Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

DeGraw, Amanda J.; Hast, Michael A.; Xu, Juhua; Mullen, Daniel G.; Beese, L.S.; Bárány, George; Distefano, Mark D.

doi:10.1111/j.1747-0285.2008.00698.x

Cited by 22 publications

(24 citation statements)

References 63 publications

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“…For these assays, CnFTase was compared with rat FTase, which was prepared as described previously (45). Rat FTase is 100% identical with hFTase in the active site in sequence and structure (46), therefore, making it a suitable substitute for hFTase.…”

Section: Journal Of Biological Chemistry 35151mentioning

confidence: 99%

“…Rat FTase is 100% identical with hFTase in the active site in sequence and structure (46), therefore, making it a suitable substitute for hFTase. Rat FTase was chosen for these assays because of the availability of a convenient His 6 -tagged recombinant expression system for the enzyme (45). Assay results are summarized in Table 2.…”

Section: Journal Of Biological Chemistry 35151mentioning

confidence: 99%

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Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

Hast

Nichols

Armstrong

et al. 2011

Journal of Biological Chemistry

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Cryptococcus neoformans is a fungal pathogen that causes lifethreatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes posttranslational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

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Section: Journal Of Biological Chemistry 35151mentioning

confidence: 99%

Section: Journal Of Biological Chemistry 35151mentioning

confidence: 99%

Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

Hast

Nichols

Armstrong

et al. 2011

Journal of Biological Chemistry

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“…Recombinant PFTase was overexpressed in E. coli and purified using previously published procedures . The activity of the PFTase enzyme was measured to be 1.6 μmol/min per mg using a previously reported continuous fluorescence assay .…”

Section: Methodsmentioning

confidence: 99%

Site‐Specific Labeling of Proteins and Peptides with Trans‐cyclooctene Containing Handles Capable of Tetrazine Ligation

Wollack

Monson²,

Dozier

et al. 2014

Chem Biol Drug Des

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There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper’s toxicity.1 Here we report the synthesis of trans-cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. Since tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.

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“…A frozen stock of BL21(DE3)pLysS E. coli cells containing yeast PFTase on a CDF-Duet1 vector, created by the Lorena Beese lab using a design previously employed for the mammalian PFTase[23], was used to inoculate a small culture of LB containing 50 µg/mL of streptomycin and grown overnight at 37°C with shaking at 240 rpm. The next morning, flasks containing 1 L LB media were inoculated with 10 mL of the overnight culture and grown to an OD 600 of approx.…”

Section: Methodsmentioning

confidence: 99%

An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

Dozier

Distefano

2012

Analytical Biochemistry

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Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays use either radiolabeled substrates and are discontinuous, or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format and that it can reproduce IC50 values for several previously reported FDPS inhibitors. This new method offers a simple, safe and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

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Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

Cited by 22 publications

References 63 publications

Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

Site‐Specific Labeling of Proteins and Peptides with Trans‐cyclooctene Containing Handles Capable of Tetrazine Ligation

An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

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