Frequent loss of BTG1 activity and impaired interactions with the Caf1 subunit of the Ccr4-Not deadenylase in non-Hodgkin lymphoma.

Mutations in the highly similar genes B-cell translocation gene 1 (BTG1) and BTG2 are identified in approximately 10-15% of non-Hodgkin lymphoma cases, which may suggest a direct involvement of BTG1 and BTG2 in malignant transformation. However, it is unclear whether or how disease-associated mutations impair the function of these genes. Therefore, we selected 16 BTG1 variants based on in silico analysis. We then evaluated (i) the ability of these variants to interact with the known protein-binding partners CNOT7 and CNOT8, which encode the Caf1 catalytic subunit of the Ccr4-Not deadenylase complex; (ii) the activity of the variant proteins in cell cycle progression; (iii) translational repression; and (iv) mRNA degradation. Based on these analyses, we conclude that mutations in BTG1 may contribute to malignant transformation and tumor cell proliferation by interfering with its anti-proliferative activity and ability to interact with CNOT7 and CNOT8.

1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg2+-dependent binding.

In eukaryotic cells, cytoplasmic mRNA is characterised by a 3' poly(A) tail. The shortening and removal of poly(A) tails (deadenylation) by the Ccr4-Not nuclease complex leads to reduced translational efficiency and RNA degradation. Using recombinant human Caf1 (CNOT7) enzyme as a screening tool, we recently described the discovery and synthesis of a series of substituted 1-hydroxy-3,7-dihydro-1H-purine-2,6-diones (1-hydroxy-xanthines) as inhibitors of the Caf1 catalytic subunit of the Ccr4-Not complex. Here, we used a chemiluminescence-based AMP detection assay to show that active 1-hydroxy-xanthines inhibit both isolated Caf1 enzyme and human Caf1-containing complexes that also contain the second nuclease subunit Ccr4 (CNOT6L) to a similar extent, indicating that the active site of the Caf1 nuclease subunit does not undergo substantial conformational change when bound to other Ccr4-Not subunits. Using differential scanning fluorimetry, we also show that binding of active 1-hydroxy-xanthines requires the presence of Mg2+ ions, which are present in the active site of Caf1.

The central region of CNOT1 and CNOT9 stimulates deadenylation by the Ccr4-Not nuclease module.

Regulated degradation of cytoplasmic mRNA is important for the accurate execution of gene expression programmes in eukaryotic cells. A key step in this process is the shortening and removal of the mRNA poly(A) tail, which can be achieved by the recruitment of the multi-subunit Ccr4-Not nuclease complex via sequence-specific RNA-binding proteins or the microRNA machinery. The Ccr4-Not complex contains several modules that are attached to its large subunit CNOT1. Modules include the nuclease module, which associates with the MIF4G domain of CNOT1 and contains the catalytic subunits Caf1 and Ccr4, as well as the module containing the non-catalytic CNOT9 subunit, which binds to the DUF3819 domain of CNOT1. To understand the contributions of the individual modules to the activity of the complex, we have started to reconstitute sub-complexes of the human Ccr4-Not complex containing one or several functional modules. Here, we report the reconstitution of a pentameric complex including a BTG2-Caf1-Ccr4 nuclease module, CNOT9 and the central region of CNOT1 encompassing the MIF4G and DUF3819 domains. By comparing the biochemical activities of the pentameric complex and the nuclease module, we conclude that the CNOT1-CNOT9 components stimulate deadenylation by the nuclease module. In addition, we show that a pentameric complex containing the melanoma-associated CNOT9 P131L variant is able to support deadenylation similar to a complex containing the wild-type CNOT9 protein.

Anti-Inflammatory and Anti-Nociceptive Activities of Stem-Bark Extracts and Fractions of Carpolobia Lutea (Polygalaceae).

