Identification and Specificity Profiling of Protein Prenyltransferase Inhibitors Using New Fluorescent Phosphoisoprenoids

Dursina, Beatrice; Reents, R.; Delon, Christine; Wu, Yao‐Wen; Kulharia, Mahesh; Thutewohl, Michael; Veligodsky, Alexei; Kalinin, Alexandr; Evstifeev, Vladimir; Ciobanu, Doina; Szedlacsek, Stefan E.; Waldmann, Herbert; Goody, Roger S.; Alexandrov, Kirill

doi:10.1021/ja052196e

Cited by 76 publications

(108 citation statements)

References 56 publications

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“…NBD-farnesyl pyrophosphate was produced as described elsewhere (29). Prenyltransferase, Rab protein and substrate were mixed in a 0.5∶1 : 5 ratio and incubated at room temperature for 1.5 h. GGTase I and farnesyl-NBD were preincubated for 30 min before addition of Rab protein.…”

Section: Methodsmentioning

confidence: 99%

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor

Oesterlin

Goody

Itzen

2012

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

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Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.T he intracellular activity of most signaling proteins is regulated at the temporal and spatial level. In the regulation of vesicular trafficking, small Ras-like GTPases of the Rab subfamily are controlled in their distribution between the cytosol and intracellular membranes, which is in turn coupled to the binding to either GDP or GTP. Cycling between the cytosol and membranes is dependent on the activation state of the Rab protein. In the active GTP-bound form, Rabs are associated with membranes, where they interact with effector proteins that promote diverse steps in vesicular trafficking. When bound to GDP, Rabs are defined as inactive and are predominantly distributed in the cytosol bound to GDI. In order to associate with the cytosolic surface of intracellular membranes, most Rab proteins possess two hydrophobic prenyl (geranylgeranyl) moieties at their structurally flexible C terminus that are posttranslationally attached via the concerted action of Rab geranylgeranyl transferase (RabGGTase) and Rab escort protein utilizing geranylgeranyl pyrophosphate as a cosubstrate. These geranylgeranyl groups mediate stable peripheral binding of Rabs to a cognate membrane by inserting the hydrophobic anchors into the lipid bilayer (1). Activation of Rab proteins is catalyzed by GDP/GTP exchange factors...

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Section: Methodsmentioning

confidence: 99%

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor

Oesterlin

Goody

Itzen

2012

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

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“…S. cerevisiae FTase was expressed and purified as described for mammalian FTase (36). For expression of GSTPex19p in S. cerevisiae, strains were grown in SD-ura to midlog phase and induced by 0.5 mM copper sulfate for 2 h. Cells were harvested and resuspended in phosphate-buffered saline containing 1 mM dithiothreitol, Roche Applied Science complete protease inhibitor mixture, and 1 mM phenylmethylsulfonyl fluoride.…”

Section: Methodsmentioning

confidence: 99%

Farnesylation of Pex19p Is Required for Its Structural Integrity and Function in Peroxisome Biogenesis

Rucktäschel

Thoms

Sidorovitch

et al. 2009

Journal of Biological Chemistry

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The conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.

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“…Substrate utilization assays in which FTase activity was measured by incorporation of fluorescent FPP analog NBD-GPP (Jena Biosciences) into these full-length Ras substrates were performed essentially as described (44). For these assays, CnFTase was compared with rat FTase, which was prepared as described previously (45).…”

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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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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Identification and Specificity Profiling of Protein Prenyltransferase Inhibitors Using New Fluorescent Phosphoisoprenoids

Cited by 76 publications

References 56 publications

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor

Farnesylation of Pex19p Is Required for Its Structural Integrity and Function in Peroxisome Biogenesis

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

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