Reversible Inhibition of Protein Splicing by Zinc Ion

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC 50 of ϳ2 M, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ϳ40 M, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.Tuberculosis remains a leading cause of death worldwide (1, 2), and the global emergence of multidrug-resistant Mycobacterium tuberculosis portends further challenges in TB Several microbial pathogens, including M. tuberculosis, contain protein self-splicing elements called inteins, which interrupt critical genes and their protein products. These inteins are therefore potential novel targets for antibiotic development, particularly because no inteins are present in the human genome. In addition to M. tuberculosis, self-splicing inteins reside in critical proteins of Mycobacterium leprae, Coxiella burnetii, and Cryptococcus neoformans, the etiological agents of leprosy, Q fever, and cryptococcosis, respectively (6 -8). For these pathogens to survive, the inteins must catalyze a multistep reaction in which they are excised from the precursor protein and the two flanking sequences, called exteins, must be ligated to form a functional product protein (9 -11). Because inteins exert control, via splicing, over the function of pathogen-specific proteins, these self-splicing elements are attractive candidates for inhibition studies. The pursuit of intein inhibitors, whether as mechanistic probes or as an avenue for developing antibacterials, does face a unique challenge; unlike a conventional enzyme inhibitor, an effective antagonist of intein splicing must outcompete a substrate that is covalently tethered to the enzyme.M. tuberculosis has inteins in three distinct genes: in the dnaB helicase gene, in the recA recombinase gene,...

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“…Zinc could also inhibit the trans splicing of the RecA intein (19). The zinc inhibition was reversed by EDTA in both studies.…”

Section: In Vitro Inhibition Of the Mtu Reca Intein By Cisplatin-mentioning

confidence: 85%

Cisplatin Inhibits Protein Splicing, Suggesting Inteins as Therapeutic Targets in Mycobacteria

Zhang

Zheng

Callahan

et al. 2011

Journal of Biological Chemistry

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“…Interestingly, attempts to determine whether the intein was inhibited by the presence of zinc ion, as observed with other inteins (22,28,29), were unsuccessful because incubation of each of the PolII intein fusion proteins with zinc chloride also resulted in protein precipitation (data not shown).…”

Section: ϫ6mentioning

confidence: 99%

Protein Splicing of a Pyrococcus abyssi Intein with a C-terminal Glutamine

Mills

Manning

Garcı́a

et al. 2004

Journal of Biological Chemistry

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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°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.Protein splicing is a post-translational auto-processing event by which an intervening polypeptide sequence, the intein, facilitates both its own excision from the flanking polypeptides, or exteins, as well as the ligation of the these segments (reviewed in Ref. 1). All of the catalytic groups required for protein splicing have been shown to lie within the intein and the two flanking amino acids (1).The splicing mechanism of the majority of inteins has been described in detail (1). The first step of splicing involves an N-O or N-S acyl rearrangement of the peptide bond at the intein N terminus to an ester or thioester (Fig. 1, step A). The second step is a transesterification between the nucleophilic residue at the N terminus of the C-extein 1 (Cys, Ser, or Thr) and the newly formed ester or thioester at the intein N terminus (Fig.

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“…For instance, its replacement by mutation can inhibit protein splicing (4,5) or lead to accumulation of branched ester intermediate (6). However, other inteins undergo efficient protein splicing with penultimate residues other than His, and the replacement of the these residues with His in these inteins can either reduce, enhance, or have no measurable effect on the extent of splicing (7)(8)(9).…”

Section: Step 5)mentioning

confidence: 99%

Kinetic Analysis of the Individual Steps of Protein Splicing for the Pyrococcus abyssi PolII Intein

Mills

Dorval²,

Lewandowski³

2005

Journal of Biological Chemistry

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Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed and purified as an unspliced precursor, which allows for a detailed in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a noncanonical Gln at its C terminus, is 9.3 ؋ 10 ؊6 s ؊1 at 60°C. The rate constant for splicing increases 3-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of dithiothreitol-dependent N-terminal cleavage is 1 ؋ 10 ؊4 s ؊1 . The first order rate constant of C-terminal cleavage is 1.2 ؋ 10 ؊5 s ؊1 with Gln at the C-terminal position, 2.8 ؋ 10 ؊4 s ؊1 with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5 and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.Protein splicing involves the post-translational, self-directed excision of an intervening polypeptide, or intein, from flanking polypeptides, or exteins. The splicing process also involves the specific ligation of the extein segments by an amide bond, concomitant with the excision of the intein (1).The canonical mechanism of protein splicing consists of four separate but coordinated chemical steps each directed by the intein without the assistance of other proteins or cofactors (1). The first step of splicing is an N-S or N-O acyl rearrangement of the amide bond linking the N-extein and intein (see Fig. 1, step 1). This is followed by a transesterification that transfers the N-extein to the side chain of the N-terminal Cys, Ser, or Thr of the C-extein (see Fig. 1, step 2). The third step of splicing couples the cyclization of the C-terminal Asn of the intein to peptide bond cleavage, and excises the intein from the newly ligated exteins (see Fig. 1, step 3). Finally, the ester bond linking the exteins is rearranged to an amide (see Fig. 1, step 4), and the C-terminal aminosuccinimide of the intein is presumably hydrolyzed to either Asn or iso-Asn (see Fig. 1, step 5).Inteins that splice via variations of the canonical mechanism have previously been described. For instance, our group recently described the protein splicing of the intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi (Pab), 1 which is able to facilitate protein splicing with a Cterminal Gln residue (2). These results are complementary to those of Pietrokovski and coworkers (3), who demonstrated that the intein that interrupts the ribonucleotide reductase (RNR) of the Chilo iridescent virus can facilitate protein splicing with a C-terminal Gln and that the intein interrupting the RNR of Carboxydothermus hydrogenoformans can facilitate protein splicing w...

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Reversible Inhibition of Protein Splicing by Zinc Ion

Cited by 68 publications

References 20 publications

Cisplatin Inhibits Protein Splicing, Suggesting Inteins as Therapeutic Targets in Mycobacteria

Cisplatin Inhibits Protein Splicing, Suggesting Inteins as Therapeutic Targets in Mycobacteria

Protein Splicing of a Pyrococcus abyssi Intein with a C-terminal Glutamine

Kinetic Analysis of the Individual Steps of Protein Splicing for the Pyrococcus abyssi PolII Intein

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