The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used in the treatment of leukaemia. Incorporation of deoxythioguanosine nucleotides (dGs) into the DNA of thiopurine-treated cells causes cell death but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Since the growth of MTAP-deleted tumour cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukaemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP−ve) transfected to express MTAP constitutively (A549-MTAP+ve). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intra-cellularly to MeTIMP, was markedly higher in both cell lines under MTAP−ve conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.
Methylthioadenosine phosphorylase; purine synthesis; 6-mercaptopurine; 6-thioguanine; cancer; 9p-deletion
The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (MtaplacZ) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten+/−.
Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of MtaplacZ/+ and Mtap+/+ mice.
Survival in both models was significantly decreased in MtaplacZ/+ compared to Mtap+/+ mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap+/+ mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc MtaplacZ/+ and Eµ-myc Mtap+/+ animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap+/+ and MtaplacZ/+ animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer.
Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the catabolism of 5′-deoxy-5′-methylthioadenosine (MTA), catalyzes the formation of adenine and 5-methylthioribose-1-phosphate. MTAP is expressed in all cells throughout the body, but a significant percentage of human tumors have lost MTAP expression, thereby making MTAP-loss a potential therapeutic target. Here, we have tested an MTAP-targeting strategy based on the idea that MTAP-expressing cells can be protected from toxic purine and uracil analogs by addition of MTA, but MTAP-deleted tumor cells cannot. Addition of as little as 10 μM MTA could entirely protect isogenic MTAP+, but not MTAP-, HT1080 cells from toxicity caused by the chemotherapy agents 6-thioguanine (6TG) or 5-fluorouracil (5FU). Inhibitor studies showed that MTA protection requires functional MTAP activity. Addition of adenine protected both MTAP+ and MTAP- cells from 6TG and 5FU, consistent with the idea that adenine produced from the MTAP reaction competes with 6TG and 5FU for a rate limiting pool of phosphoribosyl-1-pyrophosphate (PRPP), which is required for the conversion of purine and uracil bases into nucleotides. Extracellular MTA can also protect mouse mesothelioma cells from killing by 6-TG or the drug L-alanosine in an MTAP-dependent manner. In addition, MTA can protect non-transformed MTAP+ mouse embryo fibroblasts from 6TG toxicity. Taken together, our data suggest that the addition of MTA to anti-purine-based chemotherapy may greatly increase the therapeutic index of this class of drugs if used specifically to treat MTAP- tumors.
chemotherapy; mesothelioma; methionine; osteosarcoma; purine
To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease.
MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chromatography-tandem mass spectrometry.
MTAP was downregulated in hepatocytes in murine fibrosis models and in patients with chronic liver disease, leading to a concomitant increase in MTA levels. In contrast, activated hepatic stellate cells (HSCs) showed strong MTAP expression in cirrhotic livers. However, also MTA levels in activated HSCs were significantly higher than in hepatocytes, and there was a significant correlation between MTA levels and collagen expression in diseased human liver tissue indicating that activated HSCs significantly contribute to elevated MTA in diseased livers. MTAP suppression by siRNA resulted in increased MTA levels, NFκB activation and apoptosis resistance, while overexpression of MTAP caused the opposite effects in HSCs. The anti-apoptotic effect of low MTAP expression and high MTA levels, respectively, was mediated by induced expression of survivin, while inhibition of survivin abolished the anti-apoptotic effect of MTA on HSCs. Treatment with a DNA demethylating agent induced MTAP and reduced survivin expression, while oxidative stress reduced MTAP levels but enhanced survivin expression in HSCs.
MTAP mediated regulation of MTA links polyamine metabolism with NFκB activation and apoptosis in HSCs. MTAP and MTAP modulating mechanisms appear as promising prognostic markers and therapeutic targets for hepatic fibrosis.
