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.Ras plays a central role in cellular signal transduction by distributing many different extracellular signals into distinct cellular responses through activation of various downstream pathways (1). In its resting state, Ras is in a GDP-bound form and becomes activated by stimulated exchange of GDP for GTP (2). The GTP form of Ras binds more tightly to the so-called effector molecules than the GDP form and as a result of this interaction initiates, in the case of Raf, a protein phosphorylation cascade (3) that eventually leads to cell proliferation. Alternatively, other activities may be induced, such as Ral-GDS 1 (4) or phosphatidylinositol 3-kinase (5). Using pull-down or two-hybrid assays, many other proteins were identified as Ras targets by their GTP-dependent association, all of which are sensitive to mutations in the effector-binding region of Ras (6). However, for many of these Ras targets an assigned biological function is yet to be determined. For instance, no sequence homology and no functional relationship has been ascribed to the putative Ras effectors AF6 (7) and Byr2 (8), the Schizosaccharomyces pombe homologue of the mammalian MEKK1 (9). Another avenu...

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The ubiquitin superfold appears to be the general structural motif for Ras binding domains even in cases with vanishing sequence identity. However, details of the 3D structure and the interacting interface are different, thereby determining the specifity of the recognition of Ras and Ras-related proteins.

AIM:To characterize the IFN-response and its modulation by the antiviral compound lamivudine in HBVtransfected HepG2.2.15 cells. METHODS:H e p G 2 . 2 . 1 5 a n d H e p G 2 c e l l s w e r e stimulated with various concentrations of IFN-a2a in the presence or absence of lamivudine. Then, total RNA was extracted and analysed by customised cDNA arrays and northern blot for interferon-inducible genes (ISGs). In addition, cellular proteins were extracted for EMSA and western blot. HBV replication was assessed by southern blot or ELISAs for HBsAg and HBeAg. RESULTS:Two genes (MxA, Cig5) with completely abolished and 4 genes (IFITM1, -2, -3, and 6-16) with partially reduced IFN-responses were identified in HepG2.2.15 cells. In 2 genes (IFITM1, 6-16), the response to IFN-a could be restored by treatment with lamivudine. This effect could not be explained by a direct modulation of the Jak/Stat signalling pathway since EMSA and western blot experiments revealed no suppression of Stat1 activation and ISGF3 formation after stimulation with IFN-a in HepG2.2.15 compared to HepG2 cells. CONCLUSION:These results are consistent with the assumption that chronic hepatitis B may specifically modulate the cellular response to IFN by a selective blockage of some ISGs. Antiviral treatment with lamivudine may partially restore ISG expression by reducing HBV gene expression and replication.

The structure of the Ras-Byr2 complex confirms the Ras binding module as a communication element mediating Ras-effector interactions; the Ras-Byr2 complex is also conserved in a lower eukaryotic system like yeast, which is in contrast to other small GTPase families. The extra helical segment might be involved in kinase activation.