Journal articles: 'Cleaved' – Grafiati (2024)

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Abstract In cucumber cotyledons, both C6- and C9- aldehyde were formed via hydroperoxide (HPO) lyase activity. Because it has not been elucidated whether these activities are attributed to one enzyme which can cleave both 13-and 9-HPO or to two or more enzymes each of which specifically cleaves 13-or 9-HPO , an attempt to separate HPO lyase activity was done. Ion exchange chromatography separated this activity into two fractions, one of which specifically cleaved 13-hydroperoxylinoleic acid and the other specifically cleaved the 9-isomer. 13-HPO-specific activity was most active at pH 8.0 and 9-HPO-specific one was at pH 6.5. SH -reagents inhibited both the lyases but to different extents.

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Abstract ADAMTS13 is a member of the A Disintergrin And Metalloprotease with ThromboSpondin type I repeat family, and it cleaves the Tyr1605-Met1606 bond in the von Willebrand factor (VWF) central A2 domain, thereby decreasing platelet adhesion mediated by VWF. Recently a minimal substrate for ADAMTS13 was characterized that consists of GST linked to Asp1596-Arg1668 with a C-terminal 6×His tag (VWF73). Further removal of 9 amino acids that comprise a predicted C-terminal α-helix (VWF64) appeared to eliminate cleavage by plasma ADAMTS13, suggesting a critical role for this helix in substrate recognition. We obtained similar results, but VWF64 was cleaved significantly at long reaction times. For example, plasma ADAMTS13 (0.3 nM, one-tenth of normal plasma) cleaved 50% of 65 nM VWF73 in 2 hours and 50% of 62 nM VWF64 in 24 hours. Similar results were obtained in either 50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.1 μM ZnCl2, or 5 mM Tris-HCl, pH 8.0, 10 mM BaCl2. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. Using 3 nM enzyme for 30 min, VWF73 was cleaved ~2-fold faster by ADAMTS13 truncated after the spacer domain (MDTCS) than by ADAMTS13 truncated after the cysteine-rich domain (MDTC). Conversely, VWF64 was cleaved ~3-fold faster by MDTC than by MDTCS. Also, in 30 min MDTCS cleaved 70% of VWF73 and <10% of VWF64, whereas MDTC cleaved 40% of VWF73 and 30% of VWF64. ADAMTS13 truncated after the first TSP1 repeat (MDT) or the disintegrin domain (MD) had markedly reduced activity, but with prolonged incubation (24 h) at increased concentration (30 nM) both enzymes cleaved most of VWF64 and VWF73 at the expected site. No aberrant cleavage products were detected by Western blotting. The metalloproteinase domain alone (M) was inactive. The selective effects of deleting the cysteine-rich or spacer domain suggest that the C-terminal α-helix of the VWF A2 domain is not essential but facilitates substrate recognition by interacting with specific C-terminal domains of ADAMTS13, particularly the cysteine-rich and spacer domains.

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There is great interest in being able to spread beneficial traits throughout wild populations in ways that are self-sustaining. Here, we describe a chromosomal selfish genetic element,CleaveR[Cleave and Rescue (ClvR)], able to achieve this goal.ClvRcomprises two linked chromosomal components. One, germline-expressed Cas9 and guide RNAs (gRNAs)—the Cleaver—cleaves and thereby disrupts endogenous copies of a gene whose product is essential. The other, a recoded version of the essential gene resistant to cleavage and gene conversion with cleaved copies—the Rescue—provides essential gene function.ClvRenhances its transmission, and that of linked genes, by creating conditions in which progeny lackingClvRdie because they have no functional copies of the essential gene. In contrast, those who inheritClvRsurvive, resulting in an increase inClvRfrequency.ClvRis predicted to spread to fixation under diverse conditions. To test these predictions, we generated aClvRelement inDrosophila melanogaster.ClvRtkois located on chromosome 3 and uses Cas9 and four gRNAs to disruptmelanogaster technical knockout(tko), an X-linked essential gene. Rescue activity is provided bytkofromDrosophila virilis.ClvRtkoresults in germline and maternal carryover-dependent inactivation ofmelanogaster tko(>99% per generation); lethality caused by this loss is rescued by thevirilistransgene;ClvRtkoactivities are robust to genetic diversity in strains from five continents; and uncleavable but functionalmelanogaster tkoalleles were not observed. Finally,ClvRtkospreads to transgene fixation. The simplicity ofClvRsuggests it may be useful for altering populations in diverse species.