Many solid tumors and hematologic malignancies lack expression of the enzyme methylthioadenosine phosphorylase (MTAP), due either to deletion of the MTAP gene or to methylation of the MTAP promoter. In cells that have MTAP, its natural substrate, methylthioadenosine (MTA), generated during polyamine biosynthesis, is cleaved to adenine and 5-methylthioribose-1-phosphate. The latter compound is further metabolized to methionine. Adenine and methionine are further metabolized and hence salvaged. In MTAP-deficient cells, however, MTA is not cleaved and the salvage pathway for adenine and methionine is absent. As a result, MTAP-deficient cells are more sensitive than MTAP-positive cells to inhibitors of de novo purine synthesis and to methionine deprivation. The challenge has been to take advantage of MTAP deficiency, and the changes in metabolism that follow, to design a strategy for targeted treatment. In this review, the frequency of MTAP-deficiency is presented and past and recent strategies to target such deficient cells are discussed, including one in which MTA is administered, followed by very high doses of a toxic purine or pyrimidine analog. In normal host cells, adenine, generated from MTA, blocks conversion of the analog to its toxic nucleotide. In MTAP-deficient tumor cells, conversion proceeds and the tumor cells are selectively killed. Successful mouse studies using this novel strategy were recently reported.
MTAP; MTA; adenine; 6-mercaptopurine; methotrexate
Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (MtaplacZ) have an embryonic lethal phenotype dying around day 8 post-conception. Mtap/MtaplacZ heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and FACS analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein, and unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF.
Cancer; Tumor Suppressor Gene; Methionine; Embryonic Lethal
The gene for methylthioadenosine phosphorylase (MTAP) lies on 9p21, close to the gene CDKN2A that encodes the tumor suppressor proteins p16 and p14ARF. MTAP and CDKN2A are homozygously co-deleted, with a frequency of 35 to 70%, in lung and pancreatic cancer, glioblastoma, osteosarcoma, soft-tissue sarcoma, mesothelioma, and T-cell acute lymphoblastic leukemia. In normal cells, but not in tumor cells lacking MTAP, MTAP cleaves the natural substrate, 5′-deoxy-5′-methylthioadenosine (MTA), to adenine and 5-methylthioribose-1-phosphate (MTR-1-P), which are then converted to adenine nucleotides and methionine. This distinct difference between normal MTAP-positive cells and tumor MTAP-negative cells led to several proposals for therapy. We offer a novel strategy in which both MTA and a toxic adenine analog, such as 2,6-diaminopurine (DAP), 6-methylpurine (MeP), or 2-fluoroadenine (F-Ade), are administered. In MTAP-positive cells, abundant adenine, generated from supplied MTA, competitively blocks the conversion of an analog, by adenine phosphoribosyltransferase (APRT), to its active nucleotide form. In MTAP-negative tumor cells, the supplied MTA cannot generate adenine; hence conversion of the analog is not blocked.
We show that this combination treatment – adenine analog plus MTA – kills MTAP-negative A549 lung tumor cells, while MTAP-positive human fibroblasts (HF) are protected. In co-cultures of the breast tumor cell line, MCF-7, and HF cells, MCF-7 is inhibited or killed, while HF cells proliferate robustly. 5-fluorouracil (5-FU) and 6-thioguanine (6-TG) may also be used with our strategy. Though neither analog is activated by APRT, in MTAP-positive cells, adenine produced from supplied MTA blocks conversion of 5-FU and 6-TG to their toxic nucleotide forms by competing for 5-phosphoribosyl-1-pyrophosphate (PRPP). The combination of MTA with 5-FU or 6-TG, in the treatment of MTAP-negative tumors, may produce a significantly improved therapeutic index.
We describe a selective strategy to kill tumor cells lacking MTAP.
Human 5′-methylthioadenosine phosphorylase (MTAP) links the polyamine biosynthetic and S-adenosyl-L-methionine salvage pathways and is a target for anticancer drugs. p-Cl-PhT-DADMe-ImmA is a 10 pM, slow-onset tight-binding transition state analogue inhibitor of the enzyme. Titration of homotrimeric MTAP with this inhibitor established equivalent binding and independent catalytic function of the three catalytic sites. Thermodynamic analysis of MTAP with tight-binding inhibitors revealed entropic-driven interactions with small enthalpic penalties. A large negative heat capacity change of −600 cal/mol•K upon inhibitor binding to MTAP is consistent with the loss of hydrophobic interactions and release of water. Crystal structures of apo MTAP and MTAP in complex with p-Cl-PhT-DADMe-ImmA were determined at 1.9 A and 2.0 A resolution, respectively. Inhibitor binding caused condensation of the enzyme active site, reorganization at the trimer interfaces, the release of water from the active sites and subunit interfaces, and compaction of the trimeric structure. These structural changes cause the entropy-favored binding of transition state analogues. Homotrimeric human MTAP is contrasted to the structurally related homotrimeric human purine nucleoside phosphorylase. p-Cl-PhT-DADMe- ImmA binding to MTAP involves a favorable entropy term of −17.6 kcal/mol with unfavorable enthalpy of 2.6 kcal/mol. In contrast, binding of an 8.5 pM transition state analogue to human PNP has been shown to exhibit the opposite behavior, with an unfavorable entropy term of 3.5 kcal/mol and a favorable enthalpy of −18.6 kcal/mol. Transition state analogue interactions reflect protein architecture near the transition state and the profound thermodynamic differences for MTAP and PNP suggest dramatic differences in contributions to catalysis from protein architecture.
