Rab Escort Protein

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Miguel C. Seabra - One of the best experts on this subject based on the ideXlab platform.

  • Adeno-associated virus 8-mediated gene therapy for choroideremia: preclinical studies in in vitro and in vivo models.
    The journal of gene medicine, 2014
    Co-Authors: Aaron Black, Miguel C. Seabra, Albert M. Maguire, Vidyullatha Vasireddy, Daniel C. Chung, Rajashekhar Gaddameedi, Tania Tolmachova, Jean Bennett
    Abstract:

    Background Choroideremia (CHM) is a slowly progressive X-linked retinal degeneration that results in a loss of photoreceptors, retinal pigment epithelium and choroid. CHM, the gene implicated in choroideremia, encodes Rab Escort Protein-1 (REP-1), which is involved in the post-translational activation via prenylation of Rab Proteins. Methods We evaluated AAV8.CBA.hCHM, a recombinant adeno-associated virus serotype 8 (rAAV8) vector, which targets retinal cells efficiently, for both therapeutic effect and safety in vitro and in vivo in a murine model. In vitro studies included western blot analyses and prenylation assays. In vivo studies included ophthalmoscopy, pupillometry, histology and immunofluorescence analysis. Results Infection with AAV8.CBA.hCHM induced the expression of REP-1 Protein in a dose-responsive fashion. Transduction with AAV8.CBA.hCHM reverses the biochemical and pathogenetic defects in CHM both in vitro and in vivo and showed no safety concerns in the in vivo investigations performed in the present study. Conclusions AAV8 is a promising vector for human clinical gene therapy trials for choroideremia. Copyright © 2014 John Wiley & Sons, Ltd.

  • Functional expression of Rab Escort Protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo
    Journal of Molecular Medicine, 2013
    Co-Authors: Tanya Tolmachova, Oleg E. Tolmachov, Alun R. Barnard, Samantha R. Silva, Daniel M. Lipinski, Nathan J. Walker, Robert E. Maclaren, Miguel C. Seabra
    Abstract:

    Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM / REP1 gene that encodes Rab Escort Protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials.

  • Rab geranylgeranylation occurs preferentially via the pre-formed REP–RGGT complex and is regulated by geranylgeranyl pyrophosphate
    Biochemical Journal, 2008
    Co-Authors: Rudi A. Baron, Miguel C. Seabra
    Abstract:

    Prenylation (or geranylgeranylation) of Rab GTPases is catalysed by RGGT (Rab geranylgeranyl transferase) and requires REP (Rab Escort Protein). In the classical pathway, REP associates first with unprenylated Rab, which is then prenylated by RGGT. In the alternative pathway, REP associates first with RGGT; this complex then binds and prenylates Rab Proteins. In the present paper we show that REP mutants defective in RGGT binding (REP1 F282L and REP1 F282L/V290F) are unable to compete with wild-type REP in the prenylation reaction in vitro . When over-expressed in cells, REP wild-type and mutants are unable to form stable cytosolic complexes with endogenous unprenylated Rabs. These results suggest that the alternative pathway may predominate in vivo . We also extend previous suggestions that GGPP (geranylgeranyl pyrophosphate) acts as an allosteric regulator of the prenylation reaction. We observed that REP–RGGT complexes are formed in vivo and are unstable in the absence of intracellular GGPP. RGGT increases the ability of REP to extract endogenous prenylated Rabs from membranes in vitro by stabilizing a soluble REP–RGGT–Rab-GG (geranylgeranylated Rab) complex. This effect is regulated by GGPP, which promotes the dissociation of RGGT and REP–Rab-GG to allow delivery of prenylated Rabs to membranes.

  • Rab Geranylgeranylation occurs preferentially via the pre-formed REP:RGGT complex and is regulated by geranylgeranyl pyrophosphate
    Biochemical Journal, 2008
    Co-Authors: Rudi A. Baron, Miguel C. Seabra
    Abstract:

    Prenylation (or Geranylgeranylation, GG) of Rab GTPases is catalysed by Rab Geranylgeranyl Transferase (RGGT) and requires Rab Escort Protein (REP). In the classical pathway, REP associates first with unprenylated Rab which is then prenylated by RGGT. In the alternative pathway, REP associates first with RGGT; this complex then binds and prenylates Rab Proteins. Here we show that REP mutants (REP1F282L and REP1F282L/V290F) defective in RGGT binding are unable to compete with wild-type REP in the prenylation reaction in vitro. When over-expressed in cells, REP wild type and mutants are unable to form stable cytosolic complexes with endogenous unprenylated Rabs. These results suggest that the alternative pathway may predominate in vivo. We also extend previous suggestions that GGPP acts as an allosteric regulator of the reaction. We observed that REP:RGGT complexes are formed in vivo and are unstable in absence of intracellular GGPP. RGGT decreases the ability of REP to extract endogenous prenylated Rabs from membranes in vitro by stabilising a soluble REP:RGGT:Rab-GG complex. This effect is regulated by GGPP, which promotes the dissociation of RGGT and REP:Rab-GG to allow delivery of prenylated Rabs to membranes.

