Rab Geranylgeranyl Transferase Research Articles

Assessment of Rab geranylgeranyltransferase subunit beta in amyotrophic lateral sclerosis.

Geranylgeranyltransferase Subunit Beta (RABGGTB) was expressed at higher levels in patients with Amyotrophic lateral sclerosis (ALS) compared with healthy controls. This study aims to observe the expression of RABGGTB in different cells from patients with ALS and different diseases. In this case-control study, we collected peripheral blood from patients with ALS and healthy controls, and compared the expression of RABGGTB in natural killer cells (NK), T cells and B cells between patients with ALS and healthy controls by flow cytometry. And compared the expression of RABGGTB in monocytes and monocyte-derived macrophages from patients with ALS, Parkinson's disease (PD), acute cerebrovascular disease (ACVD), and healthy controls by flow cytometry and immunofluorescence. Then flow cytometry was used to detect the expression of RABGGTB in monocytes from SOD1G93A mice and WT mice. The expression of RABGGTB was not significantly changed in NK cells, cytotoxic T cells (CTL), helper T cells (Th), regulatory T cells (Treg), and B cells from patients with ALS compared to healthy controls. And the expression of RABGGTB in monocytes and monocyte-derived macrophages was higher in the ALS group than in the PD, ACVD and control group. The expression of RABGGTB was significantly higher in monocytes of SOD1G93A mice compared to WT mice. These findings suggest that RABGGTB expression was increased in monocytes and monocyte-derived macrophages from patients with ALS, not in NK, CTL, Th, Treg, and B cells. Future studies are needed to find the clinical implication of RABGGTB in ALS.

Rab Geranylgeranyltransferase Subunit Beta as a Potential Indicator to Assess the Progression of Amyotrophic Lateral Sclerosis.

Currently, there is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder. Many biomarkers have been proposed, but because ALS is a clinically heterogeneous disease with an unclear etiology, biomarker discovery for ALS has been challenging due to the lack of specificity of these biomarkers. In recent years, the role of autophagy in the development and treatment of ALS has become a research hotspot. In our previous studies, we found that the expression of RabGGTase (low RABGGTB expression and no change in RABGGTA) is lower in the lumbar and thoracic regions of spinal cord motoneurons in SOD1G93A mice compared with WT (wild-type) mice groups, and upregulation of RABGGTB promoted prenylation modification of Rab7, which promoted autophagy to protect neurons by degrading SOD1. Given that RabGGTase is associated with autophagy and autophagy is associated with inflammation, and based on the above findings, since peripheral blood mononuclear cells are readily available from patients with ALS, we proposed to investigate the expression of RabGGTase in peripheral inflammatory cells. Information and venous blood were collected from 86 patients diagnosed with ALS between January 2021 and August 2023. Flow cytometry was used to detect the expression of RABGGTB in monocytes from peripheral blood samples collected from patients with ALS and healthy controls. Extracted peripheral blood mononuclear cells (PBMCs) were differentiated in vitro into macrophages, and then the expression of RABGGTB was detected by immunofluorescence. RABGGTB levels in patients with ALS were analyzed to determine their impact on disease progression. Using flow cytometry in monocytes and immunofluorescence in macrophages, we found that RABGGTB expression in the ALS group was significantly higher than in the control group. Age, sex, original location, disease course, C-reactive protein (CRP), and interleukin-6 (IL-6) did not correlate with the ALS functional rating scale-revised (ALSFRS-R), whereas the RABGGTB level was significantly correlated with the ALSFRS-R. In addition, multivariate analysis revealed a significant correlation between RABGGTB and ALSFRS-R score. Further analysis revealed a significant correlation between RABGGTB expression levels and disease progression levels (ΔFS). The RABGGTB level was significantly increased in patients with ALS compared with healthy controls. An elevated RABGGTB level in patients with ALS is associated with the rate of progression in ALS, suggesting that elevated RABGGTB levels in patients with ALS may serve as an indicator for tracking ALS progression.

Prenyl Transferases Regulate Secretory Protein Sorting and Parasite Morphology in Toxoplasma gondii.

Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism Toxoplasma gondii (T. gondii) using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in T. gondii and potentially in other related parasites.

