The structure of human 5'-deoxy-5'-methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis.

BACKGROUND: 5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MTA) to adenine and 5-methylthio-D-ribose-1-phosphate. MTA is a by-product of polyamine biosynthesis, which is essential for cell growth and proliferation. This salvage reaction is the principle source of free adenine in human cells. Because of its importance in coupling the purine salvage pathway to polyamine biosynthesis MTAP is a potential chemotherapeutic target. RESULTS: We have determined the crystal structure of MTAP at 1.7 A resolution using multiwavelength anomalous diffraction phasing techniques. MTAP is a trimer comprised of three identical subunits. Each subunit consists of a single alpha/beta domain containing a central eight-stranded mixed beta sheet, a smaller five-stranded mixed beta sheet and six alpha helices. The native structure revealed the presence of an adenine molecule in the purine-binding site. The structure of MTAP with methylthioadenosine and sulfate ion soaked into the active site was also determined using diffraction data to 1.7 A resolution. CONCLUSIONS: The overall quaternary structure and subunit topology of MTAP are similar to mammalian purine nucleoside phosphorylase (PNP). The structures of the MTAP-ligand complexes provide a map of the active site and suggest possible roles for specific residues in substrate binding and catalysis. Residues accounting for the differences in substrate specificity between MTAP and PNP are also identified. Detailed information about the structure and chemical nature of the MTAP active site will aid in the rational design of inhibitors of this potential chemotherapeutic target. The MTAP structure represents the first structure of a mammalian PNP that is specific for 6-aminopurines.     

Entropy-driven binding of picomolar transition state analogue inhibitors to human 5'-methylthioadenosine phosphorylase

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 altered 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 and 2.0 ? 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.     

Growth and metastases of human lung cancer are inhibited in mouse xenografts by a transition state analogue of 5'-methylthioadenosine phosphorylase

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.     

Picomolar transition state analogue inhibitors of human 5'-methylthioadenosine phosphorylase and X-ray structure with MT-immucillin-A

Methythioadenosine phosphorylase (MTAP) functions solely in the polyamine pathway of mammals to remove the methylthioadenosine (MTA) product from both spermidine synthase (2.5.1.16) and spermine synthase (2.5.1.22). Inhibition of polyamine synthesis is a validated anticancer target. We designed and synthesized chemically stable analogues for the proposed transition state of human MTAP on the basis of the known ribooxacarbenium character at all reported N-ribosyltransferase transition states [Schramm, V. L. (2003) Acc. Chem. Res. 36, 588-596]. Methylthio-immucillin-A (MT-ImmA) is an iminoribitol tight-binding transition state analogue inhibitor with an equilibrium dissociation constant of 1.0 nM. The immucillins resemble the ribooxacarbenium ion transition states of N-ribosyltransferases and are tightly bound as the N4' cations. An ion pair formed between the iminoribitol cation and phosphate anion mimics the ribooxacarbenium cation-phosphate anion pair formed at the transition state and is confirmed in the crystal structure. The X-ray crystal structure of human MTAP with bound MT-Imm-A also reveals that the 5'-methylthio group lies in a flexible hydrophobic pocket. Substitution of the 5'-methylthio group with a 5'-phenylthio group gives an equilibrium binding constant of 1.0 nM. Methylthio-DADMe-immucillin-A is a pyrrolidine analogue of the transition state with a methylene bridge between the 9-deazaadenine group and the pyrrolidine ribooxacarbenium mimic. It is a slow-onset inhibitor with a dissociation constant of 86 pM. Improved binding energy with DADMe-immucillin-A suggests that the transition state is more closely matched by increasing the distance between leaving group and ribooxacarbenium mimics, consistent with a more dissociative transition state. Increasing the hydrophobic volume near the 5'-position at the catalytic site with 5'-phenylthio-DADMe-immucillin-A gave a dissociation constant of 172 pM, slightly weaker than the 5'-methylthio group. p-Cl-phenylthio-DADMe-immucillin-A binds with a dissociation constant of 10 pM (K(m)/K(i) value of 500000), the tightest binding inhibitor reported for MTAP. These slow-onset, tight-binding transition state analogue inhibitors are the most powerful reported for MTAP and have sufficient affinity to be useful in inhibiting the polyamine pathway.     

