Selective degradation of splicing factor CAPERα by anticancer sulfonamides.

Target-protein degradation is an emerging field in drug discovery and development. In particular, the substrate-receptor proteins of the cullin-ubiquitin ligase system play a key role in selective protein degradation, which is an essential component of the anti-myeloma activity of immunomodulatory drugs (IMiDs), such as lenalidomide. Here, we demonstrate that a series of anticancer sulfonamides NSC 719239 (E7820), indisulam, and NSC 339004 (chloroquinoxaline sulfonamide, CQS) induce proteasomal degradation of the U2AF-related splicing factor coactivator of activating protein-1 and estrogen receptors (CAPERα) via CRL4DCAF15 mediated ubiquitination in human cancer cell lines. Both CRISPR-Cas9-based knockout of DCAF15 and a single amino acid substitution of CAPERα conferred resistance against sulfonamide-induced CAPERα degradation and cell-growth inhibition. Thus, these sulfonamides represent selective chemical probes for disrupting CAPERα function and designate DCAFs as promising drug targets for promoting selective protein degradation in cancer therapy.

Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p.

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesis.

Continued research toward the development of new antifungals that act via inhibition of glycosylphosphatidylinositol (GPI) biosynthesis led to the design of E1210. In this study, we assessed the selectivity of the inhibitory activity of E1210 against Candida albicans GWT1 (Orf19.6884) protein, Aspergillus fumigatus GWT1 (AFUA_1G14870) protein, and human PIG-W protein, which can catalyze the inositol acylation of GPI early in the GPI biosynthesis pathway, and then we assessed the effects of E1210 on key C. albicans virulence factors. E1210 inhibited the inositol acylation activity of C. albicans Gwt1p and A. fumigatus Gwt1p with 50% inhibitory concentrations (IC(50)s) of 0.3 to 0.6 µM but had no inhibitory activity against human Pig-Wp even at concentrations as high as 100 µM. To confirm the inhibition of fungal GPI biosynthesis, expression of ALS1 protein, a GPI-anchored protein, on the surfaces of C. albicans cells treated with E1210 was studied and shown to be significantly lower than that on untreated cells. However, the ALS1 protein levels in the crude extract and the RHO1 protein levels on the cell surface were found to be almost the same. Furthermore, E1210 inhibited germ tube formation, adherence to polystyrene surfaces, and biofilm formation of C. albicans at concentrations above its MIC. These results suggested that E1210 selectively inhibited inositol acylation of fungus-specific GPI which would be catalyzed by Gwt1p, leading to the inhibition of GPI-anchored protein maturation, and also that E1210 suppressed the expression of some important virulence factors of C. albicans, through its GPI biosynthesis inhibition.

Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

The mechanisms that regulate peripheral nervous system (PNS) gliogenesis are incompletely understood. For example, gut neural crest stem cells (NCSCs) do not respond to known gliogenic factors, suggesting that yet-unidentified factors regulate gut gliogenesis. To identify new mechanisms, we performed gene expression profiling to identify factors secreted by gut NCSCs during the gliogenic phase of development. These cells highly expressed leucine-rich glioma inactivated 4 (Lgi4) despite the fact that Lgi4 has never been implicated in stem cell function or enteric nervous system development. Lgi4 is known to regulate peripheral nerve myelination (having been identified as the mutated gene in spontaneously arising claw paw mutant mice), but Lgi4 is not known to play any role in PNS development outside of peripheral nerves. To systematically analyze Lgi4 function, we generated gene-targeted mice. Lgi4-deficient mice exhibited a more severe phenotype than claw paw mice and had gliogenic defects in sensory, sympathetic, and enteric ganglia. We found that Lgi4 is required for the proliferation and differentiation of glial-restricted progenitors throughout the PNS. Analysis of compound-mutant mice revealed that the mechanism by which Lgi4 promotes enteric gliogenesis involves binding the ADAM22 receptor. Our results identify a new mechanism regulating enteric gliogenesis as well as novel functions for Lgi4 regulating the proliferation and maturation of glial lineage cells throughout the PNS.

Adam22 is a major neuronal receptor for Lgi4-mediated Schwann cell signaling.

