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Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification
Abstract
Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt DA - 20080908IS - 0021-9258 (Print)LA - engPT - Journal ArticlePT - Research Support, N.I.H., ExtramuralRN - 52-90-4 (Cysteine)RN - EC 2.5.1.29 (Farnesyltranstransferase)RN - EC 3.4.- (Endopeptidases)RN - EC 3.4.22 (RCE1 protein, human)RN - EC 3.4.22 (Rce1 protein, mouse)RN - EC 3.6.5.2 (rho GTP-Binding Proteins)SB - IM
... Thus, to study the subcellular protein localization, transfections of cells with plasmid containing the coding sequence of RND1 were performed. In these models of RND1 overexpression, active RND1 is localized at the plasma membrane [3,14,18] due to its Nterminal extension [5] and its farnesylation [19]. Indeed, deletion of the first seven amino acids of RND1 or treatment with an inhibitor of farnesyl transferase results in a decrease of RND1 at the plasma membrane and its accumulation in the cytoplasm and the nucleus [5,19]. ...
... In these models of RND1 overexpression, active RND1 is localized at the plasma membrane [3,14,18] due to its Nterminal extension [5] and its farnesylation [19]. Indeed, deletion of the first seven amino acids of RND1 or treatment with an inhibitor of farnesyl transferase results in a decrease of RND1 at the plasma membrane and its accumulation in the cytoplasm and the nucleus [5,19]. RND1 may be also sequestrated in the cytosol, in a phosphorylated form, when it is bound to 14-3-3 protein [20], which can be considered as an inhibitor of RND1 activity. ...
... Thus, to study the subcellular protein localization, transfections of cells with plasmid containing the coding sequence of RND1 were performed. In these models of RND1 overexpression, active RND1 is localized at the plasma membrane [3,14,18] due to its N-terminal extension [5] and its farnesylation [19]. Indeed, deletion of the first seven amino acids of RND1 or treatment with an inhibitor of farnesyl transferase results in a decrease of RND1 at the plasma membrane and its accumulation in the cytoplasm and the nucleus [5,19]. ...
The Rho GTPase family can be classified into classic and atypical members. Classic members cycle between an inactive Guanosine DiPhosphate -bound state and an active Guanosine TriPhosphate-bound state. Atypical Rho GTPases, such as RND1, are predominantly in an active GTP-bound conformation. The role of classic members in oncogenesis has been the subject of numerous studies, while that of atypical members has been less explored. Besides the roles of RND1 in healthy tissues, recent data suggest that RND1 is involved in oncogenesis and response to cancer therapeutics. Here, we present the current knowledge on RND1 expression, subcellular localization, and functions in healthy tissues. Then, we review data showing that RND1 expression is dysregulated in tumors, the molecular mechanisms involved in this deregulation, and the role of RND1 in oncogenesis. For several aggressive tumors, RND1 presents the features of a tumor suppressor gene. In these tumors, low expression of RND1 is associated with a bad prognosis for the patients. Finally, we highlight that RND1 expression is induced by anticancer agents and modulates their response. Of note, RND1 mRNA levels in tumors could be used as a predictive marker of both patient prognosis and response to anticancer agents.
... The Ras GTPases are usually prenylated at the C-terminal so-called CAAX box, which is formed by a stretch of amino-acid residues at the C-terminus of the small GTPases (consensus sequence: Cysteine, followed by two aliphatic amino-acid residues, and a less defined ultimate amino-acid residue). For Ras, a 15-carbon farnesyl moiety is covalently attached to the cysteine, and subsequently the AAX peptide is removed and the cysteine becomes methylated [25]. This modification is required to confer targeting of Ras to lipid bilayers, such as the plasma membrane or endomembranes. ...
... The GTPase defective Rho GTPases comprise Rnd1-3, RhoH, and RhoBTB1-2, which have alternative amino-acid residues in positions equivalent to 12, 59, and 61 (Ras numbering), and where mutations in the equivalent positions in Ras are known to result in constitutively GTP-bound small GTPases [23]. In addition, Rnd1-3 and RhoH are farnesylated rather than geranyl-geranylated, whereas RhoBTB1-2 do not have CAAX motifs, and it is not known if these proteins contain any membrane-targeting motifs [25,27]. The GTPase activity of theses atypical Rho GTPases cannot be stimulated by GAP-domain-containing proteins, although they appear to have intact GDP/GTP exchange activities [27,28]. ...
... The exchange of GDP for GTP is ensured in most cell types by the roughly 10-fold higher levels of GTP over GDP [42]. RhoU and RhoV were shown to be palmitoylated and not prenylated [43,44], while RhoD does not appear to be farnesylated or geranyl-geranylated, which is in contrast to RhoF, which can undergo both types of prenylation [25]. The fast-cycling Rho GTPases do not appear to bind RhoGDI, and to date, no GEFs or GAPs have been identified for this group of proteins. ...
Involvement of Rho GTPases in cancer has been a matter of debate since the identification of the first members of this branch of the Ras superfamily of small GTPases. The Rho GTPases were ascribed important roles in the cell, although these were restricted to regulation of cytoskeletal dynamics, cell morphogenesis, and cell locomotion, with initially no clear indications of direct involvement in cancer progression. This paradigm has been challenged by numerous observations that Rho-regulated pathways are often dysregulated in cancers. More recently, identification of point mutants in the Rho GTPases Rac1, RhoA, and Cdc42 in human tumors has finally given rise to a new paradigm, and we can now state with confidence that Rho GTPases serve as oncogenes in several human cancers. This article provides an exposé of current knowledge of the roles of activated Rho GTPases in cancers.
... Rac1 is pleiotropic and interacts with a multitude of effector proteins that control many cellular processes. Though not an exhaustive list, the following proteins are some of Rac1's most prevalent effectors (Bustelo et al., 2007, Soriano-Castell et al., 2017: While all Rac proteins localize to the PM, Rac2 is predominantly found in the Golgi, endoplasmic reticulum, and nuclear envelope, and Rac3 primarily localizes to endomembranes (Ridley, 2006, Roberts et al., 2008. Like Ras, Rac1 undergoes several post-translational modifications on the membrane anchor of the HVR to target to the PM ( Figs. 2-3). ...
... Like Ras, Rac1 undergoes several post-translational modifications on the membrane anchor of the HVR to target to the PM ( Figs. 2-3). First, geranylgeranyltransferase type 1 covalently adds a geranylgeranyl isoprenoid chain to the cysteine residue of the CAAX motif, which is subsequently cleaved by Rce1 (Roberts et al., 2008). The now prenylated C-terminal cysteine residue is methylated by Icmt (Roberts et al., 2008). ...
... First, geranylgeranyltransferase type 1 covalently adds a geranylgeranyl isoprenoid chain to the cysteine residue of the CAAX motif, which is subsequently cleaved by Rce1 (Roberts et al., 2008). The now prenylated C-terminal cysteine residue is methylated by Icmt (Roberts et al., 2008). Like K-Ras4A and K-Ras4B, Rac1 contains a PBD of six contiguous basic residues (Fig. 3). ...
Rac1 is a small, guanine-nucleotide binding protein that cycles between an inactive GDP-bound and active GTP-bound state to regulate actin-mediated motility, migration, and adhesion. Plasma membrane (PM) localization is essential for its biological activity. Rac1 PM targeting is directed by a C-terminal membrane anchor that encompasses a geranylgeranyl-cysteine-methyl-ester, palmitoyl, and a polybasic domain (PBD) of contiguous lysine and arginine residues. Using high-resolution imaging combined with spatial mapping analysis, I found that Rac1 forms nanoclusters on the PM. Cycling between the GTP- and GDP-bound states, Rac1 forms nanoclusters that are non-overlapping, consequently undergoing guanine nucleotide-dependent spatial segregation. I further found that Rac1 selectively associates with phosphatidic acid (PA) and phosphoinositol (3,4,5)-trisphosphate (PIP3), resulting in nanoclusters enriched in these anionic lipids. I found these lipids to be structurally important as depleting the PM of PA or PIP3 impaired both Rac1 PM targeting and nanoclustering. Lipid binding specificity of Rac1 is encoded in the C-terminal PBD amino acid sequence in combination with the adjacent lipid anchors. Point mutations within the PBD, including highly conserved arginine to lysine substitutions or mutations exchanging the geranylgeranyl for farnesyl, profoundly altered Rac1 lipid binding specificity without changing electrostatics of the protein and resulted in impaired macropinocytosis and decreased cell spreading. In this thesis, I proposed that Rac1 nanoclusters act as lipid-based signaling platforms emulating the spatiotemporal organization of Ras proteins and further showed that the Rac1 PBD-prenyl anchor has a biological function that extends beyond simple electrostatic engagement with the PM.
... In contrast, the palm-Cdc42 mRNA did not support axon growth when targeted into axons to the same extent as the axonally targeted prenyl-Cdc42 mRNA. Isoprenoid lipid attachments to cysteine residues in the Cterminal CaaX motif of prenyl-CDC42 promotes its subcellular localization to the plasma membrane and other cellular membrane comparts including ER, Golgi, and endosomes (Roberts et al, 2008). Palm-CDC42 and prenyl-CDC42 proteins have been shown to have different mobilities in cell membranes so they may be targeted to different membrane domains. ...
... On first glance, our data seem to contradict a recent study that linked prenylation to slowed axon growth on myelin-associated glycoprotein (MAG), a substrate that inhibits axon growth (Li et al, 2016). In cultured neurons, highly-selective FT inhibitors were more effective than GGT inhibitors for increasing axon growth, but the combination of the two showed synergistic effects (Roberts et al, 2008). RHOA shows altered subcellular localization upon inhibition of GGT (Roberts et al, 2008), and in contrast to the growth promoting effects of prenyl-CDC42 shown here, RHOA attenuates axon growth on non-permissive substrates like MAG and the protein is synthesized in axons (Walker et al, 2012b;Wu et al, 2005). ...
... In cultured neurons, highly-selective FT inhibitors were more effective than GGT inhibitors for increasing axon growth, but the combination of the two showed synergistic effects (Roberts et al, 2008). RHOA shows altered subcellular localization upon inhibition of GGT (Roberts et al, 2008), and in contrast to the growth promoting effects of prenyl-CDC42 shown here, RHOA attenuates axon growth on non-permissive substrates like MAG and the protein is synthesized in axons (Walker et al, 2012b;Wu et al, 2005). Previous studies have shown compensatory geranylgeranylation through GGT for some farnesylated GTPases when FT is inhibited (Roberts et al, 2008), which could account for the increased efficacy of FT plus GGT inhibition seen by Li et al. (2016). ...
The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic CDC42 gene is alternatively spliced to generate mRNAs with two different 3’UTRs that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CAAX and CCAX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases in vivo during PNS axon regeneration. Isoform specific siRNA knockdowns, rescue experiments with siRNA-resistant Cdc42 isoforms, and pharmacologically targeting Cdc42 activity indicate that prenyl-Cdc42 promotes axon growth while the palmitoyl-Cdc42 has little growth promoting activity. The growth promotion by prenyl-Cdc42 requires axonal mRNA localization with localized translation and an intact C-terminal CaaX motif for localized prenylation of the encoded protein. Together, these data show that alternative splicing of the CDC42 gene product generates an axon growth promoting locally synthesized prenyl-Cdc42 protein.
