Ting-Fang Wang

Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2.

Membrane-associated guanylate kinases (MAGUKs) contain multiple protein-binding domains that allow them to assemble specific multiprotein complexes in particular regions of the cell. CASK/LIN-2, a MAGUK required for EGF receptor localization and signalling in Caenorhabditis elegans, contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3 and guanylate kinase-like domains. In adult rat brain, CASK is concentrated at neuronal synapses and binds to the cell-surface proteins neurexin and syndecan and the cytoplasmic proteins Mint/LIN-10 and Veli/LIN-7 (refs 4, 9, 10). Here we report that, through its guanylate kinase domain, CASK interacts with Tbr-1, a T-box transcription factor that is involved in forebrain development. CASK enters the nucleus and binds to a specific DNA sequence (the T-element) in a complex with Tbr-1. CASK acts as a coactivator of Tbr-1 to induce transcription of T-element containing genes, including reelin, a gene that is essential for cerebrocortical development. Our findings show that a MAGUK which is usually associated with cell junctions has a transcription regulation function.

High-throughput screening of soluble recombinant proteins

The aims of high‐throughput (HTP) protein production systems are to obtain well‐expressed and highly soluble proteins, which are preferred candidates for use in structure–function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96‐well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well‐expressed soluble fusion protein, total cell lysates of bacteria culture (∼1.5 mL) were subjected to high‐speed centrifugation in a 96‐tube format and analyzed by multiwell denaturing SDS‐PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome‐wide analysis.

The Transmembrane Domains of Ectoapyrase (CD39) Affect Its Enzymatic Activity and Quaternary Structure

Mammalian ectoapyrase (CD39) is an integral membrane protein with two transmembrane domains and a large extracellular region. The enzymatic activity of ectoapyrase is inhibited by most detergents used for membrane protein solubilization. In contrast, the enzymatic activities of soluble E-type ATPases, including potato tuber (Solanum tuberosum) apyrase and parasite ecto-ATPase, are not affected by detergents. Here we show that ectoapyrase is a tetramer and that detergents that reduce the activity of the enzyme promote dissociation of the tetramer to monomers. We expressed a secreted form of the ectoapyrase in COS-7 cells by fusing the signal peptide of murine CD4 with the extracellular domain of the ectoapyrase. The soluble ectoapyrase is catalytically active and its activity is not affected by detergents. Mutants of the ectoapyrase with only the NH2- or the COOH-terminal transmembrane domain are membrane-bound, and their activity is no longer affected by detergents. The enzymatic activity of all of the mutant proteins is less than that of the native enzyme. These results suggest that the proper contacts between the transmembrane domains of the monomers in the tetramer are necessary for full enzymatic activity.

Golgi Localization and Functional Expression of Human Uridine Diphosphatase

A full-length E(ecto)-ATPase (Plesner, L. (1995)Int. Rev. Cytol. 158, 141–214) cDNA was cloned from a human brain cDNA library; it encodes a 610-amino acid protein that contains two putative transmembrane domains. Heterologous expression of this protein in COS-7 cells caused a significant increase in intracellular membrane-bound nucleoside phosphatase activity. The activity was highest with UDP as substrate and was stimulated by divalent cations in the following order: Ca2+ ≫ Mg2+ > Mn2+. The results of immunofluorescence staining indicate that this protein is located in the Golgi apparatus. UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionophore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golgi apparatus. This is the first identification of a mammalian Golgi luminal UDPase gene. Computer-aided sequence analysis of the EATPase superfamily indicates that the human UDPase is highly similar to two hypothetical proteins of the nematodeCaenorhabditis elegans and to an unidentified 71.9-kDa yeast protein and is less related to the previously identified yeast Golgi GDPase.

Transcriptional Modification by a CASK-Interacting Nucleosome Assembly Protein

CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. Here, we report that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene.

Characterization of brain ecto-apyrase: evidence for only one ecto-apyrase (CD39) gene.

