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Biomedical subjects

G Chinnadurai

Publications and source records attributed to G Chinnadurai.

At least 55 records · Page 3Linked to original sources
PubMed1996

Deletion of a nonconserved region of Bcl-2 confers a novel gain of function: suppression of apoptosis with concomitant cell proliferation.

The Bcl-2 protein coded by the proto-oncogene bcl-2 is expressed in a variety of embryonic and postnatal tissues and is overproduced in several types of tumours. Bcl-2 expression suppresses apoptosis induced by a multitude of stimuli in diverse cell types without exerting significant effects on cell proliferation, and is believed to contribute to oncogenesis by extending cell survival. In certain B-cell lymphomas, chromosomal translocations result in a gain of function of Bcl-2 by overexpression. Here, we report that a deletion of a nonconserved region of human Bcl-2 (residues 51-85) confers a novel gain of function that not only suppresses apoptosis induced by the tumor suppressor protein p53 and the Myc oncoprotein but also permits continued cell proliferation. Our result raises the possibility that mutations within the bcl-2 gene may contribute to oncogenesis by both suppressing apoptosis and facilitating cell proliferation.

Amino Acid Sequence↗
PubMed1996

Unmasking of a proliferation-restraining activity of the anti-apoptosis protein EBV BHRF1.

The BHRF1 protein of Epstein-Barr virus (EBV) is a structural and functional homolog of the Bcl-2 protein. Both BHRF1 and Bcl-2 proteins promote the survival of cells exposed to various apoptotic stimuli. This promotion of cell survival is associated with a block in proliferation. It is believed that the Bcl-2 family of anti-apoptosis proteins contribute to oncogenesis merely by promoting cell survival. We have discovered that mutations within a regulatory domain of the BHRF1 protein not only suppress apoptosis induced by the tumor suppressor protein p53, but also permit efficient proliferation of cells that would otherwise undergo total apoptosis. These gain-of-function mutants of BHRF1 cooperate more efficiently with the E1a oncogene in transformation of primary rat kidney cells where E1A expression results in apoptosis. Our results suggest that such mutational inactivation of a proliferation-restraining activity in the BHRF1 gene may play a direct role in oncogenesis.

Amino Acid Sequence↗
PubMed1996

Identification of a cellular protein that specifically interacts with the essential cysteine region of the HIV-1 Tat transactivator.

The Tat protein of the human immunodeficiency virus (HIV) is a powerful activator of HIV gene expression. Genetic and biochemical evidence suggests that one or more cellular cofactors may be important for Tat activity. We have used two-hybrid interactive cloning in yeast to identify a partial cDNA clone (clone 10) from a human B-lymphoblastoid library that specifically interacts with the N-terminal 31 amino acids of HIV-1 Tat which contains the essential cysteine-rich portion of the Tat activation domain. The encoded protein also binds to purified Tat in vitro. Mutation of single essential cysteine residues in Tat abolishes interaction between Tat and clone 10, suggesting that interaction with the encoded protein is important for Tat activity. We have identified the full-length cDNA for the Tat binding protein and shown that overexpression of the encoded protein, Tip60 (Tat interactive protein, 60 kDa), results in a fourfold augmentation of Tat transactivation of the HIV-1 promoter in transient expression assays without increasing the basal activity of the HIV promoter or activating the heterologous RSV promoter. These data together with the genetic and in vitro binding data support the notion that Tip60 might be a cofactor of Tat involved in the regulation of HIV gene expression.

Acetyltransferases↗
PubMed1995

Functional substitution identifies a cell survival promoting domain common to adenovirus E1B 19 kDa and Bcl-2 proteins.

The adenovirus E1B 19 kDa protein provides a cell survival function during adenovirus infection and facilitates efficient virus replication by preventing premature cell death. These functions resemble those performed by the protein coded by the proto-oncogene Bcl-1. The Bcl-1 protein, which provides a survival function in cells exposed to a number of cell death-inducing stimuli, can substitute for the 19 kDa protein during adenovirus infection. Although these two survival- promoting proteins are not overtly related by primary amino acid sequence, they appear to share short homologous sequences. In order to determine if these sequence motifs constitute common functional domains, we carried out domain exchanges between the 19 kDa and Bcl-2 proteins. Our results indicate that a seven amino acid region of the Bcl-2 protein (108-YRRDFAE-114) can efficiently substitute for the corresponding 19 kDa domain (47-YKWEFEE-53). Mutagenizing this domain in Bcl-2 abolishes the survival promoting activity of Bcl-2. Substitution of the kDa sequences into Bcl-2 restores the survival promoting activity, albeit at reduced levels. Our results suggest that this domain (designated NH1) may constitute a common functional domain for the 19 kDa protein and survival promoting members of the Bcl-2 family of proteins.