The segregation and myelination of axons in the developing PNS, results from a complex series of cellular and molecular interactions between Schwann cells and axons. Previously we identified the Lgi4 gene (leucine-rich glioma-inactivated4) as an important regulator of myelination in the PNS, and its dysfunction results in arthrogryposis as observed in claw paw mice. Lgi4 is a secreted protein and a member of a small family of proteins that are predominantly expressed in the nervous system. Their mechanism of action is unknown but may involve binding to members of the Adam (A disintegrin and metalloprotease) family of transmembrane proteins, in particular Adam22. We found that Lgi4 and Adam22 are both expressed in Schwann cells as well as in sensory neurons and that Lgi4 binds directly to Adam22 without a requirement for additional membrane associated factors. To determine whether Lgi4-Adam22 function involves a paracrine and/or an autocrine mechanism of action we performed heterotypic Schwann cell sensory neuron cultures and cell type-specific ablation of Lgi4 and Adam22 in mice. We show that Schwann cells are the principal cellular source of Lgi4 in the developing nerve and that Adam22 is required on axons. Our results thus reveal a novel paracrine signaling axis in peripheral nerve myelination in which Schwann cell secreted Lgi4 functions through binding of axonal Adam22 to drive the differentiation of Schwann cells.

Biological characterization of ADAM22 variants reveals the importance of a disintegrin domain sequence in cell surface expression.

ADAM metallopeptidase domain 22 (ADAM22) is a neuronal membrane-spanning protein that is a potential receptor for leucine-rich, glioma-inactivated 1 (LGI1), and leucine-rich repeat LGI family, member 4 (LGI4). Several lines of study have shown a direct interaction between ADAM22 and LGI1, a mutation which is responsible for inherited epilepsy in humans. Both ADAM22-deficient mice and claw paw mice, congenitally deficient in LGI4, show hypomyelination in the peripheral nerves, suggesting that these molecules are involved in myelination processes. These findings mark ADAM22 as a potential target molecule for epilepsy or demyelination diseases. To investigate the relationship between ADAM22 mutation and its biological character, we designed and examined several ADAM22 variants. We discovered that the ADAM22 P81R variant, the most common polymorphic variation, works as well as the wild-type ADAM22. We also showed that mutations in the disintegrin domain cause a marked decrease in the processing of ADAM22 preproteins, and result in reduced LGI4-binding abilities. Our findings provide valuable information for mutation screening of the ADAM22 gene in patients suffering from epilepsy or demyelinating diseases.

Inhibitory effect of anti-seizure medications on ionotropic glutamate receptors: special focus on AMPA receptor subunits.

OBJECTIVE: The purpose of the current analysis was to investigate the direct inhibitory effects of perampanel and other anti-seizure medications (ASMs) on the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), and kainate glutamate receptor subtypes using electrophysiological assessments. METHODS: AMPA receptor subunit-expressing cell lines (hGluA1-4, including two kinds of Q/R RNA-editing variants of hGluA2), NMDA receptor-expressing cells (hNR1/hNR2B), and kainate receptor-expressing cells (hGluK2) were developed in house. The effects of perampanel, and other ASMs including topiramate, phenobarbital, lamotrigine, gabapentin, carbamazepine, valproate, levetiracetam, and lacosamide, on AMPA, NMDA, and kainate receptors were evaluated by automated patch-clamp technique. In the same way, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX) and GYKI 52466 were evaluated as reference compounds of AMPA receptor antagonists. For the AMPA receptor functional assay, AMPA currents were elicited by AMPA in the presence of cyclothiazide. NMDA with glycine was used as a stimulant for the NMDA receptor assays, while glutamate was used for the kainate receptor assays. The mean 50 % inhibitory concentration (IC50) values were determined based on sigmoidal-curve fitting using GraphPad Prism software. RESULTS: Perampanel inhibited functions of hGluA1-4, but did not inhibit hNR1/hNR2B and hGluK2 up to 25 µM, the maximum soluble concentration. The IC50 values were 660 nM for hGluA1, 780 nM for hGluA2(R), 1200 nM for hGluA2(Q), 1200 nM for hGluA3, and 1800 nM for hGluA4. NBQX and GYKI 52466 also inhibited the function of all AMPA receptor subunits, but did not inhibit hNR1/hNR2B and hGluK2. The IC50 values for NBQX were 880 nM for hGluA1, 290 nM for hGluA2(R), 310 nM for hGluA2(Q), 330 nM for hGluA3, and 630 nM for hGluA4. For GYKI 52466, IC50 values were 25,000 nM for hGluA1, 30,000 nM for hGluA2(R), 42,000 nM for hGluA2(Q), 28,000 nM for hGluA3, and 53,000 nM for hGluA4. Phenobarbital inhibited hGluA2(R) at an IC50 value of 730,000 nM. The majority of other ASMs evaluated in this study did not show a direct inhibitory effect on almost any of the glutamate receptor functions examined up to 1 M. However, lamotrigine and carbamazepine inhibited hNR1/hNR2B function at IC50 values of 930,000 and 1,000,000 nM, respectively. SIGNIFICANCE: Only a few ASMs evaluated in this study showed direct interaction with ionotropic glutamate receptors. Perampanel is the only ASM that had a potent inhibitory effect on all AMPA receptor subtypes, but did not inhibit NMDA or kainate receptor subunits; while phenobarbital inhibited GluA2(R), and carbamazepine and lamotrigine inhibited the NMDA receptor at high concentration ranges.

