Disease relevance of Rrm2

• The bridging Co(II) carboxylate ligand Glu-267 adopts an altered orientation compared with its counterpart Glu-238 in Escherichia coli R2 [1].
• Human cytomegalovirus (HCMV) encodes a protein related to the large (R1) subunit of ribonucleotide reductase (RR), but does not encode the corresponding small (R2) subunit [2].
• Furthermore, cells with altered R2 expression also exhibited significantly reduced subcutaneous tumor latency and increased tumor growth rates in syngeneic mice, and showed markedly elevated metastatic potential in lung metastasis assays [3].
• As previously reported, a vaccinia R1/mouse R2 hybrid has low but significant activity [4].
• Our results with inactivation of protein R2 of RR imply that the cytostatic effect of p-alkoxyphenols on melanoma cells, which has been hitherto explained by inhibition of tyrosinase [Melanoma Res. 2:295-304 (1992)], may be caused at least partly by inhibition of RR [5].

High impact information on Rrm2

• The carboxy terminus of the RNR small subunit (R2) is critical for subunit association and synthetic peptides containing these amino-acid sequences selectively inhibit the viral enzyme by preventing subunit association [6].
• The properties of the different redox states of the diiron center in protein R2 are discussed, as well as the formation of the iron/radical site and its possible involvement in long range electron transfer from the substrate binding site in protein R1 [7].
• The EPR properties of oxidized neutral tyrosyl free radicals are described, and also of tryptophan free radicals found in studies of a mutant of the R2 protein, which lacks the tyrosyl radical site [7].
• However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear [8].
• In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border [8].

Chemical compound and disease context of Rrm2

• Compared with E. coli R2, two conserved phenylalanine residues in the hydrophobic environment around the diiron center have opposing rotameric conformations, and the carboxylate ligands of the diiron center in mouse R2 appear more flexible [9].
• In E. coli R2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine [10].
• The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity [11].
• Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins [12].
• Effect of cyclic AMP on the cell cycle regulation of ribonucleotide reductase M2 subunit messenger RNA concentrations in wild-type and mutant S49 T lymphoma cells [13].

Biological context of Rrm2

• Previous reports in the literature have indicated that the S phase-specific transcription of the mammalian R2 gene is regulated by a transcriptional block, is dependent on the transcription factor E2F1, or is simply regulated by proteins that bind to promoter CCAAT boxes plus the TATA box [14].
• In situ hybridization showed that the functional Rrm2 gene was located in this particular region of chromosome 12 [15].
• The mammalian ribonucleotide reductase R2 component cooperates with a variety of oncogenes in mechanisms of cellular transformation [16].
• These results suggest that adrenergic stimulation of brown adipocyte proliferation may act at the level of gene expression of the limiting subunit for RNR activity, R2, and demonstrate a qualitative switch in the response of the R2 gene to cAMP-elevating agents as a consequence of the switch from proliferating to differentiating cell status [17].
• In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein [10].

Anatomical context of Rrm2

• Ribonucleotide reductase M2 subunit sequences mapped to four different chromosomal sites in humans and mice: functional locus identified by its amplification in hydroxyurea-resistant cell lines [18].
• In addition, we observed that elevated R2 expression conferred on c-myc-transformed NIH 3T3 cells an increased tumorigenic potential in immunoincompetent mice [16].
• In contrast, NE stimulation of confluent differentiating brown adipocytes reduced both R1 and R2 expression [17].
• Four lines of H-ras-transformed mouse 10T1/2 fibroblasts showed increased growth efficiency in soft agar after infection with the recombinant R2 expression virus vector [3].
• Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region [19].

Associations of Rrm2 with chemical compounds

• The RNR subunit R2 contains a dinuclear iron center, which in its diferrous form spontaneously reacts with O2, forming a mu-oxo-bridged diferric cluster and a stable tyrosyl radical [9].
• Norepinephrine specifically stimulates ribonucleotide reductase subunit R2 gene expression in proliferating brown adipocytes: mediation via a cAMP/PKA pathway involving Src and Erk1/2 kinases [17].
• NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA [19].
• To study the mechanism of tyrosyl radical formation, substoichiometric amounts of Fe(II) were added to recombinant mouse R2 apoprotein under strictly anaerobic conditions and then the solution was exposed to air [20].
• 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 [21].

Regulatory relationships of Rrm2

• Also, inhibitors of Src and of Erk1/2 kinases markedly reduced NE-stimulated R2 expression [17].

Other interactions of Rrm2

• Mapping of the ribonucleotide reductase genes (Rrm1, Rrm2) in the rat [22].
• We report an integrated genetic and physical map of the 650-kb region containing the cpk locus and the exclusion of Rrm2 and Idb2 as candidate cpk genes [23].
• Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis [24].

Analytical, diagnostic and therapeutic context of Rrm2

• Northern blot analysis showed that elevations in activity were accompanied by transient increases in the mRNA levels of both genes (R1 and R2) that code for ribonucleotide reductase [25].
• Western blot analysis indicated that only the protein for the limiting component for enzyme activity, R2, was significantly elevated in chlorambucil treated cultures [25].
• No R2 promoter activation was observed after treatment of mouse cells with agents reported to induce the ribonucleotide reductase genes in Saccharomyces cerevisiae such as hydroxyurea or methylmethane sulfonate [26].
• Protein R2 of RR may be considered as an additional target that could be used for future cancer chemotherapy [5].
• 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 [27].

References