BACKGROUND: In Niger Delta, ethnomedicine hydroalcoholic extract of Carpolobia lutea (CL) (Polygalaceae) is used to relieve inflammatory pains. OBJECTIVES: The purpose of this study is to evaluate the anti-inflammatory and antinociceptive effects of ethanolic stem extract (ESE) and to fractionate the ESE for the elucidation of bioactive molecules. MATERIALS AND METHODS: The antinociceptive effects for ESE were tested against two noxious stimuli; chemical (acetic acid-induced writhing and formalin-induced pain) and thermal (hot plate) stimuli. The effects of paracetamol (130 mg/kg), indomethacin (10 mg/kg), and morphine (5 mg/kg) pretreatment were investigated. To isolate the bioactive compounds with anti-inflammatory effect, two doses (86.6 and 173.2 mg/kg) of four fractions (methanol fraction MTF, ethyl acetate fraction EAF, chloroform fraction CHF, and n-hexane fraction n-HF) obtained from fractionating ESE were utilized. Carrageenan, egg albumin, and capsaicin-induced edema of the hind paw of the rats were the models adopted. Paw volume was measured by a digital vernier caliper from 0 to 6 h after injection. This was compared to standard drugs. The results were subjected to statistical analysis. RESULTS: The ESE decreased significantly (P < 0.001) the writhing of acetic acid-induced abdominal contractions and licking of formalin-induced pains but does not have any effects on the hot plate test. Of the four fractions obtained, the EAFs demonstrated a significant (P < 0.001) inflammatory inhibition of 98.97% and 41.91% at 86.6 and 173.2 mg/kg, respectively, compared to 65.75% inhibition demonstrated by the reference drug, acetylsalicylic acid (100 mg/kg) on the carrageenan model while 36.36% and 29.87% inhibition of inflammation at 86.6 and 173.2 mg/kg, respectively, on the egg albumin models; there was no significant effect on the capsaicin model. CONCLUSION: The isolation of quercetin and kaemferol from CL gave credence to its anti-inflammatory and antinociceptive effects.

Discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as inhibitors of the human poly(A)-selective ribonuclease Caf1.

Eukaryotic mRNA contains a 3' poly(A) tail, which plays important roles in the regulation of mRNA stability and translation. Well-characterized enzymes involved in the shortening of the poly(A) tail include the multi-subunit Ccr4-Not deadenylase, which contains the Caf1 (Pop2) and Ccr4 catalytic components, and poly(A)-specific ribonuclease (PARN). Two Mg(2+) ions present in the active sites of these ribonucleases are required for RNA cleavage. Here, we report the discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as (sub)micromolar inhibitors of Caf1.

The enzyme activities of Caf1 and Ccr4 are both required for deadenylation by the human Ccr4-Not nuclease module.

In eukaryotic cells, the shortening and removal of the poly(A) tail (deadenylation) of cytoplasmic mRNA is a key event in regulated mRNA degradation. A major enzyme involved in deadenylation is the Ccr4-Not deadenylase complex, which can be recruited to its target mRNA by RNA-binding proteins or the miRNA repression complex. In addition to six non-catalytic components, the complex contains two enzymatic subunits with ribonuclease activity: Ccr4 and Caf1 (Pop2). In vertebrates, each deadenylase subunit is encoded by two paralogues: Caf1, which can interact with the anti-proliferative protein BTG2, is encoded by CNOT7 and CNOT8, whereas Ccr4 is encoded by the highly similar genes CNOT6 and CNOT6L. Currently, it is unclear whether the catalytic subunits work co-operatively or whether the nuclease components have unique roles in deadenylation. We therefore developed a method to express and purify a minimal human BTG2-Caf1-Ccr4 nuclease sub-complex from bacterial cells. By using chemical inhibition and well-characterized inactivating amino acid substitutions, we demonstrate that the enzyme activities of Caf1 and Ccr4 are both required for deadenylation in vitro. These results indicate that Caf1 and Ccr4 cooperate in mRNA deadenylation and suggest that the enzyme activities of Caf1 and Ccr4 are regulated via allosteric interactions within the nuclease module.