ABSTRACT
Eukaryotic mRNA precursors are co-transcriptionally assembled into ribonucleoprotein complexes. Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are involved in mRNA translocation, stability, subcellular localization and regulation of mRNA translation. About 20 major classes of hnRNPs have been identified in mammals. In a previous work, we characterized a novel, strongly-basic, RNA-binding protein (p65) in presynaptic terminals of squid neurons presenting homology with human hnRNPA/B type proteins, likely involved in local mRNA processing. We have identified and sequenced two hnRNPA/B-like proteins associated with tissue purified squid p65: Protein 1 (36.3 kDa, IP 7.1) and Protein 2 (37.6 kDa, IP 8.9). In the present work we generated an in silico, tridimensional, structural model of squid hnRNPA/B-like Protein 2, which showed highly conserved secondary and tertiary structure of RNA recognition motifs with human hnRNPA1 protein, as well as illustrated the potential for squid Protein 2 stable homodimerization. This was supported by biophysical measurements of bacterially expressed, recombinant protein. In addition, we induced expression of squid hnRNPA/B-like Protein 2 in human neuroblastoma cells (SH-SY5Y) and observed an exclusively nuclear localization, which depended on an intact C-terminal amino acid sequence and which relocated to cytoplasm particles containing PABP when the cells were challenged with sorbitol, suggesting an involvement with stress granule function.
Subject(s)
Brain/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins/genetics , Animals , Cell Nucleus/metabolism , Cytoplasm/metabolism , Decapodiformes , Dimerization , Humans , RNA-Binding Proteins/genetics , Ribonucleoproteins/metabolismABSTRACT
Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance. The transfer of γ-phosphate from ATP to ADO by ADK involves regulation by the substrates and products, as well as by Mg2+ and inorganic phosphate. Here we present new crystal structures of mouse ADK (mADK) binary (mADK:ADO; 1.2 Å) and ternary (mADK:ADO:ADP; 1.8 Å) complexes. In accordance with the structural demonstration of ADO occupancy of the ATP binding site, kinetic studies confirmed a competitive model of auto-inhibition of ADK by ADO. In the ternary complex, a K+ ion is hexacoordinated between loops adjacent to the ATP binding site, where Asp310 connects the K+ coordination sphere to the ATP binding site through an anion hole structure. Nuclear Magnetic Resonance 2D 15N-1H HSQC experiments revealed that the binding of K+ perturbs Asp310 and residues of adjacent helices 14 and 15, engaging a transition to a catalytically productive structure. Consistent with the structural data, the mutants D310A and D310P are catalytically deficient and loose responsiveness to K+. Saturation Transfer Difference spectra of ATPγS provided evidence for an unfavorable interaction of the mADK D310P mutant for ATP. Reductions in K+ concentration diminish, whereas increases enhance the in vitro activity of mADK (maximum of 2.5-fold; apparent Kd = 10.4 mM). Mechanistically, K+ increases the catalytic turnover (Kcat) but does not affect the affinity of mADK for ADO or ATP. Depletion of intracellular K+ inhibited, while its restoration was accompanied by a full recovery of cellular ADK activity. Together, this novel dataset reveals the molecular basis of the allosteric activation of ADK by K+ and highlights the role of ADK in connecting depletion of intracellular K+ to the regulation of purine metabolism.
