Biomedical subjects

D Boivin

Publications and source records attributed to D Boivin.

At least 19 recordsLinked to original sources

PubMed2001

Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex.

Although MDM2 plays a major role in regulating the stability of the p53 tumor suppressor protein, other poorly understood MDM2-independent pathways also exist. Human adenoviruses have evolved strategies to regulate p53 function and stability to permit efficient viral replication. One mechanism involves adenovirus E1B55K and E4orf6 proteins, which collaborate to target p53 for degradation. To determine the mechanism of this process, a multiprotein E4orf6-associated complex was purified and shown to contain a novel Cullin-containing E3 ubiquitin ligase that is (1) composed of Cullin family member Cul5, Elongins B and C, and the RING-H2 finger protein Rbx1(ROC1); (2) remarkably similar to the von Hippel-Lindau tumor suppressor and SCF (Skp1-Cul1/Cdc53-F-box) E3 ubiquitin ligase complexes; and (3) capable of stimulating ubiquitination of p53 in vitro in the presence of E1/E2 ubiquitin-activating and -conjugating enzymes. Cullins are activated by NEDD8 modification; therefore, to determine whether Cullin complexes are required for adenovirus-induced p53 degradation, studies were conducted in ts41 Chinese hamster ovary cells that are temperature sensitive for the NEDD8 pathway. E4orf6/E1B55K failed to induce the degradation of p53 at the nonpermissive temperature. Thus, our results identify a novel role for the Cullin-based machinery in regulation of p53.

Adenovirus E1B Proteins↗

PubMed2001

Identification of three functions of the adenovirus e4orf6 protein that mediate p53 degradation by the E4orf6-E1B55K complex.

Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich alpha-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

Adenoviridae↗

PubMed2000

Induction of p53-independent apoptosis by the adenovirus E4orf4 protein requires binding to the Balpha subunit of protein phosphatase 2A.

Previous studies have indicated that the E4orf4 protein of human adenovirus type 2 (Ad2) induces p53-independent apoptosis. We believe that this process may play a role in cell death and viral spread at the final stages of productive infection. E4orf4 may also be of therapeutic value in treating some diseases, including cancer, through its ability to induce apoptosis when expressed individually. The only previously identified biochemical function of E4orf4 is its ability to associate with the Balpha subunit of protein phosphatase 2A (PP2A). We have used a genetic approach to determine the role of such interactions in E4orf4-induced cell death. E4orf4 deletion mutants were of only limited value, as all were highly defective. We found that E4orf4 proteins from most if not all adenovirus serotypes induced cell death, and thus point mutations were introduced that converted the majority of highly conserved residues to alanines. Such mutants were used to correlate Balpha-subunit binding, association with PP2A activity, and cell killing following the transfection of appropriate cDNAs into p53-null H1299 or C33A cells. The results indicated that binding of the Balpha subunit is essential for induction of cell death, as every mutant that failed to bind efficiently was totally defective for cell killing. This class of mutations (class I) largely involved residues between amino acids 51 and 89. Almost all E4orf4 mutant proteins that associated with PP2A killed cancer cells at high levels; however, several mutants that associated with significant levels of PP2A were defective for killing (class II). Thus, binding of E4orf4 to PP2A is essential for induction of p53-independent apoptosis, but E4orf4 may possess one or more additional functions required for cell killing.

Adenovirus E4 Proteins↗

PubMed1999

Analysis of synthesis, stability, phosphorylation, and interacting polypeptides of the 34-kilodalton product of open reading frame 6 of the early region 4 protein of human adenovirus type 5.

The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.

Adenovirus E1B Proteins↗

PubMed1998

The early region 4 orf4 protein of human adenovirus type 5 induces p53-independent cell death by apoptosis.