SUMMARY STATEMENT
Axon regeneration drives selective localization of alternatively spliced CDC42 isoform to PNS axons.
... To inactivate Rac1, GTPase activating proteins (GAPs) enhance intrinsic Rac1 GTPase activity [28]. In addition, through posttranslation modification, Rac1 can associate with the plasma membrane by undergoing prenylation, producing two separate pools of Rac1, cytosolic and membrane-bound [29]. Cytosolic Rac1 is sequestered by Rho GDP-dissociation inhibitors (Rho-GDIs) and is mostly inactive [30]. ...
... Like other small Rho GTPases, Rac1 undergoes isoprenylation due to its C-terminal CAAX motif, a posttranslational modification that anchors Rac1 to the plasma membrane [29]. A pool of inactive Rac1 exists in the cytosol where it is sequestered by RhoGDI by associating with the isoprenyl group on Rac1 [30]. ...
... Two other Rho GTPases that are structurally and functionally related to Rac1 (RhoA and CDC42) have been extensively studied in synaptogenesis and spinogenesis [43,44]. In the brain, Rac1, RhoA, and CDC42 are isoprenylated by the same geranylgeranyl transferase [29]. It has also been shown that impaired distribution of one Rho GTPase affects distribution of the others [45]. ...
Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.
... Cycling of RhoA between its inactive GDP-bound and active GTP-bound state is coordinated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) [8, 9]. In addition, guanine nucleotide dissociation inhibitors (GDIs) sequester RhoA in the cytosol and maintain it in its inactive GDP-bound state by interacting with the lipophilic moiety added post-translationally to the carboxy-terminal end of RhoA in a process known as prenylation [10][11][12]. Upon activation, RhoA is released from the GDIs and is able to translocate to the plasma membrane where it interacts with its numerous downstream effectors [12]. Additionally, RhoA is negatively regulated by phosphorylation of residue S188 by both cAMP-and cGMP-dependent kinases [13][14][15][16]as well as by tyrosine glycosylation of residue Y34 [17]. ...
... In addition, guanine nucleotide dissociation inhibitors (GDIs) sequester RhoA in the cytosol and maintain it in its inactive GDP-bound state by interacting with the lipophilic moiety added post-translationally to the carboxy-terminal end of RhoA in a process known as prenylation [10][11][12]. Upon activation, RhoA is released from the GDIs and is able to translocate to the plasma membrane where it interacts with its numerous downstream effectors [12]. Additionally, RhoA is negatively regulated by phosphorylation of residue S188 by both cAMP-and cGMP-dependent kinases [13][14][15][16]as well as by tyrosine glycosylation of residue Y34 [17]. ...
... Constitutively active RhoA (Q63L and G14V) generated higher levels of RhoA-NTF compared to WT-RhoA, whereas these levels were lower with the dominant-negative (T19N) mutant, suggesting that active RhoA is preferentially proteolysed (Fig 1D). In addition to the GDP/GTP cycling, RhoA activity is regulated by prenylation, a post-translational modification essential to its translocation to the plasma membrane [11, 12]. Non-prenylated WT, active and dominant-negative RhoA (C190A-, Q63L/C190A-and T19N/ C190A-RhoA, respectively) were weakly proteolysed compared to WT-RhoA (Fig 1D). ...
The small GTPase RhoA regulates the actin cytoskeleton to affect multiple cellular processes including endocytosis, migration and adhesion. RhoA activity is tightly regulated through several mechanisms including GDP/GTP cycling, phosphorylation, glycosylation and prenylation. Previous reports have also reported that cleavage of the carboxy-terminus inactivates RhoA. Here, we describe a novel mechanism of RhoA proteolysis that generates a stable amino-terminal RhoA fragment (RhoA-NTF). RhoA-NTF is detectable in healthy cells and tissues and is upregulated following cell stress. Overexpression of either RhoA-NTF or the carboxy-terminal RhoA cleavage fragment (RhoA-CTF) induces the formation of disorganized actin stress fibres. RhoA-CTF also promotes the formation of disorganized actin stress fibres and nuclear actin rods. Both fragments disrupt the organization of actin stress fibres formed by endogenous RhoA. Together, our findings describe a novel RhoA regulatory mechanism.
... Once lipid modified, the hypervariable region mediates recruitment of the GTPase to the appropriate membrane [26]. Addition of lipophilic groups to the C-terminus of Rho family members is essential for appropriate subcellular localization and regulation [27]. ...
... Rho family members undergo addition of lipid moieties via prenylation and palmitoylation; adding C-15 farnesyl or C-20 geranyl-geranyl isoprenoid moieties, or a palmitate acyl chain, respectively. Prenylation of the cysteine located within the C-terminal CAAX tetrapeptide motif [27] is necessary for translocation between membranes and the cytosol [35]. The additional hydrophobicity contributed by the prenyl group acts as a membrane anchor [44], but is inserted into a hydrophobic groove found on Rho GDIs when in the cytosolic state [45]. ...
... The additional hydrophobicity contributed by the prenyl group acts as a membrane anchor [44], but is inserted into a hydrophobic groove found on Rho GDIs when in the cytosolic state [45]. For most Rho family members prenylation is essential for proper function [27], directing their translocation to the appropriate membrane environment [24]; members lacking a prenyl group are misallocated to the cytosol where they are inactive [44]. While not as common, some Rho family members necessitate the covalent addition of a palmitate acyl chain to their C-terminal hypervariable domain found immediately adjacent to the CAAX box [46]. ...
Rho GTPases regulate cellular morphology and dynamics, and some are key drivers of cancer progression. This superfamily offers attractive potential targets for therapeutic intervention, with RhoA, Rac1 and Cdc42 being prime examples. The challenges in developing agents that act on these signaling enzymes include the lack of obvious druggable pockets and their membrane-bound activities. However, progress in targeting the similar Ras protein is illuminating new strategies for specifically inhibiting oncogenic GTPases. The structures of multiple signaling and regulatory states of Rho proteins have been determined, and the post-translational modifications including acylation and phosphorylation points have been mapped and their functional effects examined. The development of inhibitors to probe the significance of overexpression and mutational hyperactivation of these GTPases underscores their importance in cancer progression. The ability to integrate in silico, in vitro, and in vivo investigations of drug-like molecules indicates the growing tractability of GTPase systems for lead optimization. Although no Rho-targeted drug molecules have yet been clinically approved, this family is clearly showing increasing promise for the development of precision medicine and combination cancer therapies.
... One of the most important but unanswered questions on CDC42 splicing variants is how they obtain distinct abilities, with the slight differences in their HVRs. The HVRs of small GTPases contain polybasic sequences and CAAX motifs, which are essential for determining their membrane localizations (4,(32)(33)(34). ...
... Polybasic sequences interact with phosphoinositides, while CAAX motifs are subject to lipid modifications (4,(32)(33)(34). Since CDC42 splice variants are different in both their polybasic sequences and CAAX motifs, they are assumed to exhibit different subcellular localizations, leading to different biological outcomes. ...
The small GTPase CDC42 plays essential roles in neurogenesis and brain development. Previously, we showed that a CDC42 splice variant that has a ubiquitous tissue distribution specifically stimulates the formation of neural progenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neural progenitor cells to neurons. These specific roles of CDC42 and CDC42b in neurogenesis are ascribed to their opposing effects on mTORC1 activity. Specifically, the ubiquitous form of CDC42 stimulates mTORC1 activity and thereby up-regulates tissue-specific transcription factors that are essential for neuroprogenitor formation, whereas CDC42b works together with activated CDC42-associated kinase (ACK) to down-regulate mTOR expression. Here we demonstrate that the EGF receptor (EGFR) is an additional and important target of CDC42b and ACK which is down-regulated by their combined actions in promoting neurogenesis. The activation status of the EGFR determines the timing by which neural progenitor cells derived from P19 embryonal carcinoma terminally differentiate into neurons. By promoting EGFR degradation, we found that CDC42b and ACK stimulate autophagy, which protects emerging neurons from apoptosis and helps trigger neural progenitor cells to differentiate into neurons. Moreover, our results reveal that CDC42b is localized in phosphatidylinositol (3,4,5)-triphosphate (PIP3)-enriched microdomains on the plasma membrane, mediated through its polybasic sequence ¹⁸⁵KRK¹⁸⁷, which is essential for determining its distinct functions. Overall, these findings now highlight a molecular mechanism by which CDC42b and ACK regulate neuronal differentiation and provide new insights into the functional interplay between EGFR degradation and autophagy which occurs during embryonic neurogenesis.
... Like other SmGs, RhoA has consensus sequences for two functional domains: (1) interacting/binding with GDP/ GTP and hydrolysis of GTP to GDP + P and (2) interaction with downstream effectors [16,161] (Review: [162]) (Fig. 1). Furthermore, RhoA in mammals share a common region for posttranslational modifications (PTMs) at their carboxy-terminus (COOH) [23,49,52,104,135,161,181] (reviewed in: [162]) (Fig. 1). ...
... As depicted in Fig. 2, PTMs, which allow the anchoring of RhoA to the cell membrane, are necessary for its activation. The region for PTMs of RhoA in mammals is a "CaaX-box-motif " at the C-terminal, a common motif for prenylation (Figs. 1 and 2) [135]. The prenylation of RhoA is essential for its function, by allowing its anchoring to the cell membrane and the binding to regulatory molecules [23,52,66,104,137,181] (Review: [162]). ...
The Ras homolog gene family member A (RhoA) is the founding member of Rho GTPase superfamily originally studied in cancer cells where it was found to stimulate cell cycle progression and migration. RhoA acts as a master switch control of actin dynamics essential for maintaining cytoarchitecture of a cell. In the last two decades, however, RhoA has been coined and increasingly investigated as an essential molecule involved in signal transduction and regulation of gene transcription thereby affecting physiological functions such as cell division, survival, proliferation and migration. RhoA has been shown to play an important role in cardiac remodeling and cardiomyopathies; underlying mechanisms are however still poorly understood since the results derived from in vitro and in vivo experiments are still inconclusive. Interestingly its role in the development of cardiomyopathies or heart failure remains largely unclear due to anomalies in the current data available that indicate both cardioprotective and deleterious effects. In this review, we aimed to outline the molecular mechanisms of RhoA activation, to give an overview of its regulators , and the probable mechanisms of signal transduction leading to RhoA activation and induction of downstream effector pathways and corresponding cellular responses in cardiac (patho)physiology. Furthermore, we discuss the existing studies assessing the presented results and shedding light on the often-ambiguous data. Overall, we provide an update of the molecular, physiological and pathological functions of RhoA in the heart and its potential in cardiac therapeutics.
... However, in view of their distinct expression pattern in the adult DG (Fig. S1), we can already speculate that Rnd1 and Rnd3 might mediate different functions than Rnd2 in this process. Moreover, Rnd1 and Rnd3 have been shown, in different cell types including neurons, to be predominantly expressed at the plasma membrane whereas Rnd2 is mainly located in endosomes (Pacary et al., 2011;Roberts et al., 2008). Such differences in subcellular localization further supports our hypothesis that Rnd2 might dictate different functions than Rnd1 and Rnd3 in adult-born DGNs. ...
