Analysis of Protein Prenylation In Vitro and In Vivo Using Functionalized Phosphoisoprenoids

Nguyen, Uyen; Wu, Yao‐Wen; Goodall, Andrew; Alexandrov, Kirill

doi:10.1002/0471140864.ps1403s62

Cited by 12 publications

(14 citation statements)

References 25 publications

(30 reference statements)

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“… 11 More recently, radiolabeling was replaced by either a fluorophore or a biotin group. 17 Both approaches involved the use of a cultured cell lysate because REP1 is ubiquitously expressed in all cells and tissues. In this study, protein incorporation of biotin-containing isoprenoids (biotin-labeled geranyl pyrophosphate [B-GPP]) was used to detect prenylated proteins due to their superior sensitivity to fluorescence-based methods.…”

Section: Resultsmentioning

confidence: 99%

The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can Be Measured by In Vitro Prenylation of RAB6A

Patrício

Barnard

Cox³

et al. 2018

Molecular Therapy - Methods & Clinical Development

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Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the CHM gene. CHM is ubiquitously expressed in human cells and encodes Rab escort protein 1 (REP1). REP1 plays a key role in intracellular trafficking through the prenylation of Rab GTPases, a reaction that can be reproduced in vitro. With recent advances in adeno-associated virus (AAV) gene therapy for CHM showing gene replacement to be a promising approach, an assay to assess the biological activity of the vectors is of the uttermost importance. Here we sought to compare the response of two Rab proteins, RAB27A and RAB6A, to the incorporation of a biotinylated lipid donor in a prenylation reaction in vitro. First, we found the expression of REP1 to be proportional to the amount of recombinant AAV (rAAV)2/2-REP1 used to transduce the cells. Second, prenylation of RAB6A appeared to be more sensitive to REP1 protein expression than prenylation of RAB27A. Moreover, the method was reproducible in other cell lines. These results support the further development of a prenylation reaction using a biotinylated lipid donor and RAB6A to assess the biological activity of AAV vectors for CHM gene therapy.

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Section: Resultsmentioning

confidence: 99%

The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can Be Measured by In Vitro Prenylation of RAB6A

Patrício

Barnard

Cox³

et al. 2018

Molecular Therapy - Methods & Clinical Development

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“…Furthermore, growth rescue in the RRS depends on localisation of the reporter protein to the plasma membrane which is physiologically more relevant compared to profiling enzyme activities in vitro [ 7 , 18 – 21 ]. In addition, yeast-based genetic selection experiments are much cheaper and more versatile compared to screening chemically synthesised peptide libraries [ 7 , 18 – 21 ] and technically less challenging compared to proteomic tagging strategies [ 23 – 25 ]. For instance, it is possible to modulate expression levels with high- and low-copy plasmids as well as a range of well characterised promoter systems to fine-tune the expression levels of either the reporter gene or single-chain αβ-FTase mutants [ 47 , 48 ].…”

Section: Discussionmentioning

confidence: 99%

Towards the Systematic Mapping and Engineering of the Protein Prenylation Machinery in Saccharomyces cerevisiae

et al. 2015

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Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling functions and is frequently deregulated in disease processes such as cancer. As a result, protein prenylation has been pharmacologically targeted by numerous drug discovery programs, albeit with limited success. To a large extent, this can be attributed to an insufficient understanding of the interplay of different protein prenyltransferases and the combinatorial diversity of the prenylatable sequence space. Here, we report a high-throughput, growth-based genetic selection assay in Saccharomyces cerevisiae based on the Ras Recruitment System which, for the first time, has allowed us to create a comprehensive map of prenylatable protein sequences in S. cerevisiae. We demonstrate that potential prenylatable space is sparsely (6.2%) occupied leaving room for creation of synthetic orthogonal prenylatable sequences. To experimentally demonstrate that, we used the developed platform to engineer mutant farnesyltransferases that efficiently prenylate substrate motives that are not recognised by endogenous protein prenyltransferases. These uncoupled mutants can now be used as starting points for the systematic engineering of the eukaryotic protein prenylation machinery.

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“…An in vitro prenylation assay was performed on the freshly prepared lysate using 5 µmol/l biotin-labeled geranyl pyrophosphate (Euromedex, Souffelweyersheim, France) as a prenyl group donor, 0.5 µmol/l recombinant REP1 (Euromedex), 0.5 µmol/l recombinant Rab geranylgeranyl transferase type 2 (GGTase-II; Euromedex), and 20 µmol/l guanosine diphosphate in prenylation/lysis buffer at 37 °C for 1 hour. 49 , 50 The prenylation reaction was stopped with 6× sodium dodecyl sulfate, boiled at 90 °C for 5 minutes, and biotin incorporation analyzed by western blot analysis. The membrane was incubated with 1:5,000 horseradish peroxidase–conjugated streptavidin (Jackson ImmunoResearch, Cambridge, UK) and 1:50,000 mouse anti β -actin (Sigma Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient

Cereso

Péquignot

Robert

et al. 2014

Molecular Therapy - Methods & Clinical Development

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Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC–derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model.

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Analysis of Protein Prenylation In Vitro and In Vivo Using Functionalized Phosphoisoprenoids

Cited by 12 publications

References 25 publications

The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can Be Measured by In Vitro Prenylation of RAB6A

The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can Be Measured by In Vitro Prenylation of RAB6A

Towards the Systematic Mapping and Engineering of the Protein Prenylation Machinery in Saccharomyces cerevisiae

Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient

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