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PubMed1997

Stress protein (hsp73)-mediated, TAP-independent processing of endogenous, truncated SV40 large T antigen for Db-restricted peptide presentation.

Transporter associated with antigen processing (TAP)-competent and TAP-deficient cell lines were transfected with expression plasmids encoding either the wild-type (wt) large tumor antigen (T-Ag) of SV40, or a truncated cytoplasmic variant (cT-Ag) of this viral protein. Stable expression of comparable levels of both forms of the viral protein was observed in different transfectants. The truncated cT-Ag variant, but not the wtT-Ag was stably associated with the constitutively expressed, cytosolic heat shock protein (hsp)73 chaperone. Two Db-binding peptides and one Kb-binding peptide of T-Ag were presented to cytotoxic T lymphocyte lines (CTLL) by TAP-competent transfectants expressing either wtT-Ag or cT-Ag. TAP-deficient transfectants expressing the wtT-Ag did not present any of these epitopes to CTLL. In contrast, TAP-deficient transfectants expressing the truncated hsp73-associated cT-Ag, presented the two Db-binding epitopes, but not the Kb-binding T-Ag epitope to CTLL. Regurgitation of peptides by transfectants was not detectable. The described data indicate that a pool of post-Golgi Db molecules is available for 2-3 h in TAP-deficient transfectants for loading with peptides released during endolysosomal processing of hsp73-associated, endogenous antigen.

Animals↗

PubMed1989

Activation of c-myb by carboxy-terminal truncation: relationship to transformation of murine haemopoietic cells in vitro.

Murine retroviruses which encode c-myb proteins that have either complete or truncated carboxy (C) termini were used to infect haemopoietic cells from murine fetal liver. Using an agar colony assay, we could show that infection with the virus encoding the truncated protein resulted in the persistence of colony-forming cells well beyond the short period for which such cells are present in uninfected cultures. The resultant colonies failed to give rise to cell lines; however, clonal cell lines occasionally emerged from the original infected liquid cultures. The virus which encoded a myb protein with a complete C-terminus was virtually inactive in the colony assay; surprisingly, however, this virus could enhance proliferation in liquid cultures and has led to the generation of at least one cell line. In addition to demonstrating 'activation' of c-myb by C-terminal truncation, our results imply that an unaltered c-myb protein can also contribute to cellular transformation and that a second event is required to establish myb-transformed cells as a permanent cell line.

Animals↗

PubMed1993

The truncation that generated the v-cbl oncogene reveals an ability for nuclear transport, DNA binding and acute transformation.

The v-cbl oncogene is the transforming gene of the murine Cas NS-1 retrovirus which induces pre-B cell lymphomas and myeloid leukaemias. Sequencing of c-cbl has revealed that v-cbl was generated by a large truncation that removed 60% of the C-terminus of the corresponding protein. In this study we prepared antibodies to cbl and found that c-cbl encodes a 120 kDa protein which is localized in the cytoplasm with a cytosolic and cytoskeletal distribution. Immunofluorescence studies show a striking pattern of brightly staining vesicles in mitotic cells similar to that observed with cytokeratin antibodies. In contrast to p120c-cbl, which is exclusively cytoplasmic, the p100gag-v-cbl encoded by Cas NS-1 is localized in both the cytoplasm and the nucleus. This redistribution to the nucleus correlates with the ability of cbl to induce acute transformation. Furthermore the truncated protein encoded by v-cbl can bind DNA, unlike the full-length protein. These results suggest that the C-terminus of cbl is involved in the retention of p120c-cbl in the cytoplasm and the inhibition of DNA binding. The findings also suggest that a truncated protein encoded by c-cbl exists in the nucleus of normal cells.

3T3 Cells↗

PubMed1994

Effects of truncated activin and FGF receptors and of follistatin on the inducing activities of BVg1 and activin: does activin play a role in mesoderm induction?

