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PubMed2001

The crystal structures of glutathione S-transferases isozymes 1-3 and 1-4 from Anopheles dirus species B.

Glutathione S-transferases (GSTs) are dimeric proteins that play an important role in cellular detoxification. Four GSTs from the mosquito Anopheles dirus species B (Ad), an important malaria vector in South East Asia, are produced by alternate splicing of a single transcription product and were previously shown to have detoxifying activity towards pesticides such as DDT. We have determined the crystal structures for two of these alternatively spliced proteins, AdGST1-3 (complexed with glutathione) and AdGST1-4 (apo form), at 1.75 and 2.45 A resolution, respectively. These GST isozymes show differences from the related GST from the Australian sheep blowfly Lucilia cuprina; in particular, the presence of a C-terminal helix forming part of the active site. This helix causes the active site of the Anopheles GSTs to be enclosed. The glutathione-binding helix alpha2 and flanking residues are disordered in the AdGST1-4 (apo) structure, yet ordered in the AdGST1-3 (GSH-bound) structure, suggesting that insect GSTs operate with an induced fit mechanism similar to that found in the plant phi- and human pi-class GSTs. Despite the high overall sequence identities, the active site residues of AdGST1-4 and AdGST1-3 have different conformations.

Alternative Splicing↗
PubMed1995

Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon.

The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal beta gene products, fibroblast TM-1 and beta skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed beta gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other beta gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The beta tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.

Actins↗
PubMed2002

Assay and screening methods for bioactive substances based on cellular signaling pathways.

Assay and screening methods for bioactive substances based on cellular signaling pathways are presented. Examples include: (1) intracellular protein phosphorylation and protein-protein interaction, (1-i) a new assay method for evaluating chemical selectivity of agonists for insulin signaling pathways based on agonist-induced phosphorylation of a target peptide, (1-ii) an SPR-based screening method for agonist selectivity for insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein, (1-iii) a fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways, and (1-iv) split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing; (2) a screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling; (3) a screening method for substrates of multidrug resistance-associated protein (MRP); and (4) fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2002

Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag.

The class 1 protein (PorA) is a major component of the outer membrane of Neisseria meningitidis and functions as a cationic porin. The protein is particularly effective in generating a bactericidal immune response following infection and is therefore under investigation as a potential antigen for inclusion in new meningococcal vaccines. Studies on the vaccine potential of PorA would be facilitated by the production of pure protein, free from other components of the meningococcal outer membrane. In the current study, PorA was expressed from the heterologous host Escherichia coli as a C-terminal fusion to an inducible protein-splicing element (intein) with an N-terminal chitin-binding domain (CBD) (IMPACT-TWIN system). The CBD acted as an affinity tag and allowed binding of the fusion protein to a chitin bead column, after which self-cleavage of the intein at its C-terminus was induced, resulting in the release of mature PorA. Cleavage of the fusion protein was temperature- and time-dependent, and was optimal at pH 7.0 after 5 days of storage at 4 degrees C. Efficient cleavage was also dependent on the addition of a minimal amino acid sequence (Gly-Arg-Ala) to the N-terminus of the mature PorA protein. This represented a significant improvement on the large N-terminal sequences introduced by other expression systems previously used to prepare recombinant PorA, and the yields of PorA purified with the IMPACT-TWIN system were similar. Thus, the IMPACT-TWIN system provides a facile method for producing recombinant PorA and may also be useful for the production of other bacterial outer-membrane proteins for vaccine studies.

Affinity Labels↗
PubMedDate not supplied

[The regulatory role of the Hfq protein in bacterial cells].

Hfq (HF1) is a polyfunctional translational regulator of numerous bacterial mRNAs. Its physico-chemical properties, possible mechanisms regulating bacterial translation, and the effect of Hfq on transcription of several mRNAs are considered. A similarity of Hfq and eukaryotic mRNA splicing proteins is discussed. Data are reviewed on the roles of Hfq in poly(A) metabolism and formation of the bacterial nucleoid and on the Hfq interaction with untranslated regulatory RNAs.

Bacteria↗
PubMed1996

Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with P-32, a protein co-purified with splicing factor SF2. Hyaluronic acid-binding protein as P-32 protein, co-purified with splicing factor SF2.

