Search

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP -/- cancers, however the MTAP -/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP +/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP -/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP +/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 10 4 -fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP +/+ cancers, especially the remaining 98% of CRCs without the MTAP -/- genotype., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD + synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) catalyzes an essential step in purine salvage for parasite growth. 4'-Deaza-1'-Aza-2'-Deoxy-1'-(9-Methylene)-Immucillin-G (DADMe-ImmG) is a transition state analog inhibitor of this enzyme, and P. falciparum infections in an Aotus primate malaria model can be cleared by oral administration of DADMe-ImmG. P. falciparum cultured under increasing DADMe-ImmG drug pressure exhibited PfPNP gene amplification, increased protein expression, and point mutations involved in DADMe-ImmG binding. However, the weak catalytic properties of the M183L resistance mutation (∼17,000-fold decrease in catalytic efficiency) are inconsistent with the essential function of PfPNP. We hypothesized that M183L subunits may form mixed oligomers of native and mutant PfPNP monomers to give hybrid hexameric enzymes with properties conferring DADMe-ImmG resistance. To test this hypothesis, we designed PfPNP constructs that covalently linked native and the catalytically weak M183L mutant subunits. Engineered hybrid PfPNP yielded trimer-of-dimer hexameric protein with alternating native and catalytically weak M183L subunits. This hybrid PfPNP gave near-native K m values for substrate, but the affinity for DADMe-ImmG and catalytic efficiency were both reduced approximately ninefold relative to a similar construct of native subunits. Contact between the relatively inactive M183L and native subunits is responsible for altered properties of the hybrid protein. Thus, gene amplification of PfPNP provides adequate catalytic activity while resistance to DADMe-ImmG occurs in the hybrid oligomer to promote parasite survival. Coupled with the slow development of drug resistance, this resistance mechanism highlights the potential for DADMe-ImmG use in antimalarial combination therapies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the adenine and methionine salvage pathways, catalyzes the hydrolysis of methylthioadenosine (MTA), a compound suggested to affect pivotal cellular processes in part through the regulation of protein methylation. MTAP is expressed in a wide range of cell types and tissues, and its deletion is common to cancer cells and in liver injury. The aim of this study was to investigate the proteome and methyl proteome alterations triggered by MTAP deficiency in liver cells to define novel regulatory mechanisms that may explain the pathogenic processes of liver diseases. iTRAQ analysis resulted in the identification of 216 differential proteins (p < 0.05) that suggest deregulation of cellular pathways as those mediated by ERK or NFκB. R-methyl proteome analysis led to the identification of 74 differentially methylated proteins between SK-Hep1 and SK-Hep1+ cells, including 116 new methylation sites. Restoring normal MTA levels in SK-Hep1+ cells parallels the specific methylation of 56 proteins, including KRT8, TGF, and CTF8A, which provides a novel regulatory mechanism of their activity with potential implications in carcinogenesis. Inhibition of RNA-binding proteins methylation is especially relevant upon accumulation of MTA. As an example, methylation of quaking protein in Arg(242) and Arg(256) in SK-Hep1+ cells may play a pivotal role in the regulation of its activity as indicated by the up-regulation of its target protein p27(kip1) The phenotype associated with a MTAP deficiency was further verified in the liver of MTAP± mice. Our data support that MTAP deficiency leads to MTA accumulation and deregulation of central cellular pathways, increasing proliferation and decreasing the susceptibility to chemotherapeutic drugs, which involves differential protein methylation. Data are available via ProteomeXchange with identifier PXD002957 (http://www.ebi.ac.uk/pride/archive/projects/PXD002957)., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcomes relative to HPV-negative patients, but the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n = 27) and HPV-negative (n = 26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis, and immune response. The differential abundances of cortactin and methylthioadenosine phosphorylase were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p = 0.023 and p = 0.009, respectively). An additional 1,124 proteins were independently corroborated through comparison to a published proteomic dataset of OPC. Furthermore, utilizing the Cancer Genome Atlas, we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundances to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs. These proteins might contribute to reduced survival in these patients via their established role in radiation resistance. Through interrogation of Cancer Genome Atlas data, we demonstrated that activation of the β1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of activator protein 1 transcription factor target genes are plausible consequences of elevated cortactin protein levels., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K(i)* value of 0.6 nm. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nm. The K(m)/K(i)* value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K(m) for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5'-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.

Recent studies on the tissue distribution and developmental regulation of adenosine deaminase (ADA) activity in mice show that very high ADA levels exist in the murine alimentary tract (tongue, esophagus, forestomach, proximal small intestine) and at the fetal-maternal interface. To understand the role of ADA in these tissues, we measured the levels of three other enzymes involved in purine catabolism, purine nucleoside phosphorylase (PNP), guanine deaminase (GDA), and xanthine dehydrogenase (XDH), to see how their levels correlated with ADA activity. Our results show that the highest level of PNP, GDA, and XDH is present in the proximal small intestine. Levels of these purine catabolic enzymes are much lower in the tongue, esophagus, forestomach, and fetal-maternal interface in marked contrast to ADA distribution. We also determined mRNA levels encoding PNP, XDH, and ADA in a variety of tissues. Tissue-specific differences in PNP, XDH, and ADA activity correlated with RNA abundance, indicating that the regulation of gene expression is at the level of mRNA production. Thus, ADA is part of a purine catabolic pathway leading to the production of uric acid that is present at the highest known level in the proximal small intestine. ADA may have additional roles in other tissues.

