Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

Exploring the subsite-structure of vimelysin and thermolysin using FRETS-libraries

Vimelysin is a metalloproteinase with high activity at low temperature and an unusual resistance to organic solvents. Substrate specificities of vimelysin and thermolysin were examined using FRETS-libraries, revealing a significant difference at the P3' position: vimelysin preferred acidic amino acid residues, whereas thermolysin preferred basic residues. Homology modeling of vimelysin suggests that oppositely charged residues in the S3' subsites (R215 in vimelysin and D213 in thermolysin) may be responsible for this specificity difference. This hypothesis was confirmed by examining the R215D mutant of vimelysin, which showed a substrate specificity profile intermediate between thermolysin and vimelysin.     

Absence of leukotriene B4 receptor 1 confers resistance to airway hyperresponsiveness and Th2-type immune responses

Bronchial asthma is an increasingly common disorder that remains poorly understood and difficult to manage. The disease is characterized by airway hyperresponsiveness, chronic inflammation, and mucus overproduction. Based on the finding that leukotriene B4 receptor 1 (BLT1) is expressed highly in Th2 lymphocytes, we analyzed the roles of BLT1 using an OVA-induced bronchial asthma model. BLT1-null mice did not develop airway hyperresponsiveness, eosinophilic inflammation, and hyperplasia of goblet cells. Attenuated symptoms were accompanied by reduced IgE production, and accumulation of IL-5 and IL-13 in bronchoalveolar lavage fluid, suggesting attenuated Th2-type immune response in BLT1-null mice. Peribronchial lymph node cells of sensitized BLT1-null mice showed much attenuated proliferation and production of Th2 cytokines upon re-stimulation with Ag in vitro. Thus, LTB4-BLT1 axis is required for the development of Th2-type immune response, and blockade of LTB4 functions through BLT1 would be novel and useful in the effort to ameliorate bronchial asthma and related Th2-biased immune disorders.     

DNA polymerase iota-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers

Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only approximately 25% of mutations occur at thymine (T), whilst 75% are targeted to cytosine (C). The mutational spectra changes dramatically in XP-V cells, devoid of poleta, where approximately 45% of mutations occur at Ts and approximately 55% at Cs. At the present time, it is unclear whether the C-->T mutations actually represent true misincorporations opposite C, or perhaps occur as the result of the correct incorporation of adenine (A) opposite a C in a UV-photoproduct that had undergone deamination to uracil (U). In order to assess the role that human poliota might play, if any, in the replicative bypass of such UV-photoproducts, we have analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurs more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3'U of the T-U CPD only two-fold less frequently than the correct Watson-Crick base, A. While pol iota generally extended the G:3'U-CPD mispairs less efficiently than the correctly paired primer, pol iota-dependent extension was equal to, or greater than that observed with human pols eta and kappa and S. cerevisiae pol zeta under the same assay conditions. Thus, we hypothesize that the ability of pol iota to bypass T-U CPDs through the frequent misincorporation of G opposite the 3'U of the CPD, may provide a mechanism whereby human cells can decrease the mutagenic potential of these lesions.     

Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site

Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.     

Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.     

Arkadia complexes with clathrin adaptor AP2 and regulates EGF signalling

Arkadia is a positive regulator of transforming growth factor (TGF)-β signalling that induces ubiquitin-dependent degradation of several inhibitory proteins of TGF-β signalling through its C-terminal RING domain. We report here that, through yeast-two-hybrid screening for Arkadia-binding proteins, the μ2 subunit of clathrin-adaptor 2 (AP2) complex was identified as an interacting partner of Arkadia. Arkadia was located in both the nucleus and the cytosol in mammalian cells. The C-terminal YXXΦ-binding domain of the μ2 subunit associated with the N-terminal YALL motif of Arkadia. Arkadia ubiquitylated the μ2 subunit at Lys130. In addition, Arkadia interacted with the AP2 complex, and modified endocytosis of epidermal growth factor receptor (EGFR) induced by EGF. Arkadia thus appears to regulate EGF signalling by modulating endocytosis of EGFR through interaction with AP2 complex.     

Smad7 Inhibits Transforming Growth Factor-β Family Type I Receptors through Two Distinct Modes of Interaction

The inhibitory Smads (I-Smads), i.e. Smad6 and Smad7, are negative regulators of transforming growth factor-β (TGF-β) family signaling. I-Smads inhibit TGF-β family signaling principally through physical interaction with type I receptors (activin receptor-like kinases), so as to compete with receptor-regulated Smads (R-Smads) for activation. However, how I-Smads interact with type I receptors is not well understood. In the present study, we found that Smad7 has two modes of interaction with type I receptors. One is through a three-finger-like structure in the MH2 domain, consisting of residues 331–361, 379–387, and the L3 loop. The other is through a basic groove in the MH2 domain (Mochizuki, T., Miyazaki, H., Hara, T., Furuya, T., Imamura, T., Watabe, T., and Miyazono, K. (2004) J. Biol. Chem. 279, 31568–31574). We also found that Smad6 principally utilizes a basic groove in the MH2 domain for interaction with type I receptors. Smad7 thus has an additional mode of interaction with TGF-β family type I receptors not possessed by Smad6, which may play roles in mediating the inhibitory effects unique to Smad7.     

Liver sinusoidal endothelial cells that endocytose allogeneic cells suppress T cells with indirect allospecificity

A portal venous injection of allogeneic donor cells is known to prolong the survival of subsequently transplanted allografts. In this study, we investigated the role of liver sinusoidal endothelial cells (LSECs) in immunosuppressive effects induced by a portal injection of allogeneic cells on T cells with indirect allospecificity. To eliminate the direct CD4+ T cell response, C57BL/6 (B6) MHC class II-deficient C2tatm1Ccum (C2D) mice were used as donors. After portal injection of irradiated B6 C2D splenocytes into BALB/c mice, the host LSECs that endocytosed the irradiated allogeneic splenocytes showed enhanced expression of MHC class II molecules, CD80, and Fas ligand (FasL). Due to transmigration across the LSECs from BALB/c mice treated with a portal injection of B6 C2D splenocytes, the naive BALB/c CD4+ T cells lost their responsiveness to stimulus of BALB/c splenic APCs that endocytose donor-type B6 C2D alloantigens, while maintaining a normal response to stimulus of BALB/c splenic APCs that endocytose third-party C3H alloantigens. Similar results were not observed for naive BALB/c CD4+ T cells that transmigrated across the LSECs from BALB/c FasL-deficient mice treated with a portal injection of B6 C2D splenocytes. Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts; however, a similar effect was not observed for BALB/c FasL-deficient LSECs. These findings indicate that LSECs that had endocytosed allogeneic splenocytes have immunosuppressive effects on T cells with indirect allospecificity, at least partially via the Fas/FasL pathway.