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Abstract The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.

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Restriction endonuclease CeqI, an isoschizomer of EcoRV, exhibits ‘star’ activity, a relaxation of specificity in the presence of Mn2+, dimethyl sulphoxide or glycerol. The enzyme cleaves a set of sequences that differ from the canonical GATATC by only one nucleotide in positions 2, 3, 4 or 5. Two of these sequences are not cleaved if modified by dam methylase. A further loss of specificity can be observed in circ*mstances less favourable for the enzyme, namely low-ionic-strength buffers of pH values below 6.0 or above 9.4. This activity seems to cleave DNA at any sequence, producing a smear instead of well-defined bands. Partial renaturation of the denatured enzyme gives rise to a similar non-specific nuclease activity.

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Burkholderia cenocepacia secretes two zinc-dependent metalloproteases, designated ZmpA and ZmpB. Previously, ZmpA and ZmpB have been shown to cleave several proteins important in host defence. In this study, the ability of ZmpA and ZmpB to digest and inactivate antimicrobial peptides involved in innate immunity was examined. ZmpB but not ZmpA cleaved β-defensin-1. ZmpA but not ZmpB cleaved the cathelicidin LL-37. Both enzymes cleaved elafin and secretory leukocyte inhibitor, which are antimicrobial peptides as well as neutrophil elastase inhibitors. Both ZmpA and ZmpB cleaved protamine, a fish antimicrobial peptide, and a zmpA zmpB mutant was more sensitive to protamine killing than the parental strain. ZmpA or ZmpB cleavage of elafin inactivated its anti-protease activity. The effect of ZmpA and ZmpB on the neutrophil proteases elastase and cathepsin G was also examined but neither enzyme was active against these host proteases. These studies suggest that ZmpA and ZmpB may influence the resistance of B. cenocepacia to host antimicrobial peptides as well as alter the host protease/anti-protease balance in chronic respiratory infections.

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ABSTRACTBotulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. Thein vivoprotein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from itsin vivoprotein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated thatClostridium baratiitype F toxin did not cleave this peptide. In this study, we detail experiments demonstrating thatClostridium baratiitype F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate thatClostridium baratiitype F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.

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A new site-specific endonuclease has been isolated from Streptomyces caespitosus and named ScaI. Based on analysis of sequences around the restriction sites in pBR322 and pBR325, the recognition sequence of ScaI endonuclease was deduced to be a new hexanucleotide 5′-AGTACT-3′. The cleavage site was determined by comparing the ScaI-cleaved product of a primer-extended M13mp18-SCA DNA, which contains an AGTACT sequence, with dideoxy chain terminator ladders of the same DNA. ScaI was found to cleave the recognition sequence between the internal T and A, leaving flush ends to the cleaved fragments.

