Plasmid DNA nicking- a Novel Activity of Soybean Trypsin Inhibitor and Bovine Aprotinin.
Ontology highlight
ABSTRACT:
SUBMITTER: Islam MR
PROVIDER: S-EPMC6690959 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3' end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.
| S-EPMC8464042 | biostudies-literature
Project description:Defensins are a large family of small, cationic, cysteine-rich proteins that are part of the defense arsenal that plants use for protection against potentially damaging fungal infections. The plant defensin NaD1 from Nicotiana alata is a potent antifungal protein that inhibits growth and kills a variety of fungal pathogens that affect both plant and animal (human) hosts. Some serine protease inhibitors have also been reported to be antifungal molecules, while others have no inhibitory activity against fungi. Here we describe the synergistic activity of the plant defensin NaD1 with a selection of serine protease inhibitors against the plant pathogens Fusarium graminearum and Colletotrichum graminicola and the animal pathogen Candida albicans. The synergistic activity was not related to the protease inhibitory activity of these molecules but may arise from activation of fungal stress response pathways. The bovine pancreatic trypsin inhibitor (BPTI) displayed the most synergy with NaD1. BPTI also acted synergistically with several other antifungal molecules. The observation that NaD1 acts synergistically with protease inhibitors provides the foundation for the design of transgenic plants with improved resistance to fungal disease. It also supports the possibility of naturally occurring accessory factors that function to enhance the activity of innate immunity peptides in biological systems. IMPORTANCE This work describes the increased activity of a natural antifungal peptide in the presence of another antifungal peptide from a different family. This is termed antifungal synergy. Synergy is important for decreasing the amount of antifungal molecule needed to control the disease. Traditionally, naturally occurring antifungal molecules are assayed in isolation. Identification of synergistic interactions between antifungal peptides means that their activities in a complex biological system are likely to be different from what we observe when examining them individually. This study identified synergy between an antifungal peptide and a group of peptides that do not affect fungal growth in vitro. This provides the foundation for generation of transgenic plants with increased resistance to fungal disease and identification of antifungal accessory factors that enhance the activity of innate immune molecules but do not have an antifungal effect on their own.
| S-EPMC5646242 | biostudies-literature
Project description:Characterization of the hydrated state of a protein is crucial for understanding its structural stability and function. In the present study, we have investigated the 3D hydration structure of the protein BPTI (bovine pancreatic trypsin inhibitor) by molecular dynamics (MD) and the integral equation method in the three-dimensional reference interaction site model (3D-RISM) approach. Both methods have found a well-defined hydration layer around the protein and revealed the localization of BPTI buried water molecules corresponding to the X-ray crystallography data. Moreover, under 3D-RISM calculations, the obtained positions of waters bound firmly to the BPTI sites are in reasonable agreement with the experimental results mentioned above for the BPTI crystal form. The analysis of the 3D hydration structure (thickness of hydration shell and hydration numbers) was performed for the entire protein and its polar and non-polar parts using various cut-off distances taken from the literature as well as by a straightforward procedure proposed here for determining the thickness of the hydration layer. Using the thickness of the hydration shell from this procedure allows for calculating the total hydration number of biomolecules properly under both methods. Following this approach, we have obtained the thickness of the BPTI hydration layer of 3.6 Å with 369 water molecules in the case of MD simulation and 3.9 Å with 333 water molecules in the case of the 3D-RISM approach. The above procedure was also applied for a more detailed description of the BPTI hydration structure near the polar charged and uncharged radicals as well as non-polar radicals. The results presented for the BPTI as an example bring new knowledge to the understanding of protein hydration.
| S-EPMC9737982 | biostudies-literature
Project description:Conjugative plasmid transfer results in the spread of antibiotic resistance genes and virulence factors between bacterial cells. Plasmid transfer is dependent upon the DNA nicking activity of a plasmid-encoded relaxase enzyme. Tyrosine residues within the relaxase cleave the DNA plasmid nic site in a highly sequence-specific manner. The conjugative resistance plasmid pCU1 encodes a relaxase with four tyrosine residues surrounding its active site (Y18,19,26,27). We use activity assays to demonstrate that the pCU1 relaxase preferentially uses Y26 or a combination of Y18 + 19 to nick DNA at wild type levels, and that an adjacent aspartic acid deprotonates these tyrosines to activate them for attack. Our findings illustrate the unique modifications that the pCU1 relaxase has introduced into the traditional relaxase-mediated DNA nicking mechanism.