... As mentioned previously, Rnd2 has been shown to be expressed in endosomes (Pacary et al., 2011;Roberts et al., 2008;Tanaka et al., 2002) and to interact with molecules involved in the formation and trafficking of endocytic vesicles (Kamioka et al., 2004;Tanaka et al., 2002). ...
Despite the central role of Rho GTPases in neuronal development, their functions in adult hippocampal neurogenesis remain poorly explored. Here, by using a retrovirus-based loss-of-function approach in vivo , we show that the atypical Rho GTPase Rnd2 is crucial for the survival, positioning, somatodendritic morphogenesis and functional maturation of adult-born dentate granule neurons. Interestingly, most of these functions are specific to granule neurons generated during adulthood since the deletion of Rnd2 in neonatally-born granule neurons only affects dendritogenesis. In addition, suppression of Rnd2 in adult-born dentate granule neurons increases anxiety-like behaviour whereas its deletion in pups has no such effect, a finding supporting the adult neurogenesis hypothesis of anxiety disorders. Thus, our results provide mechanistic insight into the differential regulation of hippocampal neurogenesis during development and adulthood, and establishes a causal relationship between Rnd2 expression and anxiety.
... Les principaux effecteurs des GTPases Rho et leurs fonctions biologiques associées sont résumés dans le tableau 1. Dubash et al., 2011;Etienne-Manneville and Hall, 2002;Heasman and Ridley, 2008;Jaffe and Hall, 2005;Porter et al., 2016;Ridley, 2013Ridley, , 2015Roberts et al., 2008;Sandrock et al., 2010;Vega and Ridley, 2008 Kaina, 1997, 2001;Fritz et al., 1995;Glorian et al., 2011;Wang et al., 2014), le cisplatine, l'hydroxyurée, les agents alkylants MNU (N-méthyl-N-nitrosourée) et MMS (méthanesulfonate de méthyle) (Fritz and Kaina, 1997;Fritz et al., 1995), H2O2 (Fritz and Kaina, 1997;Srougi and Burridge, 2011), les radiations ionisantes (IR) (Ader et al., 2003;Ader et al., 2002;Srougi and Burridge, 2011) et la camptothécine (CPT) . ...
... Nous ne savons pas si RND1 est présent dans le noyau des cellules à l'état de base ou en réponse à un stress. Néanmoins, la délétion de sa partie N-terminale ou l'inhibition de la farnésylation entraine la diminution de RND1 à la membrane plasmique et l'accumulation de la protéine au niveau cytoplasmique et nucléaire (Oinuma et al., 2012;Roberts et al., 2008). De plus, il a été mise en évidence que RND3, membre le plus proche de RND1 en terme de séquence et de fonction, était présent dans le noyau de cellules de glioblastomes U87 . ...
La famille des GTPases Rho, comprenant 20 membres, contrôle la dynamique du cytosquelette d'actine et différents processus cellulaires comme la migration. En plus de leurs rôles bien établis, certaines GTPases Rho, notamment RhoB et Rac1, ont émergé en tant que gènes de réponse précoce aux dommages à l'ADN. En effet, RhoB est induite en réponse à divers stress génotoxiques, y compris la camptothécine (CPT), les UV et le cisplatine, et protège principalement les cellules de l'apoptose. Le rôle des autres GTPases Rho en réponse précoce aux génotoxiques reste largement méconnu. Dans ce projet, nous avons utilisé la camptothécine, un inhibiteur de la topoisomérase I (TOP1), qui stabilise sélectivement les complexes de clivage TOP1-ADN (TOP1cc) sur la chromatine, afin de cribler les GTPases Rho induites de façon précoce par les dommages à l'ADN. En plus de RhoB, nous avons identifié RND1 comme un gène rapidement induit par la CPT. L'induction de RND1 est réversible et étroitement corrélée à la présence de TOP1cc induit par la CPT. En accord avec ces observations, les rayons UV et le péroxyde d'hydrogène, qui stabilisent indirectement les TOP1cc, induisent également RND1. La CPT augmente la transcription de RND1 indépendamment de l'activité de son promoteur minimal. De plus, la CPT augmente l'activité de la poly ADP-ribose polymérase (PARP1), dont l'inhibition prévient la transcription de RND1. La surexpression de RND1 augmente également l'expression de PARP1, suggérant une régulation positive entre PARP1 et RND1 en réponse aux TOP1cc. Ainsi, nous proposons qu'en réponse à la CPT, les TOP1cc activent PARP1, qui à son tour favorise la transcription de RND1, initiant ainsi une boucle de rétrocontrôle positive. Enfin, nous avons montré que RND1 protège les cellules contre l'apoptose induite par la CPT et entraîne leur résistance à la CPT. L'ensemble de ces résultats ont permis d'identifier RND1 comme nouvelle GTPase Rho impliquée dans la réponse au stress et proposent un nouveau mécanisme de régulation de la transcription des gènes en réponse aux TOP1cc via l'activation de PARP1. Ces résultats suggèrent par ailleurs qu'inhiber la signalisation de RND1 pourrait sensibiliser les cellules tumorales aux dérivés cliniques de la CPT.
... We considered that GS could have a similar activity profile (Supplementary Discussion 3) and explored whether it was involved in the palmitoylation of RHOJ. Even though the cysteines at positions 3 (C3) and 11 (C11) of RHOJ were predicted by in silico methods to be high-fidelity palmitoylation sites (screened with SwissPalm 22 , data not shown), the palmitoylation of RHOJ has been poorly documented, with the exception of a few studies 23,24 . Notably, the membrane- localization and activity of RHOJ were reduced by treatment of ECs with the pan-palmitoylation inhibitor 2-bromopalmitate and by introducing point mutations in C3 and C11 (Fig. 5f, Extended Data Fig. 7k-t), providing initial evidence that RHOJ can be palmitoylated in ECs. ...
... Constructs with point mutations were generated with Stratagene's QuickChange site-directed-mutagenesis kit following the manufacturer's guidelines. The cDNA for RHOJ-eGFP (GFP-TCL) was a gift from C. Der (Addgene plasmid 23231) 23 and was used as a template to generate the N-terminal-truncated ΔN20-RHOJ-eGFP, which lacked the first 20 amino acids, and Flag-tagged RHOJ. Standard cloning techniques were used to fuse GS to the photoswitchable fluorescent protein mEOS (pRSETa-mEos2 was a gift from L. Looger; Addgene plasmid 20341) 28 . ...
Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
... CDC42-V2 differs from the canonical CDC42-V1 by only the last 10 amino acids at the C-terminus (Marks & Kwiatkowski, 1996;Nishimura & Linder, 2013;Wirth et al., 2013). While CDC42-V1 possesses a CaaX motif that can undergo a prenylation modification, CDC42-V2 contains a CCaX motif that can be both prenylated and palmitoylated (Nishimura & Linder, 2013;Roberts et al., 2008). As lipid modifications alter the subcellular locations of GTPases, we determined, through the use of three lipidation-defective CDC42-V2 mutants (C188A, C189A, and C188/189A), whether lipid modifications of CDC42 affect its interaction with CDC42EP1. ...
Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.
... It has previously been shown that GGTase-I inhibitors block YAP1/TAZ activity via Rho-GTPase signaling27,30,31 . Rho-GTPase function depends on a specific post-translational modification, the transfer of a geranylgeranyl moiety to a carboxy-terminal cysteine residue, leading to Rho-GTPase component recruitment and lipidanchoring to the plasma membrane35 . This dependency was demonstrated by BAY-856 treatment, resulting in the plasma membrane delocalization of Rho-GTPase components while the expression levels of them in cytoplasm remained unaffected as seen by immunoblotting. ...
This study describes the identification and target deconvolution of novel small molecule inhibitors of oncogenic YAP1/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex, as the direct target of YAP1/TAZ pathway inhibitors. The novel small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo .
SIGNIFICANCE
YAP1/TAZ have been shown to be aberrantly activated oncogenes in several human solid tumors, resulting in enhanced cell proliferation, metastasis and provision of a pro-tumorigenic microenvironment, making YAP1/TAZ targets for novel cancer therapies. Yet, the development of effective inhibitors of these potent oncogenes has been challenging. In this work, we break new ground in this direction through the identification of novel inhibitors of YAP1/TAZ activity.
Graphical abstract
HIGHLIGHTS
Novel YAP1/TAZ pathway inhibitors identified by phenotypic high-throughput screen
Target deconvolution identifies GGTase-I as the direct target of the novel YAP1/TAZ pathway inhibitors
GGTase-I inhibitors block Rho-GTPase signaling and downstream YAP1/TAZ
GGTase-I inhibitor BAY-593 demonstrates potent anti-tumor activity in vivo
... To create the driver ORF library, individual drivers were PCR amplified out of the Cancer Pathways kit (Addgene #1000000072) (Martz et al., 2014), individual plasmids (Addgene #9053, #85140, #82262, #82297, #82175, #61852, #39872, #23776, #23688, #10745, #23231) (Hagting et al., 1998;Hayer et al., 2016;Johannessen et al., 2010;Kim et al., 2016;Ramaswamy et al., 1999;Roberts et al., 2008), a human cDNA pool (Promega Corporation), or obtained as synthesized double-stranded DNA fragments (gBlocks, IDT Inc) with flanking sequences compatible with the BamHI restriction sites. The barcoded lentiviral backbone was digested with BamHI HF (New England Biolabs) at 37 C for 3 hours in a reaction consisting of: lentiviral backbone, 4 mg, CutSmart buffer, 5 ml, BamHI, 0.625 ml, H 2 0 up to 50 ml. ...
Deconstructing tissue-specific effects of genes and variants on proliferation is critical to understanding cellular transformation and systematically selecting cancer therapeutics. This requires scalable methods for multiplexed genetic screens tracking fitness across time, across lineages, and in a suitable niche, since physiological cues influence functional differences. Towards this, we present an approach, coupling single-cell cancer driver screens in teratomas with hit enrichment by serial teratoma reinjection, to simultaneously screen drivers across multiple lineages in vivo. Using this system, we analyzed population shifts and lineage-specific enrichment for 51 cancer associated genes and variants, profiling over 100,000 cells spanning over 20 lineages, across two rounds of serial reinjection. We confirmed that c-MYC alone or combined with myristoylated AKT1 potently drives proliferation in progenitor neural lineages, demonstrating signatures of malignancy. Additionally, mutant MEK1S218D/S222D provides a proliferative advantage in mesenchymal lineages like fibroblasts. Our method provides a powerful platform for multi-lineage longitudinal study of oncogenesis.
... Functions of small GTPases are regulated by their translocation to cellular membranes where they modulate signaling by effector molecules that ultimately impact a host of cellular processes including cell growth, proliferation, and migration 13,102 . All Ras superfamily small GTPases (which also includes Rho and Rab family GTPases) undergo prenylation on their C-terminus, the irreversible modification of cysteines with an unsaturated isoprenyl fatty acid, which is critical for their processing, trafficking, and ultimately membrane association [103][104][105] . ...
