Merlin's tumor suppression linked to inhibition of the E3 ubiquitin ligase CRL4DCAF1

The mechanism by which the FERM domain protein Merlin, encoded by the tumor suppressor NF2, restrains cell proliferation is poorly understood. Prior studies have suggested that Merlin exerts its antimitogenic effect by interacting with multiple signaling proteins located at or near the plasma membrane. We have recently observed that Merlin translocates into the nucleus and binds to and inhibits the E3 ubiquitin ligase CRL4^DCAF1. Genetic evidence indicates that inactivation of Merlin induces oncogenic gene expression, hyperproliferation, and tumorigenicity by unleashing the activity of CRL4^DCAF1. In addition to providing a potential explanation for the diverse effects that loss of Merlin exerts in multiple cell types, these findings suggest that compounds inhibiting CRL4^DCAF1 may display therapeutic efficacy in Neurofibromatosis type 2 and other cancers driven by Merlin inactivation.Key words: Merlin, NF2, E3 ubiquitin ligase, CRL4, DCAF1, FERM domain protein     

Merlin's tumor suppression linked to inhibition of the E3 ubiquitin ligase CRL4DCAF1

The mechanism by which the FERM domain protein Merlin, encoded by the tumor suppressor NF2, restrains cell proliferation is poorly understood. Prior studies have suggested that Merlin exerts its antimitogenic effect by interacting with multiple signaling proteins located at or close to the plasma membrane. We have recently observed that Merlin translocates into the nucleus and binds to and inhibits the E3 ubiquitin ligase CRL4^DCAF1. Genetic evidence indicates that inactivation of Merlin induces oncogenic gene expression, hyperproliferation, and tumorigenicity by unleashing the activity of CRL4^DCAF1. In addition to providing a potential explanation for the diverse effects that loss of Merlin exerts in multiple cell types, these findings suggest that compounds inhibiting CRL4^DCAF1 may display therapeutic efficacy in Neurofibromatosis type 2 and other cancers driven by Merlin inactivation.     

Angiomotin binding-induced activation of Merlin/NF2 in the Hippo pathway

The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4^DCAF1. Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin''s auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin''s binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin''s auto-inhibitory tail does not obviously alter Merlin''s conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.     

Merlin: a tumour suppressor with functions at the cell cortex and in the nucleus

Inhibition of proliferation by cell-to-cell contact is essential for tissue organization, and its disruption contributes to tumorigenesis. The FERM domain protein Merlin, encoded by the NF2 tumour suppressor gene, is an important mediator of contact inhibition. Merlin was thought to inhibit mitogenic signalling and activate the Hippo pathway by interacting with diverse target-effectors at or near the plasma membrane. However, recent studies highlight that Merlin pleiotropically affects signalling by migrating into the nucleus and inducing a growth-suppressive programme of gene expression through its direct inhibition of the CRL4DCAF1 E3 ubiquitin ligase. In addition, Merlin promotes the establishment of epithelial adhesion and polarity by recruiting Par3 and aPKC to E-cadherin-dependent junctions, and by ensuring the assembly of tight junctions. These recent advances suggest that Merlin acts at the cell cortex and in the nucleus in a similar, albeit antithetic, manner to the oncogene β-catenin.     

Proposal of Ensifer psoraleae sp. nov., Ensifer sesbaniae sp. nov., Ensifer morelense comb. nov. and Ensifer americanum comb. nov