Chemical genetic screening for compounds that preferentially inhibit growth of methylthioadenosine phosphorylase (MTAP)-deficient Saccharomyces cerevisiae

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP-deficient cells. To determine if MTAP inactivation could be targeted, the authors have performed a differential chemical genetic screen in isogenic MTAP(+) and MTAP(-) Saccharomyces cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP(-) yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP(-) cells exhibiting a 4-fold shift in IC(50) compared to MTAP(+) cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP(-) yeast. These compounds were also examined for growth inhibition of isogenic MTAP(+) and MTAP(-) HT1080 fibrosarcoma cells, and 4 of the 5 compounds exhibited evidence of modest but significant increased potency in MTAP(-) cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP(-) cells.     

A transition state analogue of 5'-methylthioadenosine phosphorylase induces apoptosis in head and neck cancers

Methylthio-DADMe-immucillin-A (MT-DADMe-ImmA) is an 86-pm inhibitor of human 5'-methylthioadenosine phosphorylase (MTAP). The sole function of MTAP is to recycle 5'-methylthioadenosine (MTA) to S-adenosylmethionine. Treatment of cultured cells with MT-DADMe-ImmA and MTA inhibited MTAP, increased cellular MTA concentrations, decreased polyamines, and induced apoptosis in FaDu and Cal27, two head and neck squamous cell carcinoma cell lines. The same treatment did not induce apoptosis in normal human fibroblast cell lines (CRL2522 and GM02037) or in MCF7, a breast cancer cell line with an MTAP gene deletion. MT-DADMe-ImmA alone did not induce apoptosis in any cell line, implicating MTA as the active agent. Treatment of sensitive cells caused loss of mitochondrial inner membrane potential, G(2)/M arrest, activation of mitochondria-dependent caspases, and apoptosis. Changes in cellular polyamines and MTA levels occurred in both responsive and nonresponsive cells, suggesting cell-specific epigenetic effects. A survey of aberrant DNA methylation in genomic DNA using a microarray of 12,288 CpG island clones revealed decreased CpG island methylation in treated FaDu cells compared with untreated cells. FaDu tumors in a mouse xenograft model were treated with MT-DADMe-ImmA, resulting in tumor remission. The selective action of MT-DADMe-ImmA on head and neck squamous cell carcinoma cells suggests potential as an agent for treatment of cancers sensitive to reduced CpG island methylation.     

Purification and characterization of 5′-methylthioadenosine phosphorylase from the hyperthermophilic archaeon<Emphasis Type="Italic"> Pyrococcus furiosus</Emphasis>

5'-Methylthioadenosine phosphorylase (MTAP) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The protein is a homoexamer of 180 kDa. The enzyme is highly thermoactive, with an optimum temperature of 125 degrees C, and extremely thermostable, retaining 98% residual activity after 5 h at 100 degrees C and showing a half-life of 43 min at 130 degrees C. In the presence of 100 mM phosphate, the apparent T(m) (137 degrees C) increases to 139 degrees C. The enzyme is extremely stable to proteolytic cleavage and after incubation with protein denaturants, detergents, organic solvents, and salts even at high temperature. Thiol groups are not involved in the catalytic process, whereas disulfide bond(s) are present, since incubation with 0.8 M dithiothreitol significantly reduces the thermostability of the enzyme. N-Terminal sequence analysis of the purified enzyme is 100% identical to the predicted amino acid sequence of the gene PF0016 from the partially sequenced P. furiosus genome. The deduced amino acid sequence of the gene revealed a high degree of identity (52%) with human MTAP. Nevertheless, unlike human MTAP, MTAP from P. furiosus is not specific for 5'-methylthioadenosine, since it phosphorolytically cleaves adenosine, inosine, and guanosine. The calculated k(cat)/ K(m) values for 5'-methylthioadenosine and adenosine, about 20-fold higher than for inosine and guanosine, indicate that 6-amino purine nucleosides are preferred substrates of MTAP from P. furiosus. The structural features and the substrate specificity of MTAP from P. furiosus document that it represents a 5'-methylthioadenosine-metabolizing enzyme different from those previously characterized among Archaea, Bacteria, and Eukarya. The functional and structural relationships among MTAP from P. furiosus, human MTAP, and two putative MTAPs from P. furiosus and Sulfolobus solfataricus are discussed here for the first time.     