  • Membrane Targeting of Rab GTPases Is Influenced by the Prenylation Motif
    Molecular biology of the cell, 2003
    Co-Authors: Anita Quintal Gomes, Richard F. Godfrey, Bassam R. Ali, José S. Ramalho, Duarte C. Barral, Alistair N. Hume, Miguel C. Seabra
    Abstract:

    Rab GTPases are regulators of membrane traffic. Rabs specifically associate with target membranes via the attachment of (usually) two geranylgeranyl groups in a reaction involving Rab Escort Protein and Rab geranylgeranyl transferase. In contrast, related GTPases are singly prenylated by CAAX prenyl transferases. We report that di-geranylgeranyl modification is important for targeting of Rab5a and Rab27a to endosomes and melanosomes, respectively. Transient expression of EGFP-Rab5 mutants containing two prenylatable cysteines (CGC, CC, CCQNI, and CCA) in HeLa cells did not affect endosomal targeting or function, whereas mono-cysteine mutants (CSLG, CVLL, or CVIM) were mistargeted to the endoplasmic reticulum (ER) and were nonfunctional. Similarly, Rab27aCVLL mutant is also mistargeted to the ER and transgenic expression on a Rab27a null background (Rab27a ash ) did not rescue the coat color phenotype, suggesting that Rab27aCVLL is not functional in vivo. CAAX prenyl transferase inhibition and temperature-shift experiments further suggest that Rabs, singly or doubly modified are recruited to membranes via a Rab Escort Protein/Rab geranylgeranyl transferase-dependent mechanism that is distinct from the insertion of CAAX-containing GTPases. Finally, we show that both singly and doubly modified Rabs are extracted from membranes by RabGDI and propose that the mistargeting of Rabs to the ER results from loss of targeting information.

Kirill Alexandrov - One of the best experts on this subject based on the ideXlab platform.

  • Rab GTPase prenylation hierarchy and its potential role in choroideremia disease.
    PloS one, 2013
    Co-Authors: Monika Köhnke, Roger S. Goody, Herbert Waldmann, Christine Delon, Marcus L. Hastie, Uyen T. T. Nguyen, Jeffrey J. Gorman, Kirill Alexandrov
    Abstract:

    Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab Escort Protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.

  • Organization and function of the Rab prenylation and recycling machinery
    Protein Prenylation PART A, 2011
    Co-Authors: Kirill Alexandrov, Herbert Waldmann, Wulf Blankenfeldt, Roger S. Goody
    Abstract:

    The Rab Proteins form the largest subgroup of the Ras superfamily and control multiple steps of intracellular vesicular transport. Similar to many other small GTPases, Rab Proteins are posttranslationally prenylated. Prenylation by Rab Geranylgeranyl transferase increases hydrophobicity of the RabGTPases and enables them to reversibly associate with their target membrane. Due to the vectoriality of vesicular transport, RabGTPases are recycled upon completion of their functional cycle and returned to a cytosolic intermediate. The cytosolic form of Rab Proteins is stabilized by the tightly binding chaperon termed GDP dissociation inhibitor (GDI). GDI also plays a central role in loading of prenylated RabGTPases onto the target membranes. In this chapter, we discuss available structural and biochemical data that shed light on the molecular mechanism of Rab prenylation, membrane delivery, and recycling. We discuss the important functional differences between GDI and a structurally related RabGGTase accessory factor termed Rab Escort Protein. We summarize the available data on the identity of factors mediating dissociation of the Rab:GDI complex prior to membrane loading and provide arguments for the critical role of guanine nucleotide exchange factors in this process.

  • membrane targeting mechanism of Rab gtpases elucidated by semisynthetic Protein probes
    Nature Chemical Biology, 2010
    Co-Authors: Lena K Oesterlin, Kirill Alexandrov, Herbert Waldmann, Kuithong Tan, Roger S. Goody
    Abstract:

    Post-translationally isoprenylated Proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed Protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these Protein probes, we have demonstrated that RabGDI and the related Rab Escort Protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab-GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.

  • Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic Protein probes
    Nature Chemical Biology, 2010
    Co-Authors: Yao-wen Wu, Kirill Alexandrov, Herbert Waldmann, Lena K Oesterlin, Roger S. Goody
    Abstract:

    Post-translationally isoprenylated Proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed Protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these Protein probes, we have demonstrated that RabGDI and the related Rab Escort Protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab–GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process. Semisynthetic versions of the small G Protein Rab7 in the GDP-bound form have 1,000-fold higher affinity for regulators REP1 and RabGDI because of faster dissociation rates from Rab7-GTP, directly linking nucleotide exchange to Rab membrane targeting.

  • interaction analysis of prenylated Rab gtpase with Rab Escort Protein and gdp dissociation inhibitor explains the need for both regulators
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Kuithong Tan, Herbert Waldmann, Roger S. Goody, Kirill Alexandrov
    Abstract:

    Prenylated Rab GTPases regulate intracellular vesicle trafficking in eukaryotic cells by associating with specific membranes and recruiting a multitude of Rab-specific effector Proteins. Prenylation, membrane delivery, and recycling of all 60 members of the Rab GTPase family are regulated by two related molecules, Rab Escort Protein (REP) and GDP dissociation inhibitor (GDI). Biophysical analysis of the interaction of prenylated Proteins is complicated by their low solubility in aqueous solutions. Here, we used expressed Protein ligation to construct a semisynthetic fluorescent analogue of prenylated Rab7, Rab7-NBD-farnesyl. This molecule is soluble in the absence of detergent but is otherwise similar in its behavior to naturally prenylated Rab7 GTPase. To obtain information on the interaction of natively mono- and diprenylated Rab7 GTPases with REP and GDI molecules, we stabilized the former molecules in solution by using the β-subunit of Rab geranylgeranyl transferase, which we demonstrate to function as an unspecific chaperone of prenylated Proteins. Using competitive titrations of mixtures of natively prenylated and fluorescent Rab, we demonstrate that monogeranylgeranylated Rab7 binds to the REP Protein with a Kd value of ≈70 pM. The affinity of doubly prenylated Rab7 is ≈20-fold weaker. In contrast, GDI binds both prenylated forms of Rab7 with compaRable affinities (Kd = 1–5 nM) but has extremely low affinity to unprenylated Rab molecules. The obtained data allow us to formulate a thermodynamic model for the interaction of RabGTPases with their regulators and membranes and to explain the need for both REP and GDI in Rab function.

Robert E. Maclaren - One of the best experts on this subject based on the ideXlab platform.

  • Atypical choroideremia presenting with early‐onset macular atrophy
    Acta ophthalmologica, 2019
    Co-Authors: Georgios T. Kontos, Robert E. Maclaren, Maria I. Patrício, Penny Clouston, Jennifer Kwan, Kanmin Xue, Emily Packham, Susan M. Downes
    Abstract:

    Choroideremia is an X-linked recessive retinal degeneration predominantly affecting hemizygous males. It is caused by mutations in the CHM gene that encodes the Rab Escort Protein-1. Characteristic features include early nyctalopia followed by progressive constriction of peripheral visual fields and sparing of the central vision until late in life with a distinct fundoscopic appearance. We present the case of a 17-year-old male with a c.282delT in exon 4 of CHM that has not previously been reported. Phenotypically this patient presented with an atypical choroideremia phenotype of early central macular degeneration in addition to the classic peripheral fundus characteristic findings.

  • The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can Be Measured by In Vitro Prenylation of Rab6A.
    Molecular therapy. Methods & clinical development, 2018
    Co-Authors: Maria I. Patrício, Alun R. Barnard, Christopher I. Cox, Clare Blue, Robert E. Maclaren
    Abstract:

    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.

  • Retinal Gene Therapy for Choroideremia: In Vitro Testing for Gene Augmentation Using an Adeno-Associated Viral (AAV) Vector.
    Methods in molecular biology (Clifton N.J.), 2017
    Co-Authors: Maria I. Patrício, Robert E. Maclaren
    Abstract:

    As gene therapy of choroideremia is becoming a clinical reality, there is a need for reliable and sensitive assays to determine the expression of exogenously delivered Rab Escort Protein-1 (REP1), in particular to test new gene therapy vectors and as a quality control screen for clinical vector stocks. Here we describe an in vitro protocol to test transgene expression following AAV2/2-REP1 transduction of a human cell line. Gene augmentation can be confirmed by western blot and quantification of the fold-increase of human REP1 levels over untransduced controls.