Activin and Vg1, two members of the TGF-beta family, are believed to play roles in mesoderm induction and axis formation in the amphibian embryo. Both molecules are provided maternally, either as protein (activin) or as RNA and protein (Vg1), and experiments with a truncated form of a type IIB activin receptor have led to the conclusion that activin is required for induction of mesoderm in vivo. In this paper we first show that truncated versions of two different Xenopus activin receptors also have severe effects on the activity of the mature region of Vg1, suggesting that such receptors may block the function of several members of the TGF-beta family. We go on to demonstrate that follistatin, a secreted protein which binds activin and blocks its activity, does not interfere with Vg1 signalling. Furthermore, overexpression of follistatin mRNA in Xenopus embryos does not perturb mesoderm formation. Taken together, our data show that the effects of truncated activin receptors on Xenopus development can be explained by the inhibition of Vg1 activity, while the lack of effect of follistatin argues against a function for activin in mesoderm induction.

Activin Receptors↗

PubMed2006

Truncated RAR beta isoform enhances proliferation and retinoid resistance.

We previously reported the existence of a truncated isoform of the retinoic acid receptor beta, termed beta prime. Beta prime lacks the N-terminal domains of beta 2 and beta 4, including the DNA-binding domain. However, beta prime is able to heterodimerize and interact with transcription cofactors. To determine the effects of different retinoic acid receptor isoforms on cell proliferation and apoptosis, we transduced retinoid sensitive (MCF7) and retinoid-resistant (MDA-MB-231) cells with retinoic acid receptor beta 2, beta 4, or beta prime. Expression of the truncated beta prime isoform induces resistance to retinoic acid treatment in retinoid sensitive MCF7 cells. In both retinoid sensitive and resistant cells, expression of full-length beta 2 and beta 4 isoforms results in elevated sensitivity to retinoic acid treatment and caspase-independent cell death. Cell death in beta 4 transduced MDA-MB-231 cells was accompanied by metaphase chromosome decondensation and breakage suggestive of mitotic catastrophe. Our results provide evidence that: (a) the truncated form of the retinoic acid receptor beta induces retinoid resistance rather than sensitivity; and (b) alternative pathways of cell death are mediated by different isoforms in breast cancer cells.

Antineoplastic Agents↗

PubMed1988

Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells.

The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2006

Characterization of truncated hantavirus nucleocapsid proteins and their application for serotyping.

Hemorrhagic fever with renal syndrome (HFRS) is a fulminant infectious disease characterized by fever, hemorrhage, renal impairment, and thrombocytopenia. Hantaviruses associated with this belong to different serotypes: Hantaan (HTN), Seoul (SEO), Dobrava/Belgrade (DOB), and Puumala (PUU). The first two, HTN and SEO, are endemic in China. To investigate the epidemiology of HFRS and virus transmission in China, we constructed prokaryotic plasmids encoding truncated recombinant HTN and SEO nucleocapsid proteins (NPs), which lacked 154 amino acid (aa), 99 aa, or 49 aa in the N-terminal region, respectively. After expression, the truncated rNPs were tested as serotyping antigens, particularly for use in the enzyme-linked immunosorbent assay (ELISA). In addition, 68 acute and 52 convalescent sera were collected from HFRS patients from Harbin, Lantian, and Kaifeng regions in China in 2004, which had hantavirus specific antibodies by IFA. A neutralization test was used to differentiate these, which showed that 73 were due to HTN infection, 33 to SEO infection, and 14 undetermined. By ELISA, the truncated rNPs, that lacked 99 (rNP100) or 49 (rNP50) N-terminal amino acids of the NPs of HTN and SEO, were able to differentiate HTNV and SEOV-specific immune sera, but the rNP155 could not. Particularly, the ELISAs based on the rNP50s had a result comparable to PRNT. Thus, the rNP50 is recommended as efficient serotyping antigen for hantavirus infection diagnosis by ELISA.

Animals↗

PubMed2007

Testing the equality of two survival functions with right truncated data.

To compare the survival functions based on right-truncated data, Lagakos et al. proposed a weighted logrank test based on a reverse time scale. This is in contrast to Bilker and Wang, who suggested a semi-parametric version of the Mann-Whitney test by assuming that the distribution of truncation times is known or can be estimated parametrically. The approach of Lagakos et al. is simple and elegant, but the weight function in their method depends on the underlying cumulative hazard functions even under proportional hazards models. On the other hand, a semi-parametric test may have better efficiency, but it may be sensitive to misspecification of the distribution of truncation times. Therefore, this paper proposes a non-parametric test statistic based on the integrated weighted difference between two estimated survival functions in forward time. The comparative results from a simulation study are presented and the implementation of these methods to a real data set is demonstrated.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Overexpression, purification, and kinetic characterization of a carboxyl-terminal-truncated yeast squalene synthetase.