Expression and Function of Methylthioadenosine Phosphorylase in Chronic Liver Disease

To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease.

Design

MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chromatography-tandem mass spectrometry.

Results

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.

Conclusion

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.     

<Emphasis Type="Italic">Leishmania infantum</Emphasis> 5’-Methylthioadenosine Phosphorylase presents relevant structural divergence to constitute a potential drug target

Background

The 5′-methylthioadenosine phosphorylase (MTAP), an enzyme involved in purine and polyamine metabolism and in the methionine salvage pathway, is considered as a potential drug target against cancer and trypanosomiasis. In fact, Trypanosoma and Leishmania parasites lack de novo purine pathways and rely on purine salvage pathways to meet their requirements. Herein, we propose the first comprehensive bioinformatic and structural characterization of the putative Leishmania infantum MTAP (LiMTAP), using a comparative computational approach.

Results

Sequence analysis showed that LiMTAP shared higher identity rates with the Trypanosoma brucei (TbMTAP) and the human (huMTAP) homologs as compared to the human purine nucleoside phosphorylase (huPNP). Motifs search using MEME identified more common patterns and higher relatedness of the parasite proteins to the huMTAP than to the huPNP. The 3D structures of LiMTAP and TbMTAP were predicted by homology modeling and compared to the crystal structure of the huMTAP. These models presented conserved secondary structures compared to the huMTAP, with a similar topology corresponding to the Rossmann fold. This confirmed that both LiMTAP and TbMTAP are members of the NP-I family. In comparison to the huMTAP, the 3D model of LiMTAP showed an additional α-helix, at the C terminal extremity. One peptide located in this specific region was used to generate a specific antibody to LiMTAP. In comparison with the active site (AS) of huMTAP, the parasite ASs presented significant differences in the shape and the electrostatic potentials (EPs). Molecular docking of 5′-methylthioadenosine (MTA) and 5′-hydroxyethylthio-adenosine (HETA) on the ASs on the three proteins predicted differential binding modes and interactions when comparing the parasite proteins to the human orthologue.

Conclusions

This study highlighted significant structural peculiarities, corresponding to functionally relevant sequence divergence in LiMTAP, making of it a potential drug target against Leishmania.

     

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer

5''-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.     

Methylthioadenosine phosphorylase gene expression is impaired in human liver cirrhosis and hepatocarcinoma

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine and adenine salvage pathways. In mammals, the liver plays a central role in methionine metabolism, and this essential function is lost in the progression from liver cirrhosis to hepatocarcinoma. Deficient MTAP gene expression has been recognized in many transformed cell lines and tissues. In the present work, we have studied the expression of MTAP in human and experimental liver cirrhosis and hepatocarcinoma. We observe that MTAP gene expression is significantly reduced in human hepatocarcinoma tissues and cell lines. Interestingly, MTAP gene expression was also impaired in the liver of CCl4-cirrhotic rats and cirrhotic patients. We provide evidence indicating that epigenetic mechanisms, involving DNA methylation and histone deacetylation, may play a role in the silencing of MTAP gene expression in hepatocarcinoma. Given the recently proposed tumor suppressor activity of MTAP, our observations can be relevant to the elucidation of the molecular mechanisms of multistep hepatocarcinogenesis.     

