VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites.

Mutations in the human VPS13 genes are responsible for neurodevelopmental and neurodegenerative disorders including chorea acanthocytosis (VPS13A) and Parkinson's disease (VPS13C). The mechanisms of these diseases are unknown. Genetic studies in yeast hinted that Vps13 may have a role in lipid exchange between organelles. In this study, we show that the N-terminal portion of VPS13 is tubular, with a hydrophobic cavity that can solubilize and transport glycerolipids between membranes. We also show that human VPS13A and VPS13C bind to the ER, tethering it to mitochondria (VPS13A), to late endosome/lysosomes (VPS13C), and to lipid droplets (both VPS13A and VPS13C). These findings identify VPS13 as a lipid transporter between the ER and other organelles, implicating defects in membrane lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggest lipid transport roles for these proteins as well.

Molecular basis for sterol transport by StART-like lipid transfer domains.

Lipid transport proteins at membrane contact sites, where two organelles are closely apposed, play key roles in trafficking lipids between cellular compartments while distinct membrane compositions for each organelle are maintained. Understanding the mechanisms underlying non-vesicular lipid trafficking requires characterization of the lipid transporters residing at contact sites. Here, we show that the mammalian proteins in the lipid transfer proteins anchored at a membrane contact site (LAM) family, called GRAMD1a-c, transfer sterols with similar efficiency as the yeast orthologues, which have known roles in sterol transport. Moreover, we have determined the structure of a lipid transfer domain of the yeast LAM protein Ysp2p, both in its apo-bound and sterol-bound forms, at 2.0 Å resolution. It folds into a truncated version of the steroidogenic acute regulatory protein-related lipid transfer (StART) domain, resembling a lidded cup in overall shape. Ergosterol binds within the cup, with its 3-hydroxy group interacting with protein indirectly via a water network at the cup bottom. This ligand binding mode likely is conserved for the other LAM proteins and for StART domains transferring sterols.

Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3.

Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization. While Nef-mediated CD4 downmodulation is conserved, the ability to internalize CD3 was lost in HIV-1 and its precursors. Although both functions play key roles in lentiviral replication and pathogenicity, the underlying structural requirements are poorly defined. Here, we determine the structure of SIVmac239 Nef bound to the ExxxLM motif of another Nef molecule at 2.5 Å resolution. This provides a basis for a structural model, where a hydrophobic crevice in simian immunodeficiency virus (SIV) Nef targets a dileucine motif in CD4 and a tyrosine-based motif in CD3. Introducing key residues into this crevice of HIV-1 Nef enables CD3 binding but an additional N-terminal tyrosine motif is required for internalization. Our resolution of the CD4/Nef/AP2 complex and generation of HIV-1 Nefs capable of CD3 downregulation provide insights into sorting motif interactions and target discrimination of Nef.HIV and simian immunodeficiency virus (SIV) Nef proteins both stimulate the clathrin-mediated endocytosis of CD4 but differ in downmodulation of the immune receptor CD3. Here, the authors present the structure of SIV Nef bound to the ExxxLM motif of another Nef molecule, which allows them to propose a model how Nef recognizes these motifs in CD3 and CD4.

The Legionella Anti-autophagy Effector RavZ Targets the Autophagosome via PI3P- and Curvature-Sensing Motifs.

Autophagy is a conserved membrane transport pathway used to destroy pathogenic microbes that access the cytosol of cells. The intracellular pathogen Legionella pneumophila interferes with autophagy by delivering an effector protein, RavZ, into the host cytosol. RavZ acts by cleaving membrane-conjugated Atg8/LC3 proteins from pre-autophagosomal structures. Its remarkable efficiency allows minute quantities of RavZ to block autophagy throughout the cell. To understand how RavZ targets pre-autophagosomes and specifically acts only on membrane-associated Atg8 proteins, we elucidated its structure. Revealed is a catalytic domain related in fold to Ulp family deubiquitinase-like enzymes and a C-terminal PI3P-binding module. RavZ targets the autophagosome via the PI3P-binding module and a catalytic domain helix, and it preferentially binds high-curvature membranes, intimating localization to highly curved domains in autophagosome intermediate membranes. RavZ-membrane interactions enhance substrate affinity, providing a mechanism for interfacial activation that also may be used by host autophagy proteins engaging only lipidated Atg8 proteins.

Legionella pneumophila subversion of host vesicular transport by SidC effector proteins.

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover monoubiquitination of Rab1 as a new SidC-dependent activity. The crystal structure of the SidC N-terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.

Exogenous nef induces proinflammatory signaling events in murine macrophages.

