PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (37)
 

Clipboard (0)
None

Select a Filter Below

Year of Publication
more »
1.  Expression of OA1 limits the fusion of a subset of MVBs with lysosomes – a mechanism potentially involved in the initial biogenesis of melanosomes 
Journal of Cell Science  2013;126(22):5143-5152.
Summary
Multivesicular endosomes/bodies (MVBs) deliver proteins, such as activated EGF receptor (EGFR), to the lysosome for degradation, and, in pigmented cells, MVBs containing PMEL are an initial stage in melanosome biogenesis. The mechanisms regulating numbers and fate of different populations of MVB are unclear. Here, we focus on the role of the G-protein-coupled receptor OA1 (also known as GPR143), which is expressed exclusively in pigmented cells and mutations in which cause the most common type of ocular albinism. When exogenously expressing PMEL, HeLa cells have been shown to form MVBs resembling early stage melanosomes. To focus on the role of OA1 in the initial stages of melanosome biogenesis we take advantage of the absence of the later stages of melanosome maturation in HeLa cells to determine whether OA1 activity can regulate MVB number and fate. Expression of wild-type but not OA1 mutants carrying inactivating mutations or deletions causes MVB numbers to increase. Whereas OA1 expression has no effect on delivery of EGFR-containing MVBs to the lysosome, it inhibits the lysosomal delivery of PMEL and PMEL-containing MVBs accumulate. We propose that OA1 activity delays delivery of PMEL-containing MVBs to the lysosome to allow time for melanin synthesis and commitment to melanosome biogenesis.
doi:10.1242/jcs.128561
PMCID: PMC3828590  PMID: 24006264
Lysosomes; Multivesicular bodies; OA1
2.  Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo 
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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1006-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00109-013-1006-4
PMCID: PMC3695676  PMID: 23756766
Rab escort protein 1; Gene therapy; Choroideremia; Rab GTPase; Retinitis pigmentosa; AAV
3.  Correction: Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium 
PLoS ONE  2013;8(5):10.1371/annotation/83a88285-e6a0-41fb-ae67-4315c21e5090.
doi:10.1371/annotation/83a88285-e6a0-41fb-ae67-4315c21e5090
PMCID: PMC3654017
4.  Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium 
PLoS ONE  2013;8(2):e57769.
The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (ChmFlox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the ChmFlox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.
doi:10.1371/journal.pone.0057769
PMCID: PMC3584022  PMID: 23460904
5.  Novel functions for Rab GTPases in multiple aspects of tumour progression 
Biochemical Society Transactions  2012;40(Pt 6):1398-1403.
Rab GTPases are master regulators of intracellular trafficking and, in recent years, their role in the control of different aspects of tumour progression has emerged. In the present review, we show that Rab GTPases are disregulated in many cancers and have central roles in tumour cell migration, invasion, proliferation, communication with stromal cells and the development of drug resistance. As a consequence, Rab proteins may be novel potential candidates for the development of anticancer drugs and, in this context, the preliminary results obtained with an inhibitor of Rab function are also discussed.
doi:10.1042/BST20120199
PMCID: PMC3554041  PMID: 23176488
cancer; exosome; invasion; migration; Rab GTPase; tumour microenvironment; CAF, cancer-associated fibroblast; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; GAP, GTPase-activating protein; GEF, guanine-nucleotide-exchange factor; MDR, multidrug resistance; miRNA, microRNA; MMP, matrix metalloproteinase; MT1-MMP, membrane-type 1 MMP; P-gp, P-glycoprotein; RabGGTase, Rab geranylgeranyltransferase; RCP, Rab-coupling protein; Shh, Sonic Hedgehog
6.  Rab27a-mediated protease release regulates neutrophil recruitment by allowing uropod detachment 
Journal of Cell Science  2012;125(7):1652-1656.
