Guanylate-binding protein 1 inhibits nuclear delivery of pseudorabies virus by disrupting structure of actin filaments.

The alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, responsible for severe economic losses to the swine industry worldwide. The interferon-inducible GTPase guanylate-binding protein 1 (GBP1) exhibits antiviral immunity. Our findings show that there is a robust upregulation in the expression of porcine GBP1 during PRV infection. GBP1 knockout promotes PRV infection, while GBP1 overexpression restricts it. Importantly, we found that GBP1 impeded the normal structure of actin filaments in a GTPase-dependent manner, preventing PRV virions from reaching the nucleus. We also discovered that viral US3 protein bound GBP1 to interfere with its GTPase activity. Finally, the interaction between US3 and GBP1 requires US3 serine/threonine kinase activity sites and the GTPase domain (aa 1 to 308) of GBP1. Taken together, this study offers fresh perspectives on how PRV manipulates the host's antiviral immune system.

Rabaptin5 acts as a key regulator for Rab7l1-mediated phagosome maturation process.

Phagosome maturation is an important innate defence mechanism of macrophages against pathogen infections. Phagosome-lysosome (P-L) fusion is a highly regulated process. Different RabGTPases are involved in P-L fusion. Rab7l1 is shown to regulate P-L fusion process. In this study, we demonstrate that Rabaptin5 is a guanine nucleotide exchange factor (GEF) for Rab7l1. We reveal that Rabaptin5 interacts with Rab7l1-GTP form and promotes its recruitment to phagosome. In the absence of Rabaptin5, localization of P-L markers like EEA1, Rab7, LAMP1 and LAMP2 was found to be poorer. Thus, our data suggest that Rabaptin5 works upstream to Rab7l1 and triggers Rab7l1 activation for further recruitment of P-L markers and downstream regulation of phagosomal maturation process.

Metal-Binding Propensity in the Mitochondrial Dynamin-Related Protein 1.

Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Metal binding, therefore, represents a potential strategy to deplete or purify Drp1 from native tissue lysates. Importantly, high concentrations of the metal in solution inhibit GTP hydrolysis and renders Drp1 inactive in membrane fission. Together, our results emphasize a metal-binding propensity, which could significantly impact Drp1 functions.

Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism.

BACKGROUND: Parkinson's disease (PD) is one of the most important neurodegenerative disorders in elderly people. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are found in a large proportion of the patients with sporadic and familial PD. Mutations can occur at different locations in the LRRK2. Patients with LRRK2 ROC-COR mutations face an increased risk of typical motor symptoms of PD, along with cognitive decline. An animal model with a monogenic LRRK2 gene mutation is a suitable model for exploring the pathophysiology of PD and identifying potential drug therapies. However, the effect of homozygous (HOM) LRRK2 in PD pathophysiology is unclear. METHODS: We established human LRRK2 (hLRRK2) R1441G HOM transgenic (Tg) mice to explore the phenotype and pathological features that are associated with hLRRK2 R1441G Tg mouse models and discuss the potential clinical relevance. The open field test (OFT) was performed to examine motor and nonmotor behaviors. A CatWalk analysis system was used to study gait function. [18F]FDOPA PET was used to investigate functional changes in the nigrostriatal pathway in vivo. Transmission electron microscopy was used to examine the morphological changes in mitochondria and lysosomes in the substantia nigra. RESULTS: The R1441G HOM Tg mice demonstrated gait disturbance and exhibited less anxiety-related behavior and exploratory behavior than mice with hLRRK2 at 12 months old. Additionally, [18F]FDOPA PET showed a reduction in FDOPA uptake in the striatum of the HOM Tg mice. Notably, there was significant lysosome and autophagosome accumulation in the cytoplasm of dopaminergic neurons in R1441G hemizygous (HEM) and HOM mice. Moreover, it was observed using transmission electron microscopy (TEM) that the mitochondria of R1441G Tg mice were smaller than those of hLRRK2 mice. CONCLUSION: This animal provides a novel HOM hLRRK2 R1441G Tg mouse model that reproduces some phenotype of Parkinsonism in terms of both motor and behavioral dysfunction. There is an increased level of mitochondrial fission and no change in the fusion process in the group of HOM hLRRK2 R1441G Tg mouse. This mutant animal model of PD might be used to study the mechanisms of mitochondrial dysfunction and explore potential new drug targets.