In a survey of rhizobia associated with the native legumes in Yunnan Province, China, seven and nine strains isolated from the root nodules of Psoralea corylifolia, Sesbania cannabina and Medicago lupulina were respectively classified into the novel genomic species groups I and II in the genus Ensifer (former Sinorhizobium) based on the sequence analyses of the 16S rRNA gene. Analyses of concatenated housekeeping genes (atpD, recA and glnII) further revealed that they were distinct lineages in the genus, and group I was most similar to Ensifer terangae and Ensifer garamanticus (both with 94.2% similarity), while group II was most similar to Ensifer adhaerens (94.0%). These groups could be distinguished from closely related species by DNA–DNA relatedness, MALID-TOF MS, cellular fatty acid profiles and a series of phenotypic characters. Therefore, two novel species were proposed: Ensifer psoraleae sp. nov. (seven strains, type strain CCBAU 65732^T = LMG 26835^T = HAMBI 3286^T) and Ensifer sesbaniae sp. nov. (nine strains, type strain CCBAU 65729^T = LMG 26833^T = HAMBI 3287^T). They had a DNA G + C mol% (T_m) of 58.9 and 60.4, respectively. Both of the type strains formed effective nodules on common bean (Phaseolus vulgaris) and their hosts of origin. In addition, the previously described species Sinorhizobium morelense and Sinorhizobium americanum were renamed as Ensifer morelense comb. nov. and Ensifer americanum comb. nov. according to the accumulated data from different studies.     

Two new 3D (3,8)-connected metal-organic frameworks based on zinc-triazole secondary building units and benzenetricarboxylate linkers

Two new zinc-triazole-carboxylate frameworks constructed from secondary building units (SBUs), [Zn₅(trz)₄(btc)₂(DMF)₂(H₂O)₂]·2H₂O·DMF (1) and [Zn₄(trz)₃(btc)₂(CH₃CN)(H₂O)]·5H₂O·(Bu₄N) (2), [Htrz = 1,2,4-triazole, H₃btc = 1,2,4-benzenetricarboxylate, Bu₄N = tetrabutylammonium], have been synthesized by solvothermal reactions and characterized by single-crystal X-ray diffraction analyses, X-ray power diffraction, elemental analyses, infrared spectra and thermogravimetric analyses. Both compounds 1 and 2 exhibit 3D (3,8)-connected tfz-d nets with {4³}₂{4⁶.6¹⁸.8⁴} topology symbol built from rod-shaped {[Zn₅(trz)₄]⁶⁺}_n SBUs (1) and {[Zn₄(trz)₃]⁵⁺}_n SBUs (2). In two compounds, rodlike units are connected by btc ligands via different modes. Additionally, solid state fluorescent emission spectra of two compounds show fluorescent properties at room temperature.     

NEDD4L‐mediated Merlin ubiquitination facilitates Hippo pathway activation

The tumor suppressor Merlin/NF2, a key activator of the Hippo pathway in growth control, is regulated by phosphorylation. However, it is uncertain whether additional post‐translational modifications regulate Merlin. Here, we show that ubiquitination is required to activate Merlin in the Hippo pathway. Ubiquitinated Merlin is mostly conjugated by one or two ubiquitin molecules. Such modification is promoted by serine 518 dephosphorylation in response to Ca²⁺ signaling or cell detachment. Merlin ubiquitination is mediated by the E3 ubiquitin ligase, NEDD4L, which requires a scaffold protein, AMOTL1, to approach Merlin. Several NF2‐patient‐derived Merlin mutations disrupt its binding to AMOTL1 and its regulation by the AMOTL1‐NEDD4L apparatus. Lysine (K) 396 is the major ubiquitin conjugation residue. Disruption of Merlin ubiquitination by the K396R mutation or NEDD4L depletion diminishes its binding to Lats1 and inhibits Lats1 activation. These effects are also accompanied by loss of Merlin''s anti‐mitogenic and tumor suppressive properties. Thus, we propose that dephosphorylation and ubiquitination compose an intramolecular relay to activate Merlin functions in activating the Hippo pathway during growth control.     

Shedding light on Merlin's wizardry

Inactivation of the tumor suppressor Merlin, encoded by the NF2 (Neurofibromatosis type 2) gene, contributes to malignant conversion in many cell types. Merlin is an Ezrin-Radixin-Moesin protein and localizes underneath the plasma membrane at cell-cell junctions and other actin-rich sites. Recent studies indicate that Merlin mediates contact inhibition of proliferation by blocking recruitment of Rac to the plasma membrane. In mitogen-stimulated cells, p21-activated kinase phosphorylates Ser518 in the C-terminus of Merlin, inactivating the growth suppressive function of the protein. Furthermore, the myosin phosphatase MYPT1-PP1delta, has been identified as a direct activator of Merlin and its inhibition has been linked to malignant transformation. Finally, studies in the fruit fly Drosophila melanogaster have revealed that Merlin functions together with the band 4.1 protein Expanded to promote [corrected] the endocytosis of many signaling receptors, limiting [corrected] their accumulation at the plasma membrane, and to activate [corrected] the Hippo signaling pathway. Here, we review these recent findings and their relevance to the tumor suppressor function of Merlin.     