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Abstract ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, first thrombospondin type 1 repeat (TSP1), a Cys-rich domain and a spacer domain. The carboxyl terminus of ADAMTS13 has seven more TSP1 repeats and two CUB domains. ADAMTS13 cleaves the Tyr1605-Met1606 bond at the central A2 domain of von Willebrand factor (VWF). To determine the domains of ADAMTS13 required for substrate recognition and specificity, we have prepared a recombinant VWF73 peptidyl substrate from E. coli BL21Ai cells. This substrate consists of 73 amino acid residues (D1596~R1668) derived from the central A2 domain of VWF, flanked at its N-terminus by a glutathione s-transferase (GST) protein and at its C-terminus by a 6xHis epitope. It is highly specific and more sensitive than plasma VWF for determination of the proteolytic activity of ADAMTS13. Purified VWF73 substrate (200 nM) was incubated with the conditioned media from COS7 cells containing 14 nM of full-length ADAMTS13 and its various variants in 50 mM Tris-HCl and 150 mM NaCl at 37 °C for 3 hours. The cleavage of VWF73 at the specific Tyr-Met bond was determined by Western blotting with anti-GST. We showed that full-length ADAMTS13 and ADAMTS13 variants that were deleted after the TSP1 2 to 8 repeats (del1), spacer domain (del2), Cys-rich domain (del3) and first TSP1 repeat (del4) were all proteolytically active toward VWF73. ADAMTS13 variants deleted after disintegrin domain (del5) and after metalloprotease domain (del6) did not cleave VWF73 within 3 hours, but did cleave VWF73 upon a prolonged incubation (14~24 hours). Surprisingly, the del4 and del6 variants preferentially cleaved VWF73 at a site other than the Tyr-Met bond, resulting in a generation of an N-terminal fragment that was approximately 5 kDa shorter in length than expected. The cleavage of this alternative peptidyl bond by del4 and del6 was metal ion dependent and could be inhibited by an addition of 5 mM EDTA into the reaction. The conditioned medium from vector-transfected COS7 cells did not cleave VWF73 after 24 hours of incubation in the same condition. Time-course studies showed that full-length ADAMTS13, del1, del2 and del3 variants cleaved VWF73 with a similar efficiency, with an approximately 50% of VWF73 being cleaved in five minutes at 37 °C. The cleavage of VWF73 by ADAMTS13 variants del4, del5 and del6, however, was relatively slow with a less than 50% of VWF73 substrate being cleaved after 24 hours of incubation, suggesting that the Cys-rich/spacer domains participate in the substrate recognition to enhance the catalytic efficiency of ADAMTS13 metalloprotease. The other domains adjacent the Cys-rich/spacer domains upstream including the disintegrin domain and/or the first TSP1 repeat may also be critical for collaborative recoginition of the substrates, as the ADAMTS13 variants del5 and MT (consisting of only the metalloprotease domain and the first TSP1 repeat) cleaved VWF73 very slowly. In addition, the ADAMTS13 variant MCS that was deleted after the spacer domain, but lacking the disintegrin and the first TSP1 repeat was not proteolytically active toward VWF73 at all. We therefore conclude that the disintegrin domain, the first TSP1 repeat, the Cys-rich domain and the spacer domain are all important for substrate recognition to enhance the catalytic efficiency of ADAMTS13 metalloprotease domain and to improve substrate specificity.

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ABSTRACT The endonuclease dicer cleaves RNAs that are 100% double stranded and certain RNAs with extensive but <100% pairing to release ∼21-nucleotide (nt) fragments. Circular 1,679-nt genomic and antigenomic RNAs of human hepatitis delta virus (HDV) can fold into a rod-like structure with 74% pairing. However, during HDV replication in hepatocytes of human, woodchuck, and mouse origin, no ∼21-nt RNAs were detected. Likewise, in vitro, purified recombinant dicer gave <0.2% cleavage of unit-length HDV RNAs. Similarly, rod-like RNAs of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) were only 0.5% cleaved. Furthermore, when a 66-nt hairpin RNA with 79% pairing, the putative precursor to miR-122, which is an abundant liver micro-RNA, replaced one end of HDV genomic RNA, it was poorly cleaved, both in vivo and in vitro. In contrast, this 66-nt hairpin, in the absence of appended HDV sequences, was >80% cleaved in vitro. Other 66-nt hairpins derived from one end of genomic HDV, PSTVd, or ASBVd RNAs were also cleaved. Apparently, for unit-length RNAs of HDV, PSTVd, and ASBVd, it is the extended structure with <100% base pairing that confers significant resistance to dicer action.