Porcine reproductive and respiratory syndrome virus non-structural protein 4 cleaves guanylate-binding protein 1 via its cysteine proteinase activity to antagonize GBP1 antiviral effect.

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1-69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.

Conformational change and GTPase activity of human tubulin: A comparative study on Alzheimer's disease and healthy brain.

In Alzheimer's disease (AD), the most common form of dementia, microtubules (MTs) play a pivotal role through their highly dynamic structure and instability. They mediate axonal transport that is crucial to synaptic viability. MT assembly, dynamic instability and stabilization are modulated by tau proteins, whose detachment initiates MT disintegration. Albeit extensive research, the role of GTPase activity in molecular mechanism of stability remains controversial. We hypothesized that GTPase activity is altered in AD leading to microtubule dynamic dysfunction and ultimately to neuronal death. In this paper, fresh tubulin was purified by chromatography from normal young adult, normal aged, and Alzheimer's brain tissues. Polymerization pattern, assembly kinetics and dynamics, critical concentration, GTPase activity, interaction with tau, intermolecular geometry, and conformational changes were explored via Förster Resonance Energy Transfer (FRET) and various spectroscopy methods. Results showed slower MT assembly process in samples from the brains of people with AD compared with normal young and aged brains. This observation was characterized by prolonged lag phase and increased critical and inactive concentration of tubulin. In addition, the GTPase activity in samples from AD brains was significantly higher than in both normal young and normal aged samples, concurrent with profound conformational changes and contracted intermolecular MT-tau distances as revealed by FRET. These alterations were partially restored in the presence of a microtubule stabilizer, paclitaxel. We proposed that alterations of both tubulin function and GTPase activity may be involved in the molecular neuropathogenesis of AD, thus providing new avenues for therapeutic approaches.

The Parkinson's disease-associated mutation N1437H impairs conformational dynamics in the G domain of LRRK2.

Parkinson disease-associated mutations within the GTPase domain Ras of complex proteins (ROC) of leucine rich repeat kinase 2 (LRRK2) result in an abnormal over-activation of its kinase domain. However, the mechanisms involved remain unclear. Recent study has shown that LRRK2 G-domain cycles between monomeric and dimeric conformations upon binding to GTP or guanosine diphosphate, and that the Parkinson's disease (PD)-associated R1441C/G/H mutations impair the G-domain monomer-dimer dynamics and trap the G-domain in a constitutive monomeric conformation. That led us to question whether other disease-associated mutations in G-domain would also affect its conformation. Here, we report that another PD-associated N1437H mutation also impairs its monomer-dimer conformational dynamics and GTPase activity. In contrast with mutations at R1441, ROCN1437H was found to be locked in a stable dimeric conformation in solution and its GTPase activity was ∼4-fold lower than that of the wild-type. Furthermore, the N1437H mutation reduced the GTP binding affinity by ∼2.5-fold when compared with other pathogenic G-domain mutations. Moreover, ROCN1437H was found to have a slower GTP dissociation rate, indicating that N1437H might interrupt the nucleotide exchange cycle. Taken together, our data support that conformational dynamics is important for LRRK2 GTPase activity and that the N1437H mutation impairs GTPase activity by locking the ROC domain in a persistently dimeric state.-Huang, X., Wu, C., Park, Y., Long, X., Hoang, Q. Q., Liao, J. The Parkinson's disease-associated mutation N1437H impairs conformational dynamics in the G domain of LRRK2.

Antiparasitic dibenzalacetone inhibits the GTPase activity of Rab6 protein of Leishmania donovani (LdRab6), a potential target for its antileishmanial effect.

