The ribosome accelerates the rate of peptide bond formation by at least 10(7)-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.
Epigenetic modification of DNA leads to changes in gene expression. DNA methyltransferases (DNMTs) comprise a family of nuclear enzymes that catalyze the methylation of CpG dinucleotides, resulting in an epigenetic methylome distinguished between normal cells and those in disease states such as cancer. Disrupting gene expression patterns through promoter methylation has been implicated in many malignancies and supports DNMTs as attractive therapeutic targets. This review focuses on the rationale of targeting DNMTs in cancer, the historical approach to DNMT inhibition, and current marketed hypomethylating therapeutics azacytidine and decitabine. In addition, we address novel DNMT inhibitory agents emerging in development, including CP-4200 and SGI-110, analogs of azacytidine and decitabine, respectively; the oligonucleotides MG98 and miR29a; and a number of reversible inhibitors, some of which appear to be selective against particular DNMT isoforms. Finally, we discuss future opportunities and challenges for next-generation therapeutics.
Inactivation of the M2 form of pyruvate kinase (PKM2) in cancer cells is associated with increased tumorigenicity. To test the hypothesis that tumor growth may be inhibited through the PKM2 pathway, we generated a series of small-molecule PKM2 activators. The compounds exhibited low nanomolar activity in both biochemical and cell-based PKM2 activity assays. These compounds did not affect the growth of cancer cell lines under normal conditions in vitro, but strongly inhibited the proliferation of multiple lung cancer cell lines when serine was absent from the cell culture media. In addition, PKM2 activators inhibited the growth of an aggressive lung adenocarcinoma xenograft. These findings show that PKM2 activation by small molecules influences the growth of cancer cells in vitro and in vivo, and suggest that such compounds may augment cancer therapies.
Among the clinically used nucleoside analogue inhibitors that target human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), there is little detailed mechanistic information on the interactions of 2,3-didehydro-2,3-dideoxythymidine-5-triphosphate (d4TTP) with the enzyme ⅐ primer-template complex and how these interactions compare with those of the natural substrate, dTTP. Using a pre-steady-state kinetic analysis, we found that d4TTP was incorporated by HIV-1 RT just as efficiently as dTTP during both DNA-and RNA-dependent DNA synthesis. To our knowledge, these results represent the first observation of a 3-modified nucleoside triphosphate analogue that has an incorporation efficiency comparable to that observed for the natural substrate during DNA synthesis by HIV-1 RT. This information provides a mechanistic basis for understanding the inhibition of HIV-1 RT by d4TTP as well as insight into the clinically observed lack of d4T resistance mutations in HIV-1 RT isolated from AIDS patients.The replication of human immunodeficiency virus (HIV), which causes AIDS, requires the virally encoded enzyme reverse transcriptase (RT). RT converts the single-stranded HIV RNA genome to a double-stranded DNA copy by catalyzing both DNA-dependent and RNA-dependent DNA polymerization as well as RNase H cleavage activity to remove the RNA template once the DNA has been synthesized. Because of its unique catalytic properties, RT has been the target enzyme for many antiviral therapeutic agents used in the treatment of AIDS, including nucleoside and nonnucleoside analogues (2-4, 8, 26). The nucleoside analogues that are used clinically lack a 3Ј hydroxyl group and are metabolically activated by host cellular kinases to their corresponding 5Ј-triphosphate forms, which are subsequently incorporated into DNA by HIV type 1 (HIV-1) RT and which act as chain terminators of DNA synthesis. Among the nucleoside inhibitors currently used in the clinic, two compounds are deoxythymidine (compound 1) analogues: 3Ј-azido-3Ј-deoxythymidine (AZT; compound 2) and 2Ј,3Ј-didehydro-2Ј,3Ј-dideoxythymidine (d4T; compound 3) (Fig. 1). The structures of d4T and abacavir (36) are unique among the U.S. Food and Drug Administration (FDA)-approved nucleoside analogues currently used, in that they contain a 2Ј,3Ј-unsaturated bond. An X-ray crystallographic analysis of d4T has shown that the unusual unsaturation in the ribose ring provides a novel ring conformation (11). However, the structure of d4T triphosphate (d4TTP) bound to the active site of HIV-1 RT in the presence of a primer-template substrate is not available.While AZT was the first compound approved by the FDA in 1987 for the treatment of AIDS, d4T was also shown to have antiretroviral activity (24) and was approved more recently in 1994. From a therapeutic standpoint, d4T is less toxic, particularly to bone marrow cells, than AZT (35) and has a more predictable pharmacokinetic profile in forming the biologically active triphosphate (27). Because of its high degree of oral bioavailabi...