Adenovirus E1B Proteins↗
PubMed1995

A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions.

Regulation of the cell death program involves physical interactions between different members of the Bcl-2 family that either promote or suppress apoptosis. The Bcl-2 homolog, Bak, promotes apoptosis and binds anti-apoptotic family members including Bcl-2 and Bcl-xL. We have identified a domain in Bak that is both necessary and sufficient for cytotoxic activity and binding to Bcl-xL. Sequences similar to this domain were identified in Bax and Bip1, two other proteins that promote apoptosis and interact with Bcl-xL, and were likewise critical for their capacity to kill cells and bind Bcl-xL. Thus, the domain is of central importance in mediating the function of multiple cell death-regulatory proteins that interact with Bcl-2 family members.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of a cellular phosphoprotein that interacts with a conserved C-terminal domain of adenovirus E1A involved in negative modulation of oncogenic transformation.

The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP). We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.

Adenovirus E1A Proteins↗
PubMed1995

p53-independent apoptotic and necrotic cell deaths induced by adenovirus infection: suppression by E1B 19K and Bcl-2 proteins.

Adenovirus E1B 19K protein prevents premature death of adenovirus-infected cells. Viral mutants (19K mutants) defective in the 19K protein induce enhanced cell death, resulting in fragmentation of viral and cellular DNA. The 19K protein can also suppress the effects of certain external cell death-inducing stimuli, such as tumor necrosis factor alpha and various DNA-damaging agents that induce apoptosis. We have examined viral infection of permissive human cells and nonpermissive rat cells to determine if the 19K mutant induces apoptotic or necrotic type of cell death. Infection of normal rat kidney cells with an adenovirus type 2 19K deletion mutant induces internucleosomal DNA fragmentation and condensation of nuclear chromatin. Electron microscopic examination of these cells revealed the presence of condensed subnuclear bodies characteristic of apoptosis. In contrast, infection of human A549 cells induces random DNA fragmentation, and these cells do not exhibit characteristic condensation of the nuclear chromatin but contain enlarged nuclei loaded with virus particles. Therefore, it appears that adenovirus infection induces both apoptotic and necrotic types of cell death, depending on the cell type. Both types of cell death can be suppressed by E1B 19K protein. Similarly, a recombinant adenovirus expressing the human Bcl-2 protein but lacking the E1B proteins can efficiently suppress both apoptotic and necrotic cell death induced by adenovirus infection. The requirement of p53 tumor suppressor protein in adenovirus-induced cell death was investigated by infection of human Saos2 and mouse p53 nullizygous (p53-/-) cells lacking p53 tumor suppressor protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviridae Infections↗
PubMed1994

Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins.

Adenovirus E1B 19 kDa protein protects against cell death induced by viral infection and certain external stimuli. The Bcl-2 protein can functionally substitute for the E1B 19 kDa protein. To identify cellular targets for the 19 kDa protein, we used the two-hybrid screen in yeast. We have isolated cDNAs for three different proteins, designated Nip1, Nip2, and Nip3, that interact with the 19 kDa protein. Mutational analysis indicates that these proteins do not associate with 19 kDa mutants defective in suppression of cell death, suggesting a correlation between interaction of these proteins and suppression of cell death. These proteins also associate with discrete sequence motifs in the Bcl-2 protein that are homologous to motifs of the 19 kDa protein. Our results suggest that two diverse proteins, the E1B 19 kDa and the Bcl-2 proteins, promote cell survival through interaction with a common set of cellular proteins.

Adenovirus E1B Proteins↗
PubMed1994

The activation region of the Tat protein of human immunodeficiency virus type-1 functions in yeast.

The N-terminal 48 amino acids of the Tat protein of human immunodeficiency virus type (HIV)-1 constitute its activation region. This region can autonomously activate transcription when targeted to the HIV-1 long terminal repeat or certain heterologous promoters either through DNA binding sites located upstream of the transcription initiation site or via downstream RNA binding sites in mammalian cells. To determine whether the Tat activation region can function in yeast, we have assayed the effect of a chimeric gene (GAL-Tat48) expressing the DNA binding domain of the yeast transcription factor Gal4 (residues 1-147) and the activation region of Tat on GAL1 promoter-directed expression of the lacZ reporter gene in Saccharomyces cerevisiae. Our results indicate that the Gal-Tat48 fusion protein can induce significant activation of the GAL1 promoter. Analysis of a number of Tat mutants located within the activation region indicate that the amino acid residues of Tat essential for trans-activation in mammalian cells are also required for transactivation in yeast. Our results suggest that Tat-mediated transcriptional activation may involve a mechanism conserved among yeast and mammalian cells.

