Copine-I represses NF-kappaB transcription by endoproteolysis of p65.

Nuclear factor-kappaB (NF-kappaB) is a dynamic transcription factor that regulates important biological processes involved in cancer initiation and progression. Identifying regulators that control the half-life of NF-kappaB is important to understanding molecular processes that control the duration of transcriptional responses. In this study we identify copine-I, a calcium phospholipid-binding protein, as a novel repressor that physically interacts with p65 to inhibit NF-kappaB transcription. Knockdown of copine-I by siRNA increases tumor necrosis factor alpha-stimulated NF-kappaB transcription, while copine-I expression blocks endogenous transcription. Copine-I abolishes NF-kappaB transcription by inducing endoprotease processing of the N-terminus of p65, a process antagonized by IkappaB alpha. Copine-I stimulates endoproteolysis of p65 within a conserved region that is required for base-specific contact with DNA. p65 proteins lacking the N-terminus fail to bind to DNA and act as dominant-negative molecules that inhibit NF-kappaB transcription. Our work provides evidence that copine-I regulates the half-life of NF-kappaB transcriptional responses through a novel mechanism that involves endoproteolysis of the p65 protein.

Characterization of the yeast tricalbins: membrane-bound multi-C2-domain proteins that form complexes involved in membrane trafficking.

In a survey of yeast genomic sequences encoding calcium- and phospholipid-binding C2 domains, three homologous genes were identified that encode proteins that each have three C2 domains and an apparent transmembrane domain near the N terminus. The name tricalbins is suggested for these proteins, corresponding to the open reading frames YOR086c (TCB1), YNL087w (TCB2), and YML072c (TCB3). An antiserum was raised against the C-terminal portion of tricalbin 2 and used on Western blots to demonstrate that the corresponding protein is expressed in yeast and appears as a high-molecular-weight band at 130 kDa with smaller fragments at 39 kDa and 46 kDa. A fusion protein consisting of full length tricalbin 2 fused to the green fluorescent protein was expressed in cells and found to traffic from the cell surface to intracellular vesicles near the vacuole. A two-hybrid interaction screen with the C-terminal portion of tricalbin 2 indicated that tricalbin 2 binds the C-terminal portions of tricalbins 1 and 3 suggesting that the tricalbins may form heterodimers in vivo. Tricalbin 2 also interacted with the activation domain of the pleiotropic drug resistance transcription factor Pdr1p. Combinatorial disruptions of the tricalbin genes revealed that tcb2 single mutants or tcb1, tcb3 double mutants have an altered vacuole morphology and are hypersensitive to cycloheximide. A screen for single-copy suppressors of the cycloheximide sensitivity of tricalbin mutants yielded RSP5, which encodes a C2-domain-containing, ubiquitin-conjugating ligase essential for receptor-mediated and fluid phase endocytosis. The results suggest that the tricalbins function as multimers in membrane-trafficking events and may provide insights into the roles of multi-C2-domain proteins, such as the synaptotagmins, in other organisms.

Copines: a ubiquitous family of Ca(2+)-dependent phospholipid-binding proteins.

The copines are a novel family of ubiquitous Ca(2+)-dependent, phospholipid-binding proteins. They contain two Ca(2+)- and phospholipid-binding domains known as 'C2 domains' present in proteins such as protein kinase C, phospholipase C and synaptotagmin. Copines are thought to be involved in membrane-trafficking phenomena because of their phospholipid-binding properties. They may also be involved in protein-protein interactions since they contain a domain similar to the protein-binding 'A domain' of integrins. The biochemistry, gene structure, tissue distribution and possible biological roles of copines are discussed, including recent observations with Arabidopsis that indicate that copines may be involved in cell division and growth.

Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding.

