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The Small Gtpase Arf6: an Overview of Its Mechanisms of Action and of Its Role in Host–Pathogen Interactions and Innate Immunity

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The Small Gtpase Arf6: an Overview of Its Mechanisms of Action and of Its Role in Host–Pathogen Interactions and Innate Immunity International Journal of Molecular Sciences Review The Small GTPase Arf6: An Overview of Its Mechanisms of Action and of Its Role in Host–Pathogen Interactions and Innate Immunity Tim Van Acker 1,2, Jan Tavernier 1,2 and Frank Peelman 1,2,* 1 Cytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium; [email protected] (T.V.A.); [email protected] (J.T.) 2 Department of Biomolecular Medicine, Ghent University, B-9000 Ghent, Belgium * Correspondence: [email protected]; Tel.: +32-9-264-93-46 Received: 12 March 2019; Accepted: 27 April 2019; Published: 5 May 2019 Abstract: The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host–pathogen interactions. Keywords: Arf6; small GTPase; immunity; host–pathogen interaction; vesicular transport; innate immunity; toll-like receptor; phagocytosis 1. Introduction 1.1. Arf6 and Its Relatives The ADP-ribosylation factor (Arf) protein family is part of the large Ras superfamily which encompasses small GTPases [1]. Arfs received their name for their assistance in the ADP-ribosylation of the α-subunit of heterotrimeric G proteins induced by the cholera-toxin [2]. A plethora of cellular processes require Arf proteins. The human Arf family includes Arf-like proteins (Arls), Sar proteins, and six Arfs (Arf1–Arf6). The group of 6 Arf proteins is subdivided into three classes based on sequence homology: class I (Arf1–3), class II (Arf4–5) and class III (Arf6) [3]. Class I and II Arfs are regulators of vesicle trafficking between the Golgi and endoplasmatic reticulum but also have roles outside the Golgi. Arf6, the single class III member, is both in sequence and in function the outcast of the family and is active in both clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) and in endosomal traffic and endosomal recycling outside the Golgi apparatus (Figure1)[ 4]. Arf6 is located at the plasma membrane, cytosol, and endosomal membranes and is N-terminally myristoylated [5]. It associates with membranes in a GTP-dependent manner; a dissociation from the plasma membrane requires GTP hydrolysis on Arf6 [6]. Int. J. Mol. Sci. 2019, 20, 2209; doi:10.3390/ijms20092209 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 2209 2 of 23 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 23 Figure 1. The role of Arf6 in intracellular trafficking upon clathrin-mediated endocytosis and Figure 1. The role of Arf6 in intracellular trafficking upon clathrin-mediated endocytosis and clathrin- clathrin-independent endocytosis. The location of Arf6 mutants in the cell is indicated as the white text independent endocytosis. The location of Arf6 mutants in the cell is indicated as the white text on a on a black background. black background. 1.2. Activation and Inactivation of Arf6 through GEFs and GAPs 1.2. Activation and Inactivation of Arf6 through GEFs and GAPs The activation and inactivation of Arf6 are regulated by guanine nucleotide exchange factors (GEFs)The and activation GTPase-activating and inactivation proteins of (GAPs). Arf6 are GEFs regulated contain by a catalyticguanine Sec7 nucleotide domain exchange which accelerates factors (GEFs)the dissociation and GTPase-activating of GDP from the proteins Arf, allowing (GAPs). the GEFs insertion contain of GTP a catalytic in the Arf Sec7 nucleotide domain bindingwhich acceleratespocket and the switching dissociation Arfs toof anGDP activated from the state Arf, [7 –al9lowing]. GAPs the stimulate insertion GTP of hydrolysisGTP in the onArf Arfs nucleotide through bindingtheir conserved pocket and ArfGAP switching zinc fingerArfs to domain, an activated although state ELMOD1 [7–9]. GAPs and stimulate 2 are an exception GTP hydrolysis to this ruleon Arfsas they through act as their Arf6 conserved GAPs without ArfGAP a typical zinc finger ArfGAP domain, domain although [10–13 ].ELMOD1 Currently, and we 2 are have an knowledge exception toof this 8 Arf6 rule GEFsas they and act up as toArf6 15 GAPs Arf6 GAPs.without Specific a typical Arf6-mediated ArfGAP domain processes [10–13]. require Currently, distinct we have Arf6 knowledgeGAPs/GEFs of for 8 Arf6 their GEFs regulation. and up For to 15 a detailedArf6 GAPs description. Specific andArf6-mediated extensive overview processes of require the Arf6 distinct GAPs Arf6and GEFs,GAPs/GEFs the reader for their is referred regulation. to two For reviews a detailed in this description area [14, 15and]. Itextensive should beoverview noted that of the the Arf6 term GAPsactivation and /GEFs,inactivation the reader can beis referred misleading, to two as many—ifreviews in not this most—Arf6 area [14,15]. mediated It should processes be noted probablythat the termrequire activation/inactivation the Arf6 GTP/GDP cycle can ratherbe misleading, than just the as Arf6-GTPmany—if form.not most—Arf6 As Arf6 functions mediated via aprocesses regulated probablycycle of GTP require binding the Arf6 and GTP/GDP hydrolysis, cycle both rather GAPs than and GEFsjust the are Arf6-GTP necessary form. for its As functionality Arf6 functions [15]. via a regulated cycle of GTP binding and hydrolysis, both GAPs and GEFs are necessary for its functionality1.3. Research Tools[15]. to Study Arf6 Functional studies of Arf6 frequently use the following tools: (i) the silencing or gene disruption 1.3. Research Tools to Study Arf6 of Arf6, (ii) the overexpression of Arf6 or of its GAPs or GEFs; (iii) Arf6 GEF inhibitors; (iv) the overexpressionFunctional studies of Arf6 of mutants Arf6 frequently with altered use the GTP following/GDP binding tools: (i) acting the silencing as constitutively or gene disruption active or ofdominant Arf6, (ii) negative the overexpression forms—The “constitutivelyof Arf6 or of its active” GAPsArf6-Q67L or GEFs; mutant(iii) Arf6 lacks GEF GTPase inhibitors; activity (iv) and the is overexpressionassociated with of GTP Arf6 [16 ].mutantsArf6-T27N withis altered a “dominant GTP/GDP negative” binding mutant acting that as has constitutively a decreased nucleotideactive or dominantbinding and negative is often forms—Th presentede “constitutively as a mimic of active” the GDP-bound Arf6-Q67L state mutant [16]. lacks However, GTPase both activity GTP and and isGDP associated binding with are a ffGTPected, [16]. and Arf6-T27N the lack of nucleotideis a “dominant binding negative” can cause mutant denaturation that has [17 ].a Arf6-T44Ndecreased nucleotidehas a low abindingffinity for and GTP is often but retains presented a near-normal as a mimic binding of the GDP-bound of GDP, and state Arf6-T44N [16]. However, was, therefore, both GTPpresented and GDP as a betterbinding alternative are affected, for theand GDP-bound the lack of statenucleotide [17]—and binding (v) another can cause tool denaturation used in functional [17]. Arf6-T44Nstudies is a hasmyristoylated a low affinity Arf6 for peptideGTP butcorresponding retains a near-normal to the N-terminal binding of helixGDP, ofand Arf6, Arf6-T44N which can was, be therefore,transfected presented in cells byas a coupling better alternative it to penetratin for the [ 18GDP-bound–20]. The peptidestate [17]—and acts as an(v) antagonistanother tool of used Arf6, inpossibly functional via competitivestudies is a bindingmyristoylated to Arf6 Arf6 interaction peptide partners, corresponding although to the the exact N-terminal targets ofhelix this of peptide Arf6, whichhave not can been be transfected defined (to in our cells knowledge). by coupling it to penetratin [18–20]. The peptide acts as an antagonist of Arf6, possibly via competitive binding to Arf6 interaction partners, although the exact targets of this peptide have not been defined (to our knowledge). Int. J. Mol. Sci. 2019, 20, 2209 3 of 23 1.4. RolesInt. J. Mol. and Sci. Mechanisms 2018, 19, x FOR of PEER Arf6 REVIEW 3 of 23 1.4. Roles and Mechanisms of Arf6 1.4.1. Arf6 Directly Activates Lipid Modifying Enzymes Arf61.4.1. exertsArf6 Directly its effects Activates through Lipid a diverseModifying set Enzymes of effectors. It activates the lipid modifying enzymes phosphatidylinositolArf6 exerts its 4-phosphateeffects through 5-kinase a diverse (setPIP5K of effectors.) and phospholipase It activates the lipid D ( PLDmodifying), which enzymes lead to a productionphosphatidylinositol of phosphatidylinositol-4,5-bisphosphate 4-phosphate 5-kinase (PIP5K) and (PIP phospholipase2) and phosphatidic D (PLD acid), which (PA) lead (Figure to a 2A). An increaseproduction in PA of phosphatidylin concentrationositol-4,5-bisphosphate will lead to a secondary (PIP2) enhancement and phosphatidic of PIPacid2 (PA)production (Figure 2A). due to a stimulatoryAn increase effect in on PA PIP5K concentration [21–28]. Thewill lead Arf6-mediated to a secondary increase enhancement in PIP2 isof one PIP of2 production its crucial due mechanisms to a of actionstimulatory in clathrin-dependent effect on PIP5K and [21–28]. -independent The Arf6-mediated endocytosis, increase endosomal in PIP recycling,2 is one plasmaof its crucial membrane remodeling,mechanisms and actinof action polymerization. in clathrin-dependent and -independent endocytosis, endosomal recycling, plasma membrane remodeling, and actin polymerization. Figure 2. A schematic overview of some of the major effectors of Arf6: (A) Arf6 activates Figure 2.
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