Identification of a Novel DNA Methyltransferase Activity from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A DNA methyltransferase activity was identified in a strain of Bacillus thuringiensis that was found to protect DNA from cleavage by the restriction endonuclease HaeIII at overlapping sites. Site-directed mutagenesis was used to confirm the recognition sequence of the methyltransferase as ACGGC. RID= ID= <E5>Correspondence to:</E5> N. Crickmore; <E5>email:</E5> n.crickmore&commat;sussex.ac.uk Received: 13 September 2002 / Accepted: 7 October 2002     

Matriptase,HAT, and TMPRSS2 Activate the Hemagglutinin of H9N2 Influenza A Viruses

Influenza A viruses of the subtype H9N2 circulate worldwide and have become highly prevalent in poultry in many countries. Moreover, they are occasionally transmitted to humans, raising concern about their pandemic potential. Influenza virus infectivity requires cleavage of the surface glycoprotein hemagglutinin (HA) at a distinct cleavage site by host cell proteases. H9N2 viruses vary remarkably in the amino acid sequence at the cleavage site, and many isolates from Asia and the Middle East possess the multibasic motifs R-S-S-R and R-S-R-R, but are not activated by furin. Here, we investigated proteolytic activation of the early H9N2 isolate A/turkey/Wisconsin/1/66 (H9-Wisc) and two recent Asian isolates, A/quail/Shantou/782/00 (H9-782) and A/quail/Shantou/2061/00 (H9-2061), containing mono-, di-, and tribasic HA cleavage sites, respectively. All H9N2 isolates were activated by human proteases TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease). Interestingly, H9-782 and H9-2061 were also activated by matriptase, a protease widely expressed in most epithelia with high expression levels in the kidney. Nephrotropism of H9N2 viruses has been observed in chickens, and here we found that H9-782 and H9-2061 were proteolytically activated in canine kidney (MDCK-II) and chicken embryo kidney (CEK) cells, whereas H9-Wisc was not. Virus activation was inhibited by peptide-mimetic inhibitors of matriptase, strongly suggesting that matriptase is responsible for HA cleavage in these kidney cells. Our data demonstrate that H9N2 viruses with R-S-S-R or R-S-R-R cleavage sites are activated by matriptase in addition to HAT and TMPRSS2 and, therefore, can be activated in a wide range of tissues what may affect virus spread, tissue tropism and pathogenicity.     

Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor.

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I-labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage ( approximately 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317-367) was replaced with the corresponding domain of the TSHR (residues 363-418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317-319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino-acid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.     

Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P

The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.     

The effect of cleavage at site 1 of gizzard HMM in the interaction with skeletal muscle actin

Two different classes of gizzard heavy meromyosins (HMMs) were prepared from phosphorylated myosin by chymotryptic digestion in the presence and absence of ATP and were compared with respect to their actin-activated Mg2+-ATPase reactions. One class of HMM, named HMM(+), had a cleavage at site 1 in the N terminal portion of the heavy chain and the other class of HMM, named HMM(-), had no cleavage at this site. Maximum turnover rate (Vmax) of the skeletal acto-gizzard HMM Mg2+-ATPase reaction was obviously different between HMM(+) and HMM(-). The Vmax value of HMM(+) was 2.5-fold larger than that of HMM(-). On the other hand, the apparent association constants (Ka) of skeletal muscle actin for both HMMs which were deduced from double reciprocal plots (v-1 versus [actin]-1) seemed to be identical. The difference in Vmax value was attributed to the cleavage at site 1 since a following chymotryptic cleavage of HMM(-) at site 1 caused a 2.5-fold increase in the Vmax value. That site 1 in the N terminal portion of the gizzard myosin heavy chain was the key locus for the actin-myosin interaction was shown in addition to our previous finding of the effects of cleavage at site 1 on the ATPase activity and nucleotide binding ability of gizzard HMM (Okamoto, Y. & Sekine, T. (1981) J. Biochem. 90, 833-843; 843-949).     

Detailed analysis of base preferences at the cleavage site of a trans-acting HDV ribozyme: a mutation that changes cleavage site specificity.