This study investigated mechanisms controlling the nuclear-cytoplasmic partitioning of annexin II (AnxII). AnxII and its ligand, p11, were localized by immunofluorescence to the cytoplasmic compartment of U1242MG cells, with minimal AnxII or p11 detected within nuclei. Similarly, GFP-AnxII and GFP-p11 chimeras localized to the endogenous proteins. Likewise, GFP-AnxII(1-22) was excluded from nuclei, whereas GFP-AnxII(23-338) and GFP alone were distributed throughout the cells. Immunoprecipitation and biochemical studies showed that GFP-AnxII did not form heteromeric complexes with endogenous p11 and AnxII. Thus, the AnxII N-tail is necessary and sufficient to cause nuclear exclusion of the GFP fusion protein but this does not involve p11 binding. A nuclear export signal consensus sequence was found in the AnxII 3-12 region. The consensus mutant GFP-AnxII(L10A/L12A) confirmed that these residues are necessary for nuclear exclusion. The nuclear exclusion of GFP-AnxII(1-22) was temperature-dependent and reversible, and the nuclear export inhibitor leptomycin B (LmB) caused GFP-AnxII or overexpressed AnxII monomer to accumulate in nuclei. Therefore, AnxII monomer can enter the nucleus and is actively exported. However, LmB had little effect on the localization of AnxII/p11 complex in U1242MG cells, indicating that the complex is sequestered in the cytoplasm. By contrast, LmB treatment of v-src-transformed fibroblasts caused endogenous AnxII to accumulate in nuclei. The LmB-induced nuclear accumulation of AnxII was accelerated by pervanadate and inhibited by genistein, suggesting that phosphorylation promotes nuclear entry of AnxII. Thus, nuclear exclusion of AnxII results from nuclear export of the monomer and sequestration of AnxII/p11 complex, and may be modulated by phosphorylation.

Domain formation in a fluid mixed lipid bilayer modulated through binding of the C2 protein motif.

The role and mechanism of formation of lipid domains in a functional membrane have generally received limited attention. Our approach, based on the hypothesis that thermodynamic coupling between lipid-lipid and protein-lipid interactions can lead to domain formation, uses a combination of an experimental lipid bilayer model system and Monte Carlo computer simulations of a simple model of that system. The experimental system is a fluid bilayer composed of a binary mixture of phosphatidylcholine (PC) and phosphatidylserine (PS), containing 4% of a pyrene-labeled anionic phospholipid. Addition of the C2 protein motif (a structural domain found in proteins implicated in eukaryotic signal transduction and cellular trafficking processes) to the bilayer first increases and then decreases the excimer/monomer ratio of the pyrene fluorescence. We interpret this to mean that protein binding induces anionic lipid domain formation until the anionic lipid becomes saturated with protein. Monte Carlo simulations were performed on a lattice representing the lipid bilayer to which proteins were added. The important parameters are an unlike lipid-lipid interaction term and an experimentally derived preferential protein-lipid interaction term. The simulations support the experimental conclusion and indicate the existence of a maximum in PS domain size as a function of protein concentration. Thus, lipid-protein coupling is a possible mechanism for both lipid and protein clustering on a fluid bilayer. Such domains could be precursors of larger lipid-protein clusters ('rafts'), which could be important in various biological processes such as signal transduction at the level of the cell membrane.

Differential lipid specificities of the repeated domains of annexin IV.

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.

Biochemical characterization of copine: a ubiquitous Ca2+-dependent, phospholipid-binding protein.

The copines are a novel group of Ca(2+)-dependent, phospholipid-binding proteins first isolated from Paramecium tetraurelia [Creutz, C. E., et al. (1998) J. Biol. Chem. 273, 1393-1402] and found in a wide range of organisms, from plants to humans. They have a Ca(2+) and phospholipid-binding domain consisting of two C2 domains and a core domain in the C-terminal portion that is homologous to the A domain found in certain integrins. We provide here the first description of the properties and distribution of a native mammalian copine, copine I. This protein is expressed in all major adult rat organs as demonstrated by probing Western blots of rat organ homogenates with anticopine antibodies. The highest levels of copine are found in the spleen. A protocol for purifying copine to homogeneity from bovine spleen is described. Purified native copine is a 58 kDa monomer that exhibits Ca(2+) self-association to form higher-order multimers, and Ca(2+)-dependent, phospholipid binding activity with preference for negatively charged phospholipids over neutral phospholipids and selectivity for Ca(2+) over Mg(2+). Half-maximal association with vesicles enriched in phosphatidylserine occurs at Ca(2+) concentrations between 1 and 10 microM. Copine I exhibits Mn(2+) binding activity that is strongly competed by Mg(2+) and partially competed by Ca(2+), suggesting that the copine I A domain may be a functional MIDAS metal binding site similar to that found in integrins [Lee, J. O., et al. (1995) Cell 80, 631-638]. Roles for copine in binding membranes and target proteins or small molecules are discussed.