Castrate resistant prostate cancer (CRPC) and neuroendocrine carcinoma of the prostate are invariably fatal diseases for which only palliative therapies exist. As part of a prostate tumour sequencing program, a patient tumour was analyzed using Illumina genome sequencing and a matched renal capsule tumour xenograft was generated. Both tumour and xenograft had a homozygous 9p21 deletion spanning the MTAP, CDKN2 and ARF genes. It is rare for this deletion to occur in primary prostate tumours yet approximately 10% express decreased levels of MTAP mRNA. Decreased MTAP expression is a prognosticator for poor outcome. Moreover, it appears that this deletion is more common in CRPC than in primary prostate cancer. We show for the first time that treatment with methylthioadenosine and high dose 6-thioguanine causes marked inhibition of a patient derived neuroendocrine xenograft growth while protecting the host from 6-thioguanine toxicity. This therapeutic approach can be applied to other MTAP-deficient human cancers since deletion or hypermethylation of the MTAP gene occurs in a broad spectrum of tumours at high frequency. The combination of genome sequencing and patient-derived xenografts can identify candidate therapeutic agents and evaluate them for personalized oncology.
massively parallel sequencing; MTAP; patient-derived xenograft; genitourinary cancers: prostate; animal models of cancer; gene expression profiling; functional genomics; xenograft models
Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathological and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (SNP 6.0, Affymetrix) with gene expression array (U133A 2.0, Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N=114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P<0.05, Spearman’s rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene methylthioadenosine phosphorylase (MTAP) in SSM resulted in reduced cell growth. The differential expression of another metabolic related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM.
melanoma; nodular; genomics; SNP array; DNA copy number
AIM: To evaluate the methylation status of CDH1, FHIT, MTAP and PLAGL1 promoters and the association of these findings with clinico-pathological characteristics.
METHODS: Methylation-specific PCR (MSP) assay was performed in 13 nonneoplastic gastric adenocarcinoma, 30 intestinal-type gastric adenocarcinoma and 35 diffuse-type gastric adenocarcinoma samples from individuals in Northern Brazil. Statistical analyses were performed using the chi-square or Fisher's exact test to assess associations between methylation status and clinico-pathological characteristics.
RESULTS: Hypermethylation frequencies of CDH1, FHIT, MTAP and PLAGL1 promoter were 98.7%, 53.9%, 23.1% and 29.5%, respectively. Hypermethylation of three or four genes revealed a significant association with diffuse-type gastric cancer compared with nonneoplastic cancer. A higher hypermethylation frequency was significantly associated with H pylori infection in gastric cancers, especially with diffuse-type. Cancer samples without lymph node metastasis showed a higher FHIT hypermethylation frequency. MTAP hypermethylation was associated with H pylori in gastric cancer samples, as well as with diffuse-type compared with intestinal-type. In diffuse-type, MTAP hypermethylation was associated with female gender.
CONCLUSION: Our findings show differential gene methylation in tumoral tissue, which allows us to conclude that hypermethylation is associated with gastric carcinogenesis. MTAP promoter hypermethylation can be characterized as a marker of diffuse-type gastric cancer, especially in women and may help in diagnosis, prognosis and therapies. The H pylori infectious agent was present in 44.9% of the samples. This infection may be correlated with the carcinogenic process through the gene promoter hypermethylation, especially the MTAP promoter in diffuse-type. A higher H pylori infection in diffuse-type may be due to greater genetic predisposition.