Previous studies by our group showed that infection of human and rodent cells by human adenovirus type 5 (Ad5) results in the induction of p53-independent apoptosis and cell death that are dependent upon transactivation of early region 4 (E4). To identify which E4 products are involved, studies were conducted with p53-deficient human SAOS-2 cells infected with various Ad5 E4 mutants. An E4orf6-deficient mutant was defective in cell killing, whereas another that expressed only E4orf6 and E4orf4 killed like wild-type virus, suggesting that E4orf6 may be responsible for cytotoxicity; however, a mutant expressing only E4orf4 induced high levels of cell death, indicating that this E4 product may also be able to induce cytotoxicity. To define the E4 cell death-inducing functions more precisely, cDNAs encoding individual E4 products were introduced into cells by DNA transfection in the absence of other Ad5 proteins. In cotransfections with a cDNA encoding firefly luciferase, enzymatic activity was high in all cases except with E4orf4, where luciferase levels were less than 20% of those in controls. In addition, drug selection of several cell types following transfection with retroviral vector DNA encoding individual E4 products as well as puromycin resistance yielded a large number of cell colonies except when E4orf4 was expressed. These data demonstrated that E4orf4 is the only E4 product capable of independent cell killing. Cell death induced by E4orf4 was due to apoptosis, as evidenced by 4',6-diamidino-2-phenylindole (DAPI) staining of cell nuclei in E4orf4-expressing cells. Thus, although E4orf6 may play some role, these results suggested that E4orf4 may be the major E4 product responsible for induction of p53-independent apoptosis.

Adenovirus E4 Proteins↗

PubMed1997

Essential arginine residues in isoprenylcysteine protein carboxyl methyltransferase.

We used specific amino acid modifying reagents to characterize the isoprenylcysteine carboxyl methyltransferase in kidney membranes. The enzyme was inactivated by reagents specific for arginine, histidine, cysteine, and tryptophan residues. Protection by the product and inhibitor S-adenosyl-L-homocysteine was observed for arginine modification by phenylglyoxal and tryptophan modification by N-bromosuccinimide. We focused on modification by phenylglyoxal, a highly specific modifier of arginine residues. The inactivation of methyltransferase by phenylglyoxal follows pseudo-first-order kinetics and the order of the reaction, n, with respect to phenylglyoxal was 1.2. The inactivation increased with the alkalinity of the preincubation medium and was maximal at pH 10. Kinetic analysis showed that the K(m) for S-adenosyl-L-methionine is not significantly affected by treatment with phenylglyoxal but that the Vmax is reduced p-Hydroxyphenylglyoxal, a more hydrophilic derivative of phenylglyoxal, was a less potent inactivator of methyltransferase than phenylglyoxal, suggesting that arginine residues modified are in a hydrophobic environment. The methyltransferase is protected from phenylglyoxal modification by S-adenosyl-L-homocysteine but not S-adenosyl-L-methionine, sinefungin, N-acetyl-S-farnesyl-L-cysteine, or farnesylthioacetate. The arginine residue modified may thus be located either at the active site or at another additional binding site for S-adenosyl-L-homocysteine. These results indicate that arginine residues are essential for the enzymatic activity of isoprenylcysteine carboxyl methyltransferase.

Adenosine↗

PubMed1996

Regulation of cytoskeletal functions by Rho small GTP-binding proteins in normal and cancer cells.

The actin cytoskeleton is involved in numerous cellular functions such as cell motility, mitogenesis, morphology, muscle contraction, cytokinesis, and establishment of cell polarity. The members of the Rho subfamily of small GTP-binding proteins emerge as key regulators of cytokeleton organization. Rho, Rac, and CDC42 are implicated in the regulation of actin microfilament organization of different cell structures, such as stress fibers linked to focal adhesions and membrane ruffles induced by extracellular stimuli. Rho proteins also regulate the activity of several enzymes involved in the formation of phospholipid derivatives, which could mediate their effect on the cytoskeleton. The activity of Rho proteins is regulated by many nucleotide exchange factors and GTPase-activating proteins, some of which are oncogene products, and other disease-associated proteins. The potential role of these small GTP-binding proteins in carcinogenesis is suggested by the actin reorganization seen in transforming cells and by the need for functional Rho proteins in Ras mitogenic activation.

Amino Acid Sequence↗

PubMed1995

Immunochemical characterization of L-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues.