S-palmitoylation is a reversible lipid modification that regulates trafficking, localization, activity, and/or stability of protein substrates by serving as a fatty acid anchor to cell membranes. However, S-palmitoylation-dependent control of signal transduction in cardiomyocytes and its effects on cardiac physiology are not well understood. We performed an in vivo gain-of-function screen of zinc finger Asp-His-His-Cys (zDHHC) family S-acyl transferases that catalyze S-palmitoylation and identified the Golgi-localized enzyme zDHHC3 as a critical regulator of cardiac maladaptation. The closely-related enzyme, zDHHC7, also induced severe cardiomyopathy but this effect was not observed with overexpression of plasma membrane enzyme zDHHC5, endoplasmic reticulum enzyme zDHHC6, or Golgi enzyme zDHHC13. To identify effectors that may underlie zDHHC3-induced cardiomyopathy we performed quantitative site-specific S-acyl proteomics in zDHHC3-overexpressing cells that revealed the small GTPase Rac1 as a novel substrate. We generated cardiomyocyte-specific transgenic mice overexpressing zDHHC3, which develop severe cardiac disease. Cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice are preceded by enhanced S-palmitoylation of Rac1 and induction of additional Rho family small GTPases including RhoA, Cdc42, and the Rho family-specific chaperone RhoGDI. In contrast, transgenic mice overexpressing an enzymatically-dead mutant of zDHHC3 do not exhibit this profound induction of RhoGTPase signaling or develop cardiac disease. Rac1 S-palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts. Taken together, these data suggest inhibition of zDHHC3/7 S-acyl transferase activity at the cardiomyocyte Golgi or disruption of Rac1 S-palmitoylation as novel therapeutic strategies to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.
... Indeed, localization of all three Rnd is extremely sensitive to FT inhibitors (FTI) but not to GGTase-I inhibitors (GGTI) and resulted in the loss of plasma membrane localization, and increased cytoplasmic and nuclear accumulation of all three Rnd proteins. Thus, the subcellular localization of Rnd1 and Rnd3 proteins is predominantly plasma membrane-associated while Rnd2 lacks this association and appears to be in the cytosol and endosomes [6], directly bound to vacuolar protein sorting 1-A (Vps4-A), the central protein regulating early endosome trafficking [7] (Table 2). Interestingly, replacement of Rnd2 C-terminal domain with that of Rnd3 allowed Rnd2 to be localized at the plasma membrane and compensate for Rnd3 loss of function in cortical neurons [8]. ...
Rnd proteins constitute a subfamily of Rho GTPases represented in mammals by Rnd1, Rnd2 and Rnd3. Despite their GTPase structure, their specific feature is the inability to hydrolyse GTP-bound nucleotide. This aspect makes them atypical among Rho GTPases. Rnds are regulated for their expression at the transcriptional or post-transcriptional levels and they are activated through post-translational modifications and interactions with other proteins. Rnd proteins are mainly involved in the regulation of the actin cytoskeleton and cell proliferation. Whereas Rnd3 is ubiquitously expressed, Rnd1 and 2 are tissue-specific. Increasing data has described their important role during development and diseases. Herein, we describe their involvement in physiological and pathological conditions with a focus on the neuronal and vascular systems, and summarize their implications in tumorigenesis.
... Which proteins are locally lipid modified in sympathetic axons in response to NGF? The Rac1 GTPase is a known GGTase I substrate (Roberts et al., 2008). Rac1 is also a key effector of NGF trophic signaling, TrkA trafficking, and axonal morphology (Harrington et al., 2011) making it an attractive candidate for NGFregulated prenylation in axons. ...
Compartmentalized signaling is critical for cellular organization and specificity of functional outcomes in neurons. Here, we report that post-translational lipidation of newly synthesized proteins in axonal compartments allows for short-term and autonomous responses to extrinsic cues. Using conditional mutant mice, we found that protein prenylation is essential for sympathetic axon innervation of target organs. We identify a localized requirement for prenylation in sympathetic axons to promote axonal growth in response to the neurotrophin, nerve growth factor (NGF). NGF triggers prenylation of proteins including the Rac1 GTPase in axons, counter to the canonical view of prenylation as constitutive, and strikingly, in a manner dependent on axonal protein synthesis. Newly prenylated proteins localize to TrkA-harboring endosomes in axons and promote receptor trafficking necessary for axonal growth. Thus, coupling of prenylation to local protein synthesis presents a mechanism for spatially segregated cellular functions during neuronal development.
... A possible explanation for this can be the possible involvement of RhoB. RhoB has been reported to be able to switch from geranylgeranylation to farnesylation [36], is upregulated, and replaces RhoA functionality in RhoA deficient keratinocytes [37]. However, we still do not have any experimental evidence for this function and further investigation is required to elaborate this aspect. ...
The members of Rho family of GTPases, RhoA and Rac1 regulate endothelial cytoskeleton dynamics and hence barrier integrity. The spatial activities of these GTPases are regulated by post-translational prenylation. In the present study, we investigated the effect of prenylation inhibition on the endothelial cytoskeleton and barrier properties. The study was carried out in human umbilical vein endothelial cells (HUVEC) and protein prenylation is manipulated with various pharmacological inhibitors. Inhibition of either complete prenylation using statins or specifically geranylgeranylation but not farnesylation has a biphasic effect on HUVEC cytoskeleton and permeability. Short-term treatment inhibits the spatial activity of RhoA/Rho kinase (Rock) to actin cytoskeleton resulting in adherens junctions (AJ) stabilization and ameliorates thrombin-induced barrier disruption whereas long-term inhibition results in collapse of endothelial cytoskeleton leading to increased basal permeability. These effects are reversed by supplementing the cells with geranylgeranyl but not farnesyl pyrophosphate. Moreover, long-term inhibition of protein prenylation results in basal hyper activation of RhoA/Rock signaling that is antagonized by a specific Rock inhibitor or an activation of cAMP signaling. In conclusion, inhibition of geranylgeranylation in endothelial cells (ECs) exerts biphasic effect on endothelial barrier properties. Short-term inhibition stabilizes AJs and hence barrier function whereas long-term treatment results in disruption of barrier properties.
... 80, 982-987. Roberts, P.J., Mitin, N., Keller, P.J., Chenette, E.J., Madigan, J.P., Currin, R.O.,Cox, A.D., Wilson, O., Kirschmeier, P., and Der, C.J. (2008). Rho Family GTPase modification and dependence on CAAX motif-signaled posttransla- tional modification. ...
Palmitoylation is a reversible post-translational lipid modification that facilitates vesicular transport and subcellular localization of modified proteins. This process is catalyzed by ZDHHC enzymes that are implicated in several neurological and neurodevelopmental disorders. Loss-of-function mutations in ZDHHC9 have been identified in patients with X-linked intellectual disability (XLID) and associated with increased epilepsy risk. Loss of Zdhhc9 function in hippocampal cultures leads to shorter dendritic arbors and fewer inhibitory synapses, altering the ratio of excitatory-to-inhibitory inputs formed onto Zdhhc9-deficient cells. While Zdhhc9 promotes dendrite outgrowth through the palmitoylation of the GTPase Ras, it promotes inhibitory synapse formation through the palmitoylation of another GTPase, TC10. Zdhhc9 knockout mice exhibit seizure-like activity together with increased frequency and amplitude of both spontaneous and miniature excitatory and inhibitory postsynaptic currents. These findings present a plausible mechanism for how the loss of ZDHHC9 function may contribute to XLID and epilepsy. : Shimell et al. demonstrate that the palmitoylating enzyme Zdhhc9 controls dendritic growth and maintains excitatory/inhibitory synapse balance through distinct substrates. Loss of Zdhhc9 increases network excitability and seizure activity in accordance with Zdhhc9’s association with X-linked intellectual disability and epilepsy. Keywords: Zdhhc9, palmitoylation, neuron morphology, synapse, hippocampal culture, X-linked intellectual disability, dendrite growth, dendrite retraction, Ras GTPase, TC10 GTPase, epilepsy
... An essential characteristic of GTPases to fulfil their biological functions in different subcellular compartments is their association with membranes, which is primarily achieved by a modification of the C-terminally located CAAX-motif (i.e., a cysteine residue followed by two aliphatic amino acids and a variable C-terminal residue). Processing of this motif results in removal of the three terminal amino acids, a carboxymethylation, and geranyl-geranylation of the cysteine, altogether providing a hydrophobic anchor for association with target membranes [16]. A polybasic region (PBR) preceding the CAAX motif is also present in many Rho-type GTPases, where the positive charges electrostatically interact with the negatively charged membrane phospholipids to enhance membrane association and specificity [17,18]. ...
The small GTPase Rho5 of Saccharomyces cerevisiae is required for proper regulation of different signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidative stress. We here show that proper in vivo function and intracellular distribution of Rho5 depends on its hypervariable region at the carboxyterminal end, which includes the CAAX box for lipid modification, a preceding polybasic region (PBR) carrying a serine residue, and a 98 amino acid–specific insertion only present in Rho5 of S. cerevisiae but not in its human homolog Rac1. Results from trapping GFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidative stress response. These findings are supported by heterologous expression of a codon-optimized human RAC1 gene, which can only complement a yeast rho5 deletion in a chimeric fusion with RHO5 sequences that restore the correct spatiotemporal distribution of the encoded protein.
... As a small GTPase, ras homolog family member B (RHOB) is the only member of the Rho family and can be modified by palmitoylation (24). RHOB has different functions, which are realized depending on its exact locations, for example, RHOB protects keratinocytes from UVB injury and RHOB determines tumor aggressiveness (25)(26)(27). ...
Diabetic patients with high glucose exhibit vascular smooth muscle cell (VSMC) alteration. Thrombotic disease is related to erosion of an unstable plaque, the instability of which leads to ruptures, for example, a thin fibrous cap derived from VSMCs. VSMC proliferation, migration and invasion are related to thrombotic diseases, including atherosclerosis. MicroRNA‑19a (miR‑19a) has been reported to have pleiotropic functions in cancer cell survival, apoptosis and migration. The present study aimed to investigate the effect of miR‑19a on VSMC proliferation, migration and invasion, and its mechanism. Cell Counting Kit‑8 and a propidium iodide kit were used to determine the proliferation and cycle of VSMCs. A cell migration assay was performed by scratching and Matrigel was used in a cell invasion assay. miR‑19a binding to Ras homolog family member B (RHOB), and their protein and mRNA expressions were determined by performing a dual luciferase assay, western blotting and reverse transcription‑quantitative PCR, respectively. It was demonstrated that miR‑19a promoted the proliferation, migration and invasion of VSMCs, promoted the expressions of dual specificity phosphatase Cdc25A (CDC25A), cyclinD1, matrix metalloproteinase (MMP)‑2, MMP‑9, α‑smooth muscle actin (α‑SMA) and smooth muscle 22α (SM22α), and inhibited suppressor of cytokine signaling 3 and RHOB expressions in VSMCs, while miR‑19a had no effect on the expression of T‑cell intracellular antigen‑1. The miR‑19a site bound to the RHOB gene position and inhibited RHOB to promote VSMC proliferation, invasion and migration, and increased MMP‑2, MMP‑9, α‑SMA and SM22α expressions. The present study suggested that miR‑19a could promote VSMC proliferation, migration and invasion via the cyclinD1/CDC25A and MMP/α‑SMA/SM22α signaling pathways. Moreover, miR‑19a promoted proliferation, migration and invasion via the MMP/α‑SMA/SM22α signaling pathway by inhibiting RHOB, suggesting that miR‑19a is a possible regulatory factor of RHOB.
