Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene.

We have shown that the DNA demethylation complex isolated from chicken embryos has a G(.)T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a beta-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor alpha. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.

5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence.

A 1468 bp cDNA coding for the chicken homolog of the human MBD4 G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (416 amino acids) shows 46% identity with the human MBD4 and the conserved catalytic region at the C-terminal end (170 amino acids) has 90% identity. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have G/T mismatch as well as 5-methylcytosine (5-MeC) DNA glycosylase activities. When tested by gel shift assays, human recombinant protein with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzyme has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Footprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of the proteins to the modified substrate around the CpG. As for 5-MeC DNA glycosylase purified from chicken embryos, MBD4 does not use oligonucleotides containing mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligonucleotide is a much better substrate than an A+T-poor sequence. The K:(m) of human MBD4 for hemimethylated DNA is approximately 10(-7) M with a V:(max) of approximately 10(-11) mol/h/microgram protein. Deletion mutations show that G/T mismatch and 5-MeC DNA glycosylase are located in the C-terminal conserved region. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indicate that MBD4 is only located in dividing cells of differentiating embryonic tissues.

5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex.

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.

A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase.

We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced. Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595). This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity. However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity. In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.

Inhibition of matrix metalloproteinase 2 maturation and HT1080 invasiveness by a synthetic furin inhibitor.

The close correlation observed between matrix metalloproteinase 2 (MMP-2) activation and metastatic progression in various tumors suggests that MMP-2 is a 'master switch' triggering tumor spread. Recently, membrane type 1 MMP (MT1-MMP) was identified as a potential physiological activator of MMP-2. Like all other MMPs, MT1-MMP possesses a pro-domain which must be removed for the enzyme to acquire its catalytic potential. The presence of a typical recognition motif (RXKR) for the furin-like convertases at the end of its pro-domain suggests a potential role for these proteinases in MT1-MMP processing. In order to evaluate the implication of furin in pro-MT1-MMP processing, we treated HT1080 cells with a synthetic furin inhibitor and monitored their ability to activate pro-MMP-2 as well as their invasive potential. Our results demonstrated that the furin inhibitor decreased pro-MT1-MMP processing as well as pro-MMP-2 activation and cell invasiveness. Therefore, our data bring further evidence that furin is a key factor in the maturation of MMPs associated with the invasive and metastatic potential of tumor cells.

Allosteric modulation of the activity of thrombin.

Substrates containing a P3 aspartic residue are in general cleaved poorly by thrombin. This may be partly due to an unfavourable interaction between the P3 aspartate and Glu192 in the active site of thrombin. In Protein C activation and perhaps also thrombin receptor cleavage, binding of ligands at the anion-binding exosite of thrombin seems to improve the activity of thrombin with substrates containing a P3 aspartate. To investigate the importance of Glu192 and exosite-binding in modulating thrombin's interactions with a P3 aspartate, peptidyl chloromethanes based on the sequence of the thrombin receptor (containing a P3 aspartate) have been synthesized and the kinetics of their inactivation of alpha-thrombin and the mutant Glu192-->Gln determined. The values of the inactivation rate constant (ki) for the chloromethanes containing a P3 aspartate were about two-fold higher with the Glu192-->Gln mutant. A peptide based on the sequence of hirudin (rhir52 65), which binds to the anion-binding exosite of thrombin, was an allosteric modulator of the amidolytic activity of the Glu192-->Gln mutant; a 5-fold decrease in the K(m) value for the substrate D-Phe-pipecolyl-Arg-p-nitroanilide was observed in the presence of saturating concentrations of rhir52-65. This exosite-binding peptide also increased the ki values of chloromethanes containing a P3 aspartate with both alpha-thrombin and the Glu192-->Gln mutant. However, the increases in the ki values were greater with the Glu192-->Gln mutant (5-fold compared with 2-fold for alpha-thrombin). Thus exosite binding does not seem to mitigate putative unfavourable interactions between Glu192 and the P3 aspartate. Moreover, increases in the ki caused by exosite binding were not unique to chloromethanes containing a P3 aspartate; increases of the same magnitude were also observed when the P3 position was occupied by the favourable D-phenylalanine in place of the unfavourable aspartate. The results obtained were consistent with exosite binding's causing changes in the conformation of the S2 and/or S1 site of thrombin.

Characterization of structural determinants and molecular mechanisms involved in pro-stromelysin-3 activation by 4-aminophenylmercuric acetate and furin-type convertases.

Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP) which has been implicated in cancer progression and in a number of conditions involving tissue remodelling. In contrast to other MMPs which are secreted as zymogens requiring extracellular activation, ST3 is found in the extracellular space as a potentially active mature form, suggesting that the activation of the ST3 proform differs from that of other MMPs. We show in the present study that the ST3 proform is not autocatalytically processed in the presence of 4-aminophenylmercuric acetate (APMA). By using ST3/ST2 chimeras, we demonstrate that resistance to APMA is due to properties associated with both the ST3 pro- and catalytic domains. In agreement with the observation made by Pei and Weiss [Pei and Weiss (1995) Nature (London) 375, 244-247], we find that the requirement for activation of the ST3 proform by the furin convertase is entirely contained within a stretch of 10 amino acids located at the junction between the ST3 pro- and catalytic domains. Furin cleaves human and mouse ST3 equally well. However, PACE-4, a furin-like convertase, is much more efficient on the mouse enzyme, suggesting that ST3 protein determinants other than the conserved Ala-Arg-Asn-Arg-Gln-Lys-Arg sequence preceding the furin cleavage site are implicated in PACE-4 action. Finally, we show that processing of the ST3 proform is inhibited by a furin inhibitor in human MCF7 breast cancer cells stably transfected to constitutively express a full-length human ST3 cDNA. Using brefeldin A, we demonstrate that, in these MCF7 cells, the 56 kDa precursor form of ST3 is post-translationally modified in the cis- or media-Golgi into a 62 kDa proform. Thereafter, its processing into the 47 kDa mature form occurs in the trans-Golgi network and is followed by secretion into the extracellular space.

Synthesis of tight binding inhibitors and their action on the proprotein-processing enzyme furin.

Furin is a subtilisin-like eukaryotic serine endoprotease which processes proproteins to biologically active proteins and peptides. Also, the envelope proteins of viruses, such as influenza and HIV viruses, need to be processed by furin for infectivity. This enzyme has a consensus substrate specificity for Arg-Xxx-Lys/Arg-Arg at the cleavage site. Two kinds of transition state analog peptides were designed and tested in vitro with furin. The ketomethylene series, psi (COCH2), have Ki's in the submicromolar range; the aminomethyl aminomethyl ketone series, psi(COCH2NH), have Ki's in the nanomolar range. The best inhibitor is Dec-Arg-Val-Lys-Arg-CH2-Ala-Val-Gly-NH2 (2c) with a Ki of 3.4 nM.

Internally quenched fluorogenic substrate for furin.

A new substrate for furin, Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO2)-Asp-OH, has been synthesized and characterized. The peptide is an internally quenched fluorogenic substrate. The kinetic parameters are Km = 3.8 microM, kcat = 29.3 s-1, and kcat/KM = 7,710,000 M-1 s-1. The substrate is efficiently cleaved by furin; its kcat/KM value is over 2000-fold higher than that of the commonly used substrate Boc-Arg-Val-Arg-Arg-AMC.

Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2.

The fusion (F) protein of human parainfluenza virus type 3 contains the tribasic cleavage site R-T-K-R, which was altered by site-directed mutagenesis. Wild-type F protein and various mutants were expressed by recombinant vaccinia viruses. The endogenous endoprotease present in CV-1 cells cleaves F variants containing the furin recognition motif R-X-K/R-R but not variants containing the dibasic site K-R or a single R at the cleavage site. A similar cleavage pattern was obtained when the subtilisin-like endoproteases Kex2 and furin were coexpressed with the wild type and mutants of the F protein. Peptidylchloromethylketone inhibitors mimicking basic cleavage sites prevent cleavage of the precursor Fo by the endogenous protease only when the furin-specific motif is present in the peptidyl portion. The data support the concept that furin is a cellular protease responsible for the activation of the F protein of human parainfluenza virus type 3.

Processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones.

The spike glycoproteins of many enveloped viruses are proteolytically cleaved at the carboxytermini of sequences containing the basic motif R-X-K/R-R. Cleavage is often necessary for the fusion capacity of the glycoproteins and, thus, for virus infectivity. Among these viruses are pathogenic avian influenza viruses, human parainfluenza virus, human cytomegalovirus, and human immunodeficiency virus; it has been demonstrated that these viruses can be activated by furin. Indigenous furin has been identified in T-lymphocytes, which are host cells for HIV. Furin has been localized in the TGN and on the surface of cells after vectorial expression. Peptidylchloroalkylketones have been designed that inhibit with high specificity cleavage and fusion activity of viral glycoproteins, as well as virus replication.

Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes.

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.

The synthesis of inhibitors for processing proteinases and their action on the Kex2 proteinase of yeast.

Peptidyl chloromethane and sulphonium salts containing multiple Arg and Lys residues were synthesized as potential inhibitors of prohormone and pro-protein processing proteinases. The potencies of these compounds were assayed by measuring the kinetics of inactivation of the yeast Kex2 proteinase, the prototype of a growing family of eukaryotic precursor processing proteinases. The most potent inhibitor, Pro-Nvl-Tyr-Lys-Arg-chloromethane, was based on cleavage sites in the natural Kex2 substrate pro-alpha-factor. This inhibitor exhibited a Ki of 3.7 nM and a second-order inactivation rate constant (k2/Ki) of 1.3 x 10(7) M-1.s-1 comparable with the value of kcat./Km obtained with Kex2 for the corresponding peptidyl methylcoumarinylamide substrate. The enzyme exhibited sensitivity to the other peptidyl chloromethanes over a range of concentrations, depending on peptide sequence and alpha-amino decanoylation, but was completely resistant to peptidyl sulphonium salts. Kinetics of inactivation by these new inhibitors of a set of 'control' proteinases, including members of both the trypsin and subtilisin families, underscored the apparent specificity of the compounds most active against Kex2 proteinase.

