The RING ®nger protein CNOT4 is a component of the CCR4±NOT complex. This complex is implicated in repression of RNA polymerase II transcription. Here we demonstrate that CNOT4 functions as a ubiquitin±protein ligase (E3). We show that the unique C 4 C 4 RING domain of CNOT4 interacts with a subset of ubiquitin-conjugating enzymes (E2s). Using NMR spectroscopy, we detail the interaction of CNOT4 with UbcH5B and characterize RING residues that are critical for this interaction. CNOT4 acts as a potent E3 ligase in vitro. Mutations that destabilize the E2±E3 interface abolish this activity. Based on these results, we present a model of how E3 ligase function within the CCR4±NOT complex relates to transcriptional regulation.

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT 1 ) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. 257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT 1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr 113 in TM III and between the carboxyl group and His 256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT 1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G proteincoupled receptor antagonists with inverse agonist activity.Angiotensin II (Ang II) 3 receptor antagonists (ARBs) are highly selective for the Ang II type 1 (AT 1 ) receptor, which is a member of the G protein-coupled receptor (GPCR) superfamily, and block the diverse effects of Ang II. In addition to their blood pressure-lowering effects in hypertensive patients, ARBs have been shown to promote regression of left ventricular hypertrophy and decrease cardiovascular morbidity and mortality in patients with heart failure or hypertensive diabetic nephropathy with proteinuria (1). Many ARBs are available for clinical use. Because not all ARBs have the same effects, some benefits conferred by ARBs may not be class effects (2). This notion represents an exciting new area in ARB-based therapy, which holds the promise of reducing the incidence of cardiovascular disease.Inverse agonists, such as the opioid receptor ligand ICI174864 (3), block agonist-independent signal transduction by GPCRs. Many clinically important medications have been shown to behave as inverse agonists when tested against either wild-type (WT) or mutated GPCRs; e.g. olanzapine in the 5-hydroxytryptamine 2C receptors (4) and metoprolol in the ␤-adrenoreceptor (5). Spontaneous receptor mutations leading to constitutive activity have been implicated in some human diseases (6, 7). However, such spontaneous mutations have not been reported for the AT 1 receptor, and the WT AT 1 receptor shows slight constitutive activity (2). A recent study demonstrates that the WT AT 1 receptor is activated by mechanical stretching of cultured rat myocytes without the involvement of Ang II, and this was suppressed by an inverse agonist (8). The same study also demonstrates that cardiac hypertrophy induced by constricting the transverse aorta in angiotensinogen knock-out mice was attenuated by an inverse agonist, suggesting that the WT AT 1 receptor is activated ind...

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The NOT4 protein is a component of the CCR4⅐NOT complex, a global regulator of RNA polymerase II transcription. Human NOT4 (hNOT4) contains a RING finger motif of the C 4 C 4 type. We expressed and purified the N-terminal region of hNOT4 (residues 1-78) encompassing the RING finger motif and determined the solution structure by heteronuclear NMR. NMR experiments using a 113 Cd-substituted hNOT4 RING finger showed that two metal ions are bound through cysteine residues in a cross-brace manner. The three-dimensional structure of the hNOT4 RING finger was refined with root mean square deviation values of 0.58 ؎ 0.13 Å for the backbone atoms and 1.08 ؎ 0.12 Å for heavy atoms. The hNOT4 RING finger consists of an ␣-helix and three long loops that are stabilized by zinc coordination. The overall folding of the hNOT4 RING finger is similar to that of the C 3 HC 4 RING fingers. The relative orientation of the two zinc-chelating loops and the ␣-helix is well conserved. However, for the other regions, the secondary structural elements are distinct.The CCR4⅐NOT complex was first detected in Saccharomyces cerevisiae as a global transcription regulator, affecting transcription of multiple functionally unrelated genes positively as well as negatively (1). The complex consists of CCR4 (carbon catabolite repressor 4), CAF1 (CCR4-associated factor 1, also known as POP2), the five NOT proteins (NOT1-5), and several unidentified proteins (1). The yeast NOT genes have been identified in a screen for elevated HIS3 expression (2-4). The HIS3 gene contains two core promoters, T C , a TATA-less element, and T R , a canonical TATA sequence (5, 6). Mutations in NOT genes selectively elevate transcription from T C (2-4). Besides repressing genes involved in histidine biosynthesis (HIS3 and HIS4), NOT proteins also affect transcription of genes involved in pheromone response (STE4), nuclear fusion (BIK1), and RNA polymerase II transcription (TBP) (2, 3). The CCR4 gene product regulates expression of ADH2 and other genes involved in nonfermentative growth, cell wall integrity, and ion sensitivity (7-9). CCR4 exists in a complex with other proteins (10), and two-hybrid screening with CCR4 identified CAF1 (11, 12) and DBF2 (a cell cycle-regulated kinase) (9, 13) as binding partners. Recently, it was found that CCR4 and CAF1 reside with the NOT proteins in a 1.2-MDa complex (1). Besides physical interactions between CCR4, CAF1, and NOT proteins, there is also a functional association. Mutations in the NOT, CCR4, and CAF1 genes lead to similar, but not identical, phenotypes (1,14). Interestingly, mutations in NOT1, NOT3, NOT5, and CAF1 genes suppressed a mutation in SRB4, which is an essential component of the RNA polymerase II holoenzyme and required for the expression of most protein-coding genes. This suggests that the yeast CCR4⅐NOT complex has a very general role in RNA polymerase II transcription (15).Recently, the human counterpart of the yeast CCR4⅐NOT complex has been identified (16). cDNAs for four subunits, hNOT2, 1 hNOT3, hNOT4, and...

ROS1 gene rearrangement was observed in around 1–2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1 -rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1–rearranged cancers or NTRK-rearranged cancers in vitro and in vivo . Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors.

The solution conformation of an antibacterial protein sapecin has been detennined by 'H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 1523), and two extended strands (residues 2431 and 3W). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.

Lipid transfer proteins mediate inter-organelle transport of membrane lipids at organelle contact sites in cells, playing fundamental roles in the lipidome and membrane biogenesis in eukaryotes. We previously developed a ceramide-mimetic compound as a potent inhibitor of the ceramide transport protein CERT. Here we develop CERT inhibitors with structures unrelated to ceramide. To this aim, we identify a seed compound with no ceramide-like structure but with the capability of forming a hydrogen-bonding network in the ceramidebinding START domain, by virtual screening of~3 × 10 6 compounds. We also establish a surface plasmon resonance-based system to directly determine the affinity of compounds for the START domain. Then, we subject the seed compound to a series of in silico docking simulations, efficient chemical synthesis, affinity analysis, protein-ligand co-crystallography, and various in vivo assays. This strategy allows us to obtain ceramide-unrelated compounds that potently inhibited the function of CERT in human cultured cells.

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