Minor Cleavage Sites Research Articles

Antibodies against H3 and H4 histones from the sera of HIV-infected patients catalyze site-specific degradation of these histones.

Histones and their posttranslational modified forms play pivotal roles in chromatin functioning and gene transcription. Also, histones are harmful when they enter the intercellular space; their administration to animals results in systemic inflammatory and toxic responses. Autoantibodies having enzymatic activities (abzymes) are the specific feature of several autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical proteases were purified from sera of HIV-infected patients by using several affinity chromatographies. In contrast to known canonical proteases, Abs from HIV-infected patients hydrolyzed exclusively only histones but no other control globular proteins. The H3 and H4 histone cleavage sites by antihistone IgGs were determined by matrix-assisted laser desorption/ionization mass spectrometry for the first time. Two clusters of H3 hydrolysis contain major (↕) and minor (*) cleavage sites: 18-K*Q*LA↕TK*A↕AR*KS↕A*P-30 and 34-G*VK*KPHR*YRPGTVA*L*R-50. H4 histone has only 1 cluster of cleavage sites containing additionally moderate (↓) cleavage sites: 15-A↕KR↕HR↕KVLR↓D*NIQ↓GIT*K-31. Sites of these histones cleavage correspond mainly to their known epitopes. It was surprising that most of the cleavage sites of histones are involved in the interaction with DNA of nucleosome core. Because histones act as damage-associated molecules, abzymes against H3 and H4 can play important role in pathogenesis of AIDs and probably other viral and immune diseases.

Identification of sites in apolipoprotein A-I susceptible to chymase and carboxypeptidase A digestion

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe225) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL3) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe229 and Tyr192 residues were the main cleavage sites. Interestingly, the Phe225 residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL3; however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL3 at only the N-terminus, especially at Phe33. CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe225 and Phe229 residues newly exposed by chymase, but did not cleave Tyr192. These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).

Construction of a Full Transcription Map of Human Papillomavirus Type 18 during Productive Viral Infection

Human papillomavirus type 18 (HPV18) is the second most common oncogenic HPV genotype, responsible for ∼15% of cervical cancers worldwide. In this study, we constructed a full HPV18 transcription map using HPV18-infected raft tissues derived from primary human vaginal or foreskin keratinocytes. By using 5' rapid amplification of cDNA ends (RACE), we mapped two HPV18 transcription start sites (TSS) for early transcripts at nucleotide (nt) 55 and nt 102 and the HPV18 late TSS frequently at nt 811, 765, or 829 within the E7 open reading frame (ORF) of the virus genome. HPV18 polyadenylation cleavage sites for early and late transcripts were mapped to nt 4270 and mainly to nt 7299 or 7307, respectively, by using 3' RACE. Although all early transcripts were cleaved exclusively at a single cleavage site, HPV18 late transcripts displayed the heterogeneity of 3' ends, with multiple minor cleavage sites for late RNA polyadenylation. HPV18 splice sites/splice junctions for both early and late transcripts were identified by 5' RACE and primer walking techniques. Five 5' splice sites (donor sites) and six 3' splice sites (acceptor sites) that are highly conserved in other papillomaviruses were identified in the HPV18 genome. HPV18 L1 mRNA translates a L1 protein of 507 amino acids (aa), smaller than the 568 aa residues previously predicted. Collectively, a full HPV18 transcription map constructed from this report will lead us to further understand HPV18 gene expression and virus oncogenesis.

Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

BackgroundThe voltage-gated sodium channel β2 subunit (Navβ2) is a physiological substrate of BACE1 (β-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2.ResultsWe found a major (147-148 L↓M, where ↓ indicates the cleavage site) and a minor (144145 L↓Q) BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI) and ~75% in cells expressing Navβ2 (147LM/AA) as compared to cells expressing wildtype Navβ2.ConclusionWe identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

Suppression of the Raf/MEK/ERK Signaling Cascade and Inhibition of Angiogenesis by the Carboxyl Terminus of Angiopoietin-Like Protein 4

