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J D Becherer

Publications and source records attributed to J D Becherer.

At least 19 recordsLinked to original sources
PubMed2000

Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE).

Tumour necrosis factor alpha convertase (TACE) is a metalloprotease/disintegrin involved in the ectodomain shedding of several proteins, a process thought to be important in inflammation, rheumatoid arthritis and murine development. The characterization of the intracellular maturation and subcellular localization of endogenous TACE is decribed in the present study. Similarly to other proteolytically active metalloprotease/disintegrins, two forms of TACE are found in cells; a full-length precursor and a mature form lacking the prodomain. Prodomain removal occurs in a late Golgi compartment, consistent with the proposed role of a furin type proprotein convertase in this process. An additional form of TACE, lacking the pro and cytoplasmic domains, is detected when cell lysates are prepared in the presence of EDTA instead of a hydroxamate-based metalloprotease inhibitor or 1,10-phenanthroline. This form appears to be generated by mature TACE cleaving its own cytoplasmic tail and may explain why little mature TACE has been detected in previous studies. In cell-surface labelling experiments, mature TACE was detected on the cell surface but immunofluorescence data indicate that TACE is predominantly localized to a perinuclear compartment similar to that described for tumour necrosis factor (TNF)alpha. This raises the possibility that TACE-mediated ectodomain shedding may occur in an intracellular compartment in addition to the cell surface.

ADAM Proteins↗
PubMed1999

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

ADAM Proteins↗
PubMed1999

Evidence for a role of a tumor necrosis factor-alpha (TNF-alpha)-converting enzyme-like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival.

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a member of the TNF family, is a dendritic cell survival factor and is essential for osteoclastogenesis and osteoclast activation. In this report we demonstrate (i) that TRANCE, like TNF-alpha, is made as a membrane-anchored precursor, which is released from the plasma membrane by a metalloprotease; (ii) that soluble TRANCE has potent dendritic cell survival and osteoclastogenic activity; (iii) that the metalloprotease-disintegrin TNF-alpha convertase (TACE) can cleave immunoprecipitated TRANCE in vitro in a fashion that mimics the cleavage observed in tissue culture cells; and (iv) that in vitro cleavage of a TRANCE ectodomain/CD8 fusion protein and of a peptide corresponding to the TRANCE cleavage site by TACE occurs at the same site that is used when TRANCE is shed from cells into the supernatant. We propose that the TRANCE ectodomain is released from cells by TACE or a related metalloprotease-disintegrin, and that this release is an important component of the function of TRANCE in bone and immune homeostasis.

ADAM Proteins↗
PubMed1999

Metalloprotease-disintegrin MDC9: intracellular maturation and catalytic activity.

Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that are known to function in fertilization, myoblast fusion, neurogenesis, and ectodomain shedding of tumor necrosis factor (TNF)-alpha. Here we report the analysis of the intracellular maturation and catalytic activity of the widely expressed metalloprotease disintegrin MDC9. Our results suggest that the pro-domain of MDC9 is removed by a furin-type pro-protein convertase in the secretory pathway before the protein emerges on the cell surface. The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain, a generic protease substrate, providing the first evidence that MDC9 is catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with the TNF-alpha convertase (TACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one case with up to 50-fold selectivity for MDC9 versus TACE. Peptides mimicking the predicted cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation of metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MDC9 is shown to become phosphorylated when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate, a known inducer of protein ectodomain shedding. This work implies that removal of the inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in the secretory pathway is a prerequisite for protease activity. After pro-domain removal, additional steps, such as protein kinase C-dependent phosphorylation, may be involved in regulating the catalytic activity of MDC9, which is likely to target different substrates than the related TNF-alpha-convertase.

ADAM Proteins↗
PubMed1997

Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.

ADAM Proteins↗
PubMed1997

Structural features and biochemical properties of TNF-alpha converting enzyme (TACE).

Tumor necrosis factor-alpha is a potent cytokine, secreted primarily by activated monocytes and macrophages, that possesses a broad range of immunomodulating properties. Involvement of this cytokine has been validated in disease states such as arthritis and Crohn's disease and implicated in diverse neuroimmunological pathologies such as multiple sclerosis, Alzheimers and stroke. TNF-alpha is initially synthesized as a 26 kDa precursor molecule that is subsequently processed to the mature form by cleavage of the Ala76 Val77 bond. The 17 kDa carboxy-terminal protein is then secreted to function in a paracrine manner. The enzyme that processes precursor TNF-alpha has previously been identified as a microsomal metalloprotease called TNF-alpha converting enzyme (TACE). We have now purified and partially cloned the enzyme. TACE represents a novel target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases.

