RESUMEN
BACKGROUND: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. METHODS: Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. FINDINGS: CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. INTERPRETATION: CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).
Asunto(s)
Citoesqueleto de Actina , Señalización del Calcio , Células Madre Pluripotentes Inducidas , Receptor Nicotínico de Acetilcolina alfa 7 , Humanos , Citoesqueleto de Actina/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genética , Calcio/metabolismo , Inmunidad Innata , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Monocitos/metabolismo , Fagocitosis , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genéticaRESUMEN
The combination of advanced mass spectrometry and enrichment-based sample preparation methods has enhanced analytical capabilities in clinical proteomics. In this chapter, we describe a method of proteome analysis to identify Borrelia-derived peptides in urine that includes a sample affinity enrichment method coupled with liquid chromatography tandem mass spectrometry analysis and a bioinformatic peptide authentication algorithm.