Identification of a mutated receptor-like protein tyrosine phosphatase kappa as a novel,class II HLA-restricted melanoma antigen

Recent studies increasingly point to a pivotal role of CD4(+) T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4(+) T cells recognizing the autologous tumor cells in vitro. In fact, the CD4(+) T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase kappa was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4(+) T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient's peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4(+) T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.     

Defective dendrite elongation but normal fertility in mice lacking the Rho-like GTPase activator Dbl

Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation.     

Cleavage of chromogranin A N-terminal domain by plasmin provides a new mechanism for regulating cell adhesion

It has been proposed that chromogranin A (CgA), a protein secreted by many normal and neoplastic neuroendocrine cells, can play a role as a positive or a negative modulator of cell adhesion. The mechanisms that regulate these extracellular functions of CgA are unknown. We show here that plasmin can regulate the anti/pro-adhesive activity of CgA by proteolytic cleavage of the N-terminal domain. Limited proteolytic processing decreased its anti-adhesive activity and induced pro-adhesive effects in fibronectin or serum-dependent fibroblast adhesion assays. Cleavage of Lys(77)-Lys(78) dibasic site in CgA(1-115) was relatively rapid and associated with an increase of pro-adhesive effect. In contrast, antibodies against the region 53-90 enhanced the anti-adhesive activity of CgA and CgA(1-115). Structure-activity relationship studies showed that the conserved region 47-64 (RILSILRHQNLLKELQDL) is critical for both pro- and anti-adhesive activity. These findings suggest that CgA might work on one hand as a negative modulator of cell adhesion and on the other hand as a precursor of positive modulators, the latter requiring proteolytic processing for activation. Given the importance of plasminogen activation in tissue invasion and remodeling, the interplay between CgA and plasmin could provide a novel mechanism for regulating fibroblast adhesion and function in neuroendocrine tumors.     

Human mitochondrial 5'-deoxyribonucleotidase. Overproduction in cultured cells and functional aspects

Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence for a mitochondrial 5'-nucleotidase (dNT2) with a pronounced specificity for dUMP and dTMP and suggested that the enzyme protects mitochondrial DNA replication from excess dTTP. In humans, accumulation of dTTP causes a mitochondrial genetic disease. We now establish that dNT2 in vivo indeed is located in mitochondria. The native enzyme shows the same substrate specificity and affinity for inhibitors as the recombinant dNT2. We constructed ponasterone-inducible cell lines overproducing dNT2 with and without the green fluorescent protein (GFP) linked to its C terminus. The fusion protein occurred in mitochondria mostly in an inactive truncated form, with only a short C-terminal fragment of dNT2 linked to GFP. No truncation occurred when dNT2 and GFP were not linked. The cell mitochondria then contained a large excess of active dNT2 with or without the mitochondrial presequence. After removal of ponasterone overproduced dNT2 disappeared only slowly from the cells, whereas dNT2-mRNA was lost rapidly. Overproduction of dNT2 did not lead to an increased excretion of pyrimidine deoxyribonucleosides, in contrast to overproduction of the corresponding cytosolic deoxynucleotidase, suggesting that the mitochondrial enzyme does not affect overall cellular deoxynucleotide turnover.     

Crystal structure of a human mitochondrial deoxyribonucleotidase

5' nucleotidases are ubiquitous enzymes that dephosphorylate nucleoside monophosphates and participate in the regulation of nucleotide pools. The mitochondrial 5'-(3') deoxyribonucleotidase (dNT-2) specifically dephosphorylates dUMP and dTMP, thereby protecting mitochondrial DNA replication from excess dTTP. We have solved the structure of dNT-2, the first of a mammalian 5' nucleotidase. The structure reveals a relationship to the HAD family, members of which use an aspartyl nucleophile as their common catalytic strategy, with a phosphoserine phosphatase as the most similar neighbor. A structure-based sequence alignment of dNT-2 with other 5' nucleotidases also suggests a common origin for these enzymes. Here we study the structures of dNT-2 in complex with bound phosphate and beryllium trifluoride plus thymidine as model for a phosphoenzyme-product complex. Based on these structures, determinants for substrate specificity recognition and the catalytic action of dNT-2 are outlined.     

Development of a high-throughput scintillation proximity assay for the identification of C-domain translational initiation factor 2 inhibitors

Translational initiation factor 2 (IF2) is the largest of the 3 factors required for translation initiation in prokaryotes and has been shown to be essential in Escherichia coli. It stimulates the binding of fMet-tRNA(f)(Met) to the 30S ribosomal subunit in the presence of GTP. The selectivity is achieved through specific recognition of the tRNA(f)(Met) blocked alpha-amino group. IF2 is composed of 3 structural domains: N-domain, whose function is not known; G-domain, which contains the GTP/GDP binding site and the GTPase catalytic center; and C-domain, which recognizes and binds fMet-tRNA(f)(Met). Its activity is strictly bacteria specific and highly conserved among prokaryotes. So far, antibiotics targeting IF2 function are not known, and this makes it an ideal target for new drugs with mechanisms of resistance not yet developed. A few assays have been developed in the past, which allow the detection of IF2 activity either directly or indirectly. In both instances, the assays are based on radioactive detection and do not allow for high throughput because of the need for separation or solvent extraction steps. The authors describe a novel biochemical assay for IF2 that exploits the molecular recognition of fMet-tRNA(f)(Met) by the C-domain. The assay is based on the incubation of biotinyl-IF2 with fMet-tRNA(f)(Met) and the subsequent capture of the radiolabeled complex by streptavidin-coated beads, exploiting the scintillation proximity assay (SPA) technology. The assay has been designed in an automatable, homogeneous, miniaturized fashion suitable for high-throughput screening and is rapid, sensitive, and robust to dimethyl sulfoxide (DMSO) up to 10% v/v. The assay, used to screen a limited chemical collection of about 5000 compounds and a subset of compounds originated by a 2-D substructural search, has shown to be able to detect potential IF2 inhibitors.     

Three anthrones from Rubus ulmifolius

From the aerial parts of Rubus ulmifolius Schott three new anthrones, rubanthrone A, B and C, have been isolated. Their structures were established by spectral procedures including 1D and 2D NMR techniques and chemical derivatization. Rubanthrone A showed antimicrobial activity against Staphylococcus aureus at 4.5 mg/ml.