In our previous attempt at in vitro selection of a trans - acting human hepatitis delta virus (HDV) ribozyme, we found that one of the variants, G10-68-725G, cleaved a 13 nt substrate, HDVS1, at two sites [Nishikawa,F., Kawakami,J., Chiba,A., Shirai,M., Kumar,P.K.R. and Nishikawa,S. (1996) Eur. J. Biochem., 237, 712-718]. One site was the normal cleavage site and the other site was shifted 1 nt toward the 3'-end. To clarify the interactions between nucleotides around the cleavage site of the trans -acting HDV ribozyme, we analyzed the efficiency of the reaction for every possible base pair between the substrate and the ribozyme at positions -1 (-1N:726N) and +1 (+1N:725N) relative to the cleavage site using the genomic HDV ribozyme, TdS4(Xho), and derivatives of the most active variant, G10-68. These mutagenesis analyses revealed that the +1 base of the substrate affects the structure of the catalytic core in the complex with G10-68-725G, substrate and divalent metal ions, and it shifts the cleavage site. In a comparison with other variants of the trans -acting HDV ribozyme, we found that this cleavage site shift occurred only with G10-68-725G.     

Processing of human cytomegalovirus UL37 mutant glycoproteins in the endoplasmic reticulum lumen prior to mitochondrial importation

The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues -1 and -3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic alpha-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37(M), a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH(2)-terminal fragment, pUL37(M-NH2), was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37(M-COOH), was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH(2)-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37(M), and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.     

Deuterium isotope effects and product studies for the oxidation of N(omega)-allyl-L-arginine and N(omega)-allyl-N(omega)-hydroxy-L-arginine by neuronal nitric oxide synthase

The nitric oxide synthases (NOS), which require heme, tetrahydrobiopterin, FMN, FAD, and NADPH, catalyze the O2-dependent conversion of L-arginine to L-citrulline and nitric oxide. N(omega)-Allyl-L-arginine, a mechanism-based inactivator of neuronal NOS, also is a substrate, producing L-arginine, acrolein, and H2O (Zhang, H. Q.; Dixon, R. P., Marletta, M. A.; Nikolic, D.; Van Breemen, R.; Silverman, R. B. J. Am. Chem. Soc. 1997, 119, 10888). Two possible mechanisms for this turnover are proposed, one initiated by allyl C-H bond cleavage and the other by guanidino N H cleavage, and these mechanisms are investigated with the use of N(omega)-allyl-L-arginine (1), N(omega)-[1,1-(2)H2]allyl-L-arginine (7), N(omega)-allyl-N(omega)-hydroxy-L-arginine (2) and N(omega)-[1,1-(2)H2]allyl-N(omega)-hydroxy-L-arginine (8) as substrates. Significant isotope effects on the two kinetic parameters, kcat and kcat/Km, are observed in case of 1 and 7 during turnover, but not with 2 and 8. No kinetic isotope effects are observed for either compound in their role as inactivators. These results support a mechanism involving initial C-H bond cleavage of N(omega)-allyl-L-arginine followed by hydroxylation and breakdown to products.     

Fertilization potential and electrical properties of the Xenopus laevis egg

The membrane potential of Xenopus eggs was monitored continuously from prior to fertilization until early cleavage. A rapid decay of the initial potential of -33.1 +/- 8.1 (SD) mV (N = 14) upon impalement to a value of -19.3 +/- 4.2 (SD) mV (N = 68) suggested that insertion of the first electrode caused depolarization. Outward and inward rectification were observed when the resting potential was made more positive than about 5 mV or more negative than about -30 mV. Eggs were not activated by this level of current injection. Fertilization and activation evoked a membrane depolarization which was influenced by the external Cl- concentration, the nature of the halide species, and 4,4-diisothiocyanostilbene-2,2-disulfonic acid. Smaller transient depolarizations were associated with the initial stages of the fertilization potential but not with activation. Only when the fertilization potential was significantly diminished, as in high external Cl- or in the presence of Br- or I- solutions did polyspermy ensue. The input resistance of the unfertilized egg was 13.2 +/- 9.8 M omega (N = 26) and decreased about 200-fold at the peak of the fertilization potential to 0.077 +/- 0.020 M omega (N = 9). Ninety minutes after the onset of the fertilization potential and about 6 min after the start of furrow formation the membrane began a series of cleavage cycle-associated hyperpolarizations. These were unaffected by either the external Cl- concentration or other halide species. Reduction in amplitude of the fertilization potential had no apparent effect upon the normal elevation of the fertilization envelope or upon cleavage and later development. The fast electrical block to polyspermy appears to have a lower threshold in Xenopus compared with other species and is also effective at negative membrane potentials.     

Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites.

The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the diabasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites.     

Is the alkaline cleavage of disulfide bonds in peptides an alpha-beta elimination reaction or a hydrolysis?