Angiopoietin-like protein 4 (Angptl4) is a secreted glycoprotein that has recently been implicated in the regulation of angiogenesis and metastasis. This study aimed to investigate the structural and cellular basis underlying the biological actions of Angptl4. Circulating Angptl4 was proteolytically cleaved into NH2-terminal coiled-coil domain (N-Angptl4) and COOH-terminal fibrinogen-like domain (C-Angptl4). Using amino acid sequencing analysis, we identified a major cleavage site between Lys(168) and Leu(169) and a minor cleavage site between Lys(170) and Met(171) in mouse Angptl4. C-Angptl4, but not N-Angptl4, potently inhibited both bFGF- and VEGF-induced cell proliferation, migration, and tubule formation in endothelial cells, and prevented neovascularization in mice. Treatment of C-Angptl4 with PNGase F (an N-glycosidase) ablated its N-linked glycosylation, and also significantly attenuated its antiangiogenic activities. C-Angptl4 blocked bFGF-induced activation of ERK1/2 MAP kinase, but had no obvious effect on Akt and P38 MAP kinase. Furthermore, C-Angptl4 abrogated bFGF-induced phosphorylation of Raf-1 and MEK1/2, whereas neither auto-phosphorylation of FGF receptor-1 nor activation of Ras was affected, suggesting that the blockage occurs at the level of Raf-1 activation. The carboxyl terminus of Angptl4 alone is sufficient to suppress angiogenesis, possibly through inhibiting the Raf/MEK/ERK1/2 MAP kinase pathway in endothelial cells.

Amyloid Precursor-like Protein 1 Influences Endocytosis and Proteolytic Processing of the Amyloid Precursor Protein

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.

Glycation and Post-translational Processing of Human Interferon-γ Expressed in Escherichia coli

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon-gamma (rhIFN-gamma) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-gamma contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN-gamma during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg140/Arg141, Phe137/Arg138, Met135/Leu136, and Lys131/Arg132) and two minor (Lys109/Ala110 and Arg90/Asp91) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN-gamma originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-gamma, whereas covalent dimerization completely abolished it.

Retrotransposition and cell-to-cell transfer of foamy viruses.

A remarkable feature of the prototype foamy virus (PFV) replication pathway has been reported to consist of the ability to retrotranspose intracellularly with high efficiency (M. Heinkelein, T. Pietschmann, G. Jármy, M. Dressler, H. Imrich, J. Thurow, D. Lindemann, M. Bock, A. Moebes, J. Roy, O. Herchenröder, and A. Rethwilm, EMBO J. 19:3436-3345, 2000). PFV intracellular retrotransposition (IRT) was reported to be enhanced by coexpression of fusion-defective envelope protein. To investigate the possibility of cell-to-cell transfer of PFV genomes, which could mimic IRT, we performed cocultivation experiments with cells transfected with an IRT-competent and marker gene-expressing PFV vector together with cells expressing a different marker and measured cells positive for both markers. The findings corroborated the initial report on IRT of Env-deficient PFV. Furthermore, they indicated that viral cores that have incorporated fusion-deficient Env can be transferred from cell to cell in a cell type-specific manor. One possible explanation consists of a minor alternative cleavage site in Env that can be used to expose the fusion peptide of the Env transmembrane protein, which appears to be required for virus uptake.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

Negative Regulation of Epidermal Growth Factor Signaling by Selective Proteolytic Mechanisms in the Endosome Mediated by Cathepsin B

We have investigated the relevant protease activity in rat liver, which is responsible for most of the receptor-mediated epidermal growth factor (EGF) degradation in vivo. EGF was sequentially cleaved by endosomal proteases at a limited number of sites, which were identified by high performance liquid chromatography and mass spectrometry. EGF proteolysis is initiated by hydrolysis at the C-terminal Glu(51)-Leu(52) bond. Three additional minor cleavage sites were identified at positions Arg(48)-Trp(49), Trp(49)-Trp(50), and Trp(50)-Glu(51) after prolonged incubation. Using nondenaturating immunoprecipitation and cross-linking procedures, the major proteolytic activity was identified as that of the cysteine protease cathepsin-B. The effect of injected EGF on subsequent endosomal EGF receptor (EGFR) proteolysis was further evaluated by immunoblotting. Using endosomal fractions prepared from EGF-injected rats and incubated in vitro, the EGFR was lost with a time course superimposable with the loss of phosphotyrosine content. The cathepsin-B proinhibitor CA074-Me inhibited both in vivo and in vitro the endosomal degradation of the EGFR and increased the tyrosine phosphorylation states of the EGFR protein and the molecule SHC within endosomes. The data, therefore, describe a unique pathway for the endosomal processing of internalized EGF receptor complexes, which involves the sequential function of cathepsin-B through selective degradation of both the ligand and receptor.

Mutational analysis of the hydrophobic tail of the human immunodeficiency virus type 1 p6(Gag) protein produces a mutant that fails to package its envelope protein.

The p6(Gag) protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6(Gag) between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6(Gag) proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6(Gag), site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6(Gag), Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41(TM) cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6(Gag) mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.

Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves the recombinant aggrecan substrate rAgg1mut at the 'aggrecanase' and the MMP sites. Characterization of MT1-MMP catabolic activities on the interglobular domain of aggrecan.

The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704-709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor 'aggrecanase' catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269-20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the 'aggrecanase' site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS...) and BC-14 (anti-FFGV...) to distinguish between 'aggrecanase' and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong 'aggrecanase' activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for 'aggrecanase' activity in vivo.

The distal ectodomain of angiotensin-converting enzyme regulates its cleavage-secretion from the cell surface.

Angiotensin-converting enzyme (ACE) is a type I ectoprotein that is cleaved off the cell surface by a plasma membrane-bound metalloprotease. However, CD4, another type I ectoprotein does not undergo such cleavage-secretion. In this study, we investigated the structural determinants of the ACE protein that regulate the cleavage-secretion process. Substitution and deletion mutations revealed that the cytoplasmic domain, the transmembrane domain, and the juxtamembrane region encompassing the major and the minor cleavage sites of ACE do not regulate its cleavage. Moreover, a chimeric protein containing the distal extracellular domain of CD4 and the juxtamembrane, transmembrane, and the cytoplasmic domains of ACE, although transported to the cell surface, was not cleavage-secreted. In contrast, the distal extracellular domain of ACE was shown to be the important determinant: a protein containing the distal extracellular domain of ACE and the juxtamembrane, transmembrane, and cytoplasmic domain of CD4 was efficiently cleaved off the cell surface. The chimeric protein was cleaved within the CD4 sequence and the responsible enzymatic activity was inhibited by Compound 3, a relatively specific inhibitor of the ACE secretase activity. These results demonstrate that, in a chimeric protein, the distal extracellular domain of a cleavable protein, such as ACE, can induce a proteolytic cleavage within the juxtamembrane domain of an uncleaved protein such as CD4.

Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis

Analysis of the hinge region of neutrophil collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme. The mutagenesis of the amino acid residues in the P 1′ position of the two autoproteolytically cleaved peptide bonds (Leu 243 and Ile 248) to Ala showed that the mutant enzymes were more resistant to autoproteolysis. However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala 239–Ile 240 peptide bond and the half-life of the active enzyme was increased by 50%. In contrast, mutagenesis of Pro 247→Ala (P 1 of the minor cleavage site Pro 247–Ile 248) lead to increased susceptibility of the enzyme to autoproteolysis. However, when the other P 1 position Gly 242 was altered to Ala no effect on stability was observed. The analysis of the ability of the mutant active enzymes to hydrolyse 14C-type I collagen was assessed and our results demonstrate that the hinge sequence motif of neutrophil collagenase is important for collagenolytic activity. The alteration of the Gly 242–Leu–Ser–Ser–Asn–Pro–Ile–Gln–Pro 247 sequence motif to Gly 242–Ala–Ala–Ala–Ala–Pro–Ala–Ala–Pro 247 showed that the collagenolytic activity was reduced by 68.4%. In addition, mutagenesis of the downstream sequence motif Pro 247–Thr–Gly–Pro–Ser–Thr–Pro–Lys–Pro 258 to Pro 247–Ala–Ala–Pro–Ala–Ala–Pro–Ala–Pro 258 had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%. When the Pro residues in the hinge motif (Pro 247, Pro 250, Pro 253 and Pro 256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme. © 1997 Federation of European Biochemical Societies.

Metal-catalyzed oxidation and cleavage of octopus glutathione transferase by the CU(II)-ascorbate system