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ABSTRACT Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1PRO repeats. We demonstrate here that the Drosophila hom*ologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.

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SUMMARYDetergent extracts of Trypanosoma cruzi epimastigotes catalysed the hydrolysis of a range of amino-acyl and peptidyl p-nitro-anilides and aminomethylcoumarins. At least three enzymes were detected that cleave Z–Phe–Arg–MCA. Two of these were optimally active at alkaline pH, the other at pH 4·0. Of the two enzymes with alkaline pH optima, one was a cysteine peptidase and was unable to cleave Bz–Arg–MCA readily, whilst the other cleaved Bz–Arg–MCA and was inhibited by diisopropyl fluorophosphate. The acidic enzyme was similar to cathespin L of other eukayrotes with respect to its pH profile, substrate-specificity and inhibitor-sensitivity. Evidence was presented that epimastigotes contain a cysteine-type dipeptidyl aminopeptidase, one or more aminopeptidases, and a serine peptidase that cleaves Boc–Ala–Ala–pNA. Digitonin solubilization of the activities from cells supports the hypothesis that the cathespin L-like enzyme and the dipeptidyl aminopeptidase are lysosomal, whilst the Bz–Arg–MCA hydrolase, the aminopeptidases and the Boc–Ala–Ala–pNA serine peptidase are cytosolic.

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tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.

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tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.

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The use of metabolomic based techniques to aid oocyte and embryo selection has gained attention in recent years. Previous work from our laboratory has demonstrated that the 1H NMR-based metabolic profile of follicular fluid correlates with oocyte developmental potential. Patients undergoing IVF at the Merrion Fertility Clinic had follicular fluid collected at the time of oocyte retrieval. The fatty acid composition of follicular fluid from follicles where oocytes fertilised and developed into multi-cell embryos (n=15) and from oocytes that fertilised normally but failed to cleave (n=9) (cleaved vs non-cleaved) was compared. Statistical analysis was performed on the data using univariate and multivariate techniques. Analysis of the fatty acid composition revealed that there were nine fatty acids significantly different between follicular fluid from the cleaved and the non-cleaved sample groups. Of particular interest were the higher concentration of total saturated (P=0.03) and the lower concentration of total polyunsaturated fatty acids in the non-cleaved sample group (P=0.001). Random forest classification models were used to predict successful cleavage in follicular fluid samples producing models with errors rates of <10%. Receiver operating characteristic analysis demonstrated that the model had good predictability with an area under the curve of 0.96. The panel of fatty acid biomarkers identified in this study indicates that the fatty acid composition of follicular fluid may be more predictive in comparison to other previously identified biomarkers. Following validation in a larger cohort, these biomarkers may have the potential to be used in fertility clinics to aid the selection of oocytes in the future.

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Summaryβ2-glycoprotein I (β2GPI) is a plasma glycoprotein with unknown physiological function(s). In in vitro experiments it has been demonstrated that β2GPI has both anticoagulant properties, such as the inhibition of factor X and prothrombin activation and procoagulant properties, such as the inhibition of the anticoagulant activity of activated protein C. Besides this, β2GPI bound to cardiolipin is recognized by anti-phospholipid antibodies (aPL).In this study we demonstrate that β2GPI is very sensitive for cleavage between Lys317 and Thr318 by plasmin, resulting in two immunologically different cleaved forms. In vitro experiments show that these plasmin cleaved forms of β2GPI bind to negatively charged phospho-lipids with much lower affinity compared to intact β2GPI. Similar to plasmin, trypsin and elastase can also induce this proteolytical cleavage in β2GPI, whereas thrombin and factor Xa do not cleave β2GPI. The in vivo occurrence of the proteolytical cleavage was demonstrated by the finding that in plasmas of patients with disseminated intravascular coagulation(DIC) and in plasmas of patients treated with streptokinase, significant amounts of cleaved β2GPI (up to 12 μg/ml) are present.During the development of DIC, the increase in levels of cleaved β2GPI is accompanied by a 70% decrease in the levels of intact β2GPI, whereas in streptokinase treated patients levels of intact β2GPI stay within the normal range.This study demonstrates for the first time that during in vivo activation of fibrinolysis β2GPI is cleaved, which results in the formation of a form of β2GPI with much lower affinity for negatively charged phospholipids. Plasmin is most likely responsible for this modification.