Polyclonal antibodies were raised against a synthetic peptide corresponding to a sequence of 14 amino acid residues found near the C-terminus of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PCMT). The affinity-purified antibodies were used to detect the methyltransferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in the cytosol of most tissues; co-incubation with the peptide used for immunization abolished the detection. The identity of the 27 kDa protein as a PCMT was demonstrated by renaturation of PCMT activity from SDS/polyacrylamide gels. The methyltransferase from brain cytosol was immunoprecipitated by the anti-PCMT antibodies and Protein A-agarose, indicating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and heart, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat tissues. The bovine enzyme had a greater electrophoretic mobility, suggesting small structural differences. The membrane-bound methyltransferase could be extracted with detergents above their critical micellar concentration, but not with salt, alkaline or urea solutions suggesting that the binding of the enzyme to membranes is hydrophobic by nature. Anti-PCMT antibodies provide an interesting tool for studies regarding the expression of these enzymes in both soluble and membrane fractions of various cell types.

Amino Acid Sequence↗

PubMed1995

Subcellular distribution and membrane association of Rho-related small GTP-binding proteins in kidney cortex.

We have examined the subcellular distribution of Rho-related small GTP-binding proteins in the kidney. RhoA, CDC42, and Rac1 small GTP-binding proteins were found to be expressed at high levels in rat outer kidney cortex. Western blot analysis showed that these proteins were predominantly associated with brush-border and basolateral plasma membranes, with the exception of Rac1 which was localized predominantly in the mitochondria. RhoA and CDC42 were also found in the cytosol, and a small fraction was associated with cytoskeletal elements. A GDP-dissociation inhibitor specific for the Rho family (RhoGDI) was also identified and found to be located exclusively in the cytosol. Upon fractionation of kidney cytosol with anion-exchange chromatography, RhoA and CDC42 proteins eluted in two major well-resolved peaks that coeluted with the RhoGDI protein, suggesting that they form heterodimers. Association of RhoA and CDC42 with RhoGDI was further suggested by coelution of these proteins with RhoGDI at an estimated size of approximately 45 kDa after gel-filtration chromatography. However, a second peak of RhoA eluted as a 20-kDa protein, indicating that not all RhoA is complexed to RhoGDI. Addition of RhoA- and CDC42-enriched fractions to purified membranes from kidney cortex resulted in their translocation to the membranes and their carboxyl methylation. Both processes were stimulated by guanosine 5'-O-(3-thiotriphosphate). Methylation inhibitors had no effect on the translocation of RhoA to membranes, suggesting that this covalent modification is not required for association to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Functional size of C-terminal protein carboxyl methyltransferase from kidney basolateral plasma membranes.

The functional sizes of the C-terminal isoprenylcysteine protein carboxyl methyltransferase (PCMT) from kidney cortex basolateral plasma membranes and yeast membranes have been estimated by the radiation inactivation and fragmentation method. Attempts to solubilize the methyltransferase with detergents were unsuccessful as they resulted in the irreversible denaturation of its enzymatic activity. The radiation inactivation sizes of the methyltransferases were 98 and 24 kDa for kidney and yeast, respectively. Kinetic experiments showed that irradiation affects the Vmax of the reaction but not the apparent Km for either S-adenosyl-L-methionine and N-acetyl farnesylcysteine. The functional size reported here for the kidney membrane is about 4-times larger than the size predicted for the Saccharomyces cerevisiae C-terminal PCMT deduced from the nucleotide sequence of its gene (28 kDa). These results suggest that mammalian methyltransferase has a functional size different from that of the yeast; tetramerization of monomers is one possible hypothesis for this difference.

Animals↗

PubMed1994

Asymmetrical distribution of L-isoaspartyl protein carboxyl methyltransferases in the plasma membranes of rat kidney cortex.