Neutrophil migration is vital for immunity and precedes effector functions such as pathogen killing. Here, we report that this process is regulated by the Rab27a GTPase, a protein known to control granule exocytosis. Rab27a-deficient (Rab27a KO) neutrophils exhibit migration defects in vitro and in vivo, and live-cell microscopy suggests that delayed uropod detachment causes the migratory defect. Surface expression of CD11b, a key adhesion molecule, is increased in chemokine-stimulated Rab27a KO neutrophils compared with the control, suggesting a turnover delay caused by a defect in elastase secretion from azurophilic granules at the rear of bone marrow polymorphonuclear leukocytes (BM-PMNs). We suggest that Rab27a-dependent protease secretion regulates neutrophil migration through proteolysis-dependent de-adhesion of uropods, a mechanism that could be conserved in cell migration and invasion.
doi:10.1242/jcs.100438
PMCID: PMC3346826  PMID: 22375060
Rab27a; Chemotaxis; Cell migration; Neutrophil; Uropod
7.  Loss of Rab27 function results in abnormal lung epithelium structure in mice 
Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.
doi:10.1152/ajpcell.00446.2010
PMCID: PMC3063958  PMID: 21160031
Rab27 proteins; intracellular transport; alveolar epithelium type II cell; Clara cell
8.  Effect of the Secretory Small GTPase Rab27B on Breast Cancer Growth, Invasion, and Metastasis 
Background
Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown.
Methods
Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)–positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided.
Results
Increased expression of Rab27B promoted G1 to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B–injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90α as key proinvasive growth regulator. Heat-shock protein 90α secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors.
Conclusions
Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.
doi:10.1093/jnci/djq153
PMCID: PMC2886092  PMID: 20484105
9.  Impaired prenylation of Rab GTPases in the gunmetal mouse causes defects in bone cell function 
Small GTPases  2011;2(3):131-142.
Vesicular trafficking is crucial for bone resorption by osteoclasts, in particular for formation of the ruffled border membrane and for removal of the resultant bone degradation products by transcytosis. These processes are regulated by Rab family GTPases, whose activity is dependent on post-translational prenylation by Rab geranylgeranyl transferase (RGGT). Specific pharmacological inhibition of RGGT inhibits bone resorption in vitro and in vivo, illustrating the importance of Rab prenylation for osteoclast function. The gunmetal (gm/gm) mouse bears a mutation in the catalytic subunit of RGGT, causing a loss of 75% of the activity of this enzyme and hence hypoprenylation of several Rabs in melanocytes, platelets and cytotoxic T cells. We have now found that prenylation of several Rab proteins is also defective in gm/gm osteoclasts. Moreover, while osteoclast formation and cytoskeletal polarization occurs normally, gm/gm osteoclasts exhibit a substantial reduction in resorptive activity in vitro compared with osteoclasts from +/gm mice, which do not have a prenylation defect. Surprisingly, rather than the osteosclerosis that would be expected to result from defective osteoclast function in vivo, gm/gm mice exhibited a slightly lower bone mass than +/gm mice, indicating that defects in other cell types, such as osteoblasts, in which hypoprenylation of Rabs was also detected, may contribute to the phenotype. However, gm/gm mice were partially protected from ovariectomy-induced bone loss, suggesting that levels of Rab prenylation in gm/gm osteoclasts may be sufficient to maintain normal physiological levels of activity, but not pathological levels of bone resorption in vivo.
doi:10.4161/sgtp.2.3.16488
PMCID: PMC3136943  PMID: 21776414
osteoclast; bone resorption; bone; Rab; small GTPase; prenylation; gunmetal
10.  Rab27a and Rab27b Regulate Neutrophil Azurophilic Granule Exocytosis and NADPH oxidase Activity by Independent Mechanisms 
Traffic (Copenhagen, Denmark)  2009;11(4):533-547.
Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27aash/ash), Rab27b (Rab27b knockout) or both (Rab27a/b double KO), we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27aash/ash neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27aash/ash and Rab27b knockout neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b double KO neutrophils. Rab27-deficient neutrophils, showed impaired activation of the NADPH oxidase at the plasma membrane although intraphagosomal ROS production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.
doi:10.1111/j.1600-0854.2009.01029.x
PMCID: PMC2937183  PMID: 20028487
11.  Retinal Pigment Epithelium Defects Accelerate Photoreceptor Degeneration in Cell Type–Specific Knockout Mouse Models of Choroideremia 
In this study, the authors provide insight into the pathogenesis of choroideremia, which is caused by the disruption of intracellular vesicular transport. They also touch on other issues, such as the photoreceptor-RPE relationship and aging of the RPE.
