PMC:5026484 / 1094-6552 JSONTXT

{"project":"2_test","denotations":[{"id":"27552051-11152757-26907275","span":{"begin":171,"end":175},"obj":"11152757"},{"id":"27552051-19603039-26907276","span":{"begin":506,"end":510},"obj":"19603039"},{"id":"27552051-16882731-26907277","span":{"begin":855,"end":859},"obj":"16882731"},{"id":"27552051-21936789-26907278","span":{"begin":1211,"end":1215},"obj":"21936789"},{"id":"27552051-15694364-26907279","span":{"begin":1397,"end":1401},"obj":"15694364"},{"id":"27552051-22116028-26907280","span":{"begin":1584,"end":1588},"obj":"22116028"},{"id":"27552051-23911929-26907281","span":{"begin":1602,"end":1606},"obj":"23911929"},{"id":"27552051-24212093-26907282","span":{"begin":1621,"end":1625},"obj":"24212093"},{"id":"27552051-23834433-26907283","span":{"begin":1919,"end":1923},"obj":"23834433"},{"id":"27552051-23834433-26907284","span":{"begin":2134,"end":2138},"obj":"23834433"},{"id":"27552051-9600884-26907285","span":{"begin":3349,"end":3353},"obj":"9600884"},{"id":"27552051-20573983-26907286","span":{"begin":3811,"end":3815},"obj":"20573983"},{"id":"27552051-14676205-26907287","span":{"begin":3835,"end":3839},"obj":"14676205"}],"text":"Introduction\nRab proteins, the biggest subfamily within the superfamily of small GTPases, are major regulators of vesicular trafficking in eukaryotic cells (Takai et al., 2001). Like all small GTPases, Rab proteins cycle between an inactive GDP-bound and an active GTP-bound state. The cycling is tightly regulated and mediated by two families of enzymes: guanine nucleotide exchange factors (GEFs) that catalyze the GDP/GTP exchange and GTPase activating proteins that stimulate GTP hydrolysis (Stenmark, 2009). Additionally, a variety of different effector proteins interact specifically with GTP-bound Rab proteins and mediate their versatile physiological roles in membrane trafficking, including budding of vesicles from a donor membrane, directed transport through the cell and finally tethering and fusion with a target membrane (Grosshans et al., 2006). Especially in long-distance vesicular transport processes (e.g. in neuronal axons and dendrites), directed vesicular transport along cytoskeletal tracks appears to be an obvious mechanism and, consistently, different effector proteins have been reported to link Rab proteins to the cytoskeleton (Kevenaar and Hoogenraad, 2015; Horgan and McCaffrey, 2011).\nOne such family of effector proteins that was reported to link Rab proteins and the cytoskeleton is the Mical (molecules interacting with CasL) family (Figure 1) (Fischer et al., 2005). Most of these Mical proteins contain an N-terminal monooxygenase domain that was reported to regulate actin dynamics via reversible oxidation of a methionine residue (Hung et al., 2011; Lee et al., 2013; Hung et al., 2013). Additionally, all Mical proteins except the Mical C-terminal like protein (Mical-cL) contain a calponin homology (CH) and a Lin11, Isl-1 and Mec-3 (LIM) domain that have been reported to assist the interaction with actin and other cytoskeletal proteins, respectively (Giridharan and Caplan, 2014). Finally, all except Mical-2 contain a C-terminal coiled-coil domain that is also termed domain of unknown function (DUF) 3585 and that is known to interact with different Rab proteins (Giridharan and Caplan, 2014) (Figure 1).\n10.7554/eLife.18675.002Figure 1. Domain architecture of human proteins containing bMERB domains.\nBesides their C-terminal RBD (referred to as bivalent Mical/EHBP Rab binding (bMERB) domain), most Mical proteins contain an N-terminal Monooxygenase (red), a CH- and a LIM-domain (both orange). EHBPs also contain an actin binding CH-domain and an N-terminal membrane binding C2-domain (yellow) as well as a C-terminal prenylation motif (CaaX-box) following the bMERB domain. Two proteins predicted to contain only the bMERB domains (Mical-cL and C16orf45) are also shown. For proteins with multiple known splice variants, domain boundaries are indicated for isoform 1 (Mical-1: Uniprot ID Q8TDZ2, genomic location 6q21; Mical-L1: Uniprot ID Q8N3F8, genomic location 22q13.1; Mical-L2: Uniprot ID Q8IY33, genomic location 7p22.3; Mical-3: Uniprot ID Q7RTP6, genomic location 22q11.21; Mical-cL: Uniprot ID Q6ZW33, genomic location 11p15.3; EHBP1: Uniprot ID Q8NDI1, genomic location 2p15; EHBP1L1 Uniprot ID Q8N3D4, genomic location 11q13.1). The reader is referred to the main text for further details.