ADAM Proteins↗
PubMed1996

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.

Acetylcysteine↗
PubMed1996

Dissection of CR1, factor H, membrane cofactor protein, and factor B binding and functional sites in the third complement component.

Previous studies have suggested that the residues 727-768 of human (Hu) C3 contain the binding sites for CR1, factor H, and factor B. Here, we have (1) characterized further some of the C3 structural requirements for its binding to CR1, H, and B, (2) investigated the functions associated with these C3-ligand interactions, and (3) studied the relationship of MCP-binding sites in C3 with those for CR1, H, and B. Hu C3 molecules in which residues 727-768 were deleted (designated C3delta727-768) or substituted with the corresponding segment of cobra venom factor, Xenopus, or trout C3 (chimeric C3s) were expressed in the baculovirus system and analyzed for their reactivity with C3-binding proteins. In contrast to wild-type iC3 which, in the presence of CR1, is cleaved by factor I to iC3b-a and C3c-a and C3dg, all chimeric C3s were cleaved only to iC3b-a. In addition, the cleavage of deleted (C3delta727-768) iC3 to iC3b-a by factor I in the presence of CR1 was significantly reduced, whereas it remained unaltered in the presence of MCP. Cleavage of iC3 to iC3b-a by factor I and H was similar in all expressed C3s except C3delta727-768, whose cleavage was significantly reduced. All of the expressed molecules except C3delta727-768 were capable of forming the fluid-phase alternative pathway C3 convertase, and all reacted with properdin. These results suggest that during cleavage of iC3 by factor I and CR1, or H, CR1 and H bind to at least two sites on C3 and that the MCP binding site(s) on C3b are different from those for CR1. They also indicate that some or all of the C3 residues that are directly involved in, or contribute to, the structure of one of the CR1 and H binding sites are located within residues 727-768. These studies also demonstrate that, although this segment of C3 may be involved in C3-factor B interaction, other residues in addition to 736EE (previously implicated in B binding) must also contribute significantly to this interaction.

Amino Acid Sequence↗
PubMed1995

Granzyme A is an interleukin 1 beta-converting enzyme.

Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.

Amino Acid Sequence↗
PubMed1995

Monoclonal antibodies against human collagenase and stromelysin.

Mouse monoclonal antibodies against recombinant human fibroblast procollagenase and prostromelysin have been generated and characterized. The epitope-containing domains for the antibodies have been assigned based on their immunoreactivities against recombinant proenzymes, mature enzymes, truncated collagenases, proteolytic fragments of stromelysin, and chimeric molecules constructed from different domains of the two enzymes. These antibodies can be divided into four groups: (1) antibodies that recognize the truncated 19-kDa NH2-terminal collagenase, (2) antibodies that recognize the C-terminal domain of collagenase and stromelysin, (3) antibodies that recognize a 31-kDa NH2-terminal collagenase fragment, and (4) antibodies that recognize the 19-kDa NH2-fragment of stromelysin. The prostromelysin-specific antibody 11N13 is of particular interest; it neutralizes stromelysin activity in a stromelysin peptide substrate assay, with an IC50 value of 75 nM. MAb 11N13 may be useful for in vivo and in vitro studies to validate the roles of stromelysin in tumor cell invasion, metastasis, and connective tissue disorders.

Amino Acid Sequence↗
PubMed1994

Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor.

Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.

Animals↗
PubMed1992

Segment spanning residues 727-768 of the complement C3 sequence contains a neoantigenic site and accommodates the binding of CR1, factor H, and factor B.

CR1, CR2, DAF, MCP, factor H, C4bp, factor B, and C3 are members of a family of structurally related molecules, the majority of which belong to the complement system. Several of these molecules also share functional features such as cofactor and decay/dissociation activity and compete with one another in binding to C3b. Since factor H appears to bind to multiple sites in C3, we investigated the relationship between the factor H- and CR1-binding sites in C3b. Factor H binding to C3b is inhibited by either the C3c or C3d fragments, and addition of both fragments together augments this inhibition. One monoclonal anti-C3c antibody, anti-C3-9, which recognizes a neoantigenic epitope expressed upon cleavage to C3 to C3b, inhibited both factor H and CR1 binding to EC3b cells. This monoclonal antibody (MoAb) also inhibited factor B binding to EC3b. Two observations further supported our hypothesis that these molecules bind to proximal sites in C3b. First, a synthetic peptide spanning this region of C3b (C3(727-768)) inhibited factor H binding. Second, antibodies raised against this peptide inhibited binding to CR1, factor H, and factor B to C3b. These data show that H binds to at least two sites in C3b: the site in the C3c fragment is within the identified CR1-binding domain while the site in the C3d fragment surrounds the CR2-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21).