We report a defect in splicing of precursor messenger RNA (pre-mRNA) resulting from a naturally occurring mutation of the gene encoding purine nucleoside phosphorylase (PNP) in a patient with PNP-deficient severe combined immunodeficiency. This defects results from a G to T transversion at the terminal nucleotide of exon 2 within the 5' splice site of intron 2 and causes skipping of exon 2 during processing of PNP pre-mRNA. Translation of the misspliced mRNA results in a reading frameshift at the exon 1-exon 3 junction. The predicted polypeptide encoded by the aberrant mRNA is severely truncated, terminating at 31 amino acids. Only 4 residues at the NH2 terminus of the polypeptide correspond to PNP amino acids. Otherwise the translation product of the misspliced mRNA differs completely from PNP in amino acid sequence and has no PNP activity. The finding of exon skipping in PNP is the first report of a splicing defect resulting in PNP-deficient severe combined immunodeficiency. Analysis of the genomic context of the G-1 to T mutation of the 5' splice site lends support for the exon definition model of pre-mRNA splicing and contributes to the understanding of splice site selection.

Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with a severe defect in T-lymphocyte function. The mutant gene was cloned from one PNP-deficient patient who was the offspring of a consanguineous mating. The exons and intron/exon boundaries of the mutant PNP gene were sequenced and compared with the wild-type cDNA sequence. A single base difference was found in the coding region of the mutant gene, a G to A transition in the third exon. This single base mutation alters the codon at position 89 from Glu to Lys, a result which is consistent with previously published peptide mapping data. The patient was demonstrated to be autozygous for the single base mutation on the basis of hybridization of synthetic oligomers to genomic DNA digests. A mammalian expression vector was constructed containing the entire mutant gene under the transcriptional regulation of its own promoter. In another construction, the single base mutation was reverted to the wild-type sequence by in vitro mutagenesis. An isoelectric focusing gel containing extracts of the cells transfected with the mutant and reverted PNP gene was stained histochemically for PNP activity. The proteins from a similar gel were blotted on a nitrocellulose membrane, and immunoreactive human PNP protein was visualized. Cells transfected with the mutant gene contained no human PNP activity, but expressed immunoreactive PNP which focused at an abnormally alkaline pI. Cells transfected with the reverted gene expressed human PNP activity which co-focused with human PNP from a HeLa cell control, proving that the observed single base change was responsible for the loss of catalytic function.

Purine-nucleoside phosphorylase deficiency in humans is associated with a severe defect in thymus-derived lymphocyte function. We have used the consanguineous parents of one purine-nucleoside phosphorylase-deficient patient as a source of mutant protein since the enzyme is totally lacking in the homozygous deficient child. When analyzed by denaturing two-dimensional gel electrophoresis, purified erythrocytic purine-nucleoside phosphorylase from normal individuals has four major and two minor subunits of identical molecular weight (29,700) but different isoelectric points. Similar analysis of phosphorylase purified from the parents of the purine-nucleoside phosphorylase-deficient child reveals the presence of two additional, more alkaline subunits with molecular weights significantly greater (approximately 500) than the normal subunits. Comparison of the two-dimensional thin layer peptide maps of the complete tryptic hydrolysates of the 125I-labeled subunits demonstrates that all eight subunits have a majority of their peptides in common, and thus, the structures of all are closely related. Peptide maps of the major subunits of normal purine-nucleoside phosphorylase are all identical with each other but different in several peptides from those of the normal minor subunits. Peptide maps of the mutant subunits differ from those of the normal major subunits only in the loss of one normal peptide and the presence of two new peptides. Tryptic peptide analysis of overlapping partial proteolytic cleavage fragments of the mutant and normal major subunits of purine-nucleoside phosphorylase has been used to determine the order of the iodinated tryptic peptides in the molecule. These results indicate that the mutant subunits are altered at an internal position rather than at the termini of the protein. A likely explanation of this internal alteration is an insertion of several tandem amino acid residues, perhaps resulting from an unequal genetic crossover or an intron processing abnormality.

The enzyme methylthioadenosine phosphorylase functions in both purine and polyamine metabolism is dividing mammalian cells. To determine the effects of the loss of this enzyme on cell growth and metabolism, we selected two methylthioadenosine phosphorylase-deficient mutant clones of the transplantable murine T lymphoma cell line R1.1. The first had 3.5% of wild type methylthioadenosine phosphorylase activity. The second was completely enzyme-deficient. The loss of the enzyme did not alter the growth rate, cloning efficiency, or tumor-forming ability of the T lymphoma cells. The methylthioadenosine phosphorylase-deficient clones excreted substantial amounts of methylthioadenosine into the culture medium (0.13 and 0.32 nmol/h/mg of protein, respectively) and were unable to utilize the methylthioadenosine phosphorylase substrate 2',5'-dideoxyadenosine as a purine source when de novo purine synthesis was blocked. Spermine levels were 10-20% lower in the enzyme-deficient clones than in wild type cells. The loss of methylthioadenosine phosphorylase rendered the mutants exquisitely sensitive to the antiproliferative effects of methylthioadenosine. Methylthioadenosine at 3-6 microM inhibited their growth by 50%. The toxic effects of methylthioadenosine were not attributable to inhibition of purine, pyrimidine, or polyamine synthesis.

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.