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Chembiochem : a European Journal of... Sep 2021Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of... (Review)
Review
Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of proteins for biotechnological and biomedical purposes, it is often required to enforce their tertiary structure in order to ensure sufficient stability under the conditions inherent to the application of interest. The introduction of intramolecular crosslinks has proven efficient in stabilizing native protein folds. Herein, we give an overview of methods that allow the macrocyclization of expressed proteins, discussing involved reaction mechanisms and structural implications.
Topics: Protein Splicing
PubMed: 34060202
DOI: 10.1002/cbic.202100111 -
Biochimica Et Biophysica Acta. General... May 2021Inteins are intervening proteins, which are known to perform protein splicing. The reaction results in the production of an intein domain and an inteinless protein,...
BACKGROUND
Inteins are intervening proteins, which are known to perform protein splicing. The reaction results in the production of an intein domain and an inteinless protein, which shows no trace of the insertion. BIL2 is part of the polyubiquitin locus of Tetrahymena thermophila (BUBL), where two bacterial-intein-like (BIL) domains lacking the C + 1 nucleophile, are flanked by two independent ubiquitin-like domains (ubl4/ubl5).
METHODS
We solved the X-ray structures of BIL2 in both the inactive and unprecedented, zinc-induced active, forms. Then, we characterized by mass spectrometry the BUBL splicing products in the absence and in the presence of T.thRas-GTPase. Finally, we investigated the effect of ubiquitination on T.thRas-GTPase by molecular dynamics simulations.
RESULTS
The structural analysis demonstrated that zinc-induced conformational change activates protein splicing. Moreover, mass spectrometry characterization of the splicing products shed light on the possible function of BIL2, which operates as a "single-ubiquitin-dispensing-platform", allowing the conjugation, via isopeptide bond formation (K(εNH)-C-ter), of ubl4 to either ubl5 or T.thRas-GTPase. Lastly, we demonstrated that T.thRas-GTPase ubiquitination occurs in proximity of the nucleotide binding pocket and stabilizes the protein active state.
CONCLUSIONS
We demonstrated that BIL2 is activated by zinc and that protein splicing induced by this intein does not take place through classical or aminolysis mechanisms but via formation of a covalent isopeptide bond, causing the ubiquitination of endogenous substrates such as T.thRas-GTPase.
GENERAL SIGNIFICANCE
In this "enzyme-free" ubiquitination mechanism the isopeptide formation, which canonically requires E1-E2-E3 enzymatic cascade and constitutes the alphabet of ubiquitin biology, is achieved in a single, concerted step without energy consumption.
Topics: Inteins; Models, Molecular; Polyubiquitin; Protein Domains; Protein Splicing; Tetrahymena thermophila; Ubiquitination; Zinc
PubMed: 33444728
DOI: 10.1016/j.bbagen.2021.129844 -
Proceedings of the National Academy of... Mar 1998Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation...
Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N- and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each approximately 100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N- and C-terminal intein segments.
Topics: Bacterial Proteins; Hydrogen-Ion Concentration; Ligases; Mycobacterium tuberculosis; Protein Splicing; Rec A Recombinases; Sequence Analysis; Sulfhydryl Compounds
PubMed: 9520402
DOI: 10.1073/pnas.95.7.3543 -
International Journal of Molecular... Jun 2023Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for...
Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins' performances by three-fold when compared to low-quality preparations (60-75% vs. 20-30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes.
Topics: Trans-Splicing; Protein Splicing; Inteins; Gene Transfer Techniques; Drug Packaging
PubMed: 37445701
DOI: 10.3390/ijms241310524 -
IUBMB Life Jan 2006
Topics: Inteins; Protein Splicing
PubMed: 16540435
DOI: 10.1080/15216540500531739 -
Acta Myologica : Myopathies and... Oct 2005Myotonic Dystrophy (DM), the most common form of adult-onset muscular dystrophy, comprises at least 2 subtypes, DM1 and DM2. DM1 is caused by the expansion of a CTG... (Review)
Review
Myotonic Dystrophy (DM), the most common form of adult-onset muscular dystrophy, comprises at least 2 subtypes, DM1 and DM2. DM1 is caused by the expansion of a CTG repeat located in the 3' untranslated region of the DM protein kinase (DMPK) gene. Recently, the expansion of a CCTG tetranucleotide repeat located in the first intron of the ZNF9 gene was identified as the mutation responsible for DM2. Since both DM1 and DM2 are caused by the expansion of repetitive sequences, some common factors that interact with these sequences might be involved in the pathogenesis of DM. MBNL1 is a candidate for such factors and is thought to be sequestered by the expanded forms of DM transcripts.