Preparation of cobalt sandwich diphosphine ligand [(η-C5H4Pr)Co(η-C4(PPh2)2Ph2)] and its chelated palladium complex: Application of diphosphine ligand in the preparation of mono-substituted ferrocenylarenes

The reaction of (η⁵-C₅H₄ⁱPr)Co(PPh₃)₂ with PhCCPPh₂ furnished two isomeric cyclobutadiene-substituted Cp′CoCb diphosphines, [(η⁵-C₅H₄ⁱPr)Co(η⁴-1,2-(PPh₂)₂C₄Ph₂)] (5-cis) and [(η⁵-C₅H₄ⁱPr)Co(η⁴-1,3-(PPh₂)₂C₄Ph₂)] (5-trans). Further reaction of 5-cis with one molar equivalent of Pd(COD)Cl₂ gave palladium complex [(η⁵-C₅H₄ⁱPr)Co(η⁴-1,2-(PPh₂)₂C₄Ph₂)-PdCl₂] (6) in good yield. Both of the molecular structures of 5-cis and 6 were determined by single-crystal X-ray diffraction methods. Unexpectedly, the palladium complex 6 was found to be more efficient than the combination of the commonly used Buchwald’s ligand, biphenyl-2-yl-di-tert-butyl-phosphane, with Pd(OAc)₂ as the catalytic precursor in the Suzuki-Miyaura reaction between ferroceneboronic acid and 4-bromoaldehyde. The X-ray structural analysis and DFT study of several palladium complexes containing sandwich-type diphosphine chelating ligands revealed that the variations in bite angles are much larger than those in bite distances. The more energetically favorable conformation in the Pd(II) complexes is the one with bite angle close to 90°.     

Structural Basis of the Binding of Merlin FERM Domain to the E3 Ubiquitin Ligase Substrate Adaptor DCAF1

The tumor suppressor gene Nf2 product, Merlin, plays vital roles in controlling proper development of organ sizes by specifically binding to a large number of target proteins localized both in cytoplasm and nuclei. The FERM domain of Merlin is chiefly responsible for its binding to target proteins, although the molecular basis governing these interactions are poorly understood due to lack of structural information. Here, we report the crystal structure of the Merlin FERM domain in complex with its binding domain derived from the E3 ubiquitin ligase substrate adaptor DCAF1 (also known as VPRBP). Unlike target binding modes found in ERM proteins, the Merlin-FERM binding domain of DCAF1 folds as a β-hairpin and binds to the α1/β5-groove of the F3 lobe of Merlin-FERM via extensive hydrophobic interactions. In addition to providing the first structural glimpse of a Merlin-FERM·target complex, the structure of the Merlin·DCAF1 complex is likely to be valuable for understanding the interactions of Merlin with its binding partners other than DCAF1.     

HLA class II allele frequencies in the Lebanese population

Aims

Being one of the most polymorphic genetic systems , the Human Leukocyte Antigen system is divided into class I (HLA-A, HLA-B and HLA-C) and class II (HLA-DP, -DQ and -DR). This study is the first and largest of its kind to describe the distribution of HLA-DQB1 and HLA-DRB1 alleles in Lebanon and the region.

Methods

Respectively, 560 and 563 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-DQB1 and HLA-DRB1 alleles using the polymerase chain reaction/sequence specific priming (PCR-SSP) method.

Results

Our data were compared to that of several populations with interesting common findings between the Lebanese, Jordanian, Bahraini, Saudi, Kuwaiti, Tunisian, Korean, Japanese, Thai, Irish, Bulgarian and Polish populations.