Gastric adenocarcinoma; DNA hypermethylation; CDH1; FHIT; MTAP; PLAGL1
Methionine is an essential proteogenic amino acid. In addition, it is a methyl donor for DNA and protein methylation and a propylamine donor for polyamine biosyn-thesis. Both the methyl and propylamine donation pathways involve metabolic cycles, and methods are needed to quantitate these cycles. Here, we describe an analytical approach for quantifying methionine metabolic fluxes that accounts for the mixing of intracellular and extracellular methionine pools. We observe that such mixing prevents isotope tracing experiments from reaching the steady state due to the large size of the media pools and hence precludes the use of standard stationary metabolic flux analysis. Our approach is based on feeding cells with 13C methionine and measuring the isotope-labeling kinetics of both intracellular and extracellular methionine by liquid chromatography−mass spectrometry (LC-MS). We apply this method to quantify methionine metabolism in a human fibrosarcoma cell line and study how methionine salvage pathway enzyme methylthioadenosine phosphorylase (MTAP), frequently deleted in cancer, affects methionine metabolism. We find that both transmethylation and propylamine transfer fluxes amount to roughly 15% of the net methionine uptake, with no major changes due to MTAP deletion. Our method further enables the quantification of flux through the pro-tumorigenic enzyme ornithine decarboxylase, and this flux increases 2-fold following MTAP deletion. The analytical approach used to quantify methionine metabolic fluxes is applicable for other metabolic systems affected by mixing of intracellular and extracellular metabolite pools.
Human 5′-methylthioadenosine phosphorylase (MTAP) is solely responsible for 5′-methylthioadenosine (MTA) metabolism to permit S-adenosylmethionine salvage. Transition state (TS) analogues of MTAP are in development as anticancer candidates. TS analogues of MTAP incorporate a cationic nitrogen and a protonated 9-deazaadenine leaving group, mimics of the ribocation transition state. MT-ImmA and MT-DADMe-ImmA are two examples of these TS analogues. Thermodynamic analysis of MTA, inhibitor and phosphate binding reveals the cationic nitrogen to provide −2.6 and −3.6 kcal/mol binding free energy for MT-ImmA and MT-DADMe-ImmA, respectively. The protonated deazaadenine provides an additional −1.3 (MT-ImmA ) to −1.7 kcal/mol (MT-DADMe-ImmA). MT-DADMe-ImmA is a better match in TS geometry than MT-ImmA and is thermodynamically favored. Binding of TS analogues to the MTAP:phosphate complex is fully entropic, in contrast to TS analogue binding to the related human purine nucleoside phosphorylase:phosphate complex, which is fully enthalpic. The binding thermodynamics of phosphate or TS analogues alone to MTAP are fully dominated by enthalpy. Phosphate anchored in the catalytic site forms an ion pair with the cationic TS analogue to cause stabilization of the enzyme structure in the ternary complex. The ternary-induced conformational changes convert the individual enthalpic binding energies to entropy, a presumed shift of the protein architecture toward the transition state. Formation of the ternary TS analogue complex with MTAP induces a remarkable increase in thermal stability (ΔTm 35 °C). The enthalpic, entropic and protein stability features of TS analogue binding to human MTAP are resolved in these studies.
transition state analogues; MTAP; binding energy; thermodynamics; cooperativity; protein stabilization; entropic binding
The characterization of modifier genes can provide insights into disease pathways and identify novel therapeutic targets. This study identifies Mtap1a as a modifier gene of photoreceptor loss in Tulp1 and Tub mutant mice, which are models of retinal degeneration.
To identify genes that modify photoreceptor cell loss in the retinas of homozygous Tulp1tm1Pjn and Tubtub mice, which exhibit juvenile retinitis pigmentosa.
Modifier loci were identified by genetic quantitative trait locus analysis. F2 Tulp1tm1Pjn/tm1Pjn mutant mice from a B6-Tulp1tm1Pjn/tm1Pjn × AKR/J intercross were genotyped with a panel of single nucleotide polymorphism markers and phenotyped by histology for photoreceptor nuclei remaining at 9 weeks of age. Genotype and phenotype data were correlated and examined with Pseudomarker 2.02 using 128 imputations to map modifier loci. Thresholds for the 63%, 10%, 5%, and 1% significance levels were obtained from 100 permutations. A significant, protective candidate modifier was identified by bioinformatic analysis and confirmed by crossing transgenic mice bearing a protective allele of this gene with Tulp1- and Tub-deficient mice.