Phosphoinositide and redox dysregulation by the anticancer methylthioadenosine phosphorylase transition state inhibitor

Methylthio-DADMe-immucillin-A (MTDIA) is an 86 picomolar inhibitor of 5′-methylthioadenosine phosphorylase (MTAP) with potent and specific anti-cancer efficacy. MTAP salvages S-adenosylmethionine (SAM) from 5′-methylthioadenosine (MTA), a toxic metabolite produced during polyamine biosynthesis. Changes in MTAP expression are implicated in cancer growth and development, making MTAP an appealing target for anti-cancer therapeutics. Since SAM is involved in lipid metabolism, we hypothesised that MTDIA alters the lipidomes of MTDIA-treated cells. To identify these effects, we analysed the lipid profiles of MTDIA-treated Saccharomyces cerevisiae using ultra-high resolution accurate mass spectrometry (UHRAMS). MTAP inhibition by MTDIA, and knockout of the Meu1 gene that encodes for MTAP in yeast, caused global lipidomic changes and differential abundance of lipids involved in cell signaling. The phosphoinositide kinase/phosphatase signaling network was specifically impaired upon MTDIA treatment, and was independently validated and further characterised via altered localization of proteins integral to this network. Functional consequences of dysregulated lipid metabolism included a decrease in reactive oxygen species (ROS) levels induced by MTDIA that was contemporaneous with changes in immunological response factors (nitric oxide, tumour necrosis factor-alpha and interleukin-10) in mammalian cells. These results indicate that lipid homeostasis alterations and concomitant downstream effects may be associated with MTDIA mechanistic efficacy.     

Redox regulation of methylthioadenosine phosphorylase in liver cells: molecular mechanism and functional implications

MTAP (5'-methylthioadenosine phosphorylase) catalyses the reversible phosphorolytic cleavage of methylthioadenosine leading to the production of methylthioribose-1-phosphate and adenine. Deficient MTAP activity has been correlated with human diseases including cirrhosis and hepatocellular carcinoma. In the present study we have investigated the regulation of MTAP by ROS (reactive oxygen species). The results of the present study support the inactivation of MTAP in the liver of bacterial LPS (lipopolysaccharide)-challenged mice as well as in HepG2 cells after exposure to t-butyl hydroperoxide. Reversible inactivation of purified MTAP by hydrogen peroxide results from a reduction of V(max) and involves the specific oxidation of Cys(136) and Cys(223) thiols to sulfenic acid that may be further stabilized to sulfenyl amide intermediates. Additionally, we found that Cys(145) and Cys(211) were disulfide bonded upon hydrogen peroxide exposure. However, this modification is not relevant to the mediation of the loss of MTAP activity as assessed by site-directed mutagenesis. Regulation of MTAP by ROS might participate in the redox regulation of the methionine catabolic pathway in the liver. Reduced MTA (5'-deoxy-5'-methylthioadenosine)-degrading activity may compensate for the deficient production of the precursor S-adenosylmethionine, allowing maintenance of intracellular MTA levels that may be critical to ensure cellular adaptation to physiopathological conditions such as inflammation.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

Methylthioadenosine phosphorylase regulates ornithine decarboxylase by production of downstream metabolites

The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S. A., Diegelman, P., Porter, C. W., and Kruger, W. D. (2002) Cancer Res. 62, 6639-6644). Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo. In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity. We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1delta cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools. Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines. The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis. Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels. Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors.     

Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP‐deficient lung cancer

The aggressive nature and poor prognosis of lung cancer led us to explore the mechanisms driving disease progression. Utilizing our invasive cell‐based model, we identified methylthioadenosine phosphorylase (MTAP) and confirmed its suppressive effects on tumorigenesis and metastasis. Patients with low MTAP expression display worse overall and progression‐free survival. Mechanistically, accumulation of methylthioadenosine substrate in MTAP‐deficient cells reduce the level of protein arginine methyltransferase 5 (PRMT5)‐mediated symmetric dimethylarginine (sDMA) modification on proteins. We identify vimentin as a dimethyl‐protein whose dimethylation levels drop in response to MTAP deficiency. The sDMA modification on vimentin reduces its protein abundance but trivially affects its filamentous structure. In MTAP‐deficient cells, lower sDMA modification prevents ubiquitination‐mediated vimentin degradation, thereby stabilizing vimentin and contributing to cell invasion. MTAP and PRMT5 negatively correlate with vimentin in lung cancer samples. Taken together, we propose a mechanism for metastasis involving vimentin post‐translational regulation.     