The purification of a 68-kDa hyaluronic acid-binding protein (HA-binding protein), a homodimer of 34 kDa that binds specifically to hyaluronic acid, has been reported earlier by us (Gupta, S., Batchu, R.B., and Datta, K. (1991) Eur. J. Cell Biol. 56, 58-67). Here, we report the isolation of a partial cDNA clone from a lambda gt11 cDNA expression library of human skin fibroblast by immuno-screening with HA-binding protein antiserum. The internal polypeptide sequence (83 residues) of the purified hyaluronic acid-binding protein is identical to the predicted protein sequence derived from hyaluronic acid-binding protein cDNA, suggesting the authenticity of the clone. Interestingly, this hyaluronic acid-binding protein cDNA sequence has complete homology with the cDNA sequence of a protein P-32, co-purified with the human pre-mRNA splicing factor SF2 (Krainer, A.R., Mayeda, A., Kozak, D., and Binns, G. (1991) Cell 66, 383-394). Furthermore, the data on the N-terminal sequence of hyaluronic acid-binding protein and the predicted polypeptide of P-32 revealed the identical coding sequence of 209 amino acids for both the proteins. As the identity and functional characterization of P-32 have not yet been reported, P-32 cDNA was expressed in Escherichia coli, and the recombinant P-32 protein was purified by hyaluronic acid-Sepharose affinity chromatography. The recombinant P-32 protein showed immunocross-reactivity with the polyclonal antibodies raised against HA-binding protein. The predicted amino acid sequence of the protein fulfilled the minimal criteria for binding to hyaluronic acid, i.e. two basic amino acids flanking a seven-amino acid stretch, as reported for other hyaluronic acid affinity of the recombinant P-32 protein was confirmed by biotinylated hyaluronic acid binding assay. The binding of recombinant P-32 protein to biotinylated hyaluronic acid binding assay. The binding of recombinant P-32 protein to biotinylated hyaluronic acid can be competed only with excess unlabeled hyaluronic acid, confirming its specificity toward hyaluronic acid. All these results suggest that both P-32, co-purified with the human pre-mRNA splicing factor SF2, and 34-kDa hyaluronic acid-binding protein reported by us are the same protein and that it is a new member of the hyaluronic acid-binding protein family, the "hyaladherins."

Amino Acid Sequence↗
PubMed2004

[Function of Tra2beta1 proteins in the splicing of neural-specific genes].

The in vitro studies have shown that Tra2beta1 proteins are important regulators in the alternative pre-mRNA splicing in mammalians. To date, the knowledge regarding the in vivo function of Tra2beta1 proteins, especially the function in regulating the splicing of neural-specific genes and the cell-type specificity of the functions is very limited. In the present study, the cell-type specific splicing of two neural-specific genes (GluR-B and SMN2), and the function of Tra2beta1 proteins in regulation of the splicing were studied in two types of cells (COS-1 and PFSK-1) using in vivo splicing models. The results showed that the splicing of GluR-B and SMN2 minigenes is cell-type specific, while Tra2beta1 proteins regulate the splicing of the minigenes in both cell lines in a similar pattern. It indicates that the Tra2beta1-regulated splicing of GluR-B and SMN2 minigenes regulated by Tra2beta1 is not cell-type specific.

Alternative Splicing↗
PubMed2005

Intein-mediated protein trans-splicing and transgene containment in plastids.

Transgenes in plastids are contained by stringent maternal inheritance in most cultivated plant species and their expression yields high levels of protein with bona fide structure. Nevertheless, transfer of plastid genes to the nucleus has been reported, with implications for transgene containment. The significance of these transfers will depend on the likelihood that they will become functional nuclear genes. Recently a novel approach, intein-mediated protein trans-splicing, has been demonstrated promising to yield transgenic plants with greatly reduced risk of genetic outcrossing.

Inteins↗
PubMed2004

Versatile protein biotinylation strategies for potential high-throughput proteomics.

We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo.

Biotinylation↗
PubMed2000

Differential expression of four human dynamin-like protein variants in brain tumors.

Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have suggested that it is involved in the formation of coated vesicles. In this report, the differential expression of four human dynamin-like protein splice variants (HdynIV-wildtype [WT], -11, -26, and -37) from various brain tumors was identified by reverse transcription/polymerase chain reaction (RT-PCR). One novel variant (HdynIV-11), not described previously, was identified. The four alternatively spliced variants exhibited tissue specificity in normal tissues. The HdynIV-WT was strongly expressed in the brain, whereas HdynIV-37 was expressed in all tissues examined. Moreover, HdynIV-26 was dominant in the liver and apparently overexpressed in all astrocytomas and most meningiomas and adenomas. This report suggests that HdynIV-26 may cause aberrant protein trafficking and alter vesicle formation in brain tumors. Our results also suggest that dynamin-like protein is associated with various brain tumors and, more importantly, that aberrant expression of the HdynIV-26 variant may play a role in brain tumorigenesis.

