Guanine Nucleotide Dissociation Inhibitor Research Articles

Leucine-Rich Repeat Kinases.

Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein kinase that phosphorylates a specific subset of the ∼65human Rab GTPases, which are master regulators of the secretory and endocytic pathways. After phosphorylation by LRRK2, Rabs lose the capacity to bind cognate effector proteins and guanine nucleotide exchange factors. Moreover, the phosphorylated Rabs cannot interact with their cognate prenyl-binding retrieval proteins (also known as guanine nucleotide dissociation inhibitors) and, thus, they become trapped on membrane surfaces. Instead, they gain the capacity to bind phospho-Rab-specific effector proteins, such as RILPL1, with resulting pathological consequences. Rab proteins also act upstream of LRRK2 by controlling its activation and recruitment onto membranes. LRRK2 signaling is counteracted by the phosphoprotein phosphatase PPM1H, which selectively dephosphorylates phospho-Rab proteins. We present here our current understanding of the structure, biochemical properties, and cell biology of LRRK2 and its related paralog LRRK1 and discuss how this information guides the generation of LRRK2 inhibitors for the potential benefit of patients.

Transient Structural Properties of the Rho GDP-Dissociation Inhibitor.

Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N-terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. These observations revise the long-standing textbook picture of the GTPases' mechanism of membrane extraction. Rather than by a disorder-to-order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi-step process.

Transiente Strukturelle Eigenschaften des Rho‐GDP‐Dissoziationsinhibitors

AbstractRho‐GTPasen, räumliche Hauptregulatoren einer Vielzahl zellulärer Prozesse, werden durch Komplexbildung mit Guaninnukleotid‐Dissoziationsinhibitoren (RhoGDIs) reguliert. Man nimmt bisher an, dass diese einen unstrukturierten N‐Terminus aufweisen, der den Nukleotidaustausch ihres Klienten bei der Bindung/Faltung hemmt. Mithilfe von NMR‐Analysen, Molekulardynamiksimulationen und biochemischen Tests konnten wir stattdessen relevante strukturelle Eigenschaften des Terminus nachweisen, die sowohl in Anwesenheit als auch in Abwesenheit des Klienten transient existieren, allerdings kontextspezifisch variiert werden. Diese Beobachtungen revidieren das seit Langem bestehende Bild zum Mechanismus der Membranextraktion der GTPasen. Der komplexe und hochselektive Extraktionsprozess der RhoGTPasen wird nicht durch das gleichzeitige Falten und Binden eines sterisch blockierenden Peptides, sondern durch ein dynamisches Ensemble mit vordefinierten transienten strukturellen Eigenschaften orchestriert, die durch die spezifische Umgebung entlang des mehrstufigen Prozesses noch moduliert werden können.

AGS3-based optogenetic GDI induces GPCR-independent Gβγ signaling and macrophage migration.

G protein-coupled receptors (GPCRs) are efficient Guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GαGTP, and free Gβγ, and are major disease drivers. Evidence shows that the ambient low threshold signaling required for cells is likely supplemented by signaling regulators such as non-GPCR GEFs and Guanine nucleotide Dissociation Inhibitors (GDIs). Activators of G protein Signaling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure-function relationship or signaling regulation in living cells. Here, we employed in silico structure-guided engineering of a novel optogenetic GDI, based on the AGS3's G protein regulatory (GPR) motif, to understand its GDI activity and induce standalone Gβγ signaling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases Gβγ, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signaling pathways and triggering GPCR-independent Gβγ signaling in cells and in vivo.