Yeast squalene synthetase which has been truncated by 24 amino acids at the C-terminus has been overexpressed in Escherichia coli and constitutes approximately 20% of the total soluble cell protein. For the first time, milligram quantities of this essential enzyme in the cholesterol biosynthetic pathway have been purified to near homogeneity by ammonium sulfate precipitation and Mono Q anion-exchange chromatography so that the steady-state rate constants could be measured. A combination of 10% methanol, 10% glycerol, 30 mM octyl-beta-D-glucopyranoside, 0.4% Brij-58, and 1 mM dithiothreitol in 25 mM sodium phosphate, pH 7.4, was essential for the stability and maximal enzyme activity of the near homogeneous enzyme. Kinetic analysis indicated a Km for farnesyl pyrophosphate of 2.5 microM, suggesting fairly tight binding of farnesyl pyrophosphate to truncated yeast squalene synthetase. The turnover number, kcat, for the conversion of farnesyl pyrophosphate to squalene was 0.53 s-1, and the apparent second order rate constant, kcat/Km, was 2.1 x 10(5) M-1 s-1, indicating a relatively slow conversion of farnesyl pyrophosphate to squalene and a low specificity constant for this enzyme. In addition, Km for NADPH and NADH was 0.5 and 3.6 mM, respectively. Moreover, truncated yeast squalene synthetase shows a preference for NADPH over NADH as reflected in the sevenfold higher kcat/Km value for NADPH similar to that for the native enzyme.

Chromatography, Ion Exchange↗

PubMed1995

Recombinant human dihydroorotate dehydrogenase: expression, purification, and characterization of a catalytically functional truncated enzyme.

An N-terminally truncated cDNA for human dihydroorotate dehydrogenase (DHODase) was placed under the control of the inducible T7 lac promoter in a pyrimidine auxotrophic strain of Escherichia coli lacking the endogenous enzyme. Induction of gene expression rescued growth in media lacking exogenous pyrimidines. The recombinant enzyme was purified to homogeneity from detergent extracts of bacterial membranes by two chromatographic steps. The purity of the resulting enzyme was judged to be > 95% based on SDS-PAGE with Coomassie staining. The enzyme displays an apparent molecular weight of ca. 40 kDa on SDS-PAGE and ca. 120 kDa on native size-exclusion chromatography, suggesting that the native enzyme is multimeric. Recombinant DHODase displayed a specific activity and Km for dihydroorotate that were similar to those for the enzymes from bovine and human liver tissue. The pH dependence of the activity of the recombinant enzyme was likewise similar to that of the enzyme from human liver and may indicate the involvement of a critical histidine residue in catalytic turnover; only eight histidine residues remain in the truncated version of DHODase used here. The catalytic activity of the recombinant enzyme is inhibited in a dose-dependent fashion by the histidine-selective modifying agent diethylpyrocarbonate. These results further suggest a potential role for histidine in enzyme turnover. Brequinar sodium, an experimental drug which has been shown to be a nanomolar noncompetitive inhibitor of mammalian DHODases, inhibited the activity of the purified recombinant enzyme with a Ki value similar to that for enzyme derived from human liver tissue. The recombinant DHODase thus displays enzymatic behavior similar to the 50-kDa full-length human liver enzyme, illustrating that the catalytically essential structural features of the enzyme, as well as the site of Brequinar binding, are contained within the 40-kDa truncated version of the enzyme that was expressed here.

Amino Acid Sequence↗

PubMed1998

Purification and characterization of maize starch synthase I and its truncated forms.