Adenoma↗
PubMed2000

Lobster GABA receptor subunit expressed in neural tissues.

A cDNA encoding an ionotropic gamma-aminobutyric acid (GABA) receptor subunit was isolated from a lobster (Homarus americanus) cDNA library. A longer version of this cDNA, containing a 108-bp insert, was also detected. The two cDNAs are predicted to encode alternatively spliced proteins of 485 and 521 amino acids, respectively. The sequences were most similar to the Drosophila RDL (resistance to dieldrin) GABA subunit with 54% identity, and 30-35% identity with vertebrate ionotropic GABA receptor subunits. Only the shorter clone formed functional ion channels when transfected into human embryonic kidney (HEK) 293 cells. GABA caused a Cl(-)-selective current in the presence of GABA that was blocked by picrotoxin. The GABA-induced current was weakly sensitive to the GABA(A) antagonist, bicuculline, but was enhanced by pentobarbital. Expression of the GABA receptor mRNA was highest in brain and the olfactory organ, but was not detected in leg muscle. These data suggest that the isolated cDNAs are likely to encode proteins that comprise subunits of native GABA receptors expressed in olfactory receptor neurons and projection neurons of the olfactory deutocerebrum.

Alternative Splicing↗
PubMed2004

The pre-mRNA-splicing factor SF3a66 functions as a microtubule-binding and -bundling protein.

SF3a (splicing factor 3a) complex is an essential component of U2 snRNPs (small nuclear ribonucleoprotein particles), which are involved in pre-mRNA splicing. This complex consists of three subunits: SF3a60, SF3a66 and SF3a120. Here, we report a possible non-canonical function of a well-characterized RNA-splicing factor, SF3a66. Ectopic expression experiments using each SF3a subunit in N1E 115 neuroblastoma cells reveals that SF3a66 alone can induce neurite extension, suggesting that SF3a66 functions in the regulation of cell morphology. A screen for proteins that bind to SF3a66 clarifies that SF3a66 binds to beta-tubulin, and also to microtubules, with high affinity, indicating that SF3a66 is a novel MAP (microtubule-associated protein). Electron microscopy experiments show that SF3a66 can bundle microtubules, and that bundling of microtubules is due to cross-bridging of microtubules by high-molecular-mass complexes of oligomerized SF3a66. These results indicate that SF3a66 is likely to be a novel MAP, and can function as a microtubule-bundling protein independently of RNA splicing.

Animals↗
PubMed2006

Identification of a family of DNA-binding proteins with homology to RNA splicing factors.

We describe a unique family of human proteins that are capable of binding to the cAMP regulatory element (CRE) and that are homologous to RNA splicing proteins. A human cDNA was isolated that encodes a protein with a distinctive combination of modular domain structures: 2 leucine-zipper-like domains, a DNA-binding zinc-finger-like domain, an RNA-binding zinc-finger-like domain, and 2 coiled-coil protein-protein interaction domains. It also has a serine-arginine-rich domain, commonly found in proteins involved in RNA splicing. The protein was discovered using the CRE as bait in a yeast 1-hybrid assay. It was then shown to bind specifically to the CRE in vitro using gel shift assays. We have named the protein CRE-associated protein (CREAP). We show that it is widely expressed in human tissues but is highly expressed in several fetal tissues and in several regions of the adult brain. CREAP is closely related to 2 human proteins of unknown function. CREAP shows significant homology with a small nuclear ribonucleoprotein of yeast, Luc7p, involved in 5' splice site recognition. The 3 human CREAP proteins form a unique family with the potential to act as transcription factors that link to RNA processing.

Amino Acid Sequence↗
PubMed2000

Cloning and characterization of SCHIP-1, a novel protein interacting specifically with spliced isoforms and naturally occurring mutant NF2 proteins.

The neurofibromatosis type 2 (NF2) protein, known as schwannomin or merlin, is a tumor suppressor involved in NF2-associated and sporadic schwannomas and meningiomas. It is closely related to the ezrin-radixin-moesin family members, implicated in linking membrane proteins to the cytoskeleton. The molecular mechanism allowing schwannomin to function as a tumor suppressor is unknown. In attempt to shed light on schwannomin function, we have identified a novel coiled-coil protein, SCHIP-1, that specifically associates with schwannomin in vitro and in vivo. Within its coiled-coil region, this protein is homologous to human FEZ proteins and the related Caenorhabditis elegans gene product UNC-76. Immunofluorescent staining of transiently transfected cells shows a partial colocalization of SCHIP-1 and schwannomin, beneath the cytoplasmic membrane. Surprisingly, immunoprecipitation assays reveal that in a cellular context, association with SCHIP-1 can be observed only with some naturally occurring mutants of schwannomin, or a schwannomin spliced isoform lacking exons 2 and 3, but not with the schwannomin isoform exhibiting growth-suppressive activity. Our observations suggest that SCHIP-1 interaction with schwannomin is regulated by conformational changes in schwannomin, possibly induced by posttranslational modifications, alternative splicing, or mutations.

