The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing

The novel Drosophila mutant l?chrig (loe) shows progressive neurodegeneration and neuronal cell death, in addition to a low level of cholesterol ester. loe affects a specific isoform of the gamma-subunit of AMP-activated protein kinase (AMPK), a negative regulator of hydroxymethylglutaryl (HMG)-CoA reductase and cholesterol synthesis in vertebrates. Although Drosophila cannot synthesize cholesterol de novo, the regulatory role of fly AMPK on HMG-CoA reductase is conserved. The loe phenotype is modified by the level of HMG-CoA reductase and suppressed by the inhibition of this enzyme by statin, which has been used for the treatment of Alzheimer patients. In addition, the degenerative phenotype of loe is enhanced by a mutation in amyloid precursor protein-like (APPL), the fly homolog of the human amyloid precursor protein involved in Alzheimer's disease. Western analysis revealed that the loe mutation reduces APPL processing, whereas overexpression of Loe increases it. These results describe a novel function of AMPK in neurodegeneration and APPL/APP processing which could be mediated through HMG-CoA reductase and cholesterol ester.     

Prenylation of Rho G-Proteins: a Novel Mechanism Regulating Gene Expression and Protein Stability in Human Trabecular Meshwork Cells

Endogenous prenylation with sesquiterpene or diterpene isoprenoids facilitates membrane localization and functional activation of small monomeric GTP-binding proteins. A direct effect of isoprenoids on regulation of gene expression and protein stability has also been proposed. In this study, we determined the role of sesquiterpene or diterpene isoprenoids on the regulation of Rho G-protein expression, activation, and stability in human trabecular meshwork (TM) cells. In both primary and transformed human TM cells, limiting endogenous isoprenoid synthesis with lovastatin, a potent HMG-CoA reductase inhibitor, elicited marked increases in RhoA and RhoB mRNA and protein content. The effect of lovastatin was dose-dependent with newly synthesized inactive protein accumulating in the cytosol. Supplementation with geranylgeranyl pyrophosphate (GGPP) prevented, while inhibition of geranylgeranyl transferase-I mimicked, the effects of lovastatin on RhoA and RhoB protein content. Similarly, lovastatin-dependent increases in RhoA and RhoB mRNA expression were mimicked by geranylgeranyl transferase-I inhibition. Interestingly, GGPP supplementation selectively promoted the degradation of newly synthesized Rho proteins which was mediated, in part, through the 20S proteasome. Functionally, GGPP supplementation prevented lovastatin-dependent decreases in actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from the cytosol to the membrane and increasing RhoA activation. Post-translational prenylation with geranylgeranyl diterpenes selectively facilitates the expression, membrane translocation, functional activation, and turnover of newly synthesized Rho proteins. Geranylgeranyl prenylation represents a novel mechanism by which active Rho proteins are targeted to the 20S proteasome for degradation in human TM cells.     

Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization

Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.     

Regulation of Microglial Phagocytosis by RhoA/ROCK-Inhibiting Drugs

Inflammation within the central nervous system (CNS) is a major component of many neurodegenerative diseases. The underlying mechanisms of neuronal loss are not fully understood, but the activation of CNS resident phagocytic microglia seems to be a significant element contributing to neurodegeneration. At the onset of inflammation, high levels of microglial phagocytosis may serve as an essential prerequisite for creating a favorable environment for neuronal regeneration. However, the excessive and long-lasting activation of microglia and the augmented engulfment of neurons have been suggested to eventually govern widespread neurodegeneration. Here, we investigated in a functional assay of acute inflammation how the small GTPase RhoA and its main target the Rho kinase (ROCK) influence microglial phagocytosis of neuronal debris. Using BV-2 microglia and human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA activation and microglial phagocytosis of neuronal cell fragments. Inhibition of the downstream effector ROCK with the small-molecule agents Y-27632 and Fasudil reduces the engulfment of neuronal debris and attenuates the production of the inflammatory mediator nitric oxide during stimulation with lipopolysaccharide. Our results support a therapeutic potential for RhoA/ROCK-inhibiting agents as an effective treatment of excessive inflammation and the resulting progression of microglia-mediated neurodegeneration in the CNS.     

