Ras Superfamily Of GTPases Research Articles

Identification and Functional Analysis of Ras-Related Associated with Diabetes Gene (rrad) in Edwardsiella piscicida-Resistant Individuals of Japanese Flounder (Paralichthys olivaceus).

Ras-related associated with diabetes (RRAD) is a member of the Ras GTPase superfamily that plays a role in several cellular functions, such as cell proliferation and differentiation. In particular, the superfamily acts as an NF-κB signaling pathway inhibitor and calcium regulator to participate in the immune response pathway. A recent transcriptome study revealed that rrad was expressed in the spleen of disease-resistant Japanese flounder (Paralichthys olivaceus) individuals compared with disease-susceptible individuals, and the results were also verified by qPCR. Thus, the present study aimed to explore how rrad regulates antimicrobial immunity via the NF-κB pathway. First, the coding sequence of P. olivaceus rrad was identified. The sequence was 1092 bp in length, encoding 364 amino acids. Based on phylogenetic and structural relationship analyses, P. olivaceus rrad appeared to be more closely related to teleosts. Next, rrad expression differences between disease-resistant and disease-susceptible individuals in immune-related tissues were evaluated, and the results revealed that rrad was expressed preferentially in the spleen of disease-resistant individuals. In response to Edwardsiella piscicida infection, rrad expression in the spleen changed. In vitro, co-culture was carried out to assess the hypo-methylated levels of the rrad promoter in the disease-resistant spleen, which was consistent with the high mRNA expression. The siRNA-mediated knockdown of rrad performed with the gill cell line of P. olivaceus affected many rrad-network-related genes, i.e., dcp1b, amagt, rus1, rapgef1, ralbp1, plce1, rasal1, nckipsd, prkab2, cytbc-1, sh3, and others, as well as some inflammation-related genes, such as bal2 and Il-1β. In addition, flow cytometry analysis showed that rrad overexpression was more likely to induce cell apoptosis, with establishing a link between rrad's function and its potential roles in regulating the NF-κB pathway. Thus,. the current study provided some clarity in terms of understanding the immune response about rrad gene differences between disease-resistant and disease-susceptible P. olivaceus individuals. This study provides a molecular basis for fish rrad gene functional analysis and may serve as a reference for in-depth of bacterial disease resistance of teleost.

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions.

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

Ras superfamily GTPases and signal transduction in Euglena gracilis

Biological complexity is challenging to define, but can be considered through one or more features, including overall genome size, number of genes, morphological features, multicellularity, number of life cycle stages and the ability to adapt to different environments. Euglena gracilis meets several of these criteria, with a large genome of ∼38,000 protein coding genes and a considerable ability to survive under many different conditions, some of which can be described as challenging or harsh. Potential molecular exemplars of complexity tying these aspects together are signalling pathways, including GTPases, kinases and ubiquitylation, which increase the functionality of the gene-encoded proteome manyfold. Each of these examples can modulate both protein activity and gene expression. To address the connection between genome size and complexity I have undertaken a brief, and somewhat qualitative, survey of the small ras-like GTPase superfamily of E. gracilis. Unexpectedly, apart from Rab-GTPases which control intracellular transport and organelle identify, the size of the GTPase cohort is modest, and, for example, has not scaled with gene number when compared to the close relatives, trypanosomatids. I suggest that understanding the functions of this protein family will be vital to uncovering the complexity of E. gracilis biology.

Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement.

In this article, we provide detailed protocols on using optogenetic dimerizers to acutely perturb activities of guanine nucleotide exchange factors (GEFs) specific to Ras, Rac or Rho small GTPases of the migratory networks in various mammalian and amoeba cell lines. These GEFs are crucial components of signal transduction networks which link upstream G-protein coupled receptors to downstream cytoskeletal components and help cells migrate through their dynamic microenvironment. Conventional approaches to perturb and examine these signaling and cytoskeletal networks, such as gene knockout or overexpression, are protracted which allows networks to readjust through gene expression changes. Moreover, these tools lack spatial resolution to probe the effects of local network activations. To overcome these challenges, blue light-inducible cryptochrome- and LOV domain-based dimerization systems have been recently developed to control signaling or cytoskeletal events in a spatiotemporally precise manner. We illustrate that, within minutes of global membrane recruitment of full-length GEFs or their catalytic domains only, widespread increases or decreases in F-actin rich protrusions and cell size occur, depending on the particular node in the networks targeted. Additionally, we demonstrate localized GEF recruitment as a robust assay system to study local network activation-driven changes in polarity and directed migration. Altogether, these optical tools confirmed GEFs of Ras superfamily GTPases as regulators of cell shape, actin dynamics, and polarity. Furthermore, this optogenetic toolbox may be exploited in perturbing complex signaling interactions in varied physiological contexts including mammalian embryogenesis.