Glutathione transferase (GST) from octopus hepatopancreas was rapidly inactivated by micromolar concentration of Cu(II) in the presence of ascorbate at neutral pH and 0°C. Omitting the metal ion or ascorbate, or replacing the Cu(II) with Fe(II) did not result in any inactivation. Glutathione or the conjugation product of glutathione and 1-chloro-2,4-dinitrobenzene offered complete protection of the enzyme from Cu(II)-induced inactivation. 1-Chloro-2,4-dinitrobenzene, however, did not provide any protection. The inactivation was time and Cu(II) concentration dependent. The dependence of inactivation rate on Cu(II) concentration displayed saturation kinetics, which suggests that the inactivation occurs in two steps with Cu(II) binding with the enzyme first ( K dCu = 260 μM), then the locally generated free radicals modify the essential amino acid residues in the active center, which results in enzyme inactivation. The Cu(II)-ascorbate system is, thus, an affinity reagent for the octopus GST. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native octopus GST has a subunit M r of 24,000. The inactivated enzyme was cleaved at the C-terminal domain (domain II) of the enzyme molecule and resulted in the formation of peptide fragment of M r 15,300, which has the identical N-terminal amino acid sequence as the native enzyme. The other half of the peptide with M r approximately 7700 was visible in the gels only after silver staining, which also revealed a minor cleavage site, also located at the domain II, to produce peptide fragments of M r approximately 11,300 and 8300. The oxygen carrier molecule in the cephalopods' blood is the copper-containing hemocyanin, which during turnover will release Cu(II). Our results indicate that Cu(II) catalyzes a site-specific oxidation of the essential amino acid residues at the C-terminus of GST causing enzyme inactivation. The modified-enzyme is then affinity cleaved at the putative metal binding site. The ability of octopus GST to bind with free Cu(II) may have important biological implications to enable cephalopods to avoid copper-induced cellular toxicity.

ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F0. Otherwise the F0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F1 and F0 domains of the F1F0-ATPase complex.

Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties.

A cDNA fragment which encodes the zymogen of canditropsin, the extracellular aspartic protease from the yeast Candida tropicalis (Togni,G., Sanglard, D., Falchetto, R., and Monod, M. (1991) FEBS Lett. 286, 181-185) was cloned into a T7 expression vector for the synthesis of the recombinant zymogen in Escherichia coli. Recombinant canditropsinogen (Ctg), which was expressed as inclusion bodies in the cytosol of E. coli, was refolded by dialysis from an 8 M urea solution and purified to homogeneity using chromatographies on Sephacryl S-300 and on MonoQ columns. The purified Ctg was converted into canditropsin by either acid activation or trypsin conversion. The specificity of the resulting recombinant canditropsin toward polypeptide substrates is significantly different from other aspartic proteases. Canditropsin hydrolyzes oxidized insulin B chain between Ala-Leu and many other minor cleavage sites. Canditropsin also hydrolyzes keratin and collagen, which are components of connective tissues known to be hydrolyzed by canditropsin during Candida infections. Canditropsin was strongly inhibited by the universal aspartic protease inhibitor pepstatin (Ki = 1.75 x 10(-8) M) and inactivated by two aspartic protease inactivators, DAN and EPNP. Canditropsin is weakly inhibited by leupeptin and antipain, with an apparent Ki of 1.74 x 10(-4)M and 1.5 x 10(-5) M, respectively.

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.

Functional and structural elements of the mRNA of the cIII gene of bacteriophage lambda

The bacteriophage λ cIII gene product is an early regulatory protein that participates in the lysis-lysogeny decision of the phage following infection. We have previously shown that the translation of the cIII gene is determined by two unique factors: (1) efficient expression is dependent upon the presence of RNaseIII in the cell; (2) alternative mRNA structures of the cIII coding region determine the rate of its translation initiation. In this study we demonstrate the presence of the alternative mRNA structures in vivo. The presence of minor RNaseIII cleavage sites within this region indicate that RNaseIII can differentiate between the two alternative structures. We localize by a deletion analysis the RNaseIII responsive element to the cIII coding region, and suggest that regulation of cIII translation by RNaseIII is achieved through binding to the alternative structures region of the mRNA.

Proposed secondary structure of eukaryotic U14 snRNA

U14 snRNA is a small nuclear RNA that plays a role in the processing of eukaryotic ribosomal RNA. We have investigated the folded structure of this snRNA species using comparative analysis of evolutionarily diverse U14 snRNA primary sequences coupled with nuclease digestion analysis of mouse U14 snRNA. Covariant nucleotide analysis of aligned mouse, rat, human, and yeast U14 snRNA primary sequences suggested a basic folding pattern in which the 5' and 3' termini of all U14 snRNAs were base-paired. Subsequent digestion of mouse U14 snRNA with mung bean (single-strand-specific), T2 (single-strand-preferential), and V1 (double-strand-specific) nucleases defined the major and minor cleavage sites for each nuclease. This digestion data was then utilized in concert with the comparative sequence analysis of aligned U14 snRNA primary sequences to refine the secondary structure model suggested by computer-predicted folding. The proposed secondary structure of U14 snRNA is comprised of three major hairpin/helical regions which includes the helix of base-paired 5' and 3' termini. Strict and semiconservative covariation of specific base-pairs within two of the three major helices, as well as nucleotide changes that strengthen or extend base-paired regions, support this folded conformation as the evolutionary conserved secondary structure for U14 snRNA.