... Rho GTPases are considered small GTPases and are grouped together by the conservation of their Rho insert sequences and GTPase domains. A "classical" Rho GTPase contains GTPase domains, a Rho insert, an effector binding domain, and a short C-terminal extension containing a CAAX sequence that can be post-translationally modified, usually by different forms of prenylation ( Figure 2) [7,20]. This "classical" Rho GTPase structure is exemplified by those belonging to the Cdc42, Rac, and Rho subgroups. ...
Blood vessels are required for the survival of any organism larger than the oxygen diffusion limit. Blood vessel formation is a tightly regulated event and vessel growth or changes in permeability are linked to a number of diseases. Elucidating the cell biology of endothelial cells (ECs), which are the building blocks of blood vessels, is thus critical to our understanding of vascular biology and to the development of vascular-targeted disease treatments. Small GTPases of the Rho GTPase family are known to regulate several processes critical for EC growth and maintenance. In fact, many of the 21 Rho GTPases in mammals are known to regulate EC junctional remodeling, cell shape changes, and other processes. Rho GTPases are thus an attractive target for disease treatments, as they often have unique functions in specific vascular cell types. In fact, some Rho GTPases are even expressed with relative specificity in diseased vessels. Interestingly, many Rho GTPases are understudied in ECs, despite their known expression in either developing or mature vessels, suggesting an even greater wealth of knowledge yet to be gleaned from these complex signaling pathways. This review aims to provide an overview of Rho GTPase signaling contributions to EC vasculogenesis, angiogenesis, and mature vessel barrier function. A particular emphasis is placed on so-called "alternative" Rho GTPases, as they are largely understudied despite their likely important contributions to EC biology.
... The intracellular spatiotemporal distribution of Rho GTPases is tightly controlled. In general, active Rho GTPases are targeted to the plasma membrane (or endosomal membranes) via a polybasic region and a prenyl group attached to a C-terminal cysteine residue, and several Rho GTPases are also palmitoylated [18,20,21]. Prenylated cytosolic Rho GTPases are unstable and rapidly degraded [22]. ...
Rho guanosine triphosphatases (GTPases) are key regulators in a number of cellular functions, including actin cytoskeleton remodeling and vesicle traffic. Traditionally, Rho GTPases are studied because of their function in cell migration and cancer, while their roles in metabolism are less documented. However, emerging evidence implicates Rho GTPases as regulators of processes of crucial importance for maintaining metabolic homeostasis. Thus, the time is now ripe for reviewing Rho GTPases in the context of metabolic health. Rho GTPase-mediated key processes include the release of insulin from pancreatic β cells, glucose uptake into skeletal muscle and adipose tissue, and muscle mass regulation. Through the current review, we cast light on the important roles of Rho GTPases in skeletal muscle, adipose tissue, and the pancreas and discuss the proposed mechanisms by which Rho GTPases act to regulate glucose metabolism in health and disease. We also describe challenges and goals for future research.
... Nevertheless, a complex scenario may be envisaged, since the initial view proposing that RHO GTPases play a proneoplastic role has been challenged by recent data from in vivo models and human tumors (reviewed in (21)). Furthermore, ICMT deregulation is expected to exert complex effects on RHO GTPases since the action of ICMT on specific substrates may have different and even opposing consequences on subcellular localization and/or expression levels (22)(23)(24). This evidence indicates that ICMT cannot be considered a proto-oncogene under all circumstances and that several aspects of its biological role are still underexplored. ...
Isoprenyl cysteine carboxy methyltransferase (ICMT) plays a key role in posttranslational regulation of prenylated proteins. On the basis of previous results, we hypothesized that the p53 pathway and ICMT expression may be linked in cancer cells. Here, we studied whether wt p53 and cancer-associated p53 point mutants regulate ICMT levels and whether ICMT overexpression affects tumor progression. Studying the effect of p53 variants on ICMT mRNA and protein levels in cancer cells, we found that wt p53 and p53 mutants differentially affect ICMT expression, indicating that p53 status influences ICMT levels in tumors. To investigate the underlying mechanisms, we constructed ICMT-luciferase reporters and found that wt p53 represses ICMT transcription. In contrast, p53 mutants showed a positive effect on ICMT expression. Promoter truncation analyses pinpointed the repressive effect of wt p53 to the −209 and −14 region, on the ICMT promoter, and chromatin immunoprecipitation assays indicated that wt p53 is recruited to this region. Instead, a different promoter region was identfied as responsible for the mutant p53 effect. Studying the effect of ICMT overexpression on tumor-associated phenotypes in vitro and in vivo, and analyzing breast and lung cancer databases, we identified a correlation between p53 status and ICMT expression in breast and lung cancers. Moreover, we observed that ICMT overexpression is correlated with negative clinical outcomes. Our work unveils a link between postprenylation protein processing and the p53 pathway, indicating that the functional interplay between wt and mutant p53 alters ICMT levels, thereby affecting tumor aggressiveness.
... Not only other proteins but also the localization of the Rho GTPases itself can influence their function and activity. At their C-terminus, the Rho GTPases harbor a stretch of basic amino acids, which facilitates their association with acidic membrane lipids, and the post-translational modification signal CAAX motif (C -cysteine, A -any aliphatic amino acid, X -any amino acid), which targets them for isoprenylation (Roberts et al., 2008). This means, for example in the case of Cdc42 and Rac1, that prenyltransferases add a geranylgeranyl lipid tail to the cysteine residue of the CAAX motif, and with this lipid modification, Cdc42 and Rac1 are able to insert into the plasma membrane or into intracellular membranes. ...
Precise spindle orientation during mitosis is essential for determining both cell fate and tissue organization. Proper alignment of chromosomes is a result of many processes that have to be orchestrated in a precise manner. Although some of the molecular mechanisms that underlie spindle orientation have been described recently, many aspects of this fundamental process remain unknown. Our protein of interest, MISP (mitotic interactor and substrate of Plk1, C19orf21), which was first characterized by our group as a substrate of Polo-like kinase 1 (Plk1), also seems to play a role in spindle orientation and positioning and in metaphase-to-anaphase transition (Zhu et al. 2013). In a mass spectrometrical screen aiming at identifying MISP-interacting proteins I identified IQGAP1, a multidomain scaffolding protein that is believed to link the microtubule network with the actin cytoskeleton, as a potential binding partner. By using co-immunoprecipitation experiments the interaction between MISP and IQGAP1 was confirmed both after their overexpression and endogenously. Functionally, I discovered that depletion of MISP leads to increased accumulation of IQGAP1 at the cell cortex in mitosis. The cortical accumulation of IQGAP1 seems to be dependent on Cdc42, since overexpression of Cdc42 can revert the cortical accumulation of IQGAP1. Cdc42 is a small signaling molecule belonging to the Rho family of GTPases and it is a well-characterized binding partner of IQGAP1. The altered localization of IQGAP1 also coincides with a decrease in its Cdc42 binding capacity and thereby reduced active Cdc42 levels upon MISP knock-down. Furthermore, I found that MISP shows a preferential binding to active Cdc42 similar to IQGAP1. Not surprisingly, I could show that this interaction is not direct and is indeed mediated by IQGAP1. Interestingly, overexpression of IQGAP1 can rescue the mitotic defects caused by MISP downregulation including spindle misorientation, loss of astral microtubules, prolonged mitosis and cortical accumulation of the dynactin subunit p150glued. In addition, it also restores active Cdc42 levels. Importantly, MISP-depletion leads to a reduction in active Cdc42 levels in wild-type but not in IQGAP1 knock-out cells pointing to the effector role of IQGAP1 in regulating active Cdc42 levels upon MISP depletion. Altogether, stabilization of active Cdc42 by IQGAP1 can restore mitotic defects upon MISP silencing. In conclusion, I found that IQGAP1 acts downstream of MISP in regulating active Cdc42 levels, astral microtubule dynamics and the localization of p150glued. Collectively, these results identify a novel pathway, namely that MISP regulates IQGAP1 and Cdc42 to ensure proper mitotic progression and correct spindle orientation.
... Furthermore, dose-dependent toxic effects of statins (low dose: simvastatin 20 mg or equivalent; medium dose: simvastatin 40 mg or equivalent; and high dose: simvastatin 80 mg or equivalent) on increased risk of symptomatic ICH in ischemic stroke patients undergoing thrombolysis have also been observed (Scheitz et al., 2014). HMGCR is the rate-limiting enzyme in the biosynthesis of isoprenoid pyrophosphates, which serve as substrates for the prenylation of Rho GTPases for their proper cellular localization and activation in the regulation of vascular permeability (Berzat et al., 2006;Roberts et al., 2008). In line with this, zebrafish studies have demonstrated that the HMGCR pathway plays important roles in regulating the stability of developmental cerebral vasculature via prenylation-dependent Rho GTPase signaling. ...
... The 'ON-OFF' cycle is further regulated by Rho-GDP dissociation inhibitors (GDI), which interact with GDP-bound RhoA and sequester it in its GDP-bound state. Finally, RhoA GTPases are targeted to host cell membranes thanks to a post-translational modification consisting in the covalent addition of a lipid (either a geranylgeranyl or a farnesyl) to their C-terminal CAAX motif (in which C represents cysteine, A an aliphatic amino-acid and X any amino acid) (Roberts et al. 2008). Rho-GDIs act as chaperones to extract lipidated Rho from the membrane and maintain them in an inactive cytosolic pool (Fig. 1). ...
Numerous pathogens including Clostridium difficile and Yersinia pestis have evolved toxins or effectors targeting GTPases from the RhoA subfamily (RhoA/B/C) to inhibit or hijack the host cytoskeleton dynamics. The resulting impairment of RhoA GTPases activity is sensed by the host via an innate immune complex termed the pyrin inflammasome in which caspase-1 is activated. The cascade leading to activation of the pyrin inflammasome has been recently uncovered. In this review, following a brief presentation of RhoA GTPases-modulating toxins, we present the pyrin inflammasome and its regulatory mechanisms. Furthermore, we discuss how some pathogens have developed strategies to escape detection by the pyrin inflammasome. Finally, we present five monogenic autoinflammatory diseases associated with pyrin inflammasome deregulation. The molecular insights provided by the study of these diseases and the corresponding mutations on pyrin inflammasome regulation and activation are presented.
... Methotrexate, a chemotherapeutic folate analog and teratogen, can also inhibit ICMT activity [53,54]. In addition, it had been demonstrated in cell cultures that AGGC would produce improper localization of Ras and cytoskeletal proteins subject to post-translational modification by methylation [55,56]. These data indicate that AGGC is teratogenic and suggest that the mechanism of teratogenicity underlying AGGC and ICMT teratogenicity is impaired localization of GTPases and cytoskeletal proteins that undergo post-translational modification by methylation. ...