Conclusion

These data about the Lebanese population are going to aid future researchers to study the relation of HLA-DQB1 and HLA-DRB1 alleles with major and common diseases in the Lebanese population and will add to the available international literature associated with these loci. In addition it will serve as a reference for the future national bone marrow registry program in our country. We also reviewed the literature for the described association between HLA-DRB1 and -DQB1 loci and different disease entities.     

Conservation of key members in the course of the evolution of the insulin signaling pathway

Our understanding of the evolution of the insulin signaling pathway (ISP) is still incomplete. One intriguing unanswered question is the explanation of the emergence of the glucostatic role of insulin in mammals. To find out whether this is due to the development of new sets of signaling transduction elements in these organisms, or to the establishment of new interactions between pre-existing proteins, we rebuilt putative orthologous ISPs in 17 eukaryotic organisms. Then, we computed the conservation of orthologous ISPs at different levels, from sequence similarity of orthologous proteins to co-evolution of interacting domains. We found that the emergence of glucostatic role in mammals can neither be explained by the development of new sets of signaling elements, nor by the establishment of new interactions between pre-existing proteins. The comparison of orthologous IRS molecules indicates that only in mammals have they acquired their complete functionality as efficient recruiters of effector sub-pathways.     

Limonoids from the seeds of a Godavari mangrove, Xylocarpus moluccensis

Ten limonoids, named godavarins A-J (1-7, 9-11), were isolated from seeds of an Indian mangrove (Xylocarpus moluccensis) collected in the mangrove wetlands of Godavari estuary, Andhra Pradesh. Eight known limonoids, viz. xyloccensins L (8), P (12), Q (13), mexicanolide (14), 6-deoxy-3-detigloyl-swietenine acetate (15), fissinolide (16), methyl 3β-acetoxy-1-oxomeliaca-8(30),14-dienoate (17), and methyl 3β-acetoxy-1-oxomeliaca-8(9),14-dienoate (18), were also obtained. The structures of these compounds were established on the basis of spectroscopic data or comparison with data in the literature (known compounds). The stereostructure of godavarin D was confirmed by means of single-crystal X-ray analysis. Godavarins A-C are the first mexicanolide derivatives with a C₇-C₂₈ ester-linked δ-lactone ring, while godavarins D-G are further additions to the small group of limonoids with a C₁-C₂₉ oxygen bridge. Godavarin H is a phragmalin with five acetoxy groups. Two limonoids, mexicanolide and fissinolide, were found to exhibit marked antifeedant activity against the third-instar larvae of Brontispa longissima (Gestro) at a concentration of 0.5 mg/mL. The most potent compound was mexicanolide. It also showed moderate insecticidal activity.     

Genotyping of celiac disease-related-risk haplotypes using a closed-tube polymerase chain reaction analysis of dried blood and saliva disk samples.

Expansion of molecular diagnostics more widely into clinical routines requires simplified methods allowing automation. We developed a homogeneous, multilabel polymerase chain reaction (PCR) method based on time-resolved fluorometry, and studied the use of dried disk samples in PCR. Celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 genotyping was used to verify the method with blood and saliva samples dried on S&S 903 and IsoCode sample collection papers. Three sample preparation procedures, including manufacturer’s manual elution, an automated elution, and direct use of disk samples, were compared using dried disk samples. The three procedures gave successful amplification and correct genotyping results. Owing to the simplicity of the direct use of disk samples in PCR, this method was chosen for the subsequent homogeneous analysis of blood (n = 194) and saliva (n = 30) disk samples on S&S 903 paper. The results revealed that, in addition to DNA samples (n = 29), both blood and saliva disk samples were successfully amplified and genotyped using the homogeneous PCR assays for HLA-DQA1 and HLA-DQB1. The homogeneous PCR assays developed provide a useful tool to genotype celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 alleles. Furthermore, the method provides a direct way to perform a closed-tube PCR analysis of dried blood and saliva disk samples enabling simple automation.     