Covalent Fragment Inhibits RhoA Activation by Guanine Exchange Factors.

Ras homolog gene family member (RhoA) is a GTPase and a member of the RAS superfamily of GTPases. RhoA is a master regulator of the actin cytoskeleton. It inhibits axon growth preventing repair and recovery following spinal cord and traumatic brain injuries. Despite decades of research into the biological function of Rho GTPases, there exist no small-molecule Rho inhibitors. Here, we screen a library of cysteine electrophiles to explore whether covalent bond formation at Cys-107 leads to inhibition of RhoA activation by guanine exchange factor Trio. Two fragments, propiolamide 1 (ACR-895) and acrylamide 2 (ACR-917), inhibited RhoA nucleotide exchange by Trio in a time-dependent manner. The fragments formed a covalent bond with wild-type RhoA but not Cys107Ser RhoA mutant. Time- and concentration-dependent studies led to equilibrium constants KIs and reaction rates that correspond to t1/2 values in the single-digit hour range. One fragment was selective for RhoA over Rac1 GTPase and had no effect on KRAS nucleotide exchange by SOS1. The fragments did not inhibit RhoA binding to ROCK effector protein. This work establishes Cys-107 as a suitable site for Rho GTPase inhibition and provides fragment starting points for the future development of Rho GTPase covalent inhibitors that could have profound implications in the treatment of patients with injuries of the central nervous system.

Wavelet coherence phase analysis decodes the universal switching mechanism of Ras GTPase superfamily

The Ras superfamily of GTPases regulate critical cellular processes by shuttling between GTP-bound ON and GDP-bound OFF states. This switching mechanism is attributed to the conformational changes in two loops, SWI and SWII, upon GTP binding and hydrolysis. Since these conformational changes vary across the Ras superfamily, there is no generic parameter to define their functional states. A unique wavelet coherence (WC) analysis-based approach developed here shows that the structural changes in switch regions could be mapped onto the wavelet coherence phase couplings (WPCs). Thus, WPCs could serve as unique parameters to define their functional states. Disentanglement of WPCs in oncogenic GTPases shows how breakdown of structural allostery leads to their aberrant function. These observations stand out even for simulated ensemble of switch region conformers. Overall, for the first time, we show that WPCs could unravel the latent structural deviations in Ras proteins to decode their universal switching mechanism.

Identification, classification and expression analysis of the Ras superfamily genes in the Pacific white shrimp, Litopenaeus vannamei

The Ras superfamily of small guanosine triphosphatases (GTPases) are a large group of small GTP-binding proteins, which play crucial roles in basic cellular processes in all eukaryotes. In this study, by analyzing the gene structure, temporal and spatial expression patterns, a total of 108 Ras superfamily genes were identified in the genome of the Pacific white shrimp Litopenaeus vannamei. We found these genes included not only the classical Ras GTPase superfamily members, but also some unconventional and novel Ras GTPase proteins, which have unknown functions and unique expression patterns. All Ras superfamily genes of L. vannamei were highly conserved within the core G domain and closely related in phylogeny, but they might have two different evolutionary origins. In addition, different Ras GTPase genes exhibited distinct expression patterns in different tissues, development/molting stages and WSSV infection samples of L. vannamei, suggesting that they may have a high functional specialization, and play important roles in regulating the biological processes of cell differentiation, growth and development, immune response, etc. This study provides important clues for the structure, classification, evolution and function of Ras superfamily in shrimp.

Systematic pan-cancer analysis identifies RALA as a tumor targeting immune therapeutic and prognostic marker.