  • Novel non-contiguous exon duplication in choroideremia.
    Clinical genetics, 2017
    Co-Authors: Thomas L. Edwards, Maria I. Patrício, Jonathan H. Williams, Matthew P. Simunovic, Morag E Shanks, Penny Clouston, Robert E. Maclaren
    Abstract:

    The importance of establishing a genetic diagnosis in patients with a choroideremia phenotype has been underscored by the advent of gene replacement therapy for this condition. Here, we describe a complex imbalance at the CHM locus in a male patient with classical disease. At the DNA level, this imbalance consists of 2 non-contiguous duplications (exons 1-2 and 9-12). Further characterization suggests the generation of 2 independent CHM transcriptional units, one of which may produce a deleted form of the Rab Escort Protein 1 Protein. Expression of such a type of aberrant Protein in photoreceptors may have important implications when considering gene therapy for this disorder.

  • Gene Therapy for Choroideremia Using an Adeno-Associated Viral (AAV) Vector
    2016
    Co-Authors: Alun R. Barnard, Robert E. Maclaren
    Abstract:

    Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness bymiddle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab Escort Protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no estab-lished treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-asso-ciated virus (AAV) encoding the CHM gene

Roger S. Goody - One of the best experts on this subject based on the ideXlab platform.

  • Rab GTPase prenylation hierarchy and its potential role in choroideremia disease.
    PloS one, 2013
    Co-Authors: Monika Köhnke, Roger S. Goody, Herbert Waldmann, Christine Delon, Marcus L. Hastie, Uyen T. T. Nguyen, Jeffrey J. Gorman, Kirill Alexandrov
    Abstract:

    Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab Escort Protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.

  • Organization and function of the Rab prenylation and recycling machinery
    Protein Prenylation PART A, 2011
    Co-Authors: Kirill Alexandrov, Herbert Waldmann, Wulf Blankenfeldt, Roger S. Goody
    Abstract:

    The Rab Proteins form the largest subgroup of the Ras superfamily and control multiple steps of intracellular vesicular transport. Similar to many other small GTPases, Rab Proteins are posttranslationally prenylated. Prenylation by Rab Geranylgeranyl transferase increases hydrophobicity of the RabGTPases and enables them to reversibly associate with their target membrane. Due to the vectoriality of vesicular transport, RabGTPases are recycled upon completion of their functional cycle and returned to a cytosolic intermediate. The cytosolic form of Rab Proteins is stabilized by the tightly binding chaperon termed GDP dissociation inhibitor (GDI). GDI also plays a central role in loading of prenylated RabGTPases onto the target membranes. In this chapter, we discuss available structural and biochemical data that shed light on the molecular mechanism of Rab prenylation, membrane delivery, and recycling. We discuss the important functional differences between GDI and a structurally related RabGGTase accessory factor termed Rab Escort Protein. We summarize the available data on the identity of factors mediating dissociation of the Rab:GDI complex prior to membrane loading and provide arguments for the critical role of guanine nucleotide exchange factors in this process.

  • membrane targeting mechanism of Rab gtpases elucidated by semisynthetic Protein probes
    Nature Chemical Biology, 2010
    Co-Authors: Lena K Oesterlin, Kirill Alexandrov, Herbert Waldmann, Kuithong Tan, Roger S. Goody
    Abstract:

    Post-translationally isoprenylated Proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed Protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these Protein probes, we have demonstrated that RabGDI and the related Rab Escort Protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab-GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.

  • Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic Protein probes
    Nature Chemical Biology, 2010
    Co-Authors: Yao-wen Wu, Kirill Alexandrov, Herbert Waldmann, Lena K Oesterlin, Roger S. Goody
    Abstract:

    Post-translationally isoprenylated Proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed Protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these Protein probes, we have demonstrated that RabGDI and the related Rab Escort Protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab–GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process. Semisynthetic versions of the small G Protein Rab7 in the GDP-bound form have 1,000-fold higher affinity for regulators REP1 and RabGDI because of faster dissociation rates from Rab7-GTP, directly linking nucleotide exchange to Rab membrane targeting.