PLK1 phosphorylates RhoGDI1 and promotes cancer cell migration and invasion

BackgroundRho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays an important role in diverse cellular processes by regulating Rho guanosine triphosphate (GTP)ases activity. RhoGDI1 phosphorylation regulates the spatiotemporal activation of Rho GTPases during cell migration. In this study, we identified polo-like kinase 1 (PLK1) as a novel kinase of RhoGDI1 and investigated the molecular mechanism by which the interaction between RhoGDI1 and PLK1 regulates cancer cell migration.MethodsImmunoprecipitation, GST pull-down assay, and proximity ligation assay (PLA) were performed to analyze the interaction between RhoGDI1 and PLK1. In vitro kinase assay and immunoprecipitation were performed with Phospho-(Ser/Thr) antibody. We evaluated RhoA activation using RhoGTPases activity assay. Cell migration and invasion were analyzed by transwell assays.ResultsGST pull-down assays and PLA showed that PLK1 directly interacted with RhoGDI1 in vitro and in vivo. Truncation mutagenesis revealed that aa 90-111 of RhoGDI1 are critical for interacting with PLK1. We also showed that PLK1 phosphorylated RhoGDI1 at Thr7 and Thr91, which induces cell motility. Overexpression of the GFP-tagged RhoGDI1 truncated mutant (aa 90-111) inhibited the interaction of PLK1 with RhoGDI1 and attenuated RhoA activation by PLK1. Furthermore, the overexpression of the RhoGDI1 truncated mutant reduced cancer cell migration and invasion in vitro and suppressed lung metastasis in vivo.ConclusionsCollectively, we demonstrate that the phosphorylation of RhoGDI1 by PLK1 promotes cancer cell migration and invasion through RhoA activation. This study connects the interaction between PLK1 and RhoGDI1 to the promotion of cancer cell behavior associated with malignant progression, thereby providing opportunities for cancer therapeutic interventions.

Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation

Background and aimsThe pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. MethodsFor in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. ResultsPDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αβ (PDGFRαβ) rather than PDGFRαα or PDGFRββ in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. ConclusionsThe results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.

Rho GTPase regulatory proteins contribute to podocyte morphology and function

Podocytes are a critical cellular component of the glomerular filtration barrier, whose strict permselectivity prohibits the passage of large proteins and charged species into the urine. Phenotypic variability or injury of these highly specialized cells can lead to proteinuria and has been linked with altered activity of Rho GTPases, which are strongly associated with the actin cytoskeleton. Notable regulators of these intracellular molecular switches are called guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). In this study, the roles of several GEFs in podocyte morphology and activity were investigated, including ECT2, ARHGEF2, ARHGEF26, and ARHGEF40. Results from RhoA and Rac1 G-LISA Activation Assays indicated that the absence of ARHGEF40 impairs epidermal growth factor (EGF)-stimulated RhoA and Rac1 activation, whereas knockout of ARHGEF2 and ARHGEF26 may selectively diminish RhoA activation. Furthermore, filopodia formation was hindered for the ARHGEF40 knockout. There are a number of additional investigations underway to understand Rho GTPase regulatory proteins, including the elimination of new sets of GEFs and GAPs in vivo. It is hopeful that these studies can provide insights into potential novel therapeutic strategies for proteinuria.

The Dual Function of RhoGDI2 in Immunity and Cancer.

RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) specific for the Rho family of small GTPases. It is highly expressed in hematopoietic cells but is also present in a large array of other cell types. RhoGDI2 has been implicated in multiple human cancers and immunity regulation, where it can display a dual role. Despite its involvement in various biological processes, we still do not have a clear understanding of its mechanistic functions. This review sheds a light on the dual opposite role of RhoGDI2 in cancer, highlights its underappreciated role in immunity and proposes ways to explain its intricate regulatory functions.

Abstract 1494: Differential Regulation of Rho Guanine Nucleotide Dissociation Inhibitors in Pancreatic Beta Cells Under Metabolic Stress

Abstract 1494: Differential Regulation of Rho Guanine Nucleotide Dissociation Inhibitors in Pancreatic Beta Cells Under Metabolic Stress

Genetic analysis of GEFs and GDIs in rice reveals the roles of OsGEF5, OsGDI1, and OsGEF3 in the regulation of grain size and plant height

