Search

Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (mu2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and mu2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in mu2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2.

The negative factor (Nef) is one of six accessory proteins from primate lentiviruses (HIV-1, HIV-2, and SIV). It leads to high levels of viremia and the progression to AIDS in monkeys and humans. In this study, we demonstrated that Nef from HIV-1 binds to the regulatory subunit (p85) of phosphatidylinositol-3-kinase (PI3K). This interaction depended on the C-terminus of p85 and Nef. Moreover, PI3K was required to activate the Nef-associated p21-activated kinase (PAK). Finally, inhibition of PI3K blocked the activation of PAK and decreased the production of viral particles to levels observed with the Nef-deleted provirus. We conclude that Nef assembles a multiprotein signaling complex which is required for the optimal replication of HIV-1.

Small GTPases of the Ras family are major players of signal transduction in eukaryotic cells. They receive signals from a number of receptors and transmit them to a variety of effectors. The distribution of signals to different effector molecules allows for the generation of opposing effects like proliferation and differentiation. To understand the specificity of Ras signaling, we investigated the activation of RalGDS, one of the Ras effector proteins with guanine-nucleotide exchange factor activity for Ral. We determined the GTP level on RalA and showed that the highly conserved Ras binding domain (RBD) of RalGDS, which mediates association with Ras, is important but not sufficient to explain the stimulation of the exchange factor. Although a point mutation in the RBD of RalGDS, which abrogates binding to Ras, renders RalGDS independent to activated Ras, an artificially membrane-targeted version of RalGDS lacking its RBD could still be activated by Ras. The switch II region of Ras is involved in the activation, because the mutant Y64W in this region is impaired in the RalGDS activation. Furthermore, it is shown that Rap1, which was originally identified as a Ras antagonist, can block Ras-mediated RalGDS signaling only when RalGDS contains an intact RBD. In addition, kinetic studies of the complex formation between RalGDS-RBD and Ras suggest that the fast association between RalGDS and Ras, which is analogous to the Ras/Raf case, achieves signaling specificity. Conversely, the Ras x RalGDS complex has a short lifetime of 0.1 s and Rap1 forms a long-lived complex with RalGDS, possibly explaining its antagonistic effect on Ras.

Lipid rafts, also known as detergent-resistant membranes (DRM), are microdomains in the plasma membrane enriched in sphingolipids and cholesterol (reviewed in [1, 2]). Human immunodeficiency virus 1 (HIV) buds via lipid rafts [3, 4]. However, the targeting of viral structural components to DRM and its consequences for viral replication are not understood. Moreover, the negative factor Nef from HIV increases viral infectivity (reviewed in [5, 6]). With no apparent differences in structural components and morphology between wild-type and ΔNef virons, the latter viruses display less efficient reverse transcription in target cells. As Nef is expressed abundantly early in the viral replicative cycle [7], we hypothesized that Nef could affect viral morphogenesis and budding to render viruses more infectious. In this report, we demonstrated first that Nef increases viral budding from lipid rafts. Second, in the presence of Nef, viral envelopes contain more ganglioside (GM1), which is a major component of lipid rafts. This finding correlated directly with the increased infectivity of HIV. Finally, the depletion of exogenous and endogenous cholesterol biochemically and genetically, which disrupted lipid rafts, decreased viral infectivity only in the presence of Nef. Importantly, HIV lacking the nef gene remained unaffected by these manipulations. We conclude that lipids in virions are essential for viral infectivity. Thus, HIV becomes more infectious when it buds from lipid rafts, and Nef plays a major role in this process.

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.

To understand the system of secreted proteins and receptors involved in cell-cell signaling, we produced a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins, which constitute most of the protein components of the extracellular space. Each protein was tested in a suite of assays that measured metabolic, growth, or transcriptional responses in diverse cell types. The pattern of responses across assays was analyzed for the degree of functional selectivity of each protein. One of the highly selective proteins was a previously undescribed ligand, designated interleukin-34 (IL-34), which stimulates monocyte viability but does not affect responses in a wide spectrum of other assays. In a separate functional screen, we used a collection of extracellular domains of transmembrane proteins to discover the receptor for IL-34, which was a known cytokine receptor, colony-stimulating factor 1 (also called macrophage colony-stimulating factor) receptor. This systematic approach is thus useful for discovering new ligands and receptors and assessing the functional selectivity of extracellular regulatory proteins.

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.

The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein β-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras·RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.

Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.

To identify novel protein therapeutics we have utilized an integrated screening system in which a collection of full-length human cDNA clones, encoding virtually all secreted proteins and receptors, was assembled. Soluble secreted proteins were expressed in a high-throughput format using human cells as the expression host and tested in high-throughput cell-based assays. Through this approach, a novel cytokine, FPT025, was identified in a human monocyte proliferation assay. FPT025 has no apparent sequence homology to known cytokines or any other genes and is expressed in human spleen, skin, brain, and other tissues. The purified recombinant FPT025 protein stimulated human primary monocyte proliferation and/or survival. FPT025 protein specifically bound to human primary monocytes and activated ERK1/2 phosphorylation in primary monocytes as well as in a human monocytic cell line, THP-1. FPT025 promoted formation of the myeloid lineage colonies, CFU-M, in a human bone marrow colony formation assay and enhanced proliferation of cells with myeloid cell surface markers from human monocytes. To identify the receptor of FPT025, a collection of extracellular domains (ECD) of transmembrane proteins was screened for their ability to block FPT025 activation of monocyte proliferation. In this screen, we found that FPT025 binds to the M-CSF receptor (M-CSFR) with high affinity. Furthermore, we showed that the binding of FPT025 to M-CSFR was specific and could be competed by M-CSF. The soluble ECD of M-CSFR inhibited both the binding of FPT025 to M-CSFR and the activity of FPT025 in monocyte proliferation. Therefore, FPT025 functions as a novel ligand of the M-CSF receptor and participates in the regulation of myeloid lineage differentiation, proliferation, and survival.