Visceral leishmaniasis (VL, also known as kala-azar) is a vector borne disease caused by obligate intracellular protozoan parasite Leishmania donovani. To overcome the limitations of currently available drugs for VL, molecular target-based study is a promising tool to develop new drugs to treat this neglected tropical disease. One such target we recently identified from L. donovani (Ld) genome (WGS, clinical Indian isolate; BHU 1220, AVPQ01000001) is a small GTP-binding protein, Rab6 protein. We now report a specific inhibitor of the GTPase activity of Rab6 protein of L. donovani (LdRab6) without restricting host enzyme activity. First, to understand the nature of LdRab6 protein, we generated recombinant LdRab6 mutant proteins (rLdRab6) by systematically introducing deletion (two cysteine residues at C-terminal) and mutations [single amino acid substitutions in the conserved region of GTP (Q84L)/GDP(T38N) coding sequence]. The GTPase activity of rLdRab6:GTP and rLdRab6:GDP locked mutant proteins showed ~ 8-fold and ~ 1.5-fold decreases in enzyme activity, respectively, compared to the wild type enzyme activity. The mutant protein rLdRab6:ΔC inhibited the GTPase activity. Sequence alignment analysis of Rab6 protein of L. donovani with Homo sapiens showed identical amino acids in the G conserved region (GTP/GDP-binding sites) but it differed in the C-terminal region. We then evaluated the inhibitory activity of trans-dibenzalacetone (DBA, a synthetic analog of curcumin with strong antileishmanial activity reported earlier by us) in the GTPase activity of LdRab6 protein. Comparative molecular docking analysis of DBA and specific inhibitors of Rab proteins (Lovastatin, BFA, Zoledronate, and NE10790) indicated that DBA had optimum binding affinity with LdRab6 protein. This was further confirmed by the GTPase activity of DBA-treated LdRab6 which showed a basal GTP level significantly lower than that of the wild-type rLdRab6. The results confirm that DBA inhibits the GTPase activity of LdRab6 protein from L. donovani (LdRab6), a potential target for its antileishmanial effect.

Chloroplast division protein ARC3 acts on FtsZ2 by preventing filament bundling and enhancing GTPase activity.

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (Filamentous temperature sensitive Z) proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts, except that MinC was lost and replaced by the eukaryotic ARC3 (accumulation and replication of chloroplasts). Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPase assay and transmission electron microscopy in conjunction with single-particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division, FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. Three-dimensional reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6 (paralog of ARC6)-binding MORN (Membrane Occupation and Recognition Nexus) domain are in close proximity and suggest a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data.

Biochemical characterization of purified mammalian ARL13B protein indicates that it is an atypical GTPase and ARL3 guanine nucleotide exchange factor (GEF).

Primary cilia play central roles in signaling during metazoan development. Several key regulators of ciliogenesis and ciliary signaling are mutated in humans, resulting in a number of ciliopathies, including Joubert syndrome (JS). ARL13B is a ciliary GTPase with at least three missense mutations identified in JS patients. ARL13B is a member of the ADP ribosylation factor family of regulatory GTPases, but is atypical in having a non-homologous, C-terminal domain of ∼20 kDa and at least one key residue difference in the consensus GTP-binding motifs. For these reasons, and to establish a solid biochemical basis on which to begin to model its actions in cells and animals, we developed preparations of purified, recombinant, murine Arl13b protein. We report results from assays for solution-based nucleotide binding, intrinsic and GTPase-activating protein-stimulated GTPase, and ARL3 guanine nucleotide exchange factor activities. Biochemical analyses of three human missense mutations found in JS and of two consensus GTPase motifs reinforce the atypical properties of this regulatory GTPase. We also discovered that murine Arl13b is a substrate for casein kinase 2, a contaminant in our preparation from human embryonic kidney cells. This activity, and the ability of casein kinase 2 to use GTP as a phosphate donor, may be a source of differences between our data and previously published results. These results provide a solid framework for further research into ARL13B on which to develop models for the actions of this clinically important cell regulator.

Phosphatidic acid binding inhibits RGS1 activity to affect specific signaling pathways in Arabidopsis.

Modulation of the active versus inactive forms of the Gα protein is critical for the signaling processes mediated by the heterotrimeric G-protein complex. We have recently established that in Arabidopsis, the regulator of G-protein signaling (RGS1) protein and a lipid-hydrolyzing enzyme, phospholipase Dα1 (PLDα1), both act as GTPase-activity accelerating proteins (GAPs) for the Gα protein to attenuate its activity. RGS1 and PLDα1 interact with each other, and RGS1 inhibits the activity of PLDα1 during regulation of a subset of responses. In this study, we present evidence that this regulation is bidirectional. Phosphatidic acid (PA), a second messenger typically derived from the lipid-hydrolyzing activity of PLDα1, is a molecular target of RGS1. PA binds and inhibits the GAP activity of RGS1. A conserved lysine residue in RGS1 (Lys259 ) is directly involved in RGS1-PA binding. Introduction of this RGS1 protein variant in the rgs1 mutant background makes plants hypersensitive to a subset of abscisic acid-mediated responses. Our data point to the existence of negative feedback loops between these two regulatory proteins that precisely modulate the level of active Gα, consequently generating a highly controlled signal-response output.

Modulation of dynamin function by small molecules.

Dynamins are essential as membrane remodelers in various cellular processes, like receptor-mediated endocytosis, synaptic vesicle recycling and spermatogenesis. Moreover, dynamin is involved in the internalization of numerous viruses and in the motility of several cancer cell lines. As tools for dissecting the underlying mechanisms of these important biological processes and as potential future therapeutics, small molecules have been developed in the last two decades that modulate the functions of dynamin. In this review we give an overview of the compound classes that are currently in use and describe how they affect dynamin function.

Guanylate-Binding Protein 1 Inhibits Nuclear Delivery of Kaposi's Sarcoma-Associated Herpesvirus Virions by Disrupting Formation of Actin Filament.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a typical gammaherpesvirus that establishes persistent lifelong infection in host cells. In order to establish successful infection, KSHV has evolved numerous immune evasion strategies to bypass or hijack the host immune system. However, host cells still produce immune cytokines abundantly during primary KSHV infection. Whether the immune effectors produced are able to inhibit viral infection and how KSHV successfully conquers these immune effectors remain largely unknown. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on several RNA viruses; however, its function in DNA virus infection is less well understood. In this study, we found that KSHV infection increases both the transcriptional and protein levels of GBP1 at the early stage of primary infection by activating the NF-κB pathway. The overexpression of GBP1 significantly inhibited KSHV infection, while the knockdown of GBP1 promoted KSHV infection. The GTPase activity and dimerization of GBP1 were demonstrated to be responsible for its anti-KSHV activity. Furthermore, we found that GBP1 inhibited the nuclear delivery of KSHV virions by disrupting the formation of actin filaments. Finally, we demonstrated that replication and transcription activator (RTA) promotes the degradation of GBP1 through a proteasome pathway. Taken together, these results provide a new understanding of the antiviral mechanism of GBP1, which possesses potent anti-KSHV activity, and suggest the critical role of RTA in the evasion of the innate immune response during primary infection by KSHV.IMPORTANCE GBP1 can be induced by various cytokines and exerts antiviral activities against several RNA viruses. Our study demonstrated that GBP1 can exert anti-KSHV function by inhibiting the nuclear delivery of KSHV virions via the disruption of actin filaments. Moreover, we found that KSHV RTA can promote the degradation of GBP1 through a proteasome-mediated pathway. Taken together, our results elucidate a novel mechanism of GBP1 anti-KSHV activity and emphasize the critical role of RTA in KSHV evasion of the host immune system during primary infection.

LRRK 2 gene mutations in the pathophysiology of the ROCO domain and therapeutic targets for Parkinson's disease: a review.