  • interaction analysis of prenylated Rab gtpase with Rab Escort Protein and gdp dissociation inhibitor explains the need for both regulators
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Kuithong Tan, Herbert Waldmann, Roger S. Goody, Kirill Alexandrov
    Abstract:

    Prenylated Rab GTPases regulate intracellular vesicle trafficking in eukaryotic cells by associating with specific membranes and recruiting a multitude of Rab-specific effector Proteins. Prenylation, membrane delivery, and recycling of all 60 members of the Rab GTPase family are regulated by two related molecules, Rab Escort Protein (REP) and GDP dissociation inhibitor (GDI). Biophysical analysis of the interaction of prenylated Proteins is complicated by their low solubility in aqueous solutions. Here, we used expressed Protein ligation to construct a semisynthetic fluorescent analogue of prenylated Rab7, Rab7-NBD-farnesyl. This molecule is soluble in the absence of detergent but is otherwise similar in its behavior to naturally prenylated Rab7 GTPase. To obtain information on the interaction of natively mono- and diprenylated Rab7 GTPases with REP and GDI molecules, we stabilized the former molecules in solution by using the β-subunit of Rab geranylgeranyl transferase, which we demonstrate to function as an unspecific chaperone of prenylated Proteins. Using competitive titrations of mixtures of natively prenylated and fluorescent Rab, we demonstrate that monogeranylgeranylated Rab7 binds to the REP Protein with a Kd value of ≈70 pM. The affinity of doubly prenylated Rab7 is ≈20-fold weaker. In contrast, GDI binds both prenylated forms of Rab7 with compaRable affinities (Kd = 1–5 nM) but has extremely low affinity to unprenylated Rab molecules. The obtained data allow us to formulate a thermodynamic model for the interaction of RabGTPases with their regulators and membranes and to explain the need for both REP and GDI in Rab function.

Herbert Waldmann - One of the best experts on this subject based on the ideXlab platform.

  • Rab GTPase prenylation hierarchy and its potential role in choroideremia disease.
    PloS one, 2013
    Co-Authors: Monika Köhnke, Roger S. Goody, Herbert Waldmann, Christine Delon, Marcus L. Hastie, Uyen T. T. Nguyen, Jeffrey J. Gorman, Kirill Alexandrov
    Abstract:

    Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab Escort Protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.

  • Organization and function of the Rab prenylation and recycling machinery
    Protein Prenylation PART A, 2011
    Co-Authors: Kirill Alexandrov, Herbert Waldmann, Wulf Blankenfeldt, Roger S. Goody
    Abstract:

    The Rab Proteins form the largest subgroup of the Ras superfamily and control multiple steps of intracellular vesicular transport. Similar to many other small GTPases, Rab Proteins are posttranslationally prenylated. Prenylation by Rab Geranylgeranyl transferase increases hydrophobicity of the RabGTPases and enables them to reversibly associate with their target membrane. Due to the vectoriality of vesicular transport, RabGTPases are recycled upon completion of their functional cycle and returned to a cytosolic intermediate. The cytosolic form of Rab Proteins is stabilized by the tightly binding chaperon termed GDP dissociation inhibitor (GDI). GDI also plays a central role in loading of prenylated RabGTPases onto the target membranes. In this chapter, we discuss available structural and biochemical data that shed light on the molecular mechanism of Rab prenylation, membrane delivery, and recycling. We discuss the important functional differences between GDI and a structurally related RabGGTase accessory factor termed Rab Escort Protein. We summarize the available data on the identity of factors mediating dissociation of the Rab:GDI complex prior to membrane loading and provide arguments for the critical role of guanine nucleotide exchange factors in this process.

  • membrane targeting mechanism of Rab gtpases elucidated by semisynthetic Protein probes
    Nature Chemical Biology, 2010
    Co-Authors: Lena K Oesterlin, Kirill Alexandrov, Herbert Waldmann, Kuithong Tan, Roger S. Goody
    Abstract:

    Post-translationally isoprenylated Proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed Protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these Protein probes, we have demonstrated that RabGDI and the related Rab Escort Protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab-GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.

  • Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic Protein probes
    Nature Chemical Biology, 2010
    Co-Authors: Yao-wen Wu, Kirill Alexandrov, Herbert Waldmann, Lena K Oesterlin, Roger S. Goody
    Abstract:

    Post-translationally isoprenylated Proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed Protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these Protein probes, we have demonstrated that RabGDI and the related Rab Escort Protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab–GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process. Semisynthetic versions of the small G Protein Rab7 in the GDP-bound form have 1,000-fold higher affinity for regulators REP1 and RabGDI because of faster dissociation rates from Rab7-GTP, directly linking nucleotide exchange to Rab membrane targeting.

  • Structures of RabGGTase–substrate/product complexes provide insights into the evolution of Protein prenylation
    The EMBO journal, 2008
    Co-Authors: Zhong Guo, Herbert Waldmann, Christine Delon, Debapratim Das, Janinna Cramer, Sandra Thuns, Nataliya Lupilova, Luc Brunsveld
    Abstract:

    Post-translational isoprenylation of Proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual Protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other Protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely ‘outsource' their specificity for each other to an accessory subunit, the Rab Escort Protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series ProteinProtein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that ‘learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP.