Human Human Hypoxanthine-guanine Phosphoribosyltransferase Research Articles

Synthesis and Evaluation of Asymmetric Acyclic Nucleoside Bisphosphonates as Inhibitors of Plasmodium falciparum and Human Hypoxanthine-Guanine-(Xanthine) Phosphoribosyltransferase.

Acyclic nucleoside bisphosphonates (ANbPs) have previously been shown to be good inhibitors of human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and Plasmodium falciparum (Pf) hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT). On the basis of this scaffold, a new series of ANbPs was synthesized. One of these new ANbPs, [3-(guanine-9-yl)-2-((2-phosphonoethoxy)methyl)propoxy]methylphosphonic acid, exhibited Ki values of 6 and 70 nM for human HGPRT and Pf HGXPRT, respectively. These low Ki values were achieved by inserting an extra carbon atom in the linker connecting the N9 atom of guanine to one of the phosphonate groups. The crystal structure of this ANbP in complex with human HGPRT was determined at 2.0 Å resolution and shows that it fills three key pockets in the active site. The most potent phosphoramidate prodrugs of these compounds have IC50 values in the low micromolar range in Pf lines and low toxicity in human A549 cells, demonstrating that these ANbPs are excellent antimalarial drug leads.

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-5'-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5'-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Km(app) values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.

Structural and functional studies of the human phosphoribosyltransferase domain containing protein 1

Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch-Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (k(cat)/K(m)) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.

The Crystal Structure of Free Human Hypoxanthine-guanine Phosphoribosyltransferase Reveals Extensive Conformational Plasticity Throughout the Catalytic Cycle

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-alpha-d-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP.Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg(2+).PP(i) complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9A resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasma gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T.gondii).

A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity

Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) lacks the ability to phosphoribosylate xanthine, a property exhibited by HGPRTs from many parasitic protozoa. Using random mutagenesis we have obtained a mutant, F36L, of human HGPRT that phosphoribosylates xanthine. Examination of the structure indicates that F36 does not make direct contact with the purine, but long-range modulation via loop IV, a segment contacting purine at C2 position, could influence substrate specificity. Expanded substrate specificity to include xanthine probably arises from increased flexibility of loop IV as a consequence of mutation at F36. Mutation of the corresponding residue, L44 in Plasmodium falciparum HGPRT, also results in alteration of K m and k cat for xanthine, substantiating its role in affecting purine base affinity. Our studies show that mutation of this residue in the core of the protein also affects the stability of both enzymes.

Spontaneous frequency of exon skipping in the human HPRT gene

In order to elucidate the mechanisms of mRNA splicing fidelity and the mutagenic potential of aberrant mis-spliced transcripts we have investigated the frequency of spontaneous exon skipping in the human hypoxanthine-guanine phosphoribosyl transferase ( HPRT) gene in well characterized human primary fibroblasts isolated from two different individuals. In these cells, coexisting with the WT species, we also detected three aberrant HPRT transcripts missing exon IV, VII, or VIII. We were unable to detect transcripts missing exon II, III, V, or VI. Significantly, all the exons affected by skipping do not generate new stop codons more than 50 nucleotides upstream from the 3′ most exon–exon junction. Exon VIII was the most prone to skipping with a relative frequency to WT of 0.019±0.004 (approximately one aberrant transcript per 50 WT transcripts). Exon IV exhibited a relative frequency of skipping of 0.006±0.002 ((approximately one aberrant transcript per 150 WT transcripts) and exon VII exhibited a relative frequency of skipping of 0.003±0.002 ((approximately one aberrant transcript per 300 WT transcripts). These data demonstrate that aberrant transcripts with exon skipped are generated spontaneously in humans and some appear to persist in the cell.

Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme

A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7. Expression of human HPRT has been achieved in HPRT − Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with [ 35S]methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis. Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates. Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources. The K m values of recombinant HPRT for the substrates 5-phospho-α-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT.

Yeast ARS function and nuclear matrix association coincide in a short sequence from the human HPRT locus.

