Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies.

The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.

Tyrosine glycosylation of Rho by Yersinia toxin impairs blastomere cell behaviour in zebrafish embryos.

Yersinia species cause zoonotic infections, including enterocolitis and plague. Here we studied Yersinia ruckeri antifeeding prophage 18 (Afp18), the toxin component of the phage tail-derived protein translocation system Afp, which causes enteric redmouth disease in salmonid fish species. Here we show that microinjection of the glycosyltransferase domain Afp18(G) into zebrafish embryos blocks cytokinesis, actin-dependent motility and cell blebbing, eventually abrogating gastrulation. In zebrafish ZF4 cells, Afp18(G) depolymerizes actin stress fibres by mono-O-GlcNAcylation of RhoA at tyrosine-34; thereby Afp18(G) inhibits RhoA activation by guanine nucleotide exchange factors, and blocks RhoA, but not Rac and Cdc42 downstream signalling. The crystal structure of tyrosine-GlcNAcylated RhoA reveals an open conformation of the effector loop distinct from recently described structures of GDP- or GTP-bound RhoA. Unravelling of the molecular mechanism of the toxin component Afp18 as glycosyltransferase opens new perspectives in studies of phage tail-derived protein translocation systems, which are preserved from archaea to human pathogenic prokaryotes.

Local translation of TC10 is required for membrane expansion during axon outgrowth.

The surface of developing axons expands in a process mediated by the exocyst complex. The spatio-temporal regulation of the exocyst is only partially understood. Here we report that stimulated membrane enlargement in dorsal root ganglion (DRG) axons is triggered by intra-axonal synthesis of TC10, a small GTPase required for exocyst function. Induced membrane expansion and axon outgrowth are inhibited after axon-specific knockdown of TC10 mRNA. To determine the relationship of intra-axonal TC10 synthesis with the previously described stimulus-induced translation of the cytoskeletal regulator Par3, we investigate the signalling pathways controlling their local translation in response to NGF. Phosphoinositide 3-kinase (PI3K)-dependent activation of the Rheb-mTOR pathway triggers the simultaneous local synthesis of TC10 and Par3. These results reveal the importance of local translation in the control of membrane dynamics and demonstrate that localized, mTOR-dependent protein synthesis triggers the simultaneous activation of parallel pathways.

Rheb/mTORC1 signaling promotes kidney fibroblast activation and fibrosis.

Ras homolog enriched in brain (Rheb) is a small GTPase that regulates cell growth, differentiation, and survival by upregulating mammalian target of rapamycin complex 1 (mTORC1) signaling. The role of Rheb/mTORC1 signaling in the activation of kidney fibroblasts and the development of kidney fibrosis remains largely unknown. In this study, we found that Rheb/mTORC1 signaling was activated in interstitial myofibroblasts from fibrotic kidneys. Treatment of rat kidney interstitial fibroblasts (NRK-49F cell line) with TGFß1 also activated Rheb/mTORC1 signaling. Blocking Rheb/mTORC1 signaling with rapamycin or Rheb small interfering RNA abolished TGFß1-induced fibroblast activation. In a transgenic mouse, ectopic expression of Rheb activated kidney fibroblasts. These Rheb transgenic mice exhibited increased activation of mTORC1 signaling in both kidney tubular and interstitial cells as well as progressive interstitial renal fibrosis; rapamycin inhibited these effects. Similarly, mice with fibroblast-specific deletion of Tsc1, a negative regulator of Rheb, exhibited activated mTORC1 signaling in kidney interstitial fibroblasts and increased renal fibrosis, both of which rapamycin abolished. Taken together, these results suggest that Rheb/mTORC1 signaling promotes the activation of kidney fibroblasts and contributes to the development of interstitial fibrosis, possibly providing a therapeutic target for progressive renal disease.

Mevastatin-induced neurite outgrowth of neuroblastoma cells via activation of EGFR.

Neuroblastoma cell lines are commonly used as models to study neuronal differentiation, as they retain the capacity to differentiate into a neuronal-like phenotype. Receptor tyrosine kinase (RTK) signaling is essential for neuronal differentiation during development, and cholesterol-containing lipid-rafts are important for RTK signaling. Hydroxymethylglutaryl-coenzyme A reductase inhibitors of the statin family impair cholesterol biosynthesis and are in widespread clinical use for the treatment of cardiovascular diseases. It is of great clinical interest that statin treatment also correlates with a lower incidence of malignancies. We found that mevastatin triggered neurite outgrowth of neuroblastoma cells and examined the responsible signaling pathways. Treatment of Neuro2a cells with mevastatin for 24 hr induced neurite outgrowth associated with up-regulation of the neuronal marker protein NeuN. Interestingly, we found that mevastatin triggered phosphorylation of the key kinases epidermal growth factor receptor (EGFR), ERK1/2, and Akt/protein kinase B. Inhibition of EGFR, PI3K, and the mitogen-activated protein kinase cascade blocked mevastatin-induced neurite outgrowth. Moreover, add-back experiments of cell-permeable cholesterol precursors indicated that farnesylated and geranylgeranylated proteins play a major role in statin-induced neurite outgrowth. Taken together, our results provide the first mechanistic insight into statin-triggered signaling pathways that lead to neurite outgrowth in neuroblastoma cells. Surprisingly, we revealed that mevastatin triggered the phosphorylation of the EGFR and that this was because of the inhibition of farnesylated and geranylgeranylated proteins. We propose that members of the large family of farnesylated or geranylgeranylated small GTPases (such as Rabs or Rap1) regulating the trafficking and signaling of EGFR might be responsible for the statin-induced effects on EGFR signaling.