Molecular cloning of the human methylthioadenosine phosphorylase processed pseudogene and localization to 3q28

Human methylthioadenosine phosphorylase (MTAP) is a purine and methionine metabolic enzyme present ubiquitously in all normal tissues, but often deleted in many types of cancer. The gene for this enzyme maps to chromosome 9 at band p21 where the cyclin-dependent kinase inhibitor genes for p16 and p15 also reside. During our efforts to clone this gene we also isolated a phage clone containing a processed pseudogene of MTAP. The sequence is 92% homologous to the MTAP cDNA, is flanked at its 3′ end by a repetitive element, but does not possess a poly(A) stretch. We localized this processed pseudogene to band 28 on the long arm of chromosome 3 by fluorescence in situ hybridization. All 22 malignant cell lines with deletions at 9p21 screened possessed the pseudogene.     

Structural comparison of MTA phosphorylase and MTA/AdoHcy nucleosidase explains substrate preferences and identifies regions exploitable for inhibitor design

The development of new and effective antiprotozoal drugs has been a difficult challenge because of the close similarity of the metabolic pathways between microbial and mammalian systems. 5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is thought to be an ideal target for therapeutic drug design as the enzyme is present in many microbes but not in mammals. MTA/AdoHcy nucleosidase (MTAN) irreversibly depurinates MTA or AdoHcy to form adenine and the corresponding thioribose. The inhibition of MTAN leads to a buildup of toxic byproducts that affect various microbial pathways such as quorum sensing, biological methylation, polyamine biosynthesis, and methionine recycling. The design of nucleosidase-specific inhibitors is complicated by its structural similarity to the human MTA phosphorylase (MTAP). The crystal structures of human MTAP complexed with formycin A and 5'-methylthiotubercidin have been solved to 2.0 and 2.1 A resolution, respectively. Comparisons of the MTAP and MTAN inhibitor complexes reveal size and electrostatic potential differences in the purine, ribose, and 5'-alkylthio binding sites, which account for the substrate specificity and reactions catalyzed. In addition, the differences between the two enzymes have allowed the identification of exploitable regions that can be targeted for the development of high-affinity nucleosidase-specific inhibitors. Sequence alignments of Escherichia coli MTAN, human MTAP, and plant MTA nucleosidases also reveal potential structural changes to the 5'-alkylthio binding site that account for the substrate preference of plant MTA nucleosidases.     

PRMT5 silencing selectively affects MTAP-deleted mesothelioma: In vitro evidence of a novel promising approach

Malignant mesothelioma (MM) is an aggressive asbestos-related cancer of the serous membranes. Despite intensive treatment regimens, MM is still a fatal disease, mainly due to the intrinsic resistance to current therapies and the lack of predictive markers and new valuable molecular targets. Protein arginine methyltransferase 5 (PRMT5) inhibition has recently emerged as a potential therapy against methylthioadenosine phosphorylase (MTAP)-deficient cancers, in which the accumulation of the substrate 5'-methylthioadenosine (MTA) inhibits PRMT5 activity, thus sensitizing the cells to further PRMT5 inhibition. Considering that the MTAP gene is frequently codeleted with the adjacent cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in MM, we assessed whether PRMT5 could represent a therapeutic target also for this cancer type. We evaluated PRMT5 expression, the MTAP status and MTA content in normal mesothelial and MM cell lines. We found that both administration of exogenous MTA and stable PRMT5 knock-down, by short hairpin RNAs (shRNAs), selectively reduced the growth of MTAP-deleted MM cells. We also observed that PRMT5 knock-down in MTAP-deficient MM cells reduced the expression of E2F1 target genes involved in cell cycle progression and of factors implicated in epithelial-to-mesenchymal transition. Therefore, PRMT5 targeting could represent a promising new therapeutic strategy against MTAP-deleted MMs.