We have studied the distribution of membrane-associated L-isoaspartyl protein carboxyl methyltransferases (PCMTs) in plasma membranes purified from rat kidney cortex. Addition of CHAPS to brush-border membranes (BBM) and basolateral membranes (BLM) was required to measure optimal membrane-dependent methylation of ovalbumin and TS-isoD-YSKY, substrates of L-isoaspartyl PCMTs. Extraction of both membrane-associated enzymes was achieved with detergents, but not with high-salt solutions, suggesting a strong membrane attachment. However, upon phase partitioning using Triton X-114, both enzymes were predominantly associated with the detergent-poor phase, suggesting a relatively hydrophilic nature. The enzymes showed similar catalytic properties such as substrate recognition and affinity towards the methyl donor, S-adenosyl-L-methionine. The activity of the BBM enzyme, however, was about 2-fold higher than that of the BLM enzyme. Identification of the endogenous substrates located in the two plasma membranes by acidic gel electrophoresis in the presence of a cationic detergent revealed significant differences in the methyl-accepting proteins of both membranes. The BBM-methylated proteins had sizes of 35, 50 and 54 kDa, whereas the major BLM-methylated substrates were of 97 and 100 kDa. The enzymes showed distinct behaviour on Mono Q anion-exchange chromatography. The BBM-associated PCMT did not bind to the column, being eluted in the flow-through, whereas the BLM enzyme bound to the column and was eluted at 0.15 M NaCl. Moreover, the two enzymes had different molecular masses under both denaturing and nondenaturing conditions, the BLM PCMT migrating at an apparent molecular mass of 29 kDa, compared with 27 kDa for the BBM enzyme. Taken together, these results show the presence of two distinct L-isoaspartyl PCMTs in the plasma membranes of the kidney cortex.

Animals↗

PubMed1993

Purification and characterization of a membrane-bound protein carboxyl methyltransferase from rat kidney cortex.

Class II protein carboxyl methyltransferases (EC 2.1.1.77) are known to exist predominantly in a soluble form in all cells studied so far. These enzymes have been purified to homogeneity from the cytosols of many mammalian tissues but not from membranes. We describe here the purification to apparent homogeneity of a membrane-associated protein carboxyl methyltransferase from the brush border membrane of rat kidney. The enzyme was purified by fast protein liquid chromatography on Superdex 75 and Mono-Q and by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consists of a single 27,300 polypeptide. The purified enzyme recognizes exogenous substrate proteins such as ovalbumin and gamma-globulins as well as synthetic peptides containing a L-isoaspartyl residue but not a synthetic peptide containing a farnesylated C-terminal cysteine (S-farnesyl-LARYKC). The Km for S-adenosyl-L-methionine with ovalbumin as the substrate is 1.5 microM and the purified enzyme is sensitive to inhibition by S-adenosyl-L-homocysteine (Ki = 0.3 microM). Peptide map obtained after Staphylococcus aureus V8 protease digestion of brush border membrane protein carboxyl methyltransferase showed a fragmentation pattern that was identical to that obtained for a soluble protein carboxyl methyltransferase purified according to the same procedure, indicating a high degree of homology. These results support the notion that class II protein carboxyl methyltransferases are not restricted to a cytosolic localization and show that the membrane-bound form of this enzyme shares many characteristics with known cytosolic protein carboxyl methyltransferases.

Amino Acid Sequence↗

PubMed1993

Subcellular distribution and guanine nucleotide dependency of COOH-terminal methylation in kidney cortex.

The subcellular distribution of COOH-terminal carboxyl methyltransferase and methylated substrates was studied in purified brush-border and basolateral plasma membranes, as well as in crude intracellular membranes and the cytosolic fraction isolated from rat kidney cortex. The three membrane fractions showed intrinsic carboxyl methylation of 21- to 23-kDa proteins, whereas 18- and 41-kDa methylated proteins were observed in the cytosol. In contrast, methylation activities toward N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), a synthetic farnesylated substrate, were found to be strictly associated with membranes, with no detectable level of activity in the cytosol. Methylation of all membrane-associated substrates was inhibited by AFC but remained unaffected by TS-isoD-YSKY, a synthetic isopeptide recognized by L-isoaspartyl methyltransferase, suggesting that the membrane-associated substrates were methylated on a COOH-terminal isoprenylated cysteine residue. The membrane-associated methylated proteins were tightly bound to the membranes as reflected by their extraction with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate but not with 1 M NaCl or 2 M urea. The nonhydrolyzable analogues of GTP and GDP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), markedly increased the methylation of the 21- to 23-kDa substrates, whereas ATP gamma S and ADP beta S were without effect. This effect of guanine nucleotides was restricted to endogenous 21- to 23-kDa substrates with no stimulation of methylation of the exogenous substrate, AFC. Our results show a wide distribution of both COOH-terminal protein carboxyl methyltransferase activities and associated methylated substrates in the kidney cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗

PubMed1992

Tyrosine protein kinase activity in renal brush-border membranes.

