An in Vitro Screening System for Protein Splicing Inhibitors Based on Green Fluorescent Protein as an Indicator

Gangopadhyay, Jaya; Jiang, Shuqin; Paulus, Henry

doi:10.1021/ac020756b

Cited by 38 publications

(39 citation statements)

References 16 publications

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“…It was reported that 2 mM ZnCl 2 can inhibit RecA intein splicing using the same in vitro assay system (26). Zinc could also inhibit the trans splicing of the RecA intein (19).…”

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

confidence: 93%

“…In Vitro Inhibition Assay-The splicing assay was performed on a green fluorescent protein (GFP) with the RecA intein inserted before residue 129 of GFP in E. coli strain BL21 (DE3), as described previously (26). The protein expression plasmids were generous gifts from Henry Paulus.…”

Section: Methodsmentioning

confidence: 99%

“…The assay utilizes a modified GFP with the RecA intein (GFP-RecA-In) inserted in-frame adjacent to GFP residue 129. Placement of the intein at this position imposes a temporary block on GFP chromophore formation that is relieved upon splicing (26). The recovery of GFP fluorescence is therefore a direct measure of protein splicing and provides the basis for an inhibition assay (16).…”

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

confidence: 99%

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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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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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“…It was reported that 2 mM ZnCl 2 can inhibit RecA intein splicing using the same in vitro assay system (26). Zinc could also inhibit the trans splicing of the RecA intein (19).…”

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

confidence: 93%

Section: Methodsmentioning

confidence: 99%

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

confidence: 99%

See 1 more Smart Citation

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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“…(37). The PRP8 mini-intein encoding plasmid was kindly provided by Stephanie Pöggeler (Georg August Universität, Göttingen, Germany) (38).…”

Section: Methodsmentioning

confidence: 99%

Using intein catalysis to probe the origin of major histocompatibility complex class I-presented peptides

Farfán-Arribas

Stern

Rock

2012

Proc. Natl. Acad. Sci. U.S.A.

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All vertebrate nucleated cells generate peptides from their expressed gene products and then display them at the cell surface bound to MHC class I molecules. This allows CD8 + T cells to detect and eliminate abnormal cells that are synthesizing foreign proteins, e.g., from viruses or mutations. To permit the immune system to more uniformly monitor a cell's proteins, regardless of their half-life or location, it has been thought that the products of rapid degradation of the mistakes of protein synthesis (defective ribosomal products, DRiPs) preferentially contribute to the class I-presented peptides. However, using intein catalysis to generate peptide sequences exclusively by posttranslational splicing of mature proteins, we show here that presented peptides can be generated from fully folded and functional proteins. Remarkably, the presentation of peptides from two model mature proteins is just as efficient as from newly synthesized proteins subject to errors in translation or folding. These results indicate that for the constructs we have analyzed, DRiPs are not a more efficient source of class I peptides for antigen presentation than the turnover of mature functional proteins. Accordingly, our data suggest that one of the major ways the immune system evaluates the health of cells is by monitoring the breakdown products of the proteome.

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“…Using this approach, Daugelat and Jacobs (1999) inserted a RecA intein from M. tuberculosis with a C-terminal cysteine residue next to the enzyme responsible for kanamycin resistance. This same intein was also used in an in vitro screening system for protein splicing inhibitors based on green fluorescent protein (GFP), as an indicator when expressed in E. coli (Gangopadhyay et al 2003).…”

Section: Prp8 As a Therapeutic Targetmentioning

confidence: 99%

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Theodoro

Bagagli

2009

Mem. Inst. Oswaldo Cruz

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An in Vitro Screening System for Protein Splicing Inhibitors Based on Green Fluorescent Protein as an Indicator

Cited by 38 publications

References 16 publications

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

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

Using intein catalysis to probe the origin of major histocompatibility complex class I-presented peptides

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

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