Synthesis of oligopeptide chloromethanes to investigate extended binding regions of proteinases: application to the interaction of fibrinogen with thrombin.

A method was established for the synthesis of oligopeptide chloromethanes which should be useful in the study of serine and cysteine proteinases with extended binding sites. The method involved condensation of an N-terminal peptide fragment obtained by solid-phase synthesis with a C-terminal peptide chloromethane synthesized by solution-phase chemistry. By using this procedure, oligopeptide chloromethanes of up to 16 residues were synthesized. These chloromethanes were based on the sequence of fibrinopeptide A. By using oligopeptide chloromethanes of different length, it was possible to show that the residues Asp7-Phe8-Leu9 play a crucial role in the recognition of fibrinopeptide A by thrombin. In contrast, the residues Ala1-Asp2-Ser3-Gly4-Glu5-Gly6 seem to play a minor role. Substitution of valine for Gly12, which occurs in a dysfibrinogenaemia, markedly decreased the rate of inactivation of thrombin by the oligopeptide chloromethane. The results are discussed in terms of the recently published structure of the complex between human thrombin and a chloromethane inhibitor based on fibrinopeptide A.

Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.

The interaction of thrombin with fibrinogen. A structural basis for its specificity.

The structure of the ternary complex of human alpha-thrombin with a covalently bound analogue of fibrinopeptide A and a C-terminal hirudin peptide has been determined by X-ray diffraction methods at 0.25 nm resolution. Fibrinopeptide A folds in a compact manner, bringing together hydrophobic residues that slot into the apolar binding site of human alpha-thrombin. Fibrinogen residue Phe8 occupies the aryl-binding site of thrombin, adjacent to fibrinogen residues Leu9 and Val15 in the S2 subsite. The species diversity of fibrinopeptide A is analysed with respect to its conformation and its interaction with thrombin. The non-covalently attached peptide fragment hirudin(54-65) exhibits an identical conformation to that observed in the hirudin-thrombin complex. The occupancy of the secondary fibrinogen-recognition exosite by this peptide imposes restrictions on the manner of fibrinogen binding. The surface topology of the thrombin molecule indicates positions P1'-P3', differ from those of the canonical serine-proteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to protein-C activation on thrombomodulin complex formation. The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.

Additional peptidyl diazomethyl ketones, including biotinyl derivatives, which affinity-label calpain and related cysteinyl proteinases.

Calpain, the calcium-activated cysteinyl proteinase, can be irreversibly inactivated by peptidyl diazomethyl ketones in which the peptide portion contains a penultimate leucine residue. Some new derivatives of this type have been synthesized and examined for their rates of inactivation of chicken gizzard and human platelet calpain. Two derivatives containing a C-terminal biotin residue, Biot-Aca-Leu-TyrCHN2 and Biot-Aca-Leu-Leu-TyrCHN2, have also been prepared in the expectation that their application to the study of the function of calpain and related proteases will prove fruitful.

Inactivation of calpain by peptidyl fluoromethyl ketones.

The syntheses of Z-Leu-Leu-Tyr-CH2F (1) and Z-Tyr-Ala-CH2F (3) are described. The ability of Z-Leu-Leu-Tyr-CH2F (1) and Z-Leu-Tyr-CH2F (2) to inactivate in vitro calcium-activated proteinase from chicken gizzard are compared. Like the analogous diazomethyl ketones 4 and 5, these inhibitors were also found to inactivate cathepsin L in common with other inhibitors under current investigation. However, other specific inactivators for cathepsin L are available, for example, the fluoromethyl ketone 3 and diazomethyl ketone 6 of Z-Tyr-Ala-OH, which have no effect on the calcium-activated proteinase and therefore provide control inhibitors for observations made with Z-Leu-Leu-Tyr-CH2F (1).

Synthesis of histidine-containing dipeptide affinity-labelling agents. Relative inactivation rates of cathepsins B and L.

Peptidyl diazomethyl ketones and fluoromethyl ketones containing histidine in the C-terminal position were synthesized to determine their properties as proteinase inactivators. These were examined chiefly with derivatives of Z-Ala-His. The protection of histidine during conversion of the C-terminal residue to the diazomethyl ketone required unblocking conditions which avoid acid due to the lability of this function. This was achievable with a Cbz-imidazole derivative since aminolysis provided deblocking without disturbance of the diazomethyl ketone function. In the case of the fluoromethyl ketone synthesis using fluoroacetic anhydride (Dakin-West procedure), the desired product could be isolated without ring blocking. The Z-Ala-His products showed enhanced selectivity for inactivation of cathepsin B over L when compared to analogous dipeptide inhibitors.