MPR1 as a novel selection marker in Saccharomyces cerevisiae.

L-Azetidine-2-carboxylic acid (AZC) is a toxic four-membered ring analogue of L-proline that is transported into cells by proline transporters. AZC and L-proline in the cells are competitively incorporated into nascent proteins. When AZC is present in a minimum medium, misfolded proteins are synthesized in the cells, thereby inhibiting cell growth. The MPR1 gene has been isolated from the budding yeast Saccharomyces cerevisiae Sigma1278b as a multicopy suppressor of AZC-induced growth inhibition. MPR1 encodes a novel acetyltransferase that detoxifies AZC via N-acetylation. Since MPR1 is absent in the laboratory strain of S. cerevisiae S288C, it could be a positive selection marker that confers AZC resistance in the S288C background strains. To examine the usefulness of MPR1, we constructed some plasmid vectors that harboured MPR1 under the control of various promoters and introduced them into the S288C-derived strains. The expression of MPR1 conferred AZC resistance that was largely dependent on the expression level of MPR1. In an additional experiment, the galactose-inducible MPR1 and ppr1(+), the fission yeast Schizosaccharomyces pombe homologue of MPR1, were used for gene disruption by homologous recombination, and here AZC-resistant colonies were also successfully selected. We concluded that our MPR1-AZC system provides a powerful tool for yeast transformation.

Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards.

An important challenge for proteomics is to be able to compare absolute protein levels across biological samples. Here we introduce an approach based on the use of culture-derived isotope tags (CDITs) for quantitative tissue proteome analysis. We cultured Neuro2A cells in a stable isotope-enriched medium and mixed them with mouse brain samples to serve as internal standards. Using CDITs, we identified and quantified a total of 1,000 proteins, 97-98% of which were expressed in both mouse whole brain and Neuro2A cells. CDITs also allow comprehensive and absolute protein quantification. Synthetic unlabeled peptides were used to quantify the corresponding proteins labeled with stable isotopes in Neuro2A cells, and the results were used to obtain the absolute amounts of 103 proteins in mouse whole brain. The expression levels correlated well with those in Neuro2A cells. Thus, the use of CDITs allows both relative and absolute quantitative proteome studies.

Deficits in spatial learning and motor coordination in ADAM11-deficient mice.

BACKGROUND: ADAM11 is a member of the ADAM gene family and is mainly expressed in the nervous system. It is thought to be an adhesion molecule, since it has a disintegrin-like domain related to cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM11, we generated ADAM11-deficient mice by means of gene targeting. RESULTS: ADAM11-deficient mice were apparently normal, and survived more than one year with no major histological abnormalities in the brain or spinal cord. Because ADAM11 is highly expressed in the hippocampus and cerebellum, we have examined ADAM11 mutant mice for learning using visual and hidden water maze tasks, and their motor coordination using a rotating rod task. Our results showed that their visual water maze task results are normal, but the hidden water maze and rotating rod task skills are impaired in ADAM11-deficient mice. CONCLUSION: Our results indicate that ADAM11 mutation does not affect cell migration and differentiation during development, but affects learning and motor coordination. Thus, ADAM11 might play an important signalling or structural role as a cell adhesion molecule at the synapse, and may thus participate in synaptic regulation underlying behavioural changes.

Altered nociceptive response in ADAM11-deficient mice.

Mice that lack A Disintegrin And Metalloprotease 11 (ADAM11) protein showed normal responses to stimuli in the von Frey test and the hot plate test, but showed reduced responses in the formalin paw test and acetic acid writhing test. Our results indicate that the cell adhesion-related molecule ADAM11 may play a role in pain transmission and in inflammatory regulation mechanisms underlying changes in the threshold for pain perception.

Ataxia and peripheral nerve hypomyelination in ADAM22-deficient mice.

