Lino C. Gonzalez

SNARE INTERACTIONS ARE NOT SELECTIVE : IMPLICATIONS FOR MEMBRANE FUSION SPECIFICITY

The SNARE hypothesis proposes that membrane trafficking specificity is mediated by preferential high affinity interactions between particular v (vesicle membrane)- and t (target membrane)-SNARE combinations. The specificity of interactions among a diverse set of SNAREs, however, is unknown. We have tested the SNARE hypothesis by analyzing potential SNARE complexes between five proteins of the vesicle-associated membrane protein (VAMP) family, three members of the synaptosome-associated protein-25 (SNAP-25) family and three members of the syntaxin family. All of the 21 combinations of SNAREs tested formed stable complexes. Sixteen were resistant to SDS denaturation, and most complexes thermally denatured between 70 and 90 °C. These results suggest that the specificity of membrane fusion is not encoded by the interactions between SNAREs.

Regulation of Membrane Trafficking: Structural Insights from a Rab/Effector Complex

membrane compartments (reviewed by Novick and ZeIntroduction rial, 1997). Eukaryotic cells contain a highly dynamic set of memConcomitant with the SNARE cycle, Rab proteins unbrane compartments that are responsible for packaging, dergo a intricate cycle of membrane and protein interacsorting, secreting, and recycling proteins and other moltions. Rabs are posttranslationally modified at C-termiecules. Trafficking between organelles within the secrenal cysteines by the addition of two geranylgeranyl tory pathway occurs as vesicles derived from a donor groups, which mediate membrane association when the compartment fuse with specific acceptor membranes, Rab is in the GTP-bound state (Figure 1). After guanine resulting in the directional transfer of cargo molecules. nucleotide hydrolysis occurs, the Rab is extracted from This process is tightly controlled by the Rab/Ypt family the membrane upon forming a complex with a cytosolic of proteins (reviewed by Novick and Zerial, 1997), a GDP-dissociation inhibitor (GDI). This cytosolic intermebranch of the superfamily of small GTPases. Rab prodiate is then recycled onto a newly forming vesicle, most teins regulate a variety of functions, including vesicle likely through a secondary factor termed a GDI dissociatranslocation and docking at specific fusion sites. Rabs tion factor (GDF), which displaces GDI. After the Rab may also play critical roles in higher order processes becomes membrane bound, a guanidine nucleotide exsuch as modulating the levels of neurotransmitter rechange factor (GEF) promotes release of GDP and the lease in neurons, a likely mechanism in synaptic plasticsubsequent loading of GTP. In its GTP-bound conformaity that underlies learning and memory (Geppert and tion, the Rab is then free to associate with its specific Südhof, 1998). set of effectors, which can in turn trigger events leading Small GTPases share a common three-dimensional to the eventual fusion of the vesicle with a target memfold that, in the GTP bound state, can bind a variety of brane. To complete the cycle, perhaps after or concurdownstream effector proteins. GTP hydrolysis leads to rent with membrane fusion, a GTPase activating protein a conformational change in the “switch” regions that (GAP) accelerates nucleotide hydrolysis, switching off renders the GTPase unrecognizable to its effectors. In the GTPase. The remaining GDP-bound Rab can then this way, by localizing and activating a select set of participate in a new round of fusion. effectors, a common structural motif is used to control Rab interactions with effectors are likely to regulate a wide array of distinct cellular processes. Recently, the vesicle targeting and membrane fusion in three ways first structure of a Rab/effector complex was reported (Figure 1). First, a Rab may specifically facilitate vectorial for the Rab-binding domain (RBD) of the Rab effector vesicle transport. Vesicles are transported from their protein Rabphilin-3A bound to Rab3A (Ostermeier and site of origin to acceptor compartments likely through Brünger, 1999). This structure highlights a novel RBD associations with cytoskeletal elements and transport and sheds light on the specificity of effector interactions motors. A protein has been identified with a domain within the Rab family, interactions that may be critical structure that suggests a connection between the cyfor understanding the specificity and regulation of memtoskeleton and the Rabs. This protein, called Rabbrane trafficking in cells. kinesin-6, contains a kinesin-like ATPase motor domain The Role of Rabs in Regulating followed by a coiled-coil stalk region and a RBD that Membrane Trafficking specifically binds Rab6 (Echard et al., 1998). An addiThe final steps in membrane fusion are likely to be driven tional link with the cytoskeleton is provided by the Rab by a set of proteins known as SNAREs (Figure 1). After effector, Rabphilin-3A. Rabphilin-3A has been shown in a vesicle becomes docked, the cytoplasmic domains vitro to interact with a-actinin, an actin-bundling protein, of VAMP (also termed synaptobrevin) and syntaxin on but only when not bound to Rab3A (Kato et al., 1996).

