Artemin growth factor increases nicotinic cholinergic receptor subunit expression and activity in nociceptive sensory neurons

Background: Artemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) subunits, suggesting these ion channels contribute to Artn-induced sensitivity. Here we have used gene expression, immunolabeling, patch clamp electrophysiology and behavioral testing assays to investigate the link between Artn, nicotinic subunit expression and thermal hypersensitivity.Results: Reverse transcriptase-PCR validation showed increased levels of mRNAs encoding the nAChR subunits α3 (13.3-fold), β3 (4-fold) and β4 (7.7-fold) in trigeminal ganglia and α3 (4-fold) and β4 (2.8-fold) in dorsal root ganglia (DRG) of ART-OE mice. Sensory ganglia of ART-OE mice had increased immunoreactivity for nAChRα3 and exhibited increased overlap in labeling with GFRα3-positive neurons. Patch clamp analysis of back-labeled cutaneous afferents showed that while the majority of nicotine-evoked currents in DRG neurons had biophysical and pharmacological properties of α7-subunit containing nAChRs, the Artn-induced increase in α3 and β4 subunits resulted in functional channels. Behavioral analysis of ART-OE and wildtype mice showed that Artn-induced thermal hyperalgesia can be blocked by mecamylamine or hexamethonium. Complete Freund's adjuvant (CFA) inflammation of paw skin, which causes an increase in Artn in the skin, also increased the level of nAChR mRNAs in DRG. Finally, the increase in nAChRs transcription was not dependent on the Artn-induced increase in TRPV1 or TRPA1 in ART-OE mice since nAChRs were elevated in ganglia of TRPV1/TRPA1 double knockout mice.Conclusions: These findings suggest that Artn regulates the expression and composition of nAChRs in GFRα3 nociceptors and that these changes contribute to the thermal hypersensitivity that develops in response to Artn injection and perhaps to inflammation. © 2014 Albers et al.; licensee BioMed Central Ltd

Phylogenetic Distribution and Evolutionary History of Bacterial DEAD-Box Proteins

DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation

Roles of DEAD-box proteins in RNA and RNP folding

RNAs and RNA-protein complexes (RNPs) traverse rugged energy landscapes as they fold to their native structures, and many continue to undergo conformational rearrangements as they function. Due to the inherent stability of local RNA structure, proteins are required to assist with RNA conformational transitions during initial folding and in exchange between functional structures. DEAD-box proteins are superfamily 2 RNA helicases that are ubiquitously involved in RNA-mediated processes. Some of these proteins use an ATP-dependent cycle of conformational changes to disrupt RNA structure nonprocessively, accelerating structural transitions of RNAs and RNPs in a manner that bears a strong resemblance to the activities of certain groups of protein chaperones. This review summarizes recent work using model substrates and tractable self-splicing intron RNAs, which has given new insights into how DEAD-box proteins promote RNA folding steps and conformational transitions, and it summarizes recent progress in identifying sites and mechanisms of DEAD-box protein activity within more complex cellular targets

From unwinding to clamping - the DEAD box RNA helicase family

RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation

Taming free energy landscapes with RNA chaperones

Many non-coding RNAs fold into complex three-dimensional structures, yet the self-assembly of RNA structure is hampered by mispairing, weak tertiary interactions, electrostatic barriers, and the frequent requirement that the 5′ and 3′ ends of the transcript interact. This rugged free energy landscape for RNA folding means that some RNA molecules in a population rapidly form their native structure, while many others become kinetically trapped in misfolded conformations. Transient binding of RNA chaperone proteins destabilize misfolded intermediates and lower the transition states between conformations, producing a smoother landscape that increases the rate of folding and the probability that a molecule will find the native structure. DEAD-box proteins couple the chemical potential of ATP hydrolysis with repetitive cycles of RNA binding and release, expanding the range of conditions under which they can refold RNA structures