BACKGROUND: ADAM22 is a member of the ADAM gene family, but the fact that it is expressed only in the nervous systems makes it unique. ADAM22's sequence similarity to other ADAMs suggests it to be an integrin binder and thus to have a role in cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM22, we employed gene targeting to generate ADAM22 knockout mice. RESULTS: ADAM22-deficient mice were produced in a good accordance with the Mendelian ratio and appeared normal at birth. After one week, severe ataxia was observed, and all homozygotes died before weaning, probably due to convulsions. No major histological abnormalities were detected in the cerebral cortex or cerebellum of the homozygous mutants; however, marked hypomyelination of the peripheral nerves was observed. CONCLUSION: The results of our study demonstrate that ADAM22 is closely involved in the correct functioning of the nervous system. Further analysis of ADAM22 will provide clues to understanding the mechanisms of human diseases such as epileptic seizures and peripheral neuropathy.

Biological validation that SF3b is a target of the antitumor macrolide pladienolide.

Pladienolide is a naturally occurring macrolide that binds to the SF3b complex to inhibit mRNA splicing. It has not been fully validated whether the splicing impairment is a relevant mechanism for the potent antitumor activity of pladienolide. We established pladienolide-resistant clones from WiDr and DLD1 colorectal cancer cells that were insensitive to the inhibitory action of pladienolide on cell proliferation and splicing. An mRNA-Seq differential analysis revealed that these two cell lines have an identical mutation at Arg1074 in the gene for SF3B1, which encodes a subunit of the SF3b complex. Reverse expression of the mutant protein transferred pladienolide resistance to WiDr cells. Furthermore, immunoprecipitation analysis using a radiolabeled probe showed that the mutation impaired the binding affinity of paldienolide to its target. These results clearly demonstrate that pladienolide exerts its potent activity by targeting SF3b and also suggest that inhibition of SF3b is a promising drug target for anticancer therapy.

LGI1 and LGI4 bind to ADAM22, ADAM23 and ADAM11.

The transmembrane protein ADAM22 is expressed at high levels in the brain. From its molecular structure, ADAM22 is thought to be an adhesion molecule or a receptor because it has functional disintegrin-like and cysteine-rich sequences in its ectodomain. The phenotypic analysis of ADAM22-deficient mice has indicated the important roles played by ADAM22 in proper neuronal function and peripheral nerve development, however, the precise molecular function of ADAM22 is still unknown. To understand the function of ADAM22 on a molecular basis, we identified ADAM22 binding proteins by using immunoprecipitation and mass spectrometric analysis. This analysis revealed that Leucine-rich glioma inactivated 1 (LGI1) is the most potent ADAM22 binding protein in mouse brain. By our quantitative cell-ELISA system, we demonstrated the specific binding of LGI1 with ADAM22. Furthermore, we showed that LGI4, a putative ADAM22 ligand, also bound to ADAM22. Characterization of the binding specificity of LGI1 and LGI4 suggested that ADAM22 is not a sole receptor, because ADAM11 and ADAM23 had a significant binding ability to LGI1 or LGI4. Therefore, LGI-ADAM system seems to be regulated not only by the affinity but also by the cell-type-specific expression of each protein. Our findings provide new clues to understand the functions of LGI1 and LGI4 as an ADAMs ligand.

Splicing factor SF3b as a target of the antitumor natural product pladienolide.

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter. Despite the unique mechanisms of action and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.

GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast.

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to beta1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029-1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Deltagwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Deltagwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25-32), is due to the properties of Gwt1p itself and not to other membrane components.

Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly.

Glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins are required for the adhesion of pathogenic fungi, such as Candida albicans, to human epithelium. Small molecular inhibitors of the cell surface presentation of GPI-anchored mannoproteins would be promising candidate drugs to block the establishment of fungal infections. Here, we describe a medicinal genetics approach to identifying the gene encoding a novel target protein that is required for the localization of GPI-anchored cell wall mannoproteins. By means of a yeast cell-based screening procedure, we discovered a compound, 1-[4-butylbenzyl]isoquinoline (BIQ), that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae. Treatment of C. albicans cells with this compound resulted in reduced adherence to a rat intestine epithelial cell monolayer. A previously uncharacterized gene YJL091c, named GWT1, was cloned as a dosage-dependent suppressor of the BIQ-induced phenotypes. GWT1 knock-out cells showed similar phenotypes to BIQ-treated wild-type cells in terms of cell wall structure and transcriptional profiles. Two different mutants resistant to BIQ each contained a single missense mutation in the coding region of the GWT1 gene. These results all suggest that the GWT1 gene product is the primary target of the compound.