Comparison of the protein sequences deduced from the cDNAs of maize granule-bound starch synthase, Escherichia coli glycogen synthase, and maize starch synthase I (SSI) reveals that maize SSI contains an N-terminal extension of 93 amino acids. In order to study the properties of maize SSI and to understand the functions of the maize SSI N-terminal extension, the gene coding for full-length SSI (SSI-1) and genes coding for N-terminally truncated SSI (SSI-2 and SSI-3) were individually expressed in E. coli. Here we describe for the first time the purification of a higher plant starch synthase to apparent homogeneity. Its kinetic properties were therefore studied in the absence of interfering amylolytic enzymes. The specific activities of the purified SSI-1, SSI-2, and SSI-3 were 22.5, 33.4, and 26.3 micromol Glc/min/mg of protein, respectively, which are eight times higher than those of partially purified SSI from developing maize endosperm. The full-length recombinant enzyme SSI-1 exhibited properties similar to those of the enzyme from maize endosperm. As observed for native maize enzyme, recombinant SSI-1 exhibited "unprimed" activity without added primer in the presence of 0.5 M citrate. Our results have clearly indicated that the catalytic center of SSI is not located in its N-terminal extension. However, N-terminal truncation decreased the enzyme affinity for amylopectin, with the Km for amylopectin of the truncated SSI-3 being about 60-90% higher than that of the full-length SSI-1. These results suggest that the N-terminal extension in SSI may not be directly involved in enzyme catalysis, but may instead regulate the enzyme binding of alpha-glucans. Additionally, the N-terminal extension may play a role in determining the localization of SSI to specific portions of the starch granule or it may regulate its interactions with other enzymes involved in starch synthesis.

Amino Acid Sequence↗

PubMed1995

Stimulation of DNA replication by truncated Fos and Jun proteins in a cell-free extract of Xenopus eggs.

The effects of truncated Fos (residue 116-211) and/or Jun (residue 224-334) proteins on the DNA replication of plasmid with or without an AP-1 binding site were examined in a cell-free extract of Xenopus eggs. These truncated proteins, which are depleted of the domains necessary for transcriptional activation, stimulated semiconservative DNA replication only in combination and in the presence of plasmid with an AP-1 binding site. These results suggest that truncated Fos and Jun proteins act together to stimulate DNA replication and that activation depends on the presence of an AP-1 binding site in the Xenopus cell-free DNA replication system.

Animals↗

PubMed1995

Truncation of the receptor carboxyl terminus impairs membrane signaling but not ligand binding of human ETB endothelin receptor.

Human ETB endothelin receptor (hETBR) is a heptahelical G-protein-coupled receptor consisting of 442 amino acids whose carboxyl (C)-intracellular region has four and twelve sites for potential palmitoylation and phosphorylation, respectively. In order to elucidate the functional roles of these modification sites, we constructed a series of C-terminal truncated hETBRs and expressed them in Ltk- cells. All the truncated hETBRs showed ligand-binding profiles similar to those of the wild-type hETBR. The truncated receptors holding Cys-402 retained both normal intracellular calcium ([Ca2+]i) response and its rapid desensitization; however, the deleted receptors lacking Cys-402 failed to induce the [Ca2+]i response. These results showed that Cys-402 of hETBR is necessary for its intracellular calcium signaling and that at least ten of twelve putative phosphorylation sites are irresponsible for the agonist-induced desensitization.

Amino Acid Sequence↗

PubMed2000

TTIP is a novel protein that interacts with the truncated T1 TrkB neurotrophin receptor.

Alternative splicing of the TrkB gene produces a full length tyrosine kinase receptor as well as two truncated isoforms that contain extracellular and transmembrane domains but lack the kinase domain and have unique C terminal tails. The function of the truncated TrkB isoforms is unclear and to gain insights into their function, we have isolated a protein from 15N neuroblastoma cells that specifically binds the TrkB.T1 isoform. Pulldown experiments using a GST fusion protein containing the TrkB.T1 intracellular domain identified a 61 kDa protein from radiolabeled 15N lysates. Coimmunoprecipitation experiments showed that the 61 kDa protein interacted with epitope-tagged TrkB.T1 overexpressed in 15N cells as well as with TrkB.T1 which was endogenously expressed. Peptide competition experiments revealed that the protein, designated TTIP (for Truncated TrkB Interacting Protein), showed specific binding to the TrkB.T1 tail. MALDI MS and MS/MS analysis has revealed that TTIP is a novel protein not yet listed in the current databases.