The biogenesis of the large (60S) ribosomal subunit in eukaryotes involves nucleolar, nucleoplasmic, and cytoplasmic steps. The cytoplasmic protein Rei1, found in all eukaryotes, was previously shown to be necessary for the nuclear reimport of 60S subunit export factor Arx1. In this study we investigate the function of Reh1, a protein with high sequence similarity to Rei1. We demonstrate an overlapping function for Reh1 and Rei1 in the cytoplasmic maturation of the 60S subunit that is independent of Arx1 recycling. We observe that strains lacking both Reh1 and Rei1 accumulate salt-labile 60S subunits, suggesting that Reh1/Rei1 is necessary for the cytoplasmic 60S subunit to adopt its mature, stable form.Eukaryotic ribosomes are the products of a highly conserved assembly process involving more than 170 trans-acting biogenesis factors, around 75 ribosomal proteins (r-proteins), and four rRNAs (8,10,42). The small (40S) and large (60S) ribosomal subunits are assembled independently, first in the nucleolus and then the nucleoplasm, followed by export to the cytoplasm. Evidence suggests that the newly exported cytoplasmic pre-60S particle requires a slow maturation step before entering the pool of translating ribosomes (33,40,44). The exact nature of cytoplasmic 60S maturation is not well understood but is thought to involve the following steps: (i) the loading of a number of r-proteins, including Rpp0, Rpl7, Rpl10, and Rpl24; (ii) the dissociation and reimport of a small number of nonribosomal factors to the nucleus; and (iii) stabilizing structural rearrangements (43).The cytoplasmic protein Rei1 is necessary for the nuclear recycling of Arx1 (19, 27), a 60S subunit export factor (5, 20). Rei1 is conserved in all eukaryotes but absent from archaea and bacteria, suggesting an important eukaryote-specific cellular function (11). In Saccharomyces cerevisiae and a limited number of fungal organisms, an Rei1-related factor named Reh1 is also present. Reh1, like Rei1, is a cytoplasmic protein (18) with three U1-type C 2 H 2 zinc fingers (InterPro number IPR003604), and the two proteins share 34% sequence identity and 54% sequence similarity. Previous studies indicated a degree of functional redundancy between Reh1 and Rei1, as a double deletion of REH1 and REI1 results in a synthetic growth defect (22), and overexpression of REH1 can partially suppress the rei1⌬ cold-sensitive growth phenotype (22,27). However, the overlapping functions of Reh1 and Rei1 were suggested to be unrelated to 60S subunit ribosome biogenesis based on several observations: (i) polysome profiles and rRNA processing were not affected in the reh1⌬ strain (26, 27); (ii) depletion of Reh1 in the rei1⌬ background did not significantly increase rRNA maturation defects observed in rei1⌬ cells (26); and (iii) 60S subunit export was not affected by depletion of Reh1 in rei1⌬ cells (26).Here, we investigate the overlapping functions of Reh1 and Rei1 and find strong evidence that Reh1 is involved in cytoplasmic 60S subunit biogenesis. Like Rei1, Reh1 is as...