Role of reversible, thioredoxin-sensitive oxidative protein modifications in cardiac myocytes.

Reactive oxygen species (ROS) are important mediators of myocardial remodeling. However, the precise molecular mechanisms by which ROS exert their effects are incompletely understood. ROS induce oxidative posttranslational protein modifications that can regulate the function of structural, functional, and signaling proteins. For example, oxidative modification of free reactive thiols (S-thiolation) on the small G protein Ras increases Ras activity and thereby promotes ROS-dependent hypertrophic signaling in cardiac myocytes. By reducing thiols and restoring reversible thiol modifications, thioredoxin and glutaredoxin can act as regulators of ROS-mediated protein function. Understanding the regulation and functional relevance of oxidative protein modifications in myocardial remodeling may lead to new therapeutic strategies.

Statins and myocardial hypertrophy.

Cardiac hypertrophy is a physiological adaptive response by the heart to pressure overload. However, after prolonged periods, this initial adaptive response becomes maladaptive, leading to increased mortality and morbidity from heart failure. Recently, 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, have been shown to inhibit cardiac hypertrophy by cholesterol-independent mechanisms. Statins block the isoprenylation and activation of members of the Rho guanosine triphosphatase (GTPase) family, such as RhoA and Rac1. Since Rac1 is a requisite component of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is a major source of reactive oxygen species (ROS) in cardiovascular cells, the ability of statins to inhibit Rac1-mediated oxidative stress makes an important contribution to their inhibitory effects on cardiac hypertrophy.

Perspectives on the cardioprotective effects of statins.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme of cholesterol synthesis. In recent years, statins have become the major choice of treatment for hypercholesterolemia. Emerging evidence from both animal and human studies indicates that mechanisms independent of cholesterol lowering effects contribute to the observed clinical benefits of statins. The anti-hypertrophy effect of statins on the cardiac tissue represents one of such mechanisms. The beneficial effects of statins on cardiac hypertrophy and cardioprotection may be attributed to their functional influences on small G proteins such as Ras and Rho, resulting in an increase of endogenous nitric oxide (NO), reduction of oxidative stress, inhibition of inflammatory reaction, and decrease of the renin-angiotensin system activity as well as C-reactive protein (CRP) levels in cardiac tissues. Recent findings from in vitro and in vivo studies of statins on cardioprotective effects are summarized in this review. The unveiled novel mechanisms support the use of statins as the new mainstay therapeutic agents for various cardiovascular diseases and complications.

Identification of a glucocorticoid response element in the 3'-flanking region of the human Dexras1 gene.

The Dexras1 gene responds to glucocorticoids with a rapid and profound induction. A glucocorticoid response element (GRE) was identified in the 3'-flanking region (2.3 kb downstream of poly(A) signal) of the human Dexras1 gene. This element conferred rapid glucocorticoid responsiveness when inserted into a homologous promoter-driven luciferase reporter. A point mutation within the 15-bp GRE abolished this glucocorticoid responsiveness.

In vivo cross-linking of nm23/nucleoside diphosphate kinase to the PDGF-A gene promoter.

Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum H. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.

In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5.

The Cdc5 protein of budding yeast is a polo-like kinase that has multiple roles in mitosis including control of the mitotic exit network (MEN). MEN activity brings about loss of mitotic kinase activity so that the mitotic spindle is disassembled and cytokinesis can proceed. Activity of the MEN is regulated by a small GTPase, Tem1, which in turn is controlled by a two-component GTPase-activating protein (GAP) formed by Bfa1 and Bub2. Bfa1 has been identified as a regulatory target of Cdc5 but there are conflicting deductions from indirect in vivo assays as to whether phosphorylation inhibits or stimulates Bfa1 activity. To resolve this question, we have used direct in vitro assays to observe the effects of phosphorylation on Bfa1 activity. We show that when Bfa1 is phosphorylated by Cdc5, its GAP activity with Bub2 is inhibited although its ability to interact with Tem1 is unaffected. Thus, in vivo inactivation of Bfa1-Bub2 by Cdc5 would have a positive regulatory effect by increasing levels of Tem1-GTP so stimulating exit from mitosis.

Nod factors activate both heterotrimeric and monomeric G-proteins in Vigna unguiculata (L.) Walp.

Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]- (from Rhizobiumsp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata(L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [(35)S]GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N- N'- N"- N'"-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPgammaS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(common) antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.

Activation of the small GTP-binding protein Ras in the heart by hypertrophic agonists.

The small (21-kDa) guanine nucleotide-binding protein Ras plays a central role in the regulation of cell growth and division. In the cardiac myocyte, it has been implicated in the hypertrophic adaptation. We have recently examined the ability of hypertrophic agonists such as endothelin-1, phenylephrine and phorbol esters to increase the "activity" (GTP loading) of Ras. We have also studied the signaling events that lead to activation of Ras and the processes that respond to Ras activation. In this brief review, we describe these studies and set them within the context of the hypertrophic response.