\nDOI: http://dx.doi.org/10.7554/eLife.18675.002\nAccording to the SMART database (Schultz et al., 1998), this largely uncharacterized DUF3585 domain is present in more than 450 eukaryotic proteins (including 8 human proteins, see Figure 1). In humans, besides the Mical proteins this includes the family of EH (Eps15-homology) domain binding proteins (EHBPs) and one uncharacterized protein (C16orf45; see Figure 1). Interestingly, the EHBPs also contain a CH-domain and have been described to couple vesicular transport to the actin cytoskeleton (Shi et al., 2010; Guilherme et al., 2004).\nHitherto, the structural basis and the specificity of interaction between the Rab binding domains of Micals/EHBPs and Rab proteins remained largely unknown. In this publication, we have characterized the interaction of a number of these domains with Rab proteins extensively. Our results indicate preferential binding of this family of effector proteins to Rab proteins of the Rab8 family. Additionally, the results show that at least some of these effector domains can bind two Rab proteins simultaneously, suggesting a possible role as a Rab hub in vesicular trafficking.\nIn order to understand the structural basis of the interaction, we solved the first x-ray crystallographic structure of the RBD from the human protein Mical-3 and the first structures of different Rab proteins in complex with the RBDs of Mical-cL in a 1:1 stoichiometry and of Rab10 and the RBD of Mical-1 in a 2:1 stoichiometry.\nThis study is the first to show the structural basis of Rab proteins interacting with these RBDs and to systematically characterize the interaction with Rab proteins. Analysis of our data suggests that the second Rab binding site of these RBDs has evolved via a gene duplication event, indicating intriguing and hitherto unknown mechanisms of a concerted action of different Rab-regulated trafficking steps connected by these bivalent effector proteins, which we refer to as “bivalent Mical/EHBP Rab binding” (bMERB) domains (Figure 1). The study therefore substantially increases our understanding of Rab:effector interactions and will aid future research regarding the function of this diverse effector family."}

{"project":"MyTest","denotations":[{"id":"27552051-11152757-26907275","span":{"begin":171,"end":175},"obj":"11152757"},{"id":"27552051-19603039-26907276","span":{"begin":506,"end":510},"obj":"19603039"},{"id":"27552051-16882731-26907277","span":{"begin":855,"end":859},"obj":"16882731"},{"id":"27552051-21936789-26907278","span":{"begin":1211,"end":1215},"obj":"21936789"},{"id":"27552051-15694364-26907279","span":{"begin":1397,"end":1401},"obj":"15694364"},{"id":"27552051-22116028-26907280","span":{"begin":1584,"end":1588},"obj":"22116028"},{"id":"27552051-23911929-26907281","span":{"begin":1602,"end":1606},"obj":"23911929"},{"id":"27552051-24212093-26907282","span":{"begin":1621,"end":1625},"obj":"24212093"},{"id":"27552051-23834433-26907283","span":{"begin":1919,"end":1923},"obj":"23834433"},{"id":"27552051-23834433-26907284","span":{"begin":2134,"end":2138},"obj":"23834433"},{"id":"27552051-9600884-26907285","span":{"begin":3349,"end":3353},"obj":"9600884"},{"id":"27552051-20573983-26907286","span":{"begin":3811,"end":3815},"obj":"20573983"},{"id":"27552051-14676205-26907287","span":{"begin":3835,"end":3839},"obj":"14676205"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"Introduction\nRab proteins, the biggest subfamily within the superfamily of small GTPases, are major regulators of vesicular trafficking in eukaryotic cells (Takai et al., 2001). Like all small GTPases, Rab proteins cycle between an inactive GDP-bound and an active GTP-bound state. The cycling is tightly regulated and mediated by two families of enzymes: guanine nucleotide exchange factors (GEFs) that catalyze the GDP/GTP exchange and GTPase activating proteins that stimulate GTP hydrolysis (Stenmark, 2009). Additionally, a variety of different effector proteins interact specifically with GTP-bound Rab proteins and mediate their versatile physiological roles in membrane trafficking, including budding of vesicles from a donor membrane, directed transport through the cell and finally tethering and fusion with a target membrane (Grosshans et al., 2006). Especially in long-distance vesicular transport processes (e.g. in neuronal axons and dendrites), directed vesicular transport along cytoskeletal tracks appears to be an obvious mechanism and, consistently, different effector proteins have been reported to link Rab proteins to the cytoskeleton (Kevenaar and Hoogenraad, 2015; Horgan and McCaffrey, 2011).