The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain-like fold in solution, with two perpendicular, antiparallel, three- and five-stranded beta-sheets. In contrast to known structures with the SH3 domain fold, MIA is a single-domain protein, and contains an additional antiparallel beta-sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain-like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin alpha4beta1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.     

Targeting melanoma metastasis and immunosuppression with a new mode of melanoma inhibitory activity (MIA) protein inhibition

Melanoma is the most aggressive form of skin cancer, with fast progression and early dissemination mediated by the melanoma inhibitory activity (MIA) protein. Here, we discovered that dimerization of MIA is required for functional activity through mutagenesis of MIA which showed the correlation between dimerization and functional activity. We subsequently identified the dodecapeptide AR71, which prevents MIA dimerization and thereby acts as a MIA inhibitor. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy demonstrated the binding of AR71 to the MIA dimerization domain, in agreement with in vitro and in vivo data revealing reduced cell migration, reduced formation of metastases and increased immune response after AR71 treatment. We believe AR71 is a lead structure for MIA inhibitors. More generally, inhibiting MIA dimerization is a novel therapeutic concept in melanoma therapy.     

Melanoma inhibitory activity (MIA): an important molecule in melanoma development and progression

Cutaneous malignant melanoma is the leading cause of skin cancer death in industrialized countries. Melanoma development and progression are well defined by clinical and histopathological aspects; however, detailed analysis of molecular changes is still ongoing. The protein MIA, which is strongly expressed in melanoma cells but not in melanocytes, is likely to represent a key molecule regulating melanoma progression. Consistent with this, several in vitro and in vivo model systems indicate a direct involvement of MIA in melanoma migration and invasion, with recent studies suggesting a central role for MIA in early melanoma development by regulating important melanoma-related pathways and molecules. The latest developments in MIA research are summarized in this review, which describes recently published data related to the MIA protein structure and function, the role of MIA in melanoma development and progression, and the regulation of MIA expression. Furthermore, newly discovered MIA-homologous genes are discussed.     

Identification of an inhibitory circuit that regulates cerebellar Golgi cell activity

Here we provide evidence that revises the inhibitory circuit diagram of the cerebellar cortex. It was previously thought that Golgi cells, interneurons that are the sole source of inhibition onto granule cells, were exclusively coupled via gap junctions. Moreover, Golgi cells were believed to receive GABAergic inhibition from molecular layer interneurons (MLIs). Here we challenge these views by optogenetically activating the cerebellar circuitry to determine the timing and pharmacology of inhibition onto Golgi cells and by performing paired recordings to directly assess synaptic connectivity. In contrast to current thought, we find that Golgi cells, not MLIs, make inhibitory GABAergic synapses onto other Golgi cells. As a result, MLI feedback does not regulate the Golgi cell network, and Golgi cells are inhibited approximately 2?ms before Purkinje cells, following a mossy fiber input. Hence, Golgi cells and Purkinje cells receive unique sources of inhibition and can differentially process shared granule cell inputs.     

Isolation and characterization of a human collagen alpha 1(I)-like gene from a cosmid library

We have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends through most of the cloned DNA and must, therefore, be at least 35kb in length.     

Identification and characterization of a novel human myeloid inhibitory C-type lectin-like receptor (MICL) that is predominantly expressed on granulocytes and monocytes

Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the beta-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the beta-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function.     

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.     

Identification and characterization of adipokinetic hormone (Locusta migratoria)-like immunoreactivity in the human cerebrospinal fluid

Using an antiserum raised against locust adipokinetic hormone I, considerable quantity of adipokinetic hormone-like immunoreactivity was demonstrated in the human cerebrospinal fluid. The immunoreactivity was characterized by gel permeation and high performance liquid chromatography. The main immunoreactive component in the cerebrospinal fluid coeluted with adipokinetic hormone I. These results suggest that adipokinetic hormone may contribute to the neuronal function in the human central nervous system.     

BglII identifies a frequent biallelic DNA polymorphism of the human RT6 gene

A genomic probe of the human RT6 gene detects a frequent biallelic BglII polymorphism. Allele A has a frequency of 63%, whereas that of allele B is 37%. This restriction fragment length polymorphism provides the first known genetic marker for this gene.     

Identification of a translation inhibitory element (TIE) in the 3' untranslated region of the human interferon-beta mRNA

We have previously reported that the 3' untranslated region (UTR) of the human interferon-beta mRNA has an inhibitory effect on the mRNA translation both in vitro, in a rabbit reticulocyte lysate, and in vivo, in the Xenopus oocyte. In the present study, we identify the sequence in the 3' UTR which is responsible for this translation inhibition. We show that this sequence is located between the 100th and 161st nucleotides downstream from the translation stop codon. It contains several repeats of the A + U-rich consensus octanucleotide UUAUUUAU, which is also present in the 3' UTR of several mRNAs involved in the inflammatory response. We also demonstrate here that the inhibitory effect of the sequence on the mRNA translation does not depend on its position in relation to the termination codon. However, no inhibition of translation is observed when this sequence is inserted in the 5' UTR of the mRNA. The removal of the translation inhibitory sequence not only improves the mRNA translation in Xenopus oocytes but it also strongly decreases the IFN-beta mRNA stability in those cells. This suggests that, in this system at least, the mRNA degradation is linked to its translational efficiency.     

Molecular cloning and characterization of a novel peptidylprolyl isomerase (cyclophilin)-like gene (PPIL3) from human fetal brain.

During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated two cDNA clones encoding two novel proteins, which show 52% and 72% identity to the cyclophilin isoform 10 of C. elegans, respectively. Sequence analysis revealed these two cDNA clones are two different splicing variants of a novel cyclophilin-like gene (PPIL3). The PPIL3 gene was identified on a completely sequenced BAC (GenBank accession AC005037) from chromosome 2q33 between STS markers stSG2762 (proximal) and SHGC-3074 (distal), oriented toward the telomere. The PPIL3 gene consisted of eight exons spanning more than 18 kb of genomic DNA. RT-PCR analysis indicated that PPIL3 was ubiquitously expressed in adult human tissues.     

Identification of a zeta-crystallin (quinone reductase)-like 1 gene (CRYZL1) mapped to human chromosome 21q22.1

To identify a new gene(s) located on the yeast artificial chromosome (YAC) clone D142H8 that was mapped to human chromosome 21q22.1, purified YAC DNA from the clone was utilized directly as a probe to screen a human brain cDNA library after the suppression of human repetitive DNA. One cDNA clone hybridizing specifically to the YAC D142H8 DNA was identified. The clone has an insert of 1341 bp and the longest open reading frame of 349 amino acids. A search of GenBank revealed that the clone has a high degree of homology to zeta-crystallin (quinone reductase) at the amino acid level, and its nucleotide sequence represents the expressed sequence from the 50-kb segment of the human chromosome 21q11.1. Thus a new gene was named CRYZL1 (zeta-crystalline-like 1). Genomic Southern blot with total human and yeast DNAs suggests that CRYZL1 might be a single-copy gene. The fluorescence in situ hybridization procedure was applied, and the results showed that the gene mapped to the human chromosome 21q22.1 subband. The CRYZL1 mRNA was expressed in heart, brain, skeletal muscle, kidney, pancreas, liver, and lungs but at different levels in different tissues.