Identification of a WT1 protein‐derived peptide,WT1187, as a HLA‐A*0206‐restricted,WT1‐specific CTL epitope

The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1‐specific CTL epitopes with a restriction of HLA‐A*2402 or HLA‐A*0201 have been already identified. In the present study it has been demonstrated that a 9‐mer WT1‐derived WT1₁₈₇ peptide, which had already been shown to elicit a WT1‐specific CTL response with a restriction of HLA‐A*0201, can also elicit a CTL response with a restriction of HLA‐A*0206. In all three different HLA‐A*0206⁺ healthy donors examined, WT1₁₈₇ peptide‐specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA‐A*0206 molecules. The present study describes the first identification of a HLA‐A*0206‐restricted, WT1‐specific CTL epitope. The present results should help to broaden the application of WT1 peptide‐based immunotherapy from only HLA‐A*0201‐positive to HLA‐A*0206‐positive cancer patients as well.     

Purification and characterization of a rice class I chitinase, OsChia1b, produced in Esherichia coli

To determine the properties and structure of OsChia1b, a family 19 chitinase from Oryza sativa L. cv. Nipponbare (japonica ssp.), recombinant OsChia1b was produced in Esherichia coli cells and purified to homogeneity by chitin affinity column chromatography. OsChia1b was highly active against soluble chitinous substrate, but not against crystalline chitin, and clearly inhibited hyphal extension of Trichoderma reesei.     

Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. Most recently, the non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. We analyze the function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analysis with different Dsh mutants and Wnt11 demonstrated that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/beta-catenin pathway, to regulate gastrulation movements. We also provide evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, our results suggest that Xgly4 functions as positive regulator in non-canonical Wnt/PCP signaling during gastrulation.     

Tetraspanin CD63 promotes targeting and lysosomal proteolysis of membrane-type 1 matrix metalloproteinase

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is known to be internalized from cell surface, however, the fate of internalized MT1-MMP is still unknown. Here we demonstrate that at least a part of internalized MT1-MMP is targeted for lysosomal proteolysis. Treatment with an inhibitor of lysosomal proteinases chloroquine suppressed degradation of internalized MT1-MMP and induced accumulation of MT1-MMP in CD63-positive lysosomes. Ectopic expression of CD63 accelerated degradation of MT1-MMP, which was blocked by chloroquine. MT1-MMP, and CD63 were shown to form a complex through hemopexin-like domain of MT1-MMP and N-terminal region of CD63, and thus accelerated degradation of MT1-MMP was not observed with mutants lacking these domains. CD63 mutant lacking lysosomal targeting motif was unable to promote MT1-MMP degradation. These results suggest that CD63 regulates MT1-MMP by targeting to lysosomes.     

Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4: tolerance of a CDR peptide to conformational changes of a target

Somatic angiotensin-converting enzyme (ACE) consists of two homologous domains, each domain bearing a catalytic site. Differential scanning calorimetry of the enzyme revealed two distinct thermal transitions with melting points at 55.3 and 70.5 degrees C. which corresponded to denaturation of C- and N-domains, respectively. Different heat stability of the domains underlies the methods of acquiring either single active N-domain or active N-domain with inactive C-domain within parent somatic ACE. Selective denaturation of C-domain supports the hypothesis of independent folding of the two domains within the ACE molecule. Modeling of ACE secondary structure revealed the difference in predicted structures of the two domains, which, in turn, allowed suggestion of the region 29-133 in amino acid sequence of the N-part of the molecule as responsible for thermostability of the N-domain.     

Crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase complexed with cofactors: implications of a flexible loop movement upon substrate binding

The key enzyme in the nonmevalonate pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be an effective target of antimalarial drugs. Here we report the crystal structure of DXR complexed with NADPH and a sulfate ion from Escherichia coli at 2.2 A resolution. The structure showed the presence of an extra domain, which is absent from other NADPH-dependent oxidoreductases, in addition to the conformation of catalytic residues and the substrate binding site. A flexible loop covering the substrate binding site plays an important role in the enzymatic reaction and the determination of substrate specificity.     

CUB domain-containing protein 1 is a novel regulator of anoikis resistance in lung adenocarcinoma

Malignant tumor cells frequently achieve resistance to anoikis, a form of apoptosis induced by detachment from the basement membrane, which results in the anchorage-independent growth of these cells. Although the involvement of Src family kinases (SFKs) in this alteration has been reported, little is known about the signaling pathways involved in the regulation of anoikis under the control of SFKs. In this study, we identified a membrane protein, CUB-domain-containing protein 1 (CDCP1), as an SFK-binding phosphoprotein associated with the anchorage independence of human lung adenocarcinoma. Using RNA interference suppression and overexpression of CDCP1 mutants in lung cancer cells, we found that tyrosine-phosphorylated CDCP1 is required to overcome anoikis in lung cancer cells. An apoptosis-related molecule, protein kinase Cdelta, was found to be phosphorylated by the CDCP1-SFK complex and was essential for anoikis resistance downstream of CDCP1. Loss of CDCP1 also inhibited the metastatic potential of the A549 cells in vivo. Our findings indicate that CDCP1 is a novel target for treating cancer-specific disorders, such as metastasis, by regulating anoikis in lung adenocarcinoma.     

ANG II type 1A receptor signaling causes unfavorable scar dynamics in the postinfarct heart

Blockade of ANG II type 1A receptor (AT(1A)) is known to attenuate postinfarction [postmyocardial infarction (post-MI)] heart failure, accompanying reduction in fibrosis of the noninfarcted area. In the present study, we investigated the influence of AT(1A) blockade on the infarcted tissue itself. Consistent with earlier reports, AT(1A) knockout (AT(1A)KO) mice showed significantly attenuated left ventricular (LV) remodeling (dilatation) and dysfunction compared with wild-type (WT) mice. Morphometry revealed that the infarcted wall was thicker and had a smaller circumferential length in AT(1A)KO than WT hearts. In addition, significantly greater numbers of cells were present within infarcts in AT(1A)KO hearts 4 wk post-MI; most notably, there was an abundance of vessels and myofibroblasts. One week post-MI, the incidence of apoptosis among granulation tissue cells was fewer (3.3 +/- 0.4 vs. 4.4 +/- 0.5% in WT, P < 0.05), whereas vessel proliferation was higher in AT(1A)KO hearts, which likely explains the later abundance of cells within the scar tissue. Insulin-like growth factor receptor-I was upregulated and its downstream signal protein kinase B (Akt) was significantly activated in infarcted AT(1A)KO hearts compared with WT hearts. Inactivation of Akt with wortmannin partially but significantly prevented the benefits observed in AT(1A)KO. Collectively, in AT(1A)KO hearts, Akt-mediated granulation tissue cell proliferation and preservation resulting from antiapoptosis likely contributed to an abundant cell population that altered the infarct scar structure, thereby reducing wall stress and attenuating LV dilatation and dysfunction at the chronic stage. In conclusion, altered structural dynamics of infarct scar and increasing myocardial fibrosis may be responsible for the deleterious effects of AT(1A) signaling following MI.