The atypical Rab GTPase associated with Parkinson’s disease, Rab29, is localized to membranes

Several genetic studies have shown that the small GTPase Rab29 is involved in the pathogenesis of Parkinson’s Disease (PD). It has also been shown that overexpression of Rab29 increases the activity of leucine-rich repeat kinase 2, a protein kinase often mutated in familial PD, although the mechanism underlying this activation remains unclear. Here, we employed biochemical analyses to characterize the localization of Rab29 and found that, unlike general Rab proteins, Rab29 is predominantly fractionated into the membrane fraction by ultracentrifugation. We also found that Rab29 is resistant to extraction from membranes by GDP-dissociation inhibitors (GDIs) in vitro. Furthermore, Rab29 failed to interact with GDIs, and its membrane localization was not affected by the knockout of GDIs in cells. We show that the knockout of Rab geranylgeranyltransferase decreased the hydrophobicity of Rab29, suggesting that Rab29 is geranylgeranylated at its carboxyl terminus as is with typical Rab proteins. Notably, we demonstrated that membrane-bound Rab29 retains some hydrophilicity, indicating that mechanisms other than geranylgeranylation might also be involved in the membrane localization of Rab29. Taken together, these findings uncover the atypical nature of Rab29 among Rab proteins, which will provide important clues for understanding how Rab29 is involved in the molecular pathomechanism of PD.

Protein Prenyltransferases and Their Inhibitors: Structural and Functional Characterization.

Protein prenylation is a post-translational modification controlling the localization, activity, and protein–protein interactions of small GTPases, including the Ras superfamily. This covalent attachment of either a farnesyl (15 carbon) or a geranylgeranyl (20 carbon) isoprenoid group is catalyzed by four prenyltransferases, namely farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase-I), Rab geranylgeranyltransferase (GGTase-II), and recently discovered geranylgeranyltransferase type III (GGTase-III). Blocking small GTPase activity, namely inhibiting prenyltransferases, has been proposed as a potential disease treatment method. Inhibitors of prenyltransferase have resulted in substantial therapeutic benefits in various diseases, such as cancer, neurological disorders, and viral and parasitic infections. In this review, we overview the structure of FTase, GGTase-I, GGTase-II, and GGTase-III and summarize the current status of research on their inhibitors.

Rational design, optimization, and biological evaluation of novel α-Phosphonopropionic acids as covalent inhibitors of Rab geranylgeranyl transferase

Rab geranylgeranyltransferase (GGTase-II, RGGT) catalyses the post-translational modification of eukaryotic Rab GTPases, proteins implicated in several pathologies, including cancer, diabetes, neurodegenerative, and infectious diseases. Thus, RGGT inhibitors are believed to be a potential platform for the development of drugs and tools for studying processes related to the abnormal activity of Rab GTPases. Here, a series of new α-phosphonocarboxylates have been prepared in the first attempt of rational design of covalent inhibitors of RGGT derived from non-covalent inhibitors. These compounds were equipped with electrophilic groups capable of binding cysteines, which are present in the catalytic cavity of RGGT. A few of these analogues have shown micromolar activity against RGGT, which correlated with their ability to inhibit the proliferation of the HeLa cancer cell line. The proposed mechanism of this inhibitory activity was rationalised by molecular docking and mass spectrometric measurements, supported by stability and reactivity studies.

Impact of C-terminal truncations in the Arabidopsis Rab escort protein (REP) on REP-Rab interaction and plant fertility.

SUMMARYLipid anchors are common post‐translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C‐termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP–Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen–deuterium exchange mass spectrometry (HDX‐MS) to describe the structural basis of plant REP–Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule‐specific, and in other cases they are common for a subset of Rabs. In particular, the C‐terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C‐terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast.

The Rab Geranylgeranyl Transferase Beta Subunit Is Essential for Embryo and Seed Development in Arabidopsis thaliana.

Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.

Synthesis of the 6-Substituted Imidazo[1,2-a]Pyridine-3-yl-2- Phosphonopropionic Acids as Potential Inhibitors of Rab Geranylgeranyl Transferase.

