Native R2 Research Articles

Abstract 5037: Intrinsic susceptibility magnetic resonance imaging identifies tumors with ALKF1174L mutation in transgenic murine models of high-risk neuroblastoma.

Abstract In neuroblastoma, mutations in the anaplastic lymphoma kinase (ALK) tyrosine kinase gene have been identified in 8-10 % of primary tumors. The most common and potent ALK mutation, ALKF1174L, leads to the constitutive activation of the ALK protein and is associated preferentially with MYCN amplification, a markedly poorer prognosis, and confers resistance to the promising ALK inhibitor crizotinib. The development of more efficacious ALK inhibitors will benefit from non-invasive imaging strategies for the rapid identification of children with high-risk ALK-expressing or mutated neuroblastoma. Intrinsic susceptibility magnetic resonance imaging (IS-MRI) data was acquired from tumors arising in TH-ALKF1174L/TH-MYCN and TH-MYCN mice, two genetically engineered mouse model of high-risk neuroblastoma. The native MRI transverse relaxation rate R2*, an imaging biomarker sensitive to the concentration of paramagnetic deoxyhemoglobin, and changes in R2* induced with 100% oxygen inhalation, were quantified. Tumors in the TH-ALKF1174L/TH-MYCN mice demonstrated significantly (p<0.0001) slower native R2* rates (61 ± 3s−1, n=23) than tumors in the TH-MYCN mice (101 ± 6s−1, n=21). With hyperoxic challenge, the TH-ALKF1174L/TH-MYCN mice demonstrated a significantly (p<0.0001) lower and negligible ΔR2*oxygen-air (-3 ± 1s−1, n=10) compared with tumors in TH-MYCN mice (-26 ± 3s−1, n=12). A sensitivity of 90% and a specificity of 81% for native R2*, and a sensitivity of 90% and a specificity of 94% for hyperoxia-induced ΔR2* was determined. Histological correlates revealed a significantly (p<0.05) higher uptake of the perfusion marker Hoechst 33342, and the presence of large hemorrhagic blood lakes filled with stagnant deoxygenated erythrocytes, in tumors within the TH-MYCN mice, but not the TH-ALKF1174L/TH-MYCN model. Together these corroborate the IS-MRI findings (relatively fast native R2* and significant ΔR2*, indicative of vascular instability, in tumors in the TH-MYCN mice). IS-MRI provides a robust method to discriminate and identify TH-MYCN transgenic mice harboring the ALKF1174L mutation based on a stark differential vascular phenotype, which may impact on impaired drug delivery. IS-MRI is suitable for the scanning of young children, and quantitation of native R2* easily incorporated into existing pediatric clinical imaging protocols. This approach could provide a robust and rapid indicator of ALK genotypic status of the tumor, enabling the early identification of children with ultra high-risk neuroblastoma at the time of diagnosis. Citation Format: Yann Jamin, Laura Glass, Albert Hallsworth, Rani E. George, Dow-Mu Koh, Andrew D.J. Pearson, Louis Chesler, Simon P. Robinson. Intrinsic susceptibility magnetic resonance imaging identifies tumors with ALKF1174L mutation in transgenic murine models of high-risk neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5037. doi:10.1158/1538-7445.AM2013-5037

The Pore of the Voltage-Gated Proton Channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containinga VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 inthe transmembrane VSD to form the ion selectivity filter in the channel's open conformation.

Metal Binding and Activity of Ribonucleotide Reductase Protein R2 Mutants: Conditions for Formation of the Mixed Manganese−Iron Cofactor

Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

Chemical reduction of the diferric/radical center in protein R2 from mouse ribonucleotide reductase is independent of the proposed radical transfer pathway

The rates of reduction of the diferric/radical center in mouse ribonucleotide reductase protein R2 were studied by light absorption and EPR in the native protein and in three point mutants of conserved residues involved in the proposed radical transfer pathway (D266A, W103Y) or in the unstructured C terminal domain (Y370W). The pseudo-first order rate constants for chemical reduction of the tyrosyl radical and diferric center by hydroxyurea, sodium dithionite or the dihydro form of flavin adenine dinucleotide, were comparable with or higher (particularly D266A, by dithionite) than in native R2. Molecular modeling of the D266A mutant showed that the iron/radical site should be more accessible for external reductants in the mutant than in native R2. The results indicate that no specific pathway is required for the reduction. The dihydro form of flavin adenine dinucleotide was found to be a very efficient reductant in the studied proteins compared to dithionite alone. The EPR spectra of the mixed-valent Fe(II)Fe(III) sites formed by chemical reduction in the D266A and W103Y mutants were clearly different from the spectrum observed in the native protein, indicating that the structure of the diferric site was affected by the mutations, as also suggested by the modeling study. No difference was observed between the mixed-valent EPR spectra generated by chemical reduction in Y370W mutant and native mouse R2 protein.

Evidence by Mutagenesis that Tyr370 of the Mouse Ribonucleotide Reductase R2 Protein Is the Connecting Link in the Intersubunit Radical Transfer Pathway

Ribonucleotide reductase catalyzes all de novo synthesis of deoxyribonucleotides. The mammalian enzyme consists of two non-identical subunits, the R1 and R2 proteins, each inactive alone. The R1 subunit contains the active site, whereas the R2 protein harbors a binuclear iron center and a tyrosyl free radical essential for catalysis. It has been proposed that the radical properties of the R2 subunit are transferred approximately 35 A to the active site of the R1 protein, through a coupled electron/proton transfer along a conserved hydrogen-bonded chain, i.e. a radical transfer pathway (RTP). To gain a better insight into the properties and requirements of the proposed RTP, we have used site-directed mutagenesis to replace the conserved tyrosine 370 in the mouse R2 protein with tryptophan or phenylalanine. This residue is located close to the flexible C terminus, known to be essential for binding to the R1 protein. Our results strongly indicate that Tyr(370) links the RTP between the R1 and R2 proteins. Interruption of the hydrogen-bonded chain in Y370F inactivates the enzyme complex. Alteration of the same chain in Y370W slows down the RTP, resulting in a 58 times lower specific activity compared with the native R2 protein and a loss of the free radical during catalysis.

Evidence by site-directed mutagenesis supports long-range electron transfer in mouse ribonucleotide reductase.

Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.

Binding of the competitive inhibitor dCDP to ribonucleoside-diphosphate reductase from Escherichia coli studied by 1H NMR. Different properties of the large protein subunit and the holoenzyme.

Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two nonidentical subunits, proteins R1 and R2. The binding of the product dCDP to protein R1 and to the holoenzyme R1R2 has been studied by means of 1H-NMR spectroscopy. In presence of the effector dTTP at 25 degrees C, dCDP was found to be in rapid exchange between the binding sites and the solvent which results in a broadening of the dCDP resonances. When both proteins R1 and R2 are present, so that the complex R1R2 is formed, a smaller broadening is observed than with protein R1 alone. No further linewidth decrease was observed when the [R2]/[R1] ratio exceeded 1. The binding constant of dCDP to R1 or R1R2 is the same, Kd = 0.9 mM. The smaller broadening of the dCDP resonances observed with the complex R1R2 as compared with R1 may be explained by the combination of two effects: (a) the overall tumbling time of the protein will increase when going from R1 to R1R2, which will cause the broadening to increase correspondingly, and (b) a twofold decrease of the number of binding sites in rapid exchange, which will decrease the broadening by a factor of 0.5. The effect of R2 without iron (apoR2) is reduced compared with native R2, probably because of some denatured proteins, while a C-terminal peptide from R2 did not cause any narrowing at all.