Disease relevance of CET1

• Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-dependent RNA 5'-triphosphatases/NTPases encoded by fungi and DNA viruses; the family is defined by conserved sequence motifs A, B, and C. We tested the effects of 12 alanine substitutions and 16 conservative modifications at 18 positions of the motifs [1].
• The manganese- and cobalt-dependent nucleoside triphosphatase of Cet1p resembles the nucleoside triphosphatase activities of the baculovirus LEF-4 and vaccinia virus D1 capping enzymes [2].
• We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA) [3].

High impact information on CET1

• The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel [4].
• In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1) [5].
• Purified recombinant Cet1 catalyzes hydrolysis of the gamma phosphate of triphosphate-terminated RNA at a rate of 1 s-1 [6].
• The highly conserved C-terminal domain of Cet1 interacts with Tup1 in vitro, and Tup1-Ssn6 complexes co-purify with the Cet1 protein, indicating that in vivo interactions also occur between these proteins [7].
• Over-expression of CET1 compromised repression of an MFA2-lacZ reporter gene that is subject to Tup1-Ssn6 repression [7].

Biological context of CET1

• In this report, with the aid of partial amino acid sequences of purified yeast capping enzyme, we isolated the gene, designated CET1, encoding the S. cerevisiae capping enzyme beta subunit [8].
• Gene disruption experiment showed that CET1 is essential for yeast cell growth [8].
• Isolation and characterization of the yeast mRNA capping enzyme beta subunit gene encoding RNA 5'-triphosphatase, which is essential for cell viability [8].
• This suggests a model in which Cet1 possesses a preformed active site, and where major domain rearrangements are not required to form an active catalytic site [9].
• The kinetics of real-time metal ion binding to the Cet1 protein were also investigated, and the effects on RNA and nucleotide binding were evaluated [9].

Anatomical context of CET1

• Other phospholipids of the membranes of this organism, such as phosphatidylcholine and phosphatidylglycerol, had little effect on activation, indicating that the amino group of the phospholipids may be required for the function of TPase [10].

Associations of CET1 with chemical compounds

• The purified recombinant CET1 gene product, Cet1, exhibited an RNA 5'-triphosphatase activity which specifically removed the gamma-phosphate from the triphosphate-terminated RNA substrate, but not from nucleoside triphosphates, confirming the identity of the gene [8].
• Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function [1].
• Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replacement by glutamine inactivated Cet1p [1].
• The S429A and S429V proteins were fully active when produced in bacteria at 37 degrees C, but were inactive when produced at 17 degrees C. Replacement of Ser(429) by threonine partially suppressed the cold sensitivity of the Cet1 phosphohydrolase, but did not suppress the cs growth defect in yeast [11].
• We have used endogenous tryptophan fluorescence studies to elucidate both the nature and the role(s) of the metal ions in the Cet1-mediated phosphohydrolase reaction [9].

Physical interactions of CET1

• In contrast, neither the full-length human capping enzyme nor its TPase domain interacted with the yeast GTase [12].

Other interactions of CET1

• Physical and functional interaction of the yeast corepressor Tup1 with mRNA 5'-triphosphatase [7].
• These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity [13].
• The S. cerevisiae capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components [14].
• Cth1p activity was abolished by E87A and E89A mutations in motif A. Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo [1].

Analytical, diagnostic and therapeutic context of CET1

• To provide additional insight into the relationship between Cet1 structure and metal ion binding, we correlated the effect of ion binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators [9].
• The mRNA 5'-triphosphatase activity hydrolyzing the gamma-phosphoryl group from pppN-RNA was co-purified with mRNA guanylyltransferase activity through column chromatographies on CM-Sephadex and poly(U)-Sepharose, and centrifugation through glycerol gradients, suggesting that these two activities are physically associated [15].
• Here we show that the guanylyltransferase activity of Ceg1 is highly thermolabile in vitro (98% loss of activity after treatment for 10 min at 35 degrees C) and that binding to recombinant Cet1 protein, or a synthetic peptide Cet1(232-265), protects Ceg1 from heat inactivation at physiological temperatures [16].
• Direct evidence for the interaction of TPase and Triton X-100/phosphatidylserine mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200 [10].
• Expression was demonstrated via the highly specific nickel-coated Elisa plate method, using an anti-His antibody and 2 separate anti-Ac TPase antibodies, to Ac residues 103-465 and 189-807 [17].

References