Gene Products, tat↗
PubMed1994

Epstein-Barr virus BHRF1 protein protects against cell death induced by DNA-damaging agents and heterologous viral infection.

The BHRF1 open reading frame of Epstein-Barr virus (EBV) codes for a 191-amino-acid protein that is expressed at high levels in productively infected cells and in certain latently infected cells. The BHRF1-coding sequences are conserved among all EBV isolates examined. BHRF1 shares a distant colinear amino acid sequence homology with the protein coded by the Bcl-2 protooncogene which suppresses apoptotic cell death induced by multiple stimuli. We have established Chinese hamster ovary cell lines which constitutively express the BHRF1 protein and show that this viral protein can protect against apoptosis induced by the DNA-damaging agents cisplatin, etoposide, and mitomycin C. The BHRF1 protein also strongly suppresses DNA fragmentation induced by an adenovirus E1B 19K deletion mutant. These results suggest that the BHRF1 protein is functionally similar to Bcl-2 and adenovirus E1B 19-kDa proteins. Since BHRF1 efficiently suppresses apoptosis induced by anti-cancer agents, its expression may have important implications for the chemotherapy of EBV-induced malignancies.

Animals↗
PubMed1993

Mutational analysis of the transforming and apoptosis suppression activities of the adenovirus E1B 175R protein.

The role of the adenovirus-2 E1B 19-kDa (175R) T antigen in E1a-cooperative transformation was determined by cotransfection of plasmids expressing E1A or E1B 175R T antigens into primary rat kidney (BRK) cells. Transformed cells were selected by virtue of their resistance to the antibiotic Geneticin (G418) conferred by neo gene co-expression from plasmids coding for 175R. 175R cooperated efficiently with genomic E1a and specifically with the 289R protein coded by the 13S mRNA in the transformation of primary BRK cells. Mutational analysis of the 175R protein revealed that the N terminus and the C-terminal 30 amino acids are not essential for E1a-cooperative transformation. Several conserved sequences located in the middle of the 175R protein are essential for transformation. The effect of various mutants to suppress apoptosis (programmed cell death) induced by an anti-cancer agent, cisplatin, was examined in cells producing the E1A and E1B 175R proteins. Apoptosis was measured by flow cytometric analysis and indicates that the 175R protein efficiently prevents cisplatin-induced apoptosis. This suggests that the 175R function involved in transformation segregates with its ability to suppress cisplatin-induced apoptosis.

Adenovirus E1A Proteins↗
PubMed1993

A region in the C-terminus of adenovirus 2/5 E1a protein is required for association with a cellular phosphoprotein and important for the negative modulation of T24-ras mediated transformation, tumorigenesis and metastasis.

We have examined a series of small deletion mutants within exon 2 of the adenovirus 2/5 E1A oncogene product, the 243R protein, for immortalization, ras cooperative transformation, tumorigenesis and metastasis. Compared with wild-type 243R, various deletion mutants located between residues 193 and 243 cooperated more efficiently with ras to induce large transformed foci of less adherent cells that were tumorigenic and metastatic. However, the greatest enhancement of transformation (comparable to that obtained with a deletion of the C-terminal 67 amino acids) was observed with a mutant carrying a deletion of residues 225-238. This mutant was also more defective in immortalization. These results suggest that this 14 amino acid region may contain a function that is important for immortalization and negative modulation of tumorigenesis and metastasis. To identify cellular proteins that may associate with the exon 2-coded region of E1A (C-terminal half) and modulate its transformation potential, we constructed a chimeric gene coding for the C-terminal 68 amino acids of E1a fused to bacterial glutathione-S-transferase (GST). This fusion protein was used to purify cellular proteins that bind to the C-terminal region of E1a. A 48 kDa cellular protein doublet (designated CtBP) was found to bind specifically to the GST-E1a C-terminal fusion protein as well as to bacterially expressed full-length E1a (243R) protein. It also co-immunoprecipitated specifically with E1a. Analysis of a panel of GST-E1a C-terminal mutant proteins indicates that residues 225-238 are required for the association of E1a and CtBP, suggesting a correlation between the association of CtBP and the immortalization and transformation modulating activities of exon 2. CtBP is a phosphoprotein and the level of phosphorylation of CtBP appears to be regulated during the cell cycle, suggesting that it may play an important role during cellular proliferation.