Purpose.
Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers (photoreceptors, retinal pigment epithelium, and choroid), with a complex and still largely unclear pathogenesis. To investigate the pathophysiology of CHM, the authors engineered mice with a cell type–specific Chm/Rep1 knockout (KO).
Methods.
A mouse line carrying a conditional allele ChmFlox was crossed with the transgenic line IRBP-Cre to achieve Chm KO, specifically in the photoreceptor layer, and Tyr-Cre to produce Chm KO, specifically in the retinal pigment epithelial and other pigmented cells. ChmFlox, Tyr-Cre+ and ChmFlox, IRBP-Cre+ mice were mated to produce mice with Chm KO in both layers. All mouse lines were studied by histology, electron microscopy, electroretinography (ERG), scanning laser ophthalmoscopy (SLO), and biochemical methods.
Results.
In ChmFlox, IRBP-Cre+ mice the authors observed the progressive degeneration of photoreceptors in the presence of normal retinal pigment epithelium (RPE). ChmFlox, Tyr-Cre+ mice exhibited coat color dilution and pigment abnormalities of the RPE in the presence of an intact outer nuclear layer. In 6- to 8-month-old ChmFlox, Tyr-Cre+, IRBP-Cre+ mice, the degeneration of photoreceptors was accelerated compared with ChmFlox, IRBP-Cre+ mice but became leveled with age, such that it was comparable at 12 to 14 months. Detailed ERG and SLO analysis supported the histopathologic findings.
Conclusions.
Defects in photoreceptors and RPE can arise because of intrinsic defects caused cell autonomously by the Chm KO. However, when both photoreceptors and RPE are diseased, the dynamics of the degenerative process are altered. Photoreceptor functional deficit and cell death manifest much earlier, suggesting that the diseased RPE accelerates photoreceptor degeneration.
doi:10.1167/iovs.09-4892
PMCID: PMC3066613  PMID: 20445111
12.  The secretory small GTPase Rab27B as a marker for breast cancer progression 
Oncotarget  2010;1(4):304-308.
In contemporary oncology practice, an urgent need remains to refine the prognostic assessment of breast cancer. It is still difficult to identify patients with early breast cancer who are likely to benefit from adjuvant chemotherapy. Although invasion of cancer cells is the main prognostic denominator in tumor malignancy, our molecular understanding and diagnosis are often inadequate to cope with this activity. Therefore, deciphering molecular pathways of how tumors invade and metastasize may help in the identification of a useful prognostic marker. We recently discovered that the secretory small GTPase Rab27B, a regulator of vesicle exocytosis, delivers proinvasive signals for increased invasiveness, tumor size, and metastasis of various estrogen receptor (ER)-positive breast cancer cell lines, both in vitro and in vivo. In human breast cancer specimens, the presence of Rab27B protein proved to be associated with a low degree of differentiation and the presence of lymph node metastasis in ER-positive breast cancer.
PMCID: PMC3058367  PMID: 21304180
13.  The secretory small GTPase Rab27B as a marker for breast cancer progression 
Oncotarget  2010;1(4):304-308.
In contemporary oncology practice, an urgent need remains to refine the prognostic assessment of breast cancer. It is still difficult to identify patients with early breast cancer who are likely to benefit from adjuvant chemotherapy. Although invasion of cancer cells is the main prognostic denominator in tumor malignancy, our molecular understanding and diagnosis are often inadequate to cope with this activity. Therefore, deciphering molecular pathways of how tumors invade and metastasize may help in the identification of a useful prognostic marker. We recently discovered that the secretory small GTPase Rab27B, a regulator of vesicle exocytosis, delivers proinvasive signals for increased invasiveness, tumor size, and metastasis of various estrogen receptor (ER)-positive breast cancer cell lines, both in vitro and in vivo. In human breast cancer specimens, the presence of Rab27B protein proved to be associated with a low degree of differentiation and the presence of lymph node metastasis in ER-positive breast cancer.