| S-EPMC3086049 | biostudies-literature
Project description:The solution structure of bovine pancreatic trypsin inhibitor (BPTI) at a pressure of 2 kbar is presented. The structure was calculated as a change from an energy-minimized low-pressure structure, using (1)H chemical shifts as restraints. The structure has changed by 0.24 A RMS, and has almost unchanged volume. The largest changes as a result of pressure are in the loop 10-16, which contains the active site of BPTI, and residues 38-42, which are adjacent to buried water molecules. Hydrogen bonds are compressed by 0.029 +/- 0.117 A, with the longer hydrogen bonds, including those to internal buried water molecules, being compressed more. The hydrophobic core is also compressed, largely from reduction of packing defects. The parts of the structure that have the greatest change are close to buried water molecules, thus highlighting the importance of water molecules as the nucleation sites for volume fluctuation of proteins in native conditions.
| S-EPMC2323994 | biostudies-literature
Project description:We have used magnetic relaxation dispersion to study bovine pancreatic trypsin inhibitor (BPTI) self-association as a function of pH, salt type and concentration, and temperature. The magnetic relaxation dispersion method sensitively detects stable oligomers without being affected by other interactions. We find that BPTI decamers form cooperatively under a wide range of solution conditions with no sign of dimers or other small oligomers. Decamer formation is opposed by electrostatic repulsion among numerous cationic residues confined within a narrow channel. Accordingly, the decamer population increases with increasing pH, as cationic residues are deprotonated, and with increasing salt concentration. The salt effect cannot be described in terms of Debye screening, but involves the ion-specific sequestering of anions within the narrow channel. The lifetime of the BPTI decamer is 101 +/- 4 min at 27 degrees C. We propose that the BPTI decamer, with a heparin chain threading the decamer channel, plays a functional role in the mast cell. We also detect a higher oligomer that appears to be a subcritical nucleation cluster of 3-5 decamers. We argue that monomeric crystals form at high pH despite a high decamer population in solution, because the ion pairs that provide the critical decamer-decamer contacts are disrupted at high pH.
| S-EPMC1302976 | biostudies-literature
Project description:A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1' pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.
| S-EPMC3633903 | biostudies-literature
Project description:Inhibitors of serine proteases are not only extremely useful in the basic research but are also applied extensively in clinical settings. Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach we developed a family of novel, single-stranded DNA aptamers capable of specific trypsin inhibition. Our most potent candidate (T24) and its short version (T59) were thoroughly characterised in terms of efficacy. T24 and T59 efficiently inhibited bovine trypsin with Ki of 176 nM and 475 nM, respectively. Interestingly, in contrast to the majority of known trypsin inhibitors, the selected aptamers have superior specificity and did not interact with porcine trypsin or any human proteases tested. These included plasmin and thrombin characterised by trypsin-like substrate specificity. Our results demonstrate that SELEX may be successfully employed in the development of potent and specific DNA based protease inhibitors.
| S-EPMC6366424 | biostudies-literature
Project description:The disulfide bond between Cys14 and Cys38 of bovine pancreatic trypsin inhibitor lies on the surface of the inhibitor and forms part of the protease-binding region. The functional properties of three variants lacking this disulfide, with one or both of the Cys residues replaced with Ser, were examined, and X-ray crystal structures of the complexes with bovine trypsin were determined and refined to the 1.58-A resolution limit. The crystal structure of the complex formed with the mutant with both Cys residues replaced was nearly identical with that of the complex containing the wild-type protein, with the Ser oxygen atoms positioned to replace the disulfide bond with a hydrogen bond. The two structures of the complexes with single replacements displayed small local perturbations with alternate conformations of the Ser side chains. Despite the absence of the disulfide bond, the crystallographic temperature factors show no evidence of increased flexibility in the complexes with the mutant inhibitors. All three of the variants were cleaved by trypsin more rapidly than the wild-type inhibitor, by as much as 10,000-fold, indicating that the covalent constraint normally imposed by the disulfide contributes to the remarkable resistance to hydrolysis displayed by the wild-type protein. The rates of hydrolysis display an unusual dependence on pH over the range of 3.5-8.0, decreasing at the more alkaline values, as compared with the increased hydrolysis rates for normal substrates under these conditions. These observations can be accounted for by a model for inhibition in which an acyl-enzyme intermediate forms at a significant rate but is rapidly converted back to the enzyme-inhibitor complex by nucleophilic attack by the newly created amino group. The model suggests that a lack of flexibility in the acyl-enzyme intermediate, rather than the enzyme-inhibitor complex, may be a key factor in the ability of bovine pancreatic trypsin inhibitor and similar inhibitors to resist hydrolysis.
| S-EPMC2615049 | biostudies-literature
Project description:Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.
| S-EPMC4567993 | biostudies-literature