RESUMEN
Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.
Asunto(s)
Microscopía por Crioelectrón , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Interleucina-3 , Multimerización de Proteína , Transducción de Señal , Humanos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interleucina-3/metabolismo , Interleucina-3/química , Interleucina-3/genética , Células HEK293 , Unión Proteica , Modelos Moleculares , Interleucina-5/metabolismo , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/química , Imagen Individual de Molécula , Relación Estructura-Actividad , Sitios de Unión , Receptores de Interleucina-5/metabolismo , Receptores de Interleucina-5/genética , Receptores de Interleucina-5/químicaRESUMEN
The HIV-1 matrix protein p17 (p17) is a pleiotropic molecule impacting on different cell types. Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human specific adaptation. We previously showed the proangiogenic capability of p17. Here, by integrating functional analysis and receptor binding, we identify a functional epitope that displays molecular mimicry with human erythropoietin (EPO) and promotes angiogenesis through common beta chain receptor (ßCR) activation. The functional EPO-like epitope was found to be present in the matrix protein of HIV-1 ancestors SIV originated in chimpanzees (SIVcpz) and gorillas (SIVgor) but not in that of HIV-2 and its ancestor SIVsmm from sooty mangabeys. According to biological data, evolution of the EPO-like epitope showed a clear differentiation between HIV-1/SIVcpz-gor and HIV-2/SIVsmm branches, thus highlighting this epitope on p17 as a divergent signature discriminating HIV-1 and HIV-2 ancestors. P17 is known to enhance HIV-1 replication. Similarly to other ßCR ligands, p17 is capable of attracting and activating HIV-1 target cells and promoting a proinflammatory microenvironment. Thus, it is tempting to speculate that acquisition of an epitope on the matrix proteins of HIV-1 ancestors capable of triggering ßCR may have represented a critical step to enhance viral aggressiveness and early human-to-human SIVcpz/gor dissemination. The hypothesis that the p17/ßCR interaction and ßCR abnormal stimulation may also play a role in sustaining chronic activation and inflammation, thus marking the difference between HIV-1 and HIV-2 in term of pathogenicity, needs further investigation.
Asunto(s)
Eritropoyetina/genética , Antígenos VIH/metabolismo , VIH-1/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Células Cultivadas , Epítopos/inmunología , Eritropoyetina/metabolismo , Evolución Molecular , Antígenos VIH/genética , Seropositividad para VIH , VIH-1/genética , VIH-2 , Humanos , Imitación Molecular , Virus de la Inmunodeficiencia de los Simios , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.