PMCID: PMC3058367  PMID: 21304180
14.  A novel statin-mediated “prenylation block-and-release” assay provides insight into the membrane targeting mechanisms of small GTPases 
Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.
doi:10.1016/j.bbrc.2010.05.045
PMCID: PMC2908739  PMID: 20471365
GTPases; Prenylation; Statin; Trafficking; Rab proteins
15.  Defective cellular trafficking of missense NPR-B mutants is the major mechanism underlying acromesomelic dysplasia-type Maroteaux 
Human Molecular Genetics  2008;18(2):267-277.
Natriuretic peptides (NPs) comprise a family of structurally related but genetically distinct hormones that regulate a variety of physiological processes such as cardiac growth, blood pressure, axonal pathfinding and endochondral ossification leading to the formation of vertebrae and long bones. The biological actions of NPs are mediated by natriuretic peptide receptors (NPRs) A, B and C that are located on the cell surface. Mutations in NPR-B have been shown to cause acromesomelic dysplasia-type Maroteaux (AMDM), a growth disorder in humans and severe dwarfism in mice. We hypothesized that missense mutations of NPR-B associated with AMDM primarily affect NPR-B function by the arrest of receptor trafficking at the endoplasmic reticulum (ER), due to conformational change, rather than an impairment of ligand binding, transmission of signal through the membrane or catalytic activity. Twelve missense mutations found in AMDM patients and cn/cn mice were generated by site-directed mutagenesis and transiently overexpressed in HeLa cells. Confocal microscopy revealed that 11 out of 12 mutants were retained in the ER. Determination of the ligand-dependent cGMP response confirmed that ER-retained NPR-B mutants are non-functional. Meanwhile, the only cell surface-targeted NPR-B missense mutant (D176E) displayed greatly reduced enzymatic activity due to impaired ligand binding. Thus, in the majority of cases of AMDM associated with missense NPR-B mutation, disease appears to result from defects in the targeting of the ER receptor to the plasma membrane.
doi:10.1093/hmg/ddn354
PMCID: PMC2638773  PMID: 18945719
16.  Rab27a and MyRIP regulate the amount and multimeric state of VWF released from endothelial cells 
Blood  2009;113(20):5010-5018.
Endothelial cells contain cigar-shaped secretory organelles called Weibel-Palade bodies (WPBs) that play a crucial role in both hemostasis and the initiation of inflammation. The major cargo protein of WPBs is von Willebrand factor (VWF). In unstimulated cells, this protein is stored in a highly multimerized state coiled into protein tubules, but after secretagogue stimulation and exocytosis it unfurls, under shear force, as long platelet-binding strings. Small GTPases of the Rab family play a key role in organelle function. Using siRNA depletion in primary endothelial cells, we have identified a role for the WPB-associated Rab27a and its effector MyRIP. Both these proteins are present on only mature WPBs, and this rab/effector complex appears to anchor these WPBs to peripheral actin. Depletion of either the Rab or its effector results in a loss of peripheral WPB localization, and this destabilization is coupled with an increase in both basal and stimulated secretion. The VWF released from Rab27a-depleted cells is less multimerized, and the VWF strings seen under flow are shorter. Our results indicate that this Rab/effector complex controls peripheral distribution and prevents release of incompletely processed WPB content.
doi:10.1182/blood-2008-09-181206
PMCID: PMC2686148  PMID: 19270261
17.  Phosphonocarboxylates Inhibit the Second Geranylgeranyl Addition by Rab Geranylgeranyl Transferase* 
The Journal of Biological Chemistry  2009;284(11):6861-6868.
Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.
doi:10.1074/jbc.M806952200
PMCID: PMC2652301  PMID: 19074143
18.  Rab3GEP Is the Non-redundant Guanine Nucleotide Exchange Factor for Rab27a in Melanocytes* 
The Journal of Biological Chemistry  2008;283(34):23209-23216.
Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.
doi:10.1074/jbc.M804134200
PMCID: PMC2516999  PMID: 18559336
19.  Melanosome Maturation Defect in Rab38-deficient Retinal Pigment Epithelium Results in Instability of Immature Melanosomes during Transient Melanogenesis 
Molecular Biology of the Cell  2007;18(10):3914-3927.