RALA is a member of the small GTPase Ras superfamily and has been shown to play a role in promoting cell proliferation and migration in most tumors, and increase the resistance of anticancer drugs such as imatinib and cisplatin. Although many literatures have studied the cancer-promoting mechanism of RALA, there is a lack of relevant pan-cancer analysis. This study systematically analyzed the differential expression and mutation of RALA in pan-cancer, including different tissues and cancer cell lines, and studied the prognosis and immune infiltration associated with RALA in various cancers. Next, based on the genes co-expressed with RALA in pan-cancer, we selected 241 genes with high correlation for enrichment analysis. In terms of pan-cancer, we also analyzed the protein-protein interaction pathway of RALA and the application of small molecule drug Guanosine-5'-Diphosphate. We screened hepatocellular cancer (HCC) to further study RALA. The results indicated that RALA was highly expressed in most cancers. RALA was significantly correlated with the infiltration of B cells and macrophages, as well as the expression of immune checkpoint molecules such as CD274, CTLA4, HAVCR2 and LAG3, suggesting that RALA can be used as a kind of new pan-cancer immune marker. The main functions of 241 genes are mitosis and protein localization to nucleosome, which are related to cell cycle. For HCC, the results displayed that RALA was positively correlated with common intracellular signaling pathways such as angiogenesis and apoptosis. In summary, RALA was closely related to the clinical prognosis and immune infiltration of various tumors, and RALA was expected to become a broad-spectrum molecular immune therapeutic target and prognostic marker for pan-cancer.

Validation of a Novel Rac1 Inhibitor As Potent Secdrug Against Activating Ras Mutant and Drug-Resistant Multiple Myeloma

Multiple myeloma is the second-most common hematopoietic malignancy in the United States. Although several drugs have so far been approved by the FDA, myeloma still remains an incurable disease with dose-limiting toxicities and resistance to primary drugs like proteasome inhibitors (PIs) and Immunomodulatory drugs (IMiDs). Furthermore, activating Ras gene (NRAS, KRAS, and HRAS) mutations are known to occur in >50% of myeloma patients, which are also associated with greater tumor burden, cancer aggressiveness, high recurrence rates, and poor survival. We have recently established a novel drug development/re-purposing pipeline called secDrug that incorporates a pharmacogenomics data-driven computational algorithm to introduce several new drug candidates (secDrugs), including EHT1864 (a Rac1 (Ras superfamily GTPase) inhibitor), CCT018159 (Hsp90 inhibitor), and CP466722 (a reversible ATM inhibitor) as synergistic partners of primary drugs against drug-resistant myeloma. The overall objective of this study is to validate EHT1864 using in vitro and ex vivo models of relapsed and refractory myeloma (RRMM) patients. First, we used single-cell RNA sequencing (scRNAseq) as a novel screening tool to demonstrate that EHT1864 is potentially effective against myeloma subclones based on the enrichment of target genes (Figure 1). For in vitro validation, we used a human myeloma cell line (HMCL) panel representing drug-sensitive (FLAM76), innate/refractory resistance (LP1), and acquired/relapsed resistance (parental and clonally derived PI-resistant and IMiD-resistant HMCL pairs U266 P/VR, RPMI P/VR, MM1S P/VR/LenR). FLAM76 KRas (FLAM76-K12) and FLAM76 Nras (FLAM76-N12) cell lines were generated using Adeno-associated viral (AAV) vector-mediated delivery of CRISPR-Cas9 for genome editing in humans. Ras mutations were confirmed using Sanger DNA sequencing method. Cell viability measured using CellTiter-Glo luminescent cell viability assay showed the potency of EHT1864 as a single-agent (IC50 range 15-40µM) as well as in combination with PIs (represented by Ixazomib) and IMiDs (represented by Lenalidomide). Combination index (CI) values calculated using Chou-Talalay's CI theorem were consistently less than 0.9, while all the four synergy scores (HSA, Bliss, Loewe, and ZIP) were higher than 5, demonstrating high synergy. Cell death due to apoptosis was confirmed using flow cytometry (Annexin-V-Propidium Iodide staining), Caspase 3/7 activity assay, and immunoblotting. Interestingly, EHT1864 was most potent against drug-resistant and Ras-mutant HMCLs. Further, we showed that EHT1864 treatment also affects ROS generation and mitochondrial membrane potential, indicating that EHT1864 induces apoptosis via mitochondria-mediated pathway. Finally, we performed pre-vs-post-treatment genome-wide transcriptome analysis (tumor mRNAseq) and single-cell multi-ome analysis (single-cell RNA sequencing/scRNAseq + single-cell Assay of Transposase Accessible Chromatin sequencing/scATACseq) analysis to derive molecular signatures and pathways representing the basis of (on-target and off-target) efficacy of EHT1864 and drug synergy at the bulk tumor and subclonal levels. Finally, we will perform single-cell proteomics (high-dimensional immunophenotyping using mass cytometry/CyTOF/Cytometry time of flight) in CD138+ bone marrow cells derived from newly-diagnosed and relapsed myeloma patients (ex vivo) to demonstrate efficacy in RRMM. Thus, our work lays a framework to establish EHT1864 as a clinical trial-ready therapeutic option for the management of drug-resistant myeloma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Non-canonical Small GTPase RBJ Promotes NSCLC Progression Through the Canonical MEK/ERK Signaling Pathway.