Folic acid supplementation has been shown to significantly reduce both the occurrence and the recurrence of neural tube defects
(NTDs) in human populations, yet the underlying mechanisms for reducing the risk of NTDs continue to be debated. This
study examined genetic background and select folate metabolites as possible modifiers that may influence NTD risk. Specifically,
several folate cycle and methylation metabolites were examined for their ability to reduce the occurrence of NTDs in two congenic
mouse strains carrying targeted disruption of the folate receptor 1 (Folr1) gene. SWV-Folr1tm1Fnn and LM/Bc-Folr1tm1Fnn
mice were provided with folate or several folate pathway metabolites, or combinations thereof, to determine the ability of these
compounds to rescue nullizygous embryos from lethality and NTDs. Results demonstrated that SWV-Folr1tm1Fnn and LM/Bc-
Folr1tm1Fnn mice exhibit different dose responses to folinic acid (5-formyl-tetrahydrofolate) supplementation; however, treating
dams throughout gestation with downstream metabolites indicated that only folates rescued Folr1 nullizygous embryos from
lethality and NTDs. Chemical inhibitors of folate metabolism were used to further elucidate essential enzymatic and biochemical
metabolites. These data demonstrate that the inhibition of S-adenosyl-L-homocysteine hydrolase (AHCY) or selective inhibition
of folate responsive isoprenylcysteine carboxylmethyltransferase (ICMT) results in embryo toxicity and fetuses with anterior
NTDs. These data indicate that genetic background modifies NTD penetrance in folate-supplemented Folr1tm1Fnn mutants, while
downstream metabolites of folate metabolism are not capable of rescuing Folr1tm1Fnn mutants. Moreover, these findings support
the hypothesis that the methylation cycle and post-translational methylation of key signaling proteins such as Ras, by ICMT are
essential to neural tube closure.
... Like squalene synthase inhibitors (Fig. 1), the FTIs can theoretically increase inflammation by increasing the pool of unused endogenous FPP, which can farnesylate and activate GTPases in excess (66). FPP itself could lead to increased farnesylation of other non-Ras small GTPases (79,98), such as Rho family GTPases like RhoB (99)(100)(101), farnesylation of non-GTPase proteins or kinases (81), and shunting of FPP to GGPP, resulting in increased geranylgeranylation of Rho and/or Rab family GTPases (Fig. 11B). For example, the antibiotic and FTI manumycin competes with FPP as a substrate for the FTase enzyme. ...
Ras, a small GTPase protein, is thought to mediate Th2-dependent eosinophilic inflammation in asthma. Ras requires cell membrane association for its biological activity, and this requires the posttranslational modification of Ras with an isoprenyl group by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). We hypothesized that inhibition of FTase using FTase inhibitor (FTI)-277 would attenuate allergic asthma by depleting membrane-associated Ras. We used the OVA mouse model of allergic inflammation and human airway epithelial (HBE1) cells to determine the role of FTase in inflammatory cell recruitment. BALB/c mice were first sensitized then exposed to 1% OVA aerosol or filtered air, and half were injected daily with FTI-277 (20 mg/kg per day). Treatment of mice with FTI-277 had no significant effect on lung membrane-anchored Ras, Ras protein levels, or Ras GTPase activity. In OVA-exposed mice, FTI-277 treatment increased eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity. Human bronchial epithelial (HBE1) cells were pretreated with 5, 10, or 20 μM FTI-277 prior to and during 12 h IL-13 (20 ng/ml) stimulation. In HBE1 cells, FTase inhibition with FTI-277 had no significant effect on IL-13-induced STAT6 phosphorylation, eotaxin-3 peptide secretion, or Ras translocation. However, addition of exogenous FPP unexpectedly augmented IL-13-induced STAT6 phosphorylation and eotaxin-3 secretion from HBE1 cells without affecting Ras translocation. Pharmacological inhibition of FTase exacerbates allergic asthma, suggesting a protective role for FTase or possibly Ras farnesylation. FPP synergistically augments epithelial eotaxin-3 secretion, indicating a novel Ras-independent farnesylation mechanism or direct FPP effect that promotes epithelial eotaxin-3 production in allergic asthma.
... Furthermore, as there are over 22 members of the Rho GTPase family, the deletion of Ggpps may impact on these through altered geranylgeranylation or farnesylation. 10 For example, the activity of Cdc42 is dependent upon prenylation 11 and is essential for the establishment of adherens junctions. Embryonic deletion of Cdc42 in cardiomyocytes, caused embryonic lethality after E12.5 with heart defects including thin ventricle walls, which the authors attributed to reduced cardiomyocyte proliferation and impaired formation of adherens junction cell-cell contacts, 12 thus presenting a similar phenotype as described in this article by Chen et al. ...
... Nevertheless, such methods have demonstrated that the absence of Rce1 modulates the thermosensitivity of yeast (Boyartchuk et al. 1997). More commonly, GFP-tagging of Ras and other Ras-related GTPases is used to assess the role of Rce1 in regulating small GTPase subcellular localization (Boyartchuk et al. 1997;Choy et al. 1999;Michaelson et al. 2005;Manandhar et al. 2007;Bracha-Drori et al. 2008;Roberts et al. 2008;Mokry et al. 2009;Hanker et al. 2010;Manandhar et al. 2010;Gentry et al. 2015;Mohammed et al. 2016). In the yeast system, GFP-Ras2 is cytosolic in the absence of farnesylation and has diffuse membranous patterns of intracellular distribution in the absence of either Rce1p or the ICMT (Ste14p) (Boyartchuk et al. 1997;Manandhar et al. 2010); the absence of Ste24p does not impact Ras2p localization. ...
Ras converting enzyme 1 (Rce1) is an integral membrane endoprotease localized to the endoplasmic reticulum that mediates the cleavage of the carboxyl-terminal three amino acids from CaaX proteins, whose members play important roles in cell signaling processes. Examples include the Ras family of small GTPases, the γ-subunit of heterotrimeric GTPases, nuclear lamins, and protein kinases and phosphatases. CaaX proteins, especially Ras, have been implicated in cancer, and understanding the post-translational modifications of CaaX proteins would provide insight into their biological function and regulation. Many proteolytic mechanisms have been proposed for Rce1, but sequence alignment, mutational studies, topology, and recent crystallographic data point to a novel mechanism involving a glutamate-activated water and an oxyanion hole. Studies using in vivo and in vitro reporters of Rce1 activity have revealed that the enzyme cleaves only prenylated substrates and the identity of the a2 amino residue in the Ca1a2X sequence is most critical for recognition, preferring Ile, Leu, or Val. Substrate mimetics can be somewhat effective inhibitors of Rce1 in vitro. Small-molecule inhibitor discovery is currently limited by the lack of structural information on a eukaryotic enzyme, but a set of 8-hydroxyquinoline derivatives has demonstrated an ability to mislocalize all three mammalian Ras isoforms, giving optimism that potent, selective inhibitors might be developed. Much remains to be discovered regarding cleavage specificity, the impact of chemical inhibition, and the potential of Rce1 as a therapeutic target, not only for cancer, but also for other diseases.
... All three modification steps are necessary for many prenylated proteins to function optimally, although there can be variation in the extent of, and dependence on, these subsequent modifications. (30,31) Recently, a shunt pathway for prenylated proteins in yeast has been reported in which prenylation is the endpoint of CaaX protein modification. In this case, post-prenylation processing is not required and is actually deleterious to protein function. ...
Protein prenylation is a post-translational modification that has been most commonly associated with enabling protein trafficking to and interaction with cellular membranes. In this process, an isoprenoid group is attached to a cysteine near the C-terminus of a substrate protein by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I or II (GGTase-I and GGTase-II). FTase and GGTase-I have long been proposed to specifically recognize a four amino acid CaaX C-terminal sequence within their substrates. Surprisingly, genetic screening reveals that yeast FTase can modify sequences longer than the canonical CaaX sequence, specifically C(x)3X sequences with four amino acids downstream of the cysteine. Biochemical and cell-based studies using both peptide and protein substrates reveal that mammalian FTase orthologs can also prenylate C(x)3X sequences. As the search to identify physiologically relevant C(x)3X proteins begins, this new prenylation motif nearly doubles the number of proteins within the yeast and human proteomes that can be explored as potential FTase substrates. This work expands our understanding of prenylation's impact within the proteome, establishes the biological relevant reactivity possible with this new motif, and opens new frontiers in determining the impact of non-canonical prenylated proteins on cell function.
... However, defective palmitoylation is not behind the reduced plasma membrane tethering in the absence of ICMT activity, as Ras1 and Rho2 palmitoylation levels remained unchanged in control and mam4Δ cells. The possibility that ICMT activity favours proper membrane localization of farnesylated-, but not geranylgeranylated-, GTPases in mammalian cells has found different support (Roberts et al. 2008). Our results suggest that this is likely the case, as plasma membrane tethering of a geranylgeranylated Rho2 chimera was reduced minimally in mam4Δ cells as compared to the wild-type farnesylated GTPase. ...
Isoprenylcysteine-O-Carboxyl Methyltransferase (ICMT) catalyzes the final step in the prenylation process of different proteins including members of the Ras superfamily of GTPases. While cysteine methylation is essential in mammalian cells for growth, membrane association, and signalling by Ras and Rho GTPases, its role during signal transduction events in simple eukaryotes like yeasts appears irrelevant. By using a multidisciplinary approach our group has recently shown that, contrary to this initial assumption, in the fission yeast Schizosaccharomyces pombe ICMT activity encoded by the Mam4 gene is not only important to promote selective plasma membrane targeting of Ras and specific Rho GTPases, but also to allow precise downstream signalling to the mitogen-activated protein kinase and target of rapamycin pathways in response to diverse environmental cues. Thus, the dynamic regulation of in vivo methylation as a modulator of GTPase localization and function is an evolutionary conserved mechanism, making fission yeast an appealing model organism to study the regulation of this process.
... We also confirmed that the rate of cytokinesis failure was partially rescued in ect-2(ts); Rac(V190G) double mutants (14 of 19 embryos divide; Figure 4D). Rac(V190G) is a mild lof allele (n1993) that affects the CAAX box and thus likely affects Rac association with the plasma membrane (Kinsella et al., 1991;Reddien and Horvitz, 2000;Shakir et al., 2006;Roberts et al., 2008;Steffen et al., 2013). Although we do not observe the same high rate of cytokinesis failure in the ect-2(ts) mutant after prolonged upshifts to restrictive temperature as reported by Loria et al. (2012), possibly due to environmental effects, we observed a similar rescue of cytokinesis failure rates in ect-2(ts); Rac(RNAi) and ect-2(ts); Rac(V190G) embryos. ...
Cytokinesis is driven by constriction of an actomyosin contractile ring that is controlled by Rho family small GTPases. Rho, activated by the Guanine-nucleotide Exchange Factor (GEF) ECT-2, is upstream of both myosin-II activation and diaphanous formin-mediated filamentous actin (f-actin) assembly, which drive ring constriction. The role for Rac and its regulators is more controversial but, based on the finding that Rac inactivation can rescue cytokinesis failure when the GTPase Activating Protein (GAP) CYK-4 is disrupted, Rac activity was proposed to be inhibitory to contractile ring constriction and thus specifically inactivated by CYK-4 at the division plane. An alternative model proposes that Rac inactivation generally rescues cytokinesis failure by reducing cortical tension, thus making it easier for the cell to divide when ring constriction is compromised. In this alternative model, CYK-4 was instead proposed to activate Rho by binding ECT-2. Using a combination of time-lapse in vivo single-cell analysis and C. elegans genetics, our evidence does not support this alternative model. First, we found that Rac disruption does not generally rescue cytokinesis failure: inhibition of Rac specifically rescues cytokinesis failure due to disruption of CYK-4 or ECT-2 but does not rescue cytokinesis failure due to disruption of two other contractile ring components, the Rho effectors diaphanous formin or myosin-II. Second, if CYK-4 regulates cytokinesis through Rho rather than Rac, then CYK-4 inhibition should decrease levels of downstream targets of Rho. Inconsistent with this, we found no change in the levels of f-actin or myosin-II at the division plane when CYK-4 GAP activity was reduced, suggesting that CYK-4 is not upstream of ECT-2/Rho activation. Instead, we found that the rescue of cytokinesis in CYK-4 mutants by Rac inactivation was Cdc42-dependent. Together our data suggest that CYK-4 GAP activity opposes Rac (and perhaps Cdc42) during cytokinesis.