A significant, protective modifier locus on chromosome 2 and a suggestive locus on chromosome 13 that increases photoreceptor loss were identified in a B6-Tulp1tm1Pjn/tm1Pjn × AKR/J intercross. The chromosome 2 locus mapped near Mtap1a, which encodes a protein associated with microtubule-based intracellular transport and synapse function. The protective Mtap1a129P2/OlaHsd allele was shown to reduce photoreceptor loss in both Tulp1tm1Pjn/tm1Pjn and Tubtub/tub mice.
It was demonstrated that the gene Mtap1a, which modifies hearing loss in Tubtub/tub mice, also modifies retinal degeneration in Tubtub/tub and Tulp1tm1Pjn/tm1Pjn mice. These results suggest that functionally nonredundant members of the TULP family (TUB and TULP1) share a common functional interaction with MTAP1A.
In this paper, we present the biochemical and biological evaluation of N-arylmethyl-substituted iminoribitol derivatives as potential chemotherapeutic agents against trypanosomiasis. Previously, a library of 52 compounds was designed and synthesized as potent and selective inhibitors of Trypanosoma vivax inosine-adenosine-guanosine nucleoside hydrolase (IAG-NH). However, when the compounds were tested against bloodstream-form Trypanosoma brucei brucei, only one inhibitor, N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (UAMC-00363), displayed significant activity (mean 50% inhibitory concentration [IC50] ± standard error, 0.49 ± 0.31 μM). Validation in an in vivo model of African trypanosomiasis showed promising results for this compound. Several experiments were performed to investigate why only UAMC-00363 showed antiparasitic activity. First, the compound library was screened against T. b. brucei IAG-NH and inosine-guanosine nucleoside hydrolase (IG-NH) to confirm the previously demonstrated inhibitory effects of the compounds on T. vivax IAG-NH. Second, to verify the uptake of these compounds by T. b. brucei, their affinities for the nucleoside P1 and nucleoside/nucleobase P2 transporters of T. b. brucei were tested. Only UAMC-00363 displayed significant affinity for the P2 transporter. It was also shown that UAMC-00363 is concentrated in the cell via at least one additional transporter, since P2 knockout mutants of T. b. brucei displayed no resistance to the compound. Consequently, no cross-resistance to the diamidine or the melaminophenyl arsenical classes of trypanocides is expected. Third, three enzymes of the purine salvage pathway of procyclic T. b. brucei (IAG-NH, IG-NH, and methylthioadenosine phosphorylase [MTAP]) were investigated using RNA interference. The findings from all these studies showed that it is probably not sufficient to target only the nucleoside hydrolase activity to block the purine salvage pathway of T. b. brucei and that, therefore, it is possible that UAMC-00363 acts on an additional target.
Males homozygous for the mouse male sterility and histoincompatibility (mshi) mutation exhibit small testes and produce no sperm. In addition, mshi generates an “antigen-loss” histoincompatibility barrier, such that homozygous mutants reject skin grafts from wild-type co-isogenic BALB/cByJ donors. To facilitate the molecular characterization of the pleiotropic mshi mutation, we genetically mapped mshi into a 0.68 megabasepair region which contains fewer than 10 candidate genes. Complementation testing showed that one of these, Mtap7, is disrupted in mshi mice. Sequence analysis has revealed a 13 kilobasepair deletion in BALB/cByJ-mshi/J mice that begins in Intron 10–11 of Mtap7, and ends less than 2,000 base pairs downstream of the wild type gene. Analysis of the mutant cDNA predicts that Mtap7mshi encodes a 457 amino acid protein, the first 423 of which are identical to wild type, and the last 34 of which are due to aberrant mRNA splicing with two cryptic exons in the Mtap7 to P04Rik intergenic region. This molecular assignment for the mshi mutation further supports an essential role for microtubule stabilization in spermatogenesis and indicates a new role in allograft transplantation.