\nOne such family of effector proteins that was reported to link Rab proteins and the cytoskeleton is the Mical (molecules interacting with CasL) family (Figure 1) (Fischer et al., 2005). Most of these Mical proteins contain an N-terminal monooxygenase domain that was reported to regulate actin dynamics via reversible oxidation of a methionine residue (Hung et al., 2011; Lee et al., 2013; Hung et al., 2013). Additionally, all Mical proteins except the Mical C-terminal like protein (Mical-cL) contain a calponin homology (CH) and a Lin11, Isl-1 and Mec-3 (LIM) domain that have been reported to assist the interaction with actin and other cytoskeletal proteins, respectively (Giridharan and Caplan, 2014). Finally, all except Mical-2 contain a C-terminal coiled-coil domain that is also termed domain of unknown function (DUF) 3585 and that is known to interact with different Rab proteins (Giridharan and Caplan, 2014) (Figure 1).\n10.7554/eLife.18675.002Figure 1. Domain architecture of human proteins containing bMERB domains.\nBesides their C-terminal RBD (referred to as bivalent Mical/EHBP Rab binding (bMERB) domain), most Mical proteins contain an N-terminal Monooxygenase (red), a CH- and a LIM-domain (both orange). EHBPs also contain an actin binding CH-domain and an N-terminal membrane binding C2-domain (yellow) as well as a C-terminal prenylation motif (CaaX-box) following the bMERB domain. Two proteins predicted to contain only the bMERB domains (Mical-cL and C16orf45) are also shown. For proteins with multiple known splice variants, domain boundaries are indicated for isoform 1 (Mical-1: Uniprot ID Q8TDZ2, genomic location 6q21; Mical-L1: Uniprot ID Q8N3F8, genomic location 22q13.1; Mical-L2: Uniprot ID Q8IY33, genomic location 7p22.3; Mical-3: Uniprot ID Q7RTP6, genomic location 22q11.21; Mical-cL: Uniprot ID Q6ZW33, genomic location 11p15.3; EHBP1: Uniprot ID Q8NDI1, genomic location 2p15; EHBP1L1 Uniprot ID Q8N3D4, genomic location 11q13.1). The reader is referred to the main text for further details.\nDOI: http://dx.doi.org/10.7554/eLife.18675.002\nAccording to the SMART database (Schultz et al., 1998), this largely uncharacterized DUF3585 domain is present in more than 450 eukaryotic proteins (including 8 human proteins, see Figure 1). In humans, besides the Mical proteins this includes the family of EH (Eps15-homology) domain binding proteins (EHBPs) and one uncharacterized protein (C16orf45; see Figure 1). Interestingly, the EHBPs also contain a CH-domain and have been described to couple vesicular transport to the actin cytoskeleton (Shi et al., 2010; Guilherme et al., 2004).\nHitherto, the structural basis and the specificity of interaction between the Rab binding domains of Micals/EHBPs and Rab proteins remained largely unknown. In this publication, we have characterized the interaction of a number of these domains with Rab proteins extensively. Our results indicate preferential binding of this family of effector proteins to Rab proteins of the Rab8 family. Additionally, the results show that at least some of these effector domains can bind two Rab proteins simultaneously, suggesting a possible role as a Rab hub in vesicular trafficking.\nIn order to understand the structural basis of the interaction, we solved the first x-ray crystallographic structure of the RBD from the human protein Mical-3 and the first structures of different Rab proteins in complex with the RBDs of Mical-cL in a 1:1 stoichiometry and of Rab10 and the RBD of Mical-1 in a 2:1 stoichiometry.\nThis study is the first to show the structural basis of Rab proteins interacting with these RBDs and to systematically characterize the interaction with Rab proteins. Analysis of our data suggests that the second Rab binding site of these RBDs has evolved via a gene duplication event, indicating intriguing and hitherto unknown mechanisms of a concerted action of different Rab-regulated trafficking steps connected by these bivalent effector proteins, which we refer to as “bivalent Mical/EHBP Rab binding” (bMERB) domains (Figure 1). The study therefore substantially increases our understanding of Rab:effector interactions and will aid future research regarding the function of this diverse effector family."}