FRAX597, a Small Molecule Inhibitor of the p21-activated Kinases,Inhibits Tumorigenesis of Neurofibromatosis Type 2 (NF2)-associated Schwannomas

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.     

Synthesis of novel pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives and their inhibition against growth of A549 and H322 lung cancer cells

A series of substituted pyrazolo[1,5-a]pyrazin-4(5H)-one was synthesized by the reaction of ethyl 1-(2-oxo-2-phenylethyl)-3-phenyl-1H-pyrazole-5-carboxylate derivatives and 2-(2-aminoethoxy)ethanol or 2-morpholinoethanamine in the condition of microwave-assisted one-step and solvent-free in a good yield. The structures of the compounds were determined by IR, ¹H NMR and mass spectroscopy. In addition, a representative single-crystal structure was characterized by using X-ray diffraction analysis. Preliminary biological evaluation showed that the compounds could inhibit the growth of A549 and H322 cells in dosage-dependent manners.     

Tumour-suppressive microRNA-224 inhibits cancer cell migration and invasion via targeting oncogenic TPD52 in prostate cancer

Our recent study of the microRNA expression signature of prostate cancer (PCa) revealed that microRNA-224 (miR-224) is significantly downregulated in PCa tissues. Here, we found that restoration of miR-224 significantly inhibits PCa cell migration and invasion. Additionally, we found that oncogenic TPD52 is a direct target of miR-224 regulation. Silencing of the TPD52 gene significantly inhibits cancer cell migration and invasion. Moreover, TPD52 expression is upregulated in cancer tissues and negatively correlates with miR-224 expression. We conclude that loss of tumour-suppressive miR-224 enhances cancer cell migration and invasion in PCa through direct regulation of oncogenic TPD52.     

Genotypic and phenotypic characterization of Brazilian patients with GM1 gangliosidosis

GM1 gangliosidosis is a lysosomal disorder caused by β-galactosidase deficiency due to mutations in the GLB1 gene. It is a rare neurodegenerative disorder with an incidence of about 1:100,000–1:200,000 live births worldwide. Here we review GLB1 mutations and clinical features from 65 Brazilian GM1 gangliosidosis patients. Molecular analysis showed 17 different mutations and c.1622–1627insG was the most frequent, accounting for 50% of the alleles. Cognitive impairment was the main clinical sign, observed in 82% of patients, followed by hepatosplenomegaly observed in 56% of patients. It was possible to establish a significant correlation between age at onset of symptoms preceding the first year of life and the presence of the mutation c.1622–1627insG (p = 0.03). Overall our findings differ from literature and represent the exclusive genotypic profile found in Brazilian GM1 gangliosidosis patients.     

Merlin, a multi-suppressor from cell membrane to the nucleus

Recent evidence suggests that the neurofibromatosis type 2 (NF2) gene encoded protein merlin suppresses mitogenic signalling not only at the cell membrane but also in the nucleus. At the membrane, merlin inhibits signalling by integrins and tyrosine receptor kinases (RTKs) and the activation of downstream pathways, including the Ras/Raf/MEK/ERK, FAK/Src, PI3K/AKT, Rac/PAK/JNK, mTORC1, and Wnt/β-catenin pathways. In the nucleus, merlin suppresses the E3 ubiquitin ligase CRL4(DCAF1) to inhibit proliferation. Gene expression analysis suggested that CRL4(DCAF1) could also regulate the expression of integrins and RTKs. In this review, we explore the links between merlin function at the membrane and in the nucleus, and discuss the potential of targeting the master regulator CRL4 (DCAF1) to treat NF2 and other merlin-deficient tumours.     

Design of a potent, soluble glucokinase activator with increased pharmacokinetic half-life

The continued optimization of a series of glucokinase activators is described, including attempts to understand the interplay between molecular structure and the composite parameter of unbound clearance. These studies resulted in the discovery of a new scaffold for glucokinase activators and further exploration of this scaffold led to the identification of GKA60. GKA60 maintains an excellent balance of potency and physical properties whilst possessing a significantly different, but complimentary, pre-clinical pharmacokinetic profile compared with the previously disclosed compound GKA50.