Guanine nucleotide exchange factors (GEFs) and guanine nucleotide-dissociation inhibitors (GDIs) regulate small GTPase proteins, which function as molecular switches in various signaling pathways, but their identification and functions in plants are not well understood. Using in-silico analysis and transgenic approaches, respectively, we dissected the evolutionary relationships and functions of all GEF and GDI genes in rice. Intron-exon distribution and phylogenetic analyses identified 30 GEF and 10 GDI genes in rice that shared close evolutionary relationships with other eukaryotes. Tissue-specific expression and co-expression analyses revealed that phylogenetically related genes had similar expression patterns. GEF and GDI genes were highly expressed in panicles, hulls, and stamens. Co-expression network analysis identified panicle and stamen-specific modules of co-expressed genes in both families. Mapping of these genes in known protein interactomes further identified two and one small G-protein sub-networks. A mutant library of GEF and GDI families was constructed by CRISPR knockout of each gene, and their genotypes and phenotypes were confirmed. Phenotype changes occurred with the mutation of only three genes (OsGEF5, OsGDI1, and OsGEF3). OsGEF5 and OsGDI1 single mutants exhibited significantly reduced height and longer and thinner grains, whereas OsGEF3 mutants had reduced grain length compared to the wild type. Haplotype and eGWAS analyses showed that natural variations in the three genes affected gene expression in reproductive tissues that were significantly associated with the phenotypic variation. BiFC assays demonstrated that GDI1 and GEF3 interacted with grain-size protein GS3, pointing to a role of these genes in the regulation of grain size and plant architecture connected to heterotrimeric G-proteins in rice.

RhoGDI1 interacts with PHLDA2, suppresses the proliferation, migration, and invasion of trophoblast cells, and participates in the pathogenesis of preeclampsia.

Preeclampsia (PE) is a pregnancy-associated disease, which is the major cause of mortality on maternity and perinatal infants. It is hypothesized that PE is a consequence of the dysfunction of the trophoblast cells. Pleckstrin homology-like domain, family A, member 2 (PHLDA2) was shown to inhibit the proliferation, migration, and invasion of trophoblast cells in our previous studies. However, the mechanism by which PHLDA2 affects trophoblast cell function has not been clarified. In the current study, co-immunoprecipitation (Co-IP) with mass spectroscopy analysis was used to explore the proteins that interacted with PHLDA2. A total of 291 candidate proteins were found to be associated with PHLDA2. The interaction between PHLDA2 and Rho guanine nucleotide dissociation inhibitor (RhoGDI) 1 was identified by Co-IP and immunofluorescence staining. Western blot analysis indicated that overexpression of PHLDA2 resulted in upregulation of the RhoGDI1 protein levels, which were stabilized in the presence of cycloheximide. Similarly, overexpression of RhoGDI1 promoted PHLDA2 expression and its stability. Furthermore, pull-down and Co-IP results indicated that PHLDA2 repressed the activity of Rho guanosine triphosphate hydrolase family proteins by regulating RhoGDI1 expression. In addition, RhoGDI1 expression was upregulated in the placental tissues of patients with PE. The effects of the suppression of PHLDA2 expression on proliferation, migration, and invasion of trophoblast cells were partly abrogated following knockdown of RhoGDI1. Taken together, the data indicated that RhoGDI1 mediated regulation of PHLDA2 on the biological behavior of trophoblast cells and may participate in the pathophysiology of PE.

An optogenetic GDI drives GPCR and Gα independent macrophage migration

Activators of G protein signaling (AGS) proteins play diverse functional roles in eukaryotic cells, including asymmetric cell division and membrane protein trafficking. The group II AGS (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) consisting of four G‐protein regulatory (GPR) GoLoco motifs that bind to Gαi/o in the G protein heterotrimer. AGS3 binding should therefore release Gβγ from the heterotrimer. However, the ability of AGS3 to directly signal through Gβγ in a living cell has yet to be demonstrated. To investigate the role of AGS3 in Gβγ signaling independent of G protein‐coupled receptors, we engineered optogenetic AGS3 using one or more consensus GPR sequences. This consensus sequence has shown higher binding efficacy towards Gαi GDP comparative to the wild type. Upon expression, our Opto‐AGS3 showed localized PIP3 generation and directional migration in RAW 264.7 cells due to Gβγ generation, indicating non‐canonical Gβγ signaling of AGS3. Our results also provide GPCR and Gα independent optogenetic control of Gβγ signaling with subcellular spatial and millisecond temporal control.