The crystal structure of the ribosomal 50S subunit from Haloarcula marismortui in complex with the transition state analog CCdAphosphate-puromycin (CCdApPmn) led to a mechanistic proposal wherein the universally conversed A2451 in the ribosomal active site acts as an ''oxyanion hole'' to promote the peptidyl transferase reaction [Nissen, P., Hansen, J., Ban, N., Moore, P.B., and Steitz, T.A. (2000) Science 289, 920 -929]. In the model, close proximity (3 Å) between the A2451 N3 and the nonbridging phosphoramidate oxygen of CCdApPmn suggested that the carbonyl oxyanion formed during the tetrahedral transition state is stabilized by hydrogen bonding to the protonated A2451 N3, the pKa of which must be perturbed substantially. We characterize the contribution of the putative hydrogen bond between the N3 of A2451 and the nonbridging phosphoramidate oxygen by using chemical protection and peptidyl transfer inhibition assays. If this putative hydrogen bond makes a significant thermodynamic contribution, then CCdApPmn-binding affinity to the 50S ribosomal subunit should be strongly pH-dependent, with affinity increasing as the pH is lowered. We report that CCdApPmn binds 50S ribosomes with essentially equal affinity at all pH values between 5.0 and 8.5. These data argue against a mechanism for peptidyl transfer in which a residue with near neutral pKa stabilizes the transition-state oxyanion, at least to the extent that CCdApPmn accurately mimics the transition state.T he ribosome is a molecular machine that assembles polypeptide chains. The addition of an amino acid onto a nascent peptide chain, termed peptidyl transfer, is catalyzed by the 50S ribosomal subunit by using aminoacyl-tRNA and peptidyl-tRNA as substrates. In the course of the reaction, the peptidyl-tRNA, charged with the growing peptide chain, occupies the P site, and an aminoacyl-tRNA, activated with a single amino acid, binds the A site. Peptide bond formation occurs by a transacylation reaction mechanism wherein the ␣-amino group on the A-site tRNA nucleophilically attacks the ester linkage between the peptide chain and the 3Ј-hydroxyl of the P-site tRNA. It is expected that the reaction proceeds through a transition state that has a tetrahedral geometry at the carbonyl carbon and includes a negatively charged oxyanion. Collapse of the transition state produces a deacylated P-site tRNA and a peptide chain that is elongated by one amino acid coupled to the A-site tRNA (for review see ref. 1).Defining how this reaction is catalyzed has been a question of active research for over 30 years. Despite an early and rather indirect indication that a protein side chain might be responsible for catalysis (2, 3), biochemical evidence has identified RNA, which accounts for about two thirds of ribosomal molecular weight (4), as the most likely catalytic component. Highly conserved internal loops of the 23S rRNA domain V have been shown biochemically to interact with the 3Ј-CCA ends of the A-site and P-site tRNAs (5, 6) as well as aminoacyl residues attached to the P...
Epstein–Barr virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells—part of a phenomenon known as the “Warburg effect”— and is mediated by monocarboxylate transporters (MCTs). However, the role of MCTs has yet to be studied in EBV-associated malignancies, which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1, respectively. Functionally, MCT1 was required for early B cell proliferation, and MCT4 up-regulation promoted acquired resistance to MCT1 antagonism in LCLs. However, dual MCT1/4 inhibition led to LCL growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significantly reduced oxygen consumption rates (OCRs) and NAD+/NADH ratios, contrary to previous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/4-inhibited cancer cells. Furthermore, U-13C6–glucose labeling of MCT1/4-inhibited LCLs revealed depleted glutathione pools that correlated with elevated reactive oxygen species. Finally, we found that dual MCT1/4 inhibition also sensitized LCLs to killing by the electron transport chain complex I inhibitors phenformin and metformin. These findings were extended to viral lymphomas associated with EBV and the related gammaherpesvirus KSHV, pointing at a therapeutic approach for targeting both viral lymphomas.
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