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J Arribas

Publications and source records attributed to J Arribas.

31 records · Page 2Linked to original sources
PubMed1997

Role of the juxtamembrane domains of the transforming growth factor-alpha precursor and the beta-amyloid precursor protein in regulated ectodomain shedding.

Although regulated ectodomain shedding is a well known process that affects a large group of transmembrane molecules, it is not clear how the shedding system selects its substrates. Here we investigate the structural requirements for the regulated shedding of two substrates of the general shedding system, the transforming growth factor-alpha precursor, pro-TGF-alpha, and the beta-amyloid precursor protein, beta-APP. The ability of different regions of pro-TGF-alpha or beta-APP to confer susceptibility to the shedding system was tested using as a reporter a transmembrane molecule that is not a substrate of this shedding system. For this purpose we chose the TGF-beta accessory receptor, betaglycan, since genetic and biochemical evidence showed that betaglycan is not a substrate of the shedding system. We determined that replacement of the 14 extracellular amino acids adjacent to the transmembrane region of betaglycan with the corresponding regions of TGF-alpha or beta-APP rendered betaglycan susceptible to ectodomain shedding. These domain swap constructs were cleaved in response to protein kinase C stimulation, and cleavage was prevented by the metalloprotease inhibitor TAPI, both effects being characteristic of the general shedding system. Domain swap constructs containing the transmembrane and/or the cytoplasmic domains of pro-TGF-alpha did not undergo regulated ectodomain cleavage. We conclude that despite a lack of sequence similarity, the extracellular regions of pro-TGF-alpha and beta-APP immediately preceding their transmembrane domains are key determinants of ectodomain shedding.

Amyloid beta-Protein Precursor↗
PubMed1996

Diverse cell surface protein ectodomains are shed by a system sensitive to metalloprotease inhibitors.

The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor alpha precursor (proTGF-alpha) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-alpha, cell adhesion receptor L-selectin, interleukin 6 receptor alpha subunit, beta-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene.

Amyloid beta-Protein Precursor↗
PubMed1996

Phosphorylation of C8 and C9 subunits of the multicatalytic proteinase by casein kinase II and identification of the C8 phosphorylation sites by direct mutagenesis.

Two 29 kDa subunits of the multicatalytic proteinase (proteasome) complex, the C8 and C9 components, are phosphorylated in vivo and can be phosphorylated in vitro by casein kinase II (CKII). The major phosphate acceptor is the C8 subunit being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by Glu-C endoprotease digestion from the in vivo 29 kDa labeled subunit and from the in vitro phosphorylation of the recombinant C8 subunit with CKII are identical, suggesting that CKII is likely responsible for the in vivo phosphorylation of the C8 subunit. The in vitro stoichiometry of phosphorylation of the proteasome complex and the recombinant C9 and C8 subunits by CKII is 2-2.5, 0.2, and 2 mol of phosphate per mole, respectively. Several C8 protein constructs allow the location of the CKII phosphorylation sites to be the COOH terminal portion of the protein, and direct mutational analyses show that Ser-243 and Ser-250 are the residues of the C8 subunit phosphorylated by CKII. The in vitro phosphorylation of the proteasome by CKII does not affect its proteolytic activity (on proteins or fluorogenic synthetic peptides), therefore suggesting its involvement in the interaction of the proteasome with other cellular proteins, i.e. in the formation of the 26S complex and/or in the interaction with the nuclear translocation machinery.

Amino Acid Sequence↗
PubMed1995

Transforming growth factor-alpha and beta-amyloid precursor protein share a secretory mechanism.

Cleavage and release of membrane protein ectodomains, a regulated process that affects many cell surface proteins, remains largely uncharacterized. To investigate whether cell surface proteins are cleaved through a shared mechanism or through multiple independent mechanisms, we mutagenized Chinese hamster ovary (CHO) cells and selected clones that were unable to cleave membrane-anchored transforming growth factor alpha (TGF-alpha). The defect in TGF-alpha cleavage in these clones is most apparent upon cell treatment with the protein kinase C (PKC) activator PMA, which stimulates TGF-alpha cleavage in wild-type cells. The mutant clones do not have defects in TFG-alpha expression, transport to the cell surface or turnover. Concomitant with the loss of TGF-alpha cleavage, these clones have lost the ability to cleave many structurally unrelated membrane proteins in response to PMA. These proteins include beta-amyloid precursor protein (beta-APP), whose cleavage into a secreted form avoids conversion into the amyloidogenic peptide A beta, and a group of cell surface proteins whose release into the medium is stimulated by PMA in wild type CHO cells but not in mutants. The mutations prevent cleavage by PKC-dependent as well as PKC-independent mechanisms, and thus affect an essential component that functions downstream of these various signaling mechanisms. We propose that regulated cleavage and secretion of membrane protein ectodomains is mediated by a common system whose components respond to multiple activators and act on susceptible proteins of diverse structure and function.