Spermatogenesis; E-MAP-115; Positional cloning; Complementation testing
Transition state structures can be derived from kinetic isotope effects and computational chemistry. Molecular electrostatic potential maps of transition states serve as blueprints to guide synthesis of transition state analogue inhibitors of target enzymes. 5’-Methylthioadenosine phosphorylase (MTAP) functions in the polyamine pathway by recycling methylthioadenosine (MTA) and maintaining cellular S-adenosylmethionine (SAM). Its transition state structure was used to guide synthesis of MT-DADMe-ImmA, a picomolar inhibitor that shows anticancer effects against solid tumors. Biochemical and genomic analysis suggests that MTAP inhibition acts by altered DNA methylation and gene expression patterns. A related bacterial enzyme, 5’-methylthioadcnosine nucleosidase (MTAN), functions in pathways of quorum sensing involving AI-1 and AI-2 molecules. Transition states have been solved for several bacterial MTANs and used to guide synthesis of powerful inhibitors with dissociation constants in the femtomolar to picomolar range. BuT-DADMe-ImmA blocks quorum sensing in Vibrio cholerae without changing bacterial growth rates. Transition state analogue inhibitors show promise as anticancer and antibacterial agents.
5′-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 18.104.22.168) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in M. tuberculosis, was expressed in and purified from E. coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-L-homocysteine were poor substrates and no activity was detected with 5′-methylthioinosine, the other natural purines or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the Km value for MTA was 9.1 μM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicates that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.
5′-methylthioadenosine phosphorylase; Rv0535; purine metabolism; Mycobacterium tuberculosis
The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 22.214.171.124), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 126.96.36.199), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 188.8.131.52), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 184.108.40.206), aci-reductone dioxygenase (mtnD, EC 220.127.116.11) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.
The chromosome 9p21.3 region has been implicated in the pathogenesis of multiple cancers.
We systematically examined up to 203 tagging SNPs of 22 genes on 9p21.3 (19.9–32.8 Mb) in eight case–control studies: thyroid cancer, endometrial cancer (EC), renal cell carcinoma, colorectal cancer (CRC), colorectal adenoma (CA), oesophageal squamous cell carcinoma (ESCC), gastric cardia adenocarcinoma and osteosarcoma (OS). We used logistic regression to perform single SNP analyses for each study separately, adjusting for study-specific covariates. We combined SNP results across studies by fixed-effect meta-analyses and a newly developed subset-based statistical approach (ASSET). Gene-based P-values were obtained by the minP method using the Adaptive Rank Truncated Product program. We adjusted for multiple comparisons by Bonferroni correction.
Rs3731239 in cyclin-dependent kinase inhibitors 2A (CDKN2A) was significantly associated with ESCC (P=7 × 10−6). The CDKN2A-ESCC association was further supported by gene-based analyses (Pgene=0.0001). In the meta-analyses by ASSET, four SNPs (rs3731239 in CDKN2A, rs615552 and rs573687 in CDKN2B and rs564398 in CDKN2BAS) showed significant associations with ESCC and EC (P<2.46 × 10−4). One SNP in MTAP (methylthioadenosine phosphorylase) (rs7023329) that was previously associated with melanoma and nevi in multiple genome-wide association studies was associated with CRC, CA and OS by ASSET (P=0.007).
Our data indicate that genetic variants in CDKN2A, and possibly nearby genes, may be associated with ESCC and several other tumours, further highlighting the importance of 9p21.3 genetic variants in carcinogenesis.
common genetic variants; CDKN2A; 9p21.3
5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial enzyme involved in S-adenosylmethionine (SAM)-related quorum sensing pathways that regulates virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial communities to communicate. In humans, 5′-methylthioadenosine phosphorylase (MTAP) is involved in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-nucleoside mimics with a sulfur atom at the 5′ position that are potent E. coli MTAN and human MTAP inhibitors. Because of the possibility that the sulfur may affect bioavailability we were interested in synthesizing “sulfur-free” analogues. Herein we describe the preparation of a series of “sulfur-free” transition state analogues inhibitors, of E. coli MTAN and human MTAP that have low nano- to pico-molar dissociation constants and are potentially novel bacterial anti-infective and anti-cancer drug candidates.