Although the majority of members belonging to the small GTPase Ras superfamily have been studied in several malignancies, the function of RBJ has remained unclear, particularly in non-small cell lung cancer (NSCLC). The research aims to determine the function of RBJ in NSCLC. The levels of RBJ protein in tumor tissue and para-carcinoma normal tissue were ascertained via immunohistochemistry (IHC). The growth, migration, and invasion of NSCLC cells were assessed by 5- ethynyl-2-deoxyuridine (EdU) assay, colony formation, cell counting kit-8 (CCK8), transwell and wound healing assays. Furthermore, a nude mouse xenograft model was established to study the function of RBJ in tumorigenesis in vivo. The IHC analysis revealed that the protein levels of RBJ were notably increased in tumor tissue and positively associated with the clinical stage. In addition, the knockdown of RBJ restrained the growth, invasion, and migration of NSCLC cell lines by inhibiting the epithelial-mesenchymal transition (EMT) through the MEK/ERK signaling pathway. Accordingly, opposite results were observed when RBJ was overexpressed. In addition, the overexpression of RBJ accelerated tumor formation by A549 cells in nude mice. RBJ promoted cancer progression in NSCLC by activating EMT via the MEK/ERK signaling. Thus, RBJ could be used as a potential therapeutic against NSCLC.

Autophagy induced by Rab1a-ULK1 interaction promotes porcine reproductive and respiratory syndrome virus replication

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus from the Nidovirales order, continues to be a threat to the swine industry worldwide causing reproductive failure and respiratory disease in pigs. Previous studies have demonstrated that autophagy plays a positive role in PRRSV replication. However, its mechanism is less clearly understood. Herein, we report first that the protein level of Rab1a, a member of the Ras superfamily of GTPases, is upregulated during PRRSV infection. Subsequently, we demonstrate that Rab1a enhances PRRSV replication through an autophagy pathway as evidenced by knocking down the autophagy-related 7 (ATG7) gene, the key adaptor of autophagy. Importantly, we reveal that Rab1a interacts with ULK1 and promotes ULK1 phosphorylation dependent on its GTP-binding activity. These data indicate that PRRSV utilizes the Rab1a-ULK1 complex to initiate autophagy, which, in turn, benefits viral replication. These findings further highlight the interplay between PRRSV replication and the autophagy pathway, deepening our understanding of PRRSV infection.

The Small Ras Superfamily GTPase Rho4 of the Maize Anthracnose Fungus Colletotrichum graminicola Is Required for β-1,3-glucan Synthesis, Cell Wall Integrity, and Full Virulence.