Unprenylated RhoA Contributes to IL-1β Hypersecretion in Mevalonate Kinase Deficiency Model through Stimulation of Rac1 Activity

Protein prenylation is a post-translational modification whereby non-sterol isoprenoid lipid chains are added, thereby modifying the molecular partners with which proteins interact. The autoinflammatory disease mevalonate kinase deficiency (MKD) is characterized by a severe reduction in protein prenylation. A major class of proteins that are affected are small GTPases, including Rac1 and RhoA. It is not clear how protein prenylation of small GTPases relates to GTP hydrolysis activity and downstream signaling. Here, we investigated the contribution of RhoA prenylation to the biochemical pathways that underlie MKD-associated IL-1β hypersecretion using human cell cultures, Rac1 and RhoA protein variants, and pharmacological inhibitors. We found that when unprenylated, the GTP-bound levels of RhoA decrease, causing a reduction in GTPase activity and increased protein kinase B (PKB) phosphorylation. Cells expressing unprenylated RhoA produce increased levels of interleukin 1β mRNA. Of other phenotypic cellular changes seen in MKD, increased mitochondrial potential and mitochondrial elongation, only mitochondrial elongation was observed. Finally, we show that pharmacological inactivation of RhoA boosts Rac1 activity, a small GTPase whose activity was earlier implied in MKD pathogenesis. Together, our data show that RhoA plays a pivotal role in MKD pathogenesis through Rac1/PKB signaling toward interleukin 1β production and elucidate the effects of protein prenylation in monocytes.     

RhoGDI facilitates geranylgeranyltransferase-I-mediated RhoA prenylation

Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This modification is essential for the activity of small cellular GTPases, as it allows them to associate with intracellular membranes. Dissociated from membranes, prenylated proteins need to be transported through the aqueous cytoplasm by protein carriers that shield the hydrophobic anchor from the solvent. One such carrier is Rho GDP dissociation inhibitor (RhoGDI). Recently, it was shown that prenylated Rho proteins that are not associated with RhoGDI are subjected to proteolysis in the cell. We hypothesized that the role of RhoGDI might be not only to associate with prenylated proteins but also to regulate the prenylation process in the cell. This idea is supported by the fact that RhoGDI binds both unprenylated and prenylated Rho proteins with high affinity in vitro, and hence, these interactions may affect the kinetics of prenylation. We addressed this question experimentally and found that RhoGDI increased the catalytic efficiency of geranylgeranyl transferase-I in RhoA prenylation. Nevertheless, we did not observe formation of a ternary RhoGDI∗RhoA∗GGTase-I complex, indicating sequential operation of geranylgeranyltransferase-I and RhoGDI. Our results suggest that RhoGDI accelerates Rho prenylation by kinetically trapping the reaction product, thereby increasing the rate of product release.     

Isoprenoids and protein prenylation: implications in the pathogenesis and therapeutic intervention of Alzheimer’s disease

The mevalonate–isoprenoid–cholesterol biosynthesis pathway plays a key role in human health and disease. The importance of this pathway is underscored by the discovery that two major isoprenoids, farnesyl and geranylgeranyl pyrophosphate, are required to modify an array of proteins through a process known as protein prenylation, catalyzed by prenyltransferases. The lipophilic prenyl group facilitates the anchoring of proteins in cell membranes, mediating protein–protein interactions and signal transduction. Numerous essential intracellular proteins undergo prenylation, including most members of the small GTPase superfamily as well as heterotrimeric G proteins and nuclear lamins, and are involved in regulating a plethora of cellular processes and functions. Dysregulation of isoprenoids and protein prenylation is implicated in various disorders, including cardiovascular and cerebrovascular diseases, cancers, bone diseases, infectious diseases, progeria, and neurodegenerative diseases including Alzheimer’s disease (AD). Therefore, isoprenoids and/or prenyltransferases have emerged as attractive targets for developing therapeutic agents. Here, we provide a general overview of isoprenoid synthesis, the process of protein prenylation and the complexity of prenylated proteins, and pharmacological agents that regulate isoprenoids and protein prenylation. Recent findings that connect isoprenoids/protein prenylation with AD are summarized and potential applications of new prenylomic technologies for uncovering the role of prenylated proteins in the pathogenesis of AD are discussed.     

