Article

Prp8 intein in fungal pathogens: Target for potential antifungal drugs

Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 4H7.
FEBS Letters (Impact Factor: 3.34). 09/2004; 572(1-3):46-50. DOI: 10.1016/j.febslet.2004.07.016
Source: PubMed

ABSTRACT Inteins are self-splicing intervening sequences in proteins, and inteins of pathogenic organisms can be attractive drug targets. Here, we report an intein in important fungal pathogens including Aspergillus fumigatus, Aspergillus nidulans, Histoplasma capsulatum, and different serotypes of Cryptococcus neoformans. This intein is inside the extremely conserved and functionally essential Prp8 protein, and it varies in size from 170 aa in C. neoformans to 819 aa in A. fumigatus, which is caused by the presence or absence of an endonuclease domain and a putative tongs subdomain in the intein. Prp8 inteins of these organisms were demonstrated to do protein splicing in a recombinant protein in Escherichia coli. These findings revealed Prp8 inteins as attractive targets for potential antifungal drugs to be identified using existing selection and screening methods.

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    • "As shown in Figure 2B, nine of the tested mini-inteins produced the spliced protein, and their splicing efficiencies (percentage of the precursor protein that had spliced) ranged from ∼10% for the Ter DnaE-1 mini-intein to ∼100% for the Ter ThyX mini-intein. Splicing activities of the CneA PRP8, Ssp DnaX and Ssp DnaB mini-inteins were consistent with findings of other studies [23], [29], [30]. "
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    ABSTRACT: Protein trans-splicing by split inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split inteins need to be engineered to have a small N-intein or C-intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural inteins. These included both S1 split inteins having a very small (11-12 aa) N-intein fragment and S11 split inteins having a very small (6 aa) C-intein fragment. Four of the new S1 and S11 split inteins showed high efficiencies (85-100%) of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from ∼1.7×10(-4) s(-1) to ∼3.8×10(-4) s(-1), which are comparable to or higher than those of previously reported atypical split inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split inteins. They also have implications on understanding the structure-function relationship of atypical split inteins, particularly in terms of intein fragment complementation.
    PLoS ONE 04/2013; 8(4):e59516. DOI:10.1371/journal.pone.0059516 · 3.23 Impact Factor
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    • ".edu). The Prp8 gene of strain B05.10 carries a full-length, BciPRP8 intein (InBase; Liu and Yang, 2004), at exactly the same position as the inteins described in the Prp8 genes of other fungi such as Aspergillus fumigatus and Emericella nidulans (Butler et al., 2006). An additional 48 B. cinerea isolates (Table S1) were screened for the presence or absence of the BciPRP8 intein. "
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    ABSTRACT: Strains of Botrytis cinerea are polymorphic for the presence of an intein in the Prp8 gene (intein +/-). The intein encodes a homing endonuclease (HEG). During meiosis in an intein +/- heterozygote, the homing endonuclease initiates intein 'homing' by inducing gene conversion. In such meioses, the homing endonuclease triggers gene conversion of the intein together with its flanking sequences into the empty allele. The efficiency of gene conversion of the intein was found to be 100%. The extent of flanking sequence affected by the gene conversion varied in different meioses. A survey of the inteins and flanking sequences of a group B. cinerea isolates indicates that there are two distinct variants of the intein both of which have active HEGs. The survey also suggests that the intein has been actively homing during the evolution of the species and that the PRP8 intein may have entered the species by horizontal transfer.
    Fungal Genetics and Biology 04/2010; 47(4):392-8. DOI:10.1016/j.fgb.2010.01.003 · 3.26 Impact Factor
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    • "The ability of the PRP8 intein to self-splice from a non-native protein from E. coli was merely the first step for future studies and the identification of splicing inhibitors may constitute potential antifungal drugs. Since PRP8 inteins from different pathogens have the same corresponding insertion site in the PRP8 protein and, differently from the HEG domain, their splicing domain is highly conserved (Liu & Yang 2004), splicing inhibitor drugs may have the advantage of simultaneous action against most fungal diseases. "
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    ABSTRACT: Inteins or 'internal proteins' are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.
    Memórias do Instituto Oswaldo Cruz 06/2009; 104(3):497-504. DOI:10.1590/S0074-02762009000300017 · 1.57 Impact Factor
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