Amyloid beta-Protein Precursor↗
PubMed1994

Antibodies against the C2 COOH-terminal region discriminate the active and latent forms of the multicatalytic proteinase complex.

The mouse cDNA homologues of the rat C2, C9, and C5 subunits of the multicatalytic proteinase have been cloned and expressed in bacteria. The respective recombinant proteins were purified and used to produce specific anti-subunit antibodies. Immunoblotting of two-dimensional gels of purified rat liver multicatalytic proteinase showed that the C2 (32-kDa) and C9 (29-kDa) polypeptides are resolved into three and two isoelectric variants, respectively, likely due to post-translational modifications, i.e. phosphorylation, and the presence of two anti-C5 reacting polypeptides (25.5 and 23 kDa). Epitope mapping of the anti-C2-specific antibody with different constructs of the recombinant C2 protein allowed us to determine that one major epitope of this anti-C2 antibody is located within the last 9-11 amino acids of the C2 polypeptide. Affinity purified antibodies directed against the C2 COOH-terminal were able to discriminate the active and latent forms of the multicatalytic proteinase, supporting the conclusion that the C2 protein found in the active form of the enzyme is a polypeptide of 28 kDa, produced by the loss, at least, of the last 9-13 amino acids (DEPAEKADEPMEH) of the intact C2 (32-kDa) component. By in vitro treatment of the latent form of the enzyme with elastase, we show the conversion of the C2 (32-kDa) component to a 28-kDa protein with loss of recognition by the anti-C2 COOH-terminal affinity purified antibodies, but this limited degradation of the C2 component did not have any significant effect on the proteolytic activity (assayed with myelin basic protein and fluorogenic peptides) of the multicatalytic proteinase. It is suggested that the proteolytic cleavage of the C2 COOH-terminal region may be involved in the regulation of the interaction of the multicatalytic proteinase with other cellular proteins and/or in the translocation of the complex to the nucleus.

Animals↗
PubMed1993

Kinetic mechanism of activation by cardiolipin (diphosphatidylglycerol) of the rat liver multicatalytic proteinase.

The effect of phospholipids on the trypsin-like, chymotrypsin-like and peptidylglutamyl-peptide-hydrolysing activities of the so-called latent form of the rat liver multicatalytic proteinase was studied, assaying them with the following substrates: N-Cbz-ARR-4MNA (N-Cbz, N-benzyloxycarbonyl; 4MNA, 4-methoxy-beta-naphthylamide), N-Suc-LLVY-MCA (N-Suc, N-succinyl; MCA, methylcoumarin) and N-Cbz-LLE-beta-NA (beta-NA, beta-naphthylamide) respectively (amino acids are shown as their one-letter symbol). For the most part neither lysophospholipids nor phospholipids at 20 micrograms/ml have any effect on the activity of the enzyme (assayed at 50 microM peptide), except for phosphatidylserine, which activates 2-fold the hydrolysis of N-Suc-LLVY-MCA, and phosphatidylinositol, which inhibits by 20% the hydrolysis of N-Cbz-LLE-beta-NA. By contrast, cardiolipin (diphosphatidylglycerol) is a strong activator of the hydrolysis of N-Suc-LLVY-MCA (60-fold) and N-Cbz-LLE-beta-NA (30-fold), with half-maximal activation at concentrations of 0.15 micrograms/ml and 1.5 micrograms/ml respectively. The activation of N-Suc-LLVY-MCA hydrolysis is due to an increase of the affinity of the enzyme for the peptide and to an increase in the Vmax. (30-fold). The activation of N-Cbz-LLE-beta-NA hydrolysis is explained by suppressing the co-operativity for this substrate, producing hyperbolic kinetics with a Km of 60 microM and a 15-fold increase in the Vmax. of the enzyme. This activation by cardiolipin was completely suppressed by micromolar concentrations of fluophenazine, a drug known to inhibit other phospholipid-regulated process. Cardiolipin activation and the known activation by SDS are additive, either at suboptimal or optimal concentrations of both activators. Cardiolipin also activates the in vitro degradation of some proteins from metabolically labelled total cellular extracts by the latent multicatalytic proteinase. These results clearly show that cardiolipin is a natural positive modulator of the peptidase and proteolytic activities of the multicatalytic proteinase, probably acting through a binding site different from that of SDS.

Amino Acid Sequence↗
PubMed1993

A comparative study of the chymotrypsin-like activity of the rat liver multicatalytic proteinase and the ClpP from Escherichia coli.