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Cathepsin K (EC 3.4.22.38) is a recently described enzyme that has been shown to cleave type I collagen in its triple helix. The aim of this study was to determine if it also cleaves type II collagen in the triple helix and to identify the helical cleavage site(s) in types I and II collagens. Soluble human and bovine type II collagen, and rat type I collagen, were incubated with cathepsin K before the reaction was stopped with trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64). Analysis by SDS/PAGE of the collagen digests showed that optimal activity of cathepsin K against native type II collagen was between pH 5.0 and 5.5 and against denatured collagen between pH 4.0 and 7.0. The enzyme cleaved telopeptides as well as the α1(II) chains, generating multiple fragments in the range 90–120 kDa. The collagenolytic activity was not due to a contaminating metalloenzyme or serine proteinase as it was not inhibited by 1,10-phenanthroline, EDTA or 3,4-dichloroisocoumarin. Western blotting with anti-peptide antibodies to different regions of the α1(II) chain suggested that cathepsin K cleaved native α1(II) chains in the N-terminal region of the helical domain rather than at the well-defined collagenase cleavage site. This was confirmed by N-terminal sequencing of one of the fragments, revealing cleavage at a Gly-Lys bond, 58 residues from the N-terminus of the helical domain. By using a similar approach, cathepsin K was found to cleave native type I collagen close to the N-terminus of its triple helix. These results indicate that cathepsin K could have a role in the turnover of type II collagen, as well as type I collagen.

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Abstract Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

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Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

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Native and recombinant neutrophil collagenase (MMP-8) was shown to cleave at the E373-A374 ‘aggrecanase’ site in the interglobular domain of aggrecan. The time course of digestion in vitro showed that MMP-8 cleaved initially at N341-F342, the predominant metalloproteinase site, before cleaving at the E373-A374 site. A synthetic peptide, IPENFFG, inhibited cleavage at E373-A374 but not N341-F342 in vitro, indicating that the E373-A374 sequence was a less preferred site for MMP-8 cleavage than N341-F342. IPENFFG also inhibited release of A374 RGSVI fragments from cartilage in explant culture, suggesting that a metalloproteinase cleaved at the aggrecanase site in situ. The possibility remains that ‘aggrecanase’ may be a metalloproteinase in cartilage.

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I have found the best way to cleave [001]-oriented silicon wafers is rather different compared to GaAs or InP. The problem is that Si prefers to cleave on (111) planes rather than (110) and so one gets an angled face with the cleave, which is usually rather uneven and often doesn't run straight. This is even worse when cleaving close to an existing edge, which attracts the crack front as it propagates. Good for making low-angle cleaved specimens, but a problem otherwise. It is possible to make Si cleave on (110) by cleaving the wafer without any support.

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Abstract Abstract 2124 Poster Board II-101 Background: Protein S, a vitamin K-dependent plasma protein, is one of the molecules involved in down-regulation of the coagulation process. Protein S serves as a cofactor of activated protein C (APC) in the proteolytic inactivation of activated factor V and VIII. Protein S is also able to exert its anticoagulant activity independent of APC, e.g. by supporting the anticoagulant activity of tissue factor pathway inhibitor. In plasma, protein S circulates in two forms: a single chain molecule with full anticoagulant activity and a two-chain form with reduced anticoagulant activity. This two-chain variant is the result of cleavage in the protease sensitive loop, the so-called thrombin sensitive region (TSR). The enzyme responsible for the presence of cleaved protein S in the circulation is not known yet. In vitro, thrombin and factor Xa, both enzymes of the coagulation cascade, are able to cleave the TSR. In a recent study (Brinkman et al, J Thromb Haemost 2005; 3: 2712-2720), it was shown that these enzymes do not cleave protein S under physiological conditions. Instead, a platelet protease was found to cleave protein S in plasma during tissue factor induced clotting in vitro. We hypothesize that this platelet protease may be responsible for the presence of cleaved protein S in the circulation. Objective: To correlate cleaved protein S levels in the circulation with the activity of platelet-associated protein S cleaving protease and to evaluate the impact of protein S cleavage on its anticoagulant activity in terms of total protein S activity. Method: Protein S cleavage was evaluated by immunological methods employing a monoclonal antibody specific for uncleaved, single chain protein S. The total anticoagulant activity of protein S (APC-independent and APC-dependent) was assessed employing thrombography. Results: We observed in the in vitro thrombin generation test massive generation of thrombin in protein S deficient plasma that was dose-dependently inhibited by intact, single chain protein S. TSR-cleaved protein S had totally lost its anticoagulant activity. In plasma from healthy volunteers, cleaved protein S expressed as % of the total amount of protein S ranged between 8 and 51% with an average normal value of 26 ± 9% (mean ± SD, n=46). Levels of intact protein S correlated well with total protein S activity in the individual plasma samples, while this correlation was not observed for levels of TSR-cleaved protein S. These data suggest that not only in vitro but also in vivo, cleavage of protein S has an impact on its anticoagulant activity. A significant reduction of levels of cleaved protein S was observed in chemotherapy induced thrombocytopenia in hematological patients (see Figure). At a platelet count < 50, the average protein S cleavage was dropped to 16 ± 7% (mean ± SD, n=15). Upon platelet transfusion, cleavage of protein S dramatically increased to 36 ± 11% (mean ± SD, n=11). Levels of total protein S were similar in healthy volunteers and thrombocytopenic patients before and after platelet transfusion. It thus appears that platelets contribute to protein S cleavage in vivo. However, in healthy individuals showing a wide range of protein S cleavage (see Figure), no correlation was observed between platelet count (200-400 .109/l) and protein S cleavage. This suggests additional sources of protease that cleave protein S in vivo. We therefore examined lysates of a variety of cell types for protein S proteolytic activity. Protein S proteolytic activity was absent in lysates of erytrocytes, vascular smooth muscle cells and fibroblasts. Lysates of HUVEC did show protein S proteolytic activity similar to platelets. Conclusion: Our results strongly suggest that platelets contribute to the proteolytic modification of protein S in vivo resulting in an abolished anticoagulant activity. An additional source of protein S proteolytic activity may be the endothelium. Cleavage of protein S provoked by platelet infusion may contribute to the beneficial effect of platelet transfusion in the treatment of bleeding episodes. Disclosures: No relevant conflicts of interest to declare.

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Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a hom*ology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

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An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

