p21-activated kinase 1 plays a critical role in cellular activation by Nef

The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.     

Maintenance of immune tolerance depends on normal tissue homeostasis

Ags expressed at immune privileged sites and other peripheral tissues are able to induce T cell tolerance. In this study, we analyzed whether tolerance toward an intraocular tumor expressing a highly immunogenic CTL epitope is maintained, broken, or reverted into immunity in the event the anatomical integrity of the eye is lost. Inoculation of tumor cells into the anterior chamber of the eye of naive B6 mice leads to progressive intraocular tumor growth, an abortive form of CTL activation in the tumor-draining submandibular lymph node, and systemic tolerance as evidenced by the inability of these mice to reject an otherwise benign tumor cell inoculum. Loss of anatomical integrity of the eye as a consequence of phthisis resulted in loss of systemic tolerance and the emergence of effective antitumor immunity against an otherwise lethal tumor challenge. Phthisis was accompanied by dendritic cell maturation and preceded the induction of systemic tumor-specific CTL immunity. Our data show that normal tissue homeostasis and anatomical integrity is required for the maintenance of ocular tolerance and prevention of CTL-mediated immunity. These data also indicate that tissue injury in the absence of viral or microbial infection can act as a switch for the induction of CTL immunity.     

Negative factor from SIV binds to the catalytic subunit of the V-ATPase to internalize CD4 and to increase viral infectivity

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 SIV(mac239) for 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.     

Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts: the active-state conformation of the enzyme does not affect cell motility but is important for its secretion

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.     

Characterizing Protein Interactions Employing a Genome-Wide siRNA Cellular Phenotyping Screen

Characterizing the activating and inhibiting effect of protein-protein interactions (PPI) is fundamental to gain insight into the complex signaling system of a human cell. A plethora of methods has been suggested to infer PPI from data on a large scale, but none of them is able to characterize the effect of this interaction. Here, we present a novel computational development that employs mitotic phenotypes of a genome-wide RNAi knockdown screen and enables identifying the activating and inhibiting effects of PPIs. Exemplarily, we applied our technique to a knockdown screen of HeLa cells cultivated at standard conditions. Using a machine learning approach, we obtained high accuracy (82% AUC of the receiver operating characteristics) by cross-validation using 6,870 known activating and inhibiting PPIs as gold standard. We predicted de novo unknown activating and inhibiting effects for 1,954 PPIs in HeLa cells covering the ten major signaling pathways of the Kyoto Encyclopedia of Genes and Genomes, and made these predictions publicly available in a database. We finally demonstrate that the predicted effects can be used to cluster knockdown genes of similar biological processes in coherent subgroups. The characterization of the activating or inhibiting effect of individual PPIs opens up new perspectives for the interpretation of large datasets of PPIs and thus considerably increases the value of PPIs as an integrated resource for studying the detailed function of signaling pathways of the cellular system of interest.     

Diverse associations in the ternary systems of β-cyclodextrin, simple carbohydrates and phenyl derivatives of inorganic oxoacids

Complex formation reactions of phenylboronic, phenylphosphonic, phenylarsonic and 4-aminophenyl arsonic acids with β-cyclodextrin (cycloheptaamylose, β-CD) and some simple carbohydrates (mannitol, sorbitol, glucose) have been studied using spectrophotometric, potentiometric methods and solubility measurements, supplemented with HPLC and IR analyses of the solid samples. Equilibrium constants have been determined at ionic strength of 0.2 M (NaCl) and 25 °C. β-CD forms the most stable complexes with the neutral, undissociated forms of the acids, the stability constants are as follows: phenylboronic acid: 320 ± 36, phenylphosphonic acid: 108 ± 25, phenylarsonic acid: 97 ± 4 and 4-aminophenyl arsonic acid: 107 ± 10. The stability constants for the β–CD-complexes of the ionic forms are much lower. Ternary complexes of low stability could be detected in the case of phenylphosphonic acid and sorbitol with the undissociated form and with glucose and the dianion. In more concentrated solutions phenylboronic acid forms insoluble complexes with mannitol, sorbitol and β-CD. The solid phases obtained in the ternary systems are predominantly mixtures of ester type 3:1 complexes with the carbohydrate and 1:1 inclusion complex with the β-CD. No significant interaction has been found with glucose. The phenomena can be explained by the differences in the structures of the components and by the changes in the H-bonding network of β-CD on the complex formation.