Tyrosine protein kinase (TPK) activity was detected in rat renal brush-border membranes (BBM) using poly(Glu80Na,Tyr20) as a substrate. Maximal TPK activity required prior detergent dispersion of the membranes with 0.05% Triton X-100 and the presence of vanadate, a potent inhibitor of phosphotyrosine protein phosphatases, in the phosphorylation medium. Optimal conditions for measurement of TPK activity were 10 mM of MgCl2 and MnCl2, at 30 degrees C and pH 7.0. TPK activity was inhibited by genistein, with a IC50 value of 15 microM, while no inhibition was observed in the presence of 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H7), an inhibitor of serine-threonine kinases. TPK activity was enriched 4-fold in the BBM fraction relative to cortex homogenate. It was co-enriched with BBM enzyme markers, but not with those of the basolateral membrane (BLM). The endogenous substrates of TPK in brush-border and basolateral membranes were determined by Western blot analysis using an antiphosphotyrosine monoclonal antibody (PY20). Various phosphotyrosine-containing proteins were found in the BBM (31, 34, 46, 50, 53, 72, 90, 118 and 170 kDa) and in the BLM (37, 48, 50, 53, 72, 90, 130 and 170 kDa). Addition of exogenous insulin receptor to BBM and BLM increased the phosphorylation of most of the substrates. Solubilization of the TPK activity from BBM with 0.5% CHAPS and subsequent gel filtration on Superdex 75 yielded two peaks of tyrosine protein kinase activity with apparent molecular masses of 49 and 66 kDa. These results provide evidence for a non-receptor TPK activity associated with the renal tubular luminal membrane.

Animals↗

PubMed1990

Interhemispheric EEG coherence before and after partial callosotomy.

Measures of interhemispheric EEG coherence during REM and NREM sleep reflect the functional connectivity of the right and left hemispheres mediated by the corpus callosum. Surface recordings of interhemispheric coherence in two patients reflected fairly accurately the degree of anatomical section produced by partial callosotomy. With further development, EEG coherence may prove useful as a noninvasive method for assessing interhemispheric integration under different physiological and experimental conditions.

Adult↗

PubMed1987

Object exploration in staggerer mutant mice.

Staggerer mutant mice, which lose Purkinje and granule cells in the cerebellum, were found to explore for a longer period of time than normal mice a novel object placed in a familiar environment. There was no difference between the two groups in terms of general motor activity. These results are discussed in regard to a role for the cerebellum in behavioral inhibition and emotion.

Animals↗

PubMed1986

Nosology and diagnosis of alcoholism. Issues and perspectives in subtyping (DSM III) simple alcoholism and alcoholism associated with other psychopathologies.

The presence of psychopathological syndromes in alcoholic in-patients was assessed using the NIMH-Diagnosis. The most stricking finding of this study was the high percentage of additional psychopathological syndromes associated to alcoholism. Based on this finding, a tentative classification of alcoholism is proposed. The urgent need for a comprehensive diagnostic scheme for alcoholism is underlined.

Alcoholism↗

PubMed1986

Spontaneous alternation and habituation in a t-maze in nervous mutant mice.

Nervous mutant mice have a selective degeneration of Purkinje cells and deep cerebellar nuclei. In comparison with littermate controls, nervous mutant mice did not alternate spontaneously in a successive trial procedure (six consecutive trials per day) and did not habituate in a restricted part of a t-maze in a 4-min session. These data indicate that the cerebellum may have a role in spatial learning.

Animals↗