Parkinson's disease (PD) is the most common movement disorder and manifests as resting tremor, rigidity, bradykinesia, and postural instability. Pathologically, PD is characterized by selective loss of dopaminergic neurons in the substantia nigra and the formation of intracellular inclusions containing α-synuclein and ubiquitin called Lewy bodies. Consequently, a remarkable deficiency of dopamine in the striatum causes progressive disability of motor function. The etiology of PD remains uncertain. Genetic variability in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of sporadic and familial PD. LRRK2 encodes a large protein containing three catalytic and four protein-protein interaction domains. Patients with LRRK2 mutations exhibit a clinical and pathological phenotype indistinguishable from sporadic PD. Recent studies have shown that pathological mutations of LRRK2 can reduce the rate of guanosine triphosphate (GTP) hydrolysis, increase kinase activity and GTP binding activity, and subsequently cause cell death. The process of cell death involves several signaling pathways, including the autophagic-lysosomal pathway, intracellular trafficking, mitochondrial dysfunction, and the ubiquitin-proteasome system. This review summarizes the cellular function and pathophysiology of LRRK2 ROCO domain mutations in PD and the perspective of therapeutic approaches.

Structural Basis for the Specific Recognition of RhoA by the Dual GTPase-activating Protein ARAP3.

ARAP3 (Arf-GAP with Rho-GAP domain, ANK repeat, and PH domain-containing protein 3) is unique for its dual specificity GAPs (GTPase-activating protein) activity for Arf6 (ADP-ribosylation factor 6) and RhoA (Ras homolog gene family member A) regulated by phosphatidylinositol 3,4,5-trisphosphate and a small GTPase Rap1-GTP and is involved in regulation of cell shape and adhesion. However, the molecular interface between the ARAP3-RhoGAP domain and RhoA is unknown, as is the substrates specificity of the RhoGAP domain. In this study, we solved the crystal structure of RhoA in complex with the RhoGAP domain of ARAP3. The structure of the complex presented a clear interface between the RhoGAP domain and RhoA. By analyzing the crystal structure and in combination with in vitro GTPase activity assays and isothermal titration calorimetry experiments, we identified the crucial residues affecting RhoGAP activity and substrates specificity among RhoA, Rac1 (Ras-related C3 botulinum toxin substrate 1), and Cdc42 (cell division control protein 42 homolog).

The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis.

Nucleostemin is a nucleolar GTP-binding protein that is involved in stem cell proliferation, embryonic development, and ribosome biogenesis in mammals. Plant nucleostemin-like 1 (NSN1) plays a role in embryogenesis, and apical and floral meristem development. In this study, a nucleolar function of NSN1 in the regulation of ribosome biogenesis was identified. Green fluorescent protein (GFP)-fused NSN1 localized to the nucleolus, which was primarily determined by its N-terminal domain. Recombinant NSN1 and its N-terminal domain (NSN1-N) bound to RNA in vitro. Recombinant NSN1 expressed GTPase activity in vitro. NSN1 silencing in Arabidopsis thaliana and Nicotiana benthamiana led to growth retardation and premature senescence. NSN1 interacted with Pescadillo and EBNA1 binding protein 2 (EBP2), which are nucleolar proteins involved in ribosome biogenesis, and with several ribosomal proteins. NSN1, NSN1-N, and EBP2 co-fractionated primarily with the 60S ribosomal large subunit in vivo. Depletion of NSN1 delayed 25S rRNA maturation and biogenesis of the 60S ribosome subunit, and repressed global translation. NSN1-deficient plants exhibited premature leaf senescence, excessive accumulation of reactive oxygen species, and senescence-related gene expression. Taken together, these results suggest that NSN1 plays a crucial role in plant growth and senescence by modulating ribosome biogenesis.

A kinetic model of GPCRs: analysis of G protein activity, occupancy, coupling and receptor-state affinity constants.