A rat brain cDNA coding for ecto-(Ca,Mg)-apyrase activity was isolated using human CD39 cDNA and functionally expressed in COS-7 cells. The gene codes for a protein with high similarity to human (75% identity) and murine (90% identity) CD39. It is expressed in primary neurons and astrocytes in cell culture as well as in kidney, liver, muscle and spleen. Southern analysis of the mouse genome suggests that there may be a single copy of the ecto-apyrase gene. Interestingly, the human CD39 gene cytologically co-localizes with the susceptibility gene involved in human partial epilepsy with audiogenic symptoms; such a coincidence is consistent with reports on the deficiency of ecto-apyrase activity in the brains of humans with temporal lobe epilepsy and in those of mice with audiogenic seizures.

Widespread expression of ecto-apyrase (CD39) in the central nervous system

We have shown that ecto-apyrase protein is expressed in primary neurons and astrocytes in cell culture (T.-F. Wang, P.A. Rosenberg, G. Guidotti, 1997. Mol. Brain Res. 1997, 47: 295-302). Here we present immunohistochemical studies showing that ecto-apyrase protein is widely distributed in rat brain, as it is present in neurons of the cerebral cortex, hippocampus and cerebellum as well as in glial cells and endothelial cells. Ecto-apyrase is enriched in brain postsynaptic density membrane fractions and is localized in proximity to synaptophysin, the marker of synaptic vesicles. These results together with the observation that P2 purinergic receptors are present throughout the brain suggest that ecto-apyrase is involved in regulating synaptic transmission mediated by extracellular ATP.

An improved SUMO fusion protein system for effective production of native proteins

Expression of recombinant proteins as fusions with SUMO (small ubiquitin‐related modifier) protein has significantly increased the yield of difficult‐to‐express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky‐end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5′‐end and XhoI at the 3′‐end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His6‐Smt3‐X) in which the protein of interest (X) is fused to a hexahistidine (His6)‐tagged Smt3. Smt3 is the yeast SUMO protein. His6‐Smt3‐X was purified by Ni2+ resin. Removal of His6‐Smt3 was performed on the Ni2+ resin by an engineered SUMO protease, His6‐Ulp1403–621‐His6. Because of its dual His6 tags, His6‐Ulp1403–621‐His6 exhibits a high affinity for Ni2 resin and associates with Ni2+ resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni2+‐resin column. The eluant contains only the native target protein. Such a one‐column protocol is useful in developing a better high‐throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult‐to‐produce authentic proteins.

Identification of Tbr‐1/CASK complex target genes in neurons

Tbr‐1, a neuron‐specific T‐box transcription factor, plays a critical role in brain development. Here, we performed a computational search using the non‐palindromic T‐box binding sequence, namely the non‐palindromic T‐element, to determine the putative downstream target genes of Tbr‐1. More than 20 identified genes containing the non‐palindromic T‐element in the 5′ regulatory region were found expressed in brain. Luciferase reporter assays using cultured hippocampal neurons showed that overexpression of Tbr‐1 and CASK‐enhanced promoter activities of some of these putative target genes, including NMDAR subunit 2b (NR2b), glycine transporter, interleukin 7 receptor (IL‐7R) and OX‐2. Among these genes, NR2b promoter responded strongest to overexpression of Tbr‐1 and CASK. Deletion of the non‐palindromic T‐elements from NR2b promoter impaired the induction by Tbr‐1 and CASK. We also examined expression of these target genes in Tbr‐1 knockout mice, it was found that NR2b expression was consistently downregulated. Similarly, both RNA and protein expression levels of NMDAR subunit 1 (NR1), which also contains the non‐palindromic T‐elements in its 5′ regulatory region, were reduced in Tbr‐1 knockout mice. We suggest that Tbr‐1/CASK protein complex regulates expression of these downstream target genes and thus modulates neuronal activity and function.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

SUMMARY The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for “hot topic” research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.