Inhibition of farnesyl pyrophosphate (FPP) and/or geranylgeranyl pyrophosphate (GGPP) biosynthesis and its implication in the treatment of cancers

Dysregulation of isoprenoid biosynthesis is implicated in numerous biochemical disorders that play a role in the onset and/or progression of age-related diseases, such as hypercholesterolemia, osteoporosis, various cancers, and neurodegeneration. The mevalonate metabolic pathway is responsible for the biosynthesis of the two key isoprenoid metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Post-translational prenylation of various proteins, including the small GTP-binding proteins (GTPases), with either FPP or GGPP is vital for proper localization and activation of these proteins. Prenylated GTPases play a critical role in cell signaling, proliferation, cellular plasticity, oncogenesis, and cancer metastasis. Pre-clinical and clinical studies strongly suggest that inhibition of protein prenylation can be an effective treatment for non-skeletal cancers. In this review, we summarize the most recent drug discovery efforts focusing on blocking protein farnesylation and/or geranylgeranylation and the biochemical and structural data available in guiding the current on-going studies in drug discovery. Furthermore, we provide a summary on the biochemical association between disruption of protein prenylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR) signaling, and cancer.     

Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells

AMP-activated protein kinase (AMPK) is a key energy sensor, known to regulate energy metabolism in diverse cell types. Hypoxia is encountered frequently in the microenvironments of inflammatory lesions and is a critical regulator of function in inflammatory cells. Energy deficiency is one of the consequences of hypoxia, but its potential role in modulating leucocyte function has received little attention. Using micropore chemotaxis assays to assess migratory responses of the monocyte-like cell line U937, it was found that the AMPK activators AICAR and phenformin rapidly reduced random migration (chemokinesis) as well as chemotaxis due to stromal cell-derived factor (SDF)1alpha. There was an approximate 50% reduction in both chemokinesis and chemotaxis following 30 min preincubation with both AICAR and phenformin (P < 0.01), and this continued with up to 24 h preincubation. The binding of SDF1alpha to its receptor CXCR4 was unaltered, suggesting AMPK was acting on downstream intracellular signalling pathways important in cell migration. As AMPK and statins are known to inhibit HMG CoA reductase, and both reduce cell migration, the effect of mevastatin on U937 cells was compared with AMPK activators. Mevastatin inhibited cell migration but required 24 h preincubation. As expected, the inhibitory effect of mevastatin was associated with altered subcellular localization of the Rho GTPases, RhoA and cdc42, indicating decreased prenylation of these molecules. Although the effect of AMPK activation was partially reversed by mevalonate, this was not associated with altered subcellular localization of Rho GTPases. The data suggest that activation of AMPK has a general effect on cell movement in U937 cells, and this is not due to inhibition of HMG CoA reductase. These are the first data to show an effect of AMPK on cell movement, and suggest a fundamental role for energy deficiency in regulating cellular behaviour.     

