The c-Myc Target Gene Rcl (C6orf108) Encodes a Novel Enzyme, Deoxynucleoside 5′-monophosphate N-Glycosidase

Abstract

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.The c-Myc transcription factor plays an important role in the regulation of the cell cycle, cellular transformation, and apoptosis (1) as well as in the genesis of many human cancers (2). The deregulation of MYC expression by chromosomal translocation, amplification, or altered signal transduction is commonly found in human cancers (3, 4). To elucidate the mechanisms by which c-Myc contributes to tumorigenesis, several approaches have been used to identify its target genes, which are compiled in the data base myccancergene.org.Rcl was identified as a c-Myc target by representational difference analysis between non-adherent Rat1a fibroblasts and Rat 1a cells transformed by MYC (5). Rcl is expressed at a low level in untransformed cell lines, although it is significantly elevated in breast cancer and lymphoma cell lines (5). Moreover, Rcl is one of the most responsive targets to Myc activation in vitro and in Myc-induced transgenic lymphoma (6, 7), and Rcl has been shown to be a direct Myc target in a human lymphoma cell model (8). Serial analysis of gene expression studies revealed that Rcl is also highly expressed in human glioblastoma multiforme as compared with normal human brain (information from the UniGene data base, at NCBI). Furthermore, Rcl is among the top 50 genes whose overexpression distinguishes between benign and malignant prostate tissues (9). Functionally, Rcl has transforming activity in Rat1a cells when coexpressed with either vascular endothelial growth factor or lactate dehydrogenase (10). Altogether, these data suggest that RCL could be a prime therapeutic target.RCL appears to be a nuclear 22-kDa protein with yet unknown function. The closest relatives of RCL protein are members of the nucleoside 2-deoxyribosyltransferase family (EC 2.4.2.6). (pfam 05014), which catalyze the reversible transfer of the deoxyribosyl moiety from a deoxynucleoside donor to an acceptor nucleobase (11-13). Here, we report the purification and characteriza...

Proteomic and metabolomic responses in D-shape larval mussels Mytilus galloprovincialis exposed to cadmium and arsenic

“…Inorganic pyrophosphatase catalyzes the reversible transfer of phosphoryl group from a polyphosphate to water and this reaction provides a thermodynamic pull for the synthesis of proteins and nucleic acids [33]. Deoxynucleoside 5'-monophosphate N-glycosidase catalyzes the hydrolysis of the N-glycosidic bond of the nucleotide to give deoxyribose 5-phosphate and a nucleobase, preferentially a purine [34]. The up-regulated inorganic pyrophosphatase and deoxynucleoside 5'-monophosphate N-glycosidase suggested the disturbance in nucleic acid metabolisms induced by Cd in D-shape larval mussels.…”

Probing the Active Site of the Deoxynucleotide N-Hydrolase Rcl Encoded by the Rat Gene c6orf108

“…Construction of the Rcl Mutants-RclE93A, Rcl D69A, and Rcl D69N were previously described in Ghiorghi et al (9) and Yang et al (13) respectively. Oligonucleotides ϩ (Y13Aϩ, Y13Fϩ, S17Aϩ, S17Eϩ, R19Aϩ, H45Aϩ, S87Aϩ, S87Dϩ, E93Qϩ, S117Aϩ, S117Nϩ) and T7term, oligonucleotides Ϫ (Y13AϪ, Y13FϪ, S17AϪ, S17EϪ, R19AϪ, H45AϪ, S87AϪ, S87DϪ, E93QϪ, S117AϪ, S117NϪ) and T7prom (Table 1) were used in two separate PCR reactions using plasmid pET28aRcl as DNA template.…”

“…We have shown that Rcl is a 2Ј-deoxynucleoside 5Ј-phosphate N-hydrolase that had not been previously described (9). Rcl hydrolyzes 2Ј-deoxyribonucleoside 5Ј-monophosphate (dNMP) 4 to form a free nucleobase moiety (N) and 2-deoxyribose 5-phosphate.…”

“…NDT catalyzes the reversible transfer of the deoxyribosyl moiety from a deoxynucleoside donor to an acceptor nucleobase (11). On the contrary, Rcl is a hydrolase (or a transferase using only water molecule as an acceptor) (9). Both NDT and Rcl are specific for 2Ј-deoxyribose-containing substrates, whereas the corresponding riboside analogs are inhibitors.…”

“…Both NDT and Rcl are specific for 2Ј-deoxyribose-containing substrates, whereas the corresponding riboside analogs are inhibitors. Their amino acid sequences display an overall low level of identity but share the catalytic triad composed of a tyrosine, an aspartate, and a glutamate, suggesting a similar mode of action (9,12).…”