrimination power to recognize different ligands, which depends on selective mechanical force-induced ligand conformational changes.
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Subfamilia K de Receptores Similares a Lectina de Células NK/química , Sitios de Unión , Células Cultivadas , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Células K562 , Ligandos , Fenómenos Mecánicos , Simulación de Dinámica Molecular , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Unión Proteica , Imagen Individual de MoléculaRESUMEN
The Chimera antigen receptor (CAR)-T cell therapy has gained great success in the clinic. However, there are still major challenges for its wider applications in a variety of cancer types including lack of effectiveness due to the highly complex tumor microenvironment, and the forbiddingly high cost due to the personalized manufacturing procedures. In order to overcome these hurdles, numerous efforts have been spent focusing on optimizing Chimera antigen receptors, engineering and improving T cell capacity, exploiting features of subsets of T cell or NK cells, or making off-the-shelf universal cells. Here, we developed induced pluripotent stem cells (iPSCs)-derived, CAR-expressing macrophage cells (CAR-iMac). CAR expression confers antigen-dependent macrophage functions such as expression and secretion of cytokines, polarization toward the pro-inflammatory/anti-tumor state, enhanced phagocytosis of tumor cells, and in vivo anticancer cell activity. This technology platform for the first time provides an unlimited source of iPSC-derived engineered CAR-macrophage cells which could be utilized to eliminate cancer cells.
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Células Madre Pluripotentes Inducidas/citología , Macrófagos/citología , Receptores Quiméricos de Antígenos/genética , Células Cultivadas , Expresión Génica , Ingeniería Genética , Humanos , Inmunoterapia Adoptiva , Células K562 , Macrófagos/metabolismo , Neoplasias/terapia , Transducción GenéticaRESUMEN
MicroRNA-320d (miR-320d) is a novel cancer-related miRNA and functions as a tumor suppressor in human cancers. However, the expression pattern and function of miR-320d in breast cancer remain largely unknown. In the present study, we found that the expression level of miR-320d in breast cancer tissues and cells was significantly lower than in non-tumor tissues and MCF-10A cells. Decreased miR-320d was associated with poor overall survival in patients with breast cancer. Overexpression of miR-320d inhibited proliferation, migration, and invasion and promoted apoptosis of breast cancer cells. In addition, the long non-coding RNA, HNF1A antisense RNA 1 (HNF1A-AS1) was up-regulated in both breast cancer tissues and cell lines. HNF1A-AS1 suppressed the expression and function of miR-320d. Moreover, SRY-related HMG-box 4 (SOX4) was speculated and confirmed as a target of miR-320d. We also demonstrated that HNF1A-AS1 may function as a sponge competitive endogenous RNA for miR-320d, and thus regulate the expression of SOX4. Taken together, our study has identified a novel signaling pathway through which miR-320d exerts its anti-carcinogenic roles and suggested that the HNF1A-AS1/miR-320d/SOX4 may be a potential target for the therapy of breast cancer.
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The type IVb secretion system (T4BSS) of Legionella pneumophila is a multiple-component apparatus that delivers â¼300 virulent effector proteins into host cells. The injected effectors modulate host cellular processes to promote bacterial infection and proliferation. IcmS and IcmW are two conserved small, acidic adaptor proteins that form a binary complex to interact with many effectors and facilitate their translocation. IcmS and IcmW can also interact with DotL, an ATPase of the type IV coupling protein complex (T4CP). However, how IcmS-IcmW recognizes effectors, and what the roles of IcmS-IcmW are in T4BSSs are unclear. In this study, we found that IcmS and IcmW form a 1:1 heterodimeric complex to bind effector substrates. Both IcmS and IcmW adopt new structural folds and have no structural similarities with known effector chaperones. IcmS has a compact global structure with an α/ß fold, while IcmW adopts a fully α-folded, relatively loose architecture. IcmS stabilizes IcmW by binding to its two C-terminal α-helices. Photocrosslinking assays revealed that the IcmS-IcmW complex binds its cognate effectors via an extended hydrophobic surface, which can also interact with the C terminus of DotL. A crystal structure of the DotL-IcmS-IcmW complex reveals extensive and highly stable interactions between DotL and IcmS-IcmW. Moreover, IcmS-IcmW recruits LvgA to DotL and assembles a unique T4CP. These data suggest that IcmS-IcmW also functions as an inseparable integral component of the DotL-T4CP complex in the bacterial inner membrane. This study provides molecular insights into the dual roles of the IcmS-IcmW complex in T4BSSs.
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Chaperonas Moleculares/química , Sistemas de Secreción Tipo IV/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Legionella pneumophila/química , Legionella pneumophila/metabolismo , Chaperonas Moleculares/metabolismo , Unión Proteica , Sistemas de Secreción Tipo IV/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Bacteriophages express proteins that inactivate the CRISPR-Cas bacterial immune system. Here we report the crystal structure of the anti-CRISPR protein AcrF3 in complex with Pseudomonas aeruginosa Cas3 (PaCas3). AcrF3 forms a homodimer that locks PaCas3 in an ADP-bound form, blocks the entrance of the DNA-binding tunnel in the helicase domain, and masks the linker region and C-terminal domain of PaCas3, thereby preventing recruitment by Cascade and inhibiting the type I-F CRISPR-Cas system.