... Subcellular localization of RHO GTPases to cellular membranes is known to be critical for their biological activity. This is achieved by a hypervariable region (HVR) (66) and a lipid anchor in their C-terminal tail at a distinct cysteine residue in the CAAX motif (C is cysteine, A is any aliphatic amino acid, and X is any amino acid) (67). RHOGDI is known to dislodge RHO proteins from the plasma membrane (68). ...
IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity and transmigration. The RHO family proteins CDC42 and RAC1, have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal (CT) domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underly a multiple-step binding mechanism: (i) a high-affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1, and (ii) a very low-affinity binding of GRD and CT adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.
... A similar mechanism could be at play for localization of TCL. This would be contrary to previous studies suggesting TCL is not palmitoylated; however, it is possible the modification is transient and rapidly hydrolyzed, causing the detection of palmitoylated TCL to be more difficult (40,44). If this were the case, then TCL palmitoylation would possibly modulate GTPloading either by directly altering nucleotide loading or by bringing TCL to a favorable membrane environment for nucleotide exchange to occur. ...
TCL/RhoJ is a Cdc42-related Rho GTPase with reported activities in endothelial cell biology and angiogenesis, metastatic melanoma, and corneal epithelial cells; however, less is known about how it is inherently regulated in comparison to its closest homologues TC10 and Cdc42. TCL has an N-terminal extension of 18 amino acids in comparison to Cdc42, but the function of this amino acid sequence has not been elucidated. A truncation mutant lacking the N-terminus (?N) was found to alter TCL plasma membrane localization and nucleotide binding, and additional truncation and point mutants mapped the alterations of TCL biochemistry to amino acids 17-20. Interestingly, while the TCL ?N mutant clearly influenced nucleotide exchange, deletion of the N-terminus from its closest homologue, TC10, did not have a similar effect. Chimeras of TCL and TC10 revealed amino acids 121-129 of TCL contributed to the differences in nucleotide loading. Together, these results identify amino acids within the N-terminus and a loop region distal to the nucleotide binding pocket of TCL capable of allosterically regulating nucleotide exchange and thus influence membrane association of the protein.
Background
Statins, frequently prescribed medications, work by inhibiting the rate‐limiting enzyme HMG‐CoA reductase (HMGCR) in the mevalonate pathway to reduce cholesterol levels. Due to their multifaceted benefits, statins are being adapted for use as cost‐efficient, safe and effective anti‐cancer treatments. Several studies have shown that specific types of cancer are responsive to statin medications since they rely on the mevalonate pathway for their growth and survival.
Recent Findings
Statin are a class of drugs known for their potent inhibition of cholesterol production and are typically prescribed to treat high cholesterol levels. Nevertheless, there is growing interest in repurposing statins for the treatment of malignant neoplastic diseases, often in conjunction with chemotherapy and radiotherapy. The mechanism behind statin treatment includes targeting apoptosis through the BCL2 signaling pathway, regulating the cell cycle via the p53‐YAP axis, and imparting epigenetic modulations by altering methylation patterns on CpG islands and histone acetylation by downregulating DNMTs and HDACs respectively. Notably, some studies have suggested a potential chemo‐preventive effect, as decreased occurrence of tumor relapse and enhanced survival rate were reported in patients undergoing long‐term statin therapy. However, the definitive endorsement of statin usage in cancer therapy hinges on population based clinical studies with larger patient cohorts and extended follow‐up periods.
Conclusions
The potential of anti‐cancer properties of statins seems to reach beyond their influence on cholesterol production. Further investigations are necessary to uncover their effects on cancer promoting signaling pathways. Given their distinct attributes, statins might emerge as promising contenders in the fight against tumorigenesis, as they appear to enhance the efficacy and address the limitations of conventional cancer treatments.
Rho GTPases are critically important and are centrally positioned regulators of the actomyosin cytoskeleton. By influencing the organization and architecture of the cytoskeleton, Rho proteins play prominent roles in many cellular processes including adhesion, migration, intra‐cellular transportation, and proliferation. The most important method of Rho GTPase regulation is via the GTPase cycle; however, post‐translational modifications (PTMs) also play critical roles in Rho protein regulation. Relative to other PTMs such as lipidation or phosphorylation that have been extensively characterized, protein oxidation is a regulatory PTM that has been poorly studied. Protein oxidation primarily occurs from the reaction of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), with amino acid side chain thiols on cysteine (Cys) and methionine (Met) residues. The versatile redox modifications of cysteine residues exemplify their integral role in cell signalling processes. Here we review prominent members of the Rho GTPase family and discuss how lipidation, phosphorylation, and oxidation on conserved cysteine residues affects their regulation and function. Reactive oxygen species (ROS) can oxidize Rho GTPases on redox‐sensitive cysteine residues, resulting in reduced affinity for guanine nucleotides and consequent increased GDP dissociation rates. Binding of cellular GTP to the nucleotide‐free protein, independent of guanine nucleotide exchange factors (GEF), transitions the GTPases to active conformations that enable downstream signalling.
Objectives:
The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT.
Methods:
Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays.
Results:
After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (P<0.05). The mRNA and total protein expression levels of RhoA in the two groups were not significantly different (P>0.05). The expression levels of RhoA membrane protein, ROCK1, MMP-2, and MMP-9 decreased (P<0.05). The migration and invasion abilities were inhibited (P<0.05).
Conclusions:
The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.
Dans la plupart des régions cérébrales, les neurones sont générés pendant l’embryogénèse. A l’inverse, dans le gyrus denté (DG) de l'hippocampe, la majorité des neurones granulaires (NGs) est générée en période postnatale et cette production neuronale se poursuit tout au long de l'âge adulte. Cette découverte selon laquelle de nouveaux neurones sont générés dans le cerveau des mammifères adultes a ouvert de nouvelles perspectives pour réparer le cerveau et a conduit de nombreuses recherches, au cours des 20 dernières années, à caractériser comment les nouveaux neurones se différencient et s'intègrent aux circuits neuronaux adultes. Cependant, d'autres études sont nécessaires pour mieux comprendre les mécanismes et les cascades de signalisation impliqués dans ce processus. Dans ce contexte, nous nous sommes concentrés sur Rnd2, une RhoGTPase particulièrement enrichie dans le DG adulte et décrite comme une actrice clé dans la régulation de la neurogenèse corticale embryonnaire. Nous avons montré, in vivo, que la suppression de Rnd2 spécifiquement dans les néo-neurones hippocampiques diminue la survie de ces cellules, et dans les cellules survivantes, conduit à une hypertrophie du soma, augmente l'arborisation dendritique et induit un mauvais positionnement. De façon intéressante cette suppression augmente également le comportement anxiogène des souris, identifiant ainsi Rnd2 comme un régulateur critique de la neurogénèse adulte hippocampique. De plus, nos données montrent que Rnd2 ne joue pas les mêmes fonctions dans les NGs nés à P0, mettant en évidence une régulation différentielle de la neurogenèse développementale et adulte dans la DG. Dans le même ordre d'idées, nous démontrons également que les NGs nés en période périnatale, en particulier les neurones embryonnaires, sont morphologiquement distincts par rapport aux NGs nés plus tard. L'ensemble de ces travaux de thèse apporte donc de nouvelles connaissances sur le développement des différentes populations de NGs dans la DG, soulignant davantage la particularité de cette structure cérébrale.
Background
Psoriasis is a chronic and recurrent inflammatory skin disorder driven by a complex cascade of inflammatory mediators. The present study focused on the potential clinical significance of PGGT1B in psoriasis development.
Methods
The peripheral blood mononuclear cells (PBMCs) were isolated from 81 psoriasis patients and 84 healthy controls, and the expression levels of PGGT1B in PBMCs were examined by quantitative real‐time polymerase chain reaction (RT‐qPCR) methods. Furthermore, we tested the relationship between the level of PGGT1B in PBMCs and psoriasis severity. Also, we analyzed the potential significance of PGGT1B in psoriasis diagnosis. Finally, patients with psoriasis were divided into progressive and stable stage groups, and the differential expression of PGGT1B, TNF‐α, IL‐17, and IFN‐γ between different phases were analyzed.
Results
PGGT1B was dramatically decreased in the psoriasis patients’ PBMCs and negatively correlated with the Psoriasis Area and Severity Index (PASI). Moreover, ROC analysis showed the potential of differentially expressed PGGT1B in terms of distinguishing psoriasis patients from healthy controls. Finally, compared to the patients in the stable phase, PGGT1B was markedly reduced in patients’ PBMCs in the progressive stage, while proinflammatory cytokines TNF‐α and IL‐17 were notably increased.
Conclusions
PGGT1B was down‐regulated in psoriasis patients’ PBMCs and may serve as a potential biomarker for the diagnosis and treatment of psoriasis.
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Numerous experimental studies demonstrate that the Ras homolog family of guanosine triphosphate hydrolases (Rho GTPases) Ras homolog family member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42) are important regulators in somatosensory neurons, where they elicit changes in the cellular cytoskeleton and are involved in diverse biological processes during development, differentiation, survival and regeneration. This review summarizes the status of research regarding the expression and the role of the Rho GTPases in peripheral sensory neurons and how these small proteins are involved in development and outgrowth of sensory neurons, as well as in neuronal regeneration after injury, inflammation and pain perception. In sensory neurons, Rho GTPases are activated by various extracellular signals through membrane receptors and elicit their action through a wide range of downstream effectors, such as Rho-associated protein kinase (ROCK), phosphoinositide 3-kinase (PI3K) or mixed-lineage kinase (MLK). While RhoA is implicated in the assembly of stress fibres and focal adhesions and inhibits neuronal outgrowth through growth cone collapse, Rac1 and Cdc42 promote neuronal development, differentiation and neuroregeneration. The functions of Rho GTPases are critically important in the peripheral somatosensory system; however, their signalling interconnections and partially antagonistic actions are not yet fully understood.