Adenovirus E1A Proteins↗
PubMed1993

Functional dissection of the human spumaretrovirus transactivator identifies distinct classes of dominant-negative mutants.

The bel1 gene of human spumaretrovirus (HSRV) codes for a 300-amino-acid nuclear protein, termed Bel1, that can strongly activate transcription from the cognate long terminal repeat (LTR) by at least 200-fold. Bel1 can also activate human immunodeficiency virus type 1 (HIV-1) LTR expression. By using site-directed mutagenesis, we have identified distinct regions of Bel1 essential for HSRV LTR activation. The amino-terminal 55 residues, which comprise a highly acidic region followed by a short basic stretch, were dispensable for activation. The distribution of functionally defective mutants indicates that two distinct regions between residues 56 and 300 cooperate to confer full activator function. The larger, more amino-terminal region between residues 56 and 227 is sufficient to minimally activate the HSRV LTR. It contains a region between residues 88 and 110 that is strongly conserved between the simian and human spumavirus transactivators but otherwise lacks obvious homology to known transcriptional activators except for an Arg-rich nuclear localization sequence (NLS) between residues 211 and 225 that can be functionally substituted for by the NLS of the simian virus 40 large T antigen. The carboxy-terminal 73 residues contain two functionally redundant regions that can independently augment the activity of the more N-terminal minimal activator domain by 30- to 90-fold. Comparative analysis of the effect of Bel1 mutations on HSRV and HIV-1 LTR expression revealed a similar requirement of Bel1 domains for activation of the two LTRs. Bel1 is phosphorylated in vivo, and a nuclear localization-defective mutant lacking residues 211 to 222 was severely defective for phosphorylation, whereas various deletion mutations in residues 228 to 300 resulted in a four- to eightfold reduction in phosphate incorporation. When functionally defective bel1 mutants were examined for a dominant-negative phenotype, only mutants lacking a proline-rich basic region between residues 194 and 200 or the NLS between residues 211 and 222 that were found to occupy predominantly nuclear and cytoplasmic locations, respectively, could suppress wild-type Bel1 function efficiently. In identifying two classes of dominant-negative mutants with distinct subcellular localization phenotypes, the mutational analysis of Bel1 has revealed a feature unusual for known transcriptional activators.

Amino Acid Sequence↗
PubMed1993

Activation of a heterologous promoter by human immunodeficiency virus type 1 Tat requires Sp1 and is distinct from the mode of activation by acidic transcriptional activators.

We have previously shown that the Tat protein of the human immunodeficiency virus type 1 (HIV-1) is a modular transcriptional activator that can be targeted upstream of either a synthetic promoter or the intact HIV promoter to activate transcription. This activation was shown to be largely dependent on the presence of consensus binding sites for the cellular transcription factor Sp1. Since the use of heterologous promoters may provide further insight into Tat-mediated transactivation, we have analyzed the transactivation of the thymidine kinase promoter of herpes simplex virus by Tat and by the acidic transcriptional transactivator VP16. The effects of mutations of defined upstream promoter elements show that Tat transactivation is dependent on Sp1 binding sites in a site-specific manner. In contrast, transactivation by the acidic transactivator VP16 is completely independent of any of the defined promoter elements upstream of the TATA box. These results suggest that Tat and the classically defined modular acidic transcriptional activators have different modes of transactivation. In addition, the substitution of the HIV-1 TATA box for the thymidine kinase TATA box substantially increases Tat transactivation, indicating that Tat transactivation may also ultimately involve TATA box-associated cellular transcription factors.

Base Sequence↗
PubMed1993

The carboxy-terminal transcription enhancement region of the human spumaretrovirus transactivator contains discrete determinants of the activator function.