Amino Acid Sequence↗
PubMed1997

Chondroitin sulphate composition and structure in alternatively spliced CD44 fusion proteins.

Previous studies have indicated that CD44 isoforms, spliced with variant exons, are heterogeneously glycanated with chondroitin sulphate and heparan sulphate chains. Because such alternative splicing may regulate divergent biological effects of the specific isoforms, we analysed the consequences of this process on the composition and structure of the chondroitin-sulphate chains. Recombinant chimaeras were engineered with and without exons V3-10 or V3,8-10 and expressed as Ig fusion proteins in COS cells. In addition, the chondroitin sulphates of wild-type isoforms were contrasted with those of isoforms mutated with serine-to-alanine codon substitutions at a putative Ser-Gly-Ser-Gly glycosaminoglycan acceptor site within exon V3. The chondroitin sulphates contained both 4- and 6-sulphated galactosamine residues, although there was a high content of non-sulphated galactosamine-containing repeat units. Splicing of exons V4-7, which contain no Ser-Gly consensus motifs, resulted in increased glycanation with chondroitin-sulphate chains, as well as increased sulphation levels of the polymers. Comparison of wild-type and acceptor-site mutant isoforms showed that chondroitin-sulphate content declined by more than 60-80% in the mutant, indicating that assembly of chondroitin-sulphate chains occurs there, and a general decrease in the sulphation level of the remaining chains was observed. Undersulphation of the recombinant chondroitin sulphates was shown by parallel analyses with native human keratinocyte CD44 molecules and is most probably an artifact of transient expression in COS cells. Our data indicate that combinatorial exon splicing exerts complex and distal effects on glycanation patterns and structure, which presumably modulate those functions that may be mediated though the chondroitin-sulphate moieties, such as motility and matrix invasion.

Alternative Splicing↗
PubMed1992

Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae.

An unusual protein splicing reaction joins the N-terminal segment (A) and the C-terminal segment (C) of the 119K primary translation product (ABC) of the yeast VMA1 gene to yield a 69K vacuolar H(+)-ATPase subunit (AC) and an internal 50K polypeptide (B). This 50K protein is a site-specific DNA endonuclease that shares 34% identity with the homothallic switching endonuclease. The site cleaved by the VMA1-derived endonuclease exists in a VMA1 allele that lacks the derived endonuclease segment of the open reading frame. Cleavage at this site only occurs during meiosis and initiates 'homing', a genetic event that converts a VMA1 allele lacking the endonuclease coding sequence into one that contains it.

Alleles↗
PubMed2004

Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms.

Chronic infection with the hepatitis B virus (HBV) is a major risk factor for development of hepatocellular carcinoma (HCC). The pathogenesis of cancer in HBV infection has been extensively analyzed, and multiple factors appear to play a role. A major factor is chronic inflammation and the effects of cytokines in the development of fibrosis and liver cell proliferation. Also important is the role of integration of HBV DNA into host cellular DNA, which, in some situations, acts to disrupt or promote expression of cellular genes that are important in cell growth and differentiation. In addition, expression of HBV proteins may have a direct effect on cellular functions, and some of these gene products can favor malignant transformation. Several HBV genes have been found in infected tissues more frequently than others, including truncated pre-S2/S, hepatitis B X gene, and a novel spliced transcript of HBV, referred to as the hepatitis B spliced protein. The proteins expressed from these integrated genes have been shown to have intracellular activities that may account for their association with HCC, including effects on cellular growth and apoptosis. Finally, some patients with HCC have no detectable hepatitis B surface antigen in serum but do have low levels of HBV DNA in serum and integrated molecules of HBV DNA in tissue. Occult HBV infection may account for a proportion of cases of HCC that occur in patients without serologic markers for hepatitis B and C and may be a cofactor in HCC in patients with chronic hepatitis C who have coexistent occult HBV infection.

Apoptosis↗
PubMed2004

Homology modeling and mutational analysis of Ho endonuclease of yeast.

Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

Amino Acid Sequence↗