Effects of Bisphenols A, AF, and S on Endochondral Ossification and the Transcriptome of Murine Limb Buds.

Bisphenols are a family of chemicals commonly used to produce polycarbonate plastics and epoxy resins. Exposure to bisphenol A (BPA) is associated with a variety of adverse effects; thus, many alternatives to BPA, such as bisphenol AF (BPAF) and bisphenol S (BPS), are now emerging in consumer products. We have determined the effects of 3 bisphenols on endochondral ossification and the transcriptome in a murine limb bud culture system. Embryonic forelimbs were cultured in the presence of vehicle, BPA, BPAF, or BPS. BPA (≥10 μM), BPAF (≥1 μM), and BPS (≥50 μM) reduced the differentiation of hypertrophic chondrocytes and osteoblasts. Chondrogenesis was suppressed by exposure to ≥50 μM BPA, ≥5 μM BPAF, or 100 μM BPS and osteogenesis was almost completely arrested at 100 μM BPA or 10 μM BPAF. RNA sequencing analyses revealed that the total number of differentially expressed genes increased with time and the concentration tested. BPA exposure differentially regulated 635 genes, BPAF affected 554 genes, whereas BPS affected 95 genes. Although the genes that were differentially expressed overlapped extensively, each bisphenol also induced chemical-specific alterations in gene expression. BPA- and BPAF-treated limbs exhibited a downregulation of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) signaling genes. Exposure to BPA and BPS resulted in the upregulation of key genes involved in cholesterol biosynthesis, whereas exposure to BPAF induced an upregulation of genes involved in bone formation and in the p53 signaling pathway. These data suggest that BPAF may be more detrimental to endochondral ossification than BPA, whereas BPS is of comparable toxicity to BPA.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

2-O-Octadecylascorbic acid represses RhoGDIβ expression and ameliorates DNA damage-induced abnormal spindle orientations.

The appropriate regulation of spindle orientation maintains proper tissue homeostasis and avoids aberrant tissue repair or regeneration. Spindle misorientation due to imbalance or improper functioning leads to a loss of tissue integrity and aberrant growth, such as tissue loss or overgrowth. Pharmacological manipulation to prevent spindle misorientation will enable a better understanding of how spindle orientation is involved in physiological and pathological conditions and will provide therapeutic possibilities to treat patients associated with abnormal tissue function caused by spindle misorientation. N-terminal-deleted Rho guanine nucleotide dissociation inhibitor β (RhoGDIβ/RhoGDI2/LyGDI) produced by caspase-3 activation perturbs spindle orientation in surviving cells following exposure to either ionizing radiation or UVC. Thus, presumably, RhoGDIβ cleaved by caspase-3 activation acts as a determinant of radiation-induced spindle misorientation that promote aberrant tissue repair due to deregulation of directional organization of cell population and therefore becomes a potential target of drugs to prevent such response. The objective of this study was to screen and identify chemicals that suppress RhoGDIβ expression. We focused our attention on ascorbic acid (AA) derivatives because of their impact on the maintenance of skin tissue homeostasis. Here, we screened for AA derivatives that suppress RhoGDIβ expression in HeLa cells and identified a lipophilic derivative, 2-O-octadecylascorbic acid (2-OctadecylAA), as a novel RhoGDIβ inhibitor that ameliorated ionizing radiation-induced abnormal spindle orientations. Among all examined AA derivatives, which were also antioxidative, the inhibition activity was specific to 2-OctadecylAA. Therefore, this activity was not due to simple antioxidant properties. 2-OctadecylAA was previously shown to prevent hepatocellular carcinoma development. Our findings suggest that the anticarcinogenic effects of 2-OctadecylAA are partly due to RhoGDIβ inhibition mechanisms by which spindle orientation perturbations are attenuated. Thus, the molecular targeting features of RhoGDIβ warrant its further development for the treatment or control of spindle orientation abnormalities that affect epithelial homeostasis.

The lipid phosphatase-like protein PLPPR1 associates with RhoGDI1 to modulate RhoA activation in response to axon growth inhibitory molecules.