Pathways of melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce melanosomes. Here, we show that progression from pmel17-positive premelanosomes to tyrosinase-positive mature melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the “chocolate” (cht) mouse causes dramatically reduced numbers of melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced melanosome numbers, but a continuing low level of melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for melanosome biogenesis. In cht RPE, premelanosomes form but delivery of tyrosinase is impaired. Premelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing melanosome from the activity of lumenal acid hydrolases.
doi:10.1091/mbc.E07-03-0268
PMCID: PMC1995718  PMID: 17671165
20.  Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes 
The Journal of Cell Biology  2006;175(2):271-281.
Amutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype “chocolate” (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38G19V is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles.
doi:10.1083/jcb.200606050
PMCID: PMC2064568  PMID: 17043139
21.  A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes 
Molecular Biology of the Cell  2006;17(11):4720-4735.
Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.
doi:10.1091/mbc.E06-05-0457
PMCID: PMC1635380  PMID: 16914517
22.  Rab27b Regulates Mast Cell Granule Dynamics and Secretion 
Traffic (Copenhagen, Denmark)  2007;8(7):883-892.
The Rab GTPase family regulates membrane domain organization and vesicular transport pathways. Recent studies indicate that one member of the family, Rab27a, regulates transport of lysosome-related organelles in specialized cells, such as melanosomes and lytic granules. Very little is known about the related isoform, Rab27b. Here we used genetically modified mice to study the involvement of the Rab27 proteins in mast cells, which play key roles in allergic responses. Both Rab27a and Rab27b isoforms are expressed in bone marrow-derived mast cells (BMMC) and localize to secretory granules. Nevertheless, secretory defects as measured by β-hexosaminidase release in vitro and passive cutaneous anaphylaxis in vivo were found only in Rab27b and double Rab27 knockout (KO) mice. Immunofluorescence studies suggest that a subset of Rab27b and double Rab27-deficient BMMCs exhibit mild clustering of granules. Quantitative analysis of live-cell time-lapse imaging revealed that BMMCs derived from double Rab27 KO mice showed almost 10-fold increase in granules exhibiting fast movement (>1.5 μm/s), which could be disrupted by nocodazole. These results suggest that Rab27 proteins, particularly Rab27b, play a crucial role in mast cell degranulation and that their action regulates the transition from microtubule to actin-based motility.
doi:10.1111/j.1600-0854.2007.00571.x
PMCID: PMC2063611  PMID: 17587407
mast cell; organelle motility; Rab27; secretion
23.  The Ternary Rab27a–Myrip–Myosin VIIa Complex Regulates Melanosome Motility in the Retinal Pigment Epithelium 
Traffic (Copenhagen, Denmark)  2007;8(5):486-499.
The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a–Myrip–MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.
doi:10.1111/j.1600-0854.2007.00548.x
PMCID: PMC1920545  PMID: 17451552
melanosome motility; Myrip; Rab27a; RPE
24.  Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia 
Journal of Clinical Investigation  2006;116(2):386-394.
Choroideremia (CHM) is an X-linked degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid, caused by loss of function of the CHM/REP1 gene. REP1 is involved in lipid modification (prenylation) of Rab GTPases, key regulators of intracellular vesicular transport and organelle dynamics. To study the pathogenesis of CHM and to develop a model for assessing gene therapy, we have created a conditional mouse knockout of the Chm gene. Heterozygous-null females exhibit characteristic hallmarks of CHM: progressive degeneration of the photoreceptors, patchy depigmentation of the RPE, and Rab prenylation defects. Using tamoxifen-inducible and tissue-specific Cre expression in combination with floxed Chm alleles, we show that CHM pathogenesis involves independently triggered degeneration of photoreceptors and the RPE, associated with different subsets of defective Rabs.
doi:10.1172/JCI26617
PMCID: PMC1326146  PMID: 16410831
25.  The Role of Rab27a in the Regulation of Melanosome Distribution within Retinal Pigment Epithelial Cells 
Molecular Biology of the Cell  2004;15(5):2264-2275.
Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.
doi:10.1091/mbc.E03-10-0772
PMCID: PMC404021  PMID: 14978221

Results 1-25 (37)