A novel snare N-terminal domain revealed by the crystal structure of Sec22b.

Intra-cellular membrane fusion is facilitated by the association of SNAREs from opposite membranes into stable α-helical bundles. Many SNAREs, in addition to their α-helical regions, contain N-terminal domains that likely have essential regulatory functions. To better understand this regulation, we have determined the 2.4-Å crystal structure of the 130-amino acid N-terminal domain of mouse Sec22b (mSec22b), a SNARE involved in endoplasmic reticulum/Golgi membrane trafficking. The domain consists of a mixed α-helical/β-sheet fold that resembles a circular permutation of the actin/poly-proline binding protein, profilin, and the GAF/PAS family of regulatory modules. The structure is distinct from the previously characterized N-terminal domain of syntaxin 1A, and, unlike syntaxin 1A, the N-terminal domain of mSec22b has no effect on the rate of SNARE assembly in vitro. An analysis of surface conserved residues reveals a potential protein interaction site. Key residues in this site are distinct in two mammalian Sec22 variants that lack SNARE domains. Finally, sequence analysis indicates that a similar domain is likely present in the endosomal/lysosomal SNARE VAMP7.

Three-dimensional structure of the amino-terminal domain of syntaxin 6, a SNAP-25 C homolog

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are required for intracellular membrane fusion, and are differentially localized throughout the cell. SNAREs on vesicle and target membranes contain “SNARE motifs” which interact to form a four-helix bundle that contributes to the fusion of two membranes. SNARE motif sequences fall into four classes, homologous to the neuronal proteins syntaxin 1a, VAMP 2, and the N- and C-terminal SNARE motifs of SNAP-25 (S25N and S25C), and it is thought that one member from each class interacts to form a SNARE complex. Many SNAREs also feature N-terminal domains believed to function in regulating SNARE complex assembly or other aspects of vesicle transport. Syntaxin 6 is a SNARE found primarily in endosomal transport vesicles and whose SNARE motif shows significant homology to both syntaxin 1a and S25C. The crystal structure of the syntaxin 6 N-terminal domain reveals strong structural similarity with the N-terminal domains of syntaxin family members syntaxin 1a, Sso1p, and Vam3p, despite a very low level of sequence similarity. The syntaxin 6 SNARE motif can substitute for S25C in in vitro binding experiments, supporting the classification of syntaxin 6 as an S25C family member. Secondary structure prediction of SNARE proteins shows that the N-terminal domains of many syntaxin, S25N, and S25C family members are likely to be similar to one another, but are distinct from those of VAMP family members, indicating that syntaxin, S25N, and S25C SNAREs may have shared a common ancestor.

Defining the Ligand Specificity of the Deleted in Colorectal Cancer (DCC) Receptor

The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4–6 of DCC and binds with ∼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.

Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

Background: PILRα is an inhibitory receptor predominantly expressed in myeloid cells. Results: NPDC1 and COLEC12 are novel PILRα ligands. PILRα arginine residues 133 (mouse) and 126 (human) are critical contact residues. Conclusion: PILRα/ligand interactions involve a conserved domain in PILRα and a sialylated protein domain in the ligand. Significance: PILRα interacts with various ligands to alter myeloid cell function. Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

Murine Insulin Growth Factor-like (IGFL) and Human IGFL1 Proteins Are Induced in Inflammatory Skin Conditions and Bind to a Novel Tumor Necrosis Factor Receptor Family Member, IGFLR1

Psoriasis is a human skin condition characterized by epidermal hyperproliferation and infiltration of multiple leukocyte populations. In characterizing a novel insulin growth factor (IGF)-like (IGFL) gene in mice (mIGFL), we found transcripts of this gene to be most highly expressed in skin with enhanced expression in models of skin wounding and psoriatic-like inflammation. A possible functional ortholog in humans, IGFL1, was uniquely and significantly induced in psoriatic skin samples. In vitro IGFL1 expression was up-regulated in cultured primary keratinocytes stimulated with tumor necrosis factor α but not by other psoriasis-associated cytokines. Finally, using a secreted and transmembrane protein library, we discovered high affinity interactions between human IGFL1 and mIGFL and the TMEM149 ectodomain. TMEM149 (renamed here as IGFLR1) is an uncharacterized gene with structural similarity to the tumor necrosis factor receptor family. Our studies demonstrate that IGFLR1 is expressed primarily on the surface of mouse T cells. The connection between mIGFL and IGFLR1 receptor suggests mIGFL may influence T cell biology within inflammatory skin conditions.

The extracellular interactome of the human adenovirus family reveals diverse strategies for immunomodulation.

Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus–host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus–host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation.