scholarly journals Mapping Homing Endonuclease Cleavage Sites Using In Vitro Generated Protein

2014 ◽  
pp. 55-67 ◽  
Author(s):  
Richard P. Bonocora ◽  
Marlene Belfort
Keyword(s):  
Homing Endonuclease ◽  
Cleavage Sites

scholarly journals 3'-end-forming signals of yeast mRNA.

1995 ◽  
Vol 15 (11) ◽  
pp. 5983-5990 ◽  
Author(s):  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Sites ◽  
Yeast Saccharomyces Cerevisiae ◽  
Higher Eukaryotes ◽  
A Site

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

scholarly journals Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages of progastrin to release gastrin-34 and gastrin-17

2008 ◽  
Vol 415 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Jens F. Rehfeld ◽  
Xiaorong Zhu ◽  
Christina Norrbom ◽  
Jens R. Bundgaard ◽  
Anders H. Johnsen ◽  
...  
Keyword(s):  
Structural Factors ◽  
Cleavage Sites ◽  
Wild Type ◽  
Prohormone Convertases ◽  
Site Specific ◽  
G Cells ◽  
Processing Site ◽  
Vitro Effect

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and α-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg36Arg37 and Arg73Arg74 sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, α-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys53Lys54), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

scholarly journals Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

2020 ◽  
Author(s):  
Maria Kuzikov ◽  
Elisa Costanzi ◽  
Jeanette Reinshagen ◽  
Francesca Esposito ◽  
Laura Vangeel ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Small Molecules ◽  
Drug Target ◽  
Treatment Options ◽  
Cleavage Sites ◽  
X Ray ◽  
Binding Characteristics ◽  
Main Protease ◽  
Ic50 Values

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 uM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Angs., showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

scholarly journals Accurate processing of human pre-rRNA in vitro.

1989 ◽  
Vol 9 (10) ◽  
pp. 4422-4431 ◽  
Author(s):  
G J Hannon ◽  
P A Maroney ◽  
A Branch ◽  
B J Benenfield ◽  
H D Robertson ◽  
...  
Keyword(s):  
Hela Cell ◽  
18S Rrna ◽  
Cleavage Product ◽  
The Other ◽  
Second Step ◽  
Cleavage Sites ◽  
Initial Cleavage ◽  
External Transcribed Spacer ◽  
Cyclic Phosphate

We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.

scholarly journals Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Ida Höijer ◽  
Josefin Johansson ◽  
Sanna Gudmundsson ◽  
Chen-Shan Chin ◽  
Ignas Bunikis ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Human Fibroblasts ◽  
Target Prediction ◽  
Human Cell Line ◽  
Cleavage Sites ◽  
Guide Rna ◽  
Target Sites ◽  
Long Read ◽  
Target Activity

Abstract Background One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. Results The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Conclusions Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

scholarly journals In vitro-selected RNA cleaving DNA enzymes from a combinatorial library are potent inhibitors of HIV-1 gene expression

2000 ◽  
Vol 352 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Bandi SRIRAM ◽  
Akhil C. BANERJEA
Keyword(s):  
Gene Expression ◽  
Mammalian Cell ◽  
Specific Gene ◽  
Cleavage Sites ◽  
Dna Enzymes ◽  
Biological Tool ◽  
Catalytic Motif ◽  
Target Rna ◽  
Hiv 1

Selective inactivation of a target gene by antisense mechanisms is an important biological tool to delineate specific functions of the gene product. Approaches mediated by ribozymes and RNA-cleaving DNA enzymes (DNA enzymes) are more attractive because of their ability to catalytically cleave the target RNA. DNA enzymes have recently gained a lot of importance because they are short DNA molecules with simple structures that are expected to be stable to the nucleases present inside a mammalian cell. We have designed a strategy to identify accessible cleavage sites in HIV-1 gag RNA from a pool of random DNA enzymes, and for isolation of DNA enzymes. A pool of random sequences (all 29 nucleotides long) that contained the earlier-identified 10Ő23 catalytic motif were tested for their ability to cleave the target RNA. When the pool of random DNA enzymes was targeted to cleave between any A and U nucleotides, DNA enzyme 1836 was identified. Although several DNA enzymes were identified using a pool of DNA enzymes that was completely randomized with respect to its substrate-binding properties, DNA enzyme-1810 was selected for further characterization. Both DNA enzymes showed target-specific cleavage activities in the presence of Mg2+ only. When introduced into a mammalian cell, they showed interference with HIV-1-specific gene expression. This strategy could be applied for the selection of desired target sites in any target RNA.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

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

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Yoko Usami ◽  
Yukihiro Kobayashi ◽  
Takahiro Kameda ◽  
Akari Miyazaki ◽  
Kazuyuki Matsuda ◽  
...  
Keyword(s):  
Density Lipoprotein ◽  
Cleavage Sites ◽  
Carboxypeptidase A ◽  
Apolipoprotein A ◽  
C Terminus ◽  
N Terminus ◽  
Plaque Destabilization ◽  
New Biomarkers ◽  
A Minor

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).

scholarly journals Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells

2006 ◽  
Vol 80 (16) ◽  
pp. 7816-7831 ◽  
Author(s):  
Stanislav V. Sosnovtsev ◽  
Gaël Belliot ◽  
Kyeong-OK Chang ◽  
Victor G. Prikhodko ◽  
Larissa B. Thackray ◽  
...  
Keyword(s):  
Cell Culture ◽  
Caspase 3 ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Cleavage Sites ◽  
Murine Norovirus ◽  
Permissive Cell ◽  
Additional Processing ◽  
Infected Cells

ABSTRACT Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.

High Molecular Weight Kininogen Is Cleaved by FXIa at Three Sites: Arg409-Arg410, Lys502-Thr503 and Lys325-Lys326

2000 ◽  
Vol 83 (05) ◽  
pp. 709-714 ◽  
Author(s):  
T. Mauron ◽  
B. Lämmle ◽  
W. A. Wuillemin
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Coagulation Factor ◽  
Cleavage Sites ◽  
High Molecular Weight Kininogen ◽  
Surface Binding ◽  
Amino Acid Sequence Analysis ◽  
Sds Page ◽  
System Activation

SummaryWe investigated the cleavage of high molecular weight kininogen (HK) by activated coagulation factor XI (FXIa) in vitro. Incubation of HK with FXIa resulted in the generation of cleavage products which were subjected to SDS-Page and analyzed by silverstaining, ligandblotting and immunoblotting, respectively. Upon incubation with FXIa, bands were generated at 111, 100, 88 kDa on nonreduced and at 76, 62 and 51 kDa on reduced gels. Amino acid sequence analysis of the reaction mixtures revealed three cleavage sites at Arg409-Arg410, at Lys502-Thr503 and at Lys325-Lys326. Analysis of HK-samples incubated with FXIa for 3 min, 10 min and 120 min indicated HK to be cleaved first at Arg409-Arg410, followed by cleavage at Lys502-Thr503 and then at Lys325-Lys326.In conclusion, HK is cleaved by FXIa at three sites. Cleavage of HK by FXIa results in the loss of the surface binding site of HK, which may constitute a mechanism of inactivation of HK and of control of contact system activation.

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