A sequence that supports extrachromosomal replication of plasmids in yeast has been identified within the first intron of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene. This represents the first isolation of such an autonomously replicating sequence (ARS) from an exactly known position in the human genome. This ARS shares similarities of imparted yeast phenotype and DNA sequence with other heterologous ARSs. In addition, this sequence is found to be a matrix association region (MAR) on the basis of specific binding to nuclear matrices prepared from several mammalian cell types. It also exhibits anomalous electrophoretic behavior, characteristic of bent DNA, on polyacrylamide gels. The coincidence of these properties supports the possibility that this region may play a role in DNA replication within its normal chromosomal context.

Adult mammalian hepatocyte as target cell for retroviral gene transfer: a model for gene therapy.

Gene therapy models have recently been developed based on the repopulation of a major organ, such as mammalian bone marrow, with cells genetically modified in vitro by infection with retroviral vectors (1-4). Another important target organ for therapeutically useful gene transfer is the liver. Unfortunately, the murine leukemia viruses (MLV) used to construct most retroviral vectors do not infect newborn, adult, or regenerating liver cells in vivo (5-8), although fetal liver can be infected and continues to express retrovitally transduced genes postnatally (7, 8). We now show that adult primary rat hepatocyte cultures (9, 10) are in fact susceptible to infection with MLV-based retroviral vectors during a period of transient dedifferentiation in vitro. Primary hepatocyte cultures exhibit many adult liver-specific functions--including gluconeogenesis and the expression of albumin, alcohol dehydrogenase, and type I pyruvate kinase--during their early lag (days 0-2) and late stationary (after day 11) phases of culture (10 16). During the intervening phase of logarithmic growth, these mature liver-specific functions are transiently but markedly decreased, and fetal hepatocyte functions such as alphal-fetoprotein and type III pyruvate kinase are expressed (12). Hepatocyte cultures were exposed at varying times during their growth cycle to a retroviral vector expressing a mutagenized but fully functional human hypoxanthine guanine phosphoribosyl transferase (HPRT) (Fig. 1, LSAPALM) (J.-K. Yee et al., in preparation). Human HPRT enzymic activity was detected in isoelectric focusing polyacrylamide gels (17) under conditions that distinguish it from the endogenous rat enzyme by its altered isoelectric point. Twoto three-dayold cultures infected with 5 x 1 0 6 infectious units of the HPRT vector exhibited human HPRT enzymic activity on day 14 postplating, as indicated by the arrows in Fig. 2A (lane a). The transduced human HPRT activity was at least 10-25% of the endogenous rat HPRT enzymic activity, equivalent to the high levels achieved in primary rat fibroblasts (Fig. 2, lane c) and human lymphoblasts (18). Since the upper bands in Fig. 2A (lanes a and c) may also contain human HPRT multimers (17), the actual transduced levels of human HPRT enzymic activity may be even higher. Figure 2B shows that human HPRT activity was present only when the hepatocyte cultures were infected during the early proliferative, dedifferentiated period of growth (days 2-3). Very low levels of human HPRT expression, not evident in Fig. 2B, were also detected on days 1 and 5, but not at any other times of infection. In addition, quiescent hepatocytes on day 1 1 can also be stimulated to reinitiate DNA synthesis and undergo additional cell division by exposure to fresh medium containing rat serum or polypeptide mitogens, mimicking the regenerating adult liver following partial hepatectomy (9, 19).

An automated image analysis system for the detection of rare autoradiographically labelled cells in the human lymphocyte HGPRT variant assay.

This paper describes a fast and accurate method for the identification of autoradiographically labelled human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) variant lymphocytes on slide preparations using a high-speed computer image analysis system--the Fast Interval Processor (FIP). The system has been developed primarily for the analysis of cultured human peripheral blood lymphocyte populations in which the frequency of labelled cells may vary from less than one to more than 2,000 in every 10,000 cells. Evaluation experiments have demonstrated the excellent performance of FIP in the counting of labelled and total cells over this frequency range.