Multivalent but not monovalent CR2 ligands are required to elicit Raji cell proliferation as well as other B cell responses. It has been reported (C. Servis and J. D. Lambris, J. Immunol. 1989. 142: 2207) that the tetrameric peptide T-(C31202-1214)4, which represents the CR2-binding site in C3d, was able to support Raji cell growth. We show here that the tetrameric peptide T-(gp350(19-30)4, which contains the CR2-binding site in gp350 protein of EBV also induces Raji cell growth and this effect is inhibited by the monomeric peptides gp350(19-30) and C3(1201-1214). We also investigated the nature of the interaction between C3 fragment and CR2 in order to explain the Raji cell growth-supporting effect exerted by C3. The following findings suggest that there are multiple sites in the C3 molecule able to interact with CR2: (1) both C3c and C3d immobilized on microspheres are able to bind to Raji cells through CR2. (2) soluble C3d inhibits to a greater extent the binding of CR2 to fixed C3d than to fixed C3b, which suggests the existence of additional CR2-binding sites within C3b not present in the C3d portion of the molecule; (3) synthetic peptides C3(1187-1214), C3(741-757) and C3(295-307) which represents regions of similarity in the C3 molecule bind specifically to CR2 on Raji cells and compete with each other for binding to the receptor and (4) preincubation of microtiter plate-fixed C3b with monoclonal or polyclonal anti-peptide antibodies (C3-9, anti-C3(727-768) recognize the N terminus of the alpha chain of C3 (including residues 741-757) inhibited CR2 binding. Therefore, these data suggest that the N terminus of the alpha chain of C3 is involved in binding to CR2.

Amino Acid Sequence↗
PubMed1989

Cell surface proteins reacting with activated complement components.

The biologic activities mediated by the products of complement activation include cellular, bacterial, and viral lysis, inflammation, phagocytosis, and immunoregulation. These responses are achieved through the interaction of the activated forms of several of the complement proteins with cell membrane proteins. This report reviews aspects of the structure, ligand specificity, and function of the various complement receptors with particular emphasis on those receptors which bind to the activated fragments of C3. In addition, we briefly summarize the surface proteins on foreign particles that bind C3 and their possible role in the pathogenesis of these organisms.

Animals↗
PubMed1988

Identification of the C3b receptor-binding domain in third component of complement.

We report here that complement receptor type one (CR1) binds to a region of C3b that is contained within the NH2 terminus of the alpha' chain. In an enzyme-linked immunosorbent assay, CR1 bound to C3b, iC3b, and C3c but not to C3d, and this binding was inhibited by soluble C3b and C3c. Further attempts to generate a small C3 fragment capable of binding CR1 were unsuccessful. However, elastase degradation of C3 generated four species of C3c (C3c I-IV), two of which bound CR1. NH2-terminal sequence analysis and sodium dodecyl sulfate-gel electrophoresis of the C3cs indicated that the beta chains and the 40,000-dalton COOH-terminal alpha' chain fragments were identical; the NH2-terminal alpha' chain fragments of C3c I-IV varied from 21,000 to 27,000 daltons and accounted for the differential binding to CR1. C3c-I and II, which do not bind CR1, were missing 8 and 9 residues from the NH2 terminus of the alpha' chain when compared with the intact alpha' chain of C3b. C3c-III and IV, which bind CR1, had NH2 termini identical to the intact NH2-terminal alpha' chain of C3b. Using iodinated concanavalin A and endoglycosidase H, we showed that the NH2-terminal alpha' chains of C3c-I and III were glycosylated, while C3c-II and IV were not. Therefore, these data indicated that the amino terminus of the NH2-terminal alpha' chain fragment of C3c was responsible for binding CR1 while the COOH terminus of this fragment was not involved since the presence or absence of this region in C3c did not affect CR1 binding to C3c. Subsequently, two peptides were synthesized from the NH2-terminal alpha' chain fragment of C3c: X42, 42 residues in length from the NH2 terminus and C30, 30 residues in length from the COOH terminus. X42 inhibited binding of CR1 to C3b, and this effect was also observed with antipeptide antibodies against the X42 peptide. The C30 and other C3-derived peptides and antipeptide antibodies had no effect on the binding of CR1 to C3b.

Amino Acid Sequence↗