Biomedical subjects

R Rosin-Arbesfeld

Publications and source records attributed to R Rosin-Arbesfeld.

12 recordsLinked to original sources

PubMed2001

Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells.

Adenomatous polyposis coli (APC) is mutated in most colorectal cancers. APC downregulates nuclear beta-catenin, which is thought to be critical for its tumour suppressor function. However, APC may have additional and separate functions at the cell periphery. Here, we examine polarized MDCK and WIF-B hepatoma cells and find that APC is associated with their lateral plasma membranes. This depends on the actin cytoskeleton but not on microtubules, and drug wash-out experiments suggest that APC is delivered continuously to the plasma membrane by a dynamic actin-dependent process. In polarized MDCK cells, APC also clusters at microtubule tips in their basal-most regions. Microtubule depolymerization causes APC to relocalize from these tips to the plasma membrane, indicating two distinct peripheral APC pools that are in equilibrium with each other in these cells. Truncations of APC such as those found in APC mutant cancer cells can neither associate with the plasma membrane nor with microtubule tips. The ability of APC to reach the cell periphery may thus contribute to its tumour suppressor function in the intestinal epithelium.

Actins↗

PubMed2000

The APC tumour suppressor has a nuclear export function.

The adenomatous polpyposis coli (APC) protein is mutated in most colorectal tumours. Nearly all APC mutations are truncations, and many of these terminate in the mutation cluster region located halfway through the protein. In cancer cells expressing mutant APC, beta-catenin is stabilized and translocates into the nucleus to act as a transcriptional co-activator of T-cell factor. During normal development, APC also promotes the destabilization of beta-catenin and Drosophila Armadillo. It does so by binding to the Axin complex which earmarks beta-catenin/Armadillo for degradation by the proteasome pathway. APC has a regulatory role in this process, which is poorly understood. Here we show that APC contains highly conserved nuclear export signals 3' adjacent to the mutation cluster region that enable it to exit from the nucleus. This ability is lost in APC mutant cancer cells, and we provide evidence that beta-catenin accumulates in the nucleus as a result. Thus, the ability of APC to exit from the nucleus appears to be critical for its tumour suppressor function.

Adenomatous Polyposis Coli Protein↗

PubMed2000

Suboptimal splice sites of equine infectious anaemia virus control Rev responsiveness.

The Rev protein of equine infectious anaemia virus (EIAV) was shown previously to stimulate the expression of a heterologous CAT reporter gene when the 3' half of the EIAV genome was present downstream in cis. However, computer analysis could not reveal the existence of a stable RNA secondary structure that could be analogous to the Rev-responsive element of other lentiviruses. In the present study, the inhibitory RNA element designated the cis-acting repressing sequence (CRS) has been localized to the centre of the EIAV genome. The inhibition exerted by this element could be overcome by supplying Rev in trans. The ability of the EIAV CRS to function in a heterologous context suggests that it does not require interactions with other viral proteins. Site-directed mutagenesis showed that the various centrally located suboptimal splice sites of the EIAV genome function as CRS and confer Rev-dependence on the CRS-containing transcripts. In addition, the data suggest that in canine Cf2Th cells, which are highly permissive for EIAV replication, CRS prevents nuclear export of CRS-containing transcripts and the supply of Rev relieves this suppression.

Animals↗

PubMed1998

cDNA excision in stable retroviral cDNA transfectants is prevented by R removal.

The present study provides evidence on the occurrence of DNA rearrangement between the redundant 5' and 3' R domains of equine infectious anemia virus (EIAV) Tat cDNA. This was correlated with a gradual loss of cDNA copy number concomitantly with a decrease in gene expression. Removal of the 5' RU5 abolished rearrangement and stabilized Tat expression in EIAV tat cDNA transfectants. Our data suggest that prior removal of the 5' R from cloned retroviral cDNAs can impede DNA rearrangement, thus preventing cDNA excision that frequently occurs and hinders permanent expression of retroviral cDNAs in stable transfectants.

DNA, Complementary↗

PubMed1998

The Tat protein of equine infectious anemia virus (EIAV) activates cellular gene expression by read-through transcription.

The Tat protein of equine infectious anemia virus, EIAV, was shown to augment viral gene expression, presumably through interaction with the Tat responsive element, TAR. Recently, cell-free polyadenylation assays suggested that perturbation of the EIAV TAR secondary structure diminished polyadenylation efficiency. The present study indicates that the EIAV TAR regulates the efficiency of the 3'-end processing of viral RNA also in transfected cells. Moreover, our data suggest that the provision of the EIAV Tat protein in trans potentiates read-through transcription through the 3' viral long terminal repeat (3' LTR), thus suggesting activation of downstream-located cellular genes.

Animals↗

PubMed1996

Structural studies of the equine infectious anemia virus trans-activator protein.

Trans-activator (tat) proteins are necessary components for the completion of the T replication cycle of lentiviruses. The three-dimensional structure of the equine infectious anemia virus (EIAV) tat protein (e-tat) was studied with CD spectroscopy, NMR spectroscopy, and restrained molecular-dynamics calculations. No stable elements of regular secondary structure were detected, but the sequence regions responsible for nucleic acid binding showed helix-forming tendency, e-tat exhibits a flexible tertiary structure, and only the amino acids comprising the core sequence region form a well-defined tertiary fold. The three-dimensional structure allows discussion of biochemical data as well as data from molecular biological investigations of lentiviral tat proteins.

Amino Acid Sequence↗

PubMed1994

Biological activity and intracellular location of the Tat protein of equine infectious anemia virus.

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10(11) bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by 'scrape loading'. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.

Amino Acid Sequence↗

PubMed1994

Trifluoroethanol stabilizes a helix-turn-helix motif in equine infectious-anemia-virus trans-activator protein.