Small Ras superfamily GTPases are highly conserved regulatory factors of fungal cell wall biosynthesis and morphogenesis. Previous experiments have shown that the Rho4-like protein of the maize anthracnose fungus Colletotrichum graminicola, formerly erroneously annotated as a Rho1 protein, physically interacts with the β-1,3-glucan synthase Gls1 (Lange et al., 2014; Curr. Genet. 60:343–350). Here, we show that Rho4 is required for β-1,3-glucan synthesis. Accordingly, Δrho4 strains formed distorted vegetative hyphae with swellings, and exhibited strongly reduced rates of hyphal growth and defects in asexual sporulation. Moreover, on host cuticles, conidia of Δrho4 strains formed long hyphae with hyphopodia, rather than short germ tubes with appressoria. Hyphopodia of Δrho4 strains exhibited penetration defects and often germinated laterally, indicative of cell wall weaknesses. In planta differentiated infection hyphae of Δrho4 strains were fringy, and anthracnose disease symptoms caused by these strains on intact and wounded maize leaf segments were significantly weaker than those caused by the WT strain. A retarded disease symptom development was confirmed by qPCR analyses. Collectively, we identified the Ras GTPase Rho4 as a new virulence factor of C. graminicola.

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

SUMMARYExcessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a′s membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Deletion of murine Rhoh leads to de-repression of Bcl-6 via decreased KAISO levels and accelerates a malignancy phenotype in a murine model of lymphoma

ABSTRACT RHOH/TFF, a member of the RAS GTPase super family, has important functions in lymphopoiesis and proximal T cell receptor signalling and has been implicated in a variety of leukaemias and lymphomas. RHOH was initially identified as a translocation partner with BCL-6 in non-Hodgkin lymphoma (NHL), and aberrant somatic hypermutation (SHM) in the 5ʹ untranslated region of the RHOH gene has also been detected in Diffuse Large B-Cell Lymphoma (DLBCL). Recent data suggest a correlation between RhoH expression and disease progression in Acute Myeloid Leukaemia (AML). However, the effects of RHOH mutations and translocations on RhoH expression and malignant transformation remain unknown. We found that aged Rhoh−/− (KO) mice had shortened lifespans and developed B cell derived splenomegaly with an increased Bcl-6 expression profile in splenocytes. We utilized a murine model of Bcl-6 driven DLBCL to further explore the role of RhoH in malignant behaviour by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice. The loss of Rhoh in Bcl-6Tg mice led to a more rapid disease progression. Mechanistically, we demonstrated that deletion of Rhoh in these murine lymphoma cells was associated with decreased levels of the RhoH binding partner KAISO, a dual-specific Zinc finger transcription factor, de-repression of KAISO target Bcl-6, and downregulation of the BCL-6 target Blimp-1. Re-expression of RhoH in RhohKOBcl-6Tg lymphoma cell lines reversed these changes in expression profile and reduced proliferation of lymphoma cells in vitro. These findings suggest a previously unidentified regulatory role of RhoH in the proliferation of tumour cells via altered BCL-6 expression. (250)

Rab11 and Its Role in Neurodegenerative Diseases.

Vesicles mediate the trafficking of membranes/proteins in the endocytic and secretory pathways. These pathways are regulated by small GTPases of the Rab family. Rab proteins belong to the Ras superfamily of GTPases, which are significantly involved in various intracellular trafficking and signaling processes in the nervous system. Rab11 is known to play a key role especially in recycling many proteins, including receptors important for signal transduction and preservation of functional activities of nerve cells. Rab11 activity is controlled by GEFs (guanine exchange factors) and GAPs (GTPase activating proteins), which regulate its function through modulating GTP/GDP exchange and the intrinsic GTPase activity, respectively. Rab11 is involved in the transport of several growth factor molecules important for the development and repair of neurons. Overexpression of Rab11 has been shown to significantly enhance vesicle trafficking. On the other hand, a reduced expression of Rab11 was observed in several neurodegenerative diseases. Current evidence appears to support the notion that Rab11 and its cognate proteins may be potential targets for therapeutic intervention. In this review, we briefly discuss the function of Rab11 and its related interaction partners in intracellular pathways that may be involved in neurodegenerative processes.

Signaling Pathways that Regulate Macropinocytosis in Mammalian Cells.