CONTEXT: G protein-coupled receptors are vital macromolecules for a wide variety of physiological processes. Upon agonist binding, these receptors accelerate the exchange of GDP for GTP in G proteins coupled to them. The activated G protein interacts with effector proteins to implement downstream biological functions. OBJECTIVE: We present a kinetic, quaternary complex model, based on a system of coupled linear first-order differential equations, which accounts for the binding attributes of the ligand, receptor, G protein and two types of guanine nucleotide (GDP and GTP) as well as for GTPase activity. METHODS: We solved the model numerically to predict the extents of G protein activation, receptor occupancy by ligand and receptor coupling that result from varying the ligand concentration, presence of GDP and/or GTP, the ratio of G protein to receptor and the equilibrium constants governing receptor pre-coupling and constitutive activity. We also simulated responses downstream from G protein activation using a transducer function. RESULTS: Our model shows that agonist-induced G protein activation can occur with either a net decrease or increase in total receptor-G protein coupling. In addition, we demonstrate that affinity constants of the ligand for both the active and inactive states of the receptor can be derived to a close approximation from analysis of simulated responses downstream from receptor activation. DISCUSSION AND CONCLUSION: The latter result validates our prior methods for estimating the active state affinity constants of ligands, and our results on receptor coupling have relevance to studies investigating receptor-G protein interactions using fluorescence techniques.

KLF7 promotes neuroblastoma differentiation through the GTPase signaling pathway by upregulating neuroblast differentiation-associated protein AHNAKs and glycerophosphodiesterase GDPD5.

The arrest of neural crest-derived sympathoadrenal neuroblast differentiation contributes to neuroblastoma formation, and overriding this blocked differentiation is a clear strategy for treating high-risk neuroblastoma. A better understanding of neuroblast or neuroblastoma differentiation is essential for developing new therapeutic approaches. It has been proposed that Krueppel-like factor 7 (KLF7) is a neuroblastoma super-enhancer-associated transcription factor gene. Moreover, KLF7 was found to be intensely active in postmitotic neuroblasts of the developing nervous system during embryogenesis. However, the role of KLF7 in the differentiation of neuroblast or neuroblastoma is unknown. Here, we find a strong association between high KLF7 expression and favorable clinical outcomes in neuroblastoma. KLF7 induces differentiation of neuroblastoma cells independently of the retinoic acid (RA) pathway and acts cooperatively with RA to induce neuroblastoma differentiation. KLF7 alters the GTPase activity and multiple differentiation-related genes by binding directly to the promoters of neuroblast differentiation-associated protein (AHNAK and AHNAK2) and glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) and regulating their expression. Furthermore, we also observe that silencing KLF7 in neuroblastoma cells promotes the adrenergic-to-mesenchymal transition accompanied by changes in enhancer-mediated gene expression. Our results reveal that KLF7 is an inducer of neuroblast or neuroblastoma differentiation with prognostic significance and potential therapeutic value.

Quantification of GTPase Cycling Rates of GTPases and GTPase:Effector Mixtures Using GTPase Glo Assays.

In different cellular activities such as signal transduction, cell division, and intracellular transportation, small guanosine triphosphatases (GTPases) take on a vital role. Their function involves hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article, we explain the application of a commercially available GTPase assay-the GTPase Glo assay by Promega-for investigation of GTPase-effector interactions. We provide experimental protocols together with an analysis model and software to obtain GTPase cycling rates of GTPases and GTPase:effector mixtures. GTPase cycling rates refer to the rates by which a GTPase completes an entire GTPase cycle. These rates enable quantification of the strength of GTPase effectors in a concentration-dependent fashion, as well as quantification of the combined effect of two effectors, independent of which GTPase cycle step they are affecting. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Conducting GTPase Glo assays Support Protocol 1: Analyzing GTPase assays to correlate luminescence with remaining GTP Support Protocol 2: Fitting GTPase assay data to obtain GTPase cycling rates.

Measurement of KRAS-GTPase Activity.

Oncogenic mutations in KRAS typically impact the GAP-mediated and intrinsic GTP hydrolysis activity resulting in elevated levels of cellular KRAS-GTP. The development of biochemical assays for GTPase activity provides an opportunity to quantitatively measure the impact of these mutations on GTP hydrolysis. Here we describe a biochemical assay that measures the release of free phosphate upon hydrolysis of the GTP nucleotide and allows the measurement of intrinsic or GAP-stimulated GTP hydrolysis by KRAS. This assay can be used to measure GTPase activity under single turnover conditions.