Topics: Animals; CCAAT-Enhancer-Binding Protein-delta; Electrophoretic Mobility Shift Assay; Humans; Insulin Resistance; Protein Splicing; RNA-Binding Proteins; Surface Plasmon Resonance
PubMed: 16550919
DOI: No ID Found -
The Journal of Biological Chemistry May 2004Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking...
Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond. Most reported inteins have a C-terminal asparagine residue, and it has been shown that cyclization of this residue is coupled to peptide bond cleavage between the intein and C-extein. We show that the intein interrupting the DNA polymerase II DP2 subunit in Pyrococcus abyssi, which has a C-terminal glutamine, is capable of facilitating protein splicing. Substitution of an asparagine for the C-terminal glutamine moderately improves the rate and extent of protein splicing. However, substitution of an alanine for the penultimate histidine residue, with either asparagine or glutamine in the C-terminal position, prevents protein splicing and facilitates cleavage at the intein N terminus. The intein facilitates in vitro protein splicing only at temperatures above 30 degrees C and can be purified as a nonspliced precursor. This temperature dependence has enabled us to characterize the optimal in vitro splicing conditions and determine the rate constants for splicing as a function of temperature.
Topics: Amino Acid Sequence; Amino Acid Substitution; Asparagine; Bacterial Proteins; Base Sequence; DNA Polymerase II; DNA Primers; Glutamine; Hydrogen-Ion Concentration; Kinetics; Protein Splicing; Protein Subunits; Pyrococcus abyssi; Recombinant Fusion Proteins; Thermodynamics
PubMed: 15024006
DOI: 10.1074/jbc.M400887200 -
The Journal of Biological Chemistry Jun 1997The protein splicing element (intein) of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae catalyzes both protein splicing and site-specific DNA cleavage. It...
The protein splicing element (intein) of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae catalyzes both protein splicing and site-specific DNA cleavage. It has been demonstrated that the conserved splice junction residues are directly involved in protein splicing and the central dodecapeptide motifs are required for DNA cleavage. To examine whether the splicing activity of the intein can be structurally separated from the endonuclease motifs, we made large in-frame deletions at the central region of the intein. We demonstrate for the first time that protein splicing can proceed efficiently after the removal of the central region of the intein including the endonuclease motifs. Our results suggest that the N- and C-terminal regions of the Sce VMA intein may form a separate domain that is not only catalytically sufficient for protein splicing but also structurally independent from the endonuclease domain.
Topics: Carrier Proteins; Cells, Cultured; Chitin; Endonucleases; Frameshift Mutation; Maltose-Binding Proteins; Molecular Weight; Protein Splicing; Proton-Translocating ATPases; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Sequence Deletion; Vacuolar Proton-Translocating ATPases
PubMed: 9188443
DOI: 10.1074/jbc.272.25.15587 -
Journal of the American Chemical Society Aug 2002The use of small molecules that turn specific proteins on or off provides a level of temporal control that is difficult to achieve using standard genetic approaches....
The use of small molecules that turn specific proteins on or off provides a level of temporal control that is difficult to achieve using standard genetic approaches. Consequently, the development of small-molecule switches of protein function is a very active area of chemical biology, sometimes referred to as chemical genetics. Most studies in this area rely on the identification of small molecules that bind directly to the active site of a target protein, thereby acting as agonists or antagonists of function. Strategies have also been described in which the small molecule triggers a change in the secondary, tertiary, or ternary structure of the protein, in so doing changing the functional state of the molecule. Another approach to this problem would be to alter the primary structure of a target protein in response to a small-molecule trigger; a dramatic change in primary sequence would be directly coupled to function. In principle, this can be achieved by harnessing protein splicing, a posttranslational editing process that results in the precise removal of an internal domain (termed an intein) from two flanking sequences termed the N- and C-exteins. In this communication we introduce a technique that allows protein splicing to occur only in the presence of the small molecule, rapamycin. This approach is expected to be independent of the nature of the two exteins and so should provide a general vehicle for controlling protein function using small molecules.
Topics: Blotting, Western; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Kinetics; Protein Splicing; Proteins; Recombinant Fusion Proteins; Saccharomyces cerevisiae
PubMed: 12148996
DOI: 10.1021/ja026769o -
Scientific Reports Sep 2023A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding...
A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart's chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG-Fab-type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG-Fab (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG-Fab type bsAb will expand the bsAb production method.
Topics: Inteins; Trans-Splicing; Antibodies, Bispecific; Protein Splicing; Immunoglobulin G
PubMed: 37749185
DOI: 10.1038/s41598-023-43110-0