The solution structure of the 75-amino-acid trans-activator (Tat) protein of the equine infectious-anemia virus in trifluoroethanol-containing solution was determined by two-dimensional and three-dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear-Over-hauser-enhancement distance restraints, and restrained molecular-dynamics simulations. In contrast to the recently determined structure of this protein in trifluoroethanol-free pH 6.3 solution, the hydrophobic core and the adjacent basic RNA-binding region of the protein showed well-defined alpha-helical secondary structure in trifluoroethanol-containing solution. The helical regions comprise those parts of the molecule whose helix-forming tendencies were noted earlier in trifluoroethanol-free solution. Two helices (Gln38-Arg43 and Asp48-Ala64) are connected by a tight type-II turn centered at the strictly conserved Gly46 leading to a helix-turn-helix motif in the core and basic region of the protein. A third helix (Thr9-Asn13) is located in the less well defined N-terminal part of the protein. These observations may support the notion that the protein adopts a helical structure in the RNA-binding region on complex formation. Although the secondary-structure elements become better defined in trifluoroethanol-containing solution, the opposite is true for the hydrophobically stabilized tertiary structure. This adds a caveat to studies of protein structures in trifluoroethanol-containing solution in general.

Amino Acid Sequence↗

PubMed1994

Structure of the equine infectious anemia virus Tat protein.

Trans-activator (Tat) proteins regulate the transcription of lentiviral DNA in the host cell genome. These RNA binding proteins participate in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (HIV) or the equine infectious anemia virus (EIAV). The consensus RNA binding motifs [the trans-activation responsive element (TAR)] of HIV-1 as well as EIAV Tat proteins are well characterized. The structure of the 75-amino acid EIAV Tat protein in solution was determined by two- and three-dimensional nuclear magnetic resonance methods and molecular dynamics calculations. The protein structure exhibits a well-defined hydrophobic core of 15 amino acids that serves as a scaffold for two flexible domains corresponding to the NH2- and COOH-terminal regions. The core region is a strictly conserved sequence region among the known Tat proteins. The structural data can be used to explain several of the observed features of Tat proteins.

Amino Acid Sequence↗

PubMed1993

Equine infectious anemia virus Tat is a predominantly helical protein.

Nuclear magnetic resonance (NMR) spectroscopy revealed features of the secondary structure of the equine infectious anemia virus (EIAV) Tat protein in solution. We could show that this protein, which is required in the replication cycle of lentiviruses, forms a predominantly helical structure in trifluoroethanol/water (40% by vol.) solution. In particular, the basic RNA-binding region and the adjacent core domain, which are highly conserved among lentiviral Tat proteins, show helix-type secondary structure under these conditions. Our observations, in concert with recent biochemical data from other laboratories, suggest that the core sequence region and the basic sequence region form interdependent structural domains, both possibly necessary for correct RNA binding.

Amino Acid Sequence↗

PubMed1993

Sequence-specific resonance assignments of the 1H-NMR spectra of a synthetic, biologically active EIAV Tat protein.

The equine infectious anemia virus (EIAV) trans-activating (Tat) protein is a close homologue of the human immunodeficiency virus (HIV) Tat protein. Both of these proteins bind to an RNA trans-activation responsive element (TAR). We synthesized chemically a protein with the sequence of the 75 amino acid Tat protein from EIAV. The chemically synthesized protein was shown to be biologically active. Circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy were used to structurally characterize the synthetic protein. We obtained nearly complete resonance assignments in the 2D-NMR spectra of the protein at pH 3.0. There is at least some evidence from the experimental data that the basic TAR binding domain of the synthetic protein has a tendency to form a helix, but our experiments also indicate that the protein probably does not have an overall stable tertiary structure in aqueous solution at this pH. CD spectroscopy suggested that the protein adopts a more stable, predominantly alpha-helical structure in a trifluoroethanol/water solution.

Amino Acid Sequence↗

PubMed1993

Structural and functional characterization of rev-like transcripts of equine infectious anemia virus.

Three cDNA clones representing structurally distinct transcripts were isolated from a cDNA library prepared from cells infected with equine infectious anemia virus (EIAV) by using a probe representing the S3 open reading frame, which is thought to encode Rev. One species, designated p2/2, contained four exons and was identical to a previously described polycistronic mRNA that encodes Tat. This transcript was predicted to also direct the synthesis of a truncated form of the transmembrane protein and a putative Rev protein whose N-terminal 29 amino acids, derived from env, are linked to S3 sequences. The second cDNA, p176, also consisted of four exons which were generated by two of three of the same splicing events that occur with p2/2 but not with the Tat mRNA. The alternative splice site giving rise to the second exon of p176 results in a bicistronic message that would encode the same transmembrane and Rev proteins as p2/2. The first exon of the third transcript, p20, was identical to those of p2/2 and p176 but was spliced directly to S3. This monocistronic message could encode a second form of Rev that lacks env sequences, provided that Rev synthesis would initiate at a non-AUG codon. The coding capacity of each cDNA was assessed in a eukaryotic system using S3 antisera. Two putative Rev proteins with apparent molecular masses of 18 and 16 kDa were expressed by p2/2 and p176, while p20 expressed only a 16-kDa species. Analysis of EIAV-infected cells with S3 antisera revealed the presence of an 18-kDa protein. Surprisingly, the same protein was detected in purified virions. By using a reporter construct, the chloramphenicol acetyltransferase gene linked to EIAV env sequences, we were able to demonstrate greatly enhanced chloramphenicol acetyltransferase activity in cells cotransfected with this construct and any of the three cDNAs.

Amino Acid Sequence↗