The bel1 gene of human spumaretrovirus (HSRV) encodes a 300-amino-acid nuclear protein termed Bel1 that is a potent activator of transcription from the cognate long terminal repeat (LTR). Bel1 can also efficiently activate the human immunodeficiency virus type 1 (HIV-1) LTR. We have previously shown that the amino-terminal 227-residue region (minimal activator region) of Bel1 can activate the HSRV LTR at low levels and that two distinct domains within the carboxy-terminal 73 residues, from residues 255 to 266 and 272 to 300, that bear little sequence homology can independently enhance the activity of the minimal activator domain (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnadurai, J. Virol. 67:161-169, 1993). We now report on the further characterization of these two transcriptional enhancement regions. Mutational analysis of the region comprising residues 255 to 266 indicates that a cluster of leucine residues is critical to the function of this region. Also, residues 273 to 287, which are identical in sequence to a 15-amino-acid segment near the carboxy terminus of the simian foamy virus transcriptional activator Taf, can independently enhance the activity of the minimal activator region. To delineate the region(s) of Bel1 that could function autonomously as an activator domain, we tested the activity of chimeric proteins comprising either wild-type or functionally defective forms of Bel1 fused to the DNA binding domain, Gal4(1-147), of the yeast transcriptional activator Gal4 on a synthetic promoter comprising Gal4 DNA binding sites linked to the adenovirus E1B TATA box (minimal promoter). Gal4-Bel1 was found to activate basal transcription from the E1B TATA box at least 35-fold, and the region responsible for this activation function was localized to the carboxy-terminal 73 amino acids. When the transcriptional enhancement regions were tested for autonomous activator function as Gal4(1-147) chimeras, residues 272 to 300, but not 255 to 266, were found to activate transcription efficiently when targeted to the E1B TATA motif and also to HSRV and HIV-1 LTRs. The highly conserved region between amino acids 273 and 287 alone was found to activate transcription efficiently when targeted to the HSRV LTR but not to the E1B TATA box or the HIV-1 LTR. Thus, our results demonstrate that the carboxy-terminal 29-amino-acid region (residues 272 to 300) contributes to Bel1 transactivation by functioning as an autonomous activator of TATA motif-directed transcription in a manner similar to that of other modular transcriptional activators.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

A high-level expression vector for human cells.

We have developed a vector (pSupexp), for high-level expression of genes, that is dependent on transactivation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) by the HIV-1 transactivator protein, Tat. The foreign gene, expressed under transcriptional control of the HIV-1 LTR, and the tat gene, expressed under transcriptional control of SV40 early promoter, are expressed from the same plasmid. The vector also has the neomycin resistance-encoding gene (neo), with G418 being used as a dominant selection marker for stable expression. We have cloned the bacterial cat gene into pSupexp and measured transient CAT production in human HeLa and A549 cells. Our results indicate that pSupexpCAT expresses about 25- to 68-fold higher levels of CAT activity as compared to other standard SV40- and Rous sarcoma virus-based vectors, and three- to fivefold more activity than the cytomegalovirus-based vector. Immunoprecipitation of the CAT protein also revealed a high level of production in human cells.

Cell Line↗
PubMed1992

Functional comparison of the basic domains of the Tat proteins of human immunodeficiency virus types 1 and 2 in trans activation.

The trans-activator Tat proteins coded by human immunodeficiency virus type 1 (HIV-1) and HIV-2 appear to be similar in structure and function. However, the Tat protein of HIV-2 (Tat2) activates the HIV-1 long terminal repeat (LTR) less efficiently than Tat1 (M. Emerman, M. Guyader, L. Montagnier, D. Baltimore, and M. A. Muesing, EMBO J. 6:3755-3760, 1987). To determine the functional domain of Tat2 which contributes to this incomplete reciprocity, we have carried out domain substitution between Tat1 and Tat2 by exchanging the basic domains involved in Tat interaction with its target trans-activation-response (TAR) RNA structure. Our results indicate that Tat1 proteins containing substitutions of either 8 or 14 amino acids of the basic domain of Tat2 exhibited reduced trans activation of the HIV-1 LTR by about 1/20 or one-fourth the level induced by wt Tat1. In contrast, Tat2 containing a substitution of the 9-amino-acid basic domain of Tat1 trans activated HIV-1 LTR like native Tat1. A substitution of the highly conserved core domain of Tat2 with that of Tat1 did not have any significant effect on trans activation of the HIV-1 LTR. These results indicate that the basic domain of Tat2 contributes to its inefficient trans activation of the HIV-1 LTR. Mutation of an acidic residue (Glu) located between the core domain and the Arg-rich basic domain of Tat2 at position 77 to a Gly residue increased the activity of Tat2 substantially. These results further suggest that the presence of an acidic residue (Glu) adjacent to Arg-rich sequences may at least partially contribute to the reduced activity of the Tat2 basic domain.

Amino Acid Sequence↗