ABSTRACT
BACKGROUND: In the tolerogenic liver, inadequate or ineffective interferon signaling fails to clear chronic HBV infection. Lambda IFNs (IFNL) bind the interferon lambda receptor-1 (IFNLR1) which dimerizes with IL10RB to induce transcription of antiviral interferon-stimulated genes (ISG). IFNLR1 is expressed on hepatocytes, but low expression may limit the strength and antiviral efficacy of IFNL signaling. Three IFNLR1 transcriptional variants are detected in hepatocytes whose role in regulation of IFNL signaling is unclear: a full-length and signaling-capable form (isoform 1), a form that lacks a portion of the intracellular JAK1 binding domain (isoform 2), and a secreted form (isoform 3), the latter two predicted to be signaling defective. We hypothesized that altering expression of IFNLR1 isoforms would differentially impact the hepatocellular response to IFNLs and HBV replication. METHODS: Induced pluripotent stem-cell derived hepatocytes (iHeps) engineered to contain FLAG-tagged, doxycycline-inducible IFNLR1 isoform constructs were HBV-infected then treated with IFNL3 followed by assessment of gene expression, HBV replication, and cellular viability. RESULTS: Minimal overexpression of IFNLR1 isoform 1 markedly augmented ISG expression, induced de novo proinflammatory gene expression, and enhanced inhibition of HBV replication after IFNL treatment without adversely affecting cell viability. In contrast, overexpression of IFNLR1 isoform 2 or 3 partially augmented IFNL-induced ISG expression but did not support proinflammatory gene expression and minimally impacted HBV replication. CONCLUSIONS: IFNLR1 isoforms differentially influence IFNL-induced gene expression and HBV replication in hepatocytes. Regulated IFNLR1 expression in vivo could limit the capacity of this pathway to counteract HBV replication.
Subject(s)
Hepatitis B virus , Interferon Lambda , Interferons/pharmacology , Hepatocytes , Virus Replication , Antiviral Agents/pharmacology , Antiviral Agents/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Gene ExpressionABSTRACT
The Argonaute (AGO) family of proteins plays an essential role in the process of microRNA (miRNA)-mediated gene silencing. More specifically, they are the only known proteins to associate directly with miRNAs within the RNA-induced silencing complex (RISC). Given the importance of miRNA regulation of the transcriptome and its vast implications for human disease, it is essential to understand the molecular underpinnings of miRNA-AGO interactions. Although there are methods available to investigate mature miRNA decay and loading onto AGO2, no feasible method exists to detail the opposite process: release of miRNA from associated AGO proteins. In this chapter, we describe in detail a methodology derived from biochemical approaches, which can be used to quantify the release of any given miRNA from AGOs.
Subject(s)
Argonaute Proteins/metabolism , MicroRNAs/metabolism , Real-Time Polymerase Chain Reaction/methods , Animals , Cell Line , Humans , Kinetics , Protein BindingABSTRACT
Our previous studies implicated glycosylation of the CaV3.2 isoform of T-type Ca2+ channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant CaV3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our in vitro study we used whole-cell recordings of current-voltage relationships to confirm that CaV3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced in vivo by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and CaV3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the CaV3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native CaV3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in CaV3.2 channels in vivo directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.
ABSTRACT
Given the clinical significance of pain disorders and the relative ineffectiveness of current therapeutics, it is important to identify alternative means of modulating nociception. The most obvious pharmacological targets are the ion channels that facilitate nervous transmission from pain sensors in the periphery to the processing regions within the brain and spinal cord. In order to design effective pharmacological tools for this purpose, however, it is first necessary to understand how these channels are regulated. A growing area of research involves the investigation of the role that trace metals and endogenous redox agents play in modulating the activity of a diverse group of ion channels within the pain pathway. In the present review, the most recent literature concerning trace metal and redox regulation of T-type calcium channels, NMDA (N-methyl-D-aspartate) receptors, GABAA (γ-aminobutyric acid A) receptors and TRP (transient receptor potential) channels are described to gain a comprehensive understanding of the current state of the field as well as to provide a basis for future thought and experimentation.