Twelve phosphonopropionates derived from 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC) were synthesized and evaluated for their activity as inhibitors of protein geranylgeranylation. The nature of the substituent in the C6 position of imidazo[1,2-a]pyridine ring was responsible for the compound's activity against Rab geranylgeranyl transferase (RGGT). The most active inhibitors disrupted Rab11A prenylation in the human cervical carcinoma HeLa cell line. The esterification of carboxylic acid in the phosphonopropionate moiety turned the inhibitor into an inactive analog.

Double prenylation of SNARE protein Ykt6 is required for lysosomal hydrolase trafficking.

Ykt6 is an evolutionarily conserved SNARE protein regulating Golgi membrane fusion and other diverse membrane trafficking pathways. Unlike most SNARE proteins, Ykt6 lacks a transmembrane domain but instead has a tandem cysteine motif at the C-terminus. Recently, we have demonstrated that Ykt6 undergoes double prenylation at the C-terminal two cysteines first by farnesyltransferase and then by a newly identified protein prenyltransferase named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of a novel α subunit prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the β subunit of Rab geranylgeranyltransferase. PTAR1 knockout (KO) cells, where Ykt6 is singly prenylated with a farnesyl moiety, exhibit structural and functional abnormalities in the Golgi apparatus with delayed intra-Golgi trafficking and impaired protein glycosylation. It remains unclear whether the second prenylation of Ykt6 is required for proper trafficking of lysosomal hydrolases from Golgi to lysosomes. Here, we show that lysosomal hydrolases, cathepsin D and β-hexosaminidase, were missorted at the trans-Golgi network and secreted into the extracellular space in PTAR1 KO cells. Moreover, maturation of these hydrolases was disturbed. LC3B, an autophagy marker, was accumulated in PTAR1 KO cells, suggesting defects in cellular degradation pathways. Thus, doubly prenylated Ykt6, but not singly prenylated Ykt6, is critical for the efficient sorting and trafficking of acid hydrolases to lysosomes.

Rab-dependent vesicular traffic affects female gametophyte development in Arabidopsis.

Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the β-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule.

32.1 MECHANISMS OF ABNORMAL POSTTRANSLATIONAL PROTEIN PROCESSING IN SCHIZOPHRENIA BRAIN

32.1 MECHANISMS OF ABNORMAL POSTTRANSLATIONAL PROTEIN PROCESSING IN SCHIZOPHRENIA BRAIN

Synthesis and Biological Evaluation of Imidazole-Bearing α-Phosphonocarboxylates as Inhibitors of Rab Geranylgeranyl Transferase (RGGT).

Rab geranylgeranyl transferase (RGGT) is an interesting therapeutic target, as it ensures proper functioning of Rab GTPases, a class of enzymes responsible for the regulation of vesicle trafficking. Relying on our previous studies, we synthesized a set of new α-phosphonocarboxylic acids as potential RGGT inhibitors, with emphasis on the elaboration of imidazole-containing analogues. We identified two compounds with activity similar to that of previously reported RGGT inhibitors, showing structural similarity to imidazo[1,2-a]pyridine-containing analogues in terms of their substitution pattern. Interestingly, analogues of the N-series, derived from another phosphonocarboxylate RGGT inhibitor, 2-fluoro-3-(1H-imidazol-1-yl)-2-phosphonopropanoic acid, turned out to be inactive in our model, indicating that an additional substituent localized at positions C2 or C4 of the imidazole ring, may adversely affect the potency against the targeted enzyme.

Identification of the Privileged Position in the Imidazo[1,2-a]pyridine Ring of Phosphonocarboxylates for Development of Rab Geranylgeranyl Transferase (RGGT) Inhibitors.

Members of the Rab GTPase family are master regulators of vesicle trafficking. When disregulated, they are associated with a number of pathological states. The inhibition of RGGT, an enzyme responsible for post-translational geranylgeranylation of Rab GTPases represents one way to control the activity of these proteins. Because the number of molecules modulating RGGT is limited, we combined molecular modeling with biological assays to ascertain how modifications of phosphonocarboxylates, the first reported RGGT inhibitors, rationally improve understanding of their structure-activity relationship. We have identified the privileged position in the core scaffold of the imidazo[1,2-a]pyridine ring, which can be modified without compromising compounds' potency. Thus modified compounds are micromolar inhibitors of Rab11A prenylation, simultaneously being inactive against Rap1A/Rap1B modification, with the ability to inhibit proliferation of the HeLa cancer cell line. These findings were rationalized by molecular docking, which recognized interaction of phosphonic and carboxylic groups as decisive in phosphonocarboxylate localization in the RGGT binding site.

REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival.

Rab escort protein 1 (REP1), a component of the Rab geranyl-geranyltransferase 2 complex, plays a role in Rab protein recruitment in proper vesicles during vesicle trafficking. In addition to having well-known tissue degenerative phenotypes in the REP1 mutant, REP1 is tightly associated with cancer development and contributes to cell growth and survival. However, the functional mechanism of REP1 in cancer progression is largely uninvestigated. Here, we show that REP1 plays a crucial role in regulating mammalian target of rapamycin (mTOR) signaling and its downstream pathways, as well as autophagy and macropinocytosis, which are essential for cancer cell survival during metabolic stresses including starvation. REP1 small interfering RNA (siRNA) treatment downregulates mTORC1 activity in growing media, but blocks autophagosome formation under nutrient-depleted conditions. In contrast to the mild decrease of lysosomal enzyme activity seen in REP1 depletion, in REP1 knockdown the subcellular localization of lysosomes is altered, and localization of REP1 itself is modulated by intracellular nutrient levels and mTOR activity. Furthermore, REP1 depletion increases macro pinocytosis which may be a feedback mechanism to compensate autophagy inhibition. Concomitant treatment with macropinocytosis inhibitor and REP1siRNAresults in more significant cell death than autophagy blockade with REP1 knockdown. Therefore, REP1-mediated autophagy and lysosomal degradation processes act as novel regulatory mechanisms to support cancer cell survival, which can be further investigated as a potential cancer-targeting pathway.

REP1 inhibits FOXO3-mediated apoptosis to promote cancer cell survival

Rab escort protein 1 (REP1) is a component of Rab geranyl-geranyl transferase 2 complex. Mutations in REP1 cause a disease called choroideremia (CHM), which is an X-linked eye disease. Although it is postulated that REP1 has functions in cell survival or death of various tissues in addition to the eye, how REP1 functions in normal and cancer cells remains to be elucidated. Here, we demonstrated that REP1 is required for the survival of intestinal cells in addition to eyes or a variety of cells in zebrafish, and also has important roles in tumorigenesis. Notably, REP1 is highly expressed in colon cancer tissues and cell lines, and silencing of REP1 sensitizes colon cancer cells to serum starvation- and 5-FU-induced apoptosis. In an effort to elucidate the molecular mechanisms underlying REP1-mediated cell survival under those stress conditions, we identified FOXO3 as a binding partner of REP1 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that REP1 blocked the nuclear trans-localization of FOXO3 through physically interacting with FOXO3, thereby suppressing FOXO3-mediated apoptosis. Importantly, the inhibition of REP1 combined with 5-FU treatment could lead to significant retarded tumor growth in a xenograft tumor model of human cancer cells. Thus, our results suggest that REP1 could be a new therapeutic target in combination treatment for colon cancer patients.

Effects of Isoprenoid Biosynthetic Pathway Inhibition on Glucose Uptake in Myeloma Cells