Membrane-bound 3D structures reveal the intrinsic flexibility of annexin VI.

Several quasi-ordered arrays and three two-dimensional crystal forms of annexin VI were obtained on artificial lipid monolayers. Three-dimensional reconstructions of the crystal forms exhibit marked differences in the orientations of the two lobes, revealing flexibility of the linker between the two lobes of annexin VI. Evidence is presented that the lobes may bind the monolayer in a parallel orientation, or an antiparallel orientation, in which the second lobe is turned away from the monolayer. It is hypothesized that annexin VI may also adopt several conformations in vivo, underlying different functional roles.

Characterization of the Ca2+-dependent binding of annexin IV to surfactant protein A.

We have shown previously that surfactant protein A (SP-A) binds to annexin IV in a Ca2+-dependent manner [Sohma, Matsushima, Watanabe, Hattori, Kuroki and Akino (1995) Biochem. J. 312, 175-181]. Annexin IV is a member of the annexin family having four consensus repeats of about 70 amino acids and a unique N-terminal tail. In the present study, the functional site of both annexin IV and SP-A for the Ca2+-dependent binding was investigated using mutant proteins. SP-A bound in a Ca2+-dependent manner to an annexin-IV truncation mutant consisting of the N-terminal domain and the first three domains (T(N-1-2-3)). SP-A also bound to T3-4, but this interaction was not Ca2+-dependent. SP-A bound weakly to the other truncation mutants (T(N-1-2), T(2-3) and T(2-3-4)). Each consensus repeat of annexin IV possesses a conserved acidic amino acid residue (Glu70, Asp142, Glu226 and Asp301) that putatively ligates Ca2+. Using annexin-IV DE mutants in which one, two or three residues out of the four Asp/Glu were altered to Ala by site-directed mutagenesis [Nelson and Creutz (1995) Biochemistry 34, 3121-3132], it was revealed that Ca2+ binding in the third domain is more important than in the other Ca2+-binding sites. SP-A is a member of the animal lectin group homologous with mannose-binding protein A. The substitution of Arg197 of rat SP-A with Asp or Asn eliminated binding to annexin IV, whereas the substitution of Glu195 with Gln was silent. These results suggest that the Ca2+ binding to domain 3 of annexin IV is required for the Ca2+-dependent binding by SP-A and that Arg197 of SP-A is important in this binding.

Transcription, biochemistry and localization of nematode annexins.

The transcription of three annexin genes in the nematode, Caenorhabditis elegans, was detected by reverse transcriptase/polymerase chain reaction amplification of messenger RNAs. The highest level of expression was from the nex-1 gene, with lower levels detected for the nex-2 and nex-3 genes. The expression of nex-1 was reduced in the Dauer larval stage relative to the other annexins, correlating with the absence of the spermathecal valves, a major site of nex-1 protein localization. Recombinant nex-1 protein was expressed in yeast, isolated by calcium-dependent binding to acidic phospholipids, and its membrane binding and aggregating activities characterized using bovine chromaffin granules as a representative intracellular substrate. Binding to granule membranes was promoted by calcium with half-maximal binding seen at 630 microM calcium. Chromaffin granule aggregation was similarly promoted by the nex-1 protein at 630 microM calcium. This low sensitivity to calcium suggests the annexin can only be activated in vivo near the plasma membrane or other sources of calcium. Sequences including the nex-1 promoter were fused to the gene for green fluorescent protein and this construct was introduced into nematodes by microinjection. Examination of transgenic offspring revealed specific nex-1 promoter activity in the pharynx, the hypodermal cells, the vulva, and the spermathecal valve, locations in which the annexin may function in collagen secretion/deposition and membrane-membrane interactions. A sensitive anti-nex-1 antibody labelled with rhodamine was injected into body cavities of the nematode but did not detect extracellular nex-1 protein. Therefore, this annexin is apparently cytosolic and may function on the cytoplasmic side of the plasma membrane of the spermathecal valve to chaperon the folding of this membrane during the opening and closing of the valve.