XPLN,a guanine nucleotide exchange factor for RhoA and RhoB,but not RhoC

Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF. Like other Rho GEFs, XPLN contains a tandem Dbl homology and pleckstrin homology domain topography, but lacks homology with other known functional domains or motifs. XPLN protein is expressed in the brain, skeletal muscle, heart, kidney, platelets, and macrophage and neuronal cell lines. In vitro, XPLN stimulates guanine nucleotide exchange on RhoA and RhoB, but not RhoC, RhoG, Rac1, or Cdc42. Consistent with these data, XPLN preferentially associates with RhoA and RhoB. The specificity of XPLN for RhoA and RhoB, but not RhoC, is surprising given that they share over 85% sequence identity. We determined that the inability of XPLN to exchange RhoC is mediated by isoleucine 43 in RhoC, a position occupied by valine in RhoA and RhoB. When expressed in cells, XPLN activates RhoA and RhoB, but not RhoC, and stimulates the assembly of stress fibers and focal adhesions in a Rho kinase-dependent manner. We also found that XPLN possesses transforming activity, as determined by focus formation assays. In conclusion, here we describe a Rho family GEF that can discriminate between the closely related RhoA, RhoB, and RhoC, possibly giving insight to the divergent functions of these three proteins.     

Control of RhoA Methylation by Carboxylesterase I

A number of proteins that play key roles in cell signaling are post-translationally modified by the prenylation pathway. The final step in this pathway is methylation of the carboxyl terminus of the prenylated protein by isoprenylcysteine carboxylmethyltransferase. Due to the impact of methylation on Rho function, we sought to determine if the process was reversible and hence could control Rho function in a dynamic fashion. Elevating isoprenylcysteine carboxylmethyltransferase activity in cells has profound effects on MDA-MB-231 cell morphology, implying the presence of a pool of unmethylated prenyl proteins in these cells under normal conditions. Using a knockdown approach, we identified a specific esterase, carboxylesterase 1, whose function had a clear impact not only on the methylation status of RhoA but also RhoA activation and cell morphology. These data provide compelling evidence that C-terminal modification of prenyl proteins, rather than being purely a constitutive process, can serve as a point of regulation of function for this important class of protein.     

Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system

Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.     

A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity

Inhibiting protein prenylation is an attractive means to modulate cellular processes controlled by a variety of signaling proteins, including oncogenic proteins such as Ras and Rho GTPases. The largest class of prenylated proteins contain a so-called CaaX motif at their carboxyl termini and are subject to a maturation process initiated by the attachment of an isoprenoid lipid by either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I). Inhibitors of FTase, termed FTIs, have been the subject of intensive development in the past decade and have shown efficacy in clinical trials. Although GGTase-I inhibitors (GGTIs) have received less attention, accumulating evidence suggests GGTIs may augment therapies using FTIs and could be useful to treat a myriad of additional disease states. Here we describe the characterization of a selective, highly potent, and cell-active GGTase-I inhibitor, GGTI-DU40. Kinetic analysis revealed that inhibition by GGTI-DU40 is competitive with the protein substrate and uncompetitive with the isoprenoid substrate; the Ki for the inhibition is 0.8 nM. GGTI-DU40 is highly selective for GGTase-I both in vitro and in living cells. Studies indicate GGTI-DU40 blocks prenylation of a number of geranylgeranylated CaaX proteins. Treatment of MDA-MB-231 breast cancer cells with GGTI-DU40 inhibited thrombin-induced cell rounding via a process that involves inhibition of Rho proteins without significantly effecting parallel mobilization of calcium via Gbetagamma. These studies establish GGTI-DU40 as a prime tool for interrogating biologies associated with protein geranylgeranylation and define a novel structure for this emerging class of experimental therapeutics.     

3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1).