Multiple myeloma (MM) is a plasma cell malignancy characterized by the synthesis and secretion of high levels of monoclonal protein (MP). MM cells are dependent on glucose consumption and GLUT transporter activity and there has been interest in targeting glucose uptake as a therapeutic strategy. Our work has shown that inhibitors of the isoprenoid biosynthetic pathway (IBP) disrupt Rab GTPase geranylgeranylation thus impairing MP trafficking in MM cells, leading to an accumulation of MP in the ER and induction of the unfolded protein response and apoptosis. However, there are over 60 different Rab GTPases in mammalian cells involved in regulating a wide range of membrane trafficking events including exocytic, endocytic and lysosomal pathways, whose disruption may also be contributing to MM cell death. Global gene expression analysis of MM cells treated with the Rab geranylgeranyl transferase (GGTase) inhibitor, 3-PEHPC, revealed novel pathways impacted by Rab GTPase inhibition, including genes involved in glucose metabolism. As previous work with statins have suggested that statin-induced inhibition of glucose uptake may be a consequence of either blocking protein geranylgeranylation or cholesterol depletion, we were interested in determining whether disruption of isoprenoid synthesis and/or Rab geranylgeranylation has an impact on glucose uptake in MM cells. Notably, MM cells are unique in their dependence on additional members of the GLUT family including GLUT4 whose cell surface expression is regulated by Rabs. We therefore examined the effects of IBP inhibitors and direct prenyl transferase inhibitors on glucose uptake in MM cells (RPMI-8226, U266 and MM.1S) using flow cytometric analysis of the uptake of 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose), a fluorescent analog of D-glucose. The IBP inhibitors lovastatin (an inhibitor of HMG-CoA reductase) and digeranyl bisphosphonate (DGBP) (an inhibitor of geranylgeranyl diphosphate synthase) reduced glucose uptake in a concentration- and time-dependent manner in all cell lines (except U266 which we have previously shown to be resistant to DGBP) by 20-50% compared to untreated control cells. To determine whether the effects of these drugs on glucose uptake are a consequence of depletion of a specific isoprenoid species, experiments were performed in which cells were incubated in the presence or absence of the IBP inhibitors and intermediates in the IBP, namely mevalonate, FPP or GGPP. Co-incubation of lovastatin with mevalonate or GGPP completely prevented the effects of lovastatin on glucose uptake in MM.1S and U266 cells while GGPP prevented the block in glucose uptake induced by DGBP in MM.1S cells. However, co-incubation of lovastatin with FPP partially prevented the effects of lovastatin in U266 cells but had no effect in MM.1S cells. Treatment of U266 cells, but not RPMI-8226 or MM.1S cells, with the squalene synthase inhibitor zaragozic acid resulted in a significant decrease in glucose uptake. MM.1S and U266 cell lines were treated with individual prenyltransferase inhibitors (FTI-277, GGTI-2133 or 3-PEHPC) to distinguish between the effects of altering cellular isoprenoid levels versus directly protein prenylation. Whereas none of the prenyltransferase inhibitors had significant effects on the U266 cells, only inhibition of Rab GGTase by 3-PEHPC resulted in a significant decrease in glucose uptake in MM.1S cells. In aggregate these studies demonstrate that Rab inhibition via disruption of geranylgeranylation diminishes glucose uptake in MM cells. In addition, a cholesterol-dependent effect related to IBP inhibition was observed in the U266 cells but not in the other tested MM cells, suggesting that MM cells may vary in their dependence on glucose uptake by distinct GLUT transporters. Future studies will investigate these findings in primary MM cells and identify the specific GLUT family members which are affected by direct Rab GGTase inhibitors as well as by IBP inhibitors. Improved understanding of the mechanisms underlying the effects of these inhibitors will inform further development of therapies targeting glucose uptake in MM. DisclosuresHolstein:Amgen: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

A Comprehensive Analysis of Choroideremia: From Genetic Characterization to Clinical Practice.

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients´ fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.

P53- and mevalonate pathway-driven malignancies require Arf6 for metastasis and drug resistance.

Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial-mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy.

Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling.

Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol and cardiac risk, but many other effects are seen. We now show an effect of these drugs to induce profound changes in the step-wise synthesis of glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased several-fold in all cell lines tested, demonstrating a widespread effect. Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer; GlcNAcβ1-3Galβ1-4GlcCer) synthesis was observed in 70%. Western blot showed that GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase (Lc3S) activities were increased; however, transcript was elevated for Lc3S only. Supplementation with the isoprenoid precursor, geranylgeranyl pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl transferase, or geranylgeranyl transferase I, was sufficient to replicate statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab prenylation-dependent mechanism. While total cholesterol was unaffected, the trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS partially relocated by statins. GSL-dependent vesicular retrograde transport of Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling. These in vitro studies indicate a previously unreported link between Rab prenylation and regulation of GCS activity and GlcCer metabolism.