A comparative study of the chymotrypsin-like activity of the purified recombinant ClpP protease and the multicatalytic proteinase from rat liver is presented. The peptidase activity of both enzymes has been analyzed with several synthetic fluorogenic peptides, containing either aromatic or nonpolar amino acids in their P1 position. The respective Vmax, Km, and Vmax/Km were calculated from kinetic experiments. The substrate specificity of the multicatalytic proteinase, as expressed by Vmax/Km values, indicate the following substrate preference: N-Suc-IIW-MCA > N-Suc-LY-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > N-Cbz-GGL-beta-NA > Glut-GGF-beta-NA > FPAM-4-MNA. In the case of the ClpP the order of preference is: N-Suc-LY-MCA > N-Suc-IIW-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > or = N-Cbz-GGL-beta-NA > FPAM-4-MNA (where: N-Suc, N-succinyl-; MCA, 7-amido-4-methyl coumarin; beta-NA, beta-naphthylamide; N-Cbz, N-benzyloxycarbonyl-; 4-MNA, 4-methoxy-beta-naphthylamide; Glut, glutaryl. This similar substrate specificity is further supported by the lack of activity of both enzymes against SY-MCA and N-Suc-AAPF-MCA (known substrates of chymotrypsin), by very reduced activity against N-Suc-AAA-MCA and by no significant activity against LG-beta-NA. The results of mixed substrate experiments have shown that all the peptides that are substrates seem to be hydrolyzed by a single class of chymotrypsin-like site in both enzymes. The substrate specificity studies suggest a possible evolutionary relationship between the catalytic component of the ClpP of Escherichia coli and the multicatalytic proteinase chymotrypsin-like catalytic component. This conclusion is further supported by other circumstantial evidence: the fact that affinity-purified anti-ClpP antibodies cross-react with two polypeptide components of the rat liver multicatalytic proteinase complex, presented here and also shown previously; the known resemblance of both structures at the electron microscope level; and their reported role in the degradation of NH2-end rule substrates.

ATP-Dependent Proteases↗
PubMed1993

Modulation of the multicatalytic proteinase complex by lipids, interconversion and proteolytic processing.

In studying the modulation of multicatalytic proteinase (MCP), we have focused on three main aspects: (1) modulation of the activity of the MCP complex by lipids, showing that cardiolipin, sulfatides and gangliosides are potent activators of the enzymatic activity of the complex; (2) modulation by interconversion of MCP subunits, showing that casein kinase II is able to phosphorylate the C8 (this subunit is also be main in vivo phosphorylated subunit) and C9 subunits of the complex in vitro and that a 26-kD subunit is phosphorylated in vitro by protein kinase C, and (3) modulation by proteolytic processing, extending our previous observation of proteolytic processing of the C2 COOH terminus, the presence of enzymatic activities in different subcellular fractions able to convert the intact C2 (32 kD) subunit to a 28-kD polypeptide by removal of at least the last 9-13 amino acids of the C2 polypeptide. The data presented illustrate that the MCP complex is probably under tight and multifactorial control in vivo.

Amino Acid Sequence↗
PubMed1991

Autoantibodies against the multicatalytic proteinase in patients with systemic lupus erythematosus.

Sera from patients with systemic lupus erythematosus contain specific autoantibodies directed against different polypeptide components of the multicatalytic proteinase (also known as proteasome or prosome). These human autoantibodies, in contrast to polyclonal antibodies obtained in rabbits against the purified enzyme, recognize highly conserved epitopes of the multicatalytic proteinase polypeptides from yeast to human.

Animals↗
PubMed1990

Kinetic studies of the differential effect of detergents on the peptidase activities of the multicatalytic proteinase from rat liver.

We present here a detailed study of the effect of detergents on the three peptidase activities (hydrolysis of the LLVY, ARR, and LLE peptides) of the purified multicatalytic proteinase from rat liver. At Triton X-100 and sodium dodecyl sulfate (SDS) concentrations of 0.1%, all three peptidase activities are inhibited. Lower concentrations of the two detergents (0.01%) do not affect the hydrolysis of the ARR peptide, whereas they behave differently on the hydrolysis of the LLVY and LLE peptides. Triton X-100 inhibits and SDS strongly activates LLVY peptide hydrolysis by decreasing and increasing Vmax, respectively. In the absence of detergents, the saturation curve for the LLE peptide can be analyzed as the result of two components, one showing cooperative (nH = 1.6) with higher affinity (S0.5 = 60 microM) and lower Vmax than a second, noncooperative component (Km = 320 microM). SDS (0.01%) activates LLE peptide hydrolysis by suppressing cooperativity, slightly increasing Vmax, and decreasing the half-saturation concentration (Km = 30 microM) of the enzyme. Triton X-100 (0.01%) also suppresses the cooperativity and decreases the half-saturation concentration (Km = 25 microM) for the LLE peptide; in contrast, it reduces Vmax by inhibition of the low affinity, high Vmax component observed in the absence of detergents. Based on these observations, it can be concluded that both detergents behave like allosteric activators of peptidylglutamyl-peptide hydrolyzing activity and that the multicatalytic proteinase has at least three different classes of active sites: two independent noncooperative sites that catalyze the hydrolysis of trypsin and chymotrypsin-like substrates and one class for peptidylglutamyl-peptide hydrolysis having two components: one cooperative (two or more sites) and one noncooperative.

Amino Acid Sequence↗