Alternative Splicing↗

PubMed1997

Thioredoxin expression and localization in human cell lines: detection of full-length and truncated species.

Thioredoxin (Trx) is an intracellular multifunctional 12-kDa protein with a reduction/oxidation (redox) active disulfide constitutively expressed by most cells of the human body. Trx can also be released by cells such as lymphocytes upon activation or oxidative stress exposure and exert a cocytokine and cytoprotective activity. In addition, a truncated 10-kDa form of Trx has been reported. In order to better understand the function of full-length and truncated Trx, we have produced, for the first time, specific monoclonal antibodies, which can discriminate between the two forms. Using these novel antibodies, designated alpha Trx1 to alpha Trx4, a panel of cell lines derived from human B and T lymphocytes, monocytes, granulocytes, and melanomas was analyzed by immunochemical techniques. The cellular distribution differed between the two forms. All lines contained full-length Trx, also located to a minor extent on the cell surface. One exception was the melanoma cell line FM28.4, which did not show any Trx expression. Truncated Trx was present in most cells in minimal amounts only, whereas the monocytic cell lines THP-1 and U-937 expressed high amounts on the cell surface, as shown by flow cytometric analysis of living cells and confocal laser-scanning microscopy. The biological importance and function of the short versus long forms of Trx as detected by the antibodies are discussed.

Amino Acid Sequence↗

PubMed1999

Laminin alpha chains in colon carcinoma cell lines: detection of a truncated laminin alpha1 mRNA in Caco-2 cells.

Changes in basement membrane structure are known to accompany carcinoma formation. We analyzed laminin alpha1 and alpha5 chains in colon carcinoma cell lines. Variable levels of the Mr 380,000 alpha5 laminin protein and 11-kb alpha5 laminin mRNA were noted. In contrast, laminin alpha1 protein was not synthesized by any of the colon carcinoma cell lines tested. Northern blotting revealed expression of a 10-kb laminin alpha1 mRNA only in control cells. Unexpectedly, expression of a truncated laminin alpha1 message of approximately 8 kb was found in one cell line, the adenocarcinoma cell line Caco-2. By RT-PCR and Northern blotting a deletion in the laminin alpha1 mRNA was mapped to the 5' end, spanning nucleotides 41-1835. The deletion spans the translation start site, explaining the complete lack of the protein. Southern blotting of genomic Caco-2 DNA did not reveal any larger truncation, suggesting a point mutation manifested at the posttranscriptional level. The identified truncation is the first genetic defect described for the laminin alpha1 chain and suggests that mutations in the LAM A1 gene might underlie the observed lack of the laminin alpha1 chain in some colon carcinomas.

Caco-2 Cells↗

PubMed1994

Rapid detection of translation-terminating mutations at the adenomatous polyposis coli (APC) gene by direct protein truncation test.

Familial adenomatous polyposis (FAP) is usually associated with protein truncating mutations in the adenomatous polyposis coli (APC) gene. The APC mutations are known to play a major role in colorectal carcinogenesis. For the identification of protein truncating mutations of the APC gene, we developed a rapid, sensitive, and direct screening procedure. The technique is based on the in vitro transcription and translation of the genomic PCR products and is called the protein truncation test. Samples of DNA from individual FAP patients, members of a FAP family, colorectal tumors, and colorectal tumor-derived cell lines were used to show the effectiveness of this method.

Adenomatous Polyposis Coli↗

PubMed1994

Preliminary X-ray analysis of a truncated form of recombinant fructose-2,6-bisphosphatase.

The bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and a C-terminal 30 amino acid truncated form were expressed in high yield in Escherichia coli and purified to homogeneity. The separately expressed bisphosphatase domain and its C-terminal truncated form had kinetic properties similar to the bisphosphatase of the intact bifunctional enzyme, but their turnover numbers were fourfold higher. The truncated enzyme crystallized in space group P1 with two molecules per asymmetric unit. The determined cell dimensions are: a = 41.9 A, b = 43.5 A, c = 57.6 A, alpha = 95.2 degrees, beta = 99.3 degrees, and gamma = 106.2 degrees. These crystals diffract to 2.0 A resolution when exposed to synchrotron radiation and are suitable for crystallographic structure analysis.

Animals↗