Kinetic isotope effects (KIEs) and computer modeling using density functional theory were used to approximate the transition state of human 5′-methylthioadenosine phosphorylase (MTAP). KIEs were measured on the arsenolysis of 5′-methylthioadenosine (MTA) catalyzed by MTAP and were corrected for the forward commitment to catalysis. Intrinsic KIEs were obtained for [1′-3H], [1′-14C], [2′-3H], [4′-3H], [5′-3H], [9-15N] and [Me-3H3] MTAs. The primary intrinsic KIEs (1′-14C and 9-15N) suggest that MTAP has a dissociative SN1 transition state with cationic center at the anomeric carbon and insignificant bond order to the leaving group. The 9-15N intrinsic KIE of 1.039 also establishes an anionic character to the adenine leaving group, whereas the α-primary 1′-14C KIE of 1.031 indicates significant nucleophilic participation at the transition state. Computational matching of the calculated EIEs to the intrinsic isotope effects places the oxygen nucleophile 2.0 Å from the anomeric carbon. The 4′-3H KIE is sensitive to the polarization of the 3′-OH group. Calculations suggest that a 4′-3H KIE of 1.047 is consistent with ionization of the 3′-OH group, indicating formation of a zwitterion at the transition state. The transition state has cationic character at the anomeric carbon and is anionic at the 3′-OH oxygen, with an anionic leaving group. The isotope effects predicted a 3′-endo conformation for the ribosyl zwitterion corresponding to a H1′-C1′-C2′-H2′ torsional angle of 33°. The [Me-3H3] and [5′-3H] KIEs arise predominantly from the negative hyperconjugation of the lone pairs of sulphur with the σ* (C-H) antibonding orbitals. Human MTAP is characterized by a late, SN1 transition state with significant participation of the phosphate nucleophile.
5′-methylthioadenosine; kinetic isotope effects; 5′-methylthioadenosine phosphorylase; polyamines; MTAP; transition state; zwitterionic transition state; transition state structure
Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate ↔ adenine + alpha-d-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent Km (Km app) of 98 ± 6 μM (with Ado) and a native molecular mass of 125 ± 7 kDa. The second Ado cleavage activity was identified as 5′-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.
We compared the proteins which associate with middle T antigen (MT) of polyomavirus in human cells infected with Ad5(pymT), a recombinant adenovirus which directs the overexpression of MT, with the MT-associated proteins (MTAPs) previously identified in murine fibroblasts expressing MT. MTAPs of 27, 29, 36, and 63 kilodaltons (kDa) appeared to be fairly well conserved between the two species, as judged by comigration on two-dimensional gels. Several 61-kDa MTAP species detected in MT immunoprecipitates from both cell sources also comigrated on these gels. However, no protein comigrating precisely with the murine 85-kDa MTAP could be detected in the human cells. Furthermore, two proteins of 72 and 74 kDa associated with wild-type MT in the infected human cells but not in murine fibroblasts expressing MT. It had been previously reported for murine cells that the 70-kDa heat shock protein associates with a particular mutant MT but not with wild-type MT (G. Walter, A. Carbone, and W.J. Welch, J. Virol. 61:405-410, 1987). By the criteria of comigration on two-dimensional gels, tryptic peptide mapping, and immunoblotting, we showed that the 72- and 74-kDa proteins that associate with wild-type MT in human cells are the major human 70-kDa heat shock proteins.
The first line of defense against viral infection is the interferon (IFN) response, which culminates in the expression of hundreds of proteins with presumed antiviral activity, and must be overcome by a virus for successful replication. The nonstructural NSs protein is the primary IFN antagonist encoded by Bunyamwera virus (BUNV), the prototype of the Orthobunyavirus genus and the family Bunyaviridae. The NSs protein interferes with RNA polymerase II-mediated transcription, thereby inhibiting cellular mRNA production, including IFN mRNAs. A recombinant virus, rBUNdelNSs, that is unable to express the NSs protein does not inhibit cellular transcription and is a strong IFN inducer. We report here that cells stimulated into the antiviral state by IFN-β treatment were protected against wild-type BUNV and rBUNdelNSs infection but addition of IFN-β after infection had little effect on the replication cycle of either virus. By screening a panel of cell lines that overexpressed individual IFN-stimulated genes, we found that protein kinase R (PKR), MTAP44, and particularly viperin appreciably restricted BUNV replication. The enzymatic activities of PKR and viperin were required for their inhibitory activities. Taken together, our data show that the restriction of BUNV replication mediated by IFN is an accumulated effect of at least three IFN-stimulated genes that probably act on different stages of the viral replication cycle.