Macropinocytosis is an evolutionarily conserved endocytic pathway that mediates non-selective uptake of extracellular fluid in bulk. Macropinocytosis is initiated by localized polymerization of the actin cytoskeleton, which generates plasma membrane protrusions that enclose part of the environment into large endocytic vesicles. From amoebae to mammalian cells, the actin dynamics that drive macropinosome formation are regulated by a conserved set of intracellular signaling proteins including Ras superfamily GTPases and PI3-kinases. In mammalian cells, multiple upstream signaling pathways control activity of these core regulators in response to cell-extrinsic and cell-intrinsic stimuli. Growth factor signaling pathways play a central role in macropinocytosis induction. In addition, an increasing number of functionally diverse processes has been identified as macropinocytosis regulators, including several nutrient-sensing and developmental signaling pathways. Many of these signaling pathways have proto-oncogenic properties, and their dysregulation drives the high macropinocytic activity that is commonly observed in cancer cells. These regulatory principles illustrate how macropinocytosis is controlled by complex upstream inputs to exert diverse cellular functions in physiological and pathological contexts.

Relationship between Rab and insulin-like proteins in the nervous system of Bombyx mori

Rab proteins are small GTP-binding proteins and are the largest family in the Ras GTPase superfamily and mediate vesicular transport in cells. Diverse insulin-like peptides, such as bombyxin, are synthesized in the brain and secreted into the haemolymph by the corpus allatum (CA). In the brain of Bombyx mori, Rabs are expressed in a specific area; however, which Rabs actually link the secretion of bombyxin remains unknown. A double-staining analysis of nine Rabs (Rab1, 3, 6, 7, 14, 21, 26, 39 and X4) and bombyxin indicated that Rab3-, Rab7-, Rab39- and RabX4-immunohistochemical reactivity (ir) areas overlapped with bombyxin-ir in the brain and CA in B. mori, while Rab6-, Rab14- and Rab21-irs partially overlapped in the CA. Rab1-ir occurred in the other immunopositive areas in CA. Rab26-ir did not occur in the brain. Rab39-ir occurred in UNC104, Rab39- effector, -immunopositive neurons in the brain and CA. Thus, Rab3, 7, 39 and X4 may regulate the exocytosis of bombyxin.

Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification.

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound “OFF” state and GTP-bound “ON” state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound “ON” state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

Mechanistic Differences of Activation of Rac1P29S and Rac1A159V.

Rac1 is a small GTPase that plays key roles in actin reorganization, cell motility, and cell survival/growth as well as in various cancer types and neurodegenerative diseases. Similar to other Ras superfamily GTPases, Rac1 switches between active GTP-bound and inactive GDP-bound states. Switch I and II regions open and close during GDP/GTP exchange. P29S and A159V (paralogous to K-RasA146) mutations are the two most common somatic mutations of Rac1. Rac1P29S is a known hotspot for melanoma, whereas Rac1A159V most commonly occurs in head and neck cancer. To investigate how these substitutions induce the Rac1 dynamics, we used atomistic molecular dynamics simulations on the wild-type Rac1 and two mutant systems (P29S and A159V) in the GTP bound state, and on the wild-type Rac1 and P29S mutated system in the GDP bound state. Here, we show that P29S and A159V mutations activate Rac1 with different mechanisms. In Rac1P29S-GTP, the substitution increases the flexibility of Switch I based on RMSF and dihedral angle calculations and leads to an open conformation. We propose that the open Switch I conformation is one of the underlying reasons for rapid GDP/GTP exchange of Rac1P29S. On the other hand, in Rac1A159V-GTP, some of the contacts of the guanosine ring of GTP with Rac1 are temporarily lost, enabling the guanosine ring to move toward Switch I and subsequently close the switch. Rac1A159V-GTP adopts a Ras state 2 like conformation, where both switch regions are in closed conformation and Thr35 forms a hydrogen bond with the nucleotide.

Atomic Resolution Mechanism of GTP Recognition by GTPase

Ras superfamily GTPases act as molecular switches that generally cycle between a GDP-bound “off” state and a GTP-bound “on” state. The conformational changes associated with these different molecular states are involved in the regulation of multiple cellular processes. Despite significant efforts to directly target Ras activity, no anti-Ras drugs have been developed and taken into the clinic. Being a nightmare for drug developers, it is believed that they are “undruggable” due to high binding affinity of GTP to Ras and relatively smooth structure of Ras proteins devoid of any deep pocket where small molecule could bind.