The mechanism by which platelet-derived growth factor (PDGF) regulates vascular smooth muscle cell (SMC) DNA synthesis is unknown, but may involve isoprenoid intermediates of the cholesterol biosynthetic pathway. Inhibition of isoprenoid synthesis with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, simvastatin (Sim, 1-10 microM), inhibited PDGF-induced SMC DNA synthesis by >95%, retinoblastoma gene product hyperphosphorylation by 90%, and cyclin-dependent kinases (cdk)-2, -4, and -6 activity by 80 +/- 5, 50 +/- 3, and 48 +/- 3%, respectively. This correlated with a 20-fold increase in p27(Kip1) without changes in p16, p21(Waf1), or p53 levels compared with PDGF alone. Since Ras and Rho require isoprenoid modification for membrane localization and are implicated in cell cycle regulation, we investigated the effects of Sim on Ras and Rho. Up-regulation of p27(Kip1) and inhibition of Rho but not Ras membrane translocation by Sim were reversed by geranylgeranylpyrophosphate, but not farnesylpyrophosphate. Indeed, inhibition of Rho by Clostridium botulinum C3 transferase or overexpression of dominant-negative N19RhoA mutant increased p27(Kip1) and inhibited retinoblastoma hyperphosphorylation. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 decreased p27(Kip1) and increased SMC DNA synthesis. These findings indicate that the down-regulation of p27(Kip1) by Rho GTPase mediates PDGF-induced SMC DNA synthesis and suggest a novel direct effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors on the vascular wall.     

Regulation of isoprenoid/cholesterol biosynthesis in cells from mevalonate kinase-deficient patients

Mevalonic aciduria (MA) and hyper-IgD and periodic fever syndrome (HIDS) are two inherited disorders both caused by depressed mevalonate kinase (MK) activity. MK is the first enzyme to follow the highly regulated 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR), which catalyzes the rate-limiting step in the isoprenoid/cholesterol biosynthesis pathway. In fibroblasts of MA patients, but not of HIDS patients, HMGR activity is elevated under normal growth conditions. This activity is down-regulated when cells are supplemented with the isoprenoid precursors geraniol, farnesol, and geranylgeraniol, and a mixture of 25-hydroxycholesterol and cholesterol. This indicates that the regulation of the pathway in these cells is not disturbed. The elevated HMGR activity is probably due to a shortage of non-sterol isoprenoid end products, as indicated by normal HMGR mRNA levels in MA fibroblasts. Furthermore, the HMGR activity in MA cells was more sensitive to geranylgeraniol suppression and less sensitive to sterol suppression than the HMGR activity in low density lipoprotein receptor-deficient cells. HMGR activity in MA cells was down-regulated also by addition of its product mevalonate to the culture medium. Thus, it appears that the elevation of mevalonate levels, which are high in MA patients and moderate in HIDS patients, allows the cells to compensate for the depressed MK activity. Indeed, the isoprenylation of Ras and RhoA protein appeared normal in HIDS and MA fibroblasts under normal conditions but showed increased sensitivity toward inhibition of HMGR by simvastatin. Our results indicate that MK-deficient cells maintain the flux through the isoprenoid/cholesterol biosynthesis pathway by elevating intracellular mevalonate levels.     

Prenylated Rab acceptor protein is a receptor for prenylated small GTPases

Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.     

Tech: a RhoA GEF selectively expressed in hippocampal and cortical neurons

Recent studies implicating the Rho family of small G proteins in the regulation of neuronal morphology have focused attention on identifying key components of Rho signaling pathways in neurons. To this end, we have conducted studies aimed at defining the localization and function of Tech, a Rho guanine nucleotide exchange factor (GEF) family member that is highly enriched in brain. We have found that Tech is selectively expressed in cortical and hippocampal neurons with prominent Tech immunostaining apparent in the cell bodies and dendrites of these cells. In vitro studies with prototypical members of the major Rho subfamilies, RhoA, Rac1 and Cdc42, indicate that Tech binds selectively to and activates RhoA. To assess whether Tech may be involved in the regulation of neuronal morphology, we examined the effects of Tech constructs on the morphology of cortical neurons grown in primary culture. We found that a constitutively active Tech construct, Tech 245DeltaC, decreases the number of dendritic processes present on these neurons. This reduction appears to be mediated by activation of RhoA as it is blocked by insertion of a point mutation into the DH domain of Tech which blocks its ability to activate RhoA or coexpression of a dominant negative RhoA construct. As Tech protein levels increase during post-natal development and remain at peak levels into adulthood, these results indicate that Tech regulates RhoA signaling pathways in developing and mature forebrain neurons.