Phospholipid Phosphatase-Related Protein Type 1 (PLPPR1) is a member of a family of lipid phosphatase related proteins, integral membrane proteins characterized by six transmembrane domains. This family of proteins is enriched in the brain and recent data indicate potential pleiotropic functions in several different contexts. An inherent ability of this family of proteins is to induce morphological changes, and we have previously reported that members of this family interact with each other and may function co-operatively. However, the function of PLPPR1 is not yet understood. Here we show that the expression of PLPPR1 reduces the inhibition of neurite outgrowth of cultured mouse hippocampal neurons by chondroitin sulfate proteoglycans and the retraction of neurites of Neuro-2a cells by lysophosphatidic acid (LPA). Further, we show that PLPPR1 reduces the activation of Ras homolog family member A (RhoA) by LPA in Neuro-2a cells, and that this is because of an association of PLPPR1with the Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI1). These results establish a novel signaling pathway for the PLPPR1 protein.

Small GTPases of the Ras and Rho Families Switch on/off Signaling Pathways in Neurodegenerative Diseases.

Small guanosine triphosphatases (GTPases) of the Ras superfamily are key regulators of many key cellular events such as proliferation, differentiation, cell cycle regulation, migration, or apoptosis. To control these biological responses, GTPases activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some small GTPases also guanine nucleotide dissociation inhibitors (GDIs). Moreover, small GTPases transduce signals by their downstream effector molecules. Many studies demonstrate that small GTPases of the Ras family are involved in neurodegeneration processes. Here, in this review, we focus on the signaling pathways controlled by these small protein superfamilies that culminate in neurodegenerative pathologies, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Specifically, we concentrate on the two most studied families of the Ras superfamily: the Ras and Rho families. We summarize the latest findings of small GTPases of the Ras and Rho families in neurodegeneration in order to highlight these small proteins as potential therapeutic targets capable of slowing down different neurodegenerative diseases.

Cullin 3/KCTD5 Promotes the Ubiqutination of Rho Guanine Nucleotide Dissociation Inhibitor 1 and Regulates Its Stability.

Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays important roles in numerous cellular processes, including cell motility, adhesion, and proliferation, by regulating the activity of Rho GTPases. Its expression is altered in various human cancers and is associated with malignant progression. Here, we show that RhoGDI1 interacts with Cullin 3 (CUL3), a scaffold protein for E3 ubiquitin ligase complexes. Ectopic expression of CUL3 increases the ubiquitination of RhoGDI1. Furthermore, potassium channel tetramerization domain containing 5 (KCTD5) also binds to RhoGDI1 and increases its interaction with CUL3. Ectopic expression of KCTD5 increases the ubiquitination of RhoGDI1, whereas its knockdown by RNA interference has the opposite effect. Depletion of KCTD5 or expression of dominant-negative CUL3 (DN-CUL3) enhances the stability of RhoGDI1. Our findings reveal a previously unknown mechanism for controlling RhoGDI1 degradation that involves a CUL3/KCTD5 ubiquitin ligase complex.

Identification of Rho GEF and RhoA Activation by Pull-Down Assays.

The small GTPase RhoA participates in actin and microtubule machinery, cell migration and invasion, gene expression, vesicular trafficking and cell cycle, and its dysregulation is a determining factor in many pathological conditions. Similar to other Rho GTPases, RhoA is a key component of the wound-healing process, regulating the activity of different participating cell types. RhoA gets activated upon binding to guanine nucleotide exchange factors (GEFs), which catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). GTPase-activating proteins (GAPs) mediate the exchange of GTP to GDP, inactivating RhoA, whereas guanine nucleotide dissociation inhibitors (GDIs) preserve the inactive pool of RhoA proteins in the cytosol. RhoA and Rho GEF activation is detected by protein pull-down assays, which use chimeric proteins with Rhotekin and G17A mutant RhoA as "bait" to pull down active RhoA and RhoA GEFs, respectively. In this chapter, we describe an optimized protocol for performing RhoA and GEF pull-down assays.