RESUMEN
Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.
RESUMEN
Slo1 is a Ca2+- and voltage-activated K+ channel that underlies skeletal and smooth muscle contraction, audition, hormone secretion and neurotransmitter release. In mammals, Slo1 is regulated by auxiliary proteins that confer tissue-specific gating and pharmacological properties. This study presents cryo-EM structures of Slo1 in complex with the auxiliary protein, ß4. Four ß4, each containing two transmembrane helices, encircle Slo1, contacting it through helical interactions inside the membrane. On the extracellular side, ß4 forms a tetrameric crown over the pore. Structures with high and low Ca2+ concentrations show that identical gating conformations occur in the absence and presence of ß4, implying that ß4 serves to modulate the relative stabilities of 'pre-existing' conformations rather than creating new ones. The effects of ß4 on scorpion toxin inhibition kinetics are explained by the crown, which constrains access but does not prevent binding.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Microscopía por Crioelectrón , Humanos , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
The proteasome beta-4 subunit is required for the assembly of 20S proteasome complex, forming a pivotal component for the ubiquitin-proteasome system. Emerging evidence indicates that proteasome beta-4 subunit may be involved in underlying progression and mechanisms of malignancies. However, the role of proteasome beta-4 subunit in melanoma is currently unknown. Here, we reported that proteasome beta-4 subunit was markedly upregulated in human melanoma tissues and cells, compared with normal skin samples. High proteasome beta-4 subunit levels were significantly associated with poor overall survival in patients with melanoma. Proteasome beta-4 subunit knockdown strongly decreased melanoma cell growth in vitro and in vivo. We further identified miR-148b as a negative regulator of proteasome beta-4 subunit. Enforced expression of miR-148b resulted in vitro growth inhibition of melanoma cells, whereas this inhibition was further abolished by enforced expression of proteasome beta-4 subunit. Our findings, for the first time, indicated that the miR-148b/proteasome beta-4 subunit axis contributed to the development of melanoma, revealing novel therapeutic targets for the treatment of melanoma.
Asunto(s)
Carcinogénesis/genética , Melanoma/genética , MicroARNs/genética , Complejo de la Endopetidasa Proteasomal/genética , Adulto , Anciano , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Melanoma/patología , Persona de Mediana EdadRESUMEN
Voltage-gated sodium (Nav) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, ß-subunits modify Nav channel function and cause debilitating disorders when mutated. While investigating whether ß-subunits also influence ligand interactions, we found that ß4 dramatically alters toxin binding to Nav1.2. To explore these observations further, we solved the crystal structure of the extracellular ß4 domain and identified (58)Cys as an exposed residue that, when mutated, eliminates the influence of ß4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of ß4. Disrupting this bridge by introducing a ß1 mutation implicated in epilepsy repositions the (58)Cys-containing loop and disrupts ß4 modulation of Nav1.2. Overall, the principles emerging from this work (i) help explain tissue-dependent variations in Nav channel pharmacology; (ii) enable the mechanistic interpretation of ß-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant ß-subunit behavior.