Despite the fact that murine cells are not permissive for human immunodeficiency virus type 1 (HIV-1) infection, several investigators have constructed transgenic (Tg) mice to model HIV-1-induced diseases to overcome this restriction. The generation of Tg mice expressing selected HIV-1 genes revealed that Nef harbors a major disease determinant. HIV-1 Nef protein is a molecular adapter able to interact with several cellular partners, interfering with cellular functions. The phenotype of Nef Tg mice was extensively characterized regarding in vivo development of AIDS-like disease and the effects of Nef expression in T lymphocytes, but the functions eventually corrupted by Nef in monocytes and macrophages were less studied. Nef treatment of human monocyte-derived macrophages induces the internalization of the protein and modulates the production and secretion of different chemokines and cytokines by activating specific intracellular signaling pathways (i.e., NF-κB, MAPK, and IRF3). Therefore we set up an in vitro murine macrophage-based model using stabilized cell lines and primary peritoneal macrophages, and treated them with recombinant myristoylated Nef(SF2) (recNef). Like human cells, murine macrophages responded to Nef treatment, activating IKK-α and IKK-ß, JNK, and p38 MAP kinases. Activation of the NF-κB pathway is mandatory for the synthesis and release of a pool of cytokines and chemokines, including IFN-ß, that induce tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)-1, STAT-2, and STAT-3, in an autocrine and paracrine manner, confirming that murine macrophages respond to Nef similarly to human ones. These data extend the results previously obtained in human primary macrophages, allowing the use of murine cells in culture to study signaling events modulated by Nef in myeloid-derived cells. In particular, it may be feasible to use macrophages derived from mice knocked out in specific signaling intermediates to obtain greater insight into the mechanism of Nef-induced effects.

Nef surfaces: where to interfere with function.

The HIV-1 Nef protein is an accessory protein of 24-27 kDa mass that mediates a multitude of effector functions in infected cells. Although not essentially required for viral replication, HIV-1 Nef exhibits stimulating potential towards disease progression to AIDS and is therefore considered a pathogenic factor in retroviridae. Here we correlate sequence conservation in HIV-1 Nef with surface hydrophobicity and functionality in protein-protein interaction to identify accessible substructures on the surface of Nef that might be suitable as pharmacological target sites. Recent advances in targeting of Nef by small molecular compounds that interfere with SH3 domain binding or MHC class I down-regulation are discussed. Similarly, approaches for the use of larger molecules are introduced, such as tailored fusion proteins that simultaneously interact with multiple highly conserved sequence motifs of Nef. In addition, the design of a single domain antibody from llama that interferes with CD4 down-regulation activity and PAK2 binding is discussed. The flexibility in binding recognition is exemplarily shown for the modulation of RT-loop binding using engineered SH3 domains. The various considerations corroborate the potential of HIV-1 Nef as a promising target for the development of potent Nef inhibitors.

HIV-1 Nef induces proinflammatory state in macrophages through its acidic cluster domain: involvement of TNF alpha receptor associated factor 2.

BACKGROUND: HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNß to activate STAT1, -2 and -3. METHODOLOGY/PRINCIPAL FINDINGS: Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. CONCLUSIONS: Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNß.

Conformation of the dileucine-based sorting motif in HIV-1 Nef revealed by intermolecular domain assembly.

The human immunodeficiency virus 1 (HIV-1) Nef protein is a pathogenicity factor required for effective progression to AIDS, which modulates host cell signaling pathways and T-cell receptor internalization. We have determined the crystal structure of Nef, allele SF2, in complex with an engineered SH3 domain of human Hck showing unnaturally tight binding and inhibitory potential toward Nef. This complex provides the most complete Nef structure described today, and explains the structural basis of the high affinity of this interaction. Intriguingly, the 33-residue C-terminal flexible loop is resolved in the structure by its interactions with a highly conserved hydrophobic groove on the core domain of an adjacent Nef molecule. The loop mediates the interaction of Nef with the cellular adaptor protein machinery for the stimulated internalization of surface receptors. The endocytic dileucine-based sorting motif is exposed at the tip of the acidic loop, giving the myristoylated Nef protein a distinctly dipolar character. The intermolecular domain assembly of Nef provides insights into a possible regulation mechanism for cargo trafficking.

DLC1 activation requires lipid interaction through a polybasic region preceding the RhoGAP domain.

Deleted in Liver Cancer 1 (DLC1) is a GTPase-activating protein (GAP) with specificity for RhoA, RhoB, and RhoC that is frequently deleted in various tumor types. By inactivating these small GTPases, DLC1 controls actin cytoskeletal remodeling and biological processes such as cell migration and proliferation. Here we provide evidence that DLC1 binds to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) through a previously unrecognized polybasic region (PBR) adjacent to its RhoGAP domain. Importantly, PI(4,5)P(2)-containing membranes are shown to stimulate DLC1 GAP activity in vitro. In living cells, a DLC1 mutant lacking an intact PBR inactivated Rho signaling less efficiently and was severely compromised in suppressing cell spreading, directed migration, and proliferation. We therefore propose that PI(4,5)P(2) is an important cofactor in DLC1 regulation in vivo and that the PBR is essential for the cellular functions of the protein.