Crystal structure of bovine annexin VI in a calcium-bound state.

The crystal structure of a calcium-bound form of bovine annexin VI has been determined with X-ray diffraction data to 2.9 A by molecular replacement. Six Ca2+ ions were found, five in AB loops, one in a DE loop. Two loops (II-AB, which binds calcium, and V-AB, which does not) have conformations that differ significantly from those in calcium-free, human recombinant annexin VI. There are only small differences between the calci- and the apo-annexin VI in the rest of the molecule. Calcium by itself does not promote a major conformational change.

The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans.

In an attempt to identify proteins that might underlie membrane trafficking processes in ciliates, calcium-dependent, phospholipid-binding proteins were isolated from extracts of Paramecium tetraurelia. The major protein obtained, named copine, had a mass of 55 kDa, bound phosphatidylserine but not phosphatidylcholine at micromolar levels of calcium but not magnesium, and promoted lipid vesicle aggregation. The sequence of a 920-base pair partial cDNA revealed that copine is a novel protein that contains a C2 domain likely to be responsible for its membrane active properties. Paramecium was found to have two closely related copine genes, CPN1 and CPN2. Current sequence data bases indicate the presence of multiple copine homologs in green plants, nematodes, and humans. The full-length sequences reveal that copines consist of two C2 domains at the N terminus followed by a domain similar to the A domain that mediates interactions between integrins and extracellular ligands. A human homolog, copine I, was expressed in bacteria as a fusion protein with glutathione S-transferase. This recombinant protein exhibited calcium-dependent phospholipid binding properties similar to those of Paramecium copine. An antiserum raised against a fragment of human copine I was used to identify chromobindin 17, a secretory vesicle-binding protein, as a copine. This association with secretory vesicles, as well the general ability of copines to bind phospholipid bilayers in a calcium-dependent manner, suggests that these proteins may function in membrane trafficking.

Membrane domain formation by calcium-dependent, lipid-binding proteins: insights from the C2 motif.

We propose a novel role in cellular function for some membrane-binding proteins and, specifically, the C2 motif. The C2 motif binds phospholipid in a manner that is modulated by Ca2+ and is thought to confer membrane-binding ability on a wide variety of proteins, primarily proteins involved in signal transduction and membrane trafficking events. We hypothesize that in the absence of Ca2+ the C2 motif couples the free energy of binding to a bilayer membrane comprised of zwitterionic and negatively charged lipids to the formation of a domain enriched in the negative lipids. This in turn leads to the dynamic clustering of bound homologous or heterologous proteins incorporating the C2 motif, or other acidic lipid-binding motifs. In the presence of Ca2+, the protein clusters may be further stabilized. In support of this hypothesis we present evidence for membrane domain formation by the first C2 domain of synaptotagmin in the absence of Ca2+. Fluid state phospholipid mixtures incorporating a pyrene-labeled phospholipid probe exhibited a change in pyrene excimer/monomer fluorescence ratio consistent with domain formation upon binding the C2 domain. In addition, we present the results of simulations of the interaction of the C2 domain with the membrane that indicate that protein clusters and lipid domains form in concert.

Calcium-dependent binding of sorcin to the N-terminal domain of synexin (annexin VII).

The annexins are characterized by their ability to bind phospholipid membranes in a Ca2+-dependent manner. Sequence variability between the N-terminal domains of the family members may contribute to the specific cellular function of each annexin. To identify proteins that interact with the N-terminal domain of synexin (annexin VII), a fusion protein was constructed composed of glutathione S-transferase fused to amino acids 1-145 of human synexin. Affinity chromatography using this construct identified sorcin as a Ca2+-dependent synexin-binding protein. Overlay assays confirmed the interaction. The glutathione S-transferase construct associates with recombinant sorcin over the range of pCa2+ = 4.7-3.1 with no binding observed at pCa2+ = 5.4. Overlay assays using deletion constructs of the synexin N-terminal domain mapped the sorcin binding site to the N-terminal 31 amino acids of the synexin protein. Additionally, synexin forms a complex with sorcin and recruits this protein to chromaffin granule membranes in a Ca2+-dependent manner. Sorcin is able to inhibit synexin-mediated chromaffin granule aggregation in a manner saturable with increasing sorcin concentrations, but does not influence the Ca2+ sensitivity of synexin-mediated granule aggregation. Therefore, the interaction between sorcin and synexin may serve to regulate the functions of these proteins on membrane surfaces in a Ca2+-dependent manner.

The crystal structure of annexin VI indicates relative rotation of the two lobes upon membrane binding.

The crystal structure of bovine liver annexin VI has been determined to low resolution by molecular replacement. The first lobe (domains 1-4) is rotated about 90 degrees relative to the second lobe (domains 5-8). Since the same crystal form (P4(3), 68 X 68 X 205 A) grew from (NH4)2SO4, polyethylene glycol, and sodium acetate with and without added calcium, this probably reflects the structure in solution. When bound to a lipid monolayer both lobes of annexin VI are coplanar. This implies a significant change in conformation upon binding to membranes.

Calcium-dependent self-association of synaptotagmin I.

Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium-triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1-5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium-dependent manner. p65 1-5 may be able to aggregate membrane vesicles by a self-association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1-5, in the absence of membranes, was able to self-associate to form large aggregates in a calcium-dependent manner as shown by light-scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self-associate and aggregate phospholipid vesicles in a calcium-dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self-associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium-dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium-dependent manner in vitro suggests that multimerization may have an important function in vivo.

Calcium-dependent binding of the plasma protein apolipoprotein A-I to two members of the annexin family.

Affinity chromatography with purified annexins coupled to CNBr-activated Sepharose 4B was used to determine the capacity of proteins found in cytosolic fractions of the bovine adrenal medulla to bind to an immobilized annexin in a Ca2+-dependent manner. Several proteins were eluted from a recombinant annexin I column in the presence of 2 mM EGTA, including protein kinase C (PKC), members of the annexin family, and a 26 kDa protein that appeared as the most prominent band on SDS-PAGE. The identities of PKC, annexin I, annexin IV, annexin VI, and annexin VII were confirmed by Western blotting. The 26 kDa protein was purified by anion exchange chromatography on a Poros Q column and determined to be apolipoprotein A-I (apoA-I) by peptide sequencing. Comigration of apoA-I and chromobindin 2 on two-dimensional gels identified apoA-I as chromobindin 2. Overlay assays were performed to verify the apoA-I-annexin I interaction using apoA-I immobilized on nitrocellulose and annexin I in solution with binding detected using anti-annexin I antiserum. Additionally, the ability of biotin-labeled apoA-I in solution to bind to several purified annexins immobilized on nitrocellulose was determined by detection with horseradish peroxidase-conjugated avidin. Using these methods, it was shown that both annexin I and annexin VII bind to bovine apoA-I in a Ca2+-dependent manner. Other annexins, such as annexin IV and annexin VI, do not exhibit this binding. The results suggest that certain annexins may function as extracellular binding sites for plasma proteins.

Identification, localization, and functional implications of an abundant nematode annexin.

Cultures of the nematode C. elegans were examined for the presence of calcium-dependent, phospholipid-binding proteins of the annexin class. A single protein of apparent mass on SDS-polyacrylamide gels of 32 kD was isolated from soluble extracts of nematode cultures on the basis of its ability to bind to phospholipids in a calcium-dependent manner. After verification of the protein as an annexin by peptide sequencing, an antiserum to the protein was prepared and used to isolate a corresponding cDNA from an expression library in phage lambda gt11. The encoded protein, herein referred to as the nex-1 annexin, has a mass of 35 kD and is 36-42% identical in sequence to 10 known mammalian annexins. Several unique modifications were found in the portions of the sequence corresponding to calcium-binding sites. Possible phosphorylation sites in the NH2-terminal domain of the nematode annexin correspond to those of mammalian annexins. The gene for this annexin (nex-1) was physically mapped to chromosome III in the vicinity of the dpy-17 genetic marker. Two other annexin genes (nex-2 and nex-3) were also identified in chromosome III sequences reported by the nematode genomic sequencing project (Sulston, J., Z. Du, K. Thomas, R. Wilson, L. Hillier, R. Staden, N. Halloran, P. Green, J. Thierry-Mieg, L. Qiu, et al. 1992. Nature (Lond.). 356:37-41). The nex-1 annexin was localized in the nematode by immunofluorescence and by electron microscopy using immunogold labeling. The protein is associated with membrane systems of the secretory gland cells of the pharynx, with sites of cuticle formation in the grinder in the pharynx, with yolk granules in oocytes, with the uterine wall and vulva, and with membrane systems in the spermathecal valve. The presence of the annexin in association with the membranes of the spermathecal valve suggests a novel function of the protein in the folding and unfolding of these membranes as eggs pass through the valve. The localizations also indicate roles for the annexin corresponding to those proposed in mammalian systems in membrane trafficking, collagen deposition, and extracellular matrix formation.

Synaptotagmin II expression partially rescues the growth defect of the yeast sec15 secretory mutant.

Synaptotagmins are a family of calcium- and phospholipid-binding proteins implicated in the function of cell exocytosis. Synaptotagmins I and II are neurally expressed proteins thought to be involved in neurotransmitter release from neurons. We have expressed rat synaptotagmin II in several Saccharomyces cerevisiae temperature-sensitive secretory mutants that are defective in Golgi to plasma membrane vesicular transport. Synaptotagmin II expression was able to partially rescue the growth defect in one particular mutant, sec15. No suppression was observed when synaptotagmin II was expressed in sec1, sec2, sec4, sec5, sec6, sec8, sec9, sec14, sec17, or sec18. Two synaptotagmin II deletion mutants were also expressed in sec15 and screened for suppression. The expression of the cytoplasmic domain of synaptotagmin alone was not able to suppress the sec15 growth defect. In addition, the expression of a synaptotagmin II fragment lacking the second half of the cytoplasmic domain including the second C2 domain did not suppress sec15. We have isolated a membrane fraction enriched in post-Golgi vesicles from a sec15 strain expressing synaptotagmin II and found that synaptotagmin II co-purifies with this fraction, suggesting that the rat synaptotagmin II is targeted to membranes in yeast. Sec15p forms a large multisubunit protein complex that includes Sec6p and Sec8p. This protein complex is thought to function in a late stage of exocytosis in yeast. Sec6p and Sec8p homologs have been identified in mammalian cells. Our studies suggest that synaptotagmin may be a part of this complex or regulate its function in mammalian cells.

Yeast cysteine proteinase gene ycp1 induces resistance to bleomycin in mammalian cells.

Tumor resistance to the glycopeptide anticancer drug bleomycin (BLM) has been suggested to involve metabolic inactivation by BLM hydrolase. Direct evidence for this hypothesis is lacking due to difficulties in obtaining full-length BLM hydrolase cDNA from mammalian cells. In the present investigation, we used the yeast cysteine proteinase gene ycp1, a homologue of the mammalian BLM hydrolase gene, to provide direct evidence of the importance of BLM metabolism in BLM resistance. Transfection of ycp1 into NIH 3T3 cells induced resistance of these cells to BLM. The ycp1-transfected cells also metabolized BLM A2 to its inactive metabolite deamido-BLM A2 to a much greater extent. The ycp1-induced BLM resistance was completely reversed by the cysteine proteinase inhibitor E-64, a known inhibitor of BLM hydrolase. Transfection of NIH 3T3 cells with the plasmid pUT533-Sh ble, a bacterial BLM resistance gene that encodes a 14-kDa protein that does not metabolize BLM, also induced BLM resistance, but this resistance was not overcome by E-64. The results demonstrate that increased BLM hydrolase activity in NIH 3T3 cells causes BLM resistance and that inhibition of BLM metabolism sensitizes these cells to BLM. Thus, the molecular approach described in the present study directly implicates BLM hydrolase in BLM resistance.