The cleavage of oxidized glutathione by alkali has been studied as representative of the cleavage of protein disulfides. This process is quite different when studied in 10-4N or 2-10-1N NaOH. At low alkali concentration no spectral changes are noted; at higher hydroxyl concentration the appearance of persulfide groups (followed at 335 nm), the formation of thiocyanate (arising from cold cyanolysis of persulfide groups) and the absorbance at 240 nm follow the same kinetics. The amount of half-cystine, recovered as cysteic acid after a 3-h reaction, is significantly lower than calculated. These results confirm that oxidized glutathione undergoes beta-elimination at high pH values, and that persulfide groups absorb not only at 335 nm (as already known) but also at 240 nm where, under our conditions, the contribution of other absorbing species is not very high.     

The non-enzymatic hydrolysis of oligoribonucleotides VI. The role of biogenic polyamines.

Single-stranded oligoribonucleotides containing UA and CA phosphodiester bonds can be hydrolyzed specifically under non-enzymatic conditions in the presence of spermidine, a biogenic amine found in a wide variety of organisms. In the present study, the rate of oligonucleotide and tRNA(i)(Met)hydrolysis was measured in the presence of spermidine and other biogenic amines. It was found that spermine [H(3)N(+)(CH(2))(3)(+)NH(2)(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] and putrescine [H(3)N(+)(CH(2))(4)(+)NH(3)] can replace spermidine [H(3)N(+)-(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] to induce the hydrolysis. For all three polyamines, a bell-shaped cleavage rate versus concentration relationship was observed. The maximum rate of hydrolysis was achieved at 0.1, 1.0 and 10 mM spermine, spermidine and putrescine, respectively. Moreover, we found that the hydrolysis requires at least two linked amino groups since two aminoalcohols, 2-aminoethanol and 3-aminopropanol, were not able to induce the cleavage of the phospho-diester bond. The optimal cleavage rate of the oligo-ribonucleotides was observed when amino groups were separated by tri- or tetramethylene linkers. The methylation of the amino groups reduced the ability of diamines to induce oligoribonucleotide hydrolysis. Non-enzymatic cleavage of tRNA(i)(Met)from Lupinus luteus and tRNA(i)(Met)from Escherichia coli demonstrate that both RNAs hydrolyze as expected from principles derived from oligoribonucleotide models.     

Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine

PvuRts1I is a modification-dependent restriction endonuclease that recognizes 5-hydroxymethylcytosine (5hmC) as well as 5-glucosylhydroxymethylcytosine (5ghmC) in double-stranded DNA. Using PvuRts1I as the founding member, we define a family of homologous proteins with similar DNA modification-dependent recognition properties. At the sequence level, these proteins share a few uniquely conserved features. We show that these enzymes introduce a double-stranded cleavage at the 3'-side away from the recognized modified cytosine. The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority being 11-13 nt away in the top strand and 9-10 nt away in the bottom strand. The recognition sites of these enzymes generally require two cytosines on opposite strand around the cleavage sites, i.e. 5'-CN(11-13)↓N(9-10)G-3'/3'-GN(9-10)↓N(11-13)C-5', with at least one cytosine being modified for efficient cleavage. As one potential application for these enzymes is to provide useful tools for selectively mapping 5hmC sites, we have compared the relative selectivity of a few PvuRts1I family members towards different forms of modified cytosines. Our results show that the inherently different relative selectivity towards modified cytosines can have practical implications for their application. By using AbaSDFI, a PvuRts1I homolog with the highest relative selectivity towards 5ghmC, to analyze rat brain DNA, we show it is feasible to map genomic 5hmC sites close to base resolution. Our study offers unique tools for determining more accurate hydroxymethylomes in mammalian cells.     

Expression of Na+,K+-ATPase in Pichia pastoris: analysis of wild type and D369N mutant proteins by Fe2+-catalyzed oxidative cleavage and molecular modeling

Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg-1. Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E2 conformations. The results suggest a possible role of the charge of Asp369 on the E1 <--> E2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodi-phosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P (712VNDS) and N (near 440VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near 367CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp369 of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn351 structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.     

Effects of antineoplastic drugs on the post-strand-passage DNA cleavage/religation equilibrium of topoisomerase II.