Lipids represent a diverse array of molecules essential to the cell's structure, defense, energy, and communication. Lipid metabolism can often become dysregulated during tumor development. During cancer therapy, targeted inhibition of cell proliferation can likewise cause widespread and drastic changes in lipid composition. Molecular imaging techniques have been developed to monitor altered lipid profiles as a biomarker for cancer diagnosis and treatment response. For decades, MRS has been the dominant non‐invasive technique for studying lipid metabolite levels. Recent insights into the oncogenic transformations driving changes in lipid metabolism have revealed new mechanisms and signaling molecules that can be exploited using optical imaging, mass spectrometry imaging, and positron emission tomography. These novel imaging modalities have provided researchers with a diverse toolbox to examine changes in lipids in response to a wide array of anticancer strategies including chemotherapy, radiation therapy, signal transduction inhibitors, gene therapy, immunotherapy, or a combination of these strategies. The understanding of lipid metabolism in response to cancer therapy continues to evolve as each therapeutic method emerges, and this review seeks to summarize the current field and areas of unmet needs. Lipids play critical roles in biological systems, ranging from structural integrity to trafficking, energy, defense, and communication. This article reviews lipids and lipid metabolic pathways altered in cancer development and their changes in response to therapy that are amenable for study by imaging. We focus first on MRS, which was instrumental in defining the field of lipid imaging (figure) and still plays a major role, followed by complementary molecular imaging methods including PET, mass spectroscopic imaging, and optical imaging.
Rho guanosine triphosphatases (GTPases) are key regulators in a number of cellular functions, including actin cytoskeleton remodeling and vesicle traffic. Traditionally, Rho GTPases are studied because of their function in cell migration and cancer, while their roles in metabolism are less documented. However, emerging evidence implicates Rho GTPases as regulators of processes of crucial importance for maintaining metabolic homeostasis. Thus, the time is now ripe for reviewing Rho GTPases in the context of metabolic health. Rho GTPase-mediated key processes include the release of insulin from pancreatic β-cells, glucose uptake into skeletal muscle and adipose tissue, and muscle mass regulation. Through the current review, we cast light on the important role of Rho GTPases in skeletal muscle, adipose tissue, and the pancreas and mechanisms by which Rho GTPases act to regulate glucose metabolism in health and disease. We also describe challenges and goals for future research.
Emerging evidence suggests that crosstalk between hematologic tumor cells and the tumor microenvironment contributes to leukemia and lymphoma cell migration, survival, and proliferation. The supportive tumor cell-microenvironment interactions and the resulting cellular processes require adaptations and modulations of the cytoskeleton. The Rac subfamily of the Rho family GTPases includes key regulators of the cytoskeleton, with essential functions in both normal and transformed leukocytes. Rac proteins function downstream of receptor tyrosine kinases, chemokine receptors, and integrins, orchestrating a multitude of signals arising from the microenvironment. As such, it is not surprising that deregulation of Rac expression and activation plays a role in the development and progression of hematological malignancies. In this review, we will give an overview of the specific contribution of the deregulation of Rac GTPases in hematologic malignancies.
Rho GTPase-based signaling networks control cellular dynamics by coordinating protrusions and retractions in space and time. Here, we reveal a signaling network that generates pulses and propagating waves of cell contractions. These dynamic patterns emerge via self-organization from an activator–inhibitor network, in which the small GTPase Rho amplifies its activity by recruiting its activator, the guanine nucleotide exchange factor GEF-H1. Rho also inhibits itself by local recruitment of actomyosin and the associated RhoGAP Myo9b. This network structure enables spontaneous, self-limiting patterns of subcellular contractility that can explore mechanical cues in the extracellular environment. Indeed, actomyosin pulse frequency in cells is altered by matrix elasticity, showing that coupling of contractility pulses to environmental deformations modulates network dynamics. Thus, our study reveals a mechanism that integrates intracellular biochemical and extracellular mechanical signals into subcellular activity patterns to control cellular contractility dynamics.
Aim and objective:
Visceral leishmaniasis is a deadly disease left untreated in over 95% of cases. It is characterized by irregular bouts of fever, weight loss, enlargement of the spleen and liver, and anemia. It is highly endemic in the Indian subcontinent. CAAX prenyl proteaseI of Leishmania donovani is one of the important targets regulating the post translational modification process. Hence identifying potent drug candidate against the target is essential. This study mainly focuses on developing new and potent inhibitors against CAXX prenyl protease I of Leishmania donovani.
Materials and methods:
Pharmacophore based virtual screening was carried out using derivatives of bi-substrate analog farnesyl transferase inhibitors reported against CAAX prenyl proteases I. On the basis of ligand based pharmacophore model we have obtained 5 point pharmacophore AAADR with three hydrogen bond acceptors (A), one hydrogen bond donor (D) and one aromatic ring. The newly identified hits through pharmacophore model were further docked into the active site of the modeled protein. To get further insights of protein ligand interaction we have performed induced fit docking followed by molecular dynamics simulations. The DFT analysis depicts the electronic structure properties of the compounds. These results can be useful for the development of novel and potent CAAX prenyl protease I inhibitors.
Results:
Initially, we have obtained a large number of newly identified hits by screening four different databases further docked into the active site of the protein and 20 compounds were selected on the basis of docking score. Perhaps Induced fit docking was performed to infer protein ligand interaction in a dynamic state and top 5 compounds 7118044, 7806909, LEG12866807, 9208535, SYN 19867403 were found to have good protein ligand interactions with key amino acid residues such as Glu287, His290 and additional interaction like Ile197, Asn209 Tyr253, Phe254, Gly256, Tyr266 with better binding energy -59.794 Kcal/mol, -66.305 Kcal/mol, -70.467 Kcal/mol, -82.474 Kcal/mol, -64.045. The predicted ADME properties are in desirable range and the HOMO/LUMO gap clearly indicates the electrons behavior of the ligands. Molecular dynamics simulations of the protein ligand complex for 20 ns clearly depicts the compounds are stable throughout the simulation time.
Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph–ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB + cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB + cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion.
Abnormal Rac1 signaling is linked to a number of debilitating human diseases, including, cancer, cardiovascular diseases and neurodegenerative disorders. As such, Rac1 represents an attractive therapeutic target, yet the search for effective Rac1 inhibitors is still underway. Given the adverse effects associated with Rac1 signaling perturbation, cells have evolved several mechanisms to ensure the tight regulation of Rac1 signaling. Thus, characterizing these mechanisms can provide invaluable information regarding major cellular events that lead to aberrant Rac1 signaling. Importantly, this information can be utilized to further facilitate the development of effective pharmacological modulators that can restore normal Rac1 signaling. In this review, we focus on the pathological role of Rac1 signaling, highlighting the benefits and potential drawbacks of targeting Rac1 in a clinical setting. Additionally, we provide an overview of available compounds that target key Rac1 regulatory mechanisms and discuss future therapeutic avenues arising from our understanding of these mechanisms.
As a reversible posttranslational modification, protein palmitoylation has the potential to regulate the trafficking and function of a variety of proteins. However, the extent, function, and dynamic nature of palmitoylation are poorly resolved because of limitations in assay methods. Here, we introduce methods where hydroxylamine-mediated cleavage of the palmitoyl-thioester bond generates a free sulfhydryl, which can then be specifically labeled with sulfhydryl-reactive reagents. This methodology is more sensitive and allows for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or nonradioactive, and can be used to assay the palmitoylation of proteins expressed in vivo in brain or other tissues.
The Rho-family proteins make up a major branch of the Ras superfamily of small GTPases. To date, 22 human genes encoding at least 25 proteins have been described. The best known 'classical' members are RhoA, Rac1 and Cdc42. Highly related isoforms of these three proteins have not been studied as intensively, in part because it has been assumed that they are functionally identical to their better-studied counterparts. This now appears not to be the case. Variations in C-terminal-signaled modifications and subcellular targeting cause otherwise highly biochemically related isoforms (e.g. RhoA, RhoB and RhoC) to exhibit surprisingly divergent biological activities. Whereas the classical Rho GTPases are regulated by GDP/GTP cycling, other Rho GTPases are also regulated by other mechanisms, particularly by transcriptional regulation. Newer members of the family possess additional sequence elements beyond the GTPase domain, which suggests they exhibit yet other mechanisms of regulation.
Signaling proteins from the same family can have markedly different roles in a given cellular context. Here, we show that expression of one hundred constitutively active human small GTPases induced cell morphologies that fell into nine distinct classes. We developed an algorithm for pairs of classes that predicted amino acid positions that can be exchanged to create mutants with switched functionality. The algorithm was validated by creating switch-of-function mutants for Rac1, CDC42, H-Ras, RalA, Rap2B, and R-Ras3. Contrary to expectations, the relevant residues were mostly outside known interaction surfaces and were structurally far apart from each other. Our study shows that specificity in protein families can be explored by combining genome-wide experimental functional classification with the creation of switch-of-function mutants.
The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.
The spatial organization of plasma membrane components in discrete microdomains is thought to be a key factor in the generation of distinct signal outputs. A detailed characterization of plasma membrane microdomains, including descriptions of their size, dynamics and abundance, has proved to be a taxing problem for cell biologists and biophysicists. The use of novel techniques is providing exciting new insights into the challenging problem of plasma membrane microstructure and has allowed the visualization of domains with the characteristics expected of lipid rafts - microdomains of the plasma membrane enriched in cholesterol and sphingolipids. This review focuses on some of these recent advances and uses Ras signaling as a paradigm for understanding inner plasma membrane organization and the role of lipid rafts in cellular function.
Post-translational modifications are essential for the proper function of many proteins in the cell. The attachment of an isoprenoid lipid (a process termed prenylation) by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) is essential for the function of many signal transduction proteins involved in growth, differentiation, and oncogenesis. FTase and GGTase-I (also called the CaaX prenyltransferases) recognize protein substrates with a C-terminal tetrapeptide recognition motif called the Ca1a2X box. These enzymes possess distinct but overlapping protein substrate specificity that is determined primarily by the sequence identity of the Ca1a2X motif. To determine how the identity of the Ca1a2X motif residues and sequence upstream of this motif affect substrate binding, we have solved crystal structures of FTase and GGTase-I complexed with a total of eight cognate and cross-reactive substrate peptides, including those derived from the C termini of the oncoproteins K-Ras4B, H-Ras and TC21. These structures suggest that all peptide substrates adopt a common binding mode in the FTase and GGTase-I active site. Unexpectedly, while the X residue of the Ca1a2X motif binds in the same location for all GGTase-I substrates, the X residue of FTase substrates can bind in one of two different sites. Together, these structures outline a series of rules that govern substrate peptide selectivity; these rules were utilized to classify known protein substrates of CaaX prenyltransferases and to generate a list of hypothetical substrates within the human genome.
The discovery that Ras proteins are modified by enzymes restricted to the endoplasmic reticulum and Golgi apparatus and that, at steady state, a significant pool of Ras is localized on the Golgi has led to the hypothesis that Ras can become activated on and signal from intracellular membranes. Fluorescent probes capable of showing when and where in living cells Ras becomes activated together with studies of Ras proteins stringently tethered to intracellular membranes have confirmed this hypothesis. Thus, recent studies of Ras have contributed to the rapidly expanding field of compartmentalized signaling.
Rho GTPases are well known to regulate actin dynamics. They activate two types of actin nucleators, WASP/WAVE proteins and Diaphanous-related formins (DRFs), which induce different types of actin organization. Their ability to interact with membranes allows them to target actin polymerization to discrete sites on the plasma membrane and to intracellular membrane compartments and thereby induce membrane protrusions or regulate vesicle movement. Most studies have concentrated on just three of the 22 mammalian Rho proteins, RhoA, Rac1 and Cdc42. However, recent research indicates that several other members of the Rho family, including Rif, RhoD, TC10 and Wrch1, and also related Rho-of-plants proteins (ROPs) in plants, stimulate actin polymerization and affect plasma membrane protrusion and/or vesicular traffic.