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The genome of an extremely thermophilic bacterium, Thermus thermophilus HB8, contains a single ORF (open reading frame) encoding an RNase-HII-like sequence. Despite the presence of significant amino acid sequence identities with RNase (ribonuclease) HII enzymes, the ORF TTHA0198 could not suppress the temperature-sensitive growth defect of an RNase-H-deficient Escherichia coli mutant and the purified recombinant protein could not cleave an RNA strand of an RNA/DNA heteroduplex, suggesting that the TTHA0198 exhibited no RNase H activity both in vivo and in vitro. When oligomeric RNA–DNA/DNAs were used as a mimic substrate for Okazaki fragments, however, the protein cleaved them only at the 5′ side of the last ribonucleotide at the RNA–DNA junction. In fact, the TTHA0198 protein prefers the RNA–DNA junction to the RNA/DNA hybrid. We have referred to this activity as JRNase (junction RNase) activity, which recognizes an RNA–DNA junction of the RNA–DNA/DNA heteroduplex and cleaves it leaving a mono-ribonucleotide at the 5′ terminus of the RNA–DNA junction. E. coli and Deinococcus radiodurans RNases HII also cleaved the RNA–DNA/DNA substrates at the same site with a different metal-ion preference from that for RNase H activity, implying that the enzymes have JRNase activity as well as RNase H activity. The specialization in the JRNase activity of the RNase HII orthologue from T. thermophilus HB8 (Tth-JRNase) suggests that the JRNase activity of RNase HII enzymes might be independent of the RNase H activity.

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Abstract Abstract 3591 Poster Board III-528 Introduction Prochemerin is a 163 amino acid precursor protein with a C-terminal domain highly susceptible to proteolysis. Its chemotactic activity is unmasked upon C-terminal cleavage by proteases of the coagulation, fibrinolytic and inflammatory systems. Here, we studied whether thrombin is able to cleave prochemerin to generate active forms of chemerin. Methods The 15mer peptide prochemerin C-terminal sequence (YFPGQFAFSKALPRS) or prochemerin was incubated with thrombin at different concentrations and times. The reactions were stopped by addition of PPACK before determining their chemotactic activity in a transwell assay using CMKLR1-transfected cells and their mass by mass-spectrometry. Results Thrombin (0-100 nM) dose-dependently cleaved 15mer to 14mer (YFPGQFAFSKALPR). Over a longer reaction time, the 10mer (YFPGQFAFSK) was also detected. The 15mer was almost inert in the chemotaxis assay but thrombin-cleaved 15mer caused migration of CMKLR1 transfectants. The 14mer and 10mer at 1 μM induced CMKLR1 cell migration at a rate of 3200 and 2800 cells/ml, but 1 μM 15mer did not induce any chemotaxis. Thrombin can also cleave the 14mer to a 10mer as determined by mass spectrometry. Using selected thrombin mutants for the Na+ binding site (E229K) and the active site YPPW-insertion loop (W50A), we found that thrombin hydrolysis of the 15mer was dependent on the Na+ bound “fast” form of thrombin and active site topology. The 10mer could be further activated by carboxypeptidase N (CPN) by removing the C-terminal lysine, whereas the C-terminal arginine of 14mer could not be cleaved by CPN, which did not affect its activity. Full-length prochemerin was also activated by thrombin (100nM) and the chemotactic activity further increased by CPN (50nM) about 6 fold. Conclusions Prochemerin is a new substrate for thrombin. Thrombin-cleaved chemerins are active chemoattractants in chemotaxis. This extends the molecular link between blood coagulation and CMKLR1-mediated immune responses. Disclosures: No relevant conflicts of interest to declare.

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Abstract Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3′ end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures.

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Iron(II) and iron(III) complexes of 3,5-di-tert-butyl-o-benzoquinonemonooxime were synthesized and characterized by spectroscopic and electrochemical studies. Their ability to cleave DNA has been investigated under aerobic conditions at room temperature and in the presence and absence of H2O2 . The plasmid DNA pBR322 was effectively cleaved by these complexes in a concentration dependant manner.

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Boronic esters between 1,2-benzodiols and boronic acids are an efficient way for bioconjugation. The ester is stable at physiological condition, but it cleaves very slowly at acidic pH values found in the endosomes and lysosomes. During apoptosis, the boronic ester is cleaved, most likely due to the influx of Ca²⁺ ions and the oxidation of 1,2-benzodiols.

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SummaryThe natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

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ABSTRACT Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [35S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 (“cleavage from within”) and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions (“cleavage from without”), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases.

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Abstract An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.