The post-strand-passage DNA cleavage/religation equilibrium of Drosophila melanogaster topoisomerase II was examined. This was accomplished by including adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analogue which supports strand passage but not enzyme turnover, in assays. Levels of post-strand-passage enzyme-mediated DNA breakage were 3-5 times higher than those generated by topoisomerase II prior to the strand-passage event. This finding correlated with a decrease in the apparent first-order rate of topoisomerase II mediated DNA religation in the post-strand-passage cleavage complex. Since previous studies demonstrated that antineoplastic drugs stabilize the pre-strand-passage cleavage complex of topoisomerase II by impairing the enzyme's ability to religate cleaved DNA [Osheroff, N. (1989) Biochemistry 28, 6157-6160; Robinson, M.J., & Osheroff, N. (1990) Biochemistry 29, 2511-2515], the effects of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide on the enzyme's post-strand-passage DNA cleavage complex were characterized. Both drugs stimulated the ability of topoisomerase II to break double-stranded DNA after strand passage. As determined by two independent assay systems, m-AMSA and etoposide stabilized the enzyme's post-strand-passage DNA cleavage complex primarily by inhibiting DNA religation. These results strongly suggest that both the pre- and post-strand-passage DNA cleavage complexes of topoisomerase II serve as physiological targets for these structurally disparate antineoplastic drugs.     

Mechanism of the mutagenic action of hydroxylamine. IX. The UV-induced cleavage of the N-O bond in N4-hydroxy-and N4-methoxycytidine and N6-methoxyadenosine.

The principal UV-induced (lambda = 2546nm) reaction of N4-hydroxy- and N4methoxycytidines and N6-methoxyadenosine in neutral aqueous solutions is cleavage of the exocyclic N-O bond with the respective formation of cytidine and adenosine. Quantum yields are 2.8x10(-3) and 2.2x10(-3) M/E for the first two compounds and 9.1x10(-3) M/E for N6-methoxyadenosine.     

Site-specific phosphorylation protects glycogen synthase kinase-3β from calpain-mediated truncation of its N and C termini

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38-Thr-39 and Ile-384-Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39-420), ΔC-GSK-3β (amino acids 1-384), and ΔN/ΔC-GSK-3β (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity.     

Caspase-dependent N-terminal cleavage of influenza virus nucleocapsid protein in infected cells

The nucleocapsid protein (NP) (56 kDa) of human influenza A viruses is cleaved in infected cells into a 53-kDa form. Likewise, influenza B virus NP (64 kDa) is cleaved into a 55-kDa protein with a 62-kDa intermediate (O. P. Zhirnov and A. G. Bukrinskaya, Virology 109:174-179, 1981). We show now that an antibody specific for the N terminus of influenza A virus NP reacted with the uncleaved 56-kDa form but not with the truncated NP53 form, indicating the removal of a 3-kDa peptide from the N terminus. Amino acid sequencing revealed the cleavage sites ETD16*G for A/Aichi/68 NP and sites DID7*G and EAD61*V for B/Hong Kong/72 NP. With D at position -1, acidic amino acids at position -3, and aliphatic ones at positions -2 and +1, the NP cleavage sites show a recognition motif typical for caspases, key enzymes of apoptosis. These caspase cleavage sites demonstrated evolutionary stability and were retained in NPs of all human influenza A and B viruses. NP of avian influenza viruses, which is not cleaved in infected cells, contains G instead of D at position 16. Oligopeptide DEVD derivatives, specific caspase inhibitors, were shown to prevent the intracellular cleavage of NP. All three events, the NP cleavage, the increase of caspase activity, and the development of apoptosis, coincide in cells infected with human influenza A and B viruses. The data suggest that intracellular cleavage of NP is exerted by host caspases and is associated with the development of apoptosis at the late stages of infection.     

A proposed common structure of substrates bound to mitochondrial processing peptidase

Mitochondrial processing peptidase (MPP), a metalloendopeptidase consisting of alpha- and beta-subunits, specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Structural information of the substrate bound to MPP was obtained using fluorescence resonance energy transfer (FRET) measurement. A series of the peptide substrates, which have distal arginine residues required for effective cleavage at positions -7, -10, -14, and -17 from the cleavage site, were synthesized and covalently labeled with 7-diethyl aminocoumarin-3-carboxylic acid at the N termini and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) at position +4, as fluorescent donor and acceptor, respectively. When the peptides were bound to MPP, substantially the same distances were obtained between the two probes, irrespective of the length of the intervening sequence between the two probes. When 7-diethylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin was introduced into a single cysteine residue in beta-MPP as a donor and IANBD was coupled either at the N terminus or the +4 position of the peptide substrate as an acceptor, intermolecular FRET measurements also demonstrated that distances of the donor-acceptor pair were essentially the same among the peptides with different lengths of intervening sequences. The results indicate that the N-terminal portion and the portion around the cleavage site of the presequence interact with specific sites in the MPP molecule, irrespective of the length of the intervening sequence between the two portions, suggesting the structure of the intervening sequence is flexible when bound to the MPP.