No abstract

The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target of Cdc42 and Rac1, was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.

Naturally occurring peptides often possess macrocyclic and N-methylated backbone. These features grant them structural rigidity, high affinity to targets, proteolytic resistance, and occasionally membrane permeability. Because such peptides are produced by either nonribosomal peptide synthetases or enzymatic posttranslational modifications, it is yet a formidable challenge in degenerating sequence or length and preparing libraries for screening bioactive molecules. Here, we report a new means of synthesizing a de novo library of "natural product-like" macrocyclic N-methyl-peptides using translation machinery under the reprogrammed genetic code, which is coupled with an in vitro display technique, referred to as RaPID (random nonstandard peptides integrated discovery) system. This system allows for rapid selection of strong binders against an arbitrarily chosen therapeutic target. Here, we have demonstrated the selection of anti-E6AP macrocyclic N-methyl-peptides, one of which strongly inhibits polyubiqutination of proteins such as p53.

We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rac1 small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1 val12 (Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K.

A baculovirus (Autographa californica nucleopolyhedrovirus) vector containing a strong promoter, the CAG promoter, was developed to introduce foreign genes into mammalian cells. Recombinant baculoviruses carrying a reporter gene under the control of the CAG promoter were inoculated into various mammalian cell lines. High-level expression was observed not only in hepatocytes but also in other non-hepatic cell lines tested. Expression of the reporter gene was detected even 14 days after infection. The infectious titre of the recovered baculoviruses decreased significantly after infection, indicating that the baculoviruses did not repli-

IQGAP1, a target of Cdc42 and Rac1 small GTPases, directly interacts with ␤-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating ␣-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. Cell-cell adhesion is dynamically rearranged in various situations including the establishment of epithelial cell polarity, compaction of early embryogenesis, wound healing, cell scattering, and tumorigenesis (for reviews see Refs. 1-3). Cadherin is a well known calcium-dependent cell-cell adhesion molecule. The cytoplasmic domain of cadherin binds to ␤-catenin, and this complex is linked to the actin cytoskeleton by ␣-catenin. It is well known that this linkage is essential for the cadherinmediated cell-cell adhesion (for a review see Ref. 4). Therefore, it is likely that dynamic rearrangement of the cadherin-catenin complex is crucial for the above phenomena. However, little is known about the regulatory mechanism underlying the rearrangement of the cadherin-catenin complex.Cdc42 and Rac1, members of the Rho family, participate in the regulation of actin reorganization (for reviews see Refs. 5 and 6). Recent studies have suggested that they are required for maintaining the cadherin-mediated cell-cell adhesion (7-11). Target molecules for Cdc42 and Rac1 have been identified to be p21-activated kinase (12-14), WASP 1 (15, 16), IQGAP1 (17-19), and IQGAP2 (20), those for Cdc42 to be N-WASP (21) and MRCK-␣, -␤ (22), and those for Rac1 to be Sra1 (23), POR1 (24), and POSH (25). However, the mechanism underlying the regulation of cadherin-mediated cell-cell adhesion by Cdc42 and Rac1 has been unknown.We have recently found that IQGAP1 regulates the cadherinmediated cell-cell adhesion (10). IQGAP1 interacts with ␤-catenin and E-cadherin both in vitro and in vivo. The overexpression of IQGAP1 induces the dissociation of ␣-catenin from the cadherin-catenin complex and results in reduction of the Ecadherin-mediated cell adhesive activity in EL cells but not in L cells expressing E-cadherin mutant in which the cytoplasmic domain is deleted and replaced by the carboxyl-terminal half of ␣-catenin (nE␣CL cells) (26). The inhibitory effect of IQGAP1 on the E-cadherin-mediated cell-cell adhesion is counteracted by the coexpression of dominant active Cdc42 (Cdc42 Val12 ). Thus, IQGAP1 together with Cdc42 and Rac1 appear to regulate the cell-cell adhesion through the rearrangement of the cadherin-catenin complex. However, how Cdc42 and Rac1 regulate the IQGAP1 function remains to be clarified.In the present study, we investigated how Cdc42 and Rac1 regulate the IQGAP1 function. We found that IQGAP1 bound to the amino terminus of ␤-catenin and thereby dissociated ␣-catenin from the ␤-catenin-␣-catenin complex. GTP␥S⅐GST-

We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935Virol. 87: -1945Virol. 87: , 2006. Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).

Src family tyrosine kinases are involved in modulating various signal transduction pathways leading to the induction of DNA synthesis and cytoskeletal reorganization in response to cell-cell or cell-matrix adhesion. The critical role of these kinases in regulating cellular signaling pathways requires that their activity be tightly controlled. Src family proteins are regulated through reversible phosphorylation and dephosphorylation events that alter the conformation of the kinase. We have found evidence that Src also is regulated by ubiquitination. Activated forms of Src are less stable than either wild-type or kinase-inactive Src mutants and can be stabilized by proteasome inhibitors. In addition, poly-ubiquitinated forms of active Src have been detected in vivo. Taken together, our results establish ubiquitin-